Corynebacter

Phytochemistry, 2005 Jan, 66(2), 241 - 247
Bioactive ellagitannins from Cunonia macrophylla, an endemic Cunoniaceae from New Caledonia; Fogliani B et al.; Chemical study of Cunonia macrophylla, a New Caledonian Cunoniaceae, based on bioactive effects of a crude methanol extract of the leaves, detected bioactive tannins for the first time in this plant family . These ellagitannins have been identified as ellagic acid-4-O-beta-d-xylopyranoside (6), mallorepanin (3), mallotinic acid (1) along with corilagin (2), chebulagic acid (4), ellagic acid (5) and gallic acid (7) and have been shown to possess antimicrobial activity and to inhibit xanthine oxidase . Antimicrobial effects on bacterial human pathogens (Staphylococcus aureus, Corynebacterium accolans) and on a plant pathogen (Erwinia carotovora) as well as on a human pathogenic yeast (Candida albicans) were investigated . Activity is reported here for the first time for compounds 1, 3, 4 and 6 . The inhibitory effects of all molecules against xanthine oxidase in relation to their structure was evaluated and compared . Compound 6 presented the best activity and seems to be of considerable interest for further studies.

J Mol Biol, 2005 Feb 4, 345(5), 1111 - 8 Epub 2004 Dec 13.
Crystal Structure of the Alginate (Poly alpha-l-guluronate) Lyase from Corynebacterium sp . at 1.2A Resolution; Osawa T et al.; The crystal structure of alginate (poly alpha-l-guluronate) lyase from Corynebacterium sp . (ALY-1) was determined at 1.2A resolution using the MAD method and bromide ions . The structure of ALY-1 is abundant in beta-strands and has a deep cleft, similar to the jellyroll beta-sandwich found in 1,3-1,4-beta-glucanase . The structure suggests that alginate molecules may penetrate into the cleft to interact with the catalytic site of ALY-1 . The reported crystal structure of another type of alginate lyase, A1-III, differs from that of ALY-1 in that it consists almost entirely of alpha-helical structure . Nevertheless, the putative catalytic residues in both enzymes are positioned in space in nearly identical arrangements . This finding suggests that both alginate lyases may have evolved through convergent evolution.

Appl Environ Microbiol, 2005 Jan, 71(1), 407 - 16
Isolation and Characterization of a Native Composite Transposon, Tn14751, Carrying 17.4 Kilobases of Corynebacterium glutamicum Chromosomal DNA; Inui M et al.; A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751 . This transposon comprises two functional copies of a corynebacterial IS31831-like insertion sequence organized as converging terminal inverted repeats . This novel 20.3-kb element, Tn14751, carries 17.4 kb of C . glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins . A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn14751, transposed into the genome of C . glutamicum at an efficiency of 1.8 x 10(2) transformants per mug of DNA . Random insertion of the Tn14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization . This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.

Appl Environ Microbiol, 2005 Jan, 71(1), 207 - 13
Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum; Elisakova V et al.; Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways . The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C . glutamicum shuttle vector pECKA (5.4 kb, Km(r)) . By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN . The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome . The enzyme containing the M13 mutation was feedback resistant to all three amino acids . Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%) . We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule . The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts . The plasmid-free C . glutamicum DeltailvA DeltapanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium . The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.

Klin Oczna, 2004, 106(3 Suppl), 528 - 9
{Microbiological evaluation of conjunctival sac in Sjögren syndrome patients}; Krajka-Lauer J et al.; PURPOSE: To evaluate bacterial flora of conjunctival sac in patients with Sjogren syndrome and compare it with the flora of healthy individuals . MATERIAL AND METHODS: Bacteriological test was performed on the swabs from the conjunctival sac of 15 patients with primary Sjogren syndrome (PS), 15 patients with secondary Sjogren syndrome (SS) and 30 healthy individuals, chosen at random . RESULTS: Most of the swabs, in every group, were aseptic . The bacteria found relatively often was Staphylococcus epidermidis or described as the Staphylococcus sp . coagulasonegative . Bacterias: Staphylococcus aureus, Corynebacterium or Enterobacteriaceae were found rarely . CONCLUSIONS: There is no significant difference between bacterial flora of conjunctival sac of patients with PS and SS, when compared to healthy individuals.

J Clin Microbiol, 2005 Jan, 43(1), 223 - 8
Molecular characterization of diphtheria toxin repressor (dtxR) genes present in nontoxigenic Corynebacterium diphtheriae strains isolated in the United Kingdom; De Zoysa A et al.; Nontoxigenic strains of Corynebacterium diphtheriae represent a potential reservoir for the emergence of toxigenic C . diphtheriae strains if they possessed functional diphtheria toxin repressor (dtxR) genes . We studied the predominant strain of nontoxigenic C . diphtheriae circulating in the United Kingdom to see if they possessed dtxR genes and ascertain whether they were functional . A total of 26 nontoxigenic C . diphtheriae strains isolated in the United Kingdom during 1995 and 4 nontoxigenic strains isolated in other countries were analyzed by PCR and direct sequencing to determine the presence and intactness of the dtxR genes . The functionality of the DtxR proteins was assayed by testing for the production of siderophore in medium containing high and low concentrations of iron . PCR amplification and sequence analysis of the dtxR genes revealed four variants of the predicted DtxR protein among the nontoxigenic strains isolated in the United Kingdom . Production of siderophore in medium containing a low concentration of iron and repression of siderophore production in medium containing a high concentration of iron demonstrated that in all the strains the dtxR genes were functional . These findings demonstrate that, if lysogenised by a bacteriophage, nontoxigenic strains circulating in the United Kingdom could produce toxin and therefore represent a potential reservoir for toxigenic C . diphtheriae.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
The Acyl-AMP ligase FadD32 and AccD4-containing Acyl-CoA carboxylase are required for the synthesis of mycolic acids and essential for the mycobacterial growth . Identification of the carboxylation product and determination of the Acyl-CoA carboxylase components; Portevin D et al.; Mycolic acids are major and specific long-chain fatty acids involved in the unusual architecture and impermeability of the cell envelope of several important human pathogens such as Mycobacterium tuberculosis, M . leprae and Corynebacterium diphtheriae . We have previously shown that the pks13 gene encodes the condensase, the enzyme that performs the final condensation step of mycolic acid biosynthesis and is flanked by two genes, fadD32 and accD4 . We generated two mutants of Corynebacterium glutamicum with an insertion/deletion within either fadD32 or accD4 . The two mutant strains were deficient in mycolic acid production . Application of multiple analytical approaches to the analysis of the mutants demonstrated the accumulation of a tetradecylmalonic acid in the deltafadD32::km mutant and its absence from the deltaaccD4::km strain . The parental corynebacterial phenotype was restored upon the transfer of the wild type fadD32 and accD4 genes in the mutants . These data demonstrated that both FadD32 and AccD4-containing acyl-CoA carboxylase are required for the production of mycolic acids . They also prove that the proteins catalyze, respectively, the activation of one fatty acid substrate and the carboxylation of the other substrate, solving the long debated question of the mechanism involved in the condensation reaction . To address the question of the identity of the other subunits of the AccD4-containing acyl-CoA carboxylase we used comparative genomics, and applied a combination of molecular biology and proteomic technologies to the analysis of proteins that co-immunoprecipitated with AccD4 . This resulted in the identification of AccA3 and AccD5 as subunits of the acyl-CoA carboxylase . Finally, we used conditionally replicative plasmids to show that both the fadD32 and accD4 genes are essential for the survival of M . smegmatis . Thus, in addition to Pks13, FadD32 and AccD4 are promising targets for the development of new antimicrobial drugs against pathogenic species of mycobacteria and related microorganisms.

Am J Vet Res, 2004 Dec, 65(12), 1734 - 7
Molecular epidemiologic features of Corynebacterium pseudotuberculosis isolated from horses; Foley JE et al.; OBJECTIVE: To characterize isolates of Corynebacterium pseudotuberculosis from horses, cattle, and sheep in Colorado, Kentucky, Utah, and California in samples collected during perceived epidemics of infection (increased numbers of cases identified) in 2002 and 2003, and determine how closely isolates were related and their possible source . SAMPLE POPULATION: 54 isolates of C pseudotuberculosis from 49 horses, 4 cattle, and 1 sheep . PROCEDURES: Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) assay, PCR assay for the gene encoding the phospholipase D (PLD) toxin, biochemical analyses, and tests for susceptibility to 17 antimicrobial drugs were performed . RESULTS: All isolates reduced nitrate to nitrite, most yielded positive results for the PLD toxin gene, and all were susceptible to antimicrobial drugs . Ten genetic types were detected by use of RAPD PCR assay; types III to X were isolated from horses, cattle, or both in 1 or more states . Types III and IX were isolated from both horses and cattle . Types VII and VIII were isolated in only 1 state, but the number of isolates in these groups was small . In contrast, all other types were isolated in 2 or more states . All isolates from Utah were type III, but the other 3 states had isolates from more than 1 type . CONCLUSIONS AND CLINICAL RELEVANCE: These data are consistent with a clonally expanding epidemic of infection in Utah and an increase in number of infections caused by multiple strains of C pseudotuberculosis not derived from a single source in the other states . The increase in number of infections could be the result of reporting bias, environmental factors facilitating infection, or host factors such as greater herd susceptibility.

J Bacteriol, 2005 Jan, 187(2), 422 - 33
Analysis of a DtxR-Regulated Iron Transport and Siderophore Biosynthesis Gene Cluster in Corynebacterium diphtheriae; Kunkle CA et al.; This report describes a genetic locus associated with siderophore biosynthesis and transport in Corynebacterium diphtheriae . A BLAST search of the C . diphtheriae genome identified a seven-gene cluster that included four genes, designated ciuA, ciuB, ciuC, and ciuD, whose predicted products are related to ABC-type iron transporters . Downstream from ciuD is the ciuE gene, whose predicted product is similar to the aerobactin biosynthetic enzymes IucA and IucC . The CiuE protein, which has a predicted mass of 121,582 Da and is approximately twice the size of either IucC or IucA, is homologous to each of these proteins in both its N- and C-terminal regions . C . diphtheriae ciuE deletion mutants exhibited a defect in siderophore production, iron uptake, and growth in low-iron medium . Mutations in the ciuA gene, whose predicted product is a lipoprotein component of an iron transport system, resulted in a severe defect in iron uptake and reduced ability to use the C . diphtheriae siderophore as an iron source . Site-directed mutations in irp6A, a gene previously reported to be associated with siderophore transport, had no effect on iron uptake or the utilization of the C . diphtheriae siderophore as an iron source . Transcriptional analysis demonstrated that expression of ciuA and ciuE is DtxR and iron regulated, and DNase I protection experiments confirmed the presence of DtxR binding sites upstream from each of these genes . Thus, this iron- and DtxR-regulated gene cluster is involved in the synthesis and transport of the C . diphtheriae siderophore.

Biochemistry, 2005 Jan 11, 44(1), 40 - 51
Prolylpeptide Binding by the Prokaryotic SH3-like Domain of the Diphtheria Toxin Repressor: A Regulatory Switch(,); Wylie GP et al.; Diphtheria toxin repressor (DtxR) regulates the expression of iron-sensitive genes in Corynebacterium diphtheriae, including the diphtheria toxin gene . DtxR contains an N-terminal metal- and DNA-binding domain that is connected by a proline-rich flexible peptide segment (Pr) to a C-terminal src homology 3 (SH3)-like domain . We determined the solution structure of the intramolecular complex formed between the proline-rich segment and the SH3-like domain by use of NMR spectroscopy . The structure of the intramolecularly bound Pr segment differs from that seen in eukaryotic prolylpeptide-SH3 domain complexes . The prolylpeptide ligand is bound by the SH3-like domain in a deep crevice lined by aliphatic amino acid residues and passes through the binding site twice but does not adopt a polyprolyl type-II helix . NMR studies indicate that this intramolecular complex is present in the apo-state of the repressor . Isothermal equilibrium denaturation studies show that intramolecular complex formation contributes to the stability of the apo-repressor . The binding affinity of synthetic peptides to the SH3-like domain was determined using isothermal titration calorimetry . From the structure and the binding energies, we calculated the enhancement in binding energy for the intramolecular reaction and compared it to the energetics of dimerization . Together, the structural and biophysical studies suggest that the proline-rich peptide segment of DtxR functions as a switch that modulates the activation of repressor activity.

J Am Vet Med Assoc, 2004 Dec 1, 225(11), 1743 - 7, 1702
Infection with Corynebacterium pseudotuberculosis in five alpacas; Anderson DE et al.; Among the population of an alpaca breeding farm, 5 alpacas (22 days to 14 months old) developed focal swellings in the subcutaneous tissues of the head or neck . Infection with Corynebacterium pseudotuberculosis was confirmed on the basis of results of microbial culture of abscess material and a serum hemolysis inhibition assay to detect C . pseudotuberculosis toxin . The dams of the affected alpacas were seronegative for C . pseudotuberculosis toxin . The affected alpacas underwent surgical excision of the abscesses and were isolated from herdmates for 90 days; treatment was successful, and no other alpacas in the herd became infected . Common risk factors for sources of infection in the affected alpacas included housing in a maternity barn and a pasture . Also, the infection potentially originated from new alpacas introduced into the herd during the preceding 3 months . Infection with C . pseudotuberculosis should be considered as a differential diagnosis for camelids with peripheral lymphadenopathy or abscesses in subcutaneous tissues.

FEMS Microbiol Lett, 2005 Jan 15, 242(2), 265 - 74
A novel gnd mutation leading to increased l-lysine production in Corynebacterium glutamicum; Ohnishi J et al.; Toward more efficient l-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background . Following the creation of a new l-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway . Comparative genomic analysis for the pathway between a classically derived l-lysine producer and its parental wild-type identified several mutations . Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for l-lysine production . Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased l-lysine production . Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, d-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme . Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during l-lysine production . These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for l-lysine biosynthesis, thereby leading to improved product formation.

Bioprocess Biosyst Eng . 2004 Dec 22; {Epub ahead of print}
Dynamics of glutamate synthesis and excretion fluxes in batch and continuous cultures of temperature-triggered Corynebacterium glutamicum; Uy D et al.; Corynebacterium glutamicum 2262 strain, when triggered for glutamate excretion, experiences a rapid decrease in growth rate and increase in glutamate efflux . In order to gain a better quantitative understanding of the factors controlling the metabolic transition, the fermentation dynamics was investigated for a temperature-sensitive strain cultivated in batch and glucose-limited continuous cultures . For non-excreting cells at 33 degrees C, increasing the growth rate resulted in strong increases in the central metabolic fluxes, but the intracellular glutamate level, the oxoglutarate dehydrogenase complex (ODHC) activity and the flux distribution at the oxoglutarate node remained essentially constant . When subjected to a temperature rise to 39 degrees C, at both high- and low-metabolic activities, the bacteria showed a rapid attenuation in ODHC activity and an increase from 28% to more than 90% of the isocitrate dehydrogenase flux split towards glutamate synthesis . Simultaneously to the reduction in growth rate, the cells activated a high capacity export system capable of expelling the surplus of synthesized glutamate.

J Dairy Res, 2004 Nov, 71(4), 409 - 18
Decision tree analysis to evaluate dry cow strategies under UK conditions; Berry EA et al.; Economic decisions on animal health strategies address the cost-benefit aspect along with animal welfare and public health concerns . Decision tree analysis at an individual cow level highlighted that there is little economic difference between the use of either dry cow antibiotic or an internal teat sealant in preventing a new intramammary infection in a cow free of infection in all quarters of the mammary gland at drying off . However, a potential net loss of over l20 per cow might occur if the uninfected cow was left untreated . The only economically viable option, for a cow with one or more quarters infected at drying off, is antibiotic treatment, although a loss might still be incurred depending on the pathogen concerned and the cure rates achievable . There was a net loss for cows with quarters infected with Corynebacterium spp . at drying off, for both the teat sealant and untreated groups (l22 and l48, respectively) with only antibiotic-treated cows showing a gain.

Pathol Biol (Paris), 2004 Dec, 52(10), 566 - 574

Barbut F, Soukouna S, Lalande V, Garcia ML, Neyme D, de Gramont A, Petit JC.
Totally implantable venous access ports (TIVAP) are valuable medical devices for long-term intravenous treatment such as parenteral nutrition, cancer chemotherapy or antiviral therapy . Implantation and use of these devices are each associated with infectious or mechanical complications . Aims of the study . - To determine the frequency of complications and to analyze bacterial contamination of different parts of TIVAP (tip, septum, internal lumen of the port) . Material and methods . - Clinical charts of patients, which TIVAP was removed between April 20th to December 31st 2003, were retrospectively reviewed . Infectious complications (local and septicemic) and non-infectious complications (i.e . obstruction, thrombosis, drug extravasation...) were defined using clinical and/or microbiological criteria . Quantitative culture from different parts of the TIVAP was performed . Results . - One hundred and ten patients (age 57 +/-14-years-old, 94.3% cancers) were included, corresponding to 57,018 catheter-days: 39.1% had one or more non-infectious complications (density incidence: 0.86 for 1000 catheter-days) . Among the 49 complications, obstruction, thrombosis, extravasations and malposition accounted for 30.6%, 30.6% 4.1% and 6% of cases . Twenty-one patients (19.1%) had an infectious complication: 11 were local and 14 were systemic (density incidence 0.43 for 1000 catheter-days) . Bacteria responsible for TIVAP-associated bacteraemia were coagulase negative staphylococci (N =2), Staphylococcus aureus susceptible to methicilline (N =3), micrococci (N= 1), corynebacteria (N =1) or Gram-negative bacilli (N =8) . Comparison of quantitative culture of the different parts of TIVAP with a threshold at 10(3) CFU/ml showed that culture of tip, septum and port has a sensitivity of 47.6% 57.1% and 61.9 %, respectively and a specificity of 100% 92.1% and 92.1%, respectively for the diagnosis of TIVAP infection . Conclusion . - Complications associated to TIVAP are frequent but incidence that we have reported is comparable with previous studies . Analysis of internal lumen of the port is the most sensitive method for the diagnosis of TIVAP-associated infections.

Int J Med Microbiol, 2004 Oct, 294(6), 413 - 6
Isolations of Corynebacterium kroppenstedtii from a breast abscess; Riegel P et al.; The recently described species Corynebacterium kroppenstedtii was isolated from a breast abscess in a 38-year-old woman on two occasions . We discuss the pathogenic role of this bacteria and the methods used for its isolation.

Acta Vet Hung, 2004, 52(4), 423 - 38
Cystoscopy in cattle--a valuable additional tool for clinical examination; Franz S et al.; Based on the findings of physical examination and on laboratory findings the urinary bladder of 23 cows was examined endoscopically in order to investigate the application of cystoscopy in cattle . The endoscopic findings of all examined cows were compared with the findings of physical examination and the results of macroscopic and microscopic urinalysis and the bacteriological culture of the urine . By physical examination only 3 cows were diagnosed to have urinary tract disease, whereas all other cows were suspected of having an urinary tract disease . Bacteriological culture of the urine revealed Corynebacterium renale and Escherichia coli infection in 18 cows, while the remaining 5 cows were negative . By cystoscopy catarrhal cystitis was diagnosed in 2 cases, haemorrhagic cystitis in 5 cases, and fibrinous-purulent and fibrinous-haemorrhagic cystitis in 13 cases . Three cows showed no pathological changes of the urinary bladder mucosa by endoscopic examination . Cystoscopy facilitates diagnosis through the direct visualisation of mucosal lesions and makes it possible to give a more accurate prognosis based upon the findings.

J Urol, 2005 Jan, 173(1), 226 - 9
Renal transplantation in children with severe bladder dysfunction; Mendizabal S et al.; PURPOSE: Renal transplantation in children with bladder dysfunction carries a risk for the renal graft . We report our experience with transplantation in 15 patients 6 to 18 years old with severe abnormalities of the lower urinary tract . MATERIALS AND METHODS: A total of 18 renal transplants were performed in 15 children with bladder dysfunction secondary to myelomeningocele (3), occult spina bifida (1), malformation/agenesis of the sacrum (5), posterior urethral valves (4), female hypospadias (1) and bladder exstrophy (1) between 1979 and 2003 . Urological surgery was performed before transplantation in 14 cases-7 bladder augmentations, 5 incontinent urinary conduits/reservoirs and 2 vesicostomies . Voiding was maintained by intermittent catheterization in 9 cases and incontinent ostomies in 6 . Graft implantation was performed by extraperitoneal route with ureteral anastomosis to the native bladder in cases of bladder augmentation . Immunosuppression consisted of triple therapy with polyclonal/monoclonal antibodies . RESULTS: Urological complications consisted of urethral obstruction due to mucus hypersecretion (1), urinary fistula (1), ureterovesical obstruction (1), stone formation (3), urinary tract incrustation by Corynebacterium urealyticum (1) and pyelonephritis (2) . Graft survival rates at 1 and 5 years were 77% and 62%, respectively, with a median of 79 months (95% CI 51 to 107) . Three graft losses were related to urological disease . CONCLUSIONS: Renal transplantation in children with severe bladder dysfunction can achieve similar results to those obtained in the general population . Meticulous selection of patients and surgical reparative techniques ensuring voiding and adequate control of urinary infections are mandatory . Augmentation cystoplasty and intermittent catheterization are appropriate techniques currently used for achieving this outcome.

J Dairy Sci, 2005 Jan, 88(1), 426 - 32
Efficacy of two iodine teat dips based on reduction of naturally occurring new intramammary infections; Foret CJ et al.; The efficacy of 2 teat dips, product 1 (Della Care with 5 to 8 ppm of free iodine, used as a positive control) and product 2 (New Della Care with 12 to 16 ppm of free iodine), was compared using a natural exposure trial on dairy cattle . The trial was based on National Mastitis Council guidelines and performed over 9 mo . Both teat dips contained 0.25% iodine . Product 2 reduced the infection rate 57.6% for major pathogens and 53.7% for minor pathogens, compared with the positive control . Product 2 gave highly significant reductions for Staphylococcus aureus, coagulase-negative staphylococci, and Corynebacterium bovis . Teat skin, teat ends, and teat hyperkeratosis were evaluated during trial . No significant difference in teat condition was observed between these 2 products.

Environ Res, 2005 Mar, 97(3), 300 - 311
Effects of crude oil on phospholipid fatty acid compositions of marine hydrocarbon degraders: estimation of the bacterial membrane fluidity; Mazzella N et al.; In this study, we investigated, in vitro, the effects of petroleum hydrocarbons on the phospholipid ester-linked fatty acid composition of Corynebacterium sp . Strain 8 . The usual ratio of monounsaturated fatty acids E/Z (or trans/cis) was calculated . This ratio led to unexpected results because we found similar values for growths on either a hydrophobic substrate (crude oil) or a soluble carbon source (rich medium) . The use of such an indicator seemed limited for monitoring an environmental stress, so we proposed an index based on the homeoviscous adaptation theory . A membrane viscosity index was defined and applied to Corynebacterium sp . Strain 8 (in vitro growth) and to a sedimentary community (in situ experiment) . The results allowed us to estimate the membrane fluidity of both an isolated strain and a bacterial community in accordance with the medium hydrophobicity.

Chem Phys Lipids, 2005 Jan, 133(1), 17 - 26
Characterization of acyl-phosphatidylinositol from the opportunistic pathogen Corynebacterium amycolatum; Valero-Guillen PL et al.; The aim of the present study was to characterize a new lipid detected in the opportunistic pathogen Corynebacterium amycolatum . It was identified as acyl-phosphatidylinositol (acyl-PI), and revealed as a mixture of homologues compounds by electrospray ionization mass spectrometry, with pseudomolecular ions, (M - H)(-), observed at 1099 (the major one) 1113, and 1127 . Acyl-PI exclusively contained octadecenoyl on the inositol moiety (as 3-O-acyl), an unsaturated fatty acyl (mostly octadecenoyl) at sn-1 position of the glycerol and a saturated fatty acyl (mainly hexadecanoyl) at the sn-2 position . Acyl-PI constitutes a new natural substance and seems to be unique among the phospholipids of C . amycolatum . Other more complex molecules, previously undetected, and assigned in this work to several acyl forms of phosphatidylinositol trimannosides, lacked octadecenoyl in their polar heads . The present study reveals the existence of acyl-PI in C . amycolatum as rather unexpected finding and, additionally, gives evidence for the ability of this species to synthesize a great variety of inositol-containing phospholipids.

Biochim Biophys Acta, 2004 Dec 15, 1667(2), 229 - 40
Activity regulation of the betaine transporter BetP of Corynebacterium glutamicum in response to osmotic compensation; Botzenhardt J et al.; As a response to hyperosmotic stress bacterial cells accumulate compatible solutes by synthesis or by uptake . Beside the instant activation of uptake systems after an osmotic upshift, transport systems show also a second, equally important type of regulation . In order to adapt the pool size of compatible solutes in the cytoplasm to the actual extent of osmotic stress, cells down-regulate solute uptake when the initial osmotic stress is compensated . Here we describe the role of the betaine transporter BetP, the major uptake carrier for compatible solutes in Corynebacterium glutamicum, in this adaptation process . For this purpose, betP was expressed in cells (C . glutamicum and Escherichia coli), which lack all known uptake systems for compatible solutes . Betaine uptake mediated by BetP as well as by a truncated form of BetP, which is deregulated in its response to hyperosmotic stress, was dissected into the individual substrate fluxes of unidirectional uptake, unidirectional efflux and net uptake . We determined a strong decrease of unidirectional betaine uptake by BetP in the adaptation phase . The observed decrease in net uptake was thus mainly due to a decrease of Vmax of BetP and not a consequence of the presence of separate efflux system(s) . These results indicate that adaptation of BetP to osmotic compensation is different from activation by osmotic stress and also different from previously described adaptation mechanisms in other organisms . Cytoplasmic K+, which was shown to be responsible for activation of BetP upon osmotic stress, as well as a number of other factors was ruled out as triggers for the adaptation process . Our results thus indicate the presence of a second type of signal input in the adaptive regulation of osmoregulated carrier proteins.

Med Oral Patol Oral Cir Bucal, 2004, 9 Suppl, 15 - 8; 11-4
Microbiological basis of oral infections and sensitivity to antibiotics; Prieto-Prieto J et al.; Because oral infections are common, the physician must understand the underlying etiology, pathogeny, and other variables that determine how these processes evolve in order to choose the most appropriate antibiotic drug . The special characteristics of the oral cavity determine the make-up of the microflora that lives there . Different anaerobic species belonging to the Peptostreptococcus, Prevotella, Fusobacterium, Gemella, and Porphyromonas genera are of particular interest, as are the aerobic species Streptococcus, Staphylococcus, and Corynebacterium . Each of these microorganisms occupies a different microniche within the oral cavity, and the prevailing balance is upset when conditions become modified as a result of illness or due to dental interventions such as tooth extraction or tooth scaling and polishing . Pathogenic or opportunistic bacteria (Actinomyces, Prevotella intermedia species, etc.) can develop in these conditions, as can yeasts (Candida sp., Histoplasma capsulatum), virus (herpes simplex, papilomavirus), and parasites (Entamoeba gingivalis, Trichomonas tenax) . When infection occurs, the patients s immune system reacts by means of inborn immunity (non-specific) and acquired immunity (specific) . Empirical treatment is administered that should be based on etiological data and on the antimicrobial sensitivity of the pathogen that is causing the infection . However, oral microflora sensitivity to different antibiotics is currently declining and there is a noticeable trend towards resistances . As a consequence of all this, the treatment of oral infections must also aim to restore the ecological balance of the oral cavity and to minimize the emergence of resistance in the microorganisms present in the mouth . Hence, epidemiological oral pathogen sensitivity studies must be conducted, fostering the administration of appropriate antibiotics at proper doses and keeping specialists abreast of the latest trends . In recent decades, oral infections comprise one of the most common pathologies in the general population, due in large part to infectious complications associated with poor oral hygiene . This in turn, translates into an increased need and demand for dental care, while at the same time, it requires that the professional accurately understand the etiological factors involved, as well as the pathogeny and different variables that determine the specificity of these kinds of infections, so as to be able to choose the appropriate antimicrobial drugs for proper treatment.

Appl Environ Microbiol, 2004 Dec, 70(12), 7277 - 87
Metabolic fluxes in Corynebacterium glutamicum during lysine production with sucrose as carbon source; Wittmann C et al.; Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism . For this purpose, 13C metabolic flux analysis with parallel studies on {1-(13C)Fru}sucrose, {1-(13C)Glc}sucrose, and {13C6Fru}sucrose was carried out . C . glutamicum directed 27.4% of sucrose toward extracellular lysine . The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP) . The glucose monomer of sucrose was completely channeled into the PPP . After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate . Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP . This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS(Man) or by fructose-1,6-bisphosphatase . C . glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2% . Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190% . The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess . The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

Appl Environ Microbiol, 2004 Dec, 70(12), 7148 - 55
Cometabolism of a nongrowth substrate: L-serine utilization by Corynebacterium glutamicum; Netzer R et al.; Despite its key position in central metabolism, L-serine does not support the growth of Corynebacterium glutamicum . Nevertheless, during growth on glucose, L-serine is consumed at rates up to 19.4 +/- 4.0 nmol min(-1) (mg {dry weight})(-1), resulting in the complete consumption of 100 mM L-serine in the presence of 100 mM glucose and an increased growth yield of about 20% . Use of 13C-labeled L-serine and analysis of cellularly derived metabolites by nuclear magnetic resonance spectroscopy revealed that the carbon skeleton of L-serine is mainly converted to pyruvate-derived metabolites such as L-alanine . The sdaA gene was identified in the genome of C . glutamicum, and overexpression of sdaA resulted in (i) functional L-serine dehydratase (L-SerDH) activity, and therefore conversion of L-serine to pyruvate, and (ii) growth of the recombinant strain on L-serine as the single substrate . In contrast, deletion of sdaA decreased the L-serine cometabolism rate with glucose by 47% but still resulted in degradation of L-serine to pyruvate . Cystathionine beta-lyase was additionally found to convert L-serine to pyruvate, and the respective metC gene was induced 2.4-fold under high internal L-serine concentrations . Upon sdaA overexpression, the growth rate on glucose is reduced 36% from that of the wild type, illustrating that even with glucose as a single substrate, intracellular L-serine conversion to pyruvate might occur, although probably the weak affinity of L-SerDH (apparent Km, 11 mM) prevents substantial L-serine degradation.

Appl Microbiol Biotechnol, 2004 Dec, 66(2), 187 - 93 Epub 2004 Dec.
Effect of NADH dehydrogenase-disruption and over-expression on respiration-related metabolism in Corynebacterium glutamicum KY9714; Nantapong N et al.; The function of type II NADH dehydrogenase (NDH-2) in Gram-positive Corynebacterium glutamicum was investigated by preparing strains with ndh, the NDH-2 gene, disrupted and over-expressed . Although disruption showed no growth defects on glucose minimum medium, the growth rate of the over-expressed strain was lower compared with its parent, C . glutamicum KY9714 . Ndh-disruption and over-expression did not lead to a large change in the respiratory chain and energetics, including the cytochrome components and the H(+)/O ratio . However, in the strain that lacked NDH-2, membrane L-lactate oxidase activity increased, while NDH-2 over-expression led to decreased L-lactate and malate oxidase activities . In addition, relatively high cytoplasmic lactate dehydrogenase (LDH) activity was always present as was malate dehydrogenase, irrespective of NDH-2 level . Furthermore, L-lactate or malate-dependent NADH oxidase activity could be reproduced by reconstitution with the membranes and the cytoplasmic fraction isolated from the disruptant . These results suggest that coupling of LDH and the membrane L-lactate oxidase system, together with the malate-dependent NADH oxidase system, operates to oxidize NADH when the NDH-2 function is defective in C . glutamicum.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2191 - 5
Corynebacterium ciconiae sp . nov., isolated from the trachea of black storks (Ciconia nigra); Fernandez-Garayzabal JF et al.; Eight unidentified Gram-positive, rod-shaped organisms were recovered from the tracheas of apparently healthy black storks (Ciconia nigra) and subjected to a polyphasic taxonomic analysis . Based on cellular morphology and biochemical criteria the isolates were tentatively assigned to the genus Corynebacterium, although three of the organisms did not appear to correspond to any recognized species . Comparative 16S rRNA gene sequencing studies demonstrated that all of the isolates were phylogenetically members of the genus Corynebacterium . Five strains were genotypically identified as representing Corynebacterium falsenii, whereas the remaining three strains represented a hitherto unknown subline, associated with a small subcluster of species that includes Corynebacterium mastitidis and its close relatives . On the basis of phenotypic and phylogenetic evidence, it is proposed that the unknown isolates from black storks represent a novel species within the genus Corynebacterium, for which the Corynebacterium ciconiae sp . nov . is proposed . The type strain is CECT 5779(T) (=BS13(T)=CCUG 47525(T)).

Med Dosw Mikrobiol, 2004, 56(2), 147 - 54
{Biochemical properties of Corynabacterium amycolatum strains}; Zalas P et al.; C . amycolatum is the most commonly isolated nonlipophilic species of Corynebacterium from clinical samples . However, the lack of good commercial identification tests in microbiology laboratories causes some difficulties in C . amycolatum diagnostics . We decided to examine biochemical and enzymatic properties of isolated strains and analize occurrence of particular biochemical profiles (biotypes) . Perhaps it would let improve the identification schemes . 70 strains of C . amycolatum were analized . The estimation of biochemical properties consisted of the results of API Coryne and API ZYM tests (bioMerieux), the ability of excreting of protease, esterase, lipase and lecithinase . Analized strains had various biochemical and enzymatic properties . Almost all strains fermented glucose (98.6%) and maltose (95.7%) and producted pyrasinamidase (94.3%) . All strains producted alkaline phosphatase and phosphohydrolase, and 95.7%--acid phosphatase . Biotypes of particular strains were determined on the biochemical reactions included in the API Coryne tests . In the group of 70 strains 21 profiles were distinguished among which 3100325 biotype (35.7%) was dominant . The lipolysis was defined on Tween 20, Tween 40, Tween 60, Tween 80 medium and with the API ZYM test usage . All strains producted esterase-lipase (esterase C-8), 95.7% of strains-esterase C-4, and 21.4% lipase C-14 . Among analized strains 18.6% hydrolyzed Tween 20, 14.3% Tween 60, and 1.4% Tween 40 . None of these strains demonstrated lipase and lecithinase activity . Difficulties in concerning C . amycolatum as pathogens justify further investigations.

Med Dosw Mikrobiol, 2004, 56(2), 139 - 45
{Occurrence of Corynebacterium amycolatum strains in clinical specimens}; Zalas P et al.; C . amycolatum is poorly recognized and rarely described in the world literature . So, better recognizing and understanding biology of these bacteria may help effectivly prevent infections caused by them . The subject within the study were 70 of C . amycolatum strains which were isolated from the clinical specimens of patients hospitalized at the State Clinical Hospital in Bydgoszcz . After initial identification of examinated strains based on Gram staining results, colonial morphology, biochemical and enzymatic features included in API Coryne and API ZYM tests (bioMerieux), growth at 20 degrees C, Tween 80 requirement, DNA and tyrosine hydrolysis, occurrence in clinical specimens and origin of C . amycolatum strains were analized . The investigated strains were the most frequently isolated from wound swabs (61.5%), urine (14.3%), drain swabs (7.1%) and mainly (37.2%) came from patients treated at the departments of surgery.

Biochem Biophys Res Commun, 2004 Dec 17, 325(3), 983 - 90
Crp of Streptomyces coelicolor is the third transcription factor of the large CRP-FNR superfamily able to bind cAMP; Derouaux A et al.; The chromosomal inactivation of the unique transcription factor of Streptomyces coelicolor that displays a cyclic-nucleotide-binding domain, Crp(Sco), led to a germination-defective phenotype similar to the mutant of the adenylate cyclase gene (cya) unable to produce cAMP . By means of cAMP affinity chromatography we demonstrate the specific cAMP-binding ability of Crp(Sco), which definitely demonstrate that a Cya/cAMP/Crp system is used to trigger germination in S . coelicolor . However, electromobility shift assays with the purified Crp(Sco)-cAMP complex and the CRP-like cis-acting element of its own promoter failed . Moreover, we were unable to complement an Escherichia coli crp mutant in trans with Crp(Sco) . The fact that Vfr from Pseudomonas aeruginosa and GlxR from Corynebacterium glutamicum could complement such an E . coli mutant suggests that the way Crp(Sco) interacts with DNA should mechanistically differ from its most similar members . This hypothesis was further supported by homology modelling of Crp(Sco) that confirmed an unusual organisation of the DNA-binding domain compared to the situation observed in Crp(Eco).

Ir J Med Sci, 2004 Apr-Jun, 173(2), 96 - 8
Detection of mycobacterial DNA from sputum of patients with cystic fibrosis; Devine M et al.; BACKGROUND: Patients with cystic fibrosis (CF) are at high risk from atypical mycobacterial infections . There have been few attempts to delineate the intensity of mycobacterial infection in CF patients in Ireland . AIMS: To examine the incidence of mycobacterial DNA in an archived collection of genomic DNA extracted from the sputa of CF patients within the Northern Ireland population . METHODS: One hundred and eighty-two CF patients (66 adults and 116 children) were examined for the presence of mycobacterial DNA in their sputum by a genus specific PCR assay based on 16S rRNA, followed by direct automated sequencing of the PCR amplicons . RESULTS: One of 116 (0.9%) children and 2 of 66 adults were positive . Sequence identity revealed Mycobacterium xenopi in the paediatric patient and M . xenopi and M . chelonei in the two adult patients . False-positive results occurred in 11 patients (four adults), mainly due to Corynebacterium spp . CONCLUSIONS: There was a low prevalence of Mycobacterium spp in the CF patient population . All PCR positive results should be confirmed by direct automated sequencing and an alternative specific assay employed . Enhanced molecular screening will contribute in understanding their role as opportunistic pathogens in patients with worsening lung function.

Biologicals, 2004 Sep, 32(3), 165 - 9
Impact of manufacturing, irradiation and filtration steps to bacterial contamination of autologous fibrin sealant; Buchta C et al.; BACKGROUND: Preoperative production of autologous fibrin sealant has become a routine procedure during the last years . As a certain percentage of blood products is contaminated with bacteria, contamination of plasma used for the production of fibrin sealant cannot be excluded . Especially in the orthopaedic setting, application of contaminated fibrin sealant can cause severe infections . MATERIALS AND METHODS: We contaminated plasma with Staphylococcus epidermidis, Corynebacterium striatum, Bacillus subtilis or Escherichia coli and produced fibrin sealant by cryoprecipitation and alcohol precipitation . Additionally, the products were gamma-irradiated at a dose of 30 Gy, frozen at -55 degrees C and filtered through a 0.2 microm filter after thawing . After each preparation step, samples were drawn and numbers of colony forming units were counted after incubation on agar plates . RESULTS: Cryoprecipitation, irradiation, freezing at -55 degrees C, and alcohol precipitation have only little impact on numbers of colony forming units . Filtration through a bacterial filter results in a sterile product . CONCLUSION: Bacteria in plasma as a starting material for production of fibrin sealant survive all routine steps of production, including gamma irradiation and freezing . Filtration of the product through a qualified bacterial filter is the only safe means to provide a sterile product.

Biochem J . 2004 Nov 9; {Epub ahead of print}
Catalytic mechanism of alpha-retaining glucosyl transfer by Corynebacterium callunae starch phosphorylase: the role of His-334 examined through kinetic characterization of site-directed mutants; Schwarz A et al.; Purified site-directed mutants of Corynebacterium callunae starch phosphorylase in which histidine 334 was replaced by alanine, glutamine or asparagine were characterized through steady-state kinetic analysis of enzymic glycosyl transfer to and from phosphate, and studies of ligand binding to the active site . Compared to wild-type, the catalytic efficiencies for phosphorolysis of starch at 30 oC and pH 7.0 decreased about 150- and 50-fold in H334Q and H334N mutants, that of H334A was unchanged . In the direction of alpha-glucan synthesis, the selectivity for reaction with alpha-D-glucose 1-phosphate (G1P) over reaction with alpha-D-xylose 1-phosphate decreased from a wild-type value of about 20,000 to 2,600 and 100 in H334N and H334Q, respectively . Binding of G1P to the free enzyme was weakened between 10-fold (H334N, H334Q) and 50-fold (H334A) in the mutants whereas binding to the complex of enzyme and alpha-glucan was not affected . Quenching of fluorescence of the pyridoxal 5'-phosphate cofactor was used to examine interactions of the inhibitor D-gluconic acid 1,5-lactone (GL) with wild-type and mutant enzymes in transient and steady-state experiments . GL binding to the free enzyme and the enzyme-phosphate complex occurred in a single step . The 50-fold higher constant (K d) for GL dissociation from H334Q bound to phosphate resulted from an increased off-rate for the ligand in the mutant, compared to wild-type . A double logarithmic correlation of turnover number for phosphorolysis of starch with reciprocal K d value established a linear-free energy relationship (slope = 1.19 +/- 0.07; r 2 = 0.991) across the series of wild-type and mutant enzymes . It reveals that GL in combination with phosphate has properties of a transition state analogue; and the role of the His-334 side chain in selectively stabilising the transition state of the reaction.

Acta Vet Scand, 2004, 45(1-2), 19 - 26
Bacteriological investigation of infectious keratoconjunctivitis in Norwegian sheep; Akerstedt J et al.; Contagious keratoconjunctivitis is a rather common disease in Norwegian sheep . Since the knowledge of its aetiology is limited, the present study was performed to determine the microorganisms involved . Local veterinarians throughout the country collected conjunctival swabs from both sick (n = 43) and healthy (n = 42) sheep on 15 farms with outbreaks of ovine keratoconjunctivitis, and further from healthy sheep (n = 50) on 17 farms not showing any signs of conjunctival disease . All samples were cultivated for bacteria and mycoplasma . Listeria monocytogenes was isolated from 3 cases (1%) in one single herd . Staphylococcus aureus (5%), Corynebacterium spp . (2%) and Escherichia coli (4%) were isolated only in herds with keratoconjunctivitis, but from both sick and healthy animals . Moraxella (Branhamella) ovis was isolated from 28% of sampled animals in affected herds and from 10% of sampled animals in healthy herds . The corresponding numbers for Moraxella spp . were 9%/12%, for Pseudomonas spp . 7%/8%, for Staphylococcus spp . 22//22%, for Bacillus spp . 12%/14%, for Micrococcus spp . 6%/2% and for Streptococcus/Enterococcus spp . 2%/2% . Mycoplasma conjunctivae was isolated from 16 animals with keratoconjunctivitis (37%) and from 3 animals without clinical signs (7%) in farms with keratoconjunctivitis . In farms without clinical signs of keratoconjunctivitis, M . conjunctivae was isolated in 4 animals (8%) . To our knowledge, this is the first time M . conjunctivae has been isolated in Norway . Other predisposing agents found were Moraxella (Branhamella) ovis and Listeria monocytogenes . The etiological importance of different microorganisms in ovine keratoconjunctivitis seems to vary; some are probably only present as secondary invaders . Other possible causes of ovine keratoconjunctivitis in Norway, such as Chlamydia psittaci, remain to be investigated.

World J Gastroenterol, 2004 Dec 15, 10(24), 3683 - 7
Integration of E . coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis; Liu DX et al.; AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C . glutamicum) on the production of L-phenylalanine . METHODS: By nitrosoguanidine mutagenesis, five p-fluorophenylalanine (FP)-resistant mutants of C.glutamicum FP were selected . The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR . Kanamycin resistance gene (Km) and the P(BF) -aroG-pheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pTK and pTGAK, respectively . Then, they were transformed into C.glutamicum FP respectively by electroporation . Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis . The transformed strains were used for L-phenylalanine fermentation and enzyme assays . RESULTS: Engineering strains of C.glutamicum (Tyr(-)) were obtained . Compared with the original strain, the transformed strain C . glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold, and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, respectively . CONCLUSION: Integration of tandem aroG-pheA genes into tyrA locus of C . glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production.

J Glaucoma, 2004 Dec, 13(6), 507 - 9
Comparison of preoperative conjunctival bacterial flora in patients undergoing glaucoma or cataract surgery; de Kaspar HM et al.; PURPOSE: To assess differences in conjunctival bacterial flora between patients undergoing glaucoma and cataract surgery . PATIENTS AND METHODS: A prospective study comparing conjunctival bacterial cultures obtained from 339 patients undergoing either cataract (n = 258) or glaucoma (n = 81) surgery . All cultures were acquired during the preoperative visit, approximately three to seven days prior to surgery . The culture samples were inoculated onto blood and chocolate agar, as well as blood culture broth media . All bacterial isolates were identified and statistical analyses were performed to determine if there were differences in flora between the eyes undergoing cataract versus glaucoma surgery . RESULTS: Two hundred fifteen of 258 eyes (83%) undergoing cataract surgery were found to have positive bacterial growth, compared with 62 of 81 eyes (77%) of those undergoing glaucoma surgery (P = 0.2246) . Coagulase-negative Staphylococci, the most common bacterial isolate, was cultured from 167 eyes (65%) in the cataract group and 42 (52%) in the glaucoma group (P = 0.0514) . Among all bacterial isolates, only Corynebacterium species was found to be statistically different between the two patient groups with 92 (36%) and 11 (14%) eyes testing positive in the cataract and glaucoma groups, respectively (P = 0.0003) . CONCLUSIONS: There was no statistically significant difference in the proportion of conjunctival culture samples testing positive for bacterial growth in eyes undergoing glaucoma surgery compared with those undergoing cataract surgery . Glaucoma medications, or their preservatives, do not appear to significantly alter conjunctival flora . Techniques used for endophthalmitis prophylaxis prior to cataract surgery are likely appropriate for glaucoma surgery as well.

J Biol Chem . 2004 Nov 4; {Epub ahead of print}
Roles of distal Asp in heme oxygenase from Corynebacterium diphteriae, HmuO: A water-driven oxygen activation mechanism; Matsui T et al.; Heme oxygenases found in mammals, plants and bacteria catalyze degradation of heme using the same mechanism . Roles of distal Asp (Asp136) residue in HmuO, a heme oxygenase of Corynebacterium diphtheriae, have been investigated by site-directed mutagenesis, enzyme kinetics, resonance Raman spectroscopy and X-ray crystallography . Replacements of the Asp136 by Ala and Phe resulted in reduced heme degradation activity due to the formation of ferryl heme, showing that the distal Asp is critical in HmuO heme oxygenase activity . D136N HmuO catalyzed heme degradation at a similar efficiency to wild type and D136E HmuO, implying that the carboxylate moiety is not required for the heme catabolism by HmuO . Resonance Raman results suggest that the inactive ferryl heme formation in the HmuO mutants is induced by disruption of the interaction between a reactive Fe-OOH species and an adjacent distal pocket water molecule . Crystal structural analysis of the HmuO mutants confirms partial disappearance of this nearby water in D136A HmuO . Our results provide the first experimental evidence for the catalytic importance of the nearby water molecule that can be universally critical in heme oxygenase catalysis, and propose that the distal Asp helps in positioning the key water molecule at a position suitable for efficient activation of the Fe-OOH species.

Arch Soc Esp Oftalmol, 2004 Oct, 79(10), 485 - 91
{Chronic carriers of pathogen conjunctival bacteria . Possible risks in cataract surgery}; Fernandez Rubio E et al.; PURPOSE: To ascertain the frequency and characteristics of chronic carriers of conjunctival pathogen bacteria among patients undergoing cataract surgery in our hospital, to allow the design of studies of their postsurgical endophthalmitis risk . METHODS: Retrospective study of the preoperative conjunctival flora of 784 patients operated on for cataracts in both eyes, in two operations separated by 213 days (SD 170), from November 1993 to December 1997 . Results of both cultures for each patient were obtained from the Laboratory cataract preoperative database, by means of an auxiliary utility in dBASE-III-PLUS program . The preoperative bacteria in both surgeries were compared and the patients having the same pathogen bacteria (all except Staphylococcus coagulase negative and Corynebacterium sp.) were identified . The mean values and standard deviations were calculated using Epiinfo 6.04, and the Chi2 test was carried out using Excel 97 . RESULTS: The conjunctival flora stemming from the first preoperative culture of the 784 patients were statistically similar to those of our reference population . The pathogen bacteria decreased in the second preoperative culture; especially the Streptococcus Pneumoniae and Haemophilus sp . frequencies (p<0.05) . The same pathogen was isolated in both cultures of 31 patients among the 784 who entered the study, Staphylococcus Aureus and Proteus Mirabilis being the most frequent colonizers . The mean age of those carriers was higher than the mean age of the reference population (p<0.01) . CONCLUSIONS: 3.95% of our patients awaiting cataract surgery are usual conjunctival carriers of certain pathogen bacteria; this finding is associated with the age of the patients and possibly with some pre-existing diseases.

J Cataract Refract Surg, 2004 Nov, 30(11), 2441 - 4
Postoperative Corynebacterium macginleyi endophthalmitis; Ferrer C et al.; A 72-year-old man with chronic endophthalmitis who received steroid treatment for 3 months came to our center . Sterile endophthalmitis after cataract extraction had been diagnosed . Aqueous samples including smears, classic cultures, and polymerase chain reaction were taken for microbiological study . Amplified DNA was sequenced to identify the pathogen . Polymerase chain reaction amplification was positive for bacteria . Sequence analysis showed Corynebacterium macginleyi as the causal agent in 48 hours . The culture and smear stains from the ocular samples were negative . The patient was successfully treated with vancomycin . Polymerase chain reaction and subsequent DNA-typing were useful in detecting the microorganisms that caused the chronic endophthalmitis.

J Bacteriol, 2004 Nov, 186(22), 7645 - 52
Molecular identification of the urea uptake system and transcriptional analysis of urea transporter- and urease-encoding genes in Corynebacterium glutamicum; Beckers G et al.; The molecular identification of the Corynebacterium glutamicum urea uptake system is described . This ABC-type transporter is encoded by the urtABCDE operon, which is transcribed in response to nitrogen limitation . Expression of the urt genes is regulated by the global nitrogen regulator AmtR, and an amtR deletion strain showed constitutive expression of the urtABCDE genes . The AmtR repressor protein also controls transcription of the urease-encoding ureABCEFGD genes in C . glutamicum . The ure gene cluster forms an operon which is mainly transcribed in response to nitrogen starvation . To confirm the increased synthesis of urease subunits under nitrogen limitation, proteome analyses of cytoplasmic protein extracts from cells grown under nitrogen surplus and nitrogen limitation were carried out, and five of the seven urease subunits were identified.

J Am Geriatr Soc, 2004 Nov, 52(11), 1795 - 804
Effect of person-centered showering and the towel bath on bathing-associated aggression, agitation, and discomfort in nursing home residents with dementia: a randomized, controlled trial; Sloane PD et al.; OBJECTIVES: To evaluate the efficacy of two nonpharmacological techniques in reducing agitation, aggression, and discomfort in nursing home residents with dementia . The techniques evaluated were person-centered showering and the towel bath (a person-centered, in-bed bag-bath with no-rinse soap) . DESIGN: A randomized, controlled trial, with a usual-care control group and two experimental groups, with crossover . SETTING: Nine skilled nursing facilities in Oregon and six in North Carolina . PARTICIPANTS: Seventy-three residents with agitation during bathing (69 completed the trial) and 37 nursing assistants who bathed them . MEASUREMENTS: Agitation and aggression were measured using the Care Recipient Behavior Assessment; discomfort was measured using a modification of the Discomfort Scale for Dementia of the Alzheimer Type . Raters who were blinded to subject status coded both from videotaped baths . Secondary measures of effect included bath duration, bath completeness, skin condition, and skin microbial flora . RESULTS: All measures of agitation and aggression declined significantly in both treatment groups but not in the control group, with aggressive incidents declining 53% in the person-centered shower group (P<.001) and 60% in the towel-bath group (P<.001) . Discomfort scores also declined significantly in both intervention groups (P<.001) but not in the control group . The two interventions did not differ in agitation/aggression reduction, but discomfort was less with the towel bath (P=.003) . Average bath duration increased significantly (by a mean of 3.3 minutes) with person-centered showering but not with the towel bath . Neither intervention resulted in fewer body parts being bathed; both improved skin condition; and neither increased colonization with potentially pathogenic bacteria, corynebacteria, or Candida albicans . CONCLUSION: Person-centered showering and the towel bath constitute safe, effective methods of reducing agitation, aggression, and discomfort during bathing of persons with dementia.

Vet Dermatol, 2004 Oct, 15(5), 315 - 20
A herd level analysis of a Corynebacterium pseudotuberculosis outbreak in a dairy cattle herd; Yeruham I et al.; Corynebacterium pseudotuberculosis infection in an Israeli dairy cattle herd is described . The disease was characterized by ulcerative granulomatous lesions, which occurred in an epidemic form . Thirty-two cows and two heifers were affected, the ratio of the number affected to number at risk being 17.5 : 1 and 9.5 : 1, respectively . The culling rate was 50% of the affected animals . Most of the affected animals were cows (91.2%), with one first-calving cow (2.9%) and two heifers (5.9%) also affected . The infection occurred during the summer to autumn months (August-December), and lasted 118 days . The incubation period is about 2 months . The disease appeared in two clinical forms - cutaneous and mastitic - or as a mixed form . C . pseudotuberculosis organisms that were isolated from the ulcerative granulomatous lesions and from milk samples failed to reduce nitrate . A decrease in milk production (4%) and an increase in the bulk-milk somatic cell count from a herd mean of 240 x 10(3) mL(-1) to 460 x 10(3) mL(-1) were noted during the morbidity period . The organism was isolated from milk samples of eight animals (25%) . Clinical, epizootiological and microbiological aspects of the infection are described.

Clin Experiment Ophthalmol, 2004 Oct, 32(5), 478 - 81
Bacterial keratitis in Christchurch, New Zealand, 1997-2001; Hall RC et al.; PURPOSE: To identify which organisms cause bacterial keratitis in a local community and to determine how patients with suspected bacterial keratitis should be initially treated . METHODS: The results of all corneal scrapes performed in the ophthalmology department of Christchurch Hospital between 1997 and 2001 were reviewed . All samples were collected at the 'bedside' by a technician from the microbiology department and were processed immediately . RESULTS: Eighty-seven corneal scrapes were performed on 78 patients . There was a positive Gram stain in 43.7% (38/87) of scrapes . There was a positive culture in 58.6% (51/87) of scrapes . The commonest Gram-positive organisms were coagulase negative Staphylococci (19.4%) and Corynebacterium spp . (16.1%) . The commonest Gram-negative organisms were Moraxella spp . (19.4%) and Pseudomonas aeruginosa (3.2%) . Every Gram-positive organism was sensitive to chloramphenicol and every Gram-negative organism was sensitive to ciprofloxacin . In contrast, 89% of Gram-negative organisms were sensitive to chloramphenicol and 88% of Gram-positive organisms were sensitive to ciprofloxacin . CONCLUSION: The results are very different to those reported by other centres . Most notably, a much higher incidence of infection by Corynebacterium spp . and Moraxella spp . and a lower incidence of Pseudomonas aeruginosa was found . In this centre it appears appropriate to initially treat patients with Gram-positive organisms with chloramphenicol and patients with Gram-negative organisms with ciprofloxacin . Patients with a negative Gram stain should be treated with alternating chloramphenicol and ciprofloxacin while awaiting culture results.

J Biol Chem, 2005 Jan 7, 280(1), 585 - 95 Epub 2004 Oct 19.
Identification of AcnR, a TetR-type Repressor of the Aconitase Gene acn in Corynebacterium glutamicum; Krug A et al.; In Corynebacterium glutamicum, the activity of aconitase is 2.5-4-fold higher on propionate, citrate, or acetate than on glucose . Here we show that this variation is caused by transcriptional regulation . In search for putative regulators, a gene (acnR) encoding a TetR-type transcriptional regulator was found to be encoded immediately downstream of the aconitase gene (acn) in C . glutamicum . Deletion of the acnR gene led to a 5-fold increased acn-mRNA level and a 5-fold increased aconitase activity, suggesting that AcnR functions as repressor of acn expression . DNA microarray analyses indicated that acn is the primary target gene of AcnR in the C . glutamicum genome . Purified AcnR was shown to be a homodimer, which binds to the acn promoter in the region from -11 to -28 relative to the transcription start . It thus presumably acts by interfering with the binding of RNA polymerase . The acn-acnR organization is conserved in all corynebacteria and mycobacteria with known genome sequence and a putative AcnR consensus binding motif (CAGNACnnncGTACTG) was identified in the corresponding acn upstream regions . Mutations within this motif inhibited AcnR binding . Because the activities of citrate synthase and isocitrate dehydrogenase were previously reported not to be increased during growth on acetate, our data indicate that aconitase is a major control point of tricarboxylic acid cycle activity in C . glutamicum, and they identify AcnR as the first transcriptional regulator of a tricarboxylic acid cycle gene in the Corynebacterianeae.

Biol Chem, 2004 Sep, 385(9), 853 - 61
Comprehensive analysis of metabolites in Corynebacterium glutamicum by gas chromatography/mass spectrometry; Strelkov S et al.; An analytical method based on gas chromatography/mass spectrometry was developed for metabolome investigation of Corynebacterium glutamicum . For the first time a fast method for metabolic screening that can be automated is described for this organism . More than 1000 compounds could be detected per experiment, ca . 330 of those showed a peak area significantly above background . Out of these 164 compounds were identified so far, representing derivatives of 121 different metabolites, which were quantified in one sample . In spite of the different chemical nature of metabolites and high matrix content, a measurement reproducibility in the range of 6% error was achieved . The application of this method for the analysis of the adaptation of C . glutamicum to different growth conditions is demonstrated.

Metab Eng, 2004 Oct, 6(4), 256 - 67
Metabolic network simulation using logical loop algorithm and Jacobian matrix; Yang TH et al.; A novel method to accomplish efficient numerical simulation of metabolic networks for flux analysis was developed . The only inputs required are the set of stoichiometric balances and the atom mapping matrices of all components of the reaction network . The latter are used to automatically calculate isotopomer mapping matrices . Using the symbolic toolbox of MATLAB the analytical solution of the stoichiometric balance equation system, isotopomer balances and the analytical Jacobian matrix of the total set of stoichiometric and isotopomer balances are created automatically . The number of variables in the isotopomer distribution equation system is significantly reduced applying modified isotopomer mapping matrices . These allow lumping of several consecutive isotopomer reactions into a single one . The solution of the complete system of equations is improved by implementing an iterative logical loop algorithm and using the analytical Jacobian matrix . This new method provided quick and robust convergence to the root of such equation systems in all cases tested . The method was applied to a network of lysine producing Corynebacterium glutamicum . The resulting equation system with the dimension of 546 x 546 was directly derived from 12 isotopomer balance equations . The results obtained yielded identical labeling patterns for metabolites as compared to the relaxation method.

Perit Dial Int, 2004 Sep-Oct, 24(5), 454 - 9
Exit-site infections by non-diphtheria corynebacteria in CAPD; Schiffl H et al.; Non-diphtheria corynebacteria species cause disease in risk populations such as immunocompromised patients and patients with indwelling medical devices . Despite reports of exit-site infection and peritonitis caused by non-diphtheria corynebacteria, these organisms are frequently dismissed as contaminants . During a 10-year observation period, we prospectively identified 8 cases of exit-site/tunnel infections caused by 2 different species of corynebacteria (Corynebacterium striatum in 5 and C . jeikeium in 3 cases) . Four patients experienced a second episode of exit-site infection 3 months (2 cases), 25 months, and 40 months, respectively, after termination of an oral cephalosporin therapy of 4 to 6 weeks' duration . Non-diphtheria corynebacteria accounted for 9% of all exit-site infections during the study period . All catheter-related infections healed; no catheter had to be removed . The diagnosis of catheter-related non-diphtheria corynebacteria infection may be suspected when Gram stain shows gram-positive rods and with colony morphology and commercial biochemical identification systems . Susceptibility of non-diphtheria corynebacteria to antibiotics may vary, especially in C . jeikeium . Virtually all Corynebacterium species are sensitive to vancomycin . Empirical antibiotic therapy with vancomycin should be initiated while antibiotic susceptibility testing is being carried out . Oral cephalosporin may be an alternative treatment regimen for exit-site infections if sensitive . This study highlights the importance of non-diphtheria corynebacteria as emerging nosocomial pathogens in the population of end-stage renal disease patients on on continuous ambulatory peritoneal dialysis.

Zhonghua Er Ke Za Zhi, 2004 Sep, 42(9), 663 - 7
{Rapid diagosis of neonatal sepsis by 16SrRNA genes PCR amplification and genechip hybridization}; Tong MQ et al.; OBJECTIVE: To explore a method for rapid diagnosis of sepsis in newborn infants . METHODS: (1) The primers and oligonucleotide probes were designed and synthesized based on the sequences of bacterial 16SrRNA gene . The gene chip was prepared through the probes printed onto special glass slides . The gene chip included 18 special probes: universal probe 1, universal probe 2, Gram positive bacterial probe, Gram negative bacterial probe 1, Gram negative bacterial probe 2, Staphylococcus aureus, coagulase negative staphylococcus (CoNS) 1, CoNS 2, Escherichia coli, Hemophilus influenzae, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Bacteroides fragilis, Bacillus, Meningococcus, Corynebacterium, Propionibacterium; (2) Blood specimens from 285 cases of suspected septicemia were cultured and bacterial 16S rRNA gene was detected separately; DNA isolated from blood specimens and cerebrospinal fluid was amplified by PCR, and PCR products were hybridized with the probes on the gene chips . Hybridization results were scanned and read by laser-scanner . RESULTS: (1) Of the 285 cases, 17 were positive by PCR and the positive rate (5.96%) was significantly higher than that of blood culture (2.81%) (P < 0.01) . When blood culture was taken as control, the sensitivity of PCR was 100% and Specificity was 96.75%, the index of accurate diagnosis was 0.968 . (2) The 17 specimens which showed positive results by PCR were further hybridized on the gene chip . All were positive by universal probes . Among all of them, 5 were positive by E . coli probe; 4 were positive by Staphylococcus epidermidis; two were positive by Bacillus and Propionibacterium probes, separately; 4 were positive by CoNS . The 8 specimens which showed positive results by both PCR and blood culture, the result of gene chip hybridization coincided with the result of blood culture . CONCLUSION: Detection of the bacterial 16SrRNA genes in clinical specimens by gene chip hybridization technology can diagnose neonatal septicemia rapidly . This method has higher sensitivity and specificity than blood culture or other methods and can provide a rapid way for the etiological diagnosis of neonatal septicemia . Therefore the genechip method may be valuable and practical in early diagnosis of neonatal septicemia.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Jul-Aug, (4), 76 - 8
{Properties of Corynebacterium diphtheriae cultivated in the medium prepared from raw materials unsuitable for use as foodstuffs}; The glycosylated cell surface protein Rpf2 et al.; Lehrstuhl fur Genetik, Universitat Bielefeld, Universitatsstrasse 25, Bielefeld, GermanyThe genome of Corynebacterium glutamicum ATCC 13032 contains two genes, rpf1 and rpf2, encoding proteins with similarities to the essential resuscitation-promoting factor (Rpf) of Micrococcus luteus . Both the Rpf1 (20.4 kDa) and Rpf2 (40.3 kDa) proteins share the so-called Rpf motif, a highly conserved protein domain of approximately 70 amino acids, which is also present in Rpf-like proteins of other gram-positive bacteria with a high G+C content of the chromosomal DNA . Purification of the C . glutamicum Rpf2 protein from concentrated supernatants, SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified modified Rpf2 variants with increased or reduced mobility when compared with the calculated size of Rpf2 . A Western blot-based enzyme immunoassay demonstrated glycosylation of the Rpf2 variants with higher molecular masses . Galactose and mannose were identified as two components of the oligosaccharide portion of the Rpf2 glycoprotein by capillary gas chromatography coupled to mass spectrometry . The Rpf2 protein was localized on the surface of C . glutamicum with the use of immuno-fluorescence microscopy . C . glutamicum strains with defined deletions in the rpf1 or rpf2 gene or simultaneous deletions in both rpf genes were constructed, indicating that the rpf genes are neither individually nor collectively essential for C . glutamicum . The C . glutamicum rpf double mutant displayed slower growth and a prolonged lag phase after transfer of long-stored cells into fresh medium . The addition of supernatant from exponentially growing cultures of the rpf double mutant, the wild type or C . glutamicum strains with increased expression of the rpf1 or rpf2 gene significantly reduced the lag phase of long-stored wild-type and rpf single mutant strains, but addition of purified His-tagged Rpf1 or Rpf2 did not . In contrast, the lag phase of the C . glutamicum rpf double mutant was not affected upon addition of these culture supernatants .

Bioresour Technol, 2005 Feb, 96(3), 287 - 94
Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium; Kumar D et al.; The production of methionine by submerged fermentation using a mutant strain of Corynebacterium lilium was studied to determine suitable conditions for obtaining high productivity . The mutant strain resistant to the methionine analogues ethionine, norleucine, methionine sulfoxide and methionine methylsulfonium chloride produced 2.34 g l(-1) of methionine in minimal medium containing glucose as carbon source . The effect of cysteine on methionine production in a 15 l bioreactor was studied by supplementing cysteine intermittently during the course of fermentation . The addition of cysteine (0.75 g l(-1)h(-1)) every 2 h to the production medium increased the production of methionine to 3.39 g l(-1) . A metabolic flux analysis showed that during cysteine supplementation the ATP consumption reduced by 20% . It also showed that the increase in flux from phosphoenol pyruvate to oxaloacetate leads to higher methionine production . Results indicate that controlling the respiratory quotient close to 0.75 will produce the highest amount of methionine and that regulatory mutants also resistant to analogues of cysteine would be better methionine over producers.

Mol Microbiol, 2004 Oct, 54(2), 420 - 38
Deletion of the genes encoding the MtrA-MtrB two-component system of Corynebacterium glutamicum has a strong influence on cell morphology, antibiotics susceptibility and expression of genes involved in osmoprotection; Moker N et al.; The MtrAB two-component signal transduction system is highly conserved in sequence and genomic organization in Mycobacterium and Corynebacterium species, but its function is completely unknown . Here, the role of MtrAB was studied with C . glutamicum as model organism . In contrast to M . tuberculosis, it was possible to delete the mtrAB genes in C . glutamicum . The mutant cells showed a radically different cell morphology and were more sensitive to penicillin, vancomycin and lysozyme but more resistant to ethambutol . In order to identify the molecular basis for this pleiotropic phenotype, the mRNA profiles of mutant and wild type were compared with DNA microarrays . Three genes showed a more than threefold increased RNA level in the mutant, i.e . mepA (NCgl2411) encoding a putative secreted metalloprotease, ppmA (NCgl2737 ) encoding a putative membrane-bound protease modulator, and lpqB encoding a putative lipoprotein of unknown function . Expression of plasmid-encoded mepA in Escherichia coli led to elongated cells that were hypersensitive to an osmotic downshift, supporting the idea that peptidoglycan is the target of MepA . The mRNA level of two genes was more than fivefold decreased in the mutant, i.e . betP and proP which encode transporters for the uptake of betaine and proline respectively . The microarray results were confirmed by primer extension and RNA dot blot experiments . In the latter, the transcript level of genes involved in osmoprotection was tested before and after an osmotic upshift . The mRNA level of betP, proP and lcoP was strongly reduced or undetectable in the mutant, whereas that of mscL (mechanosensitive channel) was increased . The changes in cell morphology, antibiotics susceptibility and the mRNA levels of betP, proP, lcoP, mscL and mepA could be reversed by expression of plasmid-encoded copies of mtrAB in the DeltamtrAB mutant, confirming that these changes occurred as a consequence of the mtrAB deletion.

J Dermatol, 1977 Oct, 4(5), 193 - 202
A study on the characterization of Corynebacterium acnes; Takizawa K; Forty-nine strains of anaerobic gram-positive rods were used in a systematic study of their biochemical and physiological reactions and morphological characteristics and were also subjected to gas chromatographic analyses in an effort to classify them as strains of Corynebacterium acnes (C . acnes) . The strains were isolated both from lesions in acne vulgaris and from normal skin . According to their biochemical and physiological characters, these 49 strains were divided into six subgroups (Subgroup A-F) . They were also separated into two morphological types . The larger of these two types included gram-positive, unevently staining pleomorphic rods (35 strains); the smaller type contained shorter coccal rods similar to Peptostreptococci (14 strains) . The macroscopic appearance of the colonies of both types was the same . All strains of the smaller type showed the same biochemical and physiological characteristics which were of the saccharolytic type (Subgroup B) suggesting a close relationship between the microscopic appearance of the strains and their biochemical and physiological characteristics . Upon microscopical observation, the changing the pH of the media did not cause any transformation of the organism from one type to another . Between pH 6.0 and 6.5 all strains grew well but above pH 8.0 growth was poor . The gas chromatographic analyses demonstrated that selected sample strains from each of the six subgroups showed the same characteristic chromatograph, suggesting that they could be of the same species, i.e., C . acnes.

Mol Microbiol, 2004 Oct, 54(1), 132 - 47
Regulation of GlnK activity: modification, membrane sequestration and proteolysis as regulatory principles in the network of nitrogen control in Corynebacterium glutamicum; Strosser J et al.; P(II)-type signal transduction proteins play a central role in nitrogen regulation in many bacteria . In response to the intracellular nitrogen status, these proteins are rendered in their function and interaction with other proteins by modification/demodification events, e.g . by phosphorylation or uridylylation . In this study, we show that GlnK, the only P(II)-type protein in Corynebacterium glutamicum, is adenylylated in response to nitrogen starvation and deadenylylated when the nitrogen supply improves again . Both processes depend on the GlnD protein . As shown by mutant analyses, the modifying activity of this enzyme is located in the N-terminal part of the enzyme, while demodification depends on its C-terminal domain . Besides its modification status, the GlnK protein changes its intracellular localization in response to changes of the cellular nitrogen supply . While it is present in the cytoplasm during nitrogen starvation, the GlnK protein is sequestered to the cytoplasmic membrane in response to an ammonium pulse following a nitrogen starvation period . About 2-5% of the GlnK pool is located at the cytoplasmic membrane after ammonium addition . GlnK binding to the cytoplasmic membrane depends on the ammonium transporter AmtB, which is encoded in the same transcriptional unit as GlnK and GlnD, the amtB-glnK-glnD operon . In contrast, the structurally related methylammonium/ammonium permease AmtA does not bind GlnK . The membrane-bound GlnK protein is stable, most likely to inactivate AmtB-dependent ammonium transport in order to prevent a detrimental futile cycle under post-starvation ammonium-rich conditions, while the majority of GlnK is degraded within 2-4 min . Proteolysis in the transition period from nitrogen starvation to nitrogen-rich growth seems to be specific for GlnK; other proteins of the nitrogen metabolism, such as glutamine synthetase, or proteins unrelated to ammonium assimilation, such as enolase and ATP synthase subunit F(1)beta, are stable under these conditions . Our analyses of different mutant strains have shown that at least three different proteases influence the degradation of GlnK, namely FtsH, the ClpCP and the ClpXP protease complex.

Arthritis Rheum, 2004 Sep, 50(9), 2985 - 94
The etiologic diagnosis of infectious discitis is improved by amplification-based DNA analysis; Lecouvet F et al.; OBJECTIVE: Blood cultures and cultures of disc material are required to identify and treat bacterial agents responsible for septic spondylodiscitis, but these methods have limited sensitivities . We undertook this study to compare nonculture amplification-based DNA analysis with conventional culture of disc aspirate . METHODS: Nineteen patients with spondylodiscitis, including 11 with a history of spinal surgery, presented with negative blood cultures and underwent percutaneous disc or epidural abscess puncture for bacterial diagnosis . Amplification by polymerase chain reaction was performed on 16S ribosomal DNA universal target genes and femA staphylococci-specific target genes in all patients, and on the upstream p34 mycobacterial gene in 1 patient . Species identification relied on amplicon sequencing and comparison with templates from GenBank . Amplification of the femA gene led to subsequent testing for methicillin resistance by amplification of the mecA gene . Further assessment using a staphylococci- and methicillin resistance-specific DNA array was performed on 3 samples . RESULTS: Microbiologic and molecular assays identified the causative organism in 14 of 19 patients (74%) and 19 of 19 patients (100%), respectively . In culture-positive patients, DNA-based and microbiologic results were highly correlated . Five agents (Staphylococcus simulans, Staphylococcus sciuri, Brucella species, Actinomyces israelii, and Mycobacterium tuberculosis complex) were identified only by DNA-based methods . In 1 sample, Corynebacterium jeikeium and coagulase-negative Staphylococcus were both cultured, whereas DNA analysis identified only Staphylococcus hominis . CONCLUSION: DNA-based methods are highly sensitive and specific . They can usefully complement standard microbiologic methods for identifying the cause of infectious spondylodiscitis and contribute to species-specific therapeutic orientation in patients with negative blood and disc aspirate cultures.

J Biol Chem, 2004 Dec 17, 279(51), 53554 - 61 Epub 2004 Sep 29.
Functional studies of the Mycobacterium tuberculosis iron-dependent regulator; Chou CJ et al.; The iron-dependent regulator (IdeR) protein in Mycobacterium tuberculosis, and its better characterized homologue, the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae, are iron-dependent regulatory proteins that control gene expression in response to iron availability in bacteria . IdeR regulates several genes required for iron uptake and storage including those involved in the synthesis of transition metal chelators called siderophores that are linked to the M . tuberculosis virulence . In this study, the metal ion and binding affinities for IdeR binding to an fxbA operator duplex DNA were estimated using fluorescence assays . The Fe(2+), Co(2+), and Ni(2+) affinities of the two metal ion binding sites in IdeR that are involved in the activation of the regulator DNA binding process in vitro were independently estimated . Binding to the two metal ion binding sites is apparently cooperative and the two affinities differ significantly . Occupation of the first metal ion binding site causes dimerization of IdeR, and the metal ion affinity is about 4 microM for Ni(2+) and much less for Fe(2+) and Co(2+) . Binding of the second metal ion fully activates IdeR for binding to the fxbA operator . The equilibrium metal ion dissociation constants for IdeR-fxbA operator binding are approximately 9 microM for Fe(2+), 13 microM for Ni(2+), and 23 microM for Co(2+) . Interestingly, the natural IdeR cofactor, Fe(2+), shows high affinities toward both binding sites . These results provide insight into the possible roles for each metal binding site in IdeR activation.

BMC Microbiol . 2004 Sep 24;4(1):38.
Prediction of DtxR regulon: identification of binding sites and operons controlled by Diphtheria toxin repressor in Corynebacterium diphtheriae; Yellaboina S et al.; BACKGROUND: The diphtheria toxin repressor, DtxR, of Corynebacterium diphtheriae has been shown to be an iron-activated transcription regulator that controls not only the expression of diphtheria toxin but also of iron uptake genes . This study aims to identify putative binding sites and operons controlled by DtxR to understand the role of DtxR in patho-physiology of Corynebacterium diphtheriae . RESULT: Positional Shannon relative entropy method was used to build the DtxR-binding site recognition profile and the later was used to identify putative regulatory sites of DtxR within C . diphtheriae genome . In addition, DtxR-regulated operons were also identified taking into account the predicted DtxR regulatory sites and genome annotation . Few of the predicted motifs were experimentally validated by electrophoretic mobility shift assay . The analysis identifies motifs upstream to the novel iron-regulated genes that code for Formamidopyrimidine-DNA glycosylase (FpG), an enzyme involved in DNA-repair and starvation inducible DNA-binding protein (Dps) which is involved in iron storage and oxidative stress defense . In addition, we have found the DtxR motifs upstream to the genes that code for sortase which catalyzes anchoring of host-interacting proteins to the cell wall of pathogenic bacteria and the proteins of secretory system which could be involved in translocation of various iron-regulated virulence factors including diphtheria toxin . CONCLUSIONS: We have used an in silico approach to identify the putative binding sites and genes controlled by DtxR in Corynebacterium diphtheriae . Our analysis shows that DtxR could provide a molecular link between Fe+2-induced Fenton's reaction and protection of DNA from oxidative damage . DtxR-regulated Dps prevents lethal combination of Fe+2 and H2O2 and also protects DNA by nonspecific DNA-binding . In addition DtxR could play an important role in host interaction and virulence by regulating the levels of sortase, a potential vaccine candidate and proteins of secretory system.

Curr Microbiol, 2004 Sep, 49(3), 152 - 7
The open reading frame present in the nrdEF cluster of Corynebacterium ammoniagenes is a transcriptional regulator belonging to the GntR family; Torrents E et al.; In this work we have identified the cagntR gene, present between the nrdE and nrdF genes of Corynebacterium ammoniagenes, as a transcriptional regulator belonging to the GntR family . This gene encodes a transcriptional factor actively transcribed in the opposite direction relative to the nrdHIEF operon . It is expressed in a cell-culture-dependent fashion and, although the members of this family have been reported to regulate transcription of genes found within their vicinity, we have shown that cagntR is not involved in the transcriptional regulation of either nrdE or nrdF . The role of this regulator, however, remains unknown.

J Mol Microbiol Biotechnol, 2004, 7(4), 182 - 96
Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions; Inui M et al.; Lactate and succinate were produced from glucose by Corynebacterium glutamicum under oxygen deprivation conditions without growth . Addition of bicarbonate to the reaction mixture led not only to a 3.6-fold increase in succinate production rate, but also to a 2.3- and 2.5-fold increase, respectively, of the rates of lactate production and glucose consumption, compared to the control . Furthermore, when small amounts of pyruvate were added to the reaction mixture, acid production rates and the glucose consumption rate were multiplied by a factor ranging from 2 to 3 . These phenomena were paralleled by an increase in the NAD(+)/NADH ratio, thus corroborating the view that the efficient regeneration of NAD(+) could be triggered by the addition of either bicarbonate or pyruvate . To investigate the global metabolism of corynebacteria under oxygen deprivation conditions, we engineered several strains where the genes coding for key metabolic enzymes had been inactivated by gene disruption and replacement . A lactate dehydrogenase (LDH)-deficient mutant was not able to produce lactate, suggesting this enzyme has no other isozyme . Although a pyruvate carboxylase (pyc) mutant exhibited similar behavior to that of the wild type, phosphoenolpyruvate carboxylase (ppc) mutants were characterized by a dramatic decrease in succinate production, which was concomitant to decreased lactate production and glucose consumption rates . This set of observations corroborates the view that in coryneform bacteria under oxygen deprivation conditions the major anaplerotic reaction is driven by the ppc gene product rather than by the pyc gene product . Moreover, intracellular NADH concentrations in C . glutamicum were observed to correlate to oxygen-deprived metabolic flows .

J Med Food, 2004 Fall, 7(3), 340 - 2
Nutrient composition and antimicrobial activity of sorrel drinks (soborodo); Oboh G et al.; Aqueous extracts (1,200 mL) of roselle calyx (40 g), fortified with either orange juice or pineapple juice as sweetener and lemon grass as flavorant (sorrel drink), were analyzed with regard to their mineral composition (Na, Fe, Zn, Cu, Pb, Mn, and Ca), vitamin C content, and sensory evaluation . While the medicinal potentials were determined with respect to their inhibitory effect on the growth of Bacillus sp., Pseudomonas aeruginosa, Lactobacillus sp., and Corynebacterium sp . The results revealed that the roselle extract fortified with orange juice had higher vitamin C content than did those fortified with pineapple juice, while those fortified with pineapple juice had the best general acceptability . Zn, Na, and Ca were generally high in all the drinks; however, fortification with either pineapple or orange juice reduced the mineral content of the roselle extract . However, Pb, Cu, and Mn (toxic metals) were not detected . The antimicrobial effect of the unfortified roselle extract was low against the entire organism; however, fortification with pineapple juice and lemon grass greatly enhanced the inhibition of the growth of those organisms . They all had their highest inhibitory effect on the growth of P . aeruginosa . In view of the high Zn, Ca, Fe, Na, and vitamin C content as well as the antimicrobial activity, this cheaply produced drink from purely local materials could serve as a good replacement for expensive carbonated drinks.

Diagn Microbiol Infect Dis, 2004 Sep, 50(1), 71 - 2
Corynebacterium jeikeium sepsis after 8-methoxypsolaren photopheresis for cutaneous T-cell lymphoma; Umeh OC et al.; We describe a patient with cutaneous T-cell lymphoma who developed Corynebacterium jeikeium sepsis after experimental treatment with 8-methoxypsolaren . The epidemiology and clinical features of C . jeikeium infection are discussed . The patient was successfully treated with intravenous vancomycin without recurrence.

Chem Biol, 2004 Sep, 11(9), 1301 - 6
Discovery of picomolar slow tight-binding inhibitors of alpha-fucosidase; Chang CF et al.; Glycosidase inhibitors have shown great medicinal and pharmaceutical values as exemplified by the therapeutic treatment of influenza virus and non-insulin-dependent diabetes . We herein report the discovery of picomolar slow tight-binding inhibitors 2-5 against the alpha-fucosidase from Corynebacterium sp . by a rapid screening for an optimal aglycon attached to 1-aminomethyl fuconojirimycin (1) . The time-dependent inhibition displays the progressive tightening of enzyme-inhibitor complex from a low nanomolar K(i) to picomolar K(i)* value . Particularly compound 2 with a K(i)* of 0.46 pM represents the most potent glycosidase inhibitor to date . The effect of compound 3 on the intrinsic fluorescence of alpha-fucosidase is both time- and concentration-dependent in a saturation-type manner, which is consistent with the initial formation of a rapid equilibrium complex of enzyme and inhibitor (E.I), followed by the slower formation of a tightly bound enzyme-inhibitor complex (E.I*) . The binding affinity increases 3.5 x 10(4)-fold from 1 (K(i) = 16.3 nM) to 2 (K(i)* = 0.46 pM) . This work clearly demonstrates the effectiveness of our combinatorial approach leading to the rapid discovery of potent inhibitors.

Arch Microbiol, 2004 Nov, 182(5), 354 - 63 Epub 2004 Sep 15.
Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis; Netzer R et al.; In many bacteria, pyruvate kinase serves a well-defined function in glycolysis, catalyzing an ATP-generating reaction . However, its role during growth on carbon sources requiring glucoeneogenesis is less well investigated . We analyzed a defined pyruvate kinase gene (pyk) deletion mutant of Corynebacterium glutamicum, which is unable to grow on ribose as sole carbon source . Unexpectedly, the pyk deletion mutant was also unable to grow on acetate or citrate as sole carbon sources unless low amounts of pyruvate were added to the growth medium . A spontaneous suppressor mutant of the pyk deletion strain that regained the ability to grow on acetate was isolated . DNA microarray experiments revealed increased expression of the malic enzyme gene malE . The point mutation upstream of malE identified in this mutant was responsible for the loss of carbon-source-dependent regulation, as revealed by transcriptional fusion analysis . Overexpression of malE was sufficient to restore growth of the pyk deletion strain on acetate or citrate . The requirement of increased malic enzyme levels to re-route the carbon flux at the interface between glycolysis, gluconeogenesis and the tricarboxylic acid cycle in order to compensate for the absence of pyruvate kinase indicates a metabolic flux bifurcation at the metabolic node phosphoenolpyruvate . Whereas during growth of C . glutamicum on acetate or citrate most of the phosphoenolpyruvate generated from oxaloacetate is metabolized in gluconeogenesis, a fraction is converted by pyruvate kinase in the glycolytic direction to sustain proper pyruvate availability for biomass synthesis.

J Clin Microbiol, 2004 Sep, 42(9), 4268 - 74
Use of an oligonucleotide array for laboratory diagnosis of bacteria responsible for acute upper respiratory infections; Roth SB et al.; We developed a diagnostic array of oligonucleotide probes targeting species-specific variable regions of the genes encoding topoisomerases GyrB and ParE of respiratory bacterial pathogens . Suitable broad-range primer sequences were designed based on alignment of gyrB/parE sequences from nine different bacterial species . These species included Corynebacterium diphtheriae, Fusobacterium necrophorum, Haemophilus influenzae, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes . Specific probe sequences were selected by comparative analysis against the European Bioinformatics Database, as well as gyrB/parE sequences generated for this study . To verify specificity, at least six initial oligonucleotide probe sequences per bacterial species were tested by hybridization on a solid glass support using culture collection strains as templates . Finally, three oligonucleotide probes per bacterial species were utilized to examine 65 middle ear fluid and 29 throat swab samples . The sensitivities of the developed assay compared to classic culture from middle ear fluid samples for H . influenzae, M . catarrhalis, and S . pneumoniae were 96 (93 for culture), 73 (93 for culture), and 100% (78% for culture), respectively . No cross-reactivity with bacterial species belonging to the normal oral flora was observed when the 29 throat swab samples were studied . The sensitivity of the assay to detect S . pyogenes from these samples was 93% (80% for culture) . These results provide a proof of concept for the diagnostic use of microarray technology based on broad-range topoisomerase gene amplification, followed by hybridization and specific detection of bacterial species.

J Clin Microbiol, 2004 Sep, 42(9), 4189 - 98
Identification of some charcoal-black-pigmented CDC fermentative coryneform group 4 isolates as Rothia dentocariosa and some as Corynebacterium aurimucosum: proposal of Rothia dentocariosa emend . Georg and Brown 1967, Corynebacterium aurimucosum emend . Yassin et al . 2002, and Corynebacterium nigricans Shukla et al . 2003 pro synon . Corynebacterium aurimucosum; Daneshvar MI et al.; Sixty-three clinical isolates of charcoal-black-pigmented, gram-positive coryneform rods were received for identification by the Centers for Disease Control and Prevention (CDC) and were provisionally designated CDC fermentative coryneform group 4 (FCG4) . Forty-five of these were characterized by morphological, physiologic, antimicrobial susceptibility, cellular fatty acids, 16S rRNA gene sequencing, and DNA-DNA hybridization analyses . Nitrate reduction, cellular fatty acid analysis, 16S rRNA gene sequencing, and DNA-DNA hybridization studies segregated these strains into two groups: FCG4a (8 strains) and FCG4b (37 strains) . The FCG4a strains, only one of which was from a female genitourinary source, produced cellular fatty acid and biochemical profiles similar to those observed with reference strains of Rothia dentocariosa and Rothia mucilaginosa, while the FCG4b strains were similar to Corynebacterium species . DNA-DNA hybridization analysis demonstrated species-level relatedness among six FCG4a tested strains and showed that they were a charcoal-black-pigmented variant of R . dentocariosa . Sixteen isolates of the FCG4b group, mainly from female genitourinary tract specimens, as well as the type strains of two recently named species, Corynebacterium aurimucosum and Corynebacterium nigricans, were shown by DNA-DNA hybridization analysis and the sequencing of the 16S rRNA gene to be related at the species level and unrelated to the type strain of R . dentocariosa; therefore, the Corynebacterium-like strains were classified as a charcoal-black-pigmented variant of C . aurimucosum, because this name has nomenclatural priority over C . nigricans . These findings indicate that FCG4 represents a heterogeneous group that contains pigmented variants of both R . dentocariosa and C . aurimucosum; hence, the descriptions of both R . dentocariosa and C . aurimucosum have been amended to include charcoal-black-pigmented variants, and C . nigricans is a pro synonym of C . aurimucosum.

J Clin Microbiol, 2004 Sep, 42(9), 3925 - 31
rpoB gene sequencing for identification of Corynebacterium species; Khamis A et al.; The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria . It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing . However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification . The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods . In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity . Several clusters supported by high bootstrap values were identified . In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing . The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species.

Acta Microbiol Immunol Hung, 2004, 51(1-2), 47 - 56
A comparative study on phyllosphere nitrogen fixation by newly isolated Corynebacterium sp . & Flavobacterium sp . and their potentialities as biofertilizer; Giri S et al.; A number of nitrogen fixing bacteria has been isolated from forest phyllosphere on the basis of nitrogenase activity . Among them two best isolates are selected and identified as Corynebacterium sp . AN1 & Flavobacterium sp . TK2 able to reduce 88 and 132 n mol of acetylene (10(8)cells(-1)h(-1)) respectively . They were grown in large amount and sprayed on the phyllosphere of maize plants as a substitute for nitrogenous fertilizer . Marked improvements in growth and total nitrogen content of the plant have been observed by the application of these nitrogen-fixing bacteria . An average 30-37% increase in yield was obtained, which is nearer to chemical fertilizer treatment . Comparatively better effect was obtained by application of Flavobacterium sp.

Urology, 2004 Sep, 64(3), 569 - 73
Encrusted cystitis and pyelitis in children: an unusual condition with potentially severe consequences; Meria P et al.; OBJECTIVES: To report our experience with the management of encrusted cystitis and pyelitis (EC and EP) in the pediatric population . EC and EP are well-known entities in adults but are rarely identified in children . They consist of mucosal encrustations and are due to specific microorganisms . METHODS: Between 1996 and 2001, 4 children with a mean age of 9 years (range 4 to 13) were treated for EC (n = 2), EP (n = 1), and EC and EP (n = 1) . The latter was a kidney transplant recipient . We retrospectively evaluated the clinical characteristics of the patients and the results of conservative management . RESULTS: The delay between the beginning of the symptoms and the diagnosis was longer than 1 month in all cases . The diagnosis of EC was not evoked and was made during cystoscopy in all cases . EP was diagnosed during pyelotomy in 1 patient because it was evoked and confirmed by computed tomography scan in the kidney transplant recipient . Corynebacterium urealyticum was identified in the urine of all patients . EC was treated by antibiotics and endoscopic debulking, and EP was treated by antibiotics and local acidification . The duration of antibiotic therapy was between 1 and 6 months . The tolerance to local acidification of the kidneys was poor . Cure was achieved in 3 cases, but the treatment of EP failed in the kidney transplant recipient and graft removal was decided after 6 months of failed management because intractable febrile urinary tract infections became life threatening for the patient . CONCLUSIONS: EC and EP are uncommon in children; however, these diseases must be considered . They must be diagnosed rapidly and require, if possible, conservative management . Nevertheless, kidney loss can occur in transplant recipients with EP.

Microb Pathog, 2004 Sep, 37(3), 111 - 8
Internalization of non-toxigenic Corynebacterium diphtheriae by cultured human respiratory epithelial cells; Bertuccini L et al.; Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, and diphtheria is one of the most worried diseases, this microorganism can be associated also with invasive infections such as endocarditis, septic arthritis, and osteomyelitis . Invasive infections are usually caused by non-toxigenic C . diphtheriae strains . Over the last years severe pharyngitis/tonsillitis associated with the isolation of non-toxigenic C . diphtheriae have been described . Penicillin treatment failure of these infections could only partially be explained by penicillin tolerance of the causing strain . Thus, we examined the in vitro ability of non-toxigenic C . diphtheriae throat clinical isolates to adhere to, and enter human respiratory epithelial cells . Trasmission and scanning electron microscopy demonstrated intracellular C . diphtheriae in laryngeal (HEp-2 cells) and pharyngeal (Detroit D562 cells) tissue culture . Live intracellular bacteria were detectable up to 48 h post-infection . Using a variety of compound that act on eukariotic cell structures, the internalization of C . diphtheriae seems to occur via a zipper-like mechanism . It is likely that internalization of C . diphtheriae can be involved in throat colonization contributing to bacterial eradication failure and asymptomatic carriage.

Arch Microbiol, 2004 Oct, 182(2-3), 119 - 25 Epub 2004 Aug 31.
Overproduction of NAD+ and 5'-inosine monophosphate in the presence of 10 microM Mn2+ by a mutant of Corynebacterium ammoniagenes with thermosensitive nucleotide reduction (nrd(ts)) after temperature shift; Abbouni B et al.; Corynebacterium ammoniagenes strain CH31 is thermosensitive due to a mutation in nucleotide reduction ( nrd(ts)) . The strain was examined for nucleotide overproduction upon shifting the culture temperature to a range of elevated temperatures . No overproduction of NAD(+) was detected in the control maintained at 27 degrees C whereas NAD(+) was accumulated extracellularily by strain CH31 at 37 degrees C and at 40 degrees C . As a result of the temperature shift, division-inhibited cells displayed only limited elongation . This is a characteristic morphological feature of cell-cycle-arrested coryneform bacteria . Ribonucleotide reductase (RNR) activity was inactivated immediately after the temperature shift in the NAD(+)-proficient cultures, leading presumably to an exhaustion of deoxyribonucleotide pools and impairment of DNA replication . In contrast to the low extracellular accumulation of NAD(+), at the non-permissive temperature of 35 degrees C a distinct capacity for intracellular nucleotide overproduction was revealed by a new method using nucleotide-permeable cells . The approach of shifting the culture temperature was applied successfully to the overproduction of taste-enhancing nucleotides in the presence of 10 microM Mn(2+) . Concomitant with a dramatic loss of viability, the thermosensitive mutant CH31 accumulated 5.3 g 5'-inosine monophosphate per liter following the addition of hypoxanthine as precursor for the salvage pathway.

Plasmid, 2004 Sep, 52(2), 102 - 18
Comparative genomics identified two conserved DNA modules in a corynebacterial plasmid family present in clinical isolates of the opportunistic human pathogen Corynebacterium jeikeium; Tauch A et al.; Investigation of 62 clinical isolates of the opportunistic human pathogen Corynebacterium jeikeium revealed that 17 possessed plasmids ranging in size from 7.6 to 14.9 kb . The plasmids formed four groups on DNA restriction analysis . The complete nucleotide sequence of a representative from each group (pK43, pK64, pCJ84, and pB85766) was subsequently determined . Additionally, two plasmids (pCo455 and pCo420) were shown to be derivatives of pK43 and pK64 carrying insertion sequences of the IS3 family . Comparative genomics identified a conserved plasmid backbone consisting of two distinct DNA modules . Conserved motifs in the parAB-repA module indicated that the sequenced plasmids from C . jeikeium are new members of the pNG2 family . Recombinant derivatives of pK43 were shown to replicate in the soil bacterium Corynebacterium glutamicum and in the human pathogen Corynebacterium diphtheriae . The second plasmid module most likely encodes a novel type of DNA invertase . The respective gene is flanked by highly conserved 112-bp inverted repeats . All plasmids are 'loaded' with a characteristic set of genes encoding products of unknown function . Plasmids indistinguishable from pK43 by DNA restriction analysis were identified in different C . jeikeium strains, which revealed 16S-23S rDNA spacer length polymorphisms and specific antibiotic susceptibility profiles, implying a wide dissemination of the plasmid in clinical isolates of C . jeikeium.

J Dairy Sci, 2004 Aug, 87(8), 2433 - 41
Bovine mastitis in Finland 2001--prevalence, distribution of bacteria, and antimicrobial resistance; Pitkala A et al.; A nationwide survey was conducted in Finland to estimate prevalence of bovine mastitis, distribution of mastitis pathogens, and in vitro antimicrobial susceptibility of different mastitis pathogens . In total, 12,661 quarter milk samples were collected from 3282 dairy cows at 216 farms . These were randomly selected from a database covering all Finnish dairy farms . Quarter milk samples collected by the dairy advisors were submitted for somatic cell counting, bacteriological examination, and testing for antimicrobial susceptibility . If the milk SCC of a cow or of a quarter exceeded 300,000/mL, the cow was defined as having mastitis . The results were compared with those of a previous survey done in 1995 . The prevalence of mastitis continued to decrease from 38% in 1995 to 31% in 2001 . Compared with the study from 1995, the number of quarters with bacterial growth in 2001 increased significantly from 21.0 to 33.5% . This mainly resulted from increased prevalence of Corynebacterium bovis . Coagulase-negative staphylococci remained the most common bacterial group, comprising almost one-half of the pathogens isolated, whereas the relative number of Staphylococcus aureus isolations decreased from the time of the previous study . According to in vitro antimicrobial susceptibility testing, the enterococci demonstrated the highest level of resistance . Compared with the other Nordic countries, penicillin resistance among the staphylococci was still at a relatively high level in Finland (52.1 and 32.0% for Staphylococcus aureus and coagulase-negative staphylococci, respectively) . Streptococci isolated from mastitis were very susceptible to beta-lactam antibiotics, as also found in the previous survey in 1995.

J Dairy Sci, 2004 Aug, 87(8), 2393 - 400
Efficacy of extended ceftiofur intramammary therapy for treatment of subclinical mastitis in lactating dairy cows; Oliver SP et al.; Little research has focused on treatment of cows with subclinical mastitis during lactation . Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use that inhibits bacterial cell wall synthesis by interfering with enzymes essential for peptidoglycan synthesis . Ceftiofur should be effective against a wide range of contagious and environmental mastitis pathogens . Objectives of the present study were to evaluate the efficacy of ceftiofur for treatment of subclinical mastitis in lactating dairy cows, and to determine if extended therapy regimens enhanced efficacy of ceftiofur . Holstein and Jersey dairy cows (n = 88) from 3 dairy research herds were used . Cows were enrolled in the study based on milk somatic cell counts >400,000/mL and isolation of the same mastitis pathogen in 2 samples obtained 1 wk apart . Cows with one or more intramammary infections (IMI) were blocked by parity and DIM and allocated randomly to 1 of 3 different ceftiofur treatment regimens: 2-d (n = 49 IMI), 5-d (n = 41 IMI), and 8-d (n = 38 IMI) treatment regimens . For all groups, 125 mg of ceftiofur hydrochloride was administered via intramammary infusion . Eighteen cows with 38 IMI were included as an untreated negative control group . A bacteriological cure was defined as a treated infected mammary quarter that was bacteriologically negative for the presence of previously identified bacteria at 14 and 28 d after the last treatment . Efficacy of ceftiofur therapy against all subclinical IMI was 38.8, 53.7, and 65.8% for the 2-, 5-, and 8-d ceftiofur treatment regimens, respectively . Four of 38 (10.5%) IMI in control cows were cured spontaneously without treatment . All 3 ceftiofur treatment regimens were significantly better than the negative control, and the 8-d extended ceftiofur treatment regimen treatment group was significantly better than the standard 2-d treatment group . Pathogen groups had significantly different cure rates from one another . The cure rate for the 8-d extended ceftiofur treatment regimen was 70% for Corynebacterium bovis, 86% for coagulase-negative Staphylococcus species, 36% for Staph . aureus, 80% for Streptococcus dysgalactiae ssp . dysgalactiae, and 67% for Strep . uberis.

FEBS Lett, 2004 Aug 27, 573(1-3), 155 - 60
LcoP, an osmoregulated betaine/ectoine uptake system from Corynebacterium glutamicum; Steger R et al.; In Corynebacterium glutamicum, four uptake systems for compatible solutes have been characterized so far . DHPE (DeltabetPDeltaputPDeltaproPDeltaectP), a derivative of the C . glutamicum type strain ATCC 13032 carrying deletions in the corresponding genes, still showed a low betaine uptake rate of 1.4 nmol/(min mg cdm) . Genome analyses revealed the presence of a putative carrier, named low capacity osmoregulated permease (LcoP), which shows similarities to compatible solute transporters of the betaine/carnitine/choline transporter (BCCT)-family . Deletion of lcoP in DHPE resulted in betaine and ectoine uptake deficiency . LcoP, a betaine and ectoine permease is regulated at the expression and the activity level by the external osmolality . Addition of local anesthetics modulated the activity of BCCT-family members BetP, EctP, and LcoP in a different manner, indicating a different type of lipid-protein interaction.

Environ Res, 2004 Oct, 96(2), 228 - 34
Comparison of n-eicosane and phenanthrene removal by pure and mixed cultures of two marine bacteria; Syakti AD et al.; The biotransformation activities of two hydrocarbonoclastic marine bacteria, Corynebacterium sp . and Sphingomonas sp . 2MPII, on n-eicosane and phenanthrene were investigated . During a 56-day experiment, in pure and mixed cultures, Corynebacterium sp . and Sphingomonas sp . 2MPII removed about 70% of the initial n-eicosane and phenanthrene concentrations (1 and 0.4 g L(-1), respectively) . In pure cultures, culturable cell abundances increased over time, from 0.8 to 8.6 x 10(-11) CFU L(-1) (Corynebacterium sp.) and from 2.1 to 16 x 10(-11) CFU L(-1) (Sphingomonas sp . 2MPII ) but remained barely constant in mixed cultures . We defined a biotransformation index based on the number of culturable cells rather than the culture protein content, with the biotransformation cell yield (BCY) expressed in grams hydrocarbon CFU(-1) per day to better characterize hydrocarbon removal in pure and mixed cultures . The BCY was markedly higher in mixed than in pure cultures, increasing by a factor of 2-10.7 and 2.3-4.7 for n-eicosane and phenanthrene removal, respectively.

Infez Med, 2004 Jun, 12(2), 126 - 31
{Coryneform bacteria: their clinical significance and resistance patterns during a three-year study}; Ubaldi M et al.; We report data concerning our experience during three years (1998-2001) about isolation, identification and susceptibility towards antimicrobial agents of coryneform bacteria in infections of hospitalized/at risk patients . We isolated 54 Corynebacterium spp., with prevalence of C . striatum (8 strains) and C . amycolatum (7 strains), and 1 strain of Oerskovia spp . and 1 strain of Actinomyces neuii . 31 strains were isolated from the "exit-site" and 6 from peritoneal fluid of CAPD patients . Vancomycin and teicoplanin were always "in vitro" efficacious . Resistance rates towards other antibiotics were the following: 11% for minocycline, 12.5% for tetracycline, 20% for gentamicin and netilmicin, 61% for erythromycin and chloramphenicol, 66% for penicillin.

Ann Univ Mariae Curie Sklodowska {Med}, 2003, 58(1), 466 - 74
Microbial etiologies in community acquired pneumonia (CAP); Szmygin-Milanowska K et al.; The objective of the study was determination of bacterial etiologic factors, including antibiotic atypical pathogens, of community acquired pneumonia (CAP) in adults and of sensitivity of isolated strains . The examined group comprised 50 patients with clinical and x-ray image of pneumonia . Patients' expectoration sputum was analyzed . Amongst all isolated bacteria, the most frequent were Staphylococcus aureus - 17.9%, Haemophilus parainfluenzae - 12.5% and H . influenzae - 8.9% . Identification of Corynebacterium pseudodiphtheriticum in 8.9% of CAP cases drew our particular attention . Staphylococcus aureus was the least antibiotic sensitive microorganism . In the majority of patients (26: 52%), serologic markers of chlamydial infection were determined . Pneumonia often results from mixed typical and atypical flora infection . High percentage of atypical pathogens in the examined material suggests the necessity to administer intracellularly acting antibiotics.

Ann Univ Mariae Curie Sklodowska {Med}, 2003, 58(1), 142 - 8
The role of opportunistic species of Corynebacterium pseudodiphtheriticum in the pathogenesis of CAP (Community Acquired Pneumonia); Chudnicka A et al.; The analysis of eight cases of CAP (Community Acquired Pneumonia) was performed . The clinical samples of sputum were obtained from patients at which C . pseudodiphtheriticum strains were isolated in the quantity indicating the etiologic agent of infection . In two patients, K . pneumoniae and S . aureus were isolated simultaneously . They were considered as coexisting in the infection . C . pseudodiphtheriticum strains were highly susceptible to antibiotics . They were resistant to Erythromycin (87.5%), Clindamycin (87.5%), Lincomycin (75.5%), Trimeth./Sulfam.(37.5%), Chloramfenicol (37.5%) . In the examined group of patients (five persons), the infection with C . pneumoniae was detected as recently passed or in progress with chronic character as the high level of specific antibodies (IgG or IgG and IgA) was present . That fact could predispose to infection with the opportunistic species of C . pseudodiphtheriticum . Of all the examined patients, three were infected with C . pseudodiphtheriticum as the only species responsible for infection (CAP).

J Biol Chem, 2004 Oct 22, 279(43), 44847 - 57 Epub 2004 Aug 11.
Acyl-CoA carboxylases (accD2 and accD3), together with a unique polyketide synthase (Cg-pks), are key to mycolic acid biosynthesis in Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis; Gande R et al.; The Corynebacterianeae such as Corynebacterium glutamicum and Mycobacterium tuberculosis possess several unique and structurally diverse lipids, including the genus-specific mycolic acids . Although the function of a number of genes involved in fatty acid and mycolic acid biosynthesis is known, information relevant to the initial steps within these biosynthetic pathways is relatively sparse . Interestingly, the genomes of Corynebacterianeae possess a high number of accD genes, whose gene products resemble the beta-subunit of the acetyl-CoA carboxylase of Escherichia coli, providing the activated intermediate for fatty acid synthesis . We present here our studies on four putative accD genes found in C . glutamicum . Although growth of the accD4 mutant remained unchanged, growth of the accD1 mutant was strongly impaired and partially recovered by the addition of exogenous oleic acid . Overexpression of accD1 and accBC, encoding the carboxylase alpha-subunit, resulted in an 8-fold increase in malonyl-CoA formation from acetyl-CoA in cell lysates, providing evidence that accD1 encodes a carboxyltransferase involved in the biosynthesis of malonyl-CoA . Interestingly, fatty acid profiles remained unchanged in both our accD2 and accD3 mutants, but a complete loss of mycolic acids, either as organic extractable trehalose and glucose mycolates or as cell wall-bound mycolates, was observed . These two carboxyltransferases are also retained in all Corynebacterianeae, including Mycobacterium leprae, constituting two distinct groups of orthologs . Furthermore, carboxyl fixation assays, as well as a study of a Cg-pks deletion mutant, led us to conclude that accD2 and accD3 are key to mycolic acid biosynthesis, thus providing a carboxylated intermediate during condensation of the mero-chain and alpha-branch directed by the pks-encoded polyketide synthase . This study illustrates that the high number of accD paralogs have evolved to represent specific variations on the well known basic theme of providing carboxylated intermediates in lipid biosynthesis.

Appl Biochem Biotechnol, 2004 Jul-Sep, 118(1-3), 215 - 32
Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays; Polen T et al.; DNA microarray technology has become an important research tool for biotechnology and microbiology . It is now possible to characterize genetic diversity and gene expression in a genomewide manner . DNA microarrays have been applied extensively to study the biology of many bacteria including Escherichia coli, but only recently have they been developed for the Gram-positive Corynebacterium glutamicum . Both bacteria are widely used for biotechnological amino acid production . In this article, in addition to the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, we describe recent applications of DNA microarray technology regarding amino acid production in C . glutamicum and E . coli . We also discuss the impact of functional genomics studies on fundamental as well as applied aspects of amino acid production with C . glutamicum and E . coli.

Curr Microbiol, 2004 Aug, 49(2), 133 - 5
Enantioselective hydrolysis of butyl 2-ethylhexanoate by a strain of Nocardia corynebacteroides; Labeda DP et al.; The results of a screen for microbial esterases that have enantioselective activity for the hydrolysis of butyl 2-ethylhexanoate are described . The preliminary screen determined that a nocardioform bacterial strain, NRRL 21057, exhibited significant activity in preferentially hydrolyzing the S enantiomer of butyl 2-ethylhexanoate . Molecular systematics methods identified NRRL 21057 as a strain of Nocardia corynebacteroides . A survey of phylogenetically related species in the genera Gordonia, Rhodococcus, and Nocardia strains demonstrated that N . corynebacteroides NRRL 21057 is the most active strain known for the specific hydrolysis of the R-isomer of butyl 2-ethylhexanoate and that it provides the S-isomer of 2-ethylhexanoate in 86% enantiomeric excess within 22 h.

J Clin Microbiol, 2004 Aug, 42(8), 3566 - 9
Comparison of the BBL CHROMagar Staph aureus agar medium to conventional media for detection of Staphylococcus aureus in respiratory samples; Flayhart D et al.; Screening for Staphylococcus aureus has become routine in certain patient populations . This study is the first clinical evaluation of the BBL CHROMagar Staph aureus agar (CSA) medium (BD Diagnostics, Sparks, Md.) for detection of S . aureus in nasal surveillance cultures and in respiratory samples from cystic fibrosis (CF) patients . S . aureus colonies appear mauve on CSA . Other organisms are inhibited or produce a distinctly different colony color . S . aureus was identified from all media by slide coagulase, exogenous DNase, and mannitol fermentation assays . Susceptibility testing was performed using the agar dilution method . A total of 679 samples were evaluated . All samples were inoculated onto CSA . Nasal surveillance cultures were inoculated onto sheep blood agar (SBA) (BD Diagnostics), and samples from CF patients were inoculated onto mannitol salt agar (MSA) (BD Diagnostics) . Of the 679 samples cultured, 200 organisms produced a mauve color on CSA (suspicious for S . aureus) and 180 were positive for S . aureus on SBA or MSA . Of 200 CSA-positive samples 191 were identified as S . aureus . Nine mauve colonies were slide coagulase negative and were subsequently identified as Staphylococcus lugdunensis (one), Staphylococcus epidermidis (three), Staphylococcus haemolyticus (one), and Corynebacterium species (four) . CSA improved the ability to detect S . aureus by recovering 12 S . aureus isolates missed by conventional media . Of the 192 S . aureus isolates recovered, 122 were methicillin susceptible and 70 were methicillin resistant . Overall, the sensitivity and specificity of CSA in this study were 99.5 and 98%, respectively . There was no difference in the performance of the slide coagulase test or in susceptibility testing performed on S . aureus recovered from CSA compared to SBA or MSA . Our data support the use of CSA in place of standard culture media for detection of S . aureus in heavily contaminated respiratory samples.

Eur J Biochem, 2004 Aug, 271(16), 3319 - 29
Relationships between structure, function and stability for pyridoxal 5'-phosphate-dependent starch phosphorylase from Corynebacterium callunae as revealed by reversible cofactor dissociation studies; Griessler R et al.; Using 0.4 m imidazole citrate buffer (pH 7.5) containing 0.1 mm l-cysteine, homodimeric starch phosphorylase from Corynebacterium calluane (CcStP) was dissociated into native-like folded subunits concomitant with release of pyridoxal 5'-phosphate and loss of activity . The inactivation rate of CcStP under resolution conditions at 30 degrees C was, respectively, four- and threefold reduced in two mutants, Arg234-->Ala and Arg242-->Ala, previously shown to cause thermostabilization of CcStP {Griessler, R., Schwarz, A., Mucha, J . & Nidetzky, B . (2003) Eur . J . Biochem.270, 2126-2136} . The proportion of original enzyme activity restored upon the reconstitution of wild-type and mutant apo-phosphorylases with pyridoxal 5'-phosphate was increased up to 4.5-fold by added phosphate . The effect on recovery of activity displayed a saturatable dependence on the phosphate concentration and results from interactions with the oxyanion that are specific to the quarternary state . Arg234-->Ala and Arg242-->Ala mutants showed, respectively, eight- and > 20-fold decreased apparent affinities for phosphate (K(app)), compared to the wild-type (K(app) approximately 6 mm) . When reconstituted next to each other in solution, apo-protomers of CcStP and Escherichia coli maltodextrin phosphorylase did not detectably associate to hybrid dimers, indicating that structural complementarity among the different subunits was lacking . Pyridoxal-reconstituted CcStP was inactive but approximately 60% and 5% of wild-type activity could be rescued at pH 7.5 by phosphate (3 mm) and phosphite (5 mm), respectively . pH effects on catalytic rates were different for the native enzyme and pyridoxal-phosphorylase bound to phosphate and could reflect the differences in pK(a) values for the cofactor 5'-phosphate and the exogenous oxyanion.

Appl Microbiol Biotechnol . 2004 Jul 29; {Epub ahead of print}
Effect of NADH dehydrogenase-disruption and over-expression on respiration-related metabolism in Corynebacterium glutamicum KY9714; Nantapong N et al.; The function of type II NADH dehydrogenase (NDH-2) in Gram-positive Corynebacterium glutamicum was investigated by preparing strains with ndh, the NDH-2 gene, disrupted and over-expressed . Although disruption showed no growth defects on glucose minimum medium, the growth rate of the over-expressed strain was lower compared with its parent, C . glutamicum KY9714 . Ndh-disruption and over-expression did not lead to a large change in the respiratory chain and energetics, including the cytochrome components and the H(+)/O ratio . However, in the strain that lacked NDH-2, membrane l-lactate oxidase activity increased, while NDH-2 over-expression led to decreased l-lactate and malate oxidase activities . In addition, relatively high cytoplasmic lactate dehydrogenase (LDH) activity was always present as was malate dehydrogenase, irrespective of NDH-2 level . Furthermore, l-lactate or malate-dependent NADH oxidase activity could be reproduced by reconstitution with the membranes and the cytoplasmic fraction isolated from the disruptant . These results suggest that coupling of LDH and the membrane l-lactate oxidase system, together with the malate-dependent NADH oxidase system, operates to oxidize NADH when the NDH-2 function is defective in C . glutamicum.

Arch Microbiol . 2004 Aug 3; {Epub ahead of print}
The glycosylated cell surface protein Rpf2, containing a resuscitation-promoting factor motif, is involved in intercellular communication of Corynebacterium glutamicum; Hartmann M et al.; The genome of Corynebacterium glutamicum ATCC 13032 contains two genes, rpf1 and rpf2, encoding proteins with similarities to the essential resuscitation-promoting factor (Rpf) of Micrococcus luteus . Both the Rpf1 (20.4 kDa) and Rpf2 (40.3 kDa) proteins share the so-called Rpf motif, a highly conserved protein domain of approximately 70 amino acids, which is also present in Rpf-like proteins of other gram-positive bacteria with a high G+C content of the chromosomal DNA . Purification of the C . glutamicum Rpf2 protein from concentrated supernatants, SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified modified Rpf2 variants with increased or reduced mobility when compared with the calculated size of Rpf2 . A Western blot-based enzyme immunoassay demonstrated glycosylation of the Rpf2 variants with higher molecular masses . Galactose and mannose were identified as two components of the oligosaccharide portion of the Rpf2 glycoprotein by capillary gas chromatography coupled to mass spectrometry . The Rpf2 protein was localized on the surface of C . glutamicum with the use of immuno-fluorescence microscopy . C . glutamicum strains with defined deletions in the rpf1 or rpf2 gene or simultaneous deletions in both rpf genes were constructed, indicating that the rpf genes are neither individually nor collectively essential for C . glutamicum . The C . glutamicum rpf double mutant displayed slower growth and a prolonged lag phase after transfer of long-stored cells into fresh medium . The addition of supernatant from exponentially growing cultures of the rpf double mutant, the wild type or C . glutamicum strains with increased expression of the rpf1 or rpf2 gene significantly reduced the lag phase of long-stored wild-type and rpf single mutant strains, but addition of purified His-tagged Rpf1 or Rpf2 did not . In contrast, the lag phase of the C . glutamicum rpf double mutant was not affected upon addition of these culture supernatants.

J Biotechnol, 2004 Aug 26, 112(1-2), 177 - 93
Classification of hyper-variable Corynebacterium glutamicum surface-layer proteins by sequence analyses and atomic force microscopy; Hansmeier N et al.; The structural S-layer proteins of 28 different Corynebacterium glutamicum isolates have been analyzed systematically . Treatment of whole C . glutamicum cells with detergents resulted in the isolation of S-layer proteins with different apparent molecular masses, ranging in size from 55 to 66 kDa . The S-layer genes analyzed were characterized by coding regions ranging from 1,473 to 1,533 nucleotides coding for S-layer proteins with a size of 490-510 amino acids . Using PCR techniques, the corresponding S-layer genes of the 28 C . glutamicum isolates were all cloned and sequenced . The deduced amino acid sequences of the S-layer proteins showed identities between 69 and 98% and could be grouped into five phylogenetic classes . Furthermore, sequence analyses indicated that the S-layer proteins of the analyzed C . glutamicum isolates exhibit a mosaic structure of highly conserved and highly variable regions . Several conserved regions were assumed to play a key role in the formation of the C . glutamicum S-layers . Especially the N-terminal signal peptides and the C-terminal anchor sequences of the S-layer proteins showed a nearly perfect amino acid sequence conservation . Analyses by atomic force microscopy revealed a committed hexagonal structure . Morphological diversity of the C . glutamicum S-layers was observed in a class-specific unit cell dimension (ranging from 15.2 to 17.4 nm), which correlates with the sequence similarity-based classification . It could be demonstrated that differences in the primary structure of the S-layer proteins were reflected by the S-layer morphology.

Biochemistry, 2004 Aug 10, 43(31), 10112 - 26
1H NMR characterization of the solution active site structure of substrate-bound, cyanide-inhibited heme oxygenase from Neisseria meningitidis: comparison to crystal structures; Liu Y et al.; Heme oxygenase, HO, from the pathogenic bacterium Neisseria meningitidis catabolizes heme for the iron necessary for infection . The enzyme, labeled HemO, exhibits less sequence homology to mammalian HO than another studied HO from Corynebacterium diphtheriae . Solution 1H NMR has been utilized to define the active site molecular and electronic structure of the cyanide-inhibited, substrate-bound complex for comparison with those provided by several crystal structures . Extensive assignments by solely 1H NMR 2D methods reveal a structure that is very strongly conserved with respect to the crystal structure, although 1H/2H exchange indicates dynamically much more stable distal and proximal helices than those for other HOs . Several residues found with alternate orientations in crystal structures of water- and NO-ligated complexes were shown to occupy positions found solely in the NO complex, confirming that there are structural accommodations in response to ligating the substrate complex with a diatomic, H-bond acceptor ligand . The observed dipolar shifts allow the determination of the magnetic axes that show that the Fe-CN unit is tilted approximately 10 degrees toward the alpha-meso position, thereby facilitating the alpha-stereoselectivity of the enzyme . Numerous labile protons with larger than usual low-field bias are identified and, in common with the other HO complexes, shown to participate in an extended, distal side H-bond network . This H-bond network orders several water molecules, most, but not all, of which have been detected crystallographically . A series of three C-terminal residues, His207-Arg208-His209, are not detected in crystal structures . However, 1H NMR finds two residues, His207 and likely Arg208 in contact with pyrrole D, which in crystal structures is exposed to solvent . The nature of the NOEs leads us to propose a H-bond between the proximally oriented His207 ring and the carboxylate of Asp27 and a salt-bridge between the terminus of Arg208 and the reoriented 7-propionyl carboxylate . While numerous ordered water molecules are found near both propionates in the crystal structure, we find much larger water NOEs to the 6- than 7-propionate, suggesting that water molecules near the 7-propionate have been expelled from the cavity by the insertion of Arg208 into the distal pocket . The conversion of the 7-propionate link from the N-terminal region (Lys16) to the C-terminal region (Arg208) in the ligated substrate complex both closes the heme cavity more tightly and may facilitate product exit, the rate-limiting step in the enzyme activity.

Nefrologia, 2004, 24(3), 288 - 93
{Corynebacterium urealyticum in renal trasplantation . CT and sonography imaging characteristics of encrusted cistitis and pielitis}; Vazquez V et al.; PURPOSE: Its described three cases of Corynebacterium urealyticum (CU) infection in patients with renal transplantation and one of its most serious consequences: encrusted pyelitis and cystitis . It is explained the principal keys for its diagnosis, based in the appearance of alkaline pH in in urine analysis (alkaline urine), positives urinary cultures for CU, and the CT and US studies revealed the characteristic images of calcifications in the wall of renal pelvis and bladder . PATIENTS: Three male patients with renal transplantation and CU infection that caused encrusted pyelitis in two of the cases and encrusted cystitis in one case . RESULTS: Calcifications of the urinary tract were noticed in CT in all the patients . In two cases bladder stones were linear, and in the third case they were fundamentally coarse and placed in pelvis . The diagnosis suspicion showed by the images was confirmed by the use of prolonged urine cultures, necessary for detecting CU . All the patients were treated with vancomycin, with success in two of the cases and, finally needing surgery, and after loss of the graft, in the other case . CONCLUSION: Encrusted pyelitis and cystitis are cronic and severe infections of the urinary tract . Calcic struvite incrustations in the urothelium are characteristics of this infection . CT is a choice technique for the diagnosis and followup of the calcifications after treatment.

Indian J Exp Biol, 2004 Feb, 42(2), 202 - 7
Production of lipase in a fermentor using a mutant strain of Corynebacterium species: its partial purification and immobilization; Roy N et al.; Extracellular Corynebacterium lipase was produced using a 2.5 L Chemap fermentor using 1300 ml fermentation medium at temperature 33 degrees C, agitator speed 50 rpm, aeration rate 1 VVM having KLa 16.21 hr(-1) . Crude lipase was purified by salting out method followed by dialysis and immobilized using calcium alginate gel matrix followed by glutaraldehyde cross linking Purification process increased specific activity of enzyme from 2.76 to 114.7 IU/mg . Activity of immobilized enzyme was 107.31 IU/mg . Optimum temperature for purified and immobilized enzyme activity were 65 degrees and 50 degrees C respectively . Optimum pH was 8.0 in both the cases, Km and Vmax value for purified lipase were 111.1 micromol/min and 14.7% respectively . Ca2+ (5 mM) was found to be stimulator for enzyme activity . Immobilized lipase retained 68.18% of the original activity when stored for 40 days.

Biochim Biophys Acta, 2004 Jul 23, 1658(1-2), 31 - 6
BetP of Corynebacterium glutamicum, a transporter with three different functions: betaine transport, osmosensing, and osmoregulation; Kramer R et al.; In order to circumvent deleterious effects of hypo- and hyperosmotic conditions in its environment, Corynebacterium glutamicum has developed a number of mechanisms to counteract osmotic stress . The first response to an osmotic upshift is the activation of uptake mechanisms for the compatible solutes betaine, proline, or ectoine, namely BetP, EctP, ProP, LcoP and PutP . BetP, the most important uptake system responds to osmotic stress by regulation at the level of both protein activity and gene expression . BetP was shown to harbor three different properties, i.e . catalytic activity (betaine transport), sensing of appropriate stimuli (osmosensing) and signal transduction to the catalytic part of the carrier protein which adapts its activity to the extent of osmotic stress (osmoregulation) . BetP is comprised of 12 transmembrane segments and carries N- and C-terminal domains, which are involved in osmosensing and/or osmoregulation . Recent results on molecular properties of these domains indicate the significance of particular amino acids within the terminal 25 amino acids of the C-terminal domain of BetP for the process of osmosensing and osmoregulation.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1055 - 61
Taxonomic characterization of nine strains isolated from clinical and environmental specimens, and proposal of Corynebacterium tuberculostearicum sp . nov; Feurer C et al.; Nine unidentified Gram-positive, lipophilic corynebacteria were isolated from clinical and food samples and subjected to a polyphasic taxonomic analysis . The bacteria were distinguished from Corynebacterium species with validly published names by biochemical tests, fatty acid content and whole-cell protein analysis . Comparative 16S rRNA gene sequence analysis demonstrated unambiguously that the nine strains were related phylogenetically to the species 'Corynebacterium tuberculostearicum' and represented a distinct subline within the genus Corynebacterium . On the basis of both phenotypic and phylogenetic evidence, the formal description of Corynebacterium tuberculostearicum sp . nov . is proposed . The type strain of C . tuberculostearicum is Medalle XT (=LDC-20T=CIP 107291T=CCUG 45418T=ATCC 35529T).

Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1581 - 3
Purification and characterization of O-Acetylserine sulfhydrylase of Corynebacterium glutamicum; Wada M et al.; We highly purified O-acetylserine sulfhydrylase from the glutamate-producing bacterium Corynebacterium glutamicum . The molecular mass of the purified enzyme was 34,500 as determined by SDS-polyacrylamide gel electrophoresis, and 70,800 as determined by gel filtration chromatography . It had an apparent Km of 7.0 mM for O-acetylserine and a Vmax of 435 micromol min-1 (mg x protein)-1 . This is the first report of the cysteine biosynthetic enzyme of C . glutamicum in purified form.

Chest, 2004 Jul, 126(1), 90 - 4
Micrococcus-associated central venous catheter infection in patients with pulmonary arterial hypertension; Oudiz RJ et al.; STUDY OBJECTIVES: To determine the incidence of catheter-related infection in patients with pulmonary arterial hypertension (PAH) receiving epoprostenol (EPO), and to note an etiologic role for Micrococcus spp, which is rarely reported as a pathogen in the medical literature . DESIGN: Observational study . SETTING: Two PAH specialty treatment centers, Harbor-UCLA Medical Center (Torrance, CA), and the College of Physicians and Surgeons, Columbia University (New York, NY) . PATIENTS: A total of 192 patients with PAH receiving continuous therapy with IV EPO . INTERVENTIONS: From 1987 to 2000, 192 patients with PAH received infusions of EPO via central venous catheter . Catheter care included regular dressing changes with dry gauze using a sterile procedure, without the use of flushes . Patients were asked to report on known infections and treatments, and symptoms . All infections were verified by a telephone call to the patient, care provider, and microbiology laboratory whenever possible . MEASUREMENTS AND RESULTS: There were 335,285 catheter days (mean +/- SD, 1,325 +/- 974 catheter days) . There were 88 clinical catheter infections with 51 blood culture-positive infections, necessitating catheter removal in 38 instances . The following pathogens were isolated: Staphylococcus aureus (25); Micrococcus spp (14); mixed flora (3); coagulase-negative Staphylococcus spp (2); Corynebacterium spp (2); Serratia marcessens (1); Enterobacter spp (1); Pseudomonas aeruginosa (1); enterococci (1); and unidentified Gram-positive cocci (1) . The catheter infection rate was 0.26 per 1,000 catheter days . CONCLUSIONS: The use of long-term therapy with continuous EPO appears to be associated with a low incidence of catheter-related infections . Micrococcus spp were the second most common etiologic agent . Caregivers managing patients with PAH must be aware of the risk of catheter infection, as it may contribute to the morbidity and mortality associated with the use of EPO . When isolated, Micrococcus spp should not be viewed as a contaminant, but rather as a true pathogen that may require therapeutic intervention.

Biomacromolecules, 2004 Jul-Aug, 5(4), 1588 - 95
Identification of the Anabaena sp . strain PCC7120 cyanophycin synthetase as suitable enzyme for production of cyanophycin in gram-negative bacteria like Pseudomonas putida and Ralstonia eutropha; Voss I et al.; The cyanophycin synthetase gene cphA1 encoding the major cyanophycin synthetase (CphA) of Anabaena sp . strain PCC7120 was expressed in Escherichia coli conferring so far the highest specific CphA activity to E . coli (6.7 nmol arginine per min and mg protein) . CphA1 and cphA genes of Synechocystis sp . strains PCC6803 and PCC6308 and Synechococcus strain MA19 were also expressed in wild types and polyhydroxyalkanoate-negative (PHA) mutants of Pseudomonas putida and Ralstonia eutropha . Recombinant strains of these bacteria expressing cphA1 accumulated generally more cyanophycin (23.0 and 20.0% of cellular dry matter, CDM, respectively) than recombinants expressing any other cphA (6.8, 9.0, or 15.8% of CDM for P . putida strains and 7.3, 12.6, or 14.1% of CDM for R . eutropha) . Furthermore, PHA-negative mutants of P . putida (9.7, 10.0, 17.5, or 24.0% of CDM) and R . eutropha (8.9, 13.8, 16.0, or 22.0% of CDM) accumulated generally more cyanophycin than the corresponding PHA-positive parent strains (6.8, 9.0, 15.8, and 23.0% of CDM for P . putida strains and 7.3, 12.6, 14.1, or 20.0% of CDM for R . eutropha strains) . Recombinant strains of Gram-positive bacteria (Bacillus megaterium, Corynebacterium glutamicum) were not suitable for cyanophycin production due to accumulation of less cyanophycin and retarded release of cyanophycin . PHA-negative mutants of P . putida and R . eutropha expressing cphA1 of Anabaena sp . strain PCC7120 are therefore preferred candidates for industrial production of cyanophycin.

Appl Environ Microbiol, 2004 Jul, 70(7), 4222 - 9
Osmotic stress response: quantification of cell maintenance and metabolic fluxes in a lysine-overproducing strain of Corynebacterium glutamicum; Varela CA et al.; Osmotic stress diminishes cell productivity and may cause cell inactivation in industrial fermentations . The quantification of metabolic changes under such conditions is fundamental for understanding and describing microbial behavior during bioprocesses . We quantified the gradual changes that take place when a lysine-overproducing strain of Corynebacterium glutamicum is grown in continuous culture with saline gradients at different dilution rates . The use of compatible solutes depended on environmental conditions; certain osmolites predominated at different dilution rates and extracellular osmolalities . A metabolic flux analysis showed that at high dilution rates C . glutamicum redistributed its metabolic fluxes, favoring energy formation over growth . At low dilution rates, cell metabolism accelerated as the osmolality was steadily increased . Flexibility in the oxaloacetate node proved to be key for the energetic redistribution that occurred when cells were grown at high dilution rates . Substrate and ATP maintenance coefficients increased 30- and 5-fold, respectively, when the osmolality increased, which demonstrates that energy pool management is fundamental for sustaining viability.

Appl Environ Microbiol, 2004 Jul, 70(7), 3845 - 54
Impact of heterologous expression of Escherichia coli UDP-glucose pyrophosphorylase on trehalose and glycogen synthesis in Corynebacterium glutamicum; Padilla L et al.; Trehalose is a disaccharide with a wide range of applications in the food industry . We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum . This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors . In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C . glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E . coli ots genes (galU otsBA synthetic operon) . The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described . Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain . Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose . This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway . These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.

Biochem Biophys Res Commun, 2004 Jul 30, 320(3), 846 - 51
Low pKa lysine residues at the active site of sarcosine oxidase from Corynebacterium sp . U-96; Mukouyama EB et al.; Sarcosine oxidase from Corynebacterium sp . U-96 is inactivated by iodoacetamide with the modification of two specific residues . Comparing the amino acid sequence and mass spectra of the peptide fragments containing the modified residues with those from the native enzyme, the modified residues were identified to be lysine . The pKa of these residues were estimated to be 8.5 and 6.7 from the pH dependence of inactivation in the presence and absence of the competitive inhibitor, acetate . These estimated pKa values are much lower than that of the epsilon-amino group of lysine residue . There may be unique microenvironments around these residues that activate their -amino groups to be susceptible to iodoacetamide . A possible role of the lysine residue with pKa 6.7 is discussed.

J Bacteriol, 2004 Jul, 186(14), 4813 - 7
Transcriptional analysis of the groES-groEL1, groEL2, and dnaK genes in Corynebacterium glutamicum: characterization of heat shock-induced promoters; Barreiro C et al.; The appropriate conditions to switch on the heat shock promoters in Corynebacterium glutamicum were defined by Northern blot analysis . Transcriptional patterns were characterized for the groEL2 gene and the groES-groEL1 and dnaK operons . Transcriptional start points of these genes were determined by primer extension analysis, allowing the identification of CIRCE and HAIR boxes close to the -10 and -35 regions of the promoters . The presence of both CIRCE and HAIR sequences within a single promoter (P-groEL2) in bacteria is described for the first time . In addition, the dnaK promoter showed -10 and -35 sequences similar to those recognized by SigH of Mycobacterium and SigR of Streptomyces close to a second transcription start region with -10 and -35 boxes typical of promoters for housekeeping genes .

Int J Biol Macromol, 2004 Jun, 34(3), 181 - 9
Three-dimensional models and structure analysis of corynemycolyltransferases in Corynebacterium glutamicum and Corynebacterium efficiens; Adindla S et al.; The corynemycolyltransferase proteins were identified from Corynebacterium glutamicum and Corynebacterium efficiens genomes using computational tools available in the public domain . Three-dimensional models were constructed for corynemycolyltransferases based on the crystal structures of related mycolyltransferases in Mycobacterium tuberculosis using the comparative modeling methods . The corynemycolyltransferases share overall an alpha/beta-fold characteristic of the mycolyltransferases despite low sequence identity (<20%) shared by some of the corynemycolyltransferases . However, a significant difference is observed in the region between amino acid residues Trp82-Trp97 and Ala222-Asn223 corresponding to mycolyltransferases . The specificity pockets defined by interactions with the trehalose substrate observed in the crystal structure complex of Ag85B mycolyltransferase (PDB code: 1F0P) suggests that trehalose may not bind some corynemycolyltransferases . This is due to critical mutations in corynemycolyltransferase binding subsites that lead to loss of equivalent side-chain interactions with trehalose and unfavorable steric interactions, particularly, in the case of cmytC gene and the protein corresponding to the gene identifier CE0356 with the equivalent Ala222-Asn223 "long insertion loop" . Further, the fibronectin binding region (Phe58-Val69), in mycolyltransferases associated with mediating host-pathogen interactions in M . tuberculosis comprises amino acid residue mutations in the corresponding region in the soil bacterium--Corynebacterium corynemycolyltransferases, that suggest a different epitope and therefore possible lack of binding to fibronectin . The corynemycolyltransferase cmytA responsible for the cell shape formation and for maintaining the cell surface integrity is associated with a C-terminal domain that we have recently shown to comprise tandem amino acid sequence repeats that is likely to be associated with a regular secondary structural motif.

Mol Microbiol, 2004 Jul, 53(1), 251 - 61
Sortases and pilin elements involved in pilus assembly of Corynebacterium diphtheriae; Ton-That H et al.; Corynebacterium diphtheriae SpaA pili are composed of three pilin subunits, SpaA, SpaB and SpaC . SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals, and SpaC seems to be positioned at the pilus tip . Pilus assembly in C . diphtheriae requires the pilin motif and the C-terminal sorting signal of SpaA, and is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side-chain amino groups of pilin motif sequences to generate covalent linkages between pilin subunits . We show here that two elements of SpaA pilin precursor, the pilin motif and the sorting signal, are together sufficient to promote the polymerization of an otherwise secreted protein by a process requiring the function of the sortase A gene (srtA) . Five other sortase genes are dispensable for SpaA pilus assembly . Further, the incorporation of SpaB into SpaA pili requires a glutamic acid residue within the E box motif of SpaA, a feature that is found to be conserved in other Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs . When the main fimbrial subunit of Actinomyces naeslundii type I fimbriae, FimA, is expressed in corynebacteria, C . diphtheriae strain NCTC13129 polymerized FimA to form short fibres . Although C . diphtheriae does not depend on other actinomycetal genes for FimA polymerization, this process involves the pilin motif and the sorting signal of FimA as well as corynebacterial sortase D (SrtD) . Thus, pilus assembly in Gram-positive bacteria seems to occur by a universal mechanism of ordered cross-linking of precursor proteins, the multiple conserved features of which are recognized by designated sortase enzymes.

Mol Genet Genomics, 2004 Jul, 271(6), 729 - 41 Epub 2004 Jun 18.
Inhibitor-associated transposition events in Corynebacterium glutamicum; Garbe TR et al.; In up to 100% of all bacteria grown in the presence of initially inhibitory concentrations of five diverse inhibitors, an extra copy of the resident insertion element IS 31831 was found in specific chromosomal regions, the sites of which apparently depended on the inhibitor used . Thus, in nine out of nine independently isolated cyanide-associated transpositions, the acquired copy was located within an ORF encoding a protein related to the hypothetical but conserved protein YeiH of Escherichia coli . A putative Sox box upstream of the yeiH gene implicates superoxide as a potential regulator of the gene, a possibility further supported by the finding that superoxide dismutase (SodA) is overexpressed in cells cultured in cyanide-containing medium . Neither the cyanide-associated nor any of the other transposition mutations appeared to confer any discernible phenotypic advantage upon cells grown in the presence or absence of the inhibitors, as revealed most stringently by mixed-cell experiments . An alternative, albeit heterodox, explanation for the emergence of the mutants postulates a very high rate of transpositional activity in the presence of inhibitors . The initial emergence of the mutants was found to depend crucially upon the cell density . Thus, when growth medium was supplemented with 50 mM fluoropyruvate and inoculated to a density of 2 x 10(7) cfu/ml, single colonies with heterogeneous restriction fragment length polymorphisms (RFLPs) were routinely isolated at a frequency of 6 to 16% after 1-2 days of incubation . After 3 days, 10-36% of the colonies showed RFLPs, but the type was now dominated by the fluoropyruvate-specific RFLP, which, at higher resolution, invariably proved to be heterogeneous . This heterogeneity proved that these specific mutants were of multiple origin, indicating that clonal enrichment was irrelevant to their emergence . It is suggested that the presence of the inhibitor induces the development of hyper-transpositional activity, which is regulated by a soluble bacterial product.

Biotechnol Bioeng, 2004 Jul 5, 87(1), 1 - 6
Metabolic network analysis of lysine producing Corynebacterium glutamicum at a miniaturized scale; Wittmann C et al.; We present a straightforward approach comprising (13)C tracer experiments at 200-microL volume in 96-well microtiter plates with on-line measurement of dissolved oxygen for quantitative high-throughput metabolic network analysis at a miniaturized scale . This method was successfully applied for cultivation and (13)C metabolic flux analysis of two mutants of lysine producing Corynebacterium glutamicum (ATCC 13287 and ATCC 21543) . Microtiter-plate cultivations showed excellent accordance in kinetics and stoichiometry of growth and product formation as well as in intracellular flux distributions as compared with parallel shake-flask experiments . These cultivations further allowed clear identification of strain-specific flux differences such as increased flux toward lysine, increased flux through the pentose phosphate pathway (PPP), decreased flux through the tricarboxylic (TCA) cycle, and increased dihydroxyacetone formation in C . glutamicum ATCC 21543 compared with ATCC 13287 . The present approach has strong potential for broad quantitative screening of metabolic network activities, especially those involving high-cost tracer substrates .

J Bacteriol, 2004 Jul, 186(13), 4142 - 51
HutZ is required for efficient heme utilization in Vibrio cholerae; Wyckoff EE et al.; Vibrio cholerae, the causative agent of cholera, requires iron for growth . One mechanism by which it acquires iron is the uptake of heme, and several heme utilization genes have been identified in V . cholerae . These include three distinct outer membrane receptors, two TonB systems, and an apparent ABC transporter to transfer heme across the inner membrane . However, little is known about the fate of the heme after it enters the cell . In this report we show that a novel heme utilization protein, HutZ, is required for optimal heme utilization . hutZ (open reading frame {ORF} VCA0907) is encoded with two other genes, hutW (ORF VCA0909) and hutX (ORF VCA0908), in an operon divergently transcribed from the tonB1 operon . A hutZ mutant grew poorly when heme was provided as the sole source of iron, and the poor growth was likely due to the failure to use heme efficiently as a source of iron, rather than to heme toxicity . Heme oxygenase mutants of both Corynebacterium diphtheriae and C . ulcerans fail to use heme as an iron source . When the hutWXZ genes were expressed in the heme oxygenase mutants, growth on heme was restored, and hutZ was required for this effect . Biochemical characterization indicated that HutZ binds heme with high efficiency; however, no heme oxygenase activity was detected for this protein . HutZ may act as a heme storage protein, and it may also function as a shuttle protein that increases the efficiency of heme trafficking from the membrane to heme-containing proteins.

Am J Vet Res, 2004 Jun, 65(6), 829 - 34
Use of a real-time polymerase chain reaction-based fluorogenic 5' nuclease assay to evaluate insect vectors of Corynebacterium pseudotuberculosis infections in horses; Spier SJ et al.; OBJECTIVE: To develop and use a sensitive molecular assay for detecting the phospholipase D (PLD) exotoxin gene of Corynebacterium pseudotuberculosis in an attempt to identify insect vectors that may be important in transmission of clinical disease in horses . SAMPLE POPULATION: 2,621 flies of various species . PROCEDURE: A real-time polymerase chain reaction (PCR)-based fluorogenic 5' nuclease (TaqMan) system (ie, TaqMan PCR assay) was developed for the detection of the PLD gene in insects . Flies were collected monthly (May to November 2002) from 5 farms in northern California where C . pseudotuberculosis infection in horses is endemic . Three of the 5 farms (which housed a total of 358 horses) had diseased horses during the study period . A total of 2,621 flies of various species were tested for the PLD gene of C . pseudotuberculosis . RESULTS: Evidence of bacterial DNA for the PLD gene was detected in skin biopsy specimens from clinically affected horses and from 3 fly species collected from farms where affected horses were housed . Farms with a high incidence of diseased horses had a high proportion of insects carrying the organism . High percentages of flies with positive results for the PLD gene were observed in October, when most clinically affected horses were observed . CONCLUSIONS AND CLINICAL RELEVANCE: Our results are consistent with the hypothesis that C . pseudotuberculosis may be vectored to horses by flies . Three potential vectors were identified, including Haematobia irritans, Stomoxys calcitrans, and Musca domestica . The organism can be identified in up to 20% of house flies (Musca domestica) in the vicinity of diseased horses.

Biochemistry, 2004 Jun 22, 43(24), 7966 - 72
Structural and mutational studies of the carboxylate cluster in iron-free ribonucleotide reductase R2; Andersson ME et al.; The R2 protein of ribonucleotide reductase features a di-iron site deeply buried in the protein interior . The apo form of the R2 protein has an unusual clustering of carboxylate side chains at the empty metal-binding site . In a previous study, it was found that the loss of the four positive charge equivalents of the diferrous site in the apo protein appeared to be compensated for by the protonation of two histidine and two carboxylate side chains . We have studied the consequences of removing and introducing charged residues on the local hydrogen-bonding pattern in the region of the carboxylate cluster of Corynebacterium ammoniagenes and Escherichia coli protein R2 using site-directed mutagenesis and X-ray crystallography . The structures of the metal-free forms of wild-type C . ammoniagenes R2 and the mutant E . coli proteins D84N, S114D, E115A, H118A, and E238A have been determined and their hydrogen bonding and protonation states have been structurally assigned as far as possible . Significant alterations to the hydrogen-bonding patterns, protonation states, and hydration is observed for all mutant E . coli apo proteins as compared to wild-type apo R2 . Further structural variations are revealed by the wild-type apo C . ammoniagenes R2 structure . The protonation and hydration effects seen in the carboxylate cluster appear to be due to two major factors: conservation of the overall charge of the site and the requirement of electrostatic shielding of clustered carboxylate residues . Very short hydrogen-bonding distances between some protonated carboxylate pairs are indicative of low-barrier hydrogen bonding.

ANZ J Surg, 2004 Jun, 74(6), 439 - 41
Surgical masks: operative field contamination following visor-to-visor contact; Cocciolone RA et al.; BACKGROUND: Clashing of surgical visor masks frequently occurs when two surgeons bend over an operative field simultaneously; however, it is unknown whether this results in contamination . The purpose of the present study was to determine the potential for operative field contamination following surgical visor-mask clashes . The nature of bacterial contamination was also assessed . METHODS: Thirty sham operative procedures were performed under normal operating conditions for a specified time period . The number of surgical visor mask clashes during each procedure was determined by randomization (0, 1, 2, 3, 4 or 6 clashes) . All procedures were performed over a standard blood agar plate array . The degree of bacterial contamination was assessed by counting c.f.u . that developed after 24 h of incubation . Bacterial types were also determined . RESULTS: Surgical visor mask clashes resulted in increased contamination of the operative field; however, this was found to be independent of the number of clashes . 95% of pathogens were coagulase negative Staphylococcus species . Other bacteria included Micrococcus species, Bacillus species, Corynebacterium species, various Gram negative bacilli and Staphylococcus aureus (< 1%) . CONCLUSION: Surgical visor mask clashes increase the risk of bacterial contamination of the operative field.

Spinal Cord, 2004 Jun, 42(6), 378 - 81
Spontaneous corynebacterium discitis in a patient with chronic renal failure; Atalay B et al.; STUDY DESIGN: Case report describing spontaneous Corynebacterium diptheria discitis in a patient with chronic renal failure . OBJECTIVES: To describe this very rare form of discitis and the results of surgical and antibiotic therapy . SETTING: University Department of Neurosurgery, Turkey . CASE REPORT: A 55-year-old man with chronic renal failure presented with acute low-back pain . Lumbar magnetic resonance imaging (MRI) suggested discitis and osteomyelitis at the L5-S1 level . The L5-S1 disc was operated upon and the discectomy material was sent for pathological and microbiological analysis . RESULTS: Pathological examination revealed infection and bacterial culture grew C . diptheria . The patient was prescribed combination antibiotic therapy with vancomycin, a third-generation cephalosporin, and rifampicin . Clinical status improved after 8 weeks of therapy . Lumbar MRI revealed remission of the discitis and osteomyelitis after 10 months of follow-up . CONCLUSION: Chronic renal failure patients with low-back pain should be investigated for spinal infection . These individuals are prone to low-grade infection in the form of discitis or osteomyelitis . Corynebacterium subspecies rarely cause spontaneous discitis . This case is interesting because of the unusual causal organism and the occurrence of discitis in the setting of chronic renal failure.

FEBS Lett, 2004 Jun 4, 567(2-3), 179 - 82
A protein carboxylate coordinated oxo-centered tri-nuclear iron complex with possible implications for ferritin mineralization; Hogbom M et al.; The crystal structure of an oxo-centered tri-nuclear iron complex formed on a protein surface is presented . The cluster forms when crystals of the class Ib ribonucleotide reductase R2 protein from Corynebacterium ammoniagenes are subjected to iron soaking . The tri-iron-oxo complex is coordinated by protein-derived carboxylate ligands arranged in a motif similar to the one found on the inner surface of ferritins and may mimic an early stage in the mineralization of iron in ferritins . In addition, the structure adds to the very limited data on protein-mineral interfaces.

Biotechnol Lett, 2004 Apr, 26(7), 589 - 94
Cloning and characterization of a sigma 70 homologue gene, sigA from Corynebacterium ammoniagenes; Lee BR et al.; A sigma 70-like gene, sigA, has been identified from Corynebacterium ammoniagenes . The sigA gene encodes a polypeptide of 467 amino acids with a calculated molecular mass of 52036 Da . The deduced amino acid sequence preserves the common motifs of the primary sigma factors and shows very high similarity to those of SigA (sigmaA) homologues from high G+C Gram-positive bacteria, which suggest that the sigA gene encodes the primary sigma factor . The sigA gene is transcribed as a monocistronic mRNA of 2 kb and its mRNA occurs during the exponential growth phase and decays rapidly on entry into the stationary phase . The open reading frame encoding polyphosphate glucokinase-like protein is closely linked to the sigA gene.

Biotechnol Lett, 2004 Apr, 26(7), 575 - 80
Functional identification of the gene locus (ncg12319 and characterization of catechol 1,2-dioxygenase in Corynebacterium glutamicum; Shen XH et al.; Corynebacterium glutamicum assimilated phenol, benzoate, 4-hydroxybenzoate p-cresol and 3,4-dihydroxybenzoate . Ring cleavage was by catechol 1,2-dioxygenase when phenol or benzoate was used and by protocatechuate 3,4-dioxygenase when the others were used as substrate . The locus ncg12319 of its genome was cloned and expressed in Escherichia coli . Enzyme assays showed that ncg12319 encodes a catechol 1,2-dioxygenase . This catechol 1,2-dioxygenase was purified and accepted catechol, 3-, or 4-methylcatechols, but not chlorinated catechols, as substrates . The optimal temperature and pH for catechol cleavage catalyzed by the enzyme were 30 degrees C and 9, respectively, and the Km and Vmax were determined to be 4.24 micromol l(-1) and 3.7 micromol l(-1) min(-1) mg(-1) protein, respectively.

Mol Biol Evol, 2004 Sep, 21(9), 1683 - 91 Epub 2004 May 26.
Evolutionary process of amino Acid biosynthesis in corynebacterium at the whole genome level; Nishio Y et al.; Corynebacterium glutamicum, which is the closest relative of Corynebacterium efficiens, is widely used for the large scale production of many kinds of amino acids, particularly glutamic acid and lysine, by fermentation . Corynebacterium diphtheriae, which is well known as a human pathogen, is also closely related to these two species of Corynebacteria, but it lacks such productivity of amino acids . It is an important and interesting question to ask how those closely related bacterial species have undergone such significant functional differentiation in amino acid biosynthesis . The main purpose of the present study is to clarify the evolutionary process of functional differentiation among the three species of Corynebacteria by conducting a comparative analysis of genome sequences . When Mycobacterium and Streptomyces were used as out groups, our comparative study suggested that the common ancestor of Corynebacteria already possessed almost all of the gene sets necessary for amino acid production . However, C . diphtheriae was found to have lost the genes responsible for amino acid production . Moreover, we found that the common ancestor of C . efficiens and C . glutamicum have acquired some of genes responsible for amino acid production by horizontal gene transfer . Thus, we conclude that the evolutionary events of gene loss and horizontal gene transfer must have been responsible for functional differentiation in amino acid biosynthesis of the three species of Corynebacteria.

J Biotechnol, 2004 Jun 10, 110(3), 219 - 26
High level expression of Streptomyces mobaraensis transglutaminase in Corynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase; Date M et al.; We previously observed secretion of native-type Streptomyces mobaraensis transglutaminase (MTGase) in Corynebacterium glutamicum by co-expressing the subtilisin-like protease SAM-P45 from S . albogriseolus which processes the pro-region . In the present study, we have used a chimeric pro-region consisting of S . mobaraensis and Streptomyces cinnamoneus transglutaminases for the production of MTGase in C . glutamicum . As a result, secretion of MTGase using the chimeric pro-region is increased compared to that using the native pro-region .

J Bacteriol, 2004 Jun, 186(11), 3539 - 46
Evidence for an arginine exporter encoded by yggA (argO) that is regulated by the LysR-type transcriptional regulator ArgP in Escherichia coli; Nandineni MR et al.; The anonymous open reading frame yggA of Escherichia coli was identified in this study as a gene that is under the transcriptional control of argP (previously called iciA), which encodes a LysR-type transcriptional regulator protein . Strains with null mutations in either yggA or argP were supersensitive to the arginine analog canavanine, and yggA-lac expression in vivo exhibited argP(+)-dependent induction by arginine . Lysine supplementation phenocopied the argP null mutation in that it virtually abolished yggA expression, even in the argP+ strain . The dipeptides arginylalanine and lysylalanine behaved much like arginine and lysine, respectively, to induce and to turn off yggA transcription . Dominant missense mutations in argP (argPd) that conferred canavanine resistance and rendered yggA-lac expression constitutive were obtained . The protein deduced to be encoded by yggA shares similarity with a basic amino acid exporter (LysE) of Corynebacterium glutamicum, and we obtained evidence for increased arginine efflux from E . coli strains with either the argPd mutation or multicopy yggA+ . The null yggA mutation abolished the increased arginine efflux from the argPd strain . Our results suggest that yggA encodes an ArgP-regulated arginine exporter, and we have accordingly renamed it argO (for "arginine outward transport") . We propose that the physiological function of argO may be either to prevent the accumulation to toxic levels of canavanine (which is a plant-derived antimetabolite) or arginine or to maintain an appropriate balance between the intracellular lysine and arginine concentrations.

J Bacteriol, 2004 Jun, 186(11), 3453 - 60
Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum; Kim HJ et al.; A corynebacterial clone, previously isolated by scoring repression of lacZYA fused to the aceB promoter of Corynebacterium glutamicum, was analyzed further . In the clone, an open reading frame designated glxR, consisting of 681 nucleotides and encoding a 24,957-Da protein, was found . The molecular mass of a native GlxR protein was estimated by gel filtration column chromatography to be 44,000 Da, suggesting that the protein formed dimers . The predicted amino acid sequence contained both cyclic AMP (cAMP)- and DNA-binding motifs and was homologous with the cAMP receptor protein family of proteins . The aceB-repressing activity of the glxR clone was markedly relieved in an Escherichia coli cya mutant, but the activity was restored in growth medium containing cAMP . In glucose medium, the intracellular cAMP concentration of C . glutamicum reached 22 nmol/mg of protein in the early exponential phase and then decreased further; but in acetate medium, the intracellular cAMP concentration was only 5 nmol/mg of protein and remained low throughout the growth phase . The expression of glxR was not affected by the carbon source . Binding of purified GlxR to the promoter region of aceB could be demonstrated only in the presence of cAMP . These data suggest that GlxR may form dimers which bind to the aceB promoter region in the presence of cAMP and repress the glyoxylate bypass genes.

Arch Microbiol, 2004 Jun, 181(6), 443 - 50 Epub 2004 May 18.
Utilization of creatinine as an alternative nitrogen source in Corynebacterium glutamicum; Bendt AK et al.; In order to utilize different nitrogen sources and to survive situations of nitrogen limitation, microorganisms have developed several mechanisms to adapt their metabolism to changes in the nitrogen supply . In this communication, the use of creatinine as an alternative nitrogen source in Corynebacterium glutamicum, the identification of a membrane protein involved in creatinine uptake, the transcriptional regulation of the corresponding gene, and expression regulation of the gene encoding the creatinine deaminase are reported . As shown by mutant analyses, RNA hybridization experiments and real-time PCR, the expression of two genes, crnT and codA, is increased in response to nitrogen limitation, and regulation depends on the global nitrogen regulator AmtR . In addition, synthesis of creatinine deaminase during nitrogen starvation was shown by two-dimensional gel electrophoresis and MALDI-TOF-MS followed by peptide mass fingerprint analysis.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 925 - 8
Corynebacterium caspium sp . nov., from a Caspian seal (Phoca caspica); Collins MD et al.; A previously unknown Gram-positive, non-spore-forming, non-lipophilic, catalase-positive, irregular rod-shaped bacterium (M/106/00/5(T)) was isolated, in mixed culture, from the penis of a Caspian seal (Phoca caspica) . The strain was a facultative anaerobe that was able to grow at 22 and 42 degrees C . Comparative 16S rRNA gene sequencing showed that the organism formed a hitherto unknown subline within the genus Corynebacterium . Sequence divergence values of more than 5 % from other described Corynebacterium species, together with phenotypic differences, showed that the unidentified bacterium represents a previously unrecognized member of this genus . On the basis of phenotypic and phylogenetic considerations, it is proposed that the unknown bacterium isolated from a Caspian seal (strain M/106/00/5(T)=CCUG 44566(T)=CIP 107965(T)) be classified as the type strain of a novel species of the genus Corynebacterium, Corynebacterium caspium sp . nov.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 779 - 82
Corynebacterium halotolerans sp . nov., isolated from saline soil in the west of China; Chen HH et al.; A halotolerant, non-spore-forming actinobacterium was isolated from a soil sample from the west of China . The strain, designated YIM 70093(T) (=CCTCC AA 001024(T)=DSM 44683(T)), comprised Gram-positive, non-motile, diphtheroid and irregular rods . It grew in 0-25 % KCl (KCl could be substituted by NaCl or MgCl(2).6H(2)O), with optimum growth at 10 % KCl, and its optimal pH and cultivation temperature were 7.2 and 28 degrees C, respectively . On the basis of its morphological, physiological and phylogenetic characteristics, strain YIM 70093(T) should be classified in the genus CORYNEBACTERIUM: However, it is sufficiently different from hitherto described Corynebacterium species to be considered as a novel species, for which the name Corynebacterium halotolerans sp . nov . is proposed.

Vet Microbiol, 2004 May 20, 100(1-2), 129 - 37
Comparison of an interferon-gamma to a phospholipase D enzyme-linked immunosorbent assay for diagnosis of Corynebacterium pseudotuberculosis infection in experimentally infected goats; Menzies PI et al.; The optimal method of control of caseous lymphadenitis of goats caused by Corynebacterium pseudotuberculosis is eradication of infection by identification and removal of infected carrier animals . The objective of this study was to compare detection of C . pseudotuberculosis experimentally infected goats using a commercially available bovine interferon-gamma (IFN-gamma) whole blood enzyme-linked immunosorbent assay (ELISA) to serological response to a recombinant phospholipase D (PLD) ELISA . The tests were assessed repeatedly over 1 year in three infected and three non-infected goats . Using a IFN-gamma optical density cut-off at 0.10 as positive under the conditions used, the test accurately detected C . pseudotuberculosis experimentally infected goats over a 363 day period with a reliability of 89.2% and non-infected goats with a reliability of 97.1% . Using a cut-off value of the mean for negative samples plus two standard deviations, the PLD ELISA detected C . pseudotuberculosis experimentally infected goats over this period with a reliability of 81.0% and non-infected goats with a reliability of 97.0% . The PLD ELISA was however more predictive than the IFN-gamma ELISA of the presence of lesions observed at postmortem examination of infected goats.

Biochemistry, 2004 May 18, 43(19), 5583 - 91
The C-terminal domain of the betaine carrier BetP of Corynebacterium glutamicum is directly involved in sensing K+ as an osmotic stimulus; Schiller D et al.; The glycine betaine carrier BetP of Corynebacterium glutamicum was recently shown to function both as an osmosensor and as an osmoregulator in proteoliposomes by sensing changes in the internal K(+) concentration as a measure of hyperosmotic stress . In vivo analysis of mutants carrying deletions at the C-terminal extension of BetP indicated that this domain participates in osmostress-dependent activity regulation . To address the question, whether a putative K(+) sensor is located within the C-terminal domain, several mutants with truncations in this domain were purified and reconstituted in proteoliposomes of Escherichia coli phospholipids, since this in vitro system allowed variation of the K(+) concentration at the lumenal side . Truncation of 12 amino acids led to a partly deregulated BetP in terms of osmoregulation; however, K(+) sensitivity was not impaired in this mutant . The deletion of 25 amino acid residues at the C-terminal end of BetP led to both deregulation of the carrier activity, i.e., high activity independent of external osmolality, and loss of K(+)-dependent transport stimulation, indicating that this region of the C-terminal domain is necessary for K(+) sensing and/or K(+)-dependent carrier activation . Immunological and proteolysis analyses showed that BetP and its recombinant forms were reconstituted in a right-side-out orientation, i.e., the C-terminal domain faces the lumen of the proteoliposomes and is thus able to detect the K(+) signal at the inside . This is the first experimental demonstration of a direct connection between an osmotic stimulus, i.e., the change in internal K(+), and a putative sensor domain.

J Ind Microbiol Biotechnol, 2004 May, 31(4), 183 - 8 Epub 2004 May 06.
Glutamate as an inhibitor of phosphoenolpyruvate carboxylase activity in Corynebacterium glutamicum; Delaunay S et al.; The glutamate-producing bacterium, Corynebacterium glutamicum is known to possess two anaplerotic enzymes: pyruvate carboxylase (Pc) and phosphoenolpyruvate carboxylase (PEPc) . In vitro, this latter enzyme appeared to be inhibited by different glutamic acid salts, whereas ammonium-glutamate had no influence on Pc activity . To investigate the in vivo relevance of PEPc activity inhibition, the intracellular concentration of glutamate was determined throughout the glutamate-producing process . The intracellular concentration was then shown to be sufficient to induce a dramatic inhibition of PEPc activity during the process . As a consequence, intracellular accumulation of glutamate could be at least partially responsible for the weak participation of PEPc within the anaplerosis activity in amino-acid-producing strains of C . glutamicum.

J Clin Microbiol, 2004 May, 42(5), 2231 - 3
Corynebacterium species isolated from bone and joint infections identified by 16S rRNA gene sequence analysis; Roux V et al.; By the use of 16S rRNA gene sequence analysis we identified 28 of 31 Corynebacterium spp . isolated from bone and joint infections, including species never before isolated in such infections . Phenotypic analysis led to the correct identification of 8 of 31 . 16S rRNA gene sequence analysis appears to be a good technique for identification of clinical strains of Corynebacterium spp.

J Vet Med B Infect Dis Vet Public Health, 2001 May, 48(4), 241 - 58
Oedematous skin disease of buffalo in Egypt; Selim SA; This review covers a historical view and etiology of oedematous skin disease which affects buffalo in Egypt, the microbiology of Corynebacterium pseudotuberculosis causing the disease: its virulence; clinical signs; mechanism of pathogenesis; histopathology; mode of transmission; immunological aspects; treatment and control . It is concluded that C . pseudotuberculosis serotype II is the main cause of OSD and exotoxin phospholipase D and its lipid contents of the cell wall are the major causes of pathogenesis . After declaring the role of Hippobosca equina in transmission of the causative agent among buffaloes, control of OSD is now available.

Appl Environ Microbiol, 2004 May, 70(5), 2861 - 6
Heterologous expression of lactose- and galactose-utilizing pathways from lactic acid bacteria in Corynebacterium glutamicum for production of lysine in whey; Barrett E et al.; The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp . bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp . cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253 . The C . glutamicum strains expressing the lactose permease and beta-galactosidase genes of L . delbrueckii subsp . bulgaricus exhibited beta-galactosidase activity in excess of 1000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source . Similarly, C . glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and beta-galactosidase genes exhibited beta-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source . When grown in whey-based medium, the engineered C . glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C . glutamicum 21253 . Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability.

Prikl Biokhim Mikrobiol, 2004 Mar-Apr, 40(2), 214 - 9
{Effect of photosynthetic bacteria and compost on degradation of petroleum products in soil}; Mun TKh et al.; Addition of diesel fuel and waste engine oil to soil was found to cause biostimulation of hydrocarbon-oxidizing microorganisms . Corynebacteria constitute a large group of hydrocarbon-oxidizing microorganisms . Addition of a liquid culture of photosynthetic bacteria to soil not only facilitates degradation of petroleum products, but also stimulates growth of hydrocarbon-oxidizing microorganisms . Combined addition of photosynthetic bacteria and compost to soil polluted with petroleum products causes even a more significant increase in the count of hydrocarbon-oxidizing bacteria and substantially increases the rate of pollutant degradation.

Biochemistry, 2004 May 11, 43(18), 5222 - 38
Mixed regioselectivity in the Arg-177 mutants of Corynebacterium diphtheriae heme oxygenase as a consequence of in-plane heme disorder; Zeng Y et al.; It has been reported that the R183E and R183D mutants of rat heme oxygenase-1 (r-HO-1) produce approximately 30% delta-biliverdin {Zhou, H., et al . (2000) J . Am . Chem . Soc . 122, 8311-8312} . Two plausible mechanisms were proposed to explain the observations . (a) Electrostatic repulsion between E183 (D183) and one of the heme propionates forces the heme to rotate, thereby placing the delta-meso carbon in a position that is susceptible to oxidation . (b) Rearrangement of the distal pocket structure is triggered by the formation of a hydrogen bond between E183 (D183) and K179 . A change in the pK(a) for the Fe(III)-H(2)O to Fe(III)-OH transition of the mutants was interpreted to be consistent with rearrangement of the hydrogen bond network in the distal pocket . The large similarities between the high-frequency portion of the (1)H NMR spectra corresponding to the wild type and R183E and R183D mutants were interpreted to indicate that the heme in the mutants is not rotated to a significant extent . We have re-examined this issue by studying the corresponding R177 mutants in heme oxygenase from Corynebacterium diphtheriae (cd-HO) . Replacing R177 with E or D results in the formation of approximately 55% alpha- and 45% delta-biliverdin, whereas the R177A mutant retains alpha-regioselectivity . In addition, the K13N/Y130F/R177A triple mutant catalyzed the formation of 60% delta- and 40% alpha-biliverdin, while single mutants K13N and Y130F did not appreciably change the regioselectivity of the reaction . The pK(a) of the Fe(III)-H(2)O to Fe(III)-OH transition in wild-type cd-HO is 9.1, and those of the R177E, R177D, R177A, and K13N/Y130F/R177A mutants are 9.4, 9.5, 9.2, and 8.0, respectively . Thus, no obvious correlation exists between the changes in pK(a) and the altered regioselectivity . NMR spectroscopic studies conducted with the R177D and R177E mutants of cd-HO revealed the presence of three heme isomers: a major (M) and a minor (m) heme orientational isomer related by a 180 degrees rotation about the alpha-gamma meso axis and an alternative seating (m') which is related to m by an 85 degrees in-plane rotation of the macrocycle . The in-plane rotation of m to acquire conformation m' is triggered by electrostatic repulsion between the side chains of D or E at position 177 and heme propionate-6 . As a consequence, the delta-meso carbon in m' is placed in the position occupied by the alpha-meso carbon in m, where it is susceptible to hydroxylation and subsequent formation of delta-biliverdin.

FEMS Microbiol Rev, 2004 May, 28(2), 225 - 50
Comparative cell wall core biosynthesis in the mycolated pathogens, Mycobacterium tuberculosis and Corynebacterium diphtheriae; Dover LG et al.; The recent determination of the complete genome sequence of Corynebacterium diphtheriae, the aetiological agent of diphtheria, has allowed a detailed comparison of its physiology with that of its closest sequenced pathogenic relative Mycobacterium tuberculosis . Of major importance to the pathogenicity and resilience of the latter is its particularly complex cell envelope . The corynebacteria share many of the features of this extraordinary structure although to a lesser level of complexity . The cell envelope of M . tuberculosis has provided the molecular targets for several of the major anti-tubercular drugs . Given a backdrop of emerging multi-drug resistant strains of the organism (MDR-TB) and its continuing global threat to human health, the search for novel anti-tubercular agents is of paramount importance . The unique structure of this cell wall and the importance of its integrity to the viability of the organism suggest that the search for novel drug targets within the array of enzymes responsible for its construction may prove fruitful . Although the application of modern bioinformatics techniques to the 'mining' of the M . tuberculosis genome has already increased our knowledge of the biosynthesis and assembly of the mycobacterial cell wall, several issues remain uncertain . Further analysis by comparison with its relatives may bring clarity and aid the early identification of novel cellular targets for new anti-tuberculosis drugs . In order to facilitate this aim, this review intends to illustrate the broad similarities and highlight the structural differences between the two bacterial envelopes and discuss the genetics of their biosynthesis.

South Med J, 2004 Apr, 97(4), 395 - 7
Corynebacterium afermentans lung abscess and empyema in a patient with human immunodeficiency virus infection; Minkin R et al.; Necrotizing pleuropulmonary infection in a patient with acquired immunodeficiency syndrome developed due to Corynebacterium afermentans subspecies lipophilum . Long-term combination antibiotic therapy was successful in eradicating the infection without surgery.

Kansenshogaku Zasshi, 2004 Mar, 78(3), 277 - 82
{Three adult cases with Corynebacterium propinquum respiratory infections in a community hospital}; Motomura K et al.; Corynebacterium propinquum, which is included in Corynebacterium group ANF-3, exists as a commensal in the oral flora . This organism has not yet been fully recognized as a respiratory pathogen . We previously reported that the first case with respiratory infection caused by C . propinquum . On the other hand, Corynebacterium pseudodiphtheriticum is recognized as a causative organism in respiratory infections . Recently we experienced two cases with C . propinquum respiratory infections in our hospital . Three types of the onset such as a community-acquired infection, a hospital-acquired infection, and a nursing home acquired infections were observed . Our analysis indicated that gram staining of the purulent sputum is an essential tool to evaluate whether C . propinquum is a respiratory pathogen or not, because this organism is a commensal bacteria.

Proteins, 2004 May 15, 55(3), 613 - 9
NrdH-redoxin of Corynebacterium ammoniagenes forms a domain-swapped dimer; Stehr M et al.; NrdH-redoxins constitute a family of small redox proteins, which contain a conserved CXXC sequence motif, and are characterized by a glutaredoxin-like amino acid sequence but a thioredoxin-like activity profile . Here we report the structure of Corynebacterium ammoniagenes NrdH at 2.7 A resolution, determined by molecular replacement using E . coli NrdH as model . The structure is the first example of a domain-swapped dimer from the thioredoxin family . The domain-swapped structure is formed by an inter-chain two-stranded anti-parallel beta-sheet and is stabilized by electrostatic interactions at the dimer interface . Size exclusion chromatography, and MALDI-ESI experiments revealed however, that the protein exists as a monomer in solution . Similar to E . coli NrdH-redoxin and thioredoxin, C . ammoniagenes NrdH-redoxin has a wide hydrophobic pocket at the surface that could be involved in binding to thioredoxin reductase . However, the loop between alpha2 and beta3, which is complementary to a crevice in the reductase in the thioredoxin-thioredoxin reductase complex, is the hinge for formation of the swapped dimer in C . ammoniagenes NrdH-redoxin . C . ammoniagenes NrdH-redoxin has the highly conserved sequence motif W61-S-G-F-R-P-{DE}67 which is unique to the NrdH-redoxins and which determines the orientation of helix alpha3 . An extended hydrogen-bond network, similar to that in E . coli NrdH-redoxin, determines the conformation of the loop formed by the conserved motif .

J Lipid Res, 2004 Jul, 45(7), 1355 - 63 Epub 2004 Apr 21.
Bacterial phospholipid molecular species analysis by ion-pair reversed-phase HPLC/ESI/MS; Mazzella N et al.; This work set out to optimize the detection and separation of several phospholipid molecular species on a reversed-phase column with the use of an electrospray ionization/mass spectrometry-compatible counter-ion . An application of this technique concerned a qualitative and quantitative analysis of bacterial membrane phospholipids extracted from Corynebacterium species strain 8 . The phospholipid classes of strain 8 were identified as phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, and a peculiar lipid compound, acyl phosphatidylglycerol . Most of the molecular species structures were elucidated, and regarding phosphatidylglycerol, the fatty acid positions were clearly determined with the calculation of the sn-2/sn-1 intensity ratio of the fatty acyl chain fragments.

Environ Pollut, 1993, 82(2), 153 - 6
The microbiology of drill mud cuttings from a new off-shore oilfield in Nigeria; Nnubia C et al.; Drilling-fluid-utilising microorganisms present in drill mud cuttings collected from the Agbara oilfield were isolated on mineral salts agar plates . Thirty-two isolates were obtained, 26 of which were Gram-positive bacteria and six fungi . The isolates were identified as Bacillus sp . (10), Staphylococcus sp . (12), Micrococcus sp . (2), Corynebacterium sp . (1), Nocardia sp . (1), and Penicillium sp . (6) . Screen tests indicated that 27 (84.4%) of the isolates did not grow with any of the drilling fluids One Bacillus and three Staphylococcus spp . were strong primary utilisers of the drilling fluids.

J Bacteriol, 2004 May, 186(9), 2798 - 809
RamB, a novel transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum; Gerstmeir R et al.; The adaptation of Corynebacterium glutamicum to acetate as a carbon and energy source involves transcriptional regulation of the pta-ack operon coding for the acetate-activating enzymes phosphotransacetylase and acetate kinase and of the aceA and aceB genes coding for the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively . Deletion and mutation analysis of the respective promoter regions led to the identification of highly conserved 13-bp motifs (AA/GAACTTTGCAAA) as cis-regulatory elements for expression of the pta-ack operon and the aceA and aceB genes . By use of DNA affinity chromatography, a 53-kDa protein specifically binding to the promoter/operator region of the pta-ack operon was purified . Mass spectrometry and peptide mass fingerprinting identified the protein as a putative transcriptional regulator (which was designated RamB) . Purified His-tagged RamB protein was shown to bind specifically to both the pta-ack and the aceA/aceB promoter/operator regions . Directed deletion of the ramB gene in the genome of C . glutamicum resulted in mutant strain RG1 . Whereas the wild type of C . glutamicum showed high-level specific activities of acetate kinase, phosphotransacetylase, isocitrate lyase, and malate synthase when grown on acetate and low-level specific activities when grown on glucose as sole carbon and energy sources, mutant RG1 showed high-level specific activities with all four enzymes irrespective of the substrate . Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription . The results indicate that RamB is a negative transcriptional regulator of genes involved in acetate metabolism of C . glutamicum.

Ophthalmic Surg Lasers Imaging, 2004 Mar-Apr, 35(2), 159 - 61
Late-onset Corynebacterium endophthalmitis following laser posterior capsulotomy; Hollander DA et al.; Four months following uncomplicated cataract extraction, a patient underwent Nd:YAG laser posterior capsulotomy . Three days later, she presented with pain, hand motions vision, and severe anterior uveitis and vitritis . A coincident retinal detachment led to a delay in diagnosing the etiology of this intraocular inflammation . After recurrent episodes of inflammation that were initially responsive to corticosteroids, the patient underwent a vitrectomy, lens explantation, capsulectomy, and intravitreal antibiotic injections, which resulted in complete resolution of the intraocular inflammation with a best-corrected visual acuity of 20/60 . Corynebacterium species was ultimately cultured from the capsular tissue . The release of sequestered bacterial organisms must be considered in the differential diagnosis of persistent or unusually intense inflammation following laser posterior capsulotomy.

Bioresour Technol, 2004 Aug, 94(1), 99 - 105
Production of a novel polygalacturonic acid bioflocculant REA-11 by Corynebacterium glutamicum; He N et al.; The production of a novel polygalacturonic acid bioflocculant REA-11 from a newly isolated strain, Corynebacterium glutamicum CCTCC M201005, was investigated . Sucrose was chosen as a carbon source for REA-11 production . Complex nitrogen sources containing urea and an organic nitrogen compound enhanced both bacterial growth and REA-11 production, among which urea plus corn steep liquor was shown to be the most efficient combination . A cost-effective medium for REA-11 production mainly comprised 17 g/l sucrose, 0.45 g/l urea, and 5 ml/l corn steep liquor, under which conditions the flocculating activity reached 390 U/ml . The molar ratio of carbon to nitrogen (C/N) significantly affected REA-11 production, where a C/N ratio of 20:1 was shown to be the best . Interestingly, by simultaneously feeding sucrose and urea at a C/N ratio of 20:1 at 24 h of fermentation, REA-11 production (458 U/ml) was enhanced by 17% compared to the control . In a 10 l jar fermentor, lower dissolved oxygen tension was favorable for REA-11 production: a flocculating activity of 520 U/ml was achieved at a kappaLa of 100 h(-1) . REA-11 raw product is relatively thermo-stable at acidic pH ranges of 3.0-6.5 . Preliminary application studies showed that REA-11 had stronger flocculating activity to Kaolin clay suspension compared to chemical flocculants . In addition, the capability of decolorizing molasses wastewater indicates the industrial potential of this novel bioflocculant.

J Am Vet Med Assoc, 2004 Apr 1, 224(7), 1139 - 42, 1112
Treatment of suppurative facial cellulitis and panniculitis caused by Corynebacterium pseudotuberculosis in two horses; Farstvedt EG et al.; Two horses were examined for large head wounds suspected to be the result of trauma and characterized by extensive necrosis of the skin and subcutaneous tissue, with abundant purulent exudate . Corynebacterium pseudotuberculosis was isolated from the facial wounds in both horses . Histopathologic examination revealed severe suppurative cellulitis and panniculitis with fistulous tracts and granulation tissue in 1 horse . Both horses were treated with local wound care, nonsteroidal anti-inflammatory drugs, and administration of antimicrobials . The concept of moist wound care was used in the second horse, with products that have recently become available for veterinary wound management . Outcome in both horses was good.

J Am Vet Med Assoc, 2004 Apr 1, 224(7), 1133 - 8, 1112
Pericarditis and pleuritis caused by Corynebacterium pseudotuberculosis in a horse; Perkins SL et al.; A 13-year-old Oldenburg mare was evaluated for lethargy and signs of mild colic . Pericardial tamponade caused by fibrinoeffusive pericarditis was diagnosed . Cytologic and biochemical evaluation of pericardial fluid was consistent with a septic effusion . Corynebacterium pseudotuberculosis, the cause of pigeon fever, was identified by bacteriologic culture of pericardial fluid . Drainage and lavage of the pericardial sac, local (intrapericardial) and systemic antimicrobial treatment, and subsequent corticosteroid treatment resulted in a successful outcome in this horse . To the authors' knowledge, this is the first report of pericarditis associated with C pseudotuberculosis in a horse.

Arch Inst Pasteur Tunis, 2002, 79(1-4), 51 - 7
{Epidemiological and clinical studies of ovine caseous lymphadenitis}; Ben Said MS et al.; A field survey was undertaken, to determine epidemiological, clinical and biological data on Ovine Caseous Lymphadenitis disease in 54 flocks originated from Sfax area in Tunisia . The mean morbidity rate of the cutaneous form was 5.1% . This form affected sheep over 3 months and under 2 years old . On the other hand, the mean morbidity rate of the visceral form, encountered in abattoirs, was 11.02% . The clinical aspect of the superficial (or cutaneous) form was often corresponding to one abscess, located particularly in the lymphatic nodes of the animal's head; while visceral (or internal) form of the disease was represented by the presence of a unique abscess found in the pulmonary lymphatic nodes . The macroscopic aspect of lesions showed that the size of abscess was comprised between 4 and 10 cm in diameter . At the cut, colour of the pus was white yellowish to yellow greyish with a fluid or a thick aspect like onion peels; pus was microscopic . Lesions were characterised by a fibrous shell, a pyogenic membrane and a necrotic center . Bacteriological research revealed that Corynebacterium pseudotuberculosis was the pathogen the most frequently isolated, followed by Staphylococcus aureus subsp anaerobius which was particularly found in sheep aged between 3 months and 2 years old.

FEBS Lett, 2004 Apr 9, 563(1-3), 108 - 12
Cation specificity of osmosensing by the betaine carrier BetP of Corynebacterium glutamicum; Schiller D et al.; The Na(+)/betaine carrier BetP from Corynebacterium glutamicum was purified and reconstituted in Escherichia coli phospholipid liposomes and its osmosensory properties were studied with respect to the cation specificity of osmotic activation . To dissect the influence of the co-substrate Na(+) on the energetics of uptake from its possible role as a putative trigger of osmolality-dependent BetP activation, the internal Na(+) concentration was varied without changing DeltapNa(+) . Studying betaine uptake at increasing luminal Na(+) or K(+) revealed that BetP activity was triggered by Na(+) only to a negligible extent compared to activation by K(+) . We conclude that activation of BetP in proteoliposomes depends solely on K(+), both in mechanistic and in physiological terms.

Res Microbiol, 2004 Apr, 155(3), 174 - 84
Characterization and chromosomal organization of the murD-murC-ftsQ region of Corynebacterium glutamicum ATCC 13869; Ramos A et al.; The sequence of a 4.6-kb region of DNA from Corynebacterium glutamicum ATCC 13869 lying upstream from the ftsQ-ftsZ region has been determined . The region contains four genes with high similarity to the murD, ftsW, murG, and murC genes from different microorganisms . The products of these mur genes probably catalyse several steps in the formation of the precursors for peptidoglycan synthesis in C . glutamicum, whereas ftsW might play also a role in the stabilisation of the FtsZ ring during cell division . The murC gene product was purified to near homogeneity and its UDP-N-acetylmuramate: L-alanine adding activity was demonstrated . Northern analysis indicated that ftsW, murG and ftsQ are poorly expressed in C . glutamicum whereas murC and ftsZ are expressed at higher levels at the beginning of the exponential phase . Dicistronic (ftsQ-ftsZ) and monocistronic (murC and ftsZ) transcripts can be detected using specific probes and are in agreement with the lack of transcriptional terminators in the partially analysed dcw cluster . Disruption experiments performed in C . glutamicum using internal fragments of the ftsW, murG and murC genes allowed us to conclude that FtsW, MurG, and MurC are essential gene products in C . glutamicum.

Res Microbiol, 2004 Apr, 155(3), 162 - 6
International nomenclature for Corynebacterium diphtheriae ribotypes; Grimont PA et al.; The nomenclature of Corynebacterium diphtheriae ribotypes is presented . A total of 86 ribotypes obtained after BstEII digestion were given a geographic name chosen to reflect the place where one of the strains was isolated or studied.

Curr Microbiol, 2004 Apr, 48(4), 305 - 11
Cloning of the O-acetylhomoserine sulfhydrylase gene from the ruminal bacterium Selenomonas ruminantium HD4; Qin X et al.; The O-acetylhomoserine sulfhydrylase (OAHS) gene was cloned from a Selenomonas ruminantium HD4 Lambda ZAP II genomic library by degenerative probe hybridization and complementation . Sequence analysis revealed an 869-bp ORF with a G + C content of 53% . The ORF had significant homology with enzymes involved in homocysteine biosynthesis . A CuraBLASTN homology search showed that the ORF has 63% nucleotide identity with the OAHS of Bacillus stearothermophilus, Corynebacterium glutamicum, and Acremonium chrysogenum, and has 58% identity with met25 of Saccharomyces cerevisiae and metZ of Pseudomonas aeruginosa . The deduced amino acid sequence exhibited 45% similarity with Met25 and MetZ . Further analysis predicted that the gene product was a member of the pyridoxal phosphate enzyme family . Complementation experiments with Escherichia coli metA, metB, and metC mutant strains showed that the S . ruminantium OAHS gene can complement the metC mutation and allow for growth on minimal media that contained sodium thiosulfate as the sole source of sulfur . When the OAHS was disturbed by inserting a EZ::TN pMOD-2<Apramycin> transposon, the complementation was lost . Therefore, these results suggest that the gene functions as OAHS in S . ruminantium HD4.

Biotechnol Lett, 2004 Feb, 26(4), 331 - 6
Lead biosorption by waste biomass of Corynebacterium glutamicum generated from lysine fermentation process; Choi SB et al.; Biomass waste, mainly Corynebacterium glutamicum, is generated from large-scale lysine fermentation process . In this study, protonated C . glutamicum biomass was evaluated as a biosorbent for the removal of lead from synthetic wastewater . As Pb2+ were bound to the biomass, the solution pH deceased, indicating that protons in the biomass were exchanged with lead ions . The Corynebacterium biomass bound Pb2+ at up to 2.74 mmol g(-1) at pH 5, where lead does not precipitate . Compared with other biosorbents and conventional sorbents, such as natural zeolite, activated carbon and synthetic ion exchange resin, the protonated C . glutamicum biomass was considered to be a useful biomaterial for lead biosorption.

J Vet Diagn Invest, 2004 Mar, 16(2), 116 - 20
Development of a polymerase chain reaction test to confirm Mycobacterium avium subsp . paratuberculosis in culture; Shin SJ et al.; A polymerase chain reaction (PCR) assay for confirmation of Mycobacterium avium subsp . paratuberculosis was developed using the primer set derived from ISMav2 . The PCR product was 494 base pairs (bp) and could be digested with ClaI, which produced 311- and 183-bp fragments . No amplification of 494-bp DNA fragment was detected from DNA of other Mycobacterium spp., including Mycobacterium avium complex, other bacteria, including Escherichia coli, Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae, Leptospira interrogans serovar pomona, Corynebacterium pseudotuberculosis, Salmonella typhimurium, Borrelia burgdorferi, and Staphylococcus aureus, and the Scedosporium sp . This PCR assay could detect 5-8 genome equivalents.

Mol Microbiol, 2004 Apr, 52(1), 285 - 302
clpC and clpP1P2 gene expression in Corynebacterium glutamicum is controlled by a regulatory network involving the transcriptional regulators ClgR and HspR as well as the ECF sigma factor sigmaH; Engels S et al.; The ATP-dependent protease Clp plays important roles in the cell's protein quality control system and in the regulation of cellular processes . In Corynebacterium glutamicum, the levels of the proteolytic subunits ClpP1 and ClpP2 as well as of the corresponding mRNAs were drastically increased upon deletion of the clpC gene, coding for a Clp ATPase subunit . We identified a regulatory protein, designated ClgR, binding to a common palindromic sequence motif in front of clpP1P2 as well as of clpC . Deletion of clgR in the DeltaclpC background completely abolished the increased transcription of both operons, indicating that ClgR activates transcription of these genes . ClgR activity itself is probably controlled via ClpC-dependent regulation of its stability, as ClgR is only present in DeltaclpC and not in wild-type cells, whereas the levels of clgR mRNA are comparable in both strains . clpC, clpP1P2 and clgR expression is induced upon severe heat stress, however, independently of ClgR . Identification of the heat-responsive transcriptional start sites in front of these genes revealed the presence of sequence motifs typical for sigmaECF-dependent promoters . The ECF sigma factor sigmaH could be identified as being required for transcriptional activation of clpC, clpP1P2 and clgR in response to severe heat stress . A second heat-responsive but sigmaH-independent promoter in front of clgR could be identified that is subject to negative regulation by the transcriptional repressor HspR . Taken together, these results show that clpC and clpP1P2 expression in C . glutamicum is subject to complex regulation via both independent and hierarchically organized pathways, allowing for the integration of multiple environmental stimuli . Both the ClgR- and sigmaH-dependent regulation of clpC and clpP1P2 expression appears to be conserved in other actinomycetes.

Mem Inst Oswaldo Cruz, 2003 Dec, 98(8), 987 - 93 Epub 2004 Mar 09.
Diphtheria remains a threat to health in the developing world--an overview; Mattos-Guaraldi AL et al.; Changes in the epidemiology of diphtheria are occurring worldwide . A large proportion of adults in many industrialized and developing countries are now susceptible to diphtheria . Vaccine-induced immunity wanes over time unless periodic booster is given or exposure to toxigenic Corynebacterium diphtheriae occurs . Immunity gap in adults coupled with large numbers of susceptible children creates the potential for new extensive epidemics . Epidemic emergencies may not be long in coming in countries experiencing rapid industrialization or undergoing sociopolitical instability where many of the factors thought to be important in producing epidemic such as mass population movements and difficult hygienic and economic conditions are present . The continuous circulation of toxigenic C . diphtheriae emphasizes the need to be aware of epidemiological features, clinical signs, and symptoms of diphtheria in vaccine era so that cases can be promptly diagnosed and treated, and further public health measures can be taken to contain this serious disease . This overview focused on worldwide data obtained from diphtheria with particular emphasis to main factors leading to recent epidemics, new clinical forms of C . diphtheriae infections, expression of virulence factors, other than toxin production, control strategies, and laboratory diagnosis procedures.

J Mol Biol, 2004 Apr 9, 337(5), 1137 - 47
Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine symporter BetP of Corynebacterium glutamicum; Ziegler C et al.; The high-affinity glycine betaine uptake system BetP, an osmosensing and osmoregulated sodium-coupled symporter from Corynebacterium glutamicum, was overexpressed in Escherichia coli with an N-terminal StrepII-tag, solubilized in beta-dodecylmaltoside and purified by streptactin affinity chromatography . Analytical ultracentrifugation indicated that BetP forms trimers in detergent solution . Detergent-solubilized BetP can be reconstituted into proteoliposomes without loss of function, suggesting that BetP is a trimer in the bacterial membrane . Reconstitution with E.coli polar lipids produced 2D crystals with unit cell parameters of 182A x 154A, gamma=90 degrees exhibiting p22(1)2(1) symmetry . Electron cryo-microscopy yielded a projection map at 7.5A . The unit cell contains four non-crystallographic trimers of BetP . Within each monomer, ten to 12 density peaks characteristic of transmembrane alpha-helices surround low-density regions that define potential transport pathways . Small but significant differences between the three monomers indicate that the trimer does not have exact 3-fold symmetry . The observed differences may be due to crystal packing, or they may reflect different functional states of the transporter, related to osmosensing and osmoregulation . The projection map of BetP shows no clear resemblance to other secondary transporters of known structure.

Infect Immun, 2004 Apr, 72(4), 1885 - 95
Analysis of genes that encode DtxR-like transcriptional regulators in pathogenic and saprophytic corynebacterial species; Oram DM et al.; Metal-dependent transcriptional regulators of the diphtheria toxin repressor (DtxR) family have been identified in a wide variety of bacterial genera, where they control gene expression in response to one of two metal ions, Fe(2+) or Mn(2+) . DtxR of Corynebacterium diphtheriae is the best characterized of these important metal-dependent regulators . The genus Corynebacterium includes many phenotypically diverse species, and the prevalence of DtxR-like regulators within the genus is unknown . We assayed chromosomal DNA from 42 different corynebacterial isolates, representing 33 different species, for the presence of a highly conserved region of the dtxR gene that encodes the DNA-binding helix-turn-helix motif and metal-binding site 1 within domains 1 and 2 of DtxR . The chromosome of all of the isolates contained this conserved region of dtxR, and DNA sequencing revealed a high level of nucleotide sequence conservation within this region in all of the corynebacterial species (ranging from 62 to 100% identity and averaging 70% identity with the dtxR prototype) . The level of identity was even greater for the predicted protein sequences encoded by the dtxR-like genes, ranging from 81 to 100% identity and averaging 91% identity with DtxR . Using a DtxR-specific antiserum we confirmed the presence of a DtxR-like protein in extracts of most of the corynebacterial isolates and determined the precise amount of DtxR per cell in C . diphtheriae . The high level of identity at both DNA and protein levels suggests that all of the isolates tested encode a functional DtxR-like Fe(2+)-activated regulatory protein that can bind homologs of the DtxR operator and regulate gene expression in response to iron.

Vet Microbiol, 2004 Mar 5, 98(3-4), 329 - 32
Influence of sampling time on bacteriological diagnosis of goat intramammary infection; Sanchez A et al.; Intramammary infection in seven commercial goat herds was studied using premilking and postmilking samples for purposes as bacteriological diagnosis . Using a positive result on both premilking and postmilking samples as the definitive diagnosis, we compared the efficacy of single samples collected either premilking or postmilking . With this aim, 2268 bacteriological culture results were compared . The kappa values (0.60) showed moderate agreement between the two samples . Specificity and positive predictive value were higher for postmilking samples than for premilking samples . Specificity of postmilking samples was 99.4% for coagulase-negative staphylococci, 99.9% for Gram-negative bacilli, 100% for streptococci, 99.9% for corynebacteria and 100% for mixed cultures . Premilking samples specificity was 96.6, 99.5, 99.7, 99.8 and 99.8%, respectively . False positive diagnoses were more frequent for premilking samples . The results suggest that postmilking samples should be used to diagnosis of goat intramammary infection.

Bioinformatics, 2004 Mar 22, 20(5), 612 - 22 Epub 2004 Jan 22.
A systematic method to identify genomic islands and its applications in analyzing the genomes of Corynebacterium glutamicum and Vibrio vulnificus CMCP6 chromosome I; Zhang R et al.; MOTIVATION: Some genomic islands contain horizontally transferred genes, which play critical roles in altering the genotypes and phenotypes of organisms, and horizontal gene transfer has been recognized as a universal event throughout bacterial evolution . A windowless method to display the distribution of genomic GC content, the cumulative GC profile, is proposed to identify genomic islands in genomes whose complete genome sequences are available . Two new indices are proposed to assess the codon usage bias and amino acid usage bias in genomic islands . RESULTS: A 211 kb genomic island (CGGI-1) has been identified in the genome of Corynebacterium glutamicum, and three genomic islands VVGI-1, VVGI-2 and VVGI-3, with lengths 167, 40 and 33 kb, respectively, have been identified in the genome of Vibrio vulnificus CMCP6 chromosome I . The CGGI-1 is flanked by two approximately 500 bp direct repeats, and utilizes a Val-tRNA as the integration site . For the VVGI-1 and VVGI-2, each has an integrase gene at 5' junction . All the identified genomic islands show unusual GC content, codon usage and amino acid usage, compared with the rest of the genomes . In addition, it is found that genomic islands are fairly homogenous in terms of GC content variation . An index, h, to quantify the homogeneity of GC content for genomic islands is proposed, and it is shown that h is less than 0.1 for all the genomic islands analyzed . The cumulative GC profile, as well as various indices to assess the codon usage bias, amino acid usage bias and homogeneity of the genomic islands, will be useful in the analysis of other genomes . AVAILABILITY: Programs used in this work and numerical results are available upon request.

Anal Biochem, 2004 Apr 1, 327(1), 135 - 9
Impact of the cold shock phenomenon on quantification of intracellular metabolites in bacteria; Wittmann C et al.; In the present work the effect of quenching on quantification of intracellular metabolites in Corynebacterium glutamicum was investigated . C . glutamicum showed a high sensitivity to cold shock . Quenching of the cells by -50 degrees C buffered methanol prior to cell separation and extraction led to drastically reduced concentrations for free intracellular amino acids compared to those for nonquenched filtration . As demonstrated for glutamate and glutamine, this was clearly due to a more than 90% loss of these compounds from the cell interior into the medium during quenching . With lower methanol concentration in the quenching solution the metabolic losses were significantly lower but still amounted to about 30% . Due to the fact that quenching with ice-cold NaCl (0.9%) also resulted in significantly lower pool sizes for intracellular amino acids, a basic cold shock phenomenon is most likely the reason for the observed effects . The results clearly demonstrate that quenching combined with cell separation for concentration of the cells and removal of the medium is not applicable for intracellular metabolite analysis in C . glutamicum . Sampling by quick filtration without quenching allows complete cell separation and authentic quantification of intracellular metabolite pools exhibiting time constants significantly larger than sampling time.

Dtsch Tierarztl Wochenschr, 2004 Feb, 111(2), 67 - 9
{The prevalence of Actinobaculum suis in boars of breeding herds in the Omsk region (Russian Federation) by indirect immunofluorescence technique}; Pleschakowa V et al.; Actinobaculum suis (Corynebacterium suis, Eubacterium suis, Actinomyces suis) was detected in the preputial diverticulum of 64,8% of 162 boars investigated in 8 districts of the region Omsk (Russian Federation) by indirect immunofluorescent technique . Until yet no informations were available about the prevalence of Actinobaculum (A.) suis in swine herds of the Russian Federation . The study shows that A . suis, as a main aetiological factor of cystitis and pyelonephritis in sows, is widely spread among the boars of the region Omsk . Prevalence of A . suis was not influenced by housing conditions, age or breed of investigated boars . Indirect immunofluorescent technique for detection of A . suis provides a good method for screening investigations with high numbers of samples.

Hinyokika Kiyo, 2004 Jan, 50(1), 33 - 5
{Two cases of encrusted cystitis}; Ohara H et al.; Encrusted cystitis is a type of severe cystitis, which progresses chronically and is characterized by excessively alkaline urine and calcifications within the bladder wall . We report two cases of encrusted cystitis . Both cases were high aged and had severe anemia with chronic cystitis . They complained of gross hematuria, voiding frequency and pain upon urination . Urine pH was 8-9, and urine cytology was negative . Urine culture contained Corynebacterium Group D2 . Abdominal computed tomography and transurethral resection revealed wall bladder wall calcification and inflammatory change . We diagnosed it as encrusted cystitis . The patients underwent excision of plaques of calcified encrustation, adapted antibiotic therapy and acidification of urine . It is essential to diagnose encrusted cystitis early and to provide adequate treatment promptly.

Antonie Van Leeuwenhoek, 2004 May, 85(4), 287 - 96
Pyrolysis patterns of 5 close Corynebacterium species analyzed by artificial neural networks; Voisin S et al.; In the present study, an artificial neural network was trained with the Stuttgart Neural Networks Simulator, in order to identify Corynebacterium species by analyzing their pyrolysis patterns . An earlier study described the combination of pyrolysis, gas chromatography and atomic emission detection we used on whole cell bacteria . Carbon, sulfur and nitrogen were detected in the pyrolysis compounds . Pyrolysis patterns were obtained from 52 Corynebacterium strains belonging to 5 close species . These data were previously analyzed by Euclidean distances calculation followed by Unweighted Pair Group Method of Averages, a clustering method . With this early method, strains from 3 of the 5 species (C . xerosis, C . freneyi and C . amycolatum) were correctly characterized even if the 29 strains of C . amycolatum were grouped into 2 subgroups . Strains from the 2 remaining species (C . minutissimum and C . striatum) cannot be separated . To build an artificial neural network, able to discriminate the 5 previous species, the pyrolysis data of 42 selected strains were used as learning set and the 10 remaining strains as testing set . The chosen learning algorithm was Back-Propagation with Momentum . Parameters used to train a correct network are described here, and the results analyzed . The obtained artificial neural network has the following cone-shaped structure: 144 nodes in input, 25 and 9 nodes in 2 successive hidden layers, and then 5 outputs . It could classify all the strains in their species group . This network completes a chemotaxonomic method for Corynebacterium identification.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Jan-Feb, (1), 3 - 7
{Corynebacterium diphtheriae nontoxigenic strain carrying the gene of diphtheria toxin}; Mel'nikov VG et al.; Among 828 C . diphtheriae nontoxigenic cultures isolated in different region of Russia in 1994-2002, 114 cultures (13.8%) had the gene of diphtheria toxin (gene tox) and were thus called nontoxigenic tox-carrying (NTTC) strains . All NTTC strains were found to belong to biovar mitis and formed neither normal, nor "defective" diphtheria toxin . The most of NTTC strains (94%) belonged to ribotype "Moskva", not occurring among C . diphtheriae toxigenic strains . The incapacity of NNTC strains of forming diphtheria toxin was caused by mutation: the deletion of one nucleotide which led to the shift of the open reading frame and to the formation of the stop codon . The results of these studies are indicative of the fact that a sufficiently homogeneous and isolated group of C . diphtheriae nontoxigenic strains is spread in Russia . These strains carry the nonexpressing gene of diphtheria toxin and are of no epidemic importance in diphtheria infection.

Clin Microbiol Infect, 2004 Mar, 10(3), 247 - 54
AZD2563, a novel oxazolidinone: definition of antibacterial spectrum, assessment of bactericidal potential and the impact of miscellaneous factors on activity in vitro; Wookey A et al.; AZD2563, a new oxazolidinone targeted at Gram-positive bacteria, was evaluated and compared with linezolid and other agents against 802 aerobic bacterial isolates for spectrum of activity, bactericidal activity, and the effect of miscellaneous factors upon activity in vitro . At a concentration of 2 mg/L, AZD2563 inhibited 98% of all Gram-positive bacteria tested (100% at 4 mg/L), including susceptible and resistant isolates of Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus spp., Streptococcus pneumoniae, other Streptococcus spp . and Corynebacterium spp . Conversely, all Enterobacteriaceae and non-fermenting Gram-negative bacteria had MICs > 128 mg/L, and only a few Haemophilus or Moraxella spp . had MICs < 8 mg/L . By conventional laboratory definition, AZD2563 and linezolid were bacteriostatic against staphylococci and enterococci, with variable bactericidal activity against Strep . pneumoniae . The in-vitro activity of AZD2563 was essentially unaffected by altering pH, inoculum size, the type of testing medium, or the inclusion of human serum up to 25% v/v.

Chemosphere, 2004 Apr, 55(4), 533 - 8
Biodegradation of phthalate esters by two bacteria strains; Chang BV et al.; In this study two aerobic phthalic acid ester (PAE) degrading bacteria strains, DK4 and O18, were isolated from river sediment and petrochemical sludge, respectively . The two strains were found to rapidly degrade PAE with shorter alkyl-chains such diethyl phthalate (DEP), dipropyl phthalate (DPrP), di-n-butyl phthalate (DBP), benzylbutyl phthalate (BBP) and diphenyl phthalate (DPP) are very easily biodegraded, while PAE with longer alkyl-chains such as dicyclohexyl phthalate (DCP) and dihexyl phthalate (DHP) and di-(2-ethylhexyl) phthalate (DEHP) are poorly degraded . The degradation rates of the eight PAEs were higher for strain DK4 than for strain O18 . In the simultaneous presence of strains DK4 and O18, the degradation rates of the eight PAEs examined were enhanced . When the eight PAEs were present simultaneously, degradation rates were also enhanced . We also found that PAE degradation was delayed by the addition of nonylphenol or selected polycyclic aromatic hydrocarbons (PAHs) at a concentration of 1 microg/g in the sediment . The bacteria strains isolated, DK4 and O18, were identified as Sphigomonas sp . and Corynebacterium sp., respectively.

Protein Expr Purif, 2004 Apr, 34(2), 311 - 6
Three-phase partitioning as an efficient method for extraction/concentration of immunoreactive excreted-secreted proteins of Corynebacterium pseudotuberculosis; Paule BJ et al.; The three-phase partitioning (TPP) technique was used upstream to isolate/concentrate secreted proteins from Corynebacterium pseudotuberculosis cultured in a complex liquid medium . Several parameters of the TPP technique (15, 30, or 60% ammonium sulfate concentration; 4.0, 5.5, or 7.0 pH; and primary (n) or tertiary (t)-butanol solvent isomer) were varied to determine the optimal recovery of serologically and cellularly immunoreactive extracted proteins . A TPP extraction made with 30% ammonium sulfate and an initial pH of 4.0 gave the best humoral and cellular immunoreactivity of caseous lymphadenitis infected goats . In particular, two immunogenic secreted (16 and 125 kDa) proteins, which had not been found by other extraction methods, were identified.

Eye, 2004 Aug, 18(8), 778 - 84
Climatic influence on conjunctival bacteria of patients undergoing cataract surgery; Rubio EF; PURPOSE: To describe the monthly prevalence of conjunctival bacteria in patients undergoing cataract extraction and the possible climatic influence on it, in Madrid, in order to clarify postsurgical endophthalmitis pathogenesis . METHODS: The lower conjunctival content sample of 4432 consecutive patients awaiting cataract surgery was cultured from January 1994 to December 1996 . The dates of the operations and the rehospitalization for postsurgical endophthalmitis, if this took place, were checked . The isolated bacteria were grouped to study the statistical significance of the differences in the monthly prevalence differences (chi2 tests) . Temperature and relative humidity are given monthly for the area where our patients live . RESULTS: The total frequency of the conjunctival bacteria increases in April, May, and June, when the daily average temperature rises from 12 to 22 degrees C and the relative humidity oscillates between 45 and 60% in our area . Bacteria groups' frequency was significantly higher as follows: Staphylococci coagulase negative ( > 60%) in April, May, and June; Corynebacterium sp ( > 33%), Staphylococcus Aureus ( > 8%), and other Gram-positive bacteria ( > 2.5%) in May; Streptococcus Pneumoniae increases ( > 3.4%) in March, November, and December; Haemophilus sp ( > 3.4%) in January and April; Gram-negative Cocci ( > 3%) in April; and other Streptococcus sp ( > 6%) in April, May, and September . Our incidence of rehospitalization for endophthalmitis after cataract extraction in May and June together was 3.37 times higher than in the other months . CONCLUSION: Conjunctival bacteria of our patients undergoing cataract surgery present a seasonal prevalence pattern, which could be considered as a predisposing condition for having postsurgical endophthalmitis in certain months.

J Bacteriol, 2004 Mar, 186(6), 1769 - 84
In-depth profiling of lysine-producing Corynebacterium glutamicum by combined analysis of the transcriptome, metabolome, and fluxome; Kromer JO et al.; An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing Corynebacterium glutamicum ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production . For this purpose, 13C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isotopomer modeling were combined with expression profiling via DNA microarrays and with intracellular metabolite quantification . The phase shift from growth to lysine production was accompanied by a decrease in glucose uptake flux, the redirection of flux from the tricarboxylic acid (TCA) cycle towards anaplerotic carboxylation and lysine biosynthesis, transient dynamics of intracellular metabolite pools, such as an increase of lysine up to 40 mM prior to its excretion, and complex changes in the expression of genes for central metabolism . The integrated approach was valuable for the identification of correlations between gene expression and in vivo activity for numerous enzymes . The glucose uptake flux closely corresponded to the expression of glucose phosphotransferase genes . A correlation between flux and expression was also observed for glucose-6-phosphate dehydrogenase, transaldolase, and transketolase and for most TCA cycle genes . In contrast, cytoplasmic malate dehydrogenase expression increased despite a reduction of the TCA cycle flux, probably related to its contribution to NADH regeneration under conditions of reduced growth . Most genes for lysine biosynthesis showed a constant expression level, despite a marked change of the metabolic flux, indicating that they are strongly regulated at the metabolic level . Glyoxylate cycle genes were continuously expressed, but the pathway exhibited in vivo activity only in the later stage . The most pronounced changes in gene expression during cultivation were found for enzymes at entry points into glycolysis, the pentose phosphate pathway, the TCA cycle, and lysine biosynthesis, indicating that these might be of special importance for transcriptional control in C . glutamicum.

J Vet Med B Infect Dis Vet Public Health, 2004 Feb, 51(1), 1 - 7
Otitis in cattle, an aetiological review; Duarte ER et al.; Otitis externa in cattle has a significant impact in tropical and subtropical regions, and the aetiological agents are predominantly rhabditiform nematodes and mites of the genus Raillietia . Its prevalence is higher in mature and Zebu cattle . In advanced clinical cases there can be irreversible and fatal neural lesions . Ear infection in calves has been associated with concurrent respiratory diseases and mixed infection . The principal reported agents of otitis in calves are bacteria such as Actinomyces spp., Corynebacterium pseudotuberculosis, Escherichia coli, Haemophilus somnus, Pasteurella multocida, Mannheimia haemolytica, Pseudomonas spp., Streptococcus spp . and Mycoplasma bovis . The control and treatment of bovine otitis is not standardized and there is little evidence-based support for the diverse treatments available in the literature.

Vet Rec, 2004 Feb 14, 154(7), 204 - 6
Four-year survey of urinary tract infections in calves in Israel; Yeruham I et al.; The prevalence of urinary tract infections in calves aged seven days to three months in three dairy cattle herds ranged from 0.5 to 1.6 per cent, with an average of 1.1 per cent . The mortality rate reached 16.1 per cent . The morbidity rate of the female calves was 1.4 per cent and that of the male calves 0.8 per cent . The bacteria isolated from urine, and from vaginal and preputial swabs were Escherichia coli (35 per cent), Corynebacterium renale (14 per cent), plasma coagulase-negative Staphylococcus species (12 per cent), Pseudomonas aeruginosa (12 per cent), Proteus species (12 per cent) and Arcanobacterium pyogenes (5 per cent) . The affected calves had a significantly lower serum concentration of inorganic phosphorus (P < 0.01).

Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2506 - 11
Genetic and biophysical studies of diphtheria toxin repressor (DtxR) and the hyperactive mutant DtxR(E175K) support a multistep model of activation; Love JF et al.; The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is the prototypic member of a superfamily of transition metal ion-activated transcriptional regulators that have been isolated from Gram-positive prokaryotes . Upon binding divalent transition metal ions, the N-terminal domain of DtxR undergoes a dynamic structural organization leading to homodimerization and target DNA binding . We have used site-directed mutagenesis and NMR analysis to probe the mechanism by which apo-DtxR transits from an inactive to a fully active repressor upon metal ion binding . We demonstrate that the ancillary metal-binding site mutant DtxR(H79A) requires higher concentrations of metal ions for activation both in vivo and in vitro, providing a functional correlation to the proposed cooperativity between ancillary and primary binding sites . We also demonstrate that the C-terminal src homology 3 (SH3)-like domain of DtxR functions to modulate repressor activity by (i) binding to the polyprolyl tether region between the N- and C-terminal domains, and (ii) destabilizing the ancillary binding site, leading to full inactivation of the repressor . Finally, we show by NMR analysis that the hyperactive phenotype of DtxR(E175K) results from the stabilization of a structural intermediate in the activation process . Taken together, the data presented support a multistep model for the activation of apo-DtxR by transition metal ions.

J Biol Chem, 2004 May 14, 279(20), 21055 - 61 Epub 2004 Feb 13.
Crystal structure of the dioxygen-bound heme oxygenase from Corynebacterium diphtheriae: implications for heme oxygenase function; Unno M et al.; HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme . The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography . The oxygen association and dissociation rate constants are 5 microm(-1) s(-1) and 0.22 s(-1), respectively, yielding an O(2) affinity of 21 microm(-1), which is approximately 20 times greater than that of mammalian myoglobins . However, the affinity of HmuO for CO is only 3-4-fold greater than that for mammalian myoglobins, implying the presence of strong hydrogen bonding interactions in the distal pocket of HmuO that preferentially favor O(2) binding . Resonance Raman spectra show that the Fe-O(2) vibrations are tightly coupled to porphyrin vibrations, indicating the highly bent Fe-O-O geometry that is characteristic of the oxy forms of heme oxygenases . In the crystal structure of the oxy form the Fe-O-O angle is 110 degrees, the O-O bond is pointed toward the heme alpha-meso-carbon by direct steric interactions with Gly-135 and Gly-139, and hydrogen bonds occur between the bound O(2) and the amide nitrogen of Gly-139 and a distal pocket water molecule, which is a part of an extended hydrogen bonding network that provides the solvent protons required for oxygen activation . In addition, the O-O bond is orthogonal to the plane of the proximal imidazole side chain, which facilitates hydroxylation of the porphyrin alpha-meso-carbon by preventing premature O-O bond cleavage.

Int J Cancer, 1980 Jun 15, 25(6), 691 - 9
Augmentation of tumoricidal activity of human monocytes and macrophages by lymphokines; Mantovani A et al.; Monocytes were separated from peripheral blood mononuclear cells of normal human donors by adherence on plastic conditioned by cell lines (microexudate-coated plastic) and harvested by exposure to ethylene diamine tetra-acetic acid . Cytolytic activity was tested by incubating effector cells for 48 h with the murine SV40-transformed TU5 kidney line or the human lung cancer-derived CaLu line prelabelled with tritiated thymidine . Lymphokine-containing supernatants were obtained from in vitrocultures of lymphoid cells with phytohemagglutinin (PHA), purified protein derivative (PPD), or with Corynebacterium parvum strains CN6134 or CNS888 . The monocytes had significant levels of spontaneous cytotoxicity and exposure to lymphokine supernatants markedly enhanced their tumoricidal activity . Augmentation of monocyte-mediated cytotoxicity required a minimal exposure to lymphokine supernatants for 4 h and was maximal after 24 h of preincubation . Treatment of the effector cells with anti-human T-cell serum and complement did not affect either their spontaneous or their lymphokine-stimulated cytotoxicity, whereas silica impaired both reactivities . Supernatants of cultures with PHA, PPD and C . parvum CN6134 had significant levels of interferon (IF) . Since partially purified human fibroblast or leukocyte IF was able to stimulate monocyte-mediated cytotoxicity, the IF in these supernatants could play some role in the stimulation of the monocytes . However, C . parvum CN5888 supernatants, which had little IF, enhanced monocyte cytotoxicity as effectively as the C . parvum CN6134 supernatants, strongly suggesting that lymphocyte mediators other than IF can augment the tumoricidal activity of these effector cells . Mature macrophages obtained by in vitro cultivation of monocytes for 4-7 days retained natural cytolytic activity and showed enhanced cytotoxicity in the presence of lymphokines . However, more prolonged in vitro cultivation (> 10 days) resulted in cultures of epithelioid and multinucleated cells which had little natural cytotoxicity and were not responsive to lymphokines.

Kansenshogaku Zasshi, 2003 Dec, 77(12), 1024 - 31
{Investigate of nasopharyngeal flora in infants and children with influenza}; Uno Y; To clarify the bacteriological interpretation of nasopharyngeal flora from infants and children with influenza (n = 38), nasopharyngeal swabs were obtained . From 38 patients, 83 strains of bacterias were obtained . Chief pathogenic bacteria isolated from infants and children with influenza were B . catarrhalis (28 strains), S . pneumoniae (22 strains), H . influenzae (19 strains), S . aureus (6 strains) and chief nonpathogenic bacteria isolated from infants and children with influenza were Corynebacterium spp . and alpha-streptococcus (3 strains each) and Moraxella sp (2 strains) . From infants and children without influenza (n = 34), 83 strains were obtained . The chief pathogenic bacteria isolated from infants and children without influenza were B . catarrhalis (23 strains), H . influenzae (22 strains), S . pneumoniae (18 strains), S . aureus (7 strains) and chief nonpathogenic bacteria isolated from infants and children without influenza were Corynebacterium spp . and Moraxella sp (5 strains each), alpha-streptococcus (2 strains) and Neisseria sp (1 strain) . There was no significant difference in nasopharyngeal flora between infants and children with influenza and infants and children without influenza . In cases showing detection of multiple bacterial strains, common combinations were one or more of B . catarrhalis, S . pneumoniae, H . influenzae, S . aureus and nonpathogenic or weakly pathogenic bacteria . There was also no significant difference in combinations of nasopharyngeal flora between infants and children with influenza and those without influenza . We emphasize that we must study whether a difference in nasopharyngeal flora between infants and children with influenza and infants and children without influenza develops with time . Therefore, we must repeatedly obtain nasopharyngeal swabs from infants and children with influenza and infants and children without influenza.

J Dairy Sci, 2004 Jan, 87(1), 38 - 45
Identification of Corynebacterium bovis by endonuclease restriction analysis of the 16S rRNA gene sequence; Huxley JN et al.; Despite its high prevalence within the bovine mammary gland, Corynebacterium bovis is considered a minor pathogen and of limited clinical significance . It has been suggested that intramammary infection with C . bovis may protect quarters against subsequent infection with other pathogens . The literature has produced much conflicting data on the subject . A possible explanation for some of the divergence of opinion on the subject is incorrect identification of isolates in previous studies . This paper describes a novel method for differentiating C . bovis from other lipophilic Corynebacterium species based on endonuclease restriction analysis . The 16S rRNA gene sequences for all known lipophilic Corynebacterium species were obtained from published data and analyzed . It was predicted that endonuclease restriction with HindIII and SmaI could be used to differentiate C . bovis from all other known lipophilic Corynebacterium species . The method was successfully employed to identify 741 of 762 (97.2%) lipophilic Corynebacterium species as C . bovis . Twenty one (2.8%) were identified as species other than C . bovis . Using this technique, it was demonstrated that it is not safe to assume that all lipophilic coryneform organisms isolated from bovine milk samples are C . bovis . This method is an alternative to more traditional methods of identification in large scale studies until methods such as 16S rRNA gene sequencing become more widely available.

Acta Orthop Scand, 2003 Dec, 74(6), 704 - 8
No difference between daily and weekly pin site care: a randomized study of 50 patients with external fixation; W-Dahl A et al.; INTRODUCTION: We investigated whether there were any differences in the frequency and severity of pin site infections by performing pin site care daily or once a week . We studied patients operated on for gonarthrosis by the hemicallotasis technique, using hydroxyapatite-coated pins in the metaphyseal bone and standard pins in the diaphyseal bone . PATIENTS AND METHODS: 50 patients were prospectively randomized to daily (n = 27) or weekly (n = 23) pin site care . We evaluated pin sites, the occurrence of pain (VAS), the use of antibiotics and analgesics and complications every week . Bacterial cultures were taken from each pin site at 1, 6 and 10 weeks and from the pins on removal . RESULTS: We found no differences between daily or weekly pin site care as regards the frequency and severity of pin site infections, pain, or the use of antibiotics and analgesics . Grade I infections (Checketts-Otterburns classification) occurred around 11% of the pins and grade II infections around 4% . 70% of the bacterial cultures were negative . The most frequent bacteria were coagulase negative staphylococcus and corynebacterium . Antibiotics were given an average of 47 days . More problems occurred around the proximal pins . 5/200 (all proximal) pins were clinically loose on removal . INTERPRETATION: Pin site care once a week seems appropriate.

Biotechnol Bioeng, 2004 Mar 5, 85(5), 497 - 505
Serial flux mapping of Corynebacterium glutamicum during fed-batch L-lysine production using the sensor reactor approach; Drysch A et al.; Using our recently developed sensor reactor approach, lysine-producing, nongrowing Corynebacterium glutamicum MH20-22B cells were subjected to serial (13)C-labeling experiments for flux analysis during the leucine-limited fed-batch production phase in a 300-L bioreactor . Based on two-dimensional (2D) nuclear magnetic resonance (NMR) measurements of (13)C-labeling patterns of cytoplasmic free metabolites, metabolic flux distributions in the central metabolism were successfully determined . Focusing on the highly concentrated metabolite L-glutamate, the working hypothesis was validated that the equilibration of labeling patterns in intracellular pools was much faster (up to 9.45 min) than the labeling period (3 h) used in the experiments . Analysis of anaplerotic reactions revealed that highly selective lysine production was accompanied by a significant reduction of decarboxylating reactions from 10 mol% to only 2 mol%, whereas PEP/pyruvate-carboxylating fluxes remained constant at about 40 mol% of consumed glucose . These results support the conclusion that an optimized C . glutamicum L-lysine producer should possess increased PEP carboxylase and/or pyruvate carboxylase activity combined with downregulated, decarboxylating fluxes consuming oxaloacetate/malate . The findings also illustrate the usefulness of the sensor reactor approach in the study of industrial fermentations .

Ann Thorac Surg, 2004 Feb, 77(2), 537 - 43
Role of oral bacterial flora in calcific aortic stenosis: an animal model; Cohen DJ et al.; BACKGROUND: Calcific aortic stenosis is a major public health problem in the United States . The mechanism of calcification remains unclear . The hypothesis that low grade chronic or recurrent bacterial endocarditis with specific calcifiable bacteria is a cause of calcification of the aortic valves was investigated using an animal model . Such bacteria are typically present as part of the normal human oral flora . METHODS: Forty New Zealand white rabbits were divided into four groups: group 1, control (1 ml of normal saline); group 2, Corynebacterium matruchotti 100,000 colonies; group 3, Streptococcus sanguis II 10 colonies; and group 4, C matruchotti 100,000 colonies plus S sanguis II 10 colonies . Animals were inoculated with bacteria through a flexible catheter placed through the aortic valve through a right carotid cut down . Inoculations were repeated every 3 days the first 2 weeks and then twice a week thereafter . At postmortem examination the aortic valves were harvested, embedded in paraffin, and stained with von Kossa stain . They were also examined by scanning and transmission electron micrography . RESULTS: Group 4 had 93.3% large calcifications (confluent calcium densities that are easily recognized with minimal magnification) and 6.6% small microcalcifications (dustlike microscopic particles requiring a compound microscope to appreciate) of the aortic valves . Group 3 exhibited large calcification in 20% and small in 40% of the aortic valves . Group 1 and group 2 had no evidence of calcification . CONCLUSIONS: These results suggest that recurrent low-grade endocarditis from calcifying oral bacteria, particularly when occurring with synergistic strains, may be one cause of calcific aortic stenosis.

Arch Soc Esp Oftalmol, 2004 Jan, 79(1), 13 - 9
{Conjunctival bacteria of patients undergoing cataract surgery: changes in the last 50 years}; Fernandez Rubio E; PURPOSE: To describe the prevalence of preoperative conjunctival bacteria of patients living in Madrid, who underwent cataract surgery in our hospital, without using exclusion criteria to select the patients . METHOD: The preoperative conjunctival cultures of 4432 consecutive patients, in a three year period, were studied retrospectively . Samples of preoperative conjunctival cultures were selected from publications of the past 50 years . Our conjunctival bacteria frequencies were compared with those of other samples (chi-square test), in order to show that the bacterial isolation method is a determinant factor of the bacterial spectrum . RESULTS: 93.5% bacteria were Gram positive and 6.5% Gram negative . Each bacteria group was present in the patients as follows: 56.9% Staphylococcus coagulase negative, 30.5% Corynebacterium sp, 6.5% Staphylococcus Aureus, 2.7% Streptococcus Pneumoniae, 5.6% other Streptococcus sp, 2.8% Haemophilus sp, 3.5% other Gram negative rods, 1.2% diplococci Gram negative and 0.5% other bacteria . Pathogen bacteria were isolated in 19.1% of the patients and 21.9% of the cultures were negative . The samples shown in publications previous to 1980 (table II), present bacteria frequencies statistically different from our study . CONCLUSIONS: The conjunctival bacteria prevalence in preoperative cultures of patients awaiting cataract surgery had never been described in Madrid . By analysing other authors' results, it can be seen that bacteria prevalence is affected by the culture media used . It also seems that the size of the sample and the health status of the patients could affect this prevalence (Arch Soc Esp Oftalmol 2004; 79: 13-20).

Arch Microbiol, 2004 Mar, 181(3), 237 - 44 Epub 2004 Jan 21.
Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from Corynebacterium glutamicum; Hsu SK et al.; Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum . PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine ( mFP)-resistant mutants . Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme . When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type . The Ser99Met mutant was active in the presence of 50 microM phenylalanine, whereas the wild-type enzyme was not . The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis . Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in Km and kcat values, respectively . Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity . Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity . The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the Km value was 26-fold higher . Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the Km and kcat values . These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity.

J Dairy Sci, 2003 Dec, 86(12), 3912 - 9
Prophylactic effects of two selective dry cow strategies accounting for interdependence of quarter; Berry EA et al.; Infusion of a long-acting antibiotic preparation at drying off in dairy cows as a prophylactic therapy is usually recommended for all quarters where it is in use . Studying the effectiveness of such treatment using quarter as the unit of analysis assumes that each quarter within a cow has a risk of being infected independent of the other quarters of the cow . Failure to account for interdependence of quarters within a cow may lead to inaccurate variance estimates and errors in assessing treatment effects . Data from two trials assessing different dry-cow strategies were examined for interdependence of infection between quarters . Logistic regression with a variance inflation factor or a multilevel analysis was used to assess the effect of antibiotic and internal teat-sealant dry cow strategies . Parity and infection status at drying off were covariates in the analysis . Interdependence of the risk of quarter infections within control-group cows was demonstrated in both dry-cow antibiotic and teat-seal trials . However, cows that received either of these treatments did not demonstrate interdependence . Treated quarters in both trials were 3.0 times less likely to acquire a new infection at calving compared with the untreated controls . Quarters in cows of parity 3 or greater were also at an increased risk in the antibiotic treatment trial . In both trials, quarters with either Corynebacterium spp . or coagulase-negative staphylococci infections at drying off had an increased risk of a new intramammary infection at calving . This study has demonstrated the beneficial and comparable effects of antibiotic and teat seal dry cow strategies; both decreased the risk of intramammary infection at calving . The application of dry-cow strategies at the cow level and not the quarter level is also supported.

Appl Microbiol Biotechnol, 2004 May, 64(4), 447 - 54 Epub 2004 Jan 22.
Properties and applications of microbial transglutaminase; Yokoyama K et al.; Some properties and applications of the transglutaminase (TGase) referred to as microbial TGase (MTGase), derived from a variant of Streptomyces mobaraensis (formerly classified as Streptoverticillium mobaraense), are described . MTGase cross-linked most food proteins, such as caseins, soybean globulins, gluten, actin, myosins, and egg proteins, as efficiently as mammalian TGases by forming an epsilon-(gamma-glutamyl)lysine bond . However, unlike many other TGases, MTGase is calcium-independent and has a relatively low molecular weight . Both of these properties are of advantage in industrial applications; a number of studies have illustrated the potential of MTGase in food processing and other areas . The crystal structure of MTGase has been solved . It provides basic structural information on the MTGase and accounts well for its characteristics . Moreover, an efficient method for producing extracellular MTGase has been established using Corynebacterium glutamicum . MTGase may be expected to find many uses in both food and non-food applications .

J Biol Chem, 2004 Mar 19, 279(12), 10833 - 6 Epub 2004 Jan 19.
Spider and bacterial sphingomyelinases D target cellular lysophosphatidic acid receptors by hydrolyzing lysophosphatidylcholine; van Meeteren LA et al.; Bites by Loxosceles spiders can produce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, hemolysis, and persistent inflammation . The causative factor is a sphingomyelinase D (SMaseD) that cleaves sphingomyelin into choline and ceramide 1-phosphate . A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor . However, the molecular basis for SMaseD toxicity is not well understood, which hampers effective therapy . Here we show that the spider and bacterial SMases D hydrolyze albumin-bound lysophosphatidylcholine (LPC), but not sphingosylphosphorylcholine, with K(m) values ( approximately 20-40 microm) well below the normal LPC levels in blood . Thus, toxic SMases D have intrinsic lysophospholipase D activity toward LPC . LPC hydrolysis yields the lipid mediator lysophosphatidic acid (LPA), a known inducer of platelet aggregation, endothelial hyperpermeability, and pro-inflammatory responses . Introduction of LPA(1) receptor cDNA into LPA receptor-negative cells renders non-susceptible cells susceptible to SmaseD, but only in LPC-containing media . Degradation of circulating LPC to LPA with consequent activation of LPA receptors may have a previously unappreciated role in the pathophysiology of secreted SMases D.

Microb Pathog, 2004 Mar, 36(3), 125 - 30
Patterns of adherence to HEp-2 cells and actin polymerisation by toxigenic Corynebacterium diphtheriae strains; Hirata R Jr et al.; Corynebacterium diphtheriae strains displayed different degrees of attachment to HEp-2 cell monolayers with two distinct adherence patterns, termed localised (LA) and diffuse (DA) . The LA phenotype predominated over the DA phenotype . The non-sucrose fermenting strains expressing DA pattern adhered mostly with high index values (> or =10bact/cell) . Low adhesion index (<10bact/cell) was mainly observed among sucrose fermenting strains . The fluorescein isothiocyanate (FITC)-labelled phalloidin assay (fluorescent-actin staining test) showed positive results for microorganisms of both LA and DA phenotypes . The FITC-labelled C . diphtheriae non-fimbrial surface proteins 67-72p interacted directly with HEp-2 cell membranes . Therefore, toxigenic C . diphtheriae exhibited LA and DA adherence patterns and ability to induce actin polymerisation . The experimental evidences also pointed to 67-72p as putative adhesins of C . diphtheriae to HEp-2 cells.

Mikrobiol Z, 2003 Sep-Oct, 65(5), 30 - 5
{Microbiocenosis of lower extremities skin of healthy people and patients with diabetes mellitus}; Klymniuk SI et al.; A comparative study of the foot skin microbiocenosis of 50 healthy persons and 29 patients with diabetes mellitus and diabetic foot syndrome have been carried out . It has been revealed that various topodemes had unequal degree of aerobic microorganisms colonization . Different species of staphylococci are dominating taxons in skin . The gradient of bacterial populations density in healthy and sick persons grows in a direction: back of the foot--sole--interdigital space . Modification of skin microbiocenosis in patients with diabetes mellitus has been shown . The colonization degree by different species of bacteria decreased; the bacilli part enhanced and the corynebacteria part decreased in the biocenosis composition.

No Shinkei Geka, 2003 Dec, 31(12), 1303 - 7
{A case of brain abscess associated with asymptomatic multiple myeloma}; Nishimoto T et al.; A 55-year-old male was admitted to our hospital because of confusion and mild weakness of his left arm and leg . His condition had taken a gradual turn for the worse for several months . Computed tomography (CT) demonstrated a mixed density mass with multiple cysts and massive perifocal edema . Magnetic resonance imaging (MRI) demonstrated an irregular-shaped mass with multiple cysts sized 6 x 4 x 6 cm in the temporal lobe, which manifested mixed signal intensity on both the T1 weighted image and the T2 weighted image . MRI also revealed massive perifocal edema and marked midline shift . Gd-DTPA study showed ring-like enhancement . Angiography showed no tumor stain and a suppressed right posterior cerebral artery . A right extended temporo-occipital craniotomy was performed to extirpate the abscess subtotally . The histological examination showed brain abscess and Gram stain of the pus revealed the presence of gram-positive bacilli . The gram-positive bacillus, Corynebacterium only was subsequently cultured from the pus . After the operation his hemiparesis seemed to disappear . In spite of the treatment with multiple intravenous antibiotics, his hemiparesis worsened again . CT and MRI demonstrated recurrence of the brain abscess in the occipital lobe and marked perifocal edema . The second operation was performed and removed all the infected brain tissue with abscess . After the second operation, otorhinological and cardiovascular examinations were carried out, but no causal disease was found . Immunoelectrophoresis (total protein 12.2 g/d/) revealed the peak of M protein . Bone marrow revealed dysplasia of the plasma cell and he was diagnosed as having multiple myeloma that had made him an immunocompromised host.

Protein Sci, 2004 Feb, 13(2), 504 - 12 Epub 2004 Jan 10.
Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase; Sanli G et al.; Corynebacterium 2,5-Diketo-D-gluconic acid reductase (2,5-DKGR) catalyzes the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-Keto-L-gulonic acid (2-KLG) . 2-KLG is an immediate precursor to L-ascorbic acid (vitamin C), and 2,5-DKGR is, therefore, an important enzyme in a novel industrial method for the production of vitamin C . 2,5-DKGR, as with most other members of the aldo-keto reductase (AKR) superfamily, exhibits a preference for NADPH compared to NADH as a cofactor in the stereo-specific reduction of substrate . The application of 2,5-DKGR in the industrial production of vitamin C would be greatly enhanced if NADH could be efficiently utilized as a cofactor . A mutant form of 2,5-DKGR has previously been identified that exhibits two orders of magnitude higher activity with NADH in comparison to the wild-type enzyme, while retaining a high level of activity with NADPH . We report here an X-ray crystal structure of the holo form of this mutant in complex with NADH cofactor, as well as thermodynamic stability data . By comparing the results to our previously reported X-ray structure of the holo form of wild-type 2,5-DKGR in complex with NADPH, the structural basis of the differential NAD(P)H selectivity of wild-type and mutant 2,5-DKGR enzymes has been identified.

Am J Med, 2004 Jan 15, 116(2), 78 - 83
Electrocardiographic abnormalities in patients with diphtheria: a prospective study; Lumio JT et al.; PURPOSE: To determine the incidence of and risk factors for electrocardiographic (ECG) abnormalities in adults with diphtheria . METHODS: A prospective study was conducted involving 122 adult patients with respiratory tract diphtheria . Diphtheria was confirmed by isolation of a toxin-producing strain of Corynebacterium diphtheriae . Patients had serial clinical evaluations and ECGs for a minimum of 21 days . RESULTS: Cardiac involvement was detected in 25 (28%) of 88 evaluable patients, with a median time from symptom onset to an abnormal ECG of 9 days (range, 4 to 24 days) . In a logistic regression analysis, age (odds ratio {OR} = 4.1; 95% confidence interval {CI}: 1.6 to 11.0), shared accommodation (OR = 2.9; 95% CI: 1.0 to 8.6), fever (OR = 4.2; 95% CI: 1.1 to 16.6), and extensive respiratory tract infection with subcutaneous edema (OR = 7.0; 95% CI: 1.2 to 42.2) were independent risk factors for cardiac involvement . CONCLUSION: Cardiac involvement is a common complication of respiratory tract infection with C . diphtheriae, and occurs more often among older patients, those with lower socioeconomic status, and those with severe respiratory tract involvement.

Appl Environ Microbiol, 2004 Jan, 70(1), 370 - 6
Overproduction of trehalose: heterologous expression of Escherichia coli trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in Corynebacterium glutamicum; Padilla L et al.; Trehalose is a disaccharide with potential applications in the biotechnology and food industries . We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum . This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C . glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium . The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose . The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase . The results proved the suitability of using the heterologous expression of Ots proteins in C . glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C . glutamicum.

Appl Environ Microbiol, 2004 Jan, 70(1), 229 - 39
Comparative metabolic flux analysis of lysine-producing Corynebacterium glutamicum cultured on glucose or fructose; Kiefer P et al.; A comprehensive approach to (13)C tracer studies, labeling measurements by gas chromatography-mass spectrometry, metabolite balancing, and isotopomer modeling, was applied for comparative metabolic network analysis of lysine-producing Corynebacterium glutamicum on glucose or fructose . Significantly reduced yields of lysine and biomass and enhanced formation of dihydroxyacetone, glycerol, and lactate in comparison to those for glucose resulted on fructose . Metabolic flux analysis revealed drastic differences in intracellular flux depending on the carbon source applied . On fructose, flux through the pentose phosphate pathway (PPP) was only 14.4% of the total substrate uptake flux and therefore markedly decreased compared to that for glucose (62.0%) . This result is due mainly to (i) the predominance of phosphoenolpyruvate-dependent phosphotransferase systems for fructose uptake (PTS(Fructose)) (92.3%), resulting in a major entry of fructose via fructose 1,6-bisphosphate, and (ii) the inactivity of fructose 1,6-bisphosphatase (0.0%) . The uptake of fructose during flux via PTS(Mannose) was only 7.7% . In glucose-grown cells, the flux through pyruvate dehydrogenase (70.9%) was much less than that in fructose-grown cells (95.2%) . Accordingly, flux through the tricarboxylic acid cycle was decreased on glucose . Normalized to that for glucose uptake, the supply of NADPH during flux was only 112.4% on fructose compared to 176.9% on glucose, which might explain the substantially lower lysine yield of C . glutamicum on fructose . Balancing NADPH levels even revealed an apparent deficiency of NADPH on fructose, which is probably overcome by in vivo activity of malic enzyme . Based on these results, potential targets could be identified for optimization of lysine production by C . glutamicum on fructose, involving (i) modification of flux through the two PTS for fructose uptake, (ii) amplification of fructose 1,6-bisphosphatase to increase flux through the PPP, and (iii) knockout of a not-yet-annotated gene encoding dihydroxyacetone phosphatase or kinase activity to suppress overflow metabolism . Statistical evaluation revealed high precision of the estimates of flux, so the observed differences for metabolic flux are clearly substrate specific.

Plasmid, 2004 Jan, 51(1), 54 - 60
Characterization of the cryptic plasmid pCC1 from Corynebacterium callunae and its use for vector construction; Venkova-Canova T et al.; The complete nucleotide sequence of the cryptic plasmid pCC1 from Corynebacterium callunae (4109 bp) was determined . DNA sequence analysis revealed five open reading frames longer than 200 bp . One of the deduced polypeptides showed homology with the Rep proteins encoded by plasmids of the pIJ101/pJV1 family of plasmids replicating by the rolling-circle (RC) mechanism . Within this plasmid family, the Rep protein of pCC1 showed the highest degree of similarity to the Rep proteins of corynebacterial plasmids pAG3 and pBL1 . These data suggest that the plasmid pCC1 replicates by the RC mechanism . The Escherichia coli/Corynebacterium glutamicum shuttle cloning vector pSCCD1, carrying the pCC1 rep gene on the 2.1-kb DNA fragment and the streptomycin/spectinomycin resistance determinant, was constructed . This vector is stably maintained in population of C . glutamicum cells grown in the absence of selection pressure and it is compatible with plasmid vectors based on corynebacterial plasmids pBL1 and pSR1.

Expert Rev Vaccines, 2003 Aug, 2(4), 551 - 60
Mucosal immunization against respiratory bacterial pathogens; Foxwell AR et al.; Bacterial respiratory diseases remain a major cause of morbidity and mortality throughout the world . The young and the elderly are particularly susceptible to the pathogens that cause these diseases . Therapeutic approaches remain dependent upon antibiotics contributing to the persistent increases in antibiotic resistance . The main causes of respiratory disease discussed in this review are Mycobacterium tuberculosis, Corynebacterium diphtheriae, Bordatella pertussis, Streptococcus pneumoniae, non-typeable Haemophilus influenzae, Moraxella catarrhalis and Pseudomonas aeruginosa . All these organisms initiate disease at the mucosal surface of the respiratory tract and thus the efficacy of the host's response to infection needs to be optimal at this site . Vaccines available for diseases caused by many of these pathogens have limitations in accessibility or efficacy, highlighting the need for improvements in approaches and products . The most significant challenges in both therapy and prevention of disease induced by bacteria in the respiratory tract remain the development of non-injectable vaccines and delivery systems/immunization regimens that improve mucosal immunity.

Pol J Vet Sci, 2003, 6(4), 261 - 6
Relationships among ecology, demography and diseases of European bison (Bison bonasus); Kita J et al.; The bison population in the Bialowieza Forest in Poland has now grown to approximately 300, while the herds in the Belarusian part of the forest total about 240 bison . The first signs of a health problem in these herds appeared in 1980, when two cases of balanoposthitis were detected in two bulls (2 and 5 years of age) . Since 1980 research has been conducted to explain the cause of diseases, particularly balanoposthitis, and to monitor the health of bison in Bialowieza Forest . A total number of 614 bison (294 male and 320 female) of different ages was eliminated between 1980 and 2000 . Not all the culled bison were examined (postmortem analysis, histopathological, bacteriological, virological and toxicological examinations, serological tests, molecular research) . Based on the increase in numbers, reproduction in this population for the past 21 years is generally considered successful . Among 182 male bison eliminated during 1990-2000, only 85, or 47%, of the animals had balanoposthitis . Thus, the percentage of balanoposthitis cases went from 100% during the 1980s down to 47% in the past decade . It appears that the culling process has been a major factor leading to this decrease . It can be assumed that a set of factors is involved in the appearance of the disease (Corynebacterium spp., Bacillus sp., Pseudomonas aeruginosa, Escherichia coli, Ureoplasma spp, Fusobacterium necrophorum, Streptoccocus spp., Staphyloccocus spp.) while opportunistic infections including nematodes (Onchocerca spp.) are responsible for the occurrence of secondary lesions.

Vet Rec, 2003 Dec 13, 153(24), 746 - 50
Experimental Corynebacterium pseudotuberculosis mastitis in dairy cows; Aroch I et al.; The inoculation of 2000 colony-forming units of Corynebacterium pseudotuberculosis into one teat canal of each of three cows resulted in severe, chronic, pyogranulomatous mastitis . Within three days the cows had a reduced haematocrit, haemoglobin concentration and red cell count . The anaemia was initially normocytic, normochromic and non-regenerative, and was associated with a brief peak of neutrophilia; a regenerative response became evident two to three weeks later . Clinical signs of mastitis appeared seven to 14 days after the inoculation, with a peak of high fever, more severe anaemia, a second peak of neutrophilia and the complete cessation of milk production from all quarters; extensive and severe pyogranulomatous mastitis developed in the inoculated quarters . No other lesions were detected postmortem, and C pseudotuberculosis was cultured from the affected quarters but not from the supramammary lymph nodes and viscera.

Microbiology, 2004 Jan, 150(Pt 1), 73 - 84
Importance of mycoloyltransferases on the physiology of Corynebacterium glutamicum; Kacem R et al.; Mycoloyltransferases (Myts) play an essential role in the biogenesis of the cell envelope of members of the Corynebacterineae, a group of bacteria that includes the mycobacteria and corynebacteria . While the existence of several functional myt genes has been demonstrated in both mycobacteria and corynebacteria (cmyt), the disruption of any of these genes has at best generated cell-wall-defective but always viable strains . To investigate the importance of Myts on the physiology of members of the Corynebacterineae, a double mutant of Corynebacterium glutamicum was constructed by deleting cmytA and cmytB, and the consequences of the deletion on the viability of the mutant, the transfer of corynomycoloyl residues onto its cell-wall arabinogalactan and trehalose derivatives, and on its cell envelope ultrastructure were determined . The double mutant strain failed to grow at 34 degrees C and exhibited a growth defect and formed segmentation-defective cells at 30 degrees C . Biochemical analyses showed that the double mutant elaborated 60 % less cell-wall-bound corynomycolates and produced less crystalline surface layer proteins associated with the cell surface than the parent and cmytA-inactivated mutant strains . Freeze-fracture electron microscopy showed that the DeltacmytA DeltacmytB double mutant, unlike the wild-type and cmytA-inactivated single mutant strains, frequently exhibited an additional fracture plane that propagated within the plasma membrane and rarely exposed the S-layer protein . Ultra-thin sectioning of the double mutant cells showed that they were totally devoid of the outermost layer . Complementation of the double mutant with the wild-type cmytA or cmytB gene restored completely or partially this phenotype . The data indicate that Myts are important for the physiology of C . glutamicum and reinforce the concept that these enzymes would represent good targets for the discovery of new drugs against the pathogenic members of the Corynebacterineae.

J Med Assoc Thai, 2003 Aug, 86 Suppl 3, S696 - 700
Subacute infective endocarditis caused by Corynebacterium diphtheriae: a case report; Lolekha R et al.; The authors report an 11-year-old boy with septicemia and subacute infective endocarditis due to toxigenic-Corynebacterium diphtheriae . The patient had underlying congenital heart disease and incomplete immunization . He presented with fever, epistaxis and congestive heart failure . He received high-dose penicillin therapy and diphtheria antitoxin with clinical improvement . While he was receiving a high dose of penicillin for 1 month he developed a generalized tonic clonic seizure . A computerized tomogram revealed intracerebral and ventricular hemorrhage . Craniotomy with blood clot removal and ventriculostomy drainage were done . He died 2 days later from brain death and cardiovascular failure.

J Steroid Biochem Mol Biol, 2003 Dec, 87(4-5), 327 - 36
Production of malodorous steroids from androsta-5,16-dienes and androsta-4,16-dienes by Corynebacteria and other human axillary bacteria; Decreau RA et al.; The biotransformations of a number of steroids, chiefly 5,6,16,17-tetradehydro-androstanes, are reported . The strains investigated were Corynebacteria sp . G38, G40, G41, B, Brevis sp . CW5 and Micrococcus sp . M-DH2 . Corynebacterium sp . G41 proved remarkably efficient in effecting oxidative isomerisation of 5-ene-3-sterols into the corresponding 4-en-3-ones . The main biochemical reactions involved were oxidation at C-3; no reduction processes were observed . Conversions of 3beta-sterols into the C-3 oxo-steroids were high, but were correspondingly low for the 3alpha-sterol epimers . Androsta-4,16-dien-3-one and 5beta-androsta-16-en-3-one are crucial to the formation of malodour . The rate of formation of these compounds was measured over 72 h incubation periods using three substrates: androsta-5,16-dien-3beta-ol, androsta-4,16-dien-3beta-ol and androsta-5,16-dien-3-one . Induction studies of the transformation of the androsta-5,16-dien-3beta-ol into the very odorous compound androsta-4,16-dien-3-one showed that cells incubated with a mixture of antibiotics displayed the same extent of biotransformation as normal cells if the concentration of antibiotic was low (1, 3, 5 and 7 microg/ml), although at concentrations higher than 10 microg/ml, biotransformation yields were reduced . Pre-incubation with a 3beta-fluoro-steroid inhibited the formation of the odorous androsta-4,16-dien-3-one.

Proc Natl Acad Sci U S A, 2004 Jan 6, 101(1), 314 - 9 Epub 2003 Dec 26.
A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms; Portevin D et al.; Mycolic acids are major and specific constituents of the cell envelope of Corynebacterineae, a suborder of bacterial species including several important human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, or Corynebacterium diphtheriae . These long-chain fatty acids are involved in the unusual architecture and impermeability of the cell envelope of these bacteria . The condensase, the enzyme responsible for the final condensation step in mycolic acid biosynthesis, has remained an enigma for decades . By in silico analysis of various mycobacterial genomes, we identified a candidate enzyme, Pks13, that contains the four catalytic domains required for the condensation reaction . Orthologs of this enzyme were found in other Corynebacterineae species . A Corynebacterium glutamicum strain with a deletion in the pks13 gene was shown to be deficient in mycolic acid production whereas it was able to produce the fatty acids precursors . This mutant strain displayed an altered cell envelope structure . We showed that the pks13 gene was essential for the survival of Mycobacterium smegmatis . A conditional M . smegmatis mutant carrying its only copy of pks13 on a thermosensitive plasmid exhibited mycolic acid biosynthesis defect if grown at nonpermissive temperature . These results indicate that Pks13 is the condensase, a promising target for the development of new antimicrobial drugs against Corynebacterineae.

Pediatr Allergy Immunol, 2003 Dec, 14(6), 487 - 9
Hyper-IgM syndrome with CHARGE association; Bahillo P et al.; A girl with coloboma of the iris, sensorineural deafness, growth delay, distinctive face, and cranial nerve dysfunction was diagnosed of CHARGE association in the first year of life . She presented with repeated otitis . At 3 yr of age, the patient suffered a septicemia (Streptococcus pneumoniae, Corynebacterium sp.) . The immunoglobulin G (IgG) and IgA serum levels were decreased, IgM increased and cellular immunity parameters were normal, supporting the diagnosis of hyper-IgM (HIM) syndrome . The sequence of CD40 ligand and cytidine deaminase genes were normal . From then on, she was receiving immunoglobulin intravenously with an excellent outcome . Here, we report the first case of CHARGE association and HIM syndrome in the same patient . Although the cause could not be identified, a non-random link is likely.

Rapid Commun Mass Spectrom, 2003, 17(24), 2721 - 31
Dynamic calibration and dissolved gas analysis using membrane inlet mass spectrometry for the quantification of cell respiration; Yang TH et al.; A membrane inlet mass spectrometer connected to a miniaturized reactor was applied for dynamic dissolved gas analysis . Cell samples were taken from 7 mL shake flask cultures of Corynebacterium glutamicum ATCC 13032, and transferred to the 12 mL miniaturized reactor . There, oxygen uptake and carbon dioxide and its mass isotopomer production rates were determined using a new experimental procedure and applying nonlinear model equations . A novel dynamic method for the calibration of the membrane inlet mass spectrometer using first-order dynamics was developed . To derive total dissolved concentration of all carbon dioxide species (C(T)) from dissolved carbon dioxide concentration ({CO(2)}(aq)), the ratio of C(T) to {CO(2)}(aq) was determined by nonlinear parameter estimation, whereas the mass transfer coefficient of CO(2) was determined by the Wilke-Chang correlation . Subsequently, the suitability of the model equations for respiration measurements was examined using residual analysis and the Jarque-Bera hypothesis test . The resulting residuals were found to be random with normal distribution, which proved the adequacy of the application of the model for cell respiration analysis . Hence, dynamic changes in respiration activities could be accurately analyzed using membrane inlet mass spectrometry with the novel calibration method .

Vaccine, 2004 Jan 2, 22(3-4), 485 - 92
Protective immunity induced in mice by F0 and FII antigens purified from Paracoccidioides brasiliensis; Diniz SN et al.; Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity represents the principal mode of protection against this fungal infection . We investigated whether immunization with P . brasiliensis antigens fractionated by anionic chromatography on fast protein liquid chromatography (FPLC) could elicit protective immunity . BALB/c mice were immunized by subcutaneous injection of either 10 microg fractions 0 (F0), II (FII) or III (FIII) in the presence of 100 microg of Corynebacterium parvum and 1 mg of Al(OH)(3) and challenged with pathogenic P . brasiliensis strain . Mice immunized with F0 presented cellular and humoral immune responses with significant production of IFN-gamma, and high levels of IgG2a and IgG3 isotypes . Immunization with FII induced significant production of IFN-gamma and IL-10 associated with high levels of IgG1 and IgG2a . It was demonstrated that immunization with F0 or FII promoted significant decrease of organ colony-forming units (CFUs) in the lung after challenge infection without fungi dissemination to the spleen or liver . In contrast, FIII immunized mice develop a progressive disseminated disease to spleen and liver presented significant levels of INF-gamma, IL-10 or TGF-beta associated with high production of IgG1 and IgG2a with low production of IgG2b and IgG3 after challenge infection . Taken together, these findings suggest that antigens of F0 and FII are reliable vaccine candidates against the paracoccidioidomycosis.

Reprod Biol, 2002 Mar, 2(1), 73 - 85
Involvement of nitric oxide in inflammation of ovaries in gilts; Jana B et al.; NADPH-diaphorase (NADPH-d) and an inducible type of nitric oxide synthase (iNOS) were demonstrated in porcine ovaries after unilateral infusion of bacteria into the hilus of an ovary . In group I one ml of saline was infused into the hilus of each ovary from the 15th day to the 19th day of the estrous cycle . In group II one ml of bacterial suspension (10(9) colony forming units of Escherichia coli, Staphylococcus aureus and Corynebacterium pyogenes, in a proportion 1:1:1, respectively) in saline was infused into the hilus of one ovary on days corresponding to those of the control group (gr . I), whereas saline was infused into the contralateral ovary . The ovaries were collected on the 7th day of the next estrous cycle . In the bacteria-treated ovary, the activity of NADPH-d was higher in the endothelium of blood vessels, corpora lutea and follicular walls in comparison to that observed in the respective structures of the contralateral ovary . The highest activity of NADPH-d was found in the vascular endothelium in the bacteria-infused ovary . Vascular smooth muscle cells found in both ovaries of the bacteria-treated gilts were more intensely stained for NADPH-d than those in control animals . After bacteria administration, the intensity of NADPH-d reaction in all the structures of both ovaries in group II was higher than in control group . The strongest immunostaining for iNOS was observed in all structures of the bacteria-infused ovary . In the contralateral ovary, iNOS-immunoreactivity was weaker but still stronger than that in control group . The present results revealed that infusions of bacteria into the hilus of one ovary enhanced the activity of NADPH-d and immunoreactivity for iNOS in both porcine ovaries . However, the activity of both enzymes was higher in the bacteria-infused ovary than in the contralateral one . These data suggest that locally synthesized NO can mediate an inflammatory effect of bacteria in the porcine ovaries.

J Clin Microbiol, 2003 Dec, 41(12), 5500 - 10
Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients; Domann E et al.; Renal transplant recipients are predisposed to urinary tract infections caused by both common uropathogens and opportunistic bacteria resulting frequently in significant polymicrobial infections . In this study, a culture-independent 16S rRNA-based approach was established to identify unusual, fastidious, or anaerobic bacteria and to investigate bacterial diversity in urinary tract specimens . Similarly sized amplicons encompassing the V6 to V8 region of the 16S rRNA were analyzed with denaturing high-performance liquid chromatography (DHPLC) (WAVE System) . Artificial mixtures of single amplicons from commonly encountered uropathogenic bacteria produced distinct peak profiles whose identities were confirmed by sequencing individually collected peak products . We evaluated the application of the method on 109 urinary tract specimens from renal transplant recipients; 100% correlation was found for culture-positive specimens, and DHPLC generated peak profiles . However, for culture-negative specimens, DHPLC facilitated the detection of novel peak profiles . DNA sequencing of these individual peaks was used to identify the bacteria involved . Thus, in PCR-positive but culture-negative samples the method allowed detection of previously known uropathogens such as Corynebacterium urealyticum and Gardnerella vaginalis, but also unusual agents including Anaerococcus lactolyticus, Bacteroides vulgatus, Dialister invisus, Fusobacterium nucleatum, Lactobacillus iners, Leptotrichia amnionii, Prevotella buccalis, Prevotella ruminicola, Rahnella aquatilis, and Streptococcus intermedius were detected as single pathogens or as constituents of polymicrobial infections . The method described is reproducible and rapidly and enables both DHPLC-based profiling and sequence-based investigation of microbial communities and polymicrobial infections . A detailed understanding of infections found in recipients of renal transplants will guide antibiotic therapy regimens and provide new perspectives for decreasing the risk of graft rejection.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 2027 - 31
Corynebacterium suicordis sp . nov., from pigs; Vela AI et al.; Nineteen strains of Gram-positive, non-motile, non-spore-forming, catalase-positive, rod-shaped bacteria isolated from pigs were characterized by using biochemical, molecular chemical and molecular genetic methods . Two distinct groups of organisms were discerned, based on their colonial morphology, CAMP (Christie-Atkins-Munch-Petersen) reaction and numerical profile by using the API Coryne system . The first group (13 strains) gave a doubtful discrimination between Corynebacterium striatum and Corynebacterium amycolatum, whilst the second group (six strains) were identified tentatively as Corynebacterium urealyticum . Comparative 16S rRNA gene sequencing studies demonstrated that all of the isolates belonged phylogenetically to the genus CORYNEBACTERIUM: The first group of organisms was highly similar to Corynebacterium testudinoris with respect to 16S rRNA gene sequences and physiological characteristics, whereas the remaining six isolates formed a hitherto unknown subline within the genus, associated with a small subcluster of species that included Corynebacterium auriscanis and its close relatives . The unknown Corynebacterium sp . was distinguished readily from these and other species of the genus by biochemical tests . Based on both phenotypic and phylogenetic evidence, it is proposed that the new isolates from pigs should be classified as a novel species, Corynebacterium suicordis sp . nov . The type strain is P81/02(T) (=CECT 5724(T)=CCUG 46963(T)).

Hunan Yi Ke Da Xue Xue Bao, 2003 Jun, 28(3), 269 - 71
{Examination of urogenital tract microorganism infection and antibiotic susceptibility test}; Li WW et al.; OBJECTIVE: To isolate bacteria, mycoplasma and chlamydia from the urogenital tract, and to determine their antibiotic susceptibility . METHODS: Bacteria, mycoplasma and chlamydia were isolated from the urogenital tract secretion by artifical culture, and their antibiotic susceptibility was detected by disk diffusion . RESULTS: The common microorganisms were S . epidermidis and corynebacberium, and the minority microorganisms were G- bacteria or E . coli . Bacteria were susceptible to amikacin, cephazolin V, rifampin, gentamycin, and docycyclin . CONCLUSION: S . epidermidis and corynebacterium are important pathogens of the urogenital tract infection . Disk susceptibility test can be used to screen the susceptible antibiotic.

J Biotechnol, 2003 Dec 19, 106(2-3), 269 - 86
Development of a Corynebacterium glutamicum DNA microarray and validation by genome-wide expression profiling during growth with propionate as carbon source; Huser AT et al.; A DNA microarray was developed to analyse global gene expression of the amino acid-producing bacterium Corynebacterium glutamicum . PCR products representing 93.4% of the predicted C . glutamicum genes were prepared and spotted in quadruplicate onto 3-aminopropyltrimethoxysilane-coated glass slides . The applicability of the C . glutamicum DNA microarray was demonstrated by co-hybridisation with fluorescently labelled cDNA probes . Analysis of the technical variance revealed that C . glutamicum genes detected with different intensities resulting in ratios greater than 1.52 or smaller than -1.52 can be regarded as differentially expressed with a confidence level of greater than 95% . In a validation example, we measured changes of the mRNA levels during growth of C . glutamicum with acetate and propionate as carbon sources . Acetate-grown C . glutamicum cultures were used as reference . At the 95% confidence interval, 117 genes revealed increased transcript levels in the presence of propionate, while 43 genes showed a decreased expression compared with the acetate-grown culture . Global expression profiling confirmed the induction of the prpD2B2C2 gene cluster already known to be essential for propionate degradation via the 2-methylcitrate cycle . Besides many genes of unknown function, the paralogous prpD1B1C1 gene cluster as well as fasI-B (encoding fatty-acid synthase IB), dtsR1 and dtsR2 (components of acyl-CoA carboxylases), gluABCD (glutamate transport system), putP (proline transport system), and pyc (pyruvate carboxylase) showed significantly increased expression levels . Differential expression of these genes was confirmed by real-time reverse transcription (RT) PCR assays.

Biosci Biotechnol Biochem, 2003 Nov, 67(11), 2416 - 24
A Corynebacterium glutamicum rnhA recG double mutant showing lysozyme-sensitivity, temperature-sensitive growth, and UV-sensitivity; Hirasawa T et al.; Corynebacterium glutamicum mutant KY9707 was originally isolated for lysozyme-sensitivity, and showed temperature-sensitive growth . Two DNA fragments from a wild-type C . glutamicum chromosomal library suppressed the temperature-sensitivity of KY9707 . These clones also rescued the lysozyme-sensitivity of KY9707, although partially . One of them encodes a protein of 382 amino acid residues, the N-terminal domain of which was homologous to RNase HI . This gene suppressed the temperature-sensitive growth of an Escherichia coli rnhA rnhB double mutant . We concluded that this gene encodes a functional RNase HI of C . glutamicum and designated it as rnhA . The other gene encodes a protein of 707 amino acid residues highly homologous to RecG protein . The C . glutamicum recG gene complemented the UV-sensitivity of E . coli recG258::kan mutant . KY9707 showed increased UV-sensitivity, which was partially rescued by either the recG or rnhA gene of C . gluamicum . Point mutations were found in both recG and rnhA genes in KY9707 . These suggest that temperature-sensitive growth, UV-sensitivity, and probably lysozyme-sensitivity also, of KY9707 were caused by mutations in the genes encoding RNase HI and RecG.

Eur Spine J, 2004 Mar, 13(2), 152 - 6 Epub 2003 Nov 28.
Frozen cancellous bone allografts: positive cultures of implanted grafts in posterior fusions of the spine; Barriga A et al.; We have carried out a study on the behaviour pattern of implanted allografts initially stored in perfect conditions (aseptically processed, culture-negative and stored at -80 degrees C) but which presented positive cultures at the implantation stage . There is no information available on how to deal with this type of situation, so our aim was to set guidelines on the course of action which would be required in such a case . This was a retrospective study of 112 patients who underwent a spinal arthrodesis and in whom a total of 189 allograft pieces were used . All previous bone and blood cultures and tests for hepatitis B and C, syphilis and HIV (via PCR techniques) were negative . The allografts were stored by freezing them at -80 degrees C . A sample of the allograft was taken for culture in the operating theatre just before its implantation in all cases . The results of the cultures were obtained 3-5 days after the operation . There were 22 allografts with positive culture results (12%) after implantation . These allografts were implanted in 16 patients (14%) . Cultures were positive for staphylococci coagulase negative (ECN) in 10 grafts (46%), Pseudomonas stutzeri in two grafts (9%), Corynebacterium jeikeium in two grafts (9%), staphylococci coagulase positive in two grafts (9%) and for each of the following organisms in one case each (4%): Corynebacterium spp., Actinomyces odontolyticus, Streptococcus mitis, Peptostreptococcus spp., Rhodococcus equi and Bacillus spp . No clinical infection was seen in any of these patients . Positive cultures could be caused by non-detected contamination at harvesting, storing or during manipulation before implantation . The lack of clinical signs of infection during the follow-up of our patients may indicate that no specific treatment different from our antibiotic protocol is required in the case of positive culture results of a graft piece after implantation.

J Biol Chem, 2004 Mar 19, 279(12), 11937 - 47 Epub 2003 Nov 26.
The crystal structures of the ferric and ferrous forms of the heme complex of HmuO, a heme oxygenase of Corynebacterium diphtheriae; Hirotsu S et al.; Crystal structures of the ferric and ferrous heme complexes of HmuO, a 24-kDa heme oxygenase of Corynebacterium diphtheriae, have been refined to 1.4 and 1.5 A resolution, respectively . The HmuO structures show that the heme group is closely sandwiched between the proximal and distal helices . The imidazole group of His-20 is the proximal heme ligand, which closely eclipses the beta- and delta-meso axis of the porphyrin ring . A long range hydrogen bonding network is present, connecting the iron-bound water ligand to the solvent water molecule . This enables proton transfer from the solvent to the catalytic site, where the oxygen activation occurs . In comparison to the ferric complex, the proximal and distal helices move closer to the heme plane in the ferrous complex . Together with the kinked distal helix, this movement leaves only the alpha-meso carbon atom accessible to the iron-bound dioxygen . The heme pocket architecture is responsible for stabilization of the ferric hydroperoxo-active intermediate by preventing premature heterolytic O-O bond cleavage . This allows the enzyme to oxygenate selectively at the alpha-meso carbon in HmuO catalysis.

Arch Esp Urol, 2003 Oct, 56(8), 944 - 6
{Encrusted pyelitis in a transplanted kidney}; Alapont Alacreu JM et al.; OBJECTIVES: To emphasize the importance of early diagnosis of encrusted pyelitis in kidney transplant patients . METHODS: We report one case of encrusted pyelitis in a 10-year-old girl with a kidney graft who was treated by means of nephrostomy tube irrigation with an acidifier liquid substance . RESULTS: After 16 days of treatment there was a significant decrease of the size of the calcified pyelic plaque, keeping a good renal function afterwards . CONCLUSIONS: This disease should be thought of in every case of kidney transplant patient with negative urine cultures and alkaline pH, and the microbiologist should be alerted of the possibility of urinary tract infection by Corynebacterium.

FEMS Microbiol Rev, 2003 Dec, 27(5), 617 - 28
Ammonium assimilation and nitrogen control in Corynebacterium glutamicum and its relatives: an example for new regulatory mechanisms in actinomycetes; Burkovski A; Nitrogen is an essential component of nearly all complex macromolecules in a bacterial cell, such as proteins, nucleic acids and cell wall components . Accordingly, most prokaryotes have developed elaborate control mechanisms to provide an optimal supply of nitrogen for cellular metabolism and to cope with situations of nitrogen limitation . In this review, recent advances in our knowledge of ammonium uptake, its assimilation, and related regulatory systems in Corynebacterium glutamicum, a Gram-positive soil bacterium used for the industrial production of amino acids, are summarized and discussed with respect to the situation in the bacterial model organisms, Escherichia coli and Bacillus subtilis, and in comparison to the situation in other actinomycetes, namely in mycobacteria and streptomycetes . The regulatory network of nitrogen control in C . glutamicum seems to be a patchwork of different elements . It includes proteins similar to the UTase/GlnK pathway of E . coli and expression regulation by a repressor protein as in B . subtilis, but it lacks an NtrB/NtrC two-component signal transduction system . Furthermore, the C . glutamicum regulation network has unique features, such as a new sensing mechanism . Based on its extremely well-investigated central metabolism, well-established molecular biology tools, a public genome sequence and a newly-established proteome project, C . glutamicum seems to be a suitable model organism for other corynebacteria, such as Corynebacterium diphtheriae and Corynebacterium efficiens.

J Steroid Biochem Mol Biol, 2003 Oct, 87(1), 105 - 10
Microbial pathways leading to steroidal malodour in the axilla; Austin C et al.; Odorous steroids, specifically the 16-androstenes, 5alpha-androstenol and 5alpha-androstenone, are widely accepted as being contributors to underarm odour, but the precursors and pathways to these odorous steroids were unclear . This study demonstrated that the axillary microflora could only generate odorous 16-androstenes from precursors that already contain the C16 double bond, such as 5,16-androstadien-3-ol and 4,16-androstadien-3-one . In incubations containing 5,16-androstadien-3-ol, mixed populations of Corynebacterium spp., isolated from the axilla, could generate many different 16-androstene metabolites, several of which were odorous . Isolation of individual Corynebacterium strains, followed by pure culture incubations with 5,16-androstadien-3-ol, revealed organisms capable of efficient, rapid reactions . However, no single isolate could carry out a full complement of the observed biotransformations . 16-Androstene metabolites were identified by gas chromatography-mass spectrometry (GC-MS), either by comparison with known standards, or by prediction from molecular ion and fragmentation patterns . Based on detection of these metabolites, a metabolic map for axillary corynebacterial 16-androstene biotransformations was proposed, detailing potential enzyme activities . In summary, the formerly implicated 4,16-androstadien-3-one, 5alpha-androstenone and 5alpha-androstenol were detected, along with previously unreported hydroxy- and keto-substituted 16-androstenes, 16-androstatrienones and 16-androstatrienols . Additionally, many other metabolites with steroidal fragmentation patterns were present, but have remained unidentified.A key observation was that very low prevalences of microorganisms capable of biotransforming 16-androstenes were present on skin . For example, from a panel of 21 individuals, only 4 of 18 mixed populations of corynebacteria, and only 4 of 45 Corynebacterium isolates, could biotransform 5,16-androstadien-3-ol.This study has increased understanding of the metabolic pathways involved in steroidal malodour formation, and has demonstrated that the biotransformations are more complex than previously anticipated . However, it is clear that further research is required, both to assess the level of contribution of 16-androstenes to underarm odour, and to further elucidate the pathways and odour molecules formed by corynebacteria.

Mol Microbiol, 2003 Nov, 50(4), 1429 - 38
Assembly of pili on the surface of Corynebacterium diphtheriae; Ton-That H et al.; Pili of Gram-negative pathogens are formed from pilin precursor molecules by non-covalent association within the outer membrane envelope . Gram-positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, however, an assembly pathway for pili has not yet been revealed . We show here that pili of Corynebacterium diphtheriae are composed of three pilin subunits, SpaA, SpaB and SpaC . SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals and SpaC seems positioned at the pilus tip . Assembled pili are released from the bacterial surface by treatment with murein hydrolase, suggesting that the pilus fibres may be anchored to the cell wall envelope . All three pilin subunit proteins are synthesized as precursors carrying N-terminal signal peptides and C-terminal sorting signals . Some, but not all, of the six sortase genes encoded in the genome of C . diphtheriae are required for precursor processing, pilus assembly or cell wall envelope attachment . Pilus assembly is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits . This covalent tethering of adjacent pilin subunits appears to have evolved in many Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs.

Mol Microbiol, 2003 Nov, 50(4), 1295 - 308
Identification of an anion-specific channel in the cell wall of the Gram-positive bacterium Corynebacterium glutamicum; Costa-Riu N et al.; A cation-selective channel (porin), designated PorA, facilitates the passage of hydrophilic solutes across the cell wall of the mycolic acid-containing actinomycete Corynebacterium glutamicum . Biochemical and electrophysiological investigations of the cell wall of the mutant strain revealed the presence of an alternative channel-forming protein . This porin was purified to homogeneity and studied in lipid bilayer membranes . It forms small anion-selective channels with a diameter of about 1.4 nm and an average single-channel conductance of about 700 pS in 1 M KCl . The PorBCglut channel could be blocked by citrate in a dose-dependent manner . This result was in agreement with growth experiments in citrate as sole carbon source where growth in citrate was impaired as compared with growth in other carbon sources . The PorBCglut protein was partially sequenced and based on the resulting amino acid sequence of the corresponding gene, which was designated as porB, was identified as an unannotated 381 bp long open reading frame (ORF) in the published genome sequence of C . glutamicum ATCC13032 . PorBCglut contains 126 amino acids with an N-terminal extension of 27 amino acids . One hundred and thirty-eight base pairs downstream of porB, we found an ORF that codes for a protein with about 30% identity to PorBCglut, which was named PorCCglut . The arrangement of porB and porC on the chromosome suggested that both genes belong to the same cluster . RT-PCR from overlapping regions between genes from wild-type C . glutamicum ATCC 13032 and its ATCC 13032DeltaporA mutant demonstrated that this is the case and that porB and porC are cotranscribed . The gene products PorBCglut and PorCCglut represent obviously other permeability pathways for the transport of hydrophilic compounds through the cell wall of C . glutamicum.

P R Health Sci J, 2003 Sep, 22(3), 291 - 7
Bioassay screening of Amazonian plants; Guerrero RO et al.; OBJECTIVE: The purpose of this study was to evaluate several biological activities of thirty plant extracts collected in the North West Amazon (Ecuador) . Some of these plants are being used for their reputed medicinal properties by the natives of this region . METHODS: Five in vitro bioassays were used to screen the plant material . 1 . The brine shrimp lethality examination (BSLT) in microplate is a general test that seems capable of detecting a broad spectrum of bioactivity present in crude plant extracts . 2 . Free radical scavenging properties were studied in a colorimetric assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH) . 3 . The beta-glucosidase inhibition test is thought to be a method for the evaluation of anti-AIDS, anti-diabetic or anti-obesity compounds . 4 . The xanthine oxidase inhibition assay is used to identify potential anti-gout agents . 5 . The antibacterial activity that is being used to isolate and identify antibiotic drugs . RESULTS: In the BSLT, we found that Piscidia carthagenensis demonstrated very good activity with a LC50: 21.81 micrograms/mL . It is considered that plant extracts with low LC50 values may contain metabolites with cytotoxic, antifungal, insecticidal or pesticide activities . In the antioxidant activity bioassay, several plant extracts were confirmed to have excellent free radical scavenging properties . Rhus juglandifolia and Clusia venusta leaves exhibited an ED50: 3.12 micrograms/mL and 3.61 micrograms/mL, respectively . Piper reticulatum (84%), Inga heteroptera (77%), Clusia venusta (70.9%), and Rhus juglandifolia (70.5%) showed fairly good inhibition activity for beta-glucosidase . On the other hand, none of the plant extracts was capable of inhibiting xanthine oxidase . Finally, the Gram-positive microorganisms Staphylococcus aureus and Corynebacterium diphteriae were found to be sensitive to the majority of the plant extracts, whereas the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Salmonella typhi were proved to be resistant toward the plant extracts . CONCLUSIONS: It is important to continue investigating our plant kingdom, especially the world tropical reserves as an alternative for finding new or better drugs . It should be essential to follow-up this type of investigation to isolate and elucidate the active principles of the bio-positive plants.

J Bacteriol, 2003 Dec, 185(23), 6826 - 40
Analysis of the Corynebacterium diphtheriae DtxR regulon: identification of a putative siderophore synthesis and transport system that is similar to the Yersinia high-pathogenicity island-encoded yersiniabactin synthesis and uptake system; Kunkle CA et al.; The diphtheria toxin repressor, DtxR, is a global iron-dependent regulatory protein in Corynebacterium diphtheriae that controls gene expression by binding to 19-bp operator sequences . To further define the DtxR regulon in C . diphtheriae, a DtxR repressor titration assay (DRTA) was developed and used to identify 10 previously unknown DtxR binding sites . Open reading frames downstream from seven of the newly identified DtxR binding sites are predicted to encode proteins associated with iron or heme transport . Electrophoretic mobility shift assays indicated that DtxR was able to bind to DNA fragments carrying the 19-bp operator regions, and transcriptional analysis of putative promoter elements adjacent to the binding site sequences revealed that most of these regions displayed iron- and DtxR-regulated activity . A putative siderophore biosynthesis and transport operon located downstream from one of the DtxR binding sites, designated sid, is similar to the yersiniabactin synthesis and uptake genes encoded on the Yersinia pestis high pathogenicity island . The siderophore biosynthetic genes in the sid operon contained a large deletion in the C . diphtheriae C7 strain, but the sid genes were unaffected in four clinical isolates that are representative of the dominant strains from the recent diphtheria epidemic in the former Soviet Union . Mutations in the siderophore biosynthetic genes in a clinical strain had no effect on siderophore synthesis or growth in low-iron conditions; however, a mutation in one of the putative transport proteins, cdtP, resulted in reduced growth in iron-depleted media, which suggests that this system may have a role in iron uptake . The findings from this study indicate that C . diphtheriae contains at least 18 DtxR binding sites and that DtxR may affect the expression of as many as 40 genes.

J Pediatr Surg, 2003 Nov, 38(11), E16 - 7
Hyperammonemia with complex urinary tract anomaly: a case report; Gabra HO et al.; Hyperammonemia has been reported rarely in the pediatric age group in systemically ill patients . All cases resulted from infections with urea splitting organisms, which are more common among patients who have undergone surgical procedures on the urinary tract . The authors report for the first time in the pediatric literature, one patient who presented with hyperammonemic encephalopathy that resulted from urinary tract infection with Staphylococcus epidermidis and Corynebacterium sp.

Vojnosanit Pregl, 2003 Sep-Oct, 60(5), 613 - 20
{Chylous effusions}; Tomic I et al.; This paper presents 4 patients with chylothorax, and one patient with bilateral chylothorax and chyloperitoneum . The chylous effusions were of benign etiology, developed as a complication of miliary tuberculosis (1 patient), after L-2 vertebral body fracture (1 patient), and idiopathic (2 patients) . The diagnosis was confirmed by the presence of chylomicrons and high content of triglycerides in the effusion, ranged 11.9-29.1 mmol/l . Lymphangiography showed multiple abnormalities of lymphatic system, the obstruction of ductus thoracicus, dilatation and convulsion of lymphatic channels, but the site of lymphatic leak was not detected . The treatment included an extended period of pleural and peritoneal drainage with total parenteral nutrition (1 patient), pleurodesis using Corynebacterium parvum (2 patients), and surgical partial parietal pleurectomy with continuous drainage (1 patient) . The treatment was successful in all patients.

J Biochem (Tokyo), 2003 Oct, 134(4), 599 - 606
Mutagenesis of the dimer interface region of Corynebacterium callunae starch phosphorylase perturbs the phosphate-dependent conformational relay that enhances oligomeric stability of the enzyme; Nidetzky B et al.; We have used alanine-scanning site-directed mutagenesis of the dimer contact region of starch phosphorylase from Corynebacterium callunae to explore the relationship between a protein conformational change induced by phosphate binding and the up to 500-fold kinetic stabilization of the functional quarternary structure of this enzyme when phosphate is present . Purified mutants (at positions Ser-224, Arg-226, Arg-234, and Arg-242) were characterized by Fourier transform-infrared (FT-IR) spectroscopy and enzyme activity measurements at room temperature and under conditions of thermal denaturation . Difference FT-IR spectra of wild type and mutants in (2)H(2)O solvent revealed small changes in residual amide II band intensities at approximately 1,550 cm(-1), indicating that (1)H/(2)H exchange in the wild type is clearly perturbed by the mutations . Decreased (1)H/(2)H exchange in comparison to wild type suggests formation of a more compact protein structure in S224A, R234A, and R242A mutants and correlates with rates of irreversible thermal denaturation at 45 degrees C that are up to 10-fold smaller for the three mutants than the wild type . By contrast, the mutant R226A inactivates 2.5-fold faster at 45 degrees C and shows a higher (1)H/(2)H exchange than the wild type . Phosphate (20 mM) causes a greater change in FT-IR spectra of the wild type than in those of S224A and 234A mutants and leads to a 5-fold higher stabilization of the wild type than the two mutants . Therefore, structural effects of phosphate binding leading to kinetic stability of wild-type starch phosphorylase are partially complemented in the S224A and R234A mutants . Infrared spectroscopic measurements were used to compare thermal denaturations of the mutants and the wild type in the absence and presence of stabilizing oxyanion . The broad denaturation transition of unliganded wild type in the range 40-50 degrees C is reduced in the S224A and R234A mutants, and this reflects mainly a shift of the onset of denaturation to a 4-5 degrees C higher value.

Ear Nose Throat J, 2003 Oct, 82(10), 798 - 800, 803-4, 806
Bacteriologic findings in patients with chronic sinusitis; Merino LA et al.; We studied the bacteriology of maxillary sinus aspirates obtained from patients diagnosed with chronic sinusitis . We recovered 659 strains from 510 aspirates; of these, 572 (86.8%) were aerobes and 87 (13.2%) were anaerobes . Aerobes only were recovered from 310 of the 510 specimens (60.8%) and anaerobes only from 31 (6.1%) . Among the 572 aerobic bacteria, the most prevalent organisms were Streptococcus viridans (158 strains {27.6%}), Streptococcus pneumoniae (67 {11.7%}), Corynebacterium species (66 {11.5%}), Staphylococcus aureus (54 {9.4%}), Moraxella catarrhalis (38 {6.6%}), Hemophilus parainfluenzae (33 {5.8%}), and group C beta-hemolytic streptococci (26 {4.5%}) . Among the 87 recovered anaerobes were species of Peptostreptococcus (32 strains {36.8%}), Prevotella (22 {25.3%}), Actinomyces (13 {14.9%}), Propionibacterium (11 {12.6%}), Fusobacterium (8 {9.2%}), and Veillonella (1 {1.1%}) . Beta-lactamase production was detected in 115 of the 572 aerobic strains (20.1%) and in 10 of the 87 anaerobic strains (11.5%) . We found that the prevalence and type of organisms we identified in chronic sinusitis did not differ substantially from those reported in previous studies . Our study is one of the more extensive reports on the type and prevalence of pathogens in chronic sinusitis that has been published to date.

Gene, 2003 Oct 23, 317(1-2), 149 - 55
The genome stability in Corynebacterium species due to lack of the recombinational repair system; Nakamura Y et al.; Corynebacterium species are members of gram-positive bacteria closely related to Mycobacterium species, both of which are classified into the same taxonomic order Actinomycetales . Recently, three corynebacteria, Corynebacterium efficiens, Corynebacterium glutamicum, and Corynebacterium diphtheriae have been sequenced independently . We found that the order of orthologous genes in these species has been highly conserved though it has been disrupted in Mycobacterium species . This synteny suggests that corynebacteria have rarely undergone extensive genome rearrangements and have maintained ancestral genome structures even after the divergence of corynebacteria and mycobacteria . This is the first report that the genome structures have been conserved in free-living bacteria such as C . efficiens and C . glutamicum, although it has been reported that obligate parasites such as Mycoplasma and Chlamydia have the stable genomes . The comparison of recombinational repair systems among the three corynebacteria and Mycobacterium tuberculosis suggested that the absence of recBCD genes in corynebacteria be responsible for the suppression of genome shuffling in the species . The genome stability in Corynebacterium species will give us hints of the speciation mechanism with the non-shuffled genome, particularly the importance of horizontal gene transfer and nucleotide substitution in the genome.

Nucleic Acids Res, 2003 Nov 15, 31(22), 6516 - 23
The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129; Cerdeno-Tarraga AM et al.; Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms . The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria . This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme . However, the disease has made a dramatic return in recent years, in particular within the Eastern European region . The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s . We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak . The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids . It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins . This is in contrast to Corynebacterium's nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition . The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.

Int J Antimicrob Agents, 2003 Nov, 22(5), 492 - 6
In vitro activity of newer antibiotics against Corynebacterium jeikeium, Corynebacterium amycolatum and Corynebacterium urealyticum; Sanchez Hernandez J et al.; The in vitro activity of ciprofloxacin, erythromycin, telithromycin, teicoplanin, linezolid and quinupristin-dalfopristin was tested against human derived pathogenic corynebacteria . The MICs of these antibiotics were measured using the agar dilution method against 31 strains of Corynebacterium jeikeium, 58 Corynebacterium amycolatum (including 33 multidrug-resistant strains) and 64 Corynebacterium urealyticum clinical strains . A high resistance rate to ciprofloxacin and erythromycin was found in the three species . Telithromycin was much more active than erythromycin (MIC(90) of erythromycin >or=128 mg/l for all three species; MIC(90) of telithromycin: 4 mg/l for C . jeikeium, 64 mg/l for C . amycolatum and 1 mg/l for C . urealyticum) . There were no teicoplanin-resistant (MIC(90) 1, 0.5 and 1 mg/l, respectively) or linezolid-resistant strains (MIC(90) 1, 0.2 and 0.5 mg/l, respectively) . Quinupristin-dalfopristin was active against most strains with an activity similar to linezolid, but three C . jeikeium and one C . amycolatum showed MICs >or=4 mg/l . Telithromycin showed much better activity against corynebacteria than older macrolides . Synercid and linezolid were active against most isolates tested, including multidrug resistant strains.

Epidemiol Infect, 2003 Oct, 131(2), 947 - 55
Corynebacterium pseudotuberculosis infection in Israeli dairy cattle; Yeruham I et al.; Two forms of Corynebacterium pseudotuberculosis infection in Israeli dairy cattle herds during a survey period of 13 years (1989-2001) are described . The more common form, which was diagnosed in 45 herds, was characterized by ulcerative granulomatous lesions which occurred either sporadically--in 26 herds (with a morbidity rate of up to 5%)--or in an epidemic course in 19 herds . Most (80.6%) of the affected animals were cows; the rest were first-calving cows (16.2%) and heifers (3.2%) . The morbidity occurred mostly during the summer months . The ulcerative granulomatous lesions appeared in three clinical forms: cutaneous, mastitic and visceral . Mixed forms were also observed . The morbidity rate was 6.4% and the culling rate reached 16.3% of the affected animals . Most of the strains of C . pseudotuberculosis which were isolated from the abscesses in the cutaneous form of the disease and from milk samples failed to reduce nitrate . A decrease in milk production (6%) and an increase in bulk-milk somatic cell count were noted . Necrotic and ulcerative dermatitis on the heel of the foot occurred in an epidemic course in heifers in only two herds during the winter months, with morbidity rates of 7.5 and 76.2%, respectively . C . pseudotuberculosis isolates from skin lesions and from the soil did reduce nitrate . Clinical, epizootiological and microbiological aspects of the infection are described.

Prikl Biokhim Mikrobiol, 2003 Sep-Oct, 39(5), 565 - 70
{Properties of 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate--an intermediate in the non-mevalonate isoprenoid biosynthesis}; Ostrovskii DN et al.; Extraction and purification from the biomass of Corynebacterium ammoniagenes of 2-C-methyl-D-erhythritol 2,4-cyclopyrophosphate (MEC) was associated with its spontaneous transformation into a number of derivatives (which was due to pyrophosphate bond lability and the formation of complexes with metals) . These derivatives included 1,2-cyclophospho-4-phosphate, 2,4-diphosphate, 2,3-cyclophosphate, 1,4-diphosphate, and 3,5-diphosphate (identified by 1H, 31P, and 13C NMR spectroscopy) and accounted for about 10% MEC . When added to a solution of DNA in the presence of the Fenton reagent, MEC prevented DNA decomposition . In addition, MEC slowed down the interaction of the reagent with tempol radicals, which indicates that complexation of ferrous ions by MEC attenuates their ability to catalyze the formation of hydroxyl radicals from hydrogen peroxide . In the presence of 0.23 mM MEC, the rate of respiration of rat liver mitochondria increased 1.8 times . At 0.1-1.0 mM, MEC activated in vitro proliferation of human Vgamma9 T-cells . It is suggested that MEC acts as an endogenous stabilizing agent for bacterial cells subjected to oxidative stress and as an immunomodulator for eukaryotic hosts.

Vet Immunol Immunopathol, 2003 Dec 15, 96(3-4), 129 - 39
Experimental Corynebacterium pseudotuberculosis primary infection in goats: kinetics of IgG and interferon-gamma production, IgG avidity and antigen recognition by Western blotting; Paule BJ et al.; Corynebacterium pseudotuberculosis is the cause of caseous lymphadenitis (CLA) in small ruminants, a chronic granulomatous disease that provokes significant zootechnics losses to ovine and goat breeders in northern Brazil . The present work was conducted to analyse aspects of humoral and cellular immune responses after experimental infection . Eight goats were infected intradermally with a single dose of virulent C . pseudotuberculosis strain and specific IgG, interferon-gamma (IFN-gamma) production as well as IgG avidity and antigens pattern recognition dynamics against an excreted-secreted antigen were recorded during 20 weeks . At the end of the follow-up, animals were slaughtered and necropsied . Although no animals showed apparent clinical signs of infection at the end of the trial, IFN-gamma response, even more so than the humoral response, differentiated animals into two groups of high or medium/low response . The time-course of IFN-gamma production presented a short-lived primary response on day 5 after infection of animals of both groups, and a strong and long lasting secondary response starting on day 16 after infection in the high response group . The indirect ELISA used was able to detect a positive antibody titre between 6 and 11 days after infection in the two groups . IgG avidity index oscillated initially between 15 and 45%, and showed approximately 5% units increment during the 20 follow-up weeks . With only one individual exception, the qualitative antigens pattern recognition showed on day 11 after infection remained constant through the experiment . IgG avidity is highly correlated with IgG production, but could not be related with specific immunodominant bands . Both humoral and cellular responses kinetics presented a similar pattern of activation/deactivation but necropsy results suggested that the IFN-gamma test would be a very specific marker of CLA status.

Rev Argent Microbiol, 2003 Jul-Sep, 35(3), 167 - 70
{Bacteremia in 2 hospitals in Rosario, Argentina}; Notario R et al.; Bacteremia is associated with high morbidity and mortality . Nosocomial bacteremia is associated with an attributable mortality of 35% . In order to characterize the etiological agent of bacteremia cases during two time periods, 6605 patients were studied as follows: 1) from 1980 to 1982 (n = 2401) and 2) from 1988 to 2000 (n = 4204) . A total of 596 cases of bacteremia were detected . The most frequent agents were Staphylococcus aureus, negative coagulase (CNS) Staphylococcus, Klebsiella pneumoniae and Escherichia coli . In the second period, an increase in the frequency of bacteremia caused by gram positive bacteria, particularly gram-positive cocci, was noted . Conversely, gram negative bacteria diminished significantly in the second period, specially enterobacterial species . Methicillin-resistant S . aureus and CNS were significantly more frequently isolated in the second period . Infrequent agents of endocarditis, such as Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Corynebacterium urealyticum, were isolated.

Clin Nephrol, 2003 Oct, 60(4), 270 - 4
Corynebacterium striatum peritoneal dialysis catheter exit site infection; Crabtree JH et al.; BACKGROUND: Regarded as normal flora of the human skin and mucus membranes, non-diphtheria corynebacteria are frequently dismissed as contaminants or harmless colonizers . Recently, the pathogenic potential of C . striatum has been realized in immunocompromised patients with indwelling medical devices and previous antibiotic exposure . OBJECTIVE: We report here the diagnosis, treatment and clinical outcome of a peritoneal dialysis patient with a C . striatum infection of the catheter exit site . The aim is to present important features to assist in identifying clinically significant infections and provide guidelines for treatment . RESULTS: An immunocompromised patient with previous antimicrobial exposure developed an acute dialysis catheter exit site infection . C . striatum was isolated in pure growth . After initial treatment failure with oral antibiotics and intensified wound care, a satisfactory outcome was ultimately achieved without relapse or loss of the catheter with a 1-month course of vancomycin, 1 g intravenously, administered at 5-day intervals . CONCLUSIONS: The virulent capacity of Corynebacterium species should not be underestimated, particularly in high-risk patients . The presence of clinical signs of infection with isolation of the organism in pure culture and the presence of Gram-positive rods on direct Gram stain, especially in association with a leukocyte reaction, supports a cause and effect relationship . Because corynebacteria may be multiresistant, susceptibility testing should be performed on clinically significant isolates . Initial antibiotic selection is influenced by the severity of the infection, however, current experience favors vancomycin in significant infections.

Med Dosw Mikrobiol, 2003, 55(2), 189 - 95
{The presence of bacteria and yeast like fungi in specimens from surgical patients.}; Krzeminska-Jaskowiak E et al.; The aim of this study was to determine the incidence of Candida spp . strains in specimens obtained from surgically treated patients as well as to analyze the accompanying bacterial flora, both aerobic and anaerobic . The material came from two groups of patients . In the first group consisting of patients operated for colon and rectum carcinoma, the samples included peritoneal fluid, colon or rectum bioptates, pus, blood, and wound swabs . In the other group, biopsy material and smears from post operation wounds were taken from patients who underwent a surgical treatment of larynx carcinoma . Altogether, 282 various clinical specimens from 165 patients were analysed, and 41 Candida spp . strains were isolated: 39 strains of C . albicans and 2 strains of C . tropicalis . In 20 out of 41 specimens infected with Candida spp . (48.8%) the co-infection with bacterial aerobic flora was found . In 10 cases (24.4%), the fungi were isolated together with aerobic and anaerobic bacterial flora, whereas in 2 specimens (4.9%) the anaerobes and Candida albicans were diagnosed . The remaining 9 samples showed only the presence of Candida spp . (21.9%) . From among aerobic bacterial flora Enterococcus spp . strains (n = 17) and Gram negative rods from Enterobacteriaceae family (n = 13) were the most frequently isolated . The bacterial strains of Streptococcus spp . (n = 5), Pseudomonas spp . (n = 3), Staphylococcus spp . and Corynebacterium spp . (2 strains, both) were identified more rarely . Bacteroides spp . were the most frequent members of bacterial anaerobic flora (n = 10) . Other isolated anaerobic bacteria were classified as Fusobacterium spp . or Peptostreptococcus spp . (1 strain each) . E . coli and Enterococcus spp . strains of aerobic bacterial flora were more frequently isolated together with Candida spp . CONCLUSIONS: (i) Mixed bacterial flora was found to predominate in the clinical material from the patients after surgery . (ii) Candida spp . were most frequently found together with aerobic bacterial flora.

Antimicrob Agents Chemother, 2003 Nov, 47(11), 3519 - 24
Tylosin resistance in Arcanobacterium pyogenes is encoded by an erm X determinant; Jost BH et al.; Arcanobacterium pyogenes, a commensal on the mucous membranes of many economically important animal species, is also a pathogen, causing abscesses of the skin, joints, and visceral organs as well as mastitis and abortion . In food animals, A . pyogenes is exposed to antimicrobial agents used for growth promotion, prophylaxis, and therapy, notably tylosin, a macrolide antibiotic used extensively for the prevention of liver abscessation in feedlot cattle in the United States . Of 48 A . pyogenes isolates, 11 (22.9%) exhibited inducible or constitutive resistance to tylosin (MIC of > or = 128 microg/ml) . These isolates also exhibited resistance to other macrolide and lincosamide antibiotics, suggesting a macrolide-lincosamide resistance phenotype . Of the 11 resistant isolates, genomic DNA from nine hybridized to an erm(X)-specific probe . Cloning and nucleotide sequencing of the A . pyogenes erm(X) gene indicated that it was >95% similar to erm(X) genes from Corynebacterium and Propionibacterium spp . Eight of the erm(X)-containing A . pyogenes isolates exhibited inducible tylosin resistance, which was consistent with the presence of a putative leader peptide upstream of the erm(X) open reading frame . For at least one A . pyogenes isolate, 98-4277-2, erm(X) was present on a plasmid, pAP2, and was associated with the insertion sequence IS6100 . pAP2 also carried genes encoding the repressor-regulated tetracycline efflux system determinant Tet 33 . The repA gene from pAP2 was nonfunctional in Escherichia coli and at least one A . pyogenes isolate, suggesting that there may be host-encoded factors required for replication of this plasmid.

Protein Expr Purif, 2003 Oct, 31(2), 298 - 304
Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase; Aich S et al.; The Corynebacterium glutamicum (C . glutamicum) phosphoenolpyruvate carboxykinase (PCK) gene (pckA) was cloned into an Escherichia coli expression vector with a glutathione S-transferase (GST) tag . This recombinant DNA can produce highly overexpressed tagged protein in soluble form . This is the first report of the production of C . glutamicum PCK overexpressed in E . coli . The GST-fused PCK was purified using the glutathione-Sepharose 4B affinity column and the GST tag was removed in one-step . This one-step, easy purification method would be very useful for future mutational and structural studies . The molecular mass of the purified protein is approximately 68 kDa as confirmed by mass spectrometry and it is a monomeric enzyme . Also, the enzyme assays revealed that C . glutamicum PCK has a GTP-specific activity and that its activity is maximal in the presence of both Mn2+ and Mg2+.

Arq Gastroenterol, 2003 Jan-Mar, 40(1), 16 - 9 Epub 2003 Oct 06.
{Megaesophagus microbiota and carcinogenesis}; Pajecki D et al.; BACKGROUND: The risk of development of spin cell carcinoma of the esophagus is 33 times higher in patients with chagasic achalasia . It is possible that the production of N-nitroso compounds in the esophageal lumen by of bacterial action in the stasis liquid that reduce nitrates from diet into nitrites may play a role in this process . AIM: To analyze qualitatively and quantitatively the microbiota in chagasic megaesophagus with special attention to bacteria capable of transforming nitto reduction . PATIENTS: Fifteen patients (six men and nine women) were prospectively studied, with ages varying from 28 to 73 years . Patients were divided into three sub-groups according to Rezende et al . classification of esophageal dilation (grade I, grade II and grade III) . METHOD: The sample collection was performed using a method specially developed to avoid contamination with microorganisms of the oral cavity and oropharynx, using a Levine catheter n 14 and a 7,5 oro-traqueal tube . RESULTS: Ninety three point three percent of the cultures were positive, with great bacterial variability and predominance of a variety of aerobic Gram-positive and anaerobic bacteria . The bacterial concentrations were generally more elevated in grade III in comparison to grade I and grade II . Among the microorganisms found, Staphylococcus sp, Corynebacterium sp, Peptostreptococcus sp e a Veillonella sp were those with the capability of nitrate reduction . CONCLUSION: It was concluded that patients with megaesophagus present some bacteria in the esophageal lumen that are able to reduce nitrates intro nitrites, an important step in the formation of N-nitroso compounds.

J Clin Microbiol, 2003 Oct, 41(10), 4848 - 51
Detection of differences in the nucleotide and amino acid sequences of diphtheria toxin from Corynebacterium diphtheriae and Corynebacterium ulcerans causing extrapharyngeal infections; Sing A et al.; While Corynebacterium ulcerans can mimic classical diphtheria, extrapharyngeal infections are extremely rare . Sequencing of the diphtheria toxin (DT)-encoding tox gene of two C . ulcerans isolates from extrapharyngeal infections revealed differences from C . diphtheriae DT sequences, mainly in the translocation and receptor-binding domains . C . ulcerans supernatants were much less potent than supernatant from C . diphtheriae . A C . ulcerans DT-specific PCR is described below.

Appl Environ Microbiol, 2003 Oct, 69(10), 5907 - 13
Effects of iron limitation on adherence and cell surface carbohydrates of Corynebacterium diphtheriae strains; Moreira Lde O et al.; Iron limitation may cause bacterial pathogens to grow more slowly; however, it may also stimulate these microorganisms to produce greater tissue damage, given that many virulence factors are controlled by the iron supply in the environment . The present study investigated the influence of low iron availability on the expression of proteins and surface sugar residues of two toxigenic strains of Corynebacterium diphtheriae subsp . mitis and evaluated their adherence to human group B erythrocytes and HEp-2 cells . A comparison was made between bacteria grown in (i) Trypticase soy broth (TSB), (ii) TSB treated with dipyridyl to deplete free iron, and (iii) TSB enriched with FeCl(3) . The effects of iron concentration on adhesive properties were different for strains 241 and CDC-E8392, of the sucrose-fermenting and non-sucrose-fermenting biotypes, respectively . Iron-limited conditions enhanced interaction of strain 241 with erythrocytes and HEp-2 cells . Inhibition assays suggested the involvement of nonfimbrial protein combination 67-72p on hemagglutination of diphtheria bacilli grown under iron-limited conditions . Conversely, iron limitation inhibited adherence to glass and expression of electron-dense material on the bacterial surface . Lectin binding assays demonstrated a reduction in the number of sialic acid residues and an increase in D-mannose and D-galactose residues on the surfaces of both strains . Thus, iron exerts a regulatory role on adhesive properties of diphtheria bacilli, and low iron availability modulates the expression of C . diphtheriae surface carbohydrate moieties . The significant changes in the degree of lectin binding specific for D-mannose, D-galactose and sialic acid residues may have an effect on binding of host cells . The expression of dissimilar microbial virulence determinants may be coordinately controlled by common regulatory systems . For C . diphtheriae, the present results imply regulation of adherence and slime production as part of a global response to iron-limited environmental conditions that includes derepression of genes for the synthesis of cytotoxin and siderophores and for transport of the Fe(III)-siderophore complexes.

Biotechnol Lett, 2003 Aug, 25(16), 1311 - 6
Isolation of transcription initiation signals from Corynebacterium ammoniagenes and comparison of their gene expression levels in C . ammoniagenes and Escherichia coli; Paik JE et al.; With the aim of isolating transcription initiation signals, random Sau3AI fragments of Corynebacterium ammoniagenes ATCC 6872 were cloned into the promoter-probe plasmid, pEKplCm, and screened for promoter activity by chloramphenicol resistance of transformed Escherichia coli cells . Representative 22 promoter clones were analysed in C . ammoniagenes by its nucleotide sequences and promoter activities were measured by chloramphenicol acetyltransferase (CAT) assay . Activities of CAT were between 0.04 U and 2.85 U and several strong promoters, such as IJ59 clone and IJ73 clone, were found . The E . coli tac promoter was a strong promoter in C . ammoniagenes . IJ59 clone was part of the ferredoxin 3 gene and the promoter region contained a putative UP element, less conserved sequence at the -35 region and perfectly conserved sequence at the -10 region . The IJ73 clone contained a strong promoter that produced CAT at more than 10% of total cellular proteins in C . ammoniagenes and is therefore useful for expressing various genes in this coryneform bacteria.

Trop Anim Health Prod, 2003 Aug, 35(4), 301 - 7
Evaluation of the microbiological status of milk and various structures in mammary glands from naturally infected dairy cows; Benites NR et al.; A knowledge of the microbiological status of milk and of the different structures in the mammary glands has great importance in elucidating the pathogenesis of mammary gland infections . The objective of this study was to evaluate the microbiological status of various structures in the mammary glands from naturally infected dairy cows following slaughter . A total of 94 samples of milk, 184 samples of mammary parenchyma, 168 samples of gland cisterns, and 168 samples of teat cisterns were collected for microbiological examination . Microorganisms were detected in 59.9% of all samples, 67.0% of the milk samples, 70.1% of the mammary parenchymas, 55.9% of the gland cisterns and 48.8% of the teat cistern samples . When all samples were considered, coagulase-negative Staphylococcus were the most prevalent (35.7%) followed by coagulase-positive Staphylococcus (12.2%), Corynebacterium bovis (2.4%), Prototheca sp . (1.9%), and Streptococcus dysgalactiae (1.5%) . There was a significantly higher occurrence of microorganisms in the milk and mammary parenchyma compared to the gland cisterns and teat cisterns.

Chemosphere, 2003 Dec, 53(8), 1033 - 7
Influence and persistence of phorate and carbofuran insecticides on microorganisms in rice field; Das AC et al.; An experiment was conducted in microplots (4 m x 4 m) with two insecticides, phorate and carbofuran at rates of 1.5 and 1.0 kga.i.ha(-1) respectively, to investigate its effect on the population and distribution of bacteria, actinomycetes and fungi as well as the persistence of the insecticidal residues in rhizosphere soils of rice (Oryza sativa L., variety IR-50) . Application of the insecticides stimulated the population of bacteria, actinomycetes and fungi in the rhizosphere soils, and the stimulation was more pronounced with phorate as compared to carbofuran . Both the insecticides did not have marked effect on the numbers of Streptomyces and Nocardia in the rhizosphere soils . However, the growth of Bacillus, Escherichia, Flavobacterium, Micromonospora, Penicillium, Aspergillus and Trichoderma with phorate and that of Bacillus, Corynebacterium, Flavobacterium, Aspergillus and Phytophthora with carbofuran were increased . On the other hand, the numbers of Staphylococcus, Micrococcus, Fusarium, Humicola and Rhizopus under phorate and Pseudomonas, Staphylococcus, Micrococcus, Klebsiella, Fusarium, Humicola and Rhizopus under carbofuran were inhibited . Both the insecticides persisted in the rhizosphere soil for a short period of time and the rate of dissipation of carbofuran was higher than that of phorate in the soil depicting the half-life (T1/2) 9.1 and 10.4 days, respectively.

Bioprocess Biosyst Eng, 2003 Mar, 25(5), 291 - 8 Epub 2003 Jan 17.
Effects of the changes in enzyme activities on metabolic flux redistribution around the 2-oxoglutarate branch in glutamate production by Corynebacterium glutamicum; Shimizu H et al.; An experimental method for metabolic control analysis (MCA) was applied to the investigation of a metabolic network of glutamate production by Corynebacterium glutamicum . A metabolic reaction (MR) model was constructed and used for flux distribution analysis (MFA) . The flux distribution at a key branch point, 2-oxoglutarate, was investigated in detail . Activities of isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH), and 2-oxoglutarate dehydrogenase complex (ODHC) around this the branch point were changed, using two genetically engineered strains (one with enhanced ICDH activity and the other with enhanced GDH activity) and by controlling environmental conditions (i.e . biotin-deficient conditions) . The mole flux distribution was determined by an MR model, and the effects of the changes in the enzyme activities on the mole flux distribution were compared . Even though both GDH and ICDH activities were enhanced, the mole flux distribution was not significantly changed . When the ODHC activity was attenuated, the flux through ODHC decreased, and glutamate production was markedly increased . The flux control coefficients of the above three enzymes for glutamate production were determined based on changes in enzyme activities and the mole flux distributions . It was found that the factor with greatest impact on glutamate production in the metabolic network was obtained by attenuation of ODHC activity.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Jul-Aug, (4), 11 - 7
{Role of persisting opportunistic bacteria in the pathogenesis of the gastric and duodenum ulcer}; Bondarenko VM et al.; Dynamic study of the bacterial microflora of 122 patients with gastric and duodenal ulcer was carried out . Microflora examination of the bioptic samples of mucosa, obtained from the ulcerous zone of the patients, revealed that an open ulcer is like an infected wound needing sanitation . In the focus of lesion microorganisms of 32 genera and species, including Helicobacter pylori, fungi of the genus Candida, representatives of the genera Streptococcus, Staphylococcus, Corynebacterium, Bacteroides, etc., were detected . Opportunistic microorganisms were isolated in associations (up to 8 different cultures), possessing cytotoxic, hemolytic, antilysozyme, lecithinase, caseinolytic and RNAase activities . To inhibit the microflora, chitosan was used; 82-85% of the cultures of different bacteria under study proved to be sensitive to it . The inclusion of chitosan into the complex therapy suppressed the persistence of H . pylori, ensured the sanitation of mucosa affected by opportunistic bacteria and accelerated ulcers cicatrization.

Int J Mol Med, 2003 Oct, 12(4), 657 - 61
Influence of stannous chloride on the adhesive properties of Corynebacterium diphtheriae strains; Silva De Souza SM et al.; Stannous ion, as a chloride salt, influenced on the survival and adhesive properties of two toxigenic Corynebacterium diphtheriae of the sucrose-fermenting (241 strain) and non-sucrose-fermenting (CDC-E8392 strain) biotypes . Differences in survival fractions suggested differences in susceptibility of strains to bactericidal effect of stannous chloride (SnCl2) . A number of 0.3% bacterial cells of 241 strain and 0.02% of CDC-E8392 strain survived after 220 micro l ml(-1) SnCl2 treatment . Results of polystyrene and spontaneous autoaggregation tests showed an increase in hydrophobicity of SnCl2 treated-bacteria . Spontaneous bacterial autoaggregation was induced in the presence of SnCl2 . Stannous chloride also induced adherence to glass and totally inhibited the haemagglutinating activity of the non-sucrose-fermenting CDC-E8392 strain (original titer 32) . Decrease in haemagglutination was dependent on SnCl2 concentration used . The presence of SnCl2 exerted differences in the expression of diphtheria bacilli surface carbohydrates possibly related with differences in degrees of haemagglutination and adherence to glass . Lectin-binding assays showed increase in the expression of cell surface receptors to the lectin Canavalia ensiformis (Con A) with affinity for mannose-like residues . The occurrence of cell filamentation suggests genotoxicity of SnCl2 to diphtheria bacilli . SnCl2 treatment was capable of modifying cell morphology, hydrophobins and adhesin expression, suggesting ability of C . diphtheriae to withstand oxidative stressing environment . Therefore, the SnCl2, widely used in nuclear medicine as reducing agent in the 99mTc-labelling process, may influence the outcome of bacterial infections.

J Clin Microbiol, 2003 Sep, 41(9), 4353 - 8
Corynebacterium nigricans sp . nov.: proposed name for a black-pigmented Corynebacterium species recovered from the human female urogenital tract; Shukla SK et al.; Six independent isolates of an unusual black-pigmented Corynebacterium species (strains CN-1, CN-2, CN-3415, W70124, 91-0032, and 92-0360) were recovered from the human female urogenital tract . Four of the six source patients had complications of pregnancy, including spontaneous abortion, preterm labor, and low amniotic fluid volume at the time of the pathogen isolation . One isolate was recovered from a vaginal ulcer . All six strains yielded black-pigmented colonies on sheep blood agar, chocolate agar, and colistin-nalidixic acid agar after 24 to 48 h of incubation at 35 degrees C . The dry, adherent colonies pitted the agar surface . The cells were coccobacillary to rod-shaped, catalase positive, nonmotile, and nonlipophilic . Only five of six isolates were available for characterization . Biochemical and chemotaxonomic studies revealed that the strains belong to the genus Corynebacterium but differ from known corynebacterial species . Comparative 16S rRNA gene sequence analysis showed that the strains are closely related and form a new subline within the genus Corynebacterium . We propose the name Corynebacterium nigricans sp . nov . for this group of coryneforms . The type strain of Corynebacterium nigricans is CN-1 . It is deposited in the American Type Culture Collection (assigned strain number ATCC 700975) and in the Institute Pasteur collection (assigned strain number CIP 107346).

Clin Infect Dis, 2003 Sep 15, 37(6), 834 - 7 Epub 2003 Aug 27.
Is a black-pigmented Corynebacterium species an opportunistic pathogen during pregnancy? Literature review and report of 3 new cases; Shukla SK et al.; Isolation of black-pigmented Corynebacterium species from human clinical samples has been reported, although rarely . Review of the medical literature revealed 3 reports of culture isolation of these unusual bacteria . All were recovered from the female genital tract, and 1 case was associated with spontaneous abortion . Genetic characterization of these isolates indicates that they are similar and probably represent a novel pathogen in the genus Corynebacterium . We report 3 additional bacterial isolates sharing similar phenotypic and/or genotypic characteristics that were recovered from the genital tract of women who had complications of pregnancy . Similarities in these cases suggest that this newly recognized Corynebacterium species might be an opportunistic pathogen in pregnancy.

J Biotechnol, 2003 Sep 4, 104(1-3), 325 - 34
Function of Corynebacterium glutamicum promoters in Escherichia coli, Streptomyces lividans, and Bacillus subtilis; Patek M et al.; The function of seven promoters from Corynebacterium glutamicum, P-hom, P-leuA, P-per, P-aes1, P-aes2, P-45, and P-104, was analyzed in a heterologous background . DNA fragments carrying the promoters were cloned into shuttle promoter-probe vectors replicating in Escherichia coli and C . glutamicum (pET2), Streptomyces lividans (pGL7011) and Bacillus subtilis (pRB394) . With the exception of P-hom, P-leuA and P-104 in B . subtilis, all promoters were found to be active in all species . Non-radioactive methods of primer-extension analysis and of S1-nuclease protection assay using automatic sequencer were developed to determine the respective transcriptional start points (TSPs) . All TSPs were determined by primer extension and in two promoters (P-45 and P-hom) the main TSPs were confirmed by S1-mapping . While the main TSPs were identical in all four species, utilization of multiple TSPs varied among the species and additional TSPs were detected in S . lividans . Knowledge of the efficiency of promoters and of exact respective TSPs may be of practical value for the construction of expression systems in a heterologous background.

J Biotechnol, 2003 Sep 4, 104(1-3), 311 - 23
Promoters of Corynebacterium glutamicum; Patek M et al.; Regulation of gene expression in Corynebacterium glutamicum represents an important issue since this Gram-positive bacterium is a notable industrial amino acid producer . Transcription initiation, beginning by binding of RNA polymerase to the promoter DNA sequence, is one of the main points at which bacterial gene expression is regulated . More than 50 transcriptional promoters have so far been experimentally localized in C . glutamicum . Most of them are assumed to be promoters of vegetative genes recognized by the main sigma factor . Although transcription initiation rate defined by many of these promoters may be affected by transcription factors, which activate or repress their function, the promoter regions share common sequence features, which may be generalized in a consensus sequence . In the consensus C . glutamicum promoter, the prominent feature is a conserved extended -10 region tgngnTA(c/t)aaTgg, while the -35 region is much less conserved . Some commonly utilized heterologous promoters were shown to drive strong gene expression in C . glutamicum . Conversely, some C . glutamicum promoters were found to function in Escherichia coli and in other bacteria . These observations suggest that C . glutamicum promoters functionally conform with the common bacterial promoter scheme, although they differ in some sequence structures.

J Biotechnol, 2003 Sep 4, 104(1-3), 287 - 99
Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum; Kirchner O et al.; During the last decades, the gram-positive soil bacterium Corynebacterium glutamicum has been shown to be a very versatile microorganism for the large-scale fermentative production of L-amino acids . Up to now, a vast amount of techniques and tools for genetic engineering and amplification of relevant structural genes have been developed . The objectives of this study are to summarize the published literature on tools for genetic engineering in C . glutamicum and to focus on new sophisticated and highly efficient methods in the fields of DNA transfer techniques, cloning vectors, integrative genetic tools, and antibiotic-free self-cloning . This repertoire of C . glutamicum methodology provides an experimental basis for efficient genetic analyses of the recently completed genome sequence.

J Biotechnol, 2003 Sep 4, 104(1-3), 273 - 85
Genome-wide expression analysis in Corynebacterium glutamicum using DNA microarrays; Wendisch VF; DNA microarray technology has become an important research tool for microbiology and biotechnology as it allows for comprehensive DNA and RNA analyses to characterize genetic diversity and gene expression in a genome-wide manner . DNA microarrays have been applied extensively to study the biology of many bacteria including Mycobacterium tuberculosis, but only recently have they been used for the related high-GC Gram-positive Corynebacterium glutamicum, which is widely used for biotechnological amino acid production . Besides the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, recent applications of DNA microarray technology in C . glutamicum including the characterization of ribose-specific gene expression and the valine stress response will be described . Emerging perspectives of functional genomics to enlarge our insight into fundamental biology of C . glutamicum and their impact on applied biotechnology will be discussed.

J Biotechnol, 2003 Sep 4, 104(1-3), 261 - 72
Metabolic network analysis during fed-batch cultivation of Corynebacterium glutamicum for pantothenic acid production: first quantitative data and analysis of by-product formation; Chassagnole C et al.; A first generation genetically modified strain of Corynebacterium glutamicum has been assessed for its potential to synthesise and accumulate the vitamin pantothenic acid in the medium using fed-batch cultivation technology, with biomass concentration controlled by isoleucine limitation . Kinetic analysis of specific rates throughout the process has been used to model carbon flux through both central metabolism and the specific pathways involved in product formation . Flux towards pantothenic acid is potentially high but much of this flux is dissipated as by-products within associated pathways, notably linked to amino acid synthesis . The major limitation of vitamin production in this strain is linked to the tenfold higher flux of keto-isovalerate towards valine rather than pantothenic acid . Attempts to modify this ratio by imposing nitrogen limitation provoked carbon overflow as unidentified non-nitrogenous compounds . The observed accumulation of glycine suggests that the flux towards pantothenate production may by limited by the rate of the pathway intermediate (5,10-methylene-tetrahydrofolate) regeneration.

J Biotechnol, 2003 Sep 4, 104(1-3), 253 - 60
Ketopantoate reductase activity is only encoded by ilvC in Corynebacterium glutamicum; Merkamm M et al.; Ketopantoate reductase catalyzes the second step of the pantothenate pathway after ketoisovalerate, common intermediate in valine, leucine and pantothenate biosynthesis . We show here that the Corynebacterium glutamicum ilvC gene is able to complement a ketopantoate reductase deficient Escherichia coli mutant . Thus ilvC, encoding acetohydroxyacid isomeroreductase, involved in the common pathway for branched-chained amino acids, also exhibits ketopantoate reductase activity . Enzymatic activity was confirmed by biochemical analysis in C . glutamicum . Furthermore, inactivation of ilvC in C . glutamicum leads to auxotrophy for pantothenate, indicating that ilvC is the only ketopantoate reductase- encoding gene in C . glutamicum.

J Biotechnol, 2003 Sep 4, 104(1-3), 241 - 52
Characterisation of the enzyme activities involved in the valine biosynthetic pathway in a valine-producing strain of Corynebacterium glutamicum; Leyval D et al.; The enzyme activities of the valine biosynthetic pathway and their regulation have been studied in the valine-producing strain, Corynebacterium glutamicum 13032DeltailvApJC1ilvBNCD . In this micro-organism, this pathway might involve up to five enzyme activities: acetohydroxy acid synthase (AHAS), acetohydroxy acid isomeroreductase (AHAIR), dihydroxyacid dehydratase and transaminases B and C . For each enzyme, kinetic parameters (optimal temperature, optimal pH and affinity for substrates) were determined . The first enzyme of the pathway, AHAS, was shown to exhibit a weak affinity for pyruvate (K(m)=8.3 mM) . It appeared that valine and leucine inhibited the three first steps of the pathway (AHAS, AHAIR and DHAD) . Moreover, the AHAS activity was inhibited by isoleucine . Considering the kinetic data collected during this work, AHAS would be a key enzyme for further strain improvement intending to increase the valine production by C . glutamicum.

J Biotechnol, 2003 Sep 4, 104(1-3), 229 - 40
Genome-based analysis of biosynthetic aminotransferase genes of Corynebacterium glutamicum; McHardy AC et al.; Due to broad and overlapping substrate specificities, aminotransferases remain the last uncharacterized enzymes from most amino acid biosynthetic pathways in Corynebacterium glutamicum . We report here a complete description of all aminotransferases participating in the biosynthesis of the branched-chain amino acids and phenylalanine in C . glutamicum . We used methods of profile analysis on the newly available genome sequence to systematically search for and characterize members of the four known aminotransferase classes . This led to the discovery of sixteen new, potential aminotransferase encoding genes in the C . glutamicum genome, eleven of which were subsequently characterized experimentally with respect to their participation in different amino acid biosynthetic pathways . Disruption by insertion mutagenesis of ilvE, encoding a branched-chain amino acid aminotransferase, confirmed its function in leucine and isoleucine biosynthesis . Two double mutants lacking both ilvE and genes classified as class I aminotransferases exhibited additional auxotrophic requirements for valine and phenylalanine, respectively . In C . glutamicum the branched-chain amino acid aminotransferase thus participates in four amino acid biosynthetic pathways, for which in case of valine and phenylalanine biosynthesis two additional enzymes with overlapping substrate specificity exist . The novel protein with aminotransferase activity in valine biosynthesis belongs to the very recently described MocR subfamily of GntR-type helix-turn-helix transcriptional regulators, is located upstream of a potential operon of a newly described pyridoxine biosynthetic pathway and when disrupted, gives rise to a pyridoxine auxotrophy . The theoretical and experimental data we present should further provide a solid platform for ongoing research and understanding of the network of aminotransferases which participate in amino acid biosynthesis in C . glutamicum.

J Biotechnol, 2003 Sep 4, 104(1-3), 213 - 28
Genome-wide analysis of the L-methionine biosynthetic pathway in Corynebacterium glutamicum by targeted gene deletion and homologous complementation; Ruckert C et al.; The genome sequence of Corynebacterium glutamicum, a gram-positive soil bacterium widely used as an amino acid producer, was analyzed by a similarity-based approach to elucidate the pathway for the biosynthesis of L-methionine . The functions of candidate ORFs were derived by gene deletion and, if necessary, by homologous complementation of suitable mutants . Of nine candidate ORFs (four of which were known previously), seven ORFs (cg0754 (metX), cg0755 (metY), cg1290 (metE), cg1702 (metH), cg2383 (metF), cg2536 (aecD), and cg2687 (metB)) were demonstrated to be part of the pathway while two others (cg0961 and cg3086) could be excluded . C . glutamicum synthesizes methionine in three, respectively four steps, starting from homoserine . C . glutamicum possesses two genes with similarity to homoserine acetyltransferases but only MetX can act as such while Cg0961 catalyzes a different, unknown reaction . For the incorporation of the sulfur moiety, the known functions of MetY and MetB could be confirmed and AecD was proven to be the only functional cystathionine beta-lyase in C . glutamicum, while Cg3086 can act neither as cystathionine gamma-synthase nor as cystathionine beta-lyase . Finally, MetE and MetH, which catalyze the conversion of L-homocysteine to L-methionine, could be newly identified, together with MetF which provides the necessary N(5)-methyltetrahydrofolate.

J Biotechnol, 2003 Sep 4, 104(1-3), 199 - 211
Identification and characterization of the last two unknown genes, dapC and dapF, in the succinylase branch of the L-lysine biosynthesis of Corynebacterium glutamicum; Hartmann M et al.; The inspection of the complete genome sequence of Corynebacterium glutamicum ATCC 13032 led to the identification of dapC and dapF, the last two unknown genes of the succinylase branch of the L-lysine biosynthesis . The deduced DapF protein of C . glutamicum is characterized by a two-domain structure and a conserved diaminopimelate (DAP) epimerase signature . Overexpression of dapF resulted in an 8-fold increase of the specific epimerase activity . A defined deletion in the dapF gene led to a reduced growth of C . glutamicum in a medium with excess carbon but limited ammonium availability . The predicted DapC protein of C . glutamicum shared 29% identical amino acids with DapC from Bordetella pertussis, the only enzymatically characterized N-succinyl-aminoketopimelate aminotransferase . Overexpression of the dapC gene in C . glutamicum resulted in a 9-fold increase of the specific aminotransferase activity . A C . glutamicum mutant with deleted dapC showed normal growth characteristics with excess carbon and limited ammonium . Even a mutation of the two genes dapC and ddh, interrupting both branches of the split pathway, could be established in C . glutamicum . Overexpression of the dapF or the dapC gene in an industrial C . glutamicum strain resulted in an increased L-lysine production, indicating that both genes might be relevant targets for the development of improved production strains.

J Biotechnol, 2003 Sep 4, 104(1-3), 185 - 97
Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum; Marx A et al.; A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply . Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C . glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored . Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C . glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype . Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced . In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations . Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C . glutamicum strain DSM5715 . The genome region containing the pgi gene was analyzed . It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C . glutamicum Pgi mutants . We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain . A reduced growth rate and a biphasic growth behavior was observed . The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed . Another phosphoglucose isomerase mutant of C . glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed . This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.

J Biotechnol, 2003 Sep 4, 104(1-3), 173 - 84
Instability of glutamate production by Corynebacterium glutamicum 2262 in continuous culture using the temperature-triggered process; Uy D et al.; Kinetics and physiology of Corynebacterium glutamicum 2262 cultured for extended periods in continuous mode were investigated at 33, 39 and 41 degrees C . At 33 degrees C no glutamate production occurred whatever the dilution rates tested (ranging between 0.05 and 0.5 h(-1)) . When the continuous culture was performed at 39 degrees C and D=0.05 h(-1), the glutamate was actively produced, while the activities of 2-oxoglutarate dehydrogenase complex (ODHC) and pyruvate dehydrogenase (PDH) were, respectively completely inhibited and 35% decreased . Simultaneously, the intracellular glutamate was 62% reduced compared to the level found at 33 degrees C and the co-metabolites lactate and trehalose were excreted . The decrease in PDH activity during the glutamate production was suggested to be responsible for the accumulation of by-products and for limiting the carbon flux required for glutamate synthesis . When the culture was prolonged for more than 100 h, a cell selection occurred, in favor of growth and to the detriment of glutamate production . In fact, these selected cells presented high levels of ODHC and PDH activities even at 39 degrees C, resulting in a complete inhibition of the glutamate production after 150 h of culture . A further temperature increase till 41 degrees C restored the glutamate production and abolished the ODHC activity of these selected cells.

J Biotechnol, 2003 Sep 4, 104(1-3), 155 - 72
Industrial production of amino acids by coryneform bacteria; Hermann T; In the 1950s Corynebacterium glutamicum was found to be a very efficient producer of L-glutamic acid . Since this time biotechnological processes with bacteria of the species Corynebacterium developed to be among the most important in terms of tonnage and economical value . L-Glutamic acid and L-lysine are bulk products nowadays . L-Valine, L-isoleucine, L-threonine, L-aspartic acid and L-alanine are among other amino acids produced by Corynebacteria . Applications range from feed to food and pharmaceutical products . The growing market for amino acids produced with Corynebacteria led to significant improvements in bioprocess and downstream technology as well as in molecular biology . During the last decade big efforts were made to increase the productivity and to decrease the production costs . This review gives an overview of the world market for amino acids produced by Corynebacteria . Significant improvements in bioprocess technology, i.e . repeated fed batch or continuous production are summarised . Bioprocess technology itself was improved furthermore by application of more sophisticated feeding and automatisation strategies . Even though several amino acids developed towards commodities in the last decade, side aspects of the production process like sterility or detection of contaminants still have increasing relevance . Finally one focus of this review is on recent developments in downstream technology.

J Biotechnol, 2003 Sep 4, 104(1-3), 129 - 53
The respiratory chain of Corynebacterium glutamicum; Bott M et al.; Corynebacterium glutamicum is an aerobic bacterium that requires oxygen as exogenous electron acceptor for respiration . Recent molecular and biochemical analyses together with information obtained from the genome sequence showed that C . glutamicum possesses a branched electron transport chain to oxygen with some remarkable features . Reducing equivalents obtained by the oxidation of various substrates are transferred to menaquinone via at least eight different dehydrogenases, i.e . NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, pyruvate:quinone oxidoreductase, D-lactate dehydrogenase, L-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase and L-proline dehydrogenase . All these enzymes contain a flavin cofactor and, except succinate dehydrogenase, are single subunit peripheral membrane proteins located inside the cell . From menaquinol, the electrons are passed either via the cytochrome bc(1) complex to the aa(3)-type cytochrome c oxidase with low oxygen affinity, or to the cytochrome bd-type menaquinol oxidase with high oxygen affinity . The former branch is exceptional, in that it does not involve a separate cytochrome c for electron transfer from cytochrome c(1) to the Cu(A) center in subunit II of cytochrome aa(3) . Rather, cytochrome c(1) contains two covalently bound heme groups, one of which presumably takes over the function of a separate cytochrome c . The bc(1) complex and cytochrome aa(3) oxidase form a supercomplex in C . glutamicum . The phenotype of defined mutants revealed that the bc(1)-aa(3) branch, but not the bd branch, is of major importance for aerobic growth in minimal medium . Changes of the efficiency of oxidative phosphorylation caused by qualitative changes of the respiratory chain or by a defective F(1)F(0)-ATP synthase were found to have strong effects on metabolism and amino acid production . Therefore, the system of oxidative phosphorylation represents an attractive target for improving amino acid productivity of C . glutamicum by metabolic engineering.

J Biotechnol, 2003 Sep 4, 104(1-3), 123 - 8
Lysine synthesis control in Corynebacterium glutamicum RC 115 in mixed substrate (glucose-acetate) medium; Paegle L et al.; The effect of acetate as a glucose co-substrate on growth, lysine synthesis and experimental lysine yield from carbon substrates by Corynebacterium glutamicum RC 115 was investigated . It was found that low amounts of acetate, injected with a glucose-acetate pulse into the steady-state continuous culture in bioreactor, caused a slight decrease in the specific rates of glucose uptake and bacterial growth, but a significant increase in the cell specific rate of lysine synthesis and an increase in lysine yield . In contrast, acetate injected in high amounts was followed by a drastic decrease in the values of these parameters . A strong increase in experimental lysine yield under the latter conditions was reached in the response to pyruvate addition . Therefore it was shown that acetate in low concentrations can be used as a glucose co-substrate to increase the cell specific rate of lysine synthesis and lysine yield by C . glutamicum RC 115 . Pyruvate supplementation was found as a promising method to enhance lysine synthesis by bacterial cells grown in glucose-acetate media with an increased concentration of acetate.

J Biotechnol, 2003 Sep 4, 104(1-3), 99 - 122
Acetate metabolism and its regulation in Corynebacterium glutamicum; Gerstmeir R et al.; The amino acid producing Corynebacterium glutamicum grows aerobically on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy . Among the substrates metabolized are glucose and acetate which both can also serve as substrates for amino acid production . Based on biochemical, genetic and regulatory studies and on quantitative determination of metabolic fluxes during utilization of acetate and/or glucose, this review summarizes the present knowledge on the different steps of the fundamental pathways of acetate utilization in C . glutamicum, namely, on acetate transport, acetate activation, tricarboxylic acid (TCA) cycle, glyoxylate cycle and gluconeogenesis . It becomes evident that, although the pathways of acetate utilization follow the same theme in many bacteria, important biochemical, genetic and regulatory peculiarities exist in C . glutamicum . Recent genome wide and comparative expression analyses in C . glutamicum cells grown on glucose and on acetate substantiated previously identified transcriptional regulation of acetate activating enzymes and of glyoxylate cycle enzymes . Additionally, a variety of genes obviously also under transcriptional control in response to the presence or absence of acetate in the growth medium were uncovered . These genes, thus also belonging to the acetate stimulon of C . glutamicum, include genes coding for TCA cycle enzymes (e.g . aconitase and succinate dehydrogenase), for gluconeogenesis (phosphoenolpyruvate carboxykinase), for glycolysis (pyruvate dehydrogenase E1) and genes coding for proteins with hitherto unknown function . Although the basic mechanism of transcriptional regulation of the enzymes involved in acetate metabolism is not yet understood, some recent findings led to a better understanding of the adaptation of C . glutamicum to acetate at the molecular level.

J Biotechnol, 2003 Sep 4, 104(1-3), 87 - 97
Impact of osmotic stress on volume regulation, cytoplasmic solute composition and lysine production in Corynebacterium glutamicum MH20-22B; Ronsch H et al.; The response of the L-lysine producing Corynebacterium glutamicum strain MH20-22B to osmotic stress was studied in batch cultures . To mimic the conditions during a fermentation process the long term adaptation of cells subjected to a constant osmotic stress between 1.0 and 2.5 osM was investigated . Cytoplasmic water content and volume of C . glutamicum cells were found to depend on growth phase, extent of osmotic stress and availability of betaine . The maximal cytoplasmic volumes, which were highest at maximal growth rate, were linearily related to osmotic stress, whereas in stationary cells no active volume regulation was observed . Under severe osmotic stress proline was the prominent compatible solute in growing cells . Uptake of betaine, if available in the medium, reduced the concentration of proline from 750 to 300 mM, indicating that uptake of compatible solutes is preferred to synthesis . Furthermore, betaine was shown to have a higher efficiency to counteract osmotic stress, since the overall concentration of compatible solutes was lower in the presence of betaine . Under severe osmotic stress, the addition of betaine shifted L-lysine production in MH20-22B to earlier fermentation times and increased both product concentration and yield in these phases, but did not improve the final L-lysine yield.

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