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