Corynebacterium

J Biotechnol, 2003 Sep 4, 104(1-3), 77 - 85
Osmotic stress, glucose transport capacity and consequences for glutamate overproduction in Corynebacterium glutamicum; Gourdon P et al.; Glucose uptake by Corynebacterium glutamicum is predominantly assured by a mannose phosphotransferase system (PTS) with a high affinity for glucose (Km=0.35 mM) . Mutants selected for their resistance to 2-deoxyglucose (2DG) and lacking detectable PEP-dependent glucose-transporting activity, retained the capacity to grow on media in which glucose was the only carbon and energy source, albeit at significantly diminished rates, due to the presence of a low affinity (Ks=11 mM) non-PTS uptake system . During growth in media of different osmolarity, specific rates of glucose consumption and of growth of wild type cells were diminished . Cell samples from these cultures were shown to possess similar PTS activities when measured under standard conditions . However, when cells were resuspended in buffer solutions of different osmolarity measurable PTS activity was shown to be dependent upon osmolarity . This inhibition effect was sufficient to account for the decreased rates of both sugar uptake and growth observed in fermentation media of high osmolarity . The secondary glucose transporter was, however, not influenced by medium osmolarity . During industrial fermentation conditions with accumulation of glutamic acid and the corresponding increase in medium osmolarity, similar inhibition of the sugar transport capacity was observed . This phenomenon provokes a major process constraint since the decrease in specific rates leads to an increasing proportion of sugar catabolised for maintenance requirements with an associated decrease in product yields.

J Biotechnol, 2003 Sep 4, 104(1-3), 69 - 75
Impact of transport processes in the osmotic response of Corynebacterium glutamicum; Morbach S et al.; Osmoregulation, the adaptation of cells to changes in the external osmolarity, is an important aspect of the bacterial stress response, in particular for a soil bacterium like Corynebacterium glutamicum . Consequently, this organism is equipped with several redundant systems for coping with both hyper- and hypoosmotic stress . For the adaptation to hypoosmotic stress C . glutamicum possesses at least three different mechanosensitive (MS) channels . To overcome hyperosmotic stress C . glutamicum accumulates so-called compatible solutes either by means of biosynthesis or by uptake . Uptake of compatible solutes is in general preferred to de novo synthesis because of lower energy costs . Noticeable, only secondary transporters belonging to the MHS (ProP) or the BCCT-family (BetP, EctP and LcoP) are involved in the uptake of proline, betaine and ectoine . In contrast to Escherichia coli or Bacillus subtilis no ABC-transporters were found catalyzing uptake of compatible solutes . BetP was one of the first examples of the growing group of osmosensory proteins to be analyzed in detail . This transporter is characterized, besides the catalytic activity of betaine uptake, by the ability to sense osmotic changes (osmosensing) and to respond to the extent of osmotic stress by adaptation of transport activity (osmoregulation) . BetP detects hyperosmotic stress via an increase in the internal K(+) concentration following a hyperosmotic shift, and thus acts as a chemosensor.

J Biotechnol, 2003 Sep 4, 104(1-3), 55 - 67
Mycomembrane and S-layer: two important structures of Corynebacterium glutamicum cell envelope with promising biotechnology applications; Bayan N et al.; Corynebacteria belong to a distinct Gram-positive group of bacteria including mycobacteria and nocardia, which are characterized by the presence of mycolic acids in their cell wall . These bacteria share the property of having an unusual cell envelope structural organization close to Gram-negative bacteria . In addition to the inner membrane, the cell envelope is constituted of a thick arabinogalactan-peptidoglycan polymer covalently linked to an outer lipid layer, which is mainly composed of mycolic acids and probably organized in an outer membrane like structure . In some species, the cell is covered by a crystalline surface layer composed of a single protein species, which is anchored in the outer membrane like barrier . An increasing number of reports have led to a better understanding of the structure of the cell wall of Corynebacterium glutamicum . These works included the characterization of several cell wall proteins like S-layer protein and porins, genetic and biochemical characterization of mycolic acids biosynthesis, ultrastructural description of the cell envelope, and chemical analysis of its constituents . All these data address new aspects regarding cell wall permeability towards macromolecules and amino acids but also open new opportunities for biotechnology applications.

J Biotechnol, 2003 Sep 4, 104(1-3), 27 - 40
Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics; Tauch A et al.; Endogenous plasmids and selectable resistance markers are a fundamental prerequisite for the development of efficient recombinant DNA techniques in industrial microorganisms . In this article, we therefore summarize the current knowledge about endogenous plasmids in amino acid-producing Corynebacterium glutamicum isolates . Screening studies identified a total of 24 different plasmids ranging in size from 2.4 to 95 kb . Although most of the C . glutamicum plasmids were cryptic, four plasmids carried resistance determinants against the antibiotics chloramphenicol, tetracycline, streptomycin-spectinomycin, and sulfonamides . Considerable information is now available on the molecular genetic organization of 12 completely sequenced plasmid genomes from C . glutamicum . The deduced mechanism of plasmid DNA replication and the degree of amino acid sequence similarity among replication initiator proteins was the basis for performing a classification of the plasmids into four distinct C . glutamicum plasmid families.

J Biotechnol, 2003 Sep 4, 104(1-3), 5 - 25
The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins; Kalinowski J et al.; The complete genomic sequence of Corynebacterium glutamicum ATCC 13032, well-known in industry for the production of amino acids, e.g . of L-glutamate and L-lysine was determined . The C . glutamicum genome was found to consist of a single circular chromosome comprising 3282708 base pairs . Several DNA regions of unusual composition were identified that were potentially acquired by horizontal gene transfer, e.g . a segment of DNA from C . diphtheriae and a prophage-containing region . After automated and manual annotation, 3002 protein-coding genes have been identified, and to 2489 of these, functions were assigned by homologies to known proteins . These analyses confirm the taxonomic position of C . glutamicum as related to Mycobacteria and show a broad metabolic diversity as expected for a bacterium living in the soil . As an example for biotechnological application the complete genome sequence was used to reconstruct the metabolic flow of carbon into a number of industrially important products derived from the amino acid L-aspartate.

Appl Microbiol Biotechnol, 2004 Feb, 63(5), 592 - 601 Epub 2003 Aug 21.
A gene homologous to beta-type carbonic anhydrase is essential for the growth of Corynebacterium glutamicum under atmospheric conditions; Mitsuhashi S et al.; Carbonic anhydrase catalyzes the interconversion of CO(2) and bicarbonate . We focused on this enzyme in the amino acid-producing organism Corynebacterium glutamicum in order to assess the availability of bicarbonate for carboxylation reactions essential to growth and for those required for L-lysine overproduction . A whole-genome sequence revealed two genes encoding putative beta-type and gamma-type carbonic anhydrases in C . glutamicum . These genes encode polypeptides containing zinc ligands strictly conserved in each type of carbonic anhydrase and were designated bca and gca, respectively . Internal deletion of the chromosomal bca gene resulted in a phenotype showing severely reduced growth under atmospheric conditions (0.04% CO(2)) on both complete and minimal media . The growth defect of the Delta bca strain was restored under elevated CO(2) conditions (5% CO(2)) . Introduction of the red alga Porphyridium purpureum carbonic anhydrase gene ( pca) could compensate for the bca deletion, allowing normal growth under an atmospheric level of CO(2) . In contrast, the Delta gca strain behaved identically to the wild-type strain with respect to growth, irrespective of the CO(2) conditions . Attempts to increase the dosage of bca, gca, and pca in the defined L-lysine-producing strain C . glutamicum AHD-2 led to no discernable effects on growth and production . Northern blot analysis indicated that the bca transcript in strain AHD-2 and another L-lysine producer, C . glutamicum B-6, was present at a much higher level than in the wild-type strain, particularly during exponential growth phases . These results indicate that: (1) the bca product is essential to achieving normal growth under ordinary atmospheric conditions, and this effect is most likely due to the bca product's ability to maintain favorable intracellular bicarbonate/CO(2) levels, and (2) the expression of bca is induced during exponential growth phases and also in the case of L-lysine overproduction, both of which are conditions of higher bicarbonate demand.

Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1640 - 1 Epub 2003 Aug 19.
Crystallization and preliminary X-ray crystallographic studies of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum; Aich S et al.; Phosphoenolpyruvate carboxykinase (PCK) is a key enzyme involved in the regulation of gluconeogenesis . PCKs from higher animals require guanosine nucleotide for activity . PCK from Corynebacterium glutamicum is also GTP specific . X-ray diffraction data from a C . glutamicum PCK crystal were collected to 2.8 A resolution . The crystals were monoclinic, belonging to space group P2(1), with unit-cell parameters a = 71.7, b = 117.4, c = 161.3 A, beta = 92.9 degrees . The presence of two molecules in the crystallographic asymmetric unit gives a V(M) of 2.5 A(3) Da(-1) and a solvent content of 50.3%.

Proteomics, 2003 Aug, 3(8), 1637 - 46
Towards a phosphoproteome map of Corynebacterium glutamicum; Bendt AK et al.; In this study, the phosphoproteome of Corynebacterium glutamicum, an industrially important soil bacterium of the Corynebacterium/Mycobacterium/Nocardia (CMN) group of Gram-positive bacteria, was investigated by two different detection methods: first, by in vivo radio-labeling using {(33)P}-phosphoric acid with subsequent autoradiography and second, by immunostaining with phosphoamino acid-specific monoclonal antibodies . After two-dimensional gel electrophoresis (2-DE), around 60 {(33)P}-labeled protein spots were visualized and around 90 antibody-decorated protein spots detected; 31 of the protein spots were detected with both methods . By peptide mass fingerprinting, 41 different proteins were identified, namely 5-enolpyruvylshikimate 3-phosphate synthase, aconitase, acyl-CoA carboxylase, acyl-CoA synthetase, ATP (synthase alpha- and beta-chain), carbamoyl-phosphate synthase, citrate synthase, cysteine synthase, DnaK, the elongation factors G, P, Ts and Tu, enolase, fructose bisphosphate aldolase, fumarase, Gap dehydrogenase, glutamine synthetase I, glycine hydroxymethyltransferase, GroEL2, GTPase, heat-inducible transcriptional repressor DnaJ2, inorganic pyrophosphatase, isocitrate dehydrogenase, ketol-acid reductoisomerase, lactate dehydrogenase, leucine-tRNA ligase, lipoamide dehydrogenase, methionine synthase, O-acetylhomoserine sulfhydrylase, pyruvate carboxylase, pyruvate kinase, pyruvate oxidase, ribosomal protein S1, RNA polymerase (beta-subunit), succinyl-CoA:CoA transferase, transketolase and UDP-N-acetylmuramoyl-L-alanine ligase, besides a hypothetical 35k protein and a hypothetical glucose kinase . Both detection techniques were used to create a phosphoproteome map . Additionally, the influence of nitrogen deprivation on the phosphoproteome of C . glutamicum was investigated.

Eur J Biochem, 2003 Sep, 270(17), 3525 - 42
Systematic quantification of complex metabolic flux networks using stable isotopes and mass spectrometry; Klapa MI et al.; Metabolic fluxes provide a detailed metric of the cellular metabolic phenotype . Fluxes are estimated indirectly from available measurements and various methods have been developed for this purpose . Of particular interest are methods making use of stable isotopic tracers as they enable the estimation of fluxes at a high resolution . In this paper, we present data validating the use of mass spectrometry (MS) for the quantification of complex metabolic flux networks . In the context of the lysine biosynthesis flux network of Corynebacterium glutamicum (ATCC 21799) under glucose limitation in continuous culture, operating at 0.1 x h(-1) after the introduction of 50% {1-13C}glucose, we deploy a bioreaction network analysis methodology for flux determination from mass isotopomer measurements of biomass hydrolysates, while thoroughly addressing the issues of measurement accuracy, flux observability and data reconciliation . The analysis enabled the resolution of the involved anaplerotic activity of the microorganism using only one labeled substrate, the determination of the range of most of the exchange fluxes and the validation of the flux estimates through satisfaction of redundancies . Specifically, we determined that phosphoenolpyruvate carboxykinase and synthase do not carry flux at these experimental conditions and identified a high futile cycle between oxaloacetate and pyruvate, indicating a highly active in vivo oxaloacetate decarboxylase . Both results validated previous in vitro activity measurements . The flux estimates obtained passed the chi2 statistical test . This is a very important result considering that prior flux analyses of extensive metabolic networks from isotopic measurements have failed criteria of statistical consistency.

Vet Rec, 2003 Jul 26, 153(4), 118 - 21
Purpura haemorrhagica in 53 horses; Pusterla N et al.; The medical records of 53 horses with purpura haemorrhagica were reviewed . Seventeen of them had been exposed to or infected with Streptococcus equi, nine had been infected with Corynebacterium pseudotuberculosis, five had been vaccinated with S . equi M protein, five had had a respiratory infection of unknown aetiology, and two had open wounds; the other 15 cases had no history of recent viral or bacterial infection . The horses were between six months and 19 years of age (mean 8.4 years) . The predominant clinical signs were well demarcated subcutaneous oedema of all four limbs and haemorrhages on the visible mucous membranes; other signs included depression, anorexia, fever, tachycardia, tachypnoea, reluctance to move, drainage from lymph nodes, exudation of serum from the skin, colic, epistaxis and weight loss . Haematological and biochemical abnormalities commonly detected were anaemia, neutrophilia, hyperproteinaemia, hyperfibrinogenaemia, hyperglobulinaemia and high activities of muscle enzymes . All of the horses were treated with corticosteroids; 42 also received non-steroidal anti-inflammatory drugs and 26 received antimicrobial drugs . Selected cases received special nursing care, including hydrotherapy and bandaging of the limbs . Most of the horses were treated for more than seven days and none of them relapsed . Forty-nine of the horses survived, one died and three were euthanased, either because their severe clinical disease failed to respond to treatment or because they developed secondary complications . Two of the four non-survivors had been vaccinated against S . equi with a product containing the M protein, one had a S . equi infection and the other had a respiratory infection of undetermined aetiology.

Ophthalmology, 2003 Aug, 110(8), 1560 - 6
Open globe injuries with positive intraocular cultures: factors influencing final visual acuity outcomes; Lieb DF et al.; PURPOSE: To investigate the clinical features influencing final visual acuity outcomes of eyes with positive intraocular cultures after open globe injuries . DESIGN: Retrospective, consecutive, interventional case series . PARTICIPANTS: Thirty-seven patients . METHODS: The medical records were reviewed of all patients with positive intraocular cultures after open globe injuries treated at Bascom Palmer Eye Institute between January 1, 1995, and December 31, 2001 . MAIN OUTCOME MEASURES: Final visual acuity . Clinical features investigated included the following: (1) . presence or absence of clinical endophthalmitis; (2) . virulence of the cultured organism (coagulase-negative Staphylococci, Corynebacterium, and Propionibacterium acnes were classified as nonvirulent organisms, whereas all other organisms were classified as virulent organisms); (3) . presence of intraocular foreign body (IOFB); (4) . presence of retinal detachment; (5) . interval between ocular injury and surgical repair; (6) . severity of vision loss at presentation; (7) . zone of injury; (8) . wound length; and (9) . presence of vitreous hemorrhage . RESULTS: The study included 37 eyes of 37 patients with a mean age of 30 years (range, 18 months-85 years) and a median follow-up of 13 months (range, 1-71 months) . Study eyes were stratified into two groups: group 1 eyes (n = 16) were those in which clinical endophthalmitis did not develop, whereas group 2 eyes (n = 21) were those in which clinically diagnosed endophthalmitis developed at some point during their clinical course . Presenting visual acuity was similar in the two groups (mean logarithm of the minimum angle of resolution {logMAR} acuity, 1.91 and 2.22 {Snellen equivalents, 2/162 and 2/331} respectively; P = 0.33) . Final acuities in the two groups were different, but not to a statistically significant level (mean logMAR acuity, 1.14 and 2.05 {Snellen equivalents, 20/276 and 2/224}, respectively; P = 0.069) . In group 1, final visual acuity ranged from 20/20 to no light perception (median acuity, 20/186); 12 eyes (75.0%) achieved a final visual acuity of 20/400 or better . In group 2, final visual acuity ranged from 20/25 to no light perception (median acuity, 7/200); of 20 eyes with known final visual acuity, 10 (50.0%) retained 20/400 or better vision . In group 1, three eyes (19%) eyes had virulent organisms, and 13 eyes (81%) had nonvirulent organisms . In group 2, 12 eyes (57%) had virulent organisms, and nine eyes (43%) had nonvirulent organisms . A final acuity of 20/60 or better was achieved in 14 eyes (41%), and a final acuity of 20/400 or better was achieved in 22 eyes (59%) . Better presenting visual acuity (P = 0.038), culture of a nonvirulent organism (P = 0.011), lack of a retinal detachment (P = 0.002), absence of clinical endophthalmitis (P = 0.069), and shorter wound length (P = 0.024) were associated with better visual acuity outcome . In four of six eyes (67%) with both an IOFB and clinical endophthalmitis (group 2), the final visual acuity was no light perception (IOFB was not itself significantly associated with final visual acuity; P = 0.11) . CONCLUSIONS: Among eyes with positive intraocular cultures after open globe injury, the visual prognosis is guarded . Clinical features associated with better visual acuity outcomes include better presenting visual acuity, culture of a nonvirulent organism, lack of a retinal detachment, absence of clinical endophthalmitis, and shorter wound length.

Ann Ig, 2003 May-Jun, 15(3), 191 - 7
{Persistence of circulation of Corynebacterium diphtheriae non-toxigenic strains and low prevalence of carriers in a sample of children vaccinated against diphtheria}; Bergamini M et al.; This study was carried out with the aim to investigate the presence of carriers of diphtheria bacillus in a sample of 1970 healthy children, 6-14 years aged, residing in eight Italian towns . Three non-toxigenic strains of Corynebacterium diphtheriae biotype mitis were isolated from as many healthy children . Molecular characterization by ribotyping showed close genetic relation of two of the wild strains with the C7(b) reference strain whereas one of the wild strains showed close genetic relation with two collection strains isolated in the same geographic area (Emilia-Romagna) from diphtheria patients in the seventy years . This supports the hypothesis of the persistence of some non toxigenic C . diphtheriae strains derived from ancient endemic strains under the selective pressure of mass immunization against diphtheria . The persistence of carriers of diphtheria bacilli, although non toxigenic, suggests that high levels of immunity must be maintained, not only in children, but also in adults by booster vaccination.

Arch Microbiol, 2003 Oct, 180(4), 285 - 92 Epub 2003 Aug 01.
Fructose-1,6-bisphosphatase from Corynebacterium glutamicum: expression and deletion of the fbp gene and biochemical characterization of the enzyme; Rittmann D et al.; The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli . Purified, His-tagged fructose-1,6-bisphosphatase from C . glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer . The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K(m) value of about 14 micro M and a V(max) of about 5.4 micro mol min(-1) mg(-1) and k(cat )of about 3.2 s(-1) . Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg(2+) or Mn(2+) and was inhibited by the monovalent cation Li(+) with an inhibition constant of 140 micro M . Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C . glutamicum . The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP . Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source . Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C . glutamicum WTDelta fbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate . Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C . glutamicum.

J Biol Chem, 2003 Oct 17, 278(42), 40842 - 50 Epub 2003 Aug 06.
Disruption of Cg-Ppm1, a polyprenyl monophosphomannose synthase, and the generation of lipoglycan-less mutants in Corynebacterium glutamicum; Gibson KJ et al.; The glycosyl donor, polyprenyl monophosphomannose (PPM), has been shown to be involved in the biosynthesis of the mycobacterial lipoglycans: lipomannan and lipoarabinomannan . The mycobacterial PPM synthase (Mt-ppm1) catalyzes the transfer of mannose from GDP-mannose to polyprenyl phosphates . Based on sequence homology to Mt-ppm1, we have identified the PPM synthase from Corynebacterium glutamicum . In the present study, we demonstrate that the corynebacterial synthase is composed of two distinct domains; a catalytic domain (Cg-ppm1) and a membrane domain (Cg-ppm2) . Through the inactivation of Cg-ppm1, we observed a complex phenotype that included altered cell growth rate and inability to synthesize PPM molecules and lipoglycans . When Cg-ppm2 was deleted, no observable phenotype was noted, indicating the clear organization of the two domains . The complementation of the inactivated Cg-ppm1 strain with the corresponding mycobacterial enzyme (Mt-Ppm1/D2) led to the restoration of a wild type phenotype . The present study illustrates, for the first time, the generation of a lipoglycan-less mutant based on a molecular strategy in a member of the Corynebacterianeae family . Lipoglycans are important immunomodulatory molecules involved in determining the outcome of infection, and so the generation of defined mutants and their subsequent immunological characterization is timely.

J Bacteriol, 2003 Aug, 185(16), 4779 - 86
PorA represents the major cell wall channel of the Gram-positive bacterium Corynebacterium glutamicum; Costa-Riu N et al.; The cell wall of the gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes . The channel-forming polypeptide PorA is a 45-amino-acid acidic polypeptide with an excess of four negatively charged amino acids, which is encoded by the 138-bp gene porA . porA was deleted from the chromosome of C.glutamicum wild-type strain ATCC 13032 to obtain mutant ATCC 13032deltaporA . Southern blot analysis demonstrated that porA was deleted . Lipid bilayer experiments revealed that PorA was not present in the cell wall of the mutant strain . Searches within the known chromosome of C . glutamicum by using National Center for Biotechnology Information BLAST and reverse transcription-PCR showed that no other PorA-like protein is encoded on the chromosome or is expressed in the deletion strain . The porA deletion strain exhibited slower growth and longer growth times than the C . glutamicum wild-type strain . Experiments with different antibiotics revealed that the susceptibility of the mutant strain was much lower than that of the wild-type C . glutamicum strain . The results presented here suggest that PorA represents a major hydrophilic pathway through the cell wall and that C . glutamicum contains cell wall channels which are not related to PorA.

Int J Dermatol, 2003 Sep, 42 Suppl 1, 11 - 7
Evaluation of in vitro activity of ciclopirox olamine, butenafine HCl and econazole nitrate against dermatophytes, yeasts and bacteria; Kokjohn K et al.; BACKGROUND: In many instances, a cutaneous fungal infection may exist concomitantly with bacterial involvement . In this study we compared the in vitro activity of three antifungal agents against the dermatophytes, yeasts and bacteria recovered most commonly from cutaneous mycoses and bacterial infections . METHODS: Using a microdilution method adapted from the National Committee for Clinical Laboratory Standards (NCCLS), we determined the minimum inhibitory concentrations (MICs) of ciclopirox olamine, econazole nitrate and butenafine HCl against a panel of dermatophyte fungi and yeasts (n = 39) and bacterial isolates (n = 45) . RESULTS: All three antifungals demonstrated comparable activity against the dermatophytes tested, with a MIC range of 0.03-0.25 micro g/ml for ciclopirox, < 0.001-0.25 micro g/ml for econazole and 0.03-0.25 micro g/ml for butenafine . For yeasts, ciclopirox showed activity against all isolates, with an MIC range of 0.001-0.25 micro g/ml, whereas econazole had a broader range of 0.125-> 0.5 micro g/ml . Butenafine displayed limited activity against the yeast Candida albicans and no activity against Malassezia furfur . For the antibacterial activity studies, ciclopirox demonstrated activity against all isolates tested with a range of 0.06-2 micro g/ml, while econazole showed activity against Gram-positive bacteria only, with a MIC range of 0.004-0.25 micro g/ml . Butenafine HCl had a limited activity against bacterial isolates tested, showing activity against beta-hemolytic Streptococcus Group A and Corynebacterium only . Neither econazole nitrate nor butenafine HCl demonstrated activity against any of the Gram-negative strains evaluated in this study . CONCLUSIONS: The data suggest that ciclopirox olamine has the broadest in vitro activity, in comparison to econazole and butenafine HCl, against bacteria, yeasts and bacteria . These findings may have implications in the use of these antimycotics in the treatment of mixed cutaneous infections where bacteria or yeasts are present in addition to dermatophytes.

Biotechnol Prog, 2003 Jul-Aug, 19(4), 1387 - 90
Unstructured model for L-lysine fermentation under controlled dissolved oxygen; Ensari S et al.; An unstructured model was developed for batch cultivation of Corynebacterium lactofermentum (ATCC 21799) under controlled dissolved oxygen . The model is capable of predicting batch experiments performed at various initial substrate concentrations . By extending the batch culture model to a fed-batch model and using a heuristic approach to optimize the fed-batch cultivation, it is shown that fed-batch cultivation is superior to batch operation due to increased productivity at high substrate concentrations.

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1135 - 8
Isolation of Corynebacterium falsenii and description of Corynebacterium aquilae sp . nov., from eagles; Fernandez-Garayzabal JF et al.; Biochemical, molecular chemical and molecular genetic studies were performed on seven unidentified gram-positive, rod-shaped organisms recovered from eagles . The strains were provisionally identified as Corynebacterium jeikeium with the commercial API Coryne system, but they were able to grow under anaerobic conditions and were non-lipophilic . Comparative 16S rRNA gene sequencing studies demonstrated that the isolates belonged phylogenetically to the genus Corynebacterium . Three strains were identified genotypically as Corynebacterium falsenii; the remaining four strains corresponded to a hitherto unknown lineage within the genus Corynebacterium, associated with a small subcluster of species that included Corynebacterium diphtheriae and its close relatives . The unknown bacterial strains were readily distinguished from these and other species of the genus by biochemical tests . Based on both phenotypic and phylogenetic evidence, it is proposed that the unknown bacterial strains from eagles should be classified as Corynebacterium aquilae sp . nov . (type strain is S-613T = CECT 5993T = CCUG 46511T).

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1065 - 8
Corynebacterium atypicum sp . nov., from a human clinical source, does not contain corynomycolic acids; Hall V et al.; An unusual gram-positive, facultatively anaerobic, catalase-positive, diphtheroid-shaped organism originating from an unknown human clinical source was characterized by biochemical, molecular chemical and molecular phylogenetic methods . Based on its morphological and biochemical characteristics and the presence of a murein based on meso-diaminopimelic acid, the unidentified organism was tentatively assigned to the genus Corynebacterium . However, the unknown organism was found to lack the distinctive, short-chain corynomycolic acids that are considered to be characteristic of this genus . Despite the absence of these characteristic lipids, comparative 16S rRNA gene sequencing showed that the unknown bacterium was phylogenetically a member of the genus Corynebacterium and was distinct from all currently known species . Based on both phenotypic and 16S rRNA sequence considerations, it is proposed that the unknown organism be classified as a novel species, Corynebacterium atypicum sp . nov . The type strain of C . atypicum is strain R2070T (=CCUG 45804T=CIP 107431T).

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1009 - 12
Corynebacterium sphenisci sp . nov., isolated from wild penguins; Goyache J et al.; Six unidentified gram-positive, rod-shaped organisms recovered from the cloacae of apparently healthy wild penguins were characterized by phenotypic and molecular taxonomic methods . Chemotaxonomic investigations revealed the presence of a cell wall based on meso-diaminopimelic acid and long-chain cellular fatty acids of the straight-chain saturated and monounsaturated types, consistent with the genus Corynebacterium . Corynomycolic acids, which are characteristic of the genus, were also detected, albeit in small amounts . Comparative 16S rRNA gene sequencing studies showed that the unidentified organisms were phylogenetically related to corynebacteria and represent a novel subline associated with a small subcluster of species that includes Corynebacterium xerosis, Corynebacterium amycolatum and Corynebacterium freneyi . The unknown isolates were readily distinguished from their closest phylogenetic relatives and all other Corynebacterium species with validly published names by using a combination of biochemical and chemotaxonomic criteria . Based on both phenotypic and 16S rRNA gene sequence considerations, it is proposed that the unknown isolates recovered from penguins be classified as a novel species in the genus Corynebacterium, Corynebacterium sphenisci sp . nov . The type strain is CECT 5990T (= CCUG 46398T).

Mol Microbiol, 2003 Aug, 49(4), 1119 - 34
Three pathways for trehalose metabolism in Corynebacterium glutamicum ATCC13032 and their significance in response to osmotic stress; Wolf A et al.; Genome scanning of Corynebacterium glutamicum ATCC13032 revealed the presence of five different genes encoding enzymes belonging to three putative trehalose biosynthesis pathways (OtsAB, TreYZ, TreS) . The function of the different pathways and of trehalose as an osmoprotectant was studied by characterizing several strains defective for individual trehalose biosynthetic routes . Trehalose synthesis was shown to increase upon hyperosmotic conditions . Cytoplasmic trehalose levels varied considerably depending on kind and accessibility of carbon and nitrogen sources . In contrast to other organisms, osmoregulated trehalose synthesis in C . glutamicum is mediated by the TreYZ and not by the OtsAB pathway . Irrespective of their significance for the osmotic response, otsA and treS were upregulated at the transcriptional level after hyperosmotic shock . In vivo, TreS-mediated trehalose synthesis only occurred if maltose was used as the carbon source . In vitro, TreS catalysed the conversion of maltose into trehalose and, conversely, trehalose into maltose . As the reaction seems to be near equilibrium, TreS appears to be important for trehalose degradation rather than synthesis because a 1000-fold excess of trehalose to maltose was detected in the cytoplasm . Also, evidence is given that both the OtsAB and the TreYZ pathways are involved, but not essential, in supplying trehalose for mycolic acid biosynthesis.

Zh Mikrobiol Epidemiol Immunobiol, 2003 May-Jun, (3), 66 - 71
{Microflora of the nasal mucosa in allergic perennial and infectious rhinitis}; Romanenko EE et al.; The microbiological study of 69 patients with allergic annual rhinitis (AAR) and infectious rhinitis (IR) was carried out . In AAR the isolated representatives of 15 genera and 40 species were distributed in 2 to 7 component; in IR the isolated representatives of 16 genera and 25 species were grouped in 2 to 4-component associations . In AAR Staphylococcus aureus was found to belong to the main species and in IR, S . aureus and S . epidermidis, while the number of species regarded as occasional in AAR was 7 (S . auricularis, S . cohnii, S . hominis, S . haemolyticus, S . warneri, S . apitis, S . schleiferi) . Differences in the distribution of Neisseria, nonfermenting Gram negative bacteria, Streptococcus in associations in cases of AAR and IR were established . In AAR Corynebacterium pseudodiphthericum and in IR C . pseudotuberculosis were the dominant species.

Cornea, 2003 Aug, 22(6), 545 - 8
Effects of minocycline on the ocular flora of patients with acne rosacea or seborrheic blepharitis; Ta CN et al.; PURPOSE: To assess the effect of minocycline on the ocular flora in patients with acne rosacea or blepharitis . METHODS: A total of ten patients were enrolled in this prospective study, with six patients diagnosed with acne rosacea with concomitant meibomianitis, two patients with acne rosacea without concomitant ocular involvement, and two patients with seborrheic blepharitis . The eyelids and conjunctiva of both eyes were cultured before the initiation of systemic minocycline therapy, after 3 months of active therapy, and 3 months after the discontinuation of therapy . Isolated bacteria were identified and quantified, and antibiotic susceptibility was determined . RESULTS: The colony-forming units (CFU) isolated from the eyelids significantly decreased after a 3-month treatment with minocycline (P = 0.0013) . The CFU significantly increased to approach that of the baseline with the discontinuation of minocycline (P = 0.0275) . The most common isolated bacteria, including coagulase-negative Staphylococcus (CNS), Staphylococcus aureus (S . aureus), and Propionibacterium acne (P . acne), except for corynebacterium, had a significant decrease in bacterial count with minocycline therapy compared with baseline (P < 0.05) . There was a trend in the decrease of bacterial CFU isolated from the conjunctiva with minocycline therapy, although this was not statistically significant (P = 0.1955) . Four of the ten patients carried tetracycline-resistant CNS strains, but none of the S . aureus or P . acne isolated at baseline was resistant to tetracycline . All six patients with acne rosacea and concomitant meibomianitis had marked clinical improvement . CONCLUSION: Minocycline effectively decreased eyelid bacterial flora in patients with acne rosacea or blepharitis . One of the mechanisms of newer generation tetracycline analogues may be a decrease or elimination of bacterial flora from the eyelids.

Biotechnol Lett, 2003 Mar, 25(5), 377 - 80
Characterization and application of an optical sensor for quantification of dissolved O2 in shake-flasks; Wittmann C et al.; On-line measurement of dissolved O2 in shake-flasks was realized via immobilized sensor spots containing a fluorophore with an O2-dependent luminescent decay time . An unaffected sensor signal during 80 autoclaving cycles suggests multi-usage of sensor equipped shake-flasks . The sensor had a response time of 6 s . Quantification of gas-liquid mass transfer revealed maximum kLa values of 150 h(-1), from which maximum O2 transfer capacity of 33 mM h(-1) was calculated . Liquid volume and shaking frequency have a strong influence on kLa . Exemplified by cultivations of Corynebacterium glutamicum the importance of shaking rate for O2 supply of bacterial cultures is shown . Sampling of microbial cultures with intermittent shaking of a few minutes can cause O2 limitation . Based on the results of this work a simple and straightforward tool is now available for accurate O2 sensing in shake-flasks, which are widely used in microbial cultivations.

Biochem J, 2003 Jul 15, 373(Pt 2), 465 - 74
Transposon-5 mutagenesis transforms Corynebacterium matruchotii to synthesize novel hybrid fatty acids that functionally replace corynomycolic acid; Takayama K et al.; Enzymes within the biosynthetic pathway of mycolic acid (C(60)-C(90) a-alkyl,b-hydroxyl fatty acid) in Mycobacterium tuberculosis are attractive targets for developing new anti-tuberculosis drugs . We have turned to the simple model system of Corynebacterium matruchotii to study the terminal steps in the anabolic pathway of a C32 mycolic acid called corynomycolic acid . By transposon-5 mutagenesis, we transformed C . matruchotii into a mutant that is unable to synthesize corynomycolic acid . Instead, it synthesized two related series of novel fatty acids that were released by saponification from the cell wall fraction and from two chloroform/methanol-extractable glycolipids presumed to be analogues of trehalose mono- and di-corynomycolate . By chemical analyses and MS, we determined the general structure of the two series to be 2,4,6,8,10-penta-alkyl decanoic acid for the larger series (C(70)-C(77)) and 2,4,6,8-tetra-alkyl octanoic acid for the smaller series (C(52)-C(64)), both containing multiple keto groups, hydroxy groups and double bonds . The mutant was temperature-sensitive, aggregated extensively, grew very slowly relative to the wild type, and was resistant to the presence of lysozyme . We suggest that a regulatory protein that normally prevents the transfer of the condensation product back to b-ketoacyl synthase in the corynomycolate synthase system of the wild type was inactivated in the mutant . This will result in multiple Claisen-type condensation and the formation of two similar series of these complex hybrid fatty acids . A similar protein in M . tuberculosis would be an attractive target for new drug discovery.

Arch Microbiol, 2003 Sep, 180(3), 155 - 60 Epub 2003 Jul 15.
New ubiquitous translocators: amino acid export by Corynebacterium glutamicum and Escherichia coli; Eggeling L et al.; Molecular access to amino acid excretion by Corynebacterium glutamicum and Escherichia coli led to the identification of structurally novel carriers and novel carrier functions . The exporters LysE, RhtB, ThrE and BrnFE each represent the protoype of new transporter families, which are in part distributed throughout all of the kingdoms of life . LysE of C . glutamicum catalytes the export of basic amino acids . The expression of the carrier gene is regulated by the cell-internal concentration of basic amino acids . This serves, for example, to maintain homoeostasis if an excess of l-lysine or l-arginine inside the cell should arise during growth on complex media . RhtB is one of five paralogous systems in E . coli, of which at least two are relevant for l-threonine production . A third system is relevant for l-cysteine production . It is speculated that the physiological function of these paralogues is related to quorum sensing . ThrE of C . glutamicum exports l-threonine and l-serine . However, a ThrE domain with a putative hydrolytic function points to an as yet unknown role of this exporter . BrnFE in C . glutamicum is a two-component permease exporting branched-chained amino acids from the cell, and an orthologue in B . subtilis exports 4-azaleucine.

Scand J Infect Dis, 2003, 35(5), 315 - 7
Role of genital mycoplasmata and other bacteria in urolithiasis; Kaya S et al.; Urease-producing bacteria have been shown to affect the formation of infection stones by splitting urea into ammonia, bicarbonate and carbonate . An increase in alkaline pH results in urinary supersaturation of the ions . The increase in ammonia also causes injury to the urothelial glycosaminoglycan layer . Non-urease-producing bacteria have been speculated to form urinary stones . Midstream voided bladder urine and fractured stone nidus samples from 72 patients undergoing surgery for urolithiasis were cultured on specific media for genital mycoplasmata and on conventional media . Urine samples were obtained from a control group of 40 healthy subjects . Genital mycoplasmata and other bacteria were evaluated with regard to the composition of urinary stones . Compared with other origins of stones, the relation between isolation of Ureaplasma urealyticum and infection stone disease was statistically proven . Isolation of genital mycoplasmata was significantly higher in women than in men in the study group . The urinary stones comprised 84.7% calcium stones, 8.3% uric acid stones and 6.9% infection (magnesium ammonium phosphate) stones . Coagulase-negative Staphylococci, Escherichia coli, Corynebacterium spp., Enterobacterium spp . and U . urealyticum were cultured from stone samples . The results suggests that non-urease-producing bacteria, as well as urease-producing bacteria, may influence the formation of urinary stones.

Kansenshogaku Zasshi, 2003 Jun, 77(6), 456 - 60
{A case of community-acquired pneumonia caused by Corynebacterium propinquum}; Furumoto A et al.; Corynebacterium propinquum, which is classified as Corynebacterium ANF-3, has not yet been described as a cause of the respiratory infections . In this paper, we reported a case of 67-year-old immunocompetent male with community-acquired pneumonia caused by C . propinquum . Our study suggested that Gram staining of the pulurent sputum was the most important diagnostic tool to determine the pathogenecity of this organism.

J Bacteriol, 2003 Aug, 185(15), 4519 - 29
The phosphate starvation stimulon of Corynebacterium glutamicum determined by DNA microarray analyses; Ishige T et al.; The phosphate (P(i)) starvation stimulon of Corynebacterium glutamicum was characterized by global gene expression analysis by using DNA microarrays . Hierarchical cluster analysis of the genes showing altered expression 10 to 180 min after a shift from P(i)-sufficient to P(i)-limiting conditions led to identification of five groups comprising 92 genes . Four of these groups included genes which are not directly involved in P metabolism and changed expression presumably due to the reduced growth rate observed after the shift or to the exchange of medium . One group, however, comprised 25 genes, most of which are obviously related to phosphorus (P) uptake and metabolism and exhibited 4- to >30-fold-greater expression after the shift to P(i) limitation . Among these genes, the RNA levels of the pstSCAB (ABC-type P(i) uptake system), glpQ (glycerophosphoryldiester phosphodiesterase), ugpAEBC (ABC-type sn-glycerol 3-phosphate uptake system), phoH (unknown function), nucH (extracellular nuclease), and Cgl0328 (5'-nucleotidase or related esterase) genes were increased, and pstSCAB exhibited a faster response than the other genes . Transcriptional fusion analyses revealed that elevated expression of pstSCAB and ugpAEBC was primarily due to transcriptional regulation . Several genes also involved in P uptake and metabolism were not affected by P(i) starvation; these included the genes encoding a PitA-like P(i) uptake system and a putative Na(+)-dependent P(i) transporter and the genes involved in the metabolism of pyrophosphate and polyphosphate . In summary, a global, time-resolved picture of the response of C . glutamicum to P(i) starvation was obtained.

Microbiology, 2003 Jul, 149(Pt 7), 1675 - 85
Phospholipid composition of several clinically relevant Corynebacterium species as determined by mass spectrometry: an unusual fatty acyl moiety is present in inositol-containing phospholipids of Corynebacterium urealyticum; Yague G et al.; A comparative study on phospholipids of Corynebacterium amycolatum, Corynebacterium jeikeium and Corynebacterium urealyticum was carried out using fast-atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry . Data obtained indicate the presence of acylphosphatidylglycerol (APG), diphosphatidylglycerol, phosphatidylglycerol (PG), phosphatidylinositol (PI) and triacylphosphatidylinositol dimannosides (Ac(3)PIM(2)) in these bacteria . In general, octadecenoyl and hexadecanoyl fatty acyl moieties predominated in phospholipids of C . amycolatum, whereas high levels of hexadecenoyl were found in C . jeikeium and C . urealyticum . Mass spectra from purified APG and PG indicated that the sn-1 position of the glycerol was occupied by octadecenoyl in the three species studied . Notably, several major molecular species of PI and Ac(3)PIM(2) from C . urealyticum contained significant amounts of a moiety identified as 10-methyleneoctadecanoyl, located at the sn-1 position of these molecules . On the other hand, multiantibiotic resistant and susceptible strains of C . amycolatum differed in several minor phospholipid fatty acids of 19 carbon atoms, identified as 10-methyloctadecenoic, 10-methyloctadecanoic (tuberculostearic acid) and 10-methyleneoctadecanoic . The results demonstrate an overall similarity among the phospholipids of the different species studied but also significant differences related to the acyl chains of the glycerol moiety of these compounds, notably the high levels of an unusual fatty acyl moiety in inositol-containing phospholipids of C . urealyticum.

Microbiology, 2003 Jul, 149(Pt 7), 1659 - 73
Genetic dissection of trehalose biosynthesis in Corynebacterium glutamicum: inactivation of trehalose production leads to impaired growth and an altered cell wall lipid composition; Tzvetkov M et al.; The analysis of the available Corynebacterium genome sequence data led to the proposal of the presence of all three known pathways for trehalose biosynthesis in bacteria, i.e . trehalose synthesis from UDP-glucose and glucose 6-phosphate (OtsA-OtsB pathway), from malto-oligosaccharides or alpha-1,4-glucans (TreY-TreZ pathway), or from maltose (TreS pathway) . Inactivation of only one of the three pathways by chromosomal deletion did not have a severe impact on C . glutamicum growth, while the simultaneous inactivation of the OtsA-OtsB and TreY-TreZ pathway or of all three pathways resulted in the inability of the corresponding mutants to synthesize trehalose and to grow efficiently on various sugar substrates in minimal media . This growth defect was largely reversed by the addition of trehalose to the culture broth . In addition, a possible pathway for glycogen synthesis from ADP-glucose involving glycogen synthase (GlgA) was discovered . C . glutamicum was found to accumulate significant amounts of glycogen when grown under conditions of sugar excess . Insertional inactivation of the chromosomal glgA gene led to the failure of C . glutamicum cells to accumulate glycogen and to the abolition of trehalose production in a DeltaotsAB background, demonstrating that trehalose production via the TreY-TreZ pathway is dependent on a functional glycogen biosynthetic route . The trehalose-non-producing mutant with inactivated OtsA-OtsB and TreY-TreZ pathways displayed an altered cell wall lipid composition when grown in minimal broth in the absence of trehalose . Under these conditions, the mutant lacked both major trehalose-containing glycolipids, i.e . trehalose monocorynomycolate and trehalose dicorynomycolate, in its cell wall lipid fraction . The results suggest that a dramatically altered cell wall lipid bilayer of trehalose-less C . glutamicum mutants may be responsible for the observed growth deficiency of such strains in minimal medium . The results of the genetic and physiological dissection of trehalose biosynthesis in C . glutamicum reported here may be of general relevance for the whole phylogenetic group of mycolic-acid-containing coryneform bacteria.

FEMS Microbiol Lett, 2003 Jul 15, 224(1), 35 - 44
New insights into the biogenesis of the cell envelope of corynebacteria: identification and functional characterization of five new mycoloyltransferase genes in Corynebacterium glutamicum; De Sousa-D'Auria C et al.; Mycolic acids, the major lipid constituents of Corynebacterineae, play an essential role in maintaining the integrity of the bacterial cell envelope . We have previously characterized a corynebacterial mycoloyltransferase (PS1) homologous in its N-terminal part to the three known mycobacterial mycoloyltransferases, the so-called fibronectin-binding proteins A, B and C . The genomes of Corynebacterium glutamicum (ATCC13032 and CGL2005) and Corynebacterium diphtheriae were explored for the occurrence of other putative corynebacterial mycoloyltransferase-encoding genes (cmyt) . In addition to csp1 (renamed cmytA), five new cmyt genes (cmytB-F) were identified in the two strains of C . glutamicum and three cmyt genes in C . diphtheriae . In silico analysis showed that each of the putative cMyts contains the esterase domain, including the three key amino acids necessary for the catalysis . In C . glutamicum CGL2005 cmytE is a pseudogene . The four new cmyt genes were disrupted in this strain and overexpressed in the inactivated strains . Quantitative analyses of the mycolate content of all these mutants demonstrated that each of the new cMyt-defective strains, except cMytC, accumulated trehalose monocorynomycolate and exhibited a lower content of covalently bound corynomycolate than did the parent strain . For each mutant, the mycolate content was fully restored by complementation with the corresponding wild-type gene . Finally, complementation of the cmytA-inactivated mutant by the individual new cmyt genes established the existence of two classes of mycoloyltransferases in corynebacteria.

Urologe A, 2003 Jun, 42(6), 834 - 9 Epub 2003 Feb 07.
{Bladder rupture caused by spontaneous perforation of an infected urachal cyst}; Maruschke M et al.; Anomalies of the fetal urachus are rare . Normally, the postnatal urachus presents as a fibrous band extending from the bladder to the umbilicus . Urachal cysts may occur in postnatal life . Spontaneous perforation of urachal cysts is a very rare condition, which clinically may not be distinguishable from other acute abdominal conditions . We report a case of a 63-year-old male with a history of recurrent urinary tract infections and a bladder rupture caused by a spontaneous perforation of an infected urachal cyst . The symptomatology showed abdominal rigidity and pain, a palpable mass in the lower abdomen, and hematuria . Laboratory findings showed leukocytosis and an increased CRP level . The bladder rupture was confirmed by cystography . Bacteriologic examination identified Proteus vulgaris, Corynebacterium species, and Klebsiella pneumoniae . Most of the published cases in the literature report about intraperitoneal perforation of infected urachal cysts . In the present case, we found a spontaneous perforation of an infected urachal cyst leading to an extraperitoneal bladder rupture with an extraperitoneal limitation of the infection . The definitive therapy was complete surgical excision including a cuff of the bladder, drainage, and systemic broad-spectrum and local application of antibiotics . The further course was uneventful.

Metab Eng, 2003 Apr, 5(2), 96 - 107
Production process monitoring by serial mapping of microbial carbon flux distributions using a novel Sensor Reactor approach: II--(13)C-labeling-based metabolic flux analysis and L-lysine production; Drysch A et al.; Corynebacterium glutamicum is intensively used for the industrial large-scale (fed-) batch production of amino acids, especially glutamate and lysine . However, metabolic flux analyses based on 13C-labeling experiments of this organism have hitherto been restricted to small-scale batch conditions and carbon-limited chemostat cultures, and are therefore of questionable relevance for industrial fermentations . To lever flux analysis to the industrial level, a novel Sensor Reactor approach was developed (El Massaoudi et al., Metab . Eng., submitted), in which a 300-L production reactor and a 1-L Sensor Reactor are run in parallel master/slave modus, thus enabling 13C-based metabolic flux analysis to generate a series of flux maps that document large-scale fermentation courses in detail . We describe the successful combination of this technology with nuclear magnetic resonance (NMR) analysis, metabolite balancing methods and a mathematical description of 13C-isotope labelings resulting in a powerful tool for quantitative pathway analysis during a batch fermentation . As a first application, 13C-based metabolic flux analysis was performed on exponentially growing, lysine-producing C . glutamicum MH20-22B during three phases of a pilot-scale batch fermentation . By studying the growth, (co-) substrate consumption and (by-) product formation, the similarity of the fermentations in production and Sensor Reactor was verified . Applying a generally applicable mathematical model, which included metabolite and carbon labeling balances for the analysis of proteinogenic amino acid 13C-isotopomer labeling data, the in vivo metabolic flux distribution was investigated during subsequent phases of exponential growth . It was shown for the first time that the in vivo reverse C(4)-decarboxylation flux at the anaplerotic node in C . glutamicum significantly decreased (70%) in parallel with threefold increased lysine formation during the investigated subsequent phases of exponential growth.

Appl Microbiol Biotechnol, 2003 Oct, 62(5-6), 459 - 67 Epub 2003 Jul 04.
Methionine biosynthesis and its regulation in Corynebacterium glutamicum: parallel pathways of transsulfuration and direct sulfhydrylation; Lee HS et al.; There are two alternative pathways leading to methionine synthesis in microorganisms: The transsulfuration pathway involves cystathionine as the intermediate and utilizes cysteine as the sulfur source, but the direct sulfhydrylation pathway bypasses cystathionine and uses inorganic sulfur instead . While most microorganisms synthesize methionine via either one of these pathways, Corynebacterium glutamicum utilizes both pathways, which appear to be fully functional . In C . glutamicum, each pathway is catalyzed by independent enzymes and is tightly regulated by methionine . Although the physiological significance of parallel pathways remains to be elucidated, their presence suggests metabolic flexibility and efficient adaptation of the organism to its environment.

Genome Res, 2003 Jul, 13(7), 1572 - 9
Comparative complete genome sequence analysis of the amino acid replacements responsible for the thermostability of Corynebacterium efficiens; Nishio Y et al.; Corynebacterium efficiens is the closest relative of Corynebacterium glutamicum, a species widely used for the industrial production of amino acids . C . efficiens but not C . glutamicum can grow above 40 degrees C . We sequenced the complete C . efficiens genome to investigate the basis of its thermostability by comparing its genome with that of C . glutamicum . The difference in GC content between the species was reflected in codon usage and nucleotide substitutions . Our comparative genomic study clearly showed that there was tremendous bias in amino acid substitutions in all orthologous ORFs . Analysis of the direction of the amino acid substitutions suggested that three substitutions are important for the stability of the C . efficiens proteins: from lysine to arginine, serine to alanine, and serine to threonine . Our results strongly suggest that the accumulation of these three types of amino acid substitutions correlates with the acquisition of thermostability and is responsible for the greater GC content of C . efficiens.

J Biol Chem, 2003 Sep 19, 278(38), 36582 - 7 Epub 2003 Jul 02.
Regulation of intracellular heme levels by HMX1, a homologue of heme oxygenase, in Saccharomyces cerevisiae; Protchenko O et al.; Saccharomyces cerevisiae responds to iron deprivation by increasing the transcription of genes involved in the uptake of environmental iron and in the mobilization of vacuolar iron stores . HMX1 is also transcribed under conditions of iron deprivation and is under the control of the major iron-dependent transcription factor, Aft1p . Although Hmx1p exhibits limited homology to heme oxygenases, it has not been shown to be enzymatically active . We find that Hmx1p is a resident protein of the endoplasmic reticulum and that isolated yeast membranes contain a heme degradation activity that is dependent on HMX1 . Hmx1p facilitates the capacity of cells to use heme as a nutritional iron source . Deletion of HMX1 leads to defects in iron accumulation and to expansion of intracellular heme pools . These alterations in the regulatory pools of iron lead to activation of Aft1p and inappropriate activation of heme-dependent transcription factors . Expression of HmuO, the heme oxygenase from Corynebacterium diphtheriae, restores iron and heme levels, as well as Aft1p- and heme-dependent transcriptional activities, to those of wild type cells, indicating that the heme degradation activity associated with Hmx1p is important in mediating iron and heme homeostasis . Hmx1p promotes both the reutilization of heme iron and the regulation of heme-dependent transcription during periods of iron scarcity.

Appl Environ Microbiol, 2003 Jul, 69(7), 3849 - 57
Characterization and molecular cloning of a novel enzyme, inorganic polyphosphate/ATP-glucomannokinase, of Arthrobacter sp . strain KM; Mukai T et al.; A bacterium exhibiting activities of several inorganic polyphosphate {poly(P)}- and ATP-dependent kinases, including glucokinase, NAD kinase, mannokinase, and fructokinase, was isolated, determined to belong to the genus Arthrobacter, and designated Arthrobacter sp . strain KM . Among the kinases, a novel enzyme responsible for the poly(P)- and ATP-dependent mannokinase activities was purified 2,200-fold to homogeneity from a cell extract of the bacterium . The purified enzyme was a monomer with a molecular mass of 30 kDa . This enzyme phosphorylated glucose and mannose with a high affinity for glucose, utilizing poly(P) as well as ATP, and was designated poly(P)/ATP-glucomannokinase . The K(m) values of the enzyme for glucose, mannose, ATP, and hexametaphosphate were determined to be 0.50, 15, 0.20, and 0.02 mM, respectively . The catalytic sites for poly(P)-dependent phosphorylation and ATP-dependent phosphorylation of the enzyme were found to be shared, and the poly(P)-utilizing mechanism of the enzyme was shown to be nonprocessive . The gene encoding the poly(P)/ATP-glucomannokinase was cloned from Arthrobacter sp . strain KM, and its nucleotide sequence was determined . This gene contained an open reading frame consisting of 804 bp coding for a putative polypeptide with a calculated molecular mass of 29,480 Da . The deduced amino acid sequence of the polypeptide exhibited homology to the amino acid sequences of the poly(P)/ATP-glucokinase of Mycobacterium tuberculosis H37Rv (level of homology, 45%), ATP-dependent glucokinases of Corynebacterium glutamicum (45%), Renibacterium salmoninarum (45%), and Bacillus subtilis (35%), and proteins of bacteria belonging to the order Actinomyces whose functions are not known . Alignment of these homologous proteins revealed seven conserved regions . The mannose and poly(P) binding sites of poly(P)/ATP-glucomannokinase are discussed.

Acta Neurochir (Wien), 2003 Jun, 145(6), 513 - 7; discussion 517
Intracranial tuberculomas mimicking a malignant disease in an immunocompetent patient; Giese A et al.; We present the unusual occurrence of multiple systemic and two central nervous system tuberculomas in an immunocompetent young patient . A large left frontal epidural tuberculoma with transcalvarian extension was removed surgically and chemotherapy was initiated . The patient remained on a chemotherapy with INH, RMP, and EMB and was followed clinically and with MRI scans for 24 months . Findings . The clinical presentation and neuroimaging studies initially suggested malignant disease . Surgical resection of the left frontal lesion was required to relieve the mass effect . The histological evaluation showed a granulomatous inflammation with epithelial and Langhans giant cells, but no acid-fast bacilli . Cultures of the specimens yielded a mixed infection with Corynebacterium species and Staphylococcus epidermidis . Based on the histological findings, chemotherapy for tuberculosis was initiated . Subsequently, Mycobacterium tuberculosis was cultured from the surgical specimen and sputum . Interpretation . Parenchymal CNS tuberculosis with or without extracerebral manifestations may present as a space-occupying lesion . Because a tuberculoma is rarely suspected especially if there is atypical morphology, biopsy is required to establish the diagnosis and expedite specific treatment.

Appl Microbiol Biotechnol, 2003 Jul, 62(1), 69 - 75 Epub 2003 Feb 20.
Efficient 40 degrees C fermentation of L-lysine by a new Corynebacterium glutamicum mutant developed by genome breeding; Ohnishi J et al.; We have recently developed a new L-lysine-producing mutant of Corynebacterium glutamicum by "genome breeding" consisting of characterization and reconstitution of a mutation set essential for high-level production . The strain AHP-3 was examined for L-lysine fermentation on glucose at temperatures above 35 degrees C, at which no examples of efficient L-lysine production have been reported for this organism . We found that the strain had inherited the thermotolerance that the original coryneform bacteria was endowed with, and thereby grew and produced L-lysine efficiently up to 41 degrees C . A final titer of 85 g/l after only 28 h was achieved at temperatures around 40 degrees C, indicating the superior performance of the strain developed by genome breeding . When compared with the traditional 30 degrees C fermentation, the 40 degrees C fermentation allowed an increase in yield of about 20% with a concomitant decrease in final growth level, suggesting a significant transition of carbon flux distribution in glucose metabolism . DNA array analysis of metabolic changes between the 30 degrees C and 40 degrees C fermentations identified several differentially expressed genes in central carbon metabolism although we could not find stringent control-like global induction of amino-acid-biosynthetic genes in the 40 degrees C fermentation . Among these changes, two candidates were picked out as the potential causes of the increased production at 40 degrees C; decreased expression of the citrate synthase gene gltA and increased expression of malE, the product of which involves regeneration of pyruvate and NADPH.

Intensive Care Med, 2003 Aug, 29(8), 1376 - 9 Epub 2003 Jun 26.
Fatal septic shock caused by Corynebacterium D2; Audard V et al.; BACKGROUND: Septic shock remains one of the leading causes of mortality in critically ill patients . Optimal management depends on prompt diagnosis with identification of the causative organisms to allow appropriate antibiotic therapy . PATIENT: We report the first case of septic shock caused by Corynebacterium D2, a micro-organism that can cause encrusted cystitis and pyelitis of transplanted kidneys or, more rarely, native kidneys . Diagnosis rests on identification of risk factors, positive urine cultures, and computed tomography results . Despite optimal treatment our patient died with persistent encrusted pyelitis . CONCLUSIONS: Corynebacterium D2 is known to cause chronic inflammation of the bladder and proximal urinary tract but can also cause severe septic shock in immunocompetent patients.

Appl Microbiol Biotechnol, 2003 Dec, 63(2), 200 - 6 Epub 2003 Jun 24.
A novel polygalacturonic acid bioflocculant REA-11 produced by Corynebacterium glutamicum: a proposed biosynthetic pathway and experimental confirmation; Li Y et al.; Corynebacterium glutamicum CCTCC M201005 produces a novel polygalacturonic acid bioflocculant, REA-11, consisting of galacturonic acid as the main structural unit . A biosynthetic pathway of REA-11 in C . glutamicum CCTCC M201005 was proposed . Evidence for the biosynthetic pathway was provided by: (1) analyzing the response upon addition of UDP-glucose to the culture medium; (2) detecting the presence of several key intermediates in the pathway; and (3) correlating the activities of several key enzymes involved in the pathway with the yields of polygalacturonic acid . The production of polygalacturonic acid was improved by 24%, while the activities of UDP-galactose epimerase and UDP-galactose dehydrogenase were improved by 200% and 50%, respectively, upon addition of 100 microM UDP-glucose . In addition, the key intermediates in the proposed biosynthetic pathway, such as UDP-glucose, UDP-galactose, and UDP-glucuronic acid, were detected in cell-free extracts . Furthermore, the activities of UDP-glucose pyrophosphorylase (R2=0.97), UDP-galactose epimerase (R2=0.75) and UDP-galactose dehydrogenase (R2=0.89) were well correlated with the yields of polygalacturonic acid when different sugars were used as sole carbon sources . Therefore, the biosynthetic pathway of REA-11 in C . glutamicum CCTCC M201005 starts from phosphate-1-glucose, which was then converted to UDP-glucose by UDP-pyrophosphorylase . Predominantly, the UDP-glucose was converted to UDP-galactose by UDP-galactose epimerase; the latter was further converted to UDP-galacturonic acid by UDP-galactose dehydrogenase, which was presumably polymerized to polygalacturonic acid bioflocculant REA-11 by an unknown glucosyltransferase and a polymerase.

J Med Microbiol, 2003 Jul, 52(Pt 7), 599 - 602
Septicaemia due to Corynebacterium striatum: molecular confirmation of entry via the skin; Martin MC et al.; Septicaemia due to Corynebacterium striatum occurs infrequently . A case of C . striatum septicaemia with a known skin focus is reported in a 69-year-old male with ischaemia, refractory anaemia and treated for thyroid cancer . The characterization and typing of blood and cutaneous isolates was carried out using biochemical and DNA molecular typing methods to analyse the isolates . This is the first reported case with a documented source.

Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 705 - 9
Corynebacterium glaucum sp . nov; Yassin AF et al.; A bacterial strain, strain IMMIB R-5091(T), isolated from a cosmetic dye was characterized by phenotypic and molecular taxonomic methods . Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and short-chain mycolic acids consistent with the genus Corynebacterium . Comparative 16S rRNA gene sequencing showed that the isolate constitutes a distinct subline within the genus Corynebacterium, displaying > 2.6% sequence divergence from established species . The isolate could be distinguished from other members of the genus Corynebacterium by biochemical tests . Based on both phenotypic and phylogenetic evidence, it is proposed that strain IMMIB R-5091(T) (= DSM 44530T = NRRL B-24142(T)) be classified as the type strain of a novel species, Corynebacterium glaucum sp . nov.

Rev Soc Bras Med Trop, 2003 Mar-Apr, 36(2), 193 - 9 Epub 2003 Jun 10.
Identification and purification of immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine (Leishvacin ); Cardoso SR et al.; Immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine against American tegumentary leishmaniasis (Leishvacin ), produced by Biobr s (Biochemistry of Brazil ), Montes Claros, State of Minas Gerais, Brazil, were identified and purified by polyacrylamide electrophoresis gel and electroelution . C57BL/10 mice were vaccinated with proteins with estimated molecular weights of 42, 46, 63, 66, 73, 87, 97, and 160kDa in three doses of 30 g of each protein at 15-day intervals combined with 250 microg of Corynebacterium parvum followed by a challenge infection with 10(5) infective promastigotes from Leishmania (Leishmania) amazonensis . The ability of these proteins to induce immune response and protection was analyzed . No statistical difference was observed in the level of IFN-gamma induced by proteins in vaccinated groups in comparison with control groups . Six months after challenge infection, protection levels of 28.57; 42.86; 57.14; 42.86; 42.86, 57.14; 42.86 and 57.14% were demonstrated for each purified protein.

Trop Anim Health Prod, 2003 Jun, 35(3), 197 - 205
Bovine mastitis in selected areas of southern Ethiopia; Dego OK et al.; A study on bovine mastitis, designed to determine the causal agents, prevalence of infection and impact of risk factors in three cattle breeds, was conducted in selected areas of southern Ethiopia . A total of 307 lactating and non-lactating cows, of which 162 were indigenous Zebu, 85 Jersey and 60 Holstein-Friesian . were examined by clinical examination and the California mastitis (CMT) test . Of these, 40.4% were positive by CMT and bacteriology for clinical or subclinical mastitis, with prevalence rates of 37.1% and 62.9%, respectively . Out of 1133 quarters examined, 212 (18.7%) were found to be infected, 83 (39.21%) clinically and 129 (60.8%) subclinically . The prevalence of mastitis was significantly higher in Holstein-Friesian than in indigenous Zebu, in non-lactating cows than in lactating cows, in the early lactation stage than in the mid-lactation stage, in cows with lesions and/or tick infestation on skin of udder and/or teats than in cows without this factor, and in the wet season than in the dry season . Mastitis increased with parity number (R = 0.9) . Of 248 CMT and clinically positive udder quarter samples analysed microbiologically, 212 were culturally positive for known mastitis pathogens and 36 were negative . Of the 199 positive samples . Staphylococcus accounted for 39.2% . Streptococcus for 23.6%, coliforms for 14.1%, Micrococcus and Bacillus species for 8.0% each and Actinomyces or Arcanobacterium (Corynebacterium) for 7.0% . It was concluded that there was a high prevalence of clinical and subclinical mastitis, mainly caused by Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli, in this study area.

J Clin Microbiol, 2003 Jun, 41(6), 2777 - 8
Corynebacterium freneyi bacteremia; Auzias A et al.; Corynebacterium freneyi is a recently described alpha-glucosidase-positive species of the genus CORYNEBACTERIUM: To our knowledge, there is no description of human infection due to this species . We report on a case of bacteremia due to C . freneyi following vascular surgery.

Transpl Infect Dis, 2003 Mar, 5(1), 53 - 8
Multidrug-resistant Corynebacterium striatum pneumonia in a heart transplant recipient; Tarr PE et al.; Corynebacterium striatum is a rare, but likely underreported, cause of serious infections in immunocompromised hosts and generally is susceptible to multiple classes of antimicrobial agents . Here we report the first case of C . striatum infection in a solid organ transplant recipient . Three years after heart transplantation, a 58-year-old man developed bilateral pneumonia and pulmonary embolism . He did not improve with levofloxacin, piperacillin/tazobactam, and heparin treatment . A homogeneous population of abundant gram-positive rods was repeatedly demonstrated in sputum and bronchoalveolar lavage fluid, and C . striatum was grown in pure culture . The isolate was unusual for its multidrug-resistant (MDR) antimicrobial susceptibility pattern . The pneumonia resolved with 4 weeks of vancomycin therapy, in combination with rifampin given only during the first 2 weeks of treatment . The isolation of coryneforms ("diphtheroids") is often attributed to contamination . Their abundant presence on direct examination of specimens and/or their growth in pure culture suggest a pathogenic role, however, and indicate the need for accurate microbiological identification, particularly in immunocompromised hosts who have been hospitalized and previously treated with antibiotics . Combination therapy that includes vancomycin may be the most prudent treatment for MDR C . striatum infections.

Eur J Biochem, 2003 Jun, 270(12), 2622 - 6
31P NMR studies of energy metabolism in xanthosine-5'-monophosphate overproducing Corynebacterium ammoniagenes; Noguchi Y et al.; Corynebacterium ammoniagenes is an overproducer of xanthosine-5'-monophosphate (XMP) by consuming either glcose (glc) or glutamic acid (glu) . Its energy metabolism was studied in vivo using 31P NMR spectroscopy coupled with a circulating fermentation system (CFS) . CFS enabled us to validate directly the cellular dependency on carbon sources and changes in biomolecules produced according to alterations in the cellular energetic status . For the most efficient XMP production, the glutamic acid and glcose molar ratios (glu/glc) in the medium were adjusted to a molar ratio of 0.31 . The 31P NMR illustrated the two distinct phases of the cellular energetic status due to the availability of the substrates from the medium . In the earlier phase, both glc and glu were utilized, resulting in average ATP and ADP concentrations in cells of 0.50 +/- 0.17 micro mol.g-1 of dry cell weight (DCW) and an undetermined level, respectively . The ADP concentration in the later phase increased to 2.15 +/- 1.30 micro mol.g-1 of DCW, while the ATP concentration decreased to an undetectable level in association with a remarkable decrease in XMP production . This decrease in the XMP-producing ability was associated with an increase in production of the by-product hypoxanthine . Because glu was found to be consumed completely during the earlier phase, glc was the only available substrate in the later phases . These findings by in vivo NMR indicate that changes in the carbon metabolism profoundly affect XMP production by C . ammoniagenes.

Pneumologie, 2003 May, 57(5), 259 - 67
{Unusual gram positive rods, causing pneumonia}; Bar W et al.; In the last decade, a growing number of patients with pneumonia, caused by unusual gram positive rods have been observed . Mostly, the patients had been infected as a consequence of impaired immunity . In some cases, bioterrorist activities may also induce pneumonia by gram positive rods (B . anthracis) . In order to bring these organisms to the attention of the medical community, we present three clinical cases and describe six species of gram positive rods, known to provoke this kind of pneumonias . Case 1 was a 84 years old patient with impaired lung function . He was suspicious of tuberculosis (Tbc) . Nocardia spec . was isolated . Case 2 was an alcoholic of 46 years with pneumonia . Reactivation of Tbc was suspected . Actinomadura madurae has been isolated . Case 3 was a patient of 58 years with myelodysplastic syndrome (MDS) and pneumonia . N . asteroides was isolated . All patients shared impaired immunity (age, alcoholism, MDS) with impaired lung functions; Tbc had been suspected (Case 1 + 2) . Infection by A . madurae was contained by Clindamycin . Therapy of Nocardia with Moxifloxacin (Case 1) or Bactrim (Case 3) was only partly effective . In the appendix, six species of gram positive rods which are known to cause pneumonia, are summarized (Nocardia, Actinomyceta, Actinomadura, Rhodococcus, Corynebacterium and Bacillus).

Microbiology, 2003 Jun, 149(Pt 6), 1569 - 80
A single V317A or V317M substitution in Enzyme II of a newly identified beta-glucoside phosphotransferase and utilization system of Corynebacterium glutamicum R extends its specificity towards cellobiose; Kotrba P et al.; A catabolic system involved in the utilization of beta-glucosides in Corynebacterium glutamicum R and its spontaneous mutant variants allowing uptake of cellobiose were investigated . The system comprises a beta-glucoside-specific Enzyme IIBCA component (gene bglF) of the phosphotransferase system (PTS), a phospho-beta-glucosidase (bglA) and an antiterminator protein (bglG) from the BglG/SacY family of transcription regulators . The results suggest that transcription antitermination is involved in control of induction and carbon catabolite repression of bgl genes, which presumably form an operon . Functional analysis of the bglF and bglA products revealed that they are simultaneously required for uptake, phosphorylation and breakdown of methyl beta-glucoside, salicin and arbutin . Although cellobiose is not normally a substrate for BglF permease and is not utilized by C . glutamicum R, cellobiose-utilizing mutants can be obtained . The mutation responsible was mapped to the bgl locus and sequenced, and point mutations were found in codon 317 of bglF . These led to substitutions V317A and/or V317M near the putative PTS active-site H313 in the membrane-spanning IIC domain of BglF and allowed BglF to act on cellobiose . Such results strengthen the evidence that the IIC domains can be regarded as selectivity filters of the PTS.

J Lipid Res, 2003 Aug, 44(8), 1574 - 80 Epub 2003 Jun 01.
Sphingomyelinase D, a novel probe for cellular sphingomyelin: effects on cholesterol homeostasis in human skin fibroblasts; Subbaiah PV et al.; Sphingomyelin (SM) and free cholesterol (FC) are concentrated in the plasma membranes of eukaryotes; however, the physiological significance of their association is unclear . A common tool for studying the role of membrane SM is digestion with bacterial sphingomyelinase (SMase) C, which hydrolyzes SM to ceramide . However, it is not known whether the observed effects of SMase C treatment are due to the loss of SM per se or to the signaling effects of ceramide . In this study, we tested SMase D from Corynebacterium pseudotuberculosis, which hydrolyzes SM to ceramide phosphate, as an alternative probe . This enzyme specifically hydrolyzed SM in fibroblasts without causing accumulation of ceramide . Treatment of fibroblasts with SMase D stimulated translocation of PM FC to intracellular sites by <20% of the rate observed after SMase C digestion . The cells regenerated SM nearly completely within 5 h after SMase C treatment . However, even after 20 h, no regeneration occurred following SMase D digestion . These findings suggest that the translocation of PM FC caused by SMase C digestion is due to the cellular effects of ceramide rather than the loss of SM . Since ceramide phosphate does not appear to have such effects, we suggest that SMase D is a useful probe of membrane SM.

J Biotechnol, 2003 Jun 12, 103(1), 51 - 65
The putative transcriptional repressor McbR, member of the TetR-family, is involved in the regulation of the metabolic network directing the synthesis of sulfur containing amino acids in Corynebacterium glutamicum; Rey DA et al.; In order to isolate transcriptional regulatory proteins involved in L-methionine-dependent repression in Corynebacterium glutamicum, proteins binding to the putative promoter region upstream of the metY gene were isolated by DNA affinity chromatography . One of the isolated proteins was identified as a putative transcriptional repressor of the TetR-family by a mass spectrometry fingerprint technique based on the complete C . glutamicum genome sequence . The respective gene, designated mcbR, was deleted in the mutant strain C . glutamicum DR1 . Using 2D-PAGE, the protein contents of the C . glutamicum wild type and the mutant strain DR1 grown in media with or without L-methionine supplementation were compared and a set of six proteins was identified . Their abundance was drastically enhanced in the mutant strain and no longer influenced by L-methionine added to the growth medium . The corresponding genes were identified by mass spectrometry fingerprint analysis . They included metY encoding O-acetyl-L-homoserine sulfhydrylase, metK encoding S-adenosyl-methionine synthethase, hom encoding homoserine dehydrogenase, cysK encoding L-cysteine synthase, cysI encoding an NADPH dependant sulfite reductase, and ssuD encoding an alkanesulfonate monooxygenase . Evidently, the putative transcriptional repressor McbR is involved in the regulation of the metabolic network directing the synthesis of L-methionine in C . glutamicum . The C . glutamicum mcbR mutant can be considered to represent a first step in the construction of an L-methionine production strain.

Biochimie, 2003 Jan-Feb, 85(1-2), 153 - 66
Lipoarabinomannans: from structure to biosynthesis; Nigou J et al.; Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most effective human pathogens and the molecular basis of its virulence remains poorly understood . Here, we review our current knowledge about the structure and biosynthesis of the mycobacterial cell-wall lipoglycans, lipoarabinomannans (LAM) . LAM are ubiquitous of mycobacteria and appear as the most potent non-peptidic molecules to modulate the host immune response . Nevertheless, LAM structure differs according to the mycobacterial species and three types of LAM have been described: mannose-capped LAM (ManLAM), phospho-myo-inositol-capped LAM (PILAM) and non-capped LAM (AraLAM) . The type of capping is a major structural feature determining the ability of LAM to modulate the immune response . ManLAM, found in slow-growing mycobacteria, such as M . tuberculosis, have been demonstrated to be powerful anti-inflammatory molecules and emerge as key virulence factors that may be relevant drug targets . LAM-like molecules are not only confined to mycobacteria but are also present in actinomycetes (including the genera Rhodococcus, Corynebacterium or Gordonia) . This offers the possibility of comparative studies that should help in deciphering the structure-function relationships and biosynthesis of these complex molecules in the future.

Appl Microbiol Biotechnol, 2003 Jun, 61(5-6), 523 - 7 Epub 2003 Feb 20.
Host-vector system for phenol-degrading Rhodococcus erythropolis based on Corynebacterium plasmids; Vesely M et al.; The strain Rhodococcus erythropolis CCM2595, which was shown to degrade phenol, was chosen for genetic studies . To facilitate strain improvement using the methods of gene manipulation, the technique of genetic transfer was introduced and cloning vectors were constructed . Using the plasmid pFAJ2574, an electrotransformation procedure yielding up to 7x10(4) transformants/microg DNA was optimized . Escherichia coli- R . erythropolis shuttle vectors were constructed using the replicons pSR1 and pGA1 from Corynebacterium glutamicum . The small vector pSRK21 (5.8 kb) provides six unique cloning sites and selection of recombinant clones using alpha-complementation of beta-galactosidase in E . coli . This vector, exhibiting high segregational stability under non-selective conditions in R . erythropolis CCM2595, was applied to cloning and efficient expression of the gene coding for green fluorescent protein (gfpuv).

Eur J Biochem, 2003 May, 270(10), 2126 - 36
Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae . Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis; Griessler R et al.; Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic alpha1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5'-phosphate-containing subunits . In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure . To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C . callunae GP in Escherichia coli . By comparing multiple sequence alignment to structure-function assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C . callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability . Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis . Individual Arg-->Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 degrees C . R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively . The stabilization by 10 mm of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants . The replacement of Arg236 by Ala was functionally silent under all conditions tested . Arg234 and Arg242 thus partially destabilize the C . callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.

Metab Eng, 2003 Jan, 5(1), 32 - 41
Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression; Koffas MA et al.; Single gene overexpression in product pathways such as lysine synthesis has often been employed in metabolic engineering efforts aiming at pathway flux amplification and metabolite overproduction . This approach is limited due to metabolic flux imbalances that often lead to unpredictable physiological responses and suboptimal metabolite productivity . This deficiency can be overcome by the coordinated overexpression of more than one flux controlling genes in a production pathway selected by considering their individual contributions on the cell physiology This concept is demonstrated by the simultaneous overexpression of pyruvate carboxylase and aspartate kinase, two key enzymes in central carbon metabolism and the lysine production pathway in Corynebacterium glutamicum . Contrary to expectations based on the importance of each of these two genes in lysine production, the monocistronic overexpression of either gene results in marginal changes in the overall lysine productivity due to either reduced cell growth or reduced lysine specific productivity . In contrast, the simultaneous amplification of the activities of the two enzymes yielded more than 250% increase of the lysine specific productivity in lactate minimal medium without affecting the growth rate or final cell density of the culture . These results demonstrate that significant flux amplification in complex pathways involving central carbon metabolism is possible through coordinated overexpression of more than one gene in the pathway . This can be achieved either by external, gene expression inducing, controls or controls responding to the physiological cellular state.

J Fr Ophtalmol, 2003 Mar, 26(3), 255 - 8
{Bacterial contamination: epidemiology in cataract surgery}; Feys J et al.; PURPOSE: To evaluate anterior chamber (AC) bacterial contamination at the end of cataract surgery in a large series of patients, to determine the influence of operative technique on ocular contamination . METHODS: Retrospective study of 2,624 patients undergoing cataract extraction, 354 extracapsular cataract extraction (ECCE) and 2,270 phacoemulsification . Anterior chamber aspirates were performed on completion of surgery for microbiological studies . RESULTS: One hundred and thirty two patients (5%) had culture-positive anterior chamber aspirates . Coagulase-negative Staphylococcus, Propionibacterium sp . and Corynebacterium sp . were the most commonly isolated organisms . The AC contamination rates during ECCE (5.6%) and phacoemulsification (4.7%) were not statistically different . There was a statistically significantly higher risk of AC contamination in eyes receiving an intraocular lens (IOL) with polypropylene haptics (9.9%) than in eyes receiving the same IOL with polymethylmethacrylate haptics (4.4%) . CONCLUSION: Surgical technique had no statistically significant effect on ocular contamination . Polypropylene haptics IOLs were associated with a higher risk of bacterial contamination.

Pathology, 2003 Apr, 35(2), 109 - 19
A clinicopathological review of 34 cases of inflammatory breast disease showing an association between corynebacteria infection and granulomatous mastitis; Taylor GB et al.; AIM: Granulomatous mastitis is a rare condition of unknown aetiology . The great majority of cases has not been associated with bacterial pathogens if women with mammary tuberculosis are excluded . We noted that some women in Auckland with a histological diagnosis of granulomatous mastitis had both microbiological and histological evidence of corynebacteria infection and aimed to study this further . METHODS: Thirty-four women were reviewed who presented with inflammatory breast disease and had microbiological specimens from which corynebacteria were isolated and/or histological specimens containing coryneform bacteria . These 34 cases were compared with 28 controls with similar histology but no evidence of corynebacteria infection . RESULTS: Twenty-seven (79%) of the cases and 21 (75%) of the controls had histological and/or cytological evidence of suppurative granulomas . Fourteen of the 34 cases also had Gram-positive bacilli (GPB), recognisable as coryneform bacteria, in histological sections . In all cases the bacilli were confined to empty spaces, consistent with dissolved lipid, and were surrounded by neutrophils and, frequently, suppurative granulomas . Corynebacterium species were isolated from 52 of 116 microbiological specimens taken from the 34 cases . Forty of these 52 cultures were pure . Twenty-four of the cultures were further classified biochemically and using 16S rRNA gene sequencing . Twenty of the 24 were lipophilic Corynebacterium species and 14 were identified as Corynebacterium kroppenstedtii . The cases were more likely to present with fever or neutrophilia and more often formed sinuses than the controls but other clinical features were similar . Maori and Pacific Islanders accounted for 77% of the women across both groups . CONCLUSION: We suggest granulomatous mastitis can be associated with corynebacteria infection, particularly infection by C . kroppenstedtii . The significance of this finding, which has previously been described in only a single case report, is discussed.

Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 99 - 109 Epub 2003 May 13.
The Corynebacterium glutamicum genome: features and impacts on biotechnological processes; Ikeda M et al.; Corynebacterium glutamicum has played a principal role in the progress of the amino acid fermentation industry . The complete genome sequence of the representative wild-type strain of C . glutamicum, ATCC 13032, has been determined and analyzed to improve our understanding of the molecular biology and physiology of this organism, and to advance the development of more efficient production strains . Genome annotation has helped in elucidation of the gene repertoire defining a desired pathway, which is accelerating pathway engineering . Post genome technologies such as DNA arrays and proteomics are currently undergoing rapid development in C . glutamicum . Such progress has already exposed new regulatory networks and functions that had so far been unidentified in this microbe . The next goal of these studies is to integrate the fruits of genomics into strain development technology . A novel methodology that merges genomics with classical strain improvement has been developed and applied for the reconstruction of classically derived production strains . How can traditional fermentation benefit from the C . glutamicum genomic data? The path from genomics to biotechnological processes is presented.

Arch Microbiol, 2003 Jul, 180(1), 33 - 44 Epub 2003 May 10.
Identification and functional analysis of six mycolyltransferase genes of Corynebacterium glutamicum ATCC 13032: the genes cop1, cmt1, and cmt2 can replace each other in the synthesis of trehalose dicorynomycolate, a component of the mycolic acid layer of the cell envelope; Brand S et al.; By data mining in the sequence of the Corynebacterium glutamicum ATCC 13032 genome, six putative mycolyltransferase genes were identified that code for proteins with similarity to the N-terminal domain of the mycolic acid transferase PS1 of the related C . glutamicum strain ATCC 17965 . The genes identified were designated cop1, cmt1, cmt2, cmt3, cmt4, and cmt5 ( cmt from corynebacterium mycolyl transferases) . cop1 encodes a protein of 657 amino acids, which is larger than the proteins encoded by the cmt genes with 365, 341, 483, 483, and 411 amino acids . Using bioinformatics tools, it was shown that all six gene products are equipped with signal peptides and esterase domains . Proteome analyses of the cell envelope of C . glutamicum ATCC 13032 resulted in identification of the proteins Cop1, Cmt1, Cmt2, and Cmt4 . All six mycolyltransferase genes were used for mutational analysis . cmt4 could not be mutated and is considered to be essential . cop1 was found to play an additional role in cell shape formation . A triple mutant carrying mutations in cop1, cmt1, and cmt2 aggregated when cultivated in MM1 liquid medium . This mutant was also no longer able to synthesize trehalose di coryno mycolate (TDCM) . Since single and double mutants of the genes cop1, cmt1, and cmt2 could form TDCM, it is concluded that the three genes, cop1, cmt1, and cmt2, are involved in TDCM biosynthesis . The presence of the putative esterase domain makes it highly possible that cop1, cmt1, and cmt2 encode enzymes synthesizing TDCM from trehalose monocorynomycolate.

Neth J Med, 2003 Feb, 61(2), 54 - 6
Tension pneumopericardium caused by positive pressure ventilation complicating anaerobic pneumonia; Bleeker-Rovers CP et al.; A 22-year-old man was admitted with pneumonia . He was immediately intubated and positive pressure ventilation was initiated . Blood and sputum cultures showed Bacteroides fragilis and Corynebacterium sp., which were treated with metronidazole and clindamycin . Three weeks later his blood pressure suddenly dropped with an elevation of the central venous pressure . Chest X-ray revealed a pneumopericardium . A parasternal mediastinotomy with partial pericardiectomy was immediately performed . On opening the pericardium his blood pressure normalised . The patient gradually recovered and six weeks after admission he was extubated . Two weeks later he was discharged . A pneumopericardium without previous thorax trauma is very rare and early recognition is imperative because a tension pneumopericardium with cardiac tamponade may develop, as happened in this case . A tension pneumopericardium has to be treated with immediate pericardiocentesis followed by partial pericardiectomy to avoid recurrence.

Appl Environ Microbiol, 2003 May, 69(5), 3011 - 4
Production of native-type Streptoverticillium mobaraense transglutaminase in Corynebacterium glutamicum; Date M et al.; We previously observed secretion of active-form transglutaminase in Corynebacterium glutamicum by coexpressing the subtilisin-like protease SAM-P45 from Streptomyces albogriseolus to process the prodomain . However, the N-terminal amino acid sequence of the transglutaminase differed from that of the native Streptoverticillium mobaraense enzyme . In the present work we have used site-directed mutagenesis to generate an optimal SAM-P45 cleavage site in the C-terminal region of the prodomain . As a result, native-type transglutaminase was secreted.

Appl Environ Microbiol, 2003 May, 69(5), 2521 - 32
Global expression profiling and physiological characterization of Corynebacterium glutamicum grown in the presence of L-valine; Lange C et al.; Addition of L-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 {13032 DeltailvA DeltapanBC(pJC1ilvBNCD)} in a concentration-dependent manner . In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type . This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells . The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of L-isoleucine could relieve the valine effect on VAL1 whereas L-leucine had the same effect as valine . The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy . Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids . Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ . Interestingly, addition of external valine stimulated valine production by VAL1 . This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.

Transfusion, 1974 Mar-Apr, 14(2), 171 - 2
Platelet concentrates: sterility of 400 single units stored at room temperature; Wrenn HE et al.; Four hundred single platelet concentrates were prepared and stored at room temperature for 72 to 96 hours . Triple cultures of each concentrate at varying incubation temperatures revealed only four positive cultures . The Corynebacterium species and Staphylococcus species, coagulase negative, were the only organisms identified and were regarded as contaminants . No transfusion reactions of bacterial origin were observed in transfusing 10,024 other single platelet concentrates . Room temperature preparation and storage of single platelet concenrates is a safe and practical procedure.

Appl Microbiol Biotechnol, 2003 Sep, 62(4), 380 - 6 Epub 2003 Apr 26.
Cloning, sequence analysis, and heterologous expression of the gene encoding a (S)-specific alcohol dehydrogenase from Rhodococcus erythropolis DSM 43297; Abokitse K et al.; The gene encoding an (S)-specific NAD-dependent alcohol dehydrogenase (RE-ADH) was isolated from the genomic DNA of Rhodococcus erythropolis DSM 43297 . The nucleotide sequence of 1,047 bp, coding for 348 amino acids, was cloned in Escherichia coli cells and successfully expressed . The subunit molecular mass as deduced from the amino acid sequence was determined to be 36.026 kDa . The recombinant enzyme exhibited high thermostability, which facilitated its purification by heat treatment, followed by two column-chromatography steps . RE-ADH shows high similarity to several zinc-containing medium-chain alcohol dehydrogenases . All zinc ligands seem to be conserved except one of the catalytic zinc ligands, where Cys is probably substituted by Asp . A similarity of 84% with a phenylacetaldehyde reductase from Corynebacterium sp . ST-10 was determined . Biochemical properties such as thermostability and substrate specificity of the two enzymes were compared.

Prev Vet Med, 2003 May 30, 59(1-2), 67 - 81
Prevalence of and carcass condemnation from maedi-visna, paratuberculosis and caseous lymphadenitis in culled sheep from Quebec, Canada; Arsenault J et al.; We determined the prevalence of lung and mammary gland lesions associated with maedi-visna (MV) infection, the prevalence of paratuberculosis (PTB), and the prevalence and lesions distribution of caseous lymphadenitis (CL) in culled sheep . Total of 451 ewes and 34 rams were selected randomly from two slaughterhouses in Quebec, Canada . MV serostatus was determined by recombinant ELISA test . PTB diagnosis was based on characteristic histological lesions in the terminal ileum, ileocecal lymph node and/or ileocecal valve and CL by gross detection of abscesses and isolation of Corynebacterium pseudotuberculosis . Seroprevalence of MV was 44% (95% CI: 40, 48) . Seropositivity increased with age and was higher in ewes than in rams . The percentages of lung and mammary gland lesions in seropositive sheep were 14 and 40%, respectively, but mammary gland lesions lack specificity . The prevalence of PTB was 3% (95% CI: 2, 5) . PTB increased with age and was lower among sheep with abscesses . The prevalence of CL was >/=21% (95% CI: 17, 24) . The most-prevalent site of caseous lymphadenitis lesions was the thoracic cavity . The risk of carcass condemnation was significantly associated with region, body score and abscesses . Only the presence of abscesses was associated with an increase in trimming of carcasses.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Jul-Aug, (4 Suppl), 88 - 92
{Correction of the microbiocoenosis of the male urogenital tract in the course of hormonal therapy}; Bukharin OV et al.; The effect of androgenic preparations on the urogenital tract microflora in males of the reproductive age with chronic prostatitis has been studied . As the result of the use of androgens, the normalization of the microflora of ejaculate has been noted, which is characterized by the restoration of normal microflora (corynebacteria and lactic bacteria), a decrease in the content, or elimination, of opportunistic microorganisms and the inhibition of their persistence potential . The method for the correction of the dysbiosis of the male urogenital tract is proposed.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Jul-Aug, (4 Suppl), 31 - 6
{Adaptation mechanisms in the formation of the Corynebacterium diphtheriae carrier state}; Kvetnaia AS et al.; The complex clinico-laboratory examination of 92 children aged 3-14 years with the localized form of stomatopharyngeal diphtheria and toxigenic C . diphtheriae (TCD) carrier state was carried out . The children were hospitalized in the clinic of drop infections during the period of 1996-1998 . The hydrophobic properties, adhesive, lysozyme, antilysozyme and DNAase activities of 92 TCD strains, as well as the levels of the specific and nonspecific immunity of the macroorganism, were studied . The study revealed that the character of the infectious process in TCD carrier state was determined by the degree of the colonization activity of the infective agent . Direct correlation between the time of the persistence of TCD in the body and their hydrophobic properties, adhesive and antilysozyme activities (with r = 0.89-0.93) was established . The duration of TCD carrier state was found to be inversely related to the electrophysiological state of epithelial cells of the mucosa, the level of humoral specific antibacterial immunity and the content of local antidiphtheria IgA.

Infect Immun, 2003 May, 71(5), 2584 - 90
Characterization of the role of the divalent metal ion-dependent transcriptional repressor MntR in the virulence of Staphylococcus aureus; Ando M et al.; DtxR-type metal ion-dependent repressors, present in many bacterial pathogens, may regulate expression of virulence genes such as that encoding diphtheria toxin . SirR, a DtxR homologue initially identified in Staphylococcus epidermidis, governs the expression of the adjacent sitABC operon encoding a putative metal ion ABC transporter system . We identified a sirR homologue, mntR, in Staphylococcus aureus and demonstrated by gel shift assay that the corynebacterial repressor DtxR binds to the S . aureus mntABC operator in the presence of Fe(2+) or Mn(2+) . Since a mutant DtxR, DtxR(E175K), functions as an iron-independent hyperrepressor in certain settings, we constructed a heterodiploid S . aureus strain expressing dtxR(E175K) from the native mntR promoter . Transcription of the S . aureus mntABC operon was repressed in the presence of Fe(2+) or Mn(2+) in wild-type and heterodiploid S . aureus strains . Under metal ion-limiting conditions, mntABC transcription was reduced but not abolished in S . aureus isolates expressing dtxR(E175K) compared with an isogenic control, suggesting that DtxR(E175K) binds the S . aureus MntR box in vivo . Under all conditions tested, mntABC transcription in the dtxR(E175K)-expressing strain was reduced relative to the isogenic control, indicating that DtxR(E175K) function was constitutively active . In the mouse skin abscess model, dtxR(E175K)-expressing S . aureus recombinants showed significantly reduced CFU levels compared with the isogenic wild-type control . We conclude that the S . aureus MntR box is recognized by corynebacterial DtxR proteins and thus belongs to the DtxR family of metal-dependent operator sites . Moreover, constitutive repression by DtxR(E175K) reduces the virulence of S . aureus in the mouse skin abscess model.

Actas Urol Esp, 2003 Jan, 27(1), 47 - 54
{Acute and immediate urination syndrome after transurethral resection: a case of incrustating cystopathy}; Gonzalez Enguita C et al.; The authors present a case of acute and prompt symptomatic irritative urinary cystitis after transurethral resection (TR) of bladder cancer . The clinical presentation, like a irritative syndrome, was with a positive urine cultive to Enterococci and Staphylococcus . The physical examination, under general anesthesia (EBA), eliminated the urethral injury or the meatus trauma, so the urethral stenosis . The bladder view, in scaring processing yet, was congestive, bledding and edematous; an extensive white calcification was covering all the mucose surface bladder . The presumptive diagnosis was incrusted cystophatie (cystitis) and a transurethral resection (TR), along total bladder mucosa, was made so the result of pathological examination was sure . Intravenous and oral antimicrobial agent (Amoxicillin-Clavunan), in different ways, was instaured like a treatment, to achieve a negative urinary cultive, to eradicate the bacterial agents . We made a revision of the most important aspects in the clinical presentation, laboratory diagnosis and therapy, in this cystophatie that is not frequent, where the ureolitic bacterial agents have the responsibility, main Corynebacterium urealiticum, and where the recent urologic surgery or instrumentation, is narrowly related with the development of this cystophatie.

Arch Esp Urol, 2003 Jan-Feb, 56(1), 76 - 81
{Encrusted pyelitis in patients with urinary diversion}; Martinez Silva VM et al.; OBJECTIVE: This is a case of Encrusted Pyelitis (EP) caused by Corynebacterium urealyticum (CU) in a patient who had undergone a cystectomy and Bricker type urinary diversion 28 months beforehand . METHODS/RESULTS: After the immediate post-operative period no urinary catheterisation or any other urological procedure was performed on the patient . Before surgery, the patient presented non functional of the right kidney, secondary to a lithiasic obstructive uropathy . Clinical symptoms were deteriorated renal function, anuria, haematuria, pyrexia and left lumbar pain . It was suspected that the patient had this pathology and this was fundamental in diagnosis . Helicoid CT was the principal method used to show calcification plaques on the wall of the left renal pelvis, and selective culture of CU confirmed the diagnosis . Early commencement of treatment with vancomycin at an initial dosage of 500 mg/12 hours, and subsequent adjustment of dosage according to blood drug levels, achieved negative urine culture within a fortnight . Oral acidification was effected using acetohidroxamic acid 125 mg/12 hours, and it was continued until CT confirmed the disappearance or considerable reduction of the pyelic calcification plaques . CONCLUSION: The presence of EP in patients with urinary diversion is a matter worthy of consideration, even in patients who have not undergone recent urological procedures . Awareness of risk factors and early commencement of effective treatment may improve the prognosis of these patients.

Biochem J . 2003 Apr 14; Pt {Epub ahead of print}
Transposon-5 Mutagenesis Transforms Corynebacterium matruchotii to Synthesize Novel Hybrid Fatty Acids That Functionally Replaces Corynomycolic Acid; Takayama K et al.; Enzymes within the biosynthetic pathway of mycolic acid (C60-C90 alpha-alkyl, beta-hydroxyl fatty acid) in Mycobacterium tuberculosis are attractive targets for developing new anti-tuberculosis drugs . We have turned to the simple model system of Corynebacterium matruchotii to study the terminal steps in the anabolic pathway of a C32-mycolic acid called corynomycolic acid . By transposon-5 mutagenesis, we transformed C . matruchotii into a mutant that is unable to synthesize corynomycolic acid . Instead, it synthesized two homologous series of novel fatty acids that were released by saponification from the cell wall fraction and from two chloroform/methanol-extractable glycolipids presumed to be analogs of trehalose mono- and di-corynomycolate . By chemical analyses and mass spectrometry we determined the general structure of the two series to be 2,4,6,8,10-pentaalkyl decanoic acid for the larger series (C70-C77) and 2,4,6,8-tetraalkyl octanoic acid for the smaller series (C52-C64), both containing multiple keto groups, hydroxyl groups and double bonds . The mutant was temperature-sensitive, aggregated extensively, grew very slowly relative to the wild type and was resistant to the presence of lysozyme . We suggest that a regulatory protein that normally prevents the transfer of the condensation product back to beta-ketoacyl synthase in the corynomycolate synthase system of the wild type was inactivated in the mutant . This resulted in multiple Claisen-type condensation and the formation of two homologous series of these complex hybrid fatty acids . A similar protein in M . tuberculosis would be an attractive target for new drug discovery.

J Clin Microbiol, 2003 Apr, 41(4), 1386 - 90
Quantitative detection of Moraxella catarrhalis in nasopharyngeal secretions by real-time PCR; Greiner O et al.; The recognition of Moraxella catarrhalis as an important cause of respiratory tract infections has been protracted, mainly because it is a frequent commensal organism of the upper respiratory tract and the diagnostic sensitivity of blood or pleural fluid culture is low . Given that the amount of M . catarrhalis bacteria in the upper respiratory tract may change during infection, quantification of these bacteria in nasopharyngeal secretions (NPSs) by real-time PCR may offer a suitable diagnostic approach . Using primers and a fluorescent probe specific for the copB outer membrane protein gene, we detected DNA from serial dilutions of M . catarrhalis cells corresponding to 1 to 10(6) cells . Importantly, there was no difference in the amplification efficiency when the same DNA was mixed with DNA from NPSs devoid of M . catarrhalis . The specificity of the reaction was further confirmed by the lack of amplification of DNAs from other Moraxella species, nontypeable Haemophilus influenzae, H . influenzae type b, Streptococcus pneumoniae, Streptococcus oralis, Streptococcus pyogenes, Bordetella pertussis, Corynebacterium diphtheriae, and various Neisseria species . The assay applied to NPSs from 184 patients with respiratory tract infections performed with a sensitivity of 100% and a specificity of up to 98% compared to the culture results . The numbers of M . catarrhalis organisms detected by real-time PCR correlated with the numbers detected by semiquantitative culture . This real-time PCR assay targeting the copB outer membrane protein gene provided a sensitive and reliable means for the rapid detection and quantification of M . catarrhalis in NPSs; may serve as a tool to study changes in the amounts of M . catarrhalis during lower respiratory tract infections or following vaccination against S . pneumoniae, H . influenzae, or N . meningitidis; and may be applied to other clinical samples.

Microbiology, 2003 Apr, 149(Pt 4), 1011 - 20
Corynebacterium ammoniagenes class Ib ribonucleotide reductase: transcriptional regulation of an atypical genomic organization in the nrd cluster; Torrents E et al.; Ribonucleotide reductases (RNRs) are a family of complex enzymes that play an essential role in all organisms because they catalyse de novo synthesis of deoxyribonucleotides required for DNA replication and repair . Three different classes of RNR have been described according to their metal cofactors and organic radicals . Class Ib RNR is encoded in four different genes (nrdH, nrdI, nrdE and nrdF) organized in an operon . The authors previously cloned and sequenced the genes encoding the active class Ib RNR of Corynebacterium ammoniagenes and showed that these genes are clustered in an atypical nrdEF region, which differs from that of other known class Ib enzymes because of an intergenic sequence (1171 bp) present between nrdE and nrdF . This study investigated the transcriptional organization and regulation of this nrd region by RT-PCR . Three different and independent mRNA were found (nrdHIE, nrdF and an ORF present in the intergenic region), each one being transcribed from its own promoter and being essential for normal growth . The ratio of nrdF to nrdHIE mRNA was 9.1, as determined by competitive RT-PCR; the expression of both nrdHIE and nrdF was found to be dependent on the culture growth phase, and was induced in the presence of hydroxyurea, manganese and hydrogen peroxide . This is believed to be the first direct evidence for a manganese-dependent transcriptional regulation of nrd genes . These results suggest a common and coordinated regulation of the different nrd genes, despite their being transcribed from independent promoters.

Cardiovasc Pathol, 2003 Mar-Apr, 12(2), 98 - 101
Infectious endocarditis due to Corynebacterium xerosis; Pessanha B et al.; Corynebacterium xerosis is a rare cause of endocarditis, mainly affecting immunocompromised patients and those with predisposing cardiovascular lesions . While often considered a blood culture contaminant, serious infections by this organism have been reported . C . xerosis endocarditis is associated with poor outcomes that could be potentially improved by early recognition and treatment .

J Vet Med Sci, 2003 Mar, 65(3), 319 - 23
Lactoferrin concentrations in milk from normal and subclinical mastitic cows; Hagiwara S et al.; The concentrations of lactoferrin (Lf) in quarter milk from normal lactating cows and subclinical mastitic cows were measured to determine whether the Lf concentration in milk is influenced by the age of the cow, the stage of lactation, number of milk somatic cells and the presence of pathogens . Lf concentrations in 111 quarter milk samples from 28 normal lactating cows and 270 quarter milk samples from 198 subclinical mastitic cows were measured by means of a single radial immunodiffusion test . Lf concentrations (means +/- standard deviations; logarithmic form) in normal cows and subclinical mastitic cows were 2.23 +/- 0.39 and 2.70 +/- 0.39, respectively . The mean milk Lf concentration (log) in subclinical mastitic cows was significantly (p<0.01) higher than that in normal cows . The mean milk Lf concentration (log) in normal lactating cows aged 5 years was lower than those in normal lactating cows aged 2 years (p<0.01) and 3 years (p<0.05) . The results showed that the milk Lf concentration (log) is associated with age of the dairy cow (one-way analysis of variance test, p<0.01) . The mean milk Lf concentration (log) in the latter lactational period tended to be higher than those in the peak and middle periods . Milk Lf concentrations (log) tended to be proportional to the level of the somatic cell count (SCC) score . Mean milk Lf concentrations (log) in subclinical mastitic cows infected with Staphylococcus aureus and with other streptococci species were significantly (p<0.01) higher than those in cows infected with coagulase-negative staphylococci and with Corynebacterium bovis.

Mol Microbiol, 2003 Apr, 48(2), 495 - 506
Origins of metal ion selectivity in the DtxR/MntR family of metalloregulators; Guedon E et al.; Corynebacterium diphtheriae DtxR is an iron-specific repressor of diphtheria toxin expression and iron homeostasis functions . A homologue, MntR, serves as a manganese-specific repressor of Mn(II) uptake in Bacillus subtilis . When expressed in B . subtilis, DtxR regulates gene expression in response to either iron or manganese with comparable sensitivity . Replacement of two amino acids in the metal-sensing site with the corresponding residues from MntR results in a DtxR mutant that is highly selective for Mn(II) . However, iron responsiveness can be partially restored in a fur mutant in which iron uptake is derepressed and intracellular iron pools elevated . Conversely, if the putative metal-binding residues in MntR are altered to those in DtxR, the resulting protein responds to both iron and manganese . These results suggest that the composition and geometry of the metal-binding site plays a major role in defining the metal-selectivity in this protein family . However, the broadened selectivity of DtxR when expressed in B . subtilis, and the effects of a fur mutation, demonstrate that cellular milieu also influences metal responsiveness.

Chemotherapy, 2002, 48(6), 316 - 9
Report of a case with polymicrobial endocarditis related to multiresistant strains; Kocazeybek B et al.; We present a patient with polymicrobial endocarditis who had been operated on previously for a mycotic aneurysm and was seen at the cardiology clinic because of palpitations related to effort . A transesophageal echocardiogram revealed a 15-mm vegetation on his aortic valve . Staphylococcus epidermidis and Corynebacterium striatum were isolated from the blood cultures . Both strains were multiresistant (susceptible to 3 antibiotics at most) against chemotherapy in vitro . Microbiological eradication was not achieved from blood cultures even after applying antimicrobial therapy with effective antibiotics as determined with an antibiotic susceptibility test . For this reason, the patient underwent valve replacement . He was discharged from hospital in fairly good health .

J Bacteriol, 2003 Apr, 185(8), 2402 - 9
Control of rep gene expression in plasmid pGA1 from Corynebacterium glutamicum; Venkova-Canova T et al.; The cryptic multicopy plasmid pGA1 (4,826 bp) from Corynebacterium glutamicum LP-6 belongs to the fifth group of rolling-circle-replicating plasmids . A determinant, which negatively controls pGA1 replication, was localized in the leader region of the rep gene coding for the initiator of plasmid replication . This region, when cloned into the compatible vector pEC6, was found to cause decrease of segregational stability of the pGA1 derivative pKG48 . A promoter and a single transcriptional start site were found in the rep leader region in orientation opposite to the rep gene . These results suggest that a small countertranscribed RNA (ctRNA) (ca . 89 nucleotides in length), which might inhibit translation of pGA1 rep gene, is formed . Analysis of predicted secondary structure of the pGA1-encoded ctRNA revealed features common with the known ctRNAs in bacteria . Inactivation of the promoter P-ctRNA caused a dramatic increase of copies of the respective plasmid, which proved a negative role of the ctRNA in control of pGA1 copy number . A region between the promoters Prep and P-ctRNA with a potential to form secondary structures on both ctRNA and rep mRNA was found to cause low activity of the rep promoter even when promoter P-ctRNA was deleted . Thus, the sequence within the rep leader region itself seems to act, in addition to the ctRNA, as a second regulatory element of a novel type, negatively influencing expression of the pGA1 rep gene.

Clin Microbiol Infect, 2003 Mar, 9(3), 230 - 3
Rhodococcus equi pneumonia in a heart transplant recipient in Korea, with emphasis on microbial diagnosis; Yoo SJ et al.; Rhodococcus equi is an opportunistic pathogen that usually causes infection in immunocompromised hosts . A heart transplant recipient who had been treated with amphotericin B for pulmonary aspergillosis showed newly developed multiple nodules with a central necrotic area in the right lower lobes . Cultures of several blood samples and an aspirate of the lung nodule yielded a Gram-positive coccobacillary bacterium, which was initially reported as a Corynebacterium species, but was later identified as R . equi by API CORYNE (bioMerieux SA, Marcy l'Etoile, France) and by demonstrating the production of 'equi factor' . The identification was subsequently confirmed by an R . equi-specific polymerase chain reaction (PCR) . The patient was successfully treated with ciprofloxacin and azithromycin for 14 weeks . This is the first documented case of R . equi infection in Korea . There is a possibility of underestimation of R . equi infections due to the misidentification of the organism as a contaminating diphtheroid . Because R . equi will not respond to the conventional empirical therapy, the microbiology laboratory should identify R . equi in a timely manner . R . equi-specific PCR will be a useful confirmatory test in human infection.

Appl Microbiol Biotechnol, 2003 Feb, 60(6), 738 - 42 Epub 2002 Dec 18.
GltS, the sodium-coupled L-glutamate uptake system of Corynebacterium glutamicum: identification of the corresponding gene and impact on L-glutamate production; Trotschel C et al.; A screening procedure was established to identify Corynebacterium glutamicum transposon mutants with an altered L-glutamate excretion behaviour . By this microtiter plate-based approach seven non- or less excreting C . glutamicum strains and two hyper-excreters were found . The subsequently carried out molecular analysis of a hyper-producing clone led to the identification of the gltS gene, which codes for the sodium-coupled secondary L-glutamate uptake system in C . glutamicum . Characterization of a gltS deletion strain revealed that this transporter has a weak but significant impact on L-glutamate production induced by biotin limitation in the wild type . Obviously, GltS leads to the re-uptake of excreted L-glutamate causing a futile cycle . In accord with this hypothesis, the overexpression of gltS decreased L-glutamate production.

Ned Tijdschr Geneeskd, 2003 Mar 1, 147(9), 403 - 6
{A case of diphtheria in the Netherlands due to an infection with Corynebacterium ulcerans}; van Dam AP et al.; A 58-year-old woman was admitted due to a pseudomembranous pharyngitis . The patient had not been vaccinated against diphtheria . Corynebacterium ulcerans was cultured from a throat swab . The production of diphtheria toxin by these bacteria was demonstrated with PCR and an immunoprecipitation test . The patient was cared for in respiratory isolation and was treated with benzylpenicillin . She quickly recovered and was discharged four days after admission . A contact investigation did not reveal any dissemination of the toxin-producing C . ulcerans and a source was not found . In spite of the large-scale vaccination against diphtheria which has taken place in the Netherlands since 1953, a physician has to consider diphtheria in the differential diagnosis of patients who present with a clinical syndrome compatible with this disease . Either Corynebacterium diphtheriae or C . ulcerans could be the pathogen responsible.

Appl Microbiol Biotechnol, 2003 Mar, 61(1), 61 - 8 Epub 2002 Dec 21.
Disparity between changes in mRNA abundance and enzyme activity in Corynebacterium glutamicum: implications for DNA microarray analysis; Glanemann C et al.; The relationship between changes in mRNA abundance and enzyme activity was determined for three genes over a span of nearly 3 h during amino acid production in Corynebacterium glutamicum . Gene expression changes during C . glutamicum fermentations were examined by complementary DNA (cDNA) microarrays and by a second method for quantitating RNA levels, competitive reverse transcriptase-PCR (RT-PCR) . The results obtained independently by both methods were compared and found to be in agreement, thus validating the quantitative potential of DNA microarrays for gene expression profiling . Evidence of a disparity between mRNA abundance and enzyme activity is presented and supports our belief that it is difficult to generally predict protein activity from quantitative transcriptome data . Homoserine dehydrogenase, threonine dehydratase, and homoserine kinase are enzymes involved in the biosynthesis of l-isoleucine and other aspartate-derived amino acids in C . glutamicum . Our data suggest that different underlying regulatory mechanisms may be connected with the expression of the genes encoding each of these three enzymes . Indeed, whereas in one case the increases in enzyme activity exceeded those in the corresponding mRNA abundance, in another case large increases in the levels of gene expression were not congruent with changes in enzyme activity.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 43 - 6
Corynebacterium spheniscorum sp . nov., isolated from the cloacae of wild penguins; Goyache J et al.; Twenty unidentified Gram-positive, rod-shaped organisms were recovered from the cloacae of apparently healthy wild penguins (Spheniscus magellanicus) and subjected to a polyphasic taxonomic analysis . On the basis of cellular morphology and biochemical criteria, the isolates were tentatively assigned to the genus Corynebacterium, although the organisms did not appear to correspond to any recognized species . Lipid studies confirmed this generic placement, and comparative 16S rRNA gene sequencing showed that the unidentified organisms represent a hitherto unknown subline, associated with a small subcluster of species that includes Corynebacterium diphtheriae and its close relatives . On the basis of phenotypic and phylogenetic evidence, it is proposed that the unknown isolates from penguins be classified in the genus Corynebacterium, as Corynebacterium spheniscorum sp . nov . The type strain is strain PG 39T (=CCUG 45512T =CECT 5986T).

Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 3808 - 13 Epub 2003 Mar 24.
Metal stoichiometry and functional studies of the diphtheria toxin repressor; Spiering MM et al.; Diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor in Corynebacterium diphtheriae . DtxR is an iron sensor; metal-bound DtxR represses transcription of genes downstream of the tox operator . Wild-type DtxR {DtxR(wt)} and several mutant forms were overexpressed and purified from Escherichia coli . DtxR was isolated without bound metal . Metal reconstitution gave a binding stoichiometry of 2 per monomer for DtxR(wt) and 1 per monomer for DtxR(H79A) and DtxR(M10A) . DNA binding of DtxR(H79A) and DtxR(M10A) indicates that metal site 2 is essential for activity . Metal binding lowers the dimerization K(d) of DtxR from low micromolar to 33 nM . Gel electrophoretic mobility-shift assays show that Fe(2+) and not Fe(3+) activates DtxR for DNA binding . This finding suggests that gene regulation by DtxR may be sensitive not only to iron levels but also to redox state of the iron . Mutations in the tox operator sequence indicate that DtxR dimers binding to DNA may be highly cooperative.

Antimicrob Agents Chemother, 2003 Apr, 47(4), 1403 - 6
Activities of ABT-773 against Listeria monocytogenes and coryneform bacteria of clinical interest; Conejo Mdel C et al.; The in vitro activities of ABT-773 were evaluated against 15 Listeria monocytogenes strains and 196 coryneform bacteria isolated from clinical samples . One hundred percent of the L . monocytogenes strains were inhibited by </=0.015 micro g of ABT-773/ml . MICs of ABT-773 ( micro g/ml) at which 50% of the isolates tested were inhibited (MIC(50)s) and MIC(90)s for other organisms were 0.125 and 0.5 (Corynebacterium amycolatum), 1 and >32 (Corynebacterium jeikeium), 0.03 and >32 (Corynebacterium minutissimum), >32 and >32 (Corynebacterium pseudodiphtheriticum and Corynebacterium urealyticum), 0.125 and >32 (Corynebacterium striatum), and 0.03 and 0.5 (Rhodococcus equi), respectively.

Acta Anaesthesiol Scand, 2003 Mar, 47(3), 335 - 41
Chronic intraperitoneal endotoxin treatment in rats induces resistance to d-tubocurarine, but does not produce up-regulation of acetylcholine receptors; Hinohara H et al.; BACKGROUND: Chronic systemic inflammation resulting from intraperitoneal Eschevichia coli endotoxin administration or Corynebacterium injections induces tolerance to non-depolarizing neuromuscular blockers in rodents . Although this has been explained as up-regulation of muscle acetylcholine receptors (AChR), the numbers of involved receptors have not been documented . The aim of this study was to determine the effects of chronic endotoxin administration on rat muscle AChR . METHODS: One day after one, seven, or 14 daily intraperitoneal doses of lipopolysaccharide endotoxin (0 or 0.5 mg kg(-1)), we studied in vivo dose-response relationships for d-tubocurarine (d-Tc) and AChR binding using {125I}alpha-bungarotoxin as a ligand . RESULTS: One day after seven and 14 daily intraperitoneal doses of endotoxin, the effective dose of d-Tc required to suppress the twitch response to 50% of the control (ED50) was significantly increased compared with that of time-matched control rats (146.5 +/- 38.2 vs . 76.1 +/- 9.0 microg kg(-1) for seven doses; 116.4 +/- 51.3 vs . 74.4 +/- 9.6 micro g kg-1 for 14 doses, P < 0.05) . However, this was not associated with an increase in the number of AChR in the anterior tibial muscle or diaphragm . CONCLUSIONS: Mechanisms other than AChR up-regulation might be responsible for the increased d-Tc requirement during chronic intraperitoneal endotoxin administration.

Metab Eng, 2002 Oct, 4(4), 295 - 305
Changes of pentose phosphate pathway flux in vivo in Corynebacterium glutamicum during leucine-limited batch cultivation as determined from intracellular metabolite concentration measurements; Moritz B et al.; Corynebacterium glutamicum is an important organism for the industrial production of amino acids such as lysine . In the present study time-dependent changes in the oxidative pentose phosphate pathway activity, an important site of NADPH regeneration in C . glutamicum, are investigated, whereby intracellular metabolite concentrations and specific enzyme activities in two isogenic leucine auxotrophic strains differing only in the regulation of their aspartate kinases were compared . After leucine limitation only the strain with a feedback-resistant aspartate kinase began to excrete lysine into the culture medium . Concomitantly, the intracellular NADPH to NADP concentration ratio increased from 2 to 4 in the non-producing strain, whereas it remained constant at about 1.2 in the lysine-producing strain . From these data the in'vivo flux through the pentose phosphate pathway was calculated . These results were used to approximate the total NADPH regeneration by glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase, which agreed fairly well with the calculated demands for biomass formation and lysine biosynthesis . The analysis allowed to conclude that NADPH regeneration in the pentose phosphate pathway is essential for lysine biosynthesis in C . glutamicum.

Metab Eng, 2002 Oct, 4(4), 273 - 84
Mathematical modeling of in vitro enzymatic production of 2-Keto-L-gulonic acid using NAD(H) or NADP(H) as cofactors; Banta S et al.; A 2-Keto-L-gulonic acid (2-KLG) production process using stationary Pantoea citrea cells and a Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme has been developed which may represent an improved method of vitamin C biosynthesis . Experimental data was collected using the F22Y/A272G 2,5-DKG reductase mutant and NADP(H) as a cofactor . An extensive kinetic analysis was performed and a kinetic rate equation model for this process was developed . A recent protein engineering effort has resulted in several 2,5-DKG reductase mutants exhibiting improved activity with NADH as a cofactor . The use of NAD(H) in the bioreactor may be preferable due to its increased stability and lower cost . The kinetic parameters in the rate equation model have been replaced in order to predict 2-KLG production with NAD(H) as a cofactor . The model was also extended to predict 2-KLG production in the presence of a range of combined cofactor concentrations . This analysis suggests that the use of the F22Y/K232G/R238H/A272G 2,5-DKG reductase mutant with NAD(H) combined with a small amount of NADP(H) could provide a significant cost benefit for in vitro enzymatic 2-KLG production.

J Bacteriol, 2003 Apr, 185(7), 2251 - 8
The src homology 3-like domain of the diphtheria toxin repressor (DtxR) modulates repressor activation through interaction with the ancillary metal ion-binding site; Love JF et al.; The diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor that acts as a global regulatory element in the control of iron-sensitive genes in Corynebacterium diphtheriae . We recently described (L . Sun, J . C . vanderSpek, and J . R . Murphy, Proc . Natl . Acad . Sci . USA 95:14985-14990, 1998) the isolation and in vivo characterization of a hyperactive mutant of DtxR, DtxR(E175K), that appeared to be constitutively active . We demonstrate here that while DtxR(E175K) remains active in vivo in the presence of 300 micro M 2,2'dipyridyl, the purified repressor is, in fact, dependent upon low levels of transition metal ion to transit from the inactive apo form to the active metal ion-bound form of the repressor . Binding studies using 8-anilino-1-naphthalenesulfonic acid suggest that the E175K mutation stabilizes an intermediate of the molten-globule form of the repressor, increasing exposure of hydrophobic residues to solvent . We demonstrate that the hyperactive DtxR(E175K) phenotype is dependent upon an intact ancillary metal ion-binding site (site 1) of the repressor . These observations support the hypothesis that metal ion binding in the ancillary site facilitates the conversion of the inactive apo-repressor to its active, operator-binding conformation . Furthermore, these results support the hypothesis that the C-terminal src homology 3-like domain of DtxR plays an active role in the modulation of repressor activity.

Vet Ophthalmol, 2003 Mar, 6(1), 45 - 50
Seasonal effects on the aerobic bacterial and fungal conjunctival flora of normal thoroughbred brood mares in Florida; Andrew SE et al.; OBJECTIVE: To evaluate seasonal effects on the presence or absence of fungal and aerobic bacterial flora of the conjunctival fornix of normal Florida Thoroughbred horses . SAMPLE POPULATION: Both eyes of 100 horses . PROCEDURE: Horses with normal anterior segment ophthalmic examinations from three farms in north central Florida were included . Each animal had the ventral conjunctival fornix of each eye swabbed with sterile culturettes . Samples were taken in October, January, April, and July (1999-2000) . Aerobic and fungal cultures were plated . Bacterial cultures were reviewed at 24 and 48 h . Fungal cultures were reviewed weekly for 4 weeks . Logistic regression analysis with season as a factor and age of the horse as a covariate was performed . Statistical significance was set at P < 0.01 . RESULTS: Horses ranged from 3 to 24 years of age, with a median age of 9 years . Twenty-four genera of bacteria and 35 genera of fungi were recovered . Corynebacterium sp., Staphylococcus sp., Bacillus sp . and Moraxella sp . were the bacteria most frequently isolated . Mold species, dematiaceous mold species, Chrysosporium sp., Cladosporium sp., and Aspergillus sp . were the most frequently recovered fungi . Season did not have a significant effect on the presence of microorganisms isolated for individual horses adjusted for age . Younger horses had an increased incidence of gram-negative rods and fungal isolates . The number of bacteria and fungi isolated are not uniform across seasons . CONCLUSION: There were no significant differences between the number or type of organisms cultured during the sampling seasons in normal Florida horses . A large range of normal bacterial and fungal flora were isolated from these horses . The number of bacteria and fungi isolated are not uniform across seasons . The likelihood of detecting an organism depends on the horses' age.

Euro Surveill, 2002 Jan, 7(1), 7 - 12
Report on the Sixth International Meeting of the European Laboratory Working Group on Diphtheria, Brussels, Belgium; Lai S et al.; In addition to the Eastern European resurgence of diphtheria during the last decade, there has also been an emergence of infections caused by non-toxigenic Corynebacterium diphtheriae and non-toxigenic, toxin gene bearing C . diphtheriae . Given that these strains may manifest as symptomatic infections of differing degrees of severity, their clinical and epidemiological significance need to be assessed . The persistence of toxigenic and non-toxigenic C . diphtheriae in circulation, together with genotypic and biotype variability means that innovative measures to vaccinate populations are pertinent . The most effective method of protecting the currently most vulnerable population group (adults) is to implement a booster dose of vaccine amongst the adult populations . Furthermore, in combination with an efficient surveillance system, effective antibiotic prophylaxis and an up-to-date vaccination programme, serological studies needs to be maintained to monitor the immunity status of the population.

Folia Microbiol (Praha), 2002, 47(6), 737 - 41
Infective endocarditis caused by unusual gram-positive pathogens; Benes J et al.; Of a total of 81 patients hospitalized in the infectious diseases department in 1990-2000 with infectious endocarditis caused by Gram-positive pathogen, unusual etiological agents were found in several cases: Streptococcus pyogenes, Streptococcus pneumoniae, Corynebacterium diphtheriae, and Gemella morbillorum . Cardiac defects were present in the latter two patients: bicuspid aortic valve and tetralogy of Fallot . Two patients were successfully treated with antibiotics only and one patient with antibiotics and surgery . The patient with C . diphtheriae endocarditis died due to progressive sepsis and multiple organ failure.

Glycobiology, 2003 Apr, 13(4), 17R - 27R Epub 2003 Jan 22.
New insights on trehalose: a multifunctional molecule; Elbein AD et al.; Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an alpha,alpha-1,1-glycosidic linkage . This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates, and lower and higher plants, where it may serve as a source of energy and carbon . In yeast and plants, it may also serve as a signaling molecule to direct or control certain metabolic pathways or even to affect growth . In addition, it has been shown that trehalose can protect proteins and cellular membranes from inactivation or denaturation caused by a variety of stress conditions, including desiccation, dehydration, heat, cold, and oxidation . Finally, in mycobacteria and corynebacteria, trehalose is an integral component of various glycolipids that are important cell wall structures . There are now at least three different pathways described for the biosynthesis of trehalose . The best known and most widely distributed pathway involves the transfer of glucose from UDP-glucose (or GDP-glucose in some cases) to glucose 6-phosphate to form trehalose-6-phosphate and UDP . This reaction is catalyzed by the trehalose-P synthase (TPS here, or OtsA in Escherichia coli ) . Organisms that use this pathway usually also have a trehalose-P phosphatase (TPP here, or OtsB in E . coli) that converts the trehalose-P to free trehalose . A second pathway that has been reported in a few unusual bacteria involves the intramolecular rearrangement of maltose (glucosyl-alpha1,4-glucopyranoside) to convert the 1,4-linkage to the 1,1-bond of trehalose . This reaction is catalyzed by the enzyme called trehalose synthase and gives rise to free trehalose as the initial product . A third pathway involves several different enzymes, the first of which rearranges the glucose at the reducing end of a glycogen chain to convert the alpha1,4-linkage to an alpha,alpha1,1-bond . A second enzyme then releases the trehalose disaccharide from the reducing end of the glycogen molecule . Finally, in mushrooms there is a trehalose phosphorylase that catalyzes the phosphorolysis of trehalose to produce glucose-1-phosphate and glucose . This reaction is reversible in vitro and could theoretically give rise to trehalose from glucose-1-P and glucose . Another important enzyme in trehalose metabolism is trehalase (T), which may be involved in energy metabolism and also have a regulatory role in controlling the levels of trehalose in cells . This enzyme may be important in lowering trehalose concentrations once the stress is alleviated . Recent studies in yeast indicate that the enzymes involved in trehalose synthesis (TPS, TPP) exist together in a complex that is highly regulated at the activity level as well as at the genetic level.

J Clin Microbiol, 2003 Mar, 41(3), 1285 - 8
Genotypic and phenotypic characteristics of Corynebacterium diphtheriae strains isolated from patients in belarus during an epidemic period; Titov L et al.; One hundred two Corynebacterium diphtheriae strains (93 of the gravis biotype and nine of the mitis biotype) isolated from clinical cases during the Belarus diphtheria epidemic were characterized by biotyping, toxigenicity testing by the Elek test and an indirect hemagglutination assay, phage typing, and ribotyping . The gravis biotype strains were characterized as high and medium toxin producers, and strains of biotype mitis were characterized as low and medium toxin producers . Most strains (82 of 102) were distributed among five phage types . Seventy-two strains (64 of the gravis biotype and 8 of the mitis biotype) belonged to phage type VI ls5,34add . Hybridization of genomic DNA digested with BstEII and PvuII revealed five ribotype patterns, namely, D1, D4, D6, D7, and D13 . The majority of gravis biotype strains belonged to ribotypes D1 (49 of 93) and D4 (33 of 93) and included one clonal group of C . diphtheriae . This clone predominated in all regions in Belarus . There was a statistical association between ribotypes and phage types but not between ribotypes and levels of toxin production.

J Biol Chem, 2003 May 23, 278(21), 19387 - 95 Epub 2003 Mar 05.
The crystal structure and stereospecificity of levodione reductase from Corynebacterium aquaticum M-13; Sogabe S et al.; The (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase (LVR) of the soil isolate bacterium Corynebacterium aquaticum M-13 is a NAD(H)-linked enzyme that catalyzes reversible oxidoreduction between (4R)-hydroxy-(6R)-2,2,6-trimethylcyclohexanone (actinol) and levodione . Here the crystal structure of a ternary complex of LVR with NADH and its inhibitor 2-methyl-2,4-pentanediol has been determined by molecular replacement and refined at 1.6-A resolution with a crystallographic R factor of 0.199 . The overall structure is similar to those of other short-chain alcohol dehydrogenase/reductase enzymes . The positions of NADH and 2-methyl-2,4-pentanediol indicate the binding site of the substrate and identify residues that are likely to be important in the catalytic reaction . Modeling of the substrate binding in the active site suggests that the specificity of LVR is determined by electrostatic interactions between the negatively charged surface of Glu-103 of LVR and the positively charged surface on the re side of levodione . Mutant LVR enzymes in which Glu-103 is substituted with alanine (E103A), glutamine (E103Q), asparagines (E103N), or aspartic acid (E103D) show a 2-6-fold increase in Km values as compared with wild-type LVR and a much lower enantiomeric excess of the reaction products (60%) than the wild-type enzyme (95%) . Together, these data indicate that Glu-103 has an important role in determining the stereospecificity of LVR.

Biochim Biophys Acta, 2003 Mar 6, 1557(1-3), 125 - 31
QcrCAB operon of a nocardia-form actinomycete Rhodococcus rhodochrous encodes cytochrome reductase complex with diheme cytochrome cc subunit; Sone N et al.; Structural genes encoding quinol-cytochrome c reductase (QcR) were cloned and sequenced from nocardia-form actinomycete Rhodococcus rhodochrous . QcrC and qcrA encode diheme cytochrome cc and the Rieske Fe-S protein, respectively, while the qcrB product is a diheme cytochrome b . These amino acid sequences are similar to those of Corynebacterium and Mycobacterium, the members of high G+C content firmicutes . The presence of diheme cytochrome cc subunit as a sole c-type cytochrome in these organisms suggests the direct elecron transfer to cytochrome c oxidase . The N-terminal half of the Rieske Fe-S proteins of these bacteria has a unique structure with three transmembrane helices, while the C-terminal half sequence is conserved . A phylogenetic tree using the latter region showed that high G+C firmicutes form a clear clade with Thermus, but not with low G+C firmicutes.

J Ind Microbiol Biotechnol, 2003 Feb, 30(2), 129 - 32 Epub 2003 Jan 10.
Significance of the non-oxidative route of the pentose phosphate pathway for supplying carbon to the purine-nucleotide pathway in Corynebacterium ammoniagenes; Kamada N et al.; To evaluate the strategy of supplying ribose 5-phosphate to the purine-nucleotide pathway exclusively via the nonoxidative route, the glucose 6-phosphate dehydrogenase gene zwf was disrupted in inosine- and 5'-xanthylic acid-producers of Corynebacterium ammoniagenes . In both producers, interruption of the oxidative route caused a decrease in production yields of about 50% . Attempts to increase the capacity of the nonoxidative route through overexpression of the transketolase or transaldolase gene in the zwf mutants led to no discernable effects on production, indicating that, in C . ammoniagenes, the nonoxidative route alone cannot provide sufficient ribose 5-phosphate for high-level production, although nonoxidative synthesis of the precursor is possible.

Arch Microbiol, 2003 Mar, 179(3), 184 - 90 Epub 2003 Feb 12.
Characterization of the gene encoding glutamate dehydrogenase ( gdhA) from the ruminal bacterium Ruminococcus flavefaciens FD-1; Antonopoulos DA et al.; The gene encoding glutamate dehydrogenase ( gdhA) in the ruminal bacterium Ruminococcus flavefaciens FD-1 was cloned . A degenerate primer based on the N-terminal amino acid sequence of the purified protein was used in conjunction with genome walking to obtain the complete ORF of 1,365 bp, capable of encoding a polypeptide of 455 amino acid residues . The translated ORF contained the amino acid motifs characteristic of the subfamily GDH S_50(I) small glutamate dehydrogenases, including the catalytic site, and matched the originally deduced N-terminal amino acid sequence . BLAST search yielded high scores with other GdhA sequences from a variety of organisms, the closest match being with the GdhA sequence of Corynebacterium glutamicum (63% amino acid identity) . Classification of the GdhA enzyme from R . flavefaciens FD-1 as a GDH S_50(I) subfamily member was further supported by phylogenetic analysis . The transcript size determined by Northern blot analysis was in good agreement with the putative regulatory region of the gene and confirmed its monocistronic structure . R . flavefaciens GdhA activity appears to be regulated primarily at the level of transcription . Brief exposure to 20 mM NH(4)Cl prior to extraction did not alter the level of activity . Transcriptional regulation, studied with quantitative real-time RT-PCR, demonstrated a three-fold increase of the gdhA transcript concentration in ammonia-limited cells in comparison with an excess of ammonia in the medium . This is in agreement with the enzyme activity data obtained under ammonia- and carbon-limited growth conditions.

Am J Pathol, 2003 Mar, 162(3), 837 - 47
AIM inhibits apoptosis of T cells and NKT cells in Corynebacterium-induced granuloma formation in mice; Kuwata K et al.; Apoptosis inhibitor expressed by macrophages (AIM) inhibits apoptosis of CD4(+)CD8(+) (CD4/CD8) double-positive thymocytes, and supports the viability of these cells on the thymic selection . However, pleiotropic functions of AIM have been suggested . In this study, heat-killed Corynebacterium parvum (C . parvum) was injected into mice carrying the homozygous mutation (AIM(-/-)) and wild-type (AIM(+/+)) mice, to investigate the role of AIM in the formation of hepatic granulomas . In AIM(-/-) mice, the size and the number of hepatic granulomas were larger, and the resorption of granulomas was more delayed than in AIM(+/+) mice . The production of interleukin-12 was more prominent in AIM(-/-) mice than in AIM(+/+) mice . In the liver of AIM(+/+) mice, expression of AIM messenger ribonucleic acid (mRNA) increased after C . parvum injection . In situ hybridization demonstrated that AIM mRNA was expressed in Kupffer cells and exudate macrophages in the liver, especially in granulomas . Larger numbers of T cells and natural killer T (NKT) cells underwent apoptosis in the granulomas of AIM(-/-) mice, suggesting that AIM prevents apoptosis of NKT cells and T cells in C . parvum-induced inflammation . Recombinant AIM (rAIM) protein significantly inhibited apoptosis of NKT cells and T cells obtained from C . parvum-stimulated livers in vitro . These results indicate that AIM functions to induce resistance to apoptosis within NKT cells and T cells, and supports the host defense in granulomatous inflammation.

Urology . 2003 Feb;61(2):463.
Mortality from grossly encrusted bilateral pyelitis, ureteritis, and cystitis by Corynebacterium group D2; Chung SY et al.; This is the first report of death due to gross encrustations of the entire upper urinary tract and bladder by Corynebacterium group D2 in a man with no history of renal transplantation or prolonged catheterizations . This case demonstrates that debilitated patients with a prior endoscopic procedure are at risk for this disease process . Prolonged treatment with appropriate antibiotics, acidification of the urine, and removal of crusts is essential for proper management.

FEMS Microbiol Lett, 2003 Jan 28, 218(2), 305 - 9
Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum; Nottebrock D et al.; Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli . To elucidate the physiological role of these putative channels deletion mutants were constructed . Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate . Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift . These results led to the hypothesis that C . glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families . Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant.

Endoscopy, 2003 Mar, 35(3), 197 - 202
Standardized reprocessing of reusable colonoscopy biopsy forceps is effective: results of a German multicenter study; Jung M et al.; BACKGROUND AND STUDY AIMS: National and international guidelines recommend that a standardized protocol consisting of cleaning, ultrasound cleaning, and sterilization should be used for the reprocessing of endoscopic accessories in order to reduce the risk of transmission of microorganisms . This German multicenter study investigated the efficacy of standardized reprocessing of reusable biopsy forceps used during colonoscopy . MATERIALS AND METHODS: Ten endoscopy centers (eight hospitals and two private practices) used 330 biopsy forceps during routine colonoscopy . The forceps were used once, five times, or 20 times for colonoscopy, based on a randomization plan . The reprocessing protocol consisted of manual cleaning with an enzymatic agent, ultrasound cleaning with an enzymatic agent (30 min, 40 degrees C, 47 Hz), neutralization, drying, and sterilization (5 min, 134 degrees C) . Aldehydes were not used, and the protocol did not include a disinfection step . The biopsy forceps were sent to three microbiological institutes, based on a randomization plan, to have them tested for the presence of organisms, including identification of bacteria . RESULTS: A total of 318 of the 330 forceps were evaluable; 314 forceps (98.74 %) were sterile after use once, five times, or 20 times . Four forceps were contaminated with Staphylococcus epidermidis (n = 2), Bacillus licheniformis (n = 1) and Corynebacterium aquaticum (n = 2) . All of 25 forceps were sterile after being used 20 times . CONCLUSION: Colonoscopy biopsy forceps can be reliably reprocessed following this standardized protocol, even without aldehydes.

Plasmid, 2003 Jan, 49(1), 63 - 74
Insights into the genetic organization of the Corynebacterium diphtheriae erythromycin resistance plasmid pNG2 deduced from its complete nucleotide sequence; Tauch A et al.; The complete nucleotide sequence of the erythromycin resistance plasmid pNG2 from the human pathogen Corynebacterium diphtheriae S601 was determined . The plasmid has a total size of 15,100 bp and contains at least 17 coding regions . Comparative genomics identified conserved motifs within replication initiator proteins of corynebacterial plasmids and a novel nucleotide sequence feature, termed 22-bp box, located downstream of the repA gene . The erythromycin resistance determinant erm(X) is flanked by inverted repeats of the novel insertion sequence IS3504, which may be responsible for a spontaneous deletion of the antibiotic resistance gene region . Furthermore, pNG2 encodes a putative conjugative relaxase, a membrane protein of the natural resistance-associated macrophage protein (Nramp) family and a protein with Nudix hydrolase signature . Expression of the predicted coding regions of pNG2 in Escherichia coli JM109 was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) assays . The detailed annotation of the entire pNG2 sequence provided genetic information regarding its molecular evolution and its role in dissemination of antibiotic resistance genes by horizontal gene transfer .

Appl Environ Microbiol, 2003 Feb, 69(2), 933 - 7
Production of a doubly chiral compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, by two-step enzymatic asymmetric reduction; Wada M et al.; A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described . Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst . Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E . coli, to the corresponding alcohol . Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps . Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD(+), and glucose dehydrogenase . To our knowledge, this is the first report of the application of S . cerevisiae old yellow enzyme for the production of a useful compound.

Eur J Echocardiogr, 2003 Mar, 4(1), 68 - 70
Native valve endocarditis with aorta-to-left atrial fistula due to Corynebacterium amycolatum; Daniels C et al.; Infective endocarditis remains a pathology with a high rate of complications and mortality . One of the most dramatic complications is abscess formation . A rare evolution of abscess formation is the development of fistula . We describe an 88-year-old woman with an aortic root abscess and aorta-to-left atrial fistula . To our knowledge this has only been described with streptococcus species as causative micro-organism . In this case the abscess was caused by Corynebacterium amycolatum, which is an infrequently found micro-organism .

Arch Microbiol, 2003 Jan-Feb, 179(2), 83 - 8 Epub 2003 Jan 11.
I do it my way: Regulation of ammonium uptake and ammonium assimilation in Corynebacterium glutamicum; Burkovski A; In order to utilize different nitrogen sources and to survive situations of nitrogen limitation, microorganisms have developed several mechanisms to adapt their metabolism to changes in the nitrogen supply . In this communication, recent advances in our knowledge of ammonium uptake, its assimilation, and connected regulatory systems in Corynebacterium glutamicum are discussed with respect to the situation in the bacterial model organisms Escherichia coli and Bacillus subtilis . The regulatory network of nitrogen control in C . glutamicum differs substantially from that in these bacteria, for example, by the presence of AmtR, the unique "master regulator" of nitrogen control, the absence of a NtrB/NtrC two-component signal transduction system, and a different sensing mechanism in C . glutamicum.

Wei Sheng Wu Xue Bao, 2002 Aug, 42(4), 458 - 64
{Site-directed construction of in-frame deletion mutants of Corynebacterium glutamicum}; Ruan H et al.; This study is focused on Corynebacterium glutamicum, an important producer in amino-acids industry, by the protocol with construction of the in-frame deletion fragments of targeting genes, application of DNA recombination technique and so on . We achieved successfully the defined deletion mutants in frame of the single gene or double genes . The in-frame deletion mutants are subsequently analyzed and confirmed by polymerase chain reaction and sequencing.

Wei Sheng Wu Xue Bao, 2002 Aug, 42(4), 395 - 9
{Cloning and sequence analysis of aspartokinase genes from Corynebacterium crenatum}; Liu Y et al.; Aspartokinase genes (ask) from wild-type Corynebacterium crenatum AS1.542 and an AEC-resistant mutant Corynebacterium crenatum CD945 were cloned and sequenced . Analysis of ask sequence shows a exchange in a single base pair at position 1199 from T to C, leading to an amino acid change in the beta subunit of Aspartokinase . Leu80 in the wild-type is converted to Pro80 in the feedback-resistant enzyme . The substitution is located in ACT domain, a region regulated by concentration of lysine . The ORF sequence of ask from C . crenatum AS 1.542 shows homologies of 97.23%, 97.55% and 97.62% to those from C . glutamicum, C . flavum and B . lactofermentum . And the amino acid sequence deduced from ORF displays homologies of 99.76%, 99.52% and 99.76%, respectively . But there is much variation in the upstream sequence of C . crenatum AS 1.542 ask promoter compared to those from other Corynebacteria.

Wei Sheng Wu Xue Bao, 2002 Jun, 42(3), 326 - 30
{Analysis on integration sites of transposon-insertion mutant by transposon rescue}; Ruan H et al.; In order to define the chromosomal locus of the transposon integration in a mutant of Corynebacterium glutamicum, transposon rescue experiments were performed . By isolating parts of the transposon together with adjacent parts of the bacterial chromosome and subsequent sequencing of the adjacent parts, the mutant was found to have three transposon integrations, one just in front of the citrate synthase gene gltA, the other two in hitherto unknown open reading frames designated orfA and orfB . The transposon rescue experiments are proven to be a easy, practical and ideal way in analyzing the transposon integration sites.

Biotechnol Bioeng, 2003 Mar 30, 81(7), 829 - 36
Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation; John GT et al.; Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates . The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference . The sensor measured dissolved oxygen accurately in optically well-defined media . Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker . For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite . k(L)a values in one commercial reader were about 10 to 40 h(-1) . k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity . Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity . Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor . Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate . The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors . The resulting maximum oxygen transfer rate was 26 mM h(-1) . Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions .

Wei Sheng Wu Xue Bao, 1999 Feb, 39(1), 64 - 7
{Strain screening and fermentation conditions of a novel heparinase-producing strain}; Gao N et al.; The novel heparinase-producing bacterial strain Corynebacterium sp . was screened and isolated from soil . The optimum medium composition is (g/L): Trypticase 20, NaCl 1, K2HPO4 2.5, MgSO4 0.5, Heparin 2, maltose 20, pH 6.5 . The optimum growth temperature was 27 degrees C while, maximum enzyme production was achieved at temperature 31 degrees C . When cultured at a rotating shaker at 30 degrees C for 24 hours, 200 r/min, 40 mL medium in 500 mL flask, the Production of heparinase reached 1700 u/L.

Wei Sheng Wu Xue Bao, 1998 Feb, 38(1), 70 - 3
{Strain of screening high toxin--producing from Corynebacterium diphtheria PW8-weissensee}; Rong F et al.; Corynebacterium diphtheria PW8-weissensee was grown on different media for screening high toxin produce . Through six times of screening (selecting butyrous brilliant smooth small colonies and testing their toxin production capacity) from original strain producing toxin 160 Lf/ml, a high toxin--producing descendand culture was obtained, which produced 79% more toxin than the original strain did.

Wei Sheng Wu Xue Bao, 1998 Dec, 38(6), 435 - 40
{Cloning and expression in E . coli of 2,5-DKG reductase I from Corynebacterium}; Chen C et al.; Two different 2,5-DKG reductases was purified from Corynebacterium sp . SCB3058, then its genomic DNA was used as template, a segment containing 2,5-DKG reductase I was amplified by PCR and cloned into pGEM3zf(+) to obtain the recombinant plasmid pGEM813 . The sequence analysis showed that the cloned segment was 1107 bp in length, contained a single open reading frame of 834 nucleotides, which encoded a 34 kD protein consisted of 278 amino acids . After the primary control sequence was deleted, the expression vector pBL4 was constructed by plasmid pBL . With induction of temperature, a 34 kD protein was specifically expressed in E . coli DH5 alpha in high level . The expressed recombinant protein accounted for 20% of the total cell protein and had high specific enzyme activity . In spite of 30 degrees C, 37 degrees C or 42 degrees C induction, the specific activity of enzyme was almost the same level . These results indicate that most of the recombinant protein induced at 42 degrees C was existed with inclusion bodies . This work was the bases of constructing a recombinant Erwinia which can produce 2-KLG directly from D-glucose by one-step fermentation.

J Med Microbiol, 2003 Feb, 52(Pt 2), 181 - 8
Molecular epidemiology and characteristics of Corynebacterium diphtheriae and Corynebacterium ulcerans strains isolated in Italy during the 1990s; von Hunolstein C et al.; Five cases of diphtheria were reported in Italy between January 1990 and June 2001 . Three cases were confirmed microbiologically by the isolation of toxigenic Corynebacterium diphtheriae (two cases) and Corynebacterium ulcerans (one case) . Over the same period, 11 cases of non-toxigenic C . diphtheriae infection were reported to the Italian Public Health Institute, from which the causative organism was isolated from a skin infection in one case and from the throat in the other ten . Seven of the throat isolates were associated with fever, severe pharyngitis and tonsillitis and were all biotype gravis . Because there are no standardized breakpoints, the antimicrobial sensitivities of C . diphtheriae were determined in accordance with the National Committee for Clinical Laboratory Standards guidelines for Streptococcus spp . other than Streptococcus pneumoniae . MICs for penicillin ranged between 0.125 and 0.250 mg l(-1) and 7 out of 11 strains had a minimal bactericidal concentration (MBC)/MIC ratio >or= 32 . All strains were sensitive to clindamycin (MIC <or= 0.25 mg l(-1)), rifampicin (MIC <or= 1 mg l(-1)) and tetracycline (MIC <or= 2 mg l(-1)), and showed moderate susceptibility to cefotaxime (MIC 0.75-1.5 mg l(-1)) . Molecular typing (ribotyping) demonstrated the presence of several distinct ribotypes . The ribotype designated 'D11' has been documented amongst strains isolated in the UK, Russia, Germany, Romania and Sweden . Ribotype 'D75' has only been documented in the UK . The C . ulcerans strain had a ribotype pattern identical to that found in recent isolates from the UK.

Appl Microbiol Biotechnol, 2003 Jan, 60(5), 547 - 55 Epub 2002 Nov 07.
Metabolic flux redistribution in Corynebacterium glutamicum in response to osmotic stress; Varela C et al.; Osmotic stress constitutes a major bacterial stress factor in the soil and during industrial fermentation . In this paper, we quantified the metabolic response, in terms of metabolic flux redistribution, of a lysine-overproducing strain of Corynebacterium glutamicum grown under continuous culture, to gradually increasing osmolality . Oxygen and carbon dioxide evolution rates, and the changes in concentration of extracellular, as well as intracellular, metabolites were measured throughout the osmotic gradient . The metabolic fluxes were estimated from these measurements and from the mass balance constraints at each metabolite-node of the assumed metabolic reaction network . Our results show that formation rates of compatible solutes--trehalose first and proline at a later stage of the gradient--increased with osmotic stress to equilibrate the external osmotic pressure . Estimated flux distributions indicate that the observed increase in the glucose specific uptake rate with osmotic stress is channeled through the main energy generating pathways-- glycolysis and the tricarboxylic acid cycle--while the flux through the pentose phosphate pathway remains constant throughout the gradient . This results in a significant increase in the net specific ATP production rate, which may possibly be used to support the higher energy requirements required for cellular maintenance at high osmolalities . Finally, nodal analysis confirmed that the PEP/pyruvate node is essentially rigid and that the glucose-6-phosphate, oxaloacetate and alpha-ketoglutarate nodes are flexible and therefore adaptable to changes in osmotic pressure in C . glutamicum.

Adv Biochem Eng Biotechnol, 2003, 79, 59 - 112
Biotechnological manufacture of lysine; Pfefferle W et al.; L-Lysine has been manufactured using Corynebacterium glutamicum for more than 40 years . Nowadays production exceeds 600,000 tons per year . Based on conventionally bred strains, further improvement of lysine productivity has been achieved by genetic engineering . Pyruvate carboxylase, aspartate kinase, dihydrodipicolinate synthase, homoserine dehydrogenase and the specific lysine exporter were shown to be key enzymes for lysine production and were characterized in detail . Their combined engineering led to a striking increase in lysine formation . Pathway modeling with data emerging from 13C-isotope experiments revealed a coordinated flux through pentose phosphate cycle and tricarboxylic acid cycle and intensive futile cycling between C3 compounds of glycolysis and C4 compounds of tricarboxylic acid cycle . Process economics have been optimized by developing repeated fed-batch techniques and technical continuous fermentations . In addition, on-line metabolic pathway analysis or flow cytometry may help to improve the fermentation performance . Finally, the availability of the Corynebacterium glutamicum genome sequence has a major impact on the improvement of the biotechnological manufacture of lysine . In this context, all genes of the carbon flow from sugar uptake to lysine secretion have been identified and are accessible to manipulation . The whole sequence information gives access to post genome technologies such as transcriptome analysis, investigation of the proteome and the active metabolic network . These multi-parallel working technologies will accelerate the generation of knowledge . For the first time there is a chance of understanding the overall picture of the physiological state of lysine overproduction in a technical environment.

Adv Biochem Eng Biotechnol, 2003, 79, 37 - 57
Metabolic engineering of glutamate production; Kimura E; Since the discovery of Corynebacterium glutamicum as an efficient glutamate-overproducing microorganism in 1957, the production of L-amino acids by the fermentative method has become one of the most important research-target of industrial microbiology . Several research groups have developed metabolic engineering principles for L-amino acid-producing C . glutamicum strains over the last four decades . The mechanism of L-glutamate-overproduction by the microorganism is very unique and interesting . L-Glutamate overproduction by this bacterium, a biotin auxotroph, is induced by a biotin limitation and suppressed by an excess of biotin . Addition of a surfactant or penicillin is known to induce L-glutamate overproduction under excess biotin . After the development of the general molecular biology tools such as cloning vectors and DNA transfer technique, genes encoding biosynthetic enzymes were isolated . With those genes and tools, recombinant DNA technology can be applied in analysis of biosynthetic pathways and strain construction of C . glutamicum . In this review, key points of the L-glutamate biosynthetic pathways are summarized and the recent studies about triggering mechanism of L-glutamate overproduction by C . glutamicum are introduced . Then the metabolic flux analysis of L-glutamate overproduction is explored.

J Clin Microbiol, 2003 Jan, 41(1), 295 - 303
Culture-independent molecular analysis of microbial constituents of the healthy human outer ear; Frank DN et al.; Molecular-phylogenetic sequence analyses have provided a new perspective on microbial communities by allowing the detection and identification of constituent microorganisms in the absence of cultivation . In this study we used broad-specificity amplification of ribosomal DNA (rDNA) genes to survey organisms present in the human outer ear canal . Samples were obtained from 24 individuals, including members of three extended families, in order to survey the resident microbiota and to examine microbial population structures in individuals related by familial or household associations . To examine the stability of the microbial populations, one individual was sampled four times and another twice over a 14-month period . We found that a distinct set of microbial types was present in the majority of the subjects sampled . The two most prevalent rDNA sequence types that were identified in multiple individuals corresponded closely to those of Alloiococcus otitis and Corynebacterium otitidis, commonly thought to be associated exclusively with infections of the middle ear . Our results suggest, therefore, that the outer ear canal may serve as a reservoir for normally commensal microbes that can contribute to pathogenesis upon introduction into the middle ear . Alternatively, culture analyses of diseases of the middle ear may have been confounded by these contaminating commensal organisms.

Appl Environ Microbiol, 2003 Jan, 69(1), 358 - 66
Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus; Kikuchi Y et al.; The transglutaminase secreted by Streptoverticillium mobaraense is a useful enzyme in the food industry . A fragment of transglutaminase was secreted by Corynebacterium glutamicum when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from C . glutamicum . We analyzed the signal peptide and the pro-domain of the transglutaminase gene and found that the signal peptide consists of 31 amino acid residues and the pro-domain consists of 45 residues . When the pro-domain of the transglutaminase was used, the pro-transglutaminase was secreted efficiently by C . glutamicum but had no enzymatic activity . However, when the plasmid carrying the S . mobaraense transglutaminase also encoded SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus, the peptide bond to the C side of 41-Ser of the pro-transglutaminase was hydrolyzed, and the pro-transglutaminase was converted to an active form . Our findings suggest that C . glutamicum has potential as a host for industrial-scale protein production.

Appl Environ Microbiol, 2003 Jan, 69(1), 18 - 23
Bacterial competition for human nasal cavity colonization: role of Staphylococcal agr alleles; Lina G et al.; We examined the bacterial aerobic nasal flora of 216 healthy volunteers to identify potential competitive interactions among different species, with special emphasis on the influence of staphylococcal agr alleles . The Staphylococcus aureus colonization rate correlated negatively with the rate of colonization by Corynebacterium spp . and non-aureus staphylococci, especially S . epidermidis, suggesting that both Corynebacterium spp . and S . epidermidis antagonize S . aureus colonization . Most of the S . aureus and S . epidermidis isolates were agr typed by a PCR method . Only one S . aureus agr (agr(Sa)) allele was detected in each carrier . Multiple logistic regression of the two most prevalent agr(Sa) alleles (agr-1(Sa) and agr-2(Sa)) and the three S . epidermidis agr (agr(Se)) alleles showed a specific influence of the agr system . The results of this model did not support conclusions drawn from previous in vitro agr-specific cross-inhibition experiments . Our findings suggest that the agr alleles, which are strongly linked to the bacterial genetic background, may simply be associated with common biological properties--including mediators of bacterial interference--in the strains that bear them.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Nov-Dec, (6), 47 - 52
{Characteristics of antitoxic and antibacterial immune response in nontoxic forms of oropharyngeal diphtheria}; Anokhin VA et al.; The enzyme immunoassay system (EIA) for differentiation of antibodies in therapeutic heterogeneous antitoxic serum and antibodies to Corynebacterium diphtheriae toxigenic strains in patients and carriers was developed . The use of EIA permitted the dynamic evaluation of the characteristics of humoral antitoxic and antibacterial immune response in 50 patients with the localized and disseminated forms of stomatopharyngeal diphtheria and 14 "healthy" carriers of toxigenic C . diphtheriae . As revealed in this study, the symptoms of the disease in patients with disseminated forms of stomatopharyngeal diphtheria developed in the presence of statistically significant low quantitative values of antitoxic and antibacterial antibodies to C . diphtheriae antigens . In the group of patients with the localized forms of the disease the initially low level of antitoxic antibodies was detected with the concentration of antibacterial antibodies remaining unchanged . During the period of convalescence the levels of antitoxic antibodies in both groups reached those of healthy persons . In case of localized forms of the disease the level of antibacterial antibodies decreased as compared with healthy persons, starting from the second week of the disease . The period of convalescence in the disseminated forms was characterized by the low concentration of antibacterial antibodies . Carrier state was formed in the presence of high levels of antitoxic antibodies and significantly low levels of antibacterial ones.

Ann Pharmacother, 2003 Jan, 37(1), 61 - 5
Sensorineural hearing loss associated with intrathecal vancomycin; Klibanov OM et al.; OBJECTIVE: To report a case of nonreversible bilateral sensorineural hearing loss resulting from administration of intrathecal vancomycin . CASE SUMMARY: A 63-year-old white man with newly diagnosed pre-B-cell acute lymphocytic leukemia developed Corynebacterium jeikeium meningitis associated with an Ommaya reservoir . The patient was treated with intravenous vancomycin for several days without symptomatic improvement, and intrathecal vancomycin was added to the treatment regimen . Difficulty in the patient's hearing was noted after the first intrathecal dose and he experienced complete hearing loss after the second intrathecal dose . An audiogram was performed and the patient was diagnosed with cranial nerve VIII bilateral sensorineural hearing loss . The Ommaya reservoir was removed and the patient was successfully treated with linezolid . DISCUSSION: Ototoxicity with intravenous vancomycin has been documented in multiple case reports, but this adverse effect has not been reported with intrathecal vancomycin . Cerebrospinal fluid vancomycin concentrations were not measured in our patient, but there was 1 documented occurrence of supratherapeutic serum vancomycin concentrations . Other drug-related causes of ototoxicity were evaluated and intrathecal vancomycin-induced ototoxicity was considered to be possible according to the Naranjo probability scale . CONCLUSIONS: The strong temporal relationship that was seen in this case suggests the possibility of an association between administration of intrathecal vancomycin and hearing loss . Healthcare providers should consider the potential for this adverse reaction with the intrathecal route of vancomycin administration.

Anesthesiology, 2003 Jan, 98(1), 82 - 8
Systemic inflammation leads to resistance to atracurium without increasing membrane expression of acetylcholine receptors; Fink H et al.; BACKGROUND: Systemic inflammation may be associated with resistance to nondepolarizing neuromuscular blocking drugs, the mechanisms of which are, however, uncharacterized . The authors therefore investigated the pharmacodynamics of atracurium and its relation to the expression of nicotinic acetylcholine receptors and alpha1 -acid glycoprotein in a rat model of systemic inflammation . METHODS: To induce a systemic inflammation, male CD rats received 56 mg/kg corynebacterium parvum intravenously . On days 2, 4, 6, 8, 10, 12, 14, or 16 after infection, neuromuscular transmission was measured . The individual effective dose of atracurium was determined, followed by an atracurium infusion at a rate to establish a steady state neuromuscular block of 50% . Total and unbound plasma concentrations of atracurium for 50% paralysis were measured using high-performance liquid chromatography . Acetylcholine receptors were quantitated using 125I-alpha-bungarotoxin . alpha1 -Acid glycoprotein concentrations in the serum were measured using a competitive chemiluminescence immunoassay . RESULTS: The effective dose of atracurium was increased on days 4, 6, and 8 . Total atracurium plasma concentrations at 50% neuromuscular paralysis were increased on days 4, 6, 8, and 10, with a peak at day 8 (8.0 +/- 1.3 micro g/ml) compared with control rats (4.23 +/- 0.82 micro g/ml) . The alpha1 -acid glycoprotein concentrations were increased between days 2 and 10, with a peak on day 4 (6.52 +/- 1.45 mg/ml), and recovered to control values (0.61 +/- 0.33 mg/ml) on day 12 . Unbound plasma concentrations of atracurium to achieve 50% depression, as well as the expression of acetylcholine receptors, did not differ between groups . CONCLUSION: Resistance to atracurium during corynebacterium parvum-induced systemic inflammation is due to increased drug binding to alpha1 -acid glycoprotein and is unrelated to changes in acetylcholine receptor expression.

J Gen Appl Microbiol, 1999 Aug, 45(4), 169 - 176
Accumulation of anthranilic acid and N-glucosylanthranilic acid by a Corynebacterium glutamicum mutant resistant to DL-serine hydroxamate; Araki K et al.; During a study on the effect of DL-serine hydroxamate on Corynebacterium glutamicum (JCM1318, a wild strain), a mutant resistant to the drug, strain TO3002, was isolated . This mutant accumulated five Ehrlich's reagent positive fluorescent substances in the culture medium . Two major and one minor fluorescent products were isolated by preparative high-performance liquid chromatography following charcoal column chromatography from the culture supernatant . One major product was identified as anthranilic acid whose molecular ion was confirmed to be 137 by a measurement of liquid chromatography-mass spectrometry (LC-MS), and NMR spectrum coincided with that of anthranilic acid . LC-MS spectra of another major and the minor product showed that they had the same molecular weight of 299 . This major product was supported to be N-glucosylanthranilic acid (N-o-carboxyphenyl-1-beta-glucosylamine) by two-dimensional (1)H and (13)C NMR analyses . The minor product was speculated to be an Amadori compound derived from N-glucosylanthranilic acid . N-Glucosylanthranilic acid accumulated in the early phase, then decreased in the late phase of the culture . In contrast, the accumulation of anthranilic acid increased remarkably in the late phase of the fermentation . Based on this phenomenon, it was assumed that N-glucosylanthranilic acid once accumulated was decomposed to form anthranilic acid, at least in large part, with the progress of fermentation . The strain TO3002 showed a leaky requirement for L-tryptophan or indole (but did not for anthranilic acid) and resistance to DL-serine hydroxamate.

J Gen Appl Microbiol, 1999 Oct, 45(5), 253 - 262
Leifsonia gen . nov., a genus for 2,4-diaminobutyric acid-containing actinomycetes to accommodate "Corynebacterium aquaticum" Leifson 1962 and Clavibacter xyli subsp . cynodontis Davis et al . 1984; Suzuki KI et al.; "Corynebacterium aquaticum" was first proposed by Leifson in 1962 but not included in the approved lists of bacterial names in 1980 . This species has been left from reclassification of the genus Corynebacterium because of the unusual chemotaxonomic characteristics such as 2,4-diaminobutyric acid (DAB) in the peptidoglycan and menaquinones of MK-10 and MK-11 . A close relationship of "C . aquaticum" to the genera Agromyces and Rathayibacter has been pointed out from the viewpoint of chemotaxonomic profiles and phylogeny based on the 16S rDNA sequences . An analysis of DAB isomers of the peptidoglycan distinguished "C . aquaticum" clearly from these genera by possessing both L-DAB and D-DAB . We also found that the type strain of Clavibacter xyli subsp . cynodontis and two strains of amine-decomposing bacteria showed the similar chemotaxonomic features and formed a cluster with "C . aquaticum" in the phylogenetic tree based on 16S rDNA sequences in the family Microbacteriaceae . Considering these results, we propose a new genus Leifsonia to accommodate the four strains . The four species, Leifsonia aquatica sp . nov., nom . rev., comb . nov . (type species, type strain=JCM 1368), Leifsonia shinshuensis sp . nov . (type strain=DB102=JCM 10591), Leifsonia naganoensis sp . nov . (type strain=DB103=JCM 10592), and Leifsonia cynodontis comb . nov . (type strain=JCM 9733=ICMP 8790), were proposed here for the strains.

Ann Agric Environ Med, 2002, 9(2), 225 - 35
Exposure to airborne microorganisms and endotoxin in a potato processing plant; Dutkiewicz J et al.; Microbiological air sampling was performed in a big potato processing plant located in eastern Poland . Air samples for determination of concentrations of microorganisms, dust and endotoxin were collected at 6 sites in the division producing potato flakes and meal from dried potato pulp and at 2 sites in the division producing potato syrup from imported starch . The concentrations of total airborne microorganisms were within a range of 28.3-93.1 x 10(3) cfu/m(3) . Mesophilic bacteria were dominant at all sampling sites, forming 73.1-98.8% of the total count . Among them, distinctly prevailed corynebacteria (irregular Gram-positive rods) that accounted for 54.3-81.1% of the total airborne microflora . The most common were strains of Corynebacterium spp., followed by strains of Arthrobacter spp., Microbacterium spp., and Agromyces ramosus . The latter species, so far not reported from the air of occupational environments, abundantly develops in the parenchyma of potato tubers . Its airborne concentration increased rapidly after peeling of potatoes, and attained maximal values at cutting and blanching (steaming and sulfuration) of potatoes, and at sacking of potato meal . The proportions of Gram-negative bacteria and endospore-forming bacilli were low, respectively 0.6-7.6% and 2.0-8.1% of total count . Fungi constituted 1.2-26.9% of total count . The dominant species was Aspergillus niger that formed 99.8% of total airborne fungi . The values of the respirable fraction of airborne microflora varied between 25.3-73.2% . The concentrations of airborne dust were 1.4-26.6 mg/m(3) in the division producing potato flakes and meal and 114.9-200.5 mg/m(3) at pouring of potato and corn starch for syrup . The concentrations of airborne endotoxin were in the range of 0.011-0.089 microg/m(3) during the initial stages of potato processing (unloading, washing, peeling) and drastically increased after blanching to the extraordinarily high levels of 45.9-1893.9 microg/m(3) . At pouring of starch for syrup, the concentrations of airborne endotoxin were much lower, within a range of 0.029-0.156 microg/m(3) . In conclusion, the workers of potato processing facilities could be exposed to large concentrations of microorganisms, dust and endotoxin posing a risk of work-related respiratory disease.

Pathol Biol (Paris), 2002 Nov, 50(9), 513 - 5
{In vivo activity of amoxicillin in a non-toxigenic Corynebacterium diphtheriae rabbit endocarditis experimental model}; Grandiere-Perez L et al.; The mortality of non-toxigenic Corynebacterium diphtheriae endocarditis is high (27%) . One explanation could be tolerance to amoxicillin . The aim of this work was to evaluate in vivo the tolerance phenomenon, in a rabbit endocarditis experimental model . Two strains of non-toxigenic Corynebacterium diphtheriae were compared: a tolerant one and a non-tolerant one . EACH ANIMAL WAS RANDOMLY ASSIGNED TO ONE OF THE FOLLOWING GROUPS: no treatment, continuous infusion of amoxicillin simulating 200 mg/kg/24 h in humans, or fractionated infusion of amoxicillin simulating 66 mg/kg/8 h in humans . Surviving bacteria were counted in vegetations after one or three days of treatment.The 24 h fractionated amoxicillin infusion was more efficacious on the non-tolerant strain than on the tolerant strain . On the tolerant strain, 24 h amoxicillin was more efficacious as a continuous infusion than as a fractionated one.

Braz J Biol, 2002 May, 62(2), 363 - 9 Epub 2002 Aug 26.
Screening for exopolysaccharide-producing bacteria from sub-tropical polluted groundwater; Fusconi R et al.; A selection of exopolysaccharide (EPS)--producing bacterial strains was conducted in groundwater adjacent to an old controlled landfill in the City of Sao Carlos (Sao Paulo, Brazil) . The strains were isolated in P and E media under aerobic and microacrophilic conditions at 25 degrees C . A total of 26 strains were isolated and based on the mucoid mode of the colonies, 6 were selected and their morphological, physiological and biochemical aspects were characterized . All strains presented pigmentation, ranging from yellow to orange and from pink to salmon, with a shiny glistening aspect in all tested media . Strains Lb, Lc and Lg, which excelled the others with regard to the mucoid mode of the colonies, were selected to be cultured in E medium with alternate sucrose and glucose as carbon sources in anaerobiosis at 25 degrees C to analyze the production of EPS . Strains Lc and Lg were classified as being of order Actinomycelates, suborder Corynebacterineae . Lg strain was identified as Gordonia polyisoprenivorans and Lc strain did not correspond to a known description and therefore a more detailed study is under preparation . Considering all ecological aspects and the metabolic potential associated with the microorganisms of the environment studied, as well as the capacity to produce pigment and EPS, and the presence of G . polyisoprenivorans, a rubber degrader bacterium, the potential of the groundwater analyzed is evident as a source of microorganisms to be utilized in studies related to environmental remediation.

Pediatr Infect Dis J, 2002 Dec, 21(12), 1124 - 6
Turicella otitidis and Corynebacterium auris do not cause otitis media with effusion in children; Holzmann D et al.; BACKGROUND: The recently described coryneform bacteria and were first detected in the middle ear of patients with acute otitis media and chronic otitis media . Whether these bacteria play an essential role in the pathogenesis of otitis media with effusion (OME) is unclear . METHODS: In a prospective study 60 children with OME and 205 controls were evaluated to determine the incidence of and . Swabs from the external auditory canal (EAC) and the middle ear effusion (MEE) of OME children undergoing tympanotomy, ventilation tube insertion or both were cultured . Swabs from the EAC from healthy children served as controls . RESULTS: In control children was found in EAC swabs from 23 of 205 (11.2%) and in 32 of 205 (15.6%) . was isolated from 14 of 60 (23.3%) OME patients from the EAC only and in 6 of 60 (10.0%) OME patients from both EAC and MEE . was isolated in 2 of 60 (3.3%) from the EAC only and in 1 of 60 (1.7%) from both EAC and MEE . In no patient did or grow exclusively from MEE . CONCLUSION: and may be part of the normal bacterial flora of the EAC in some children . Neither organism seems to cause OME in children.

J Mol Evol, 2002 Dec, 55(6), 623 - 31
Verification of a novel NADH-binding motif: combinatorial mutagenesis of three amino acids in the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase; Banta S et al.; A screening method has been developed to support randomized mutagenesis of amino acids in the cofactor-binding pocket of the NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase . Such an approach could enable the isolation of an enzyme that can better catalyze the reduction of 2,5-DKG to 2-keto-L-gulonic acid (2-KLG) using NADH as a cofactor . 2-KLG is a valuable precursor to ascorbic acid, or vitamin C, and an enzyme with increased activity with NADH may be able to improve two potential vitamin C production processes . Previously we have identified three amino acid residues that can be mutated to improve activity with NADH as a cofactor . As a pilot study to show feasibility, a library was made with these three amino acids randomized, and 300 random colonies were screened for increased NADH activity . The activities of seven mutants with apparent improvements were verified using activity-stained native gels, and sequencing showed that the amino acids obtained were similar to some of those already discovered using rational design . The four most active mutants were purified and kinetically characterized . All of the new mutations resulted in apparent kcat values that were equal to or higher than that of the best mutant obtained through rational design . At saturating levels of cofactor, the best mutant obtained was almost twice as active with NADH as a cofactor as the wild-type enzyme is with NADPH . This screen is a valuable tool for improving 2,5-DKG reductase, and it could easily be modified for improving other aspects of this protein or similar enzymes.

J Biol Chem, 2003 Feb 28, 278(9), 6651 - 63 Epub 2002 Dec 11.
Solution 1H NMR investigation of the active site molecular and electronic structures of substrate-bound, cyanide-inhibited HmuO, a bacterial heme oxygenase from Corynebacterium diphtheriae; Li Y et al.; The molecular structure and dynamic properties of the active site environment of HmuO, a heme oxygenase (HO) from the pathogenic bacterium Corynebacterium diphtheriae, have been investigated by (1)H NMR spectroscopy using the human HO (hHO) complex as a homology model . It is demonstrated that not only the spatial contacts among residues and between residues and heme, but the magnetic axes that can be related to the direction and magnitude of the steric tilt of the FeCN unit are strongly conserved in the two HO complexes . The results indicate that very similar contributions of steric blockage of several meso positions and steric tilt of the attacking ligand are operative . A distal H-bond network that involves numerous very strong H-bonds and immobilized water molecules is identified in HmuO that is analogous to that previously identified in hHO (Li, Y., Syvitski, R . T., Auclair, K., Wilks, A., Ortiz de Montellano, P . R., and La Mar, G . N . (2002) J . Biol . Chem . 277, 33018-33031) . The NMR results are completely consistent with the very recent crystal structure of the HmuO.substrate complex . The H-bond network/ordered water molecules are proposed to orient the distal water molecule near the catalytically key Asp(136) (Asp(140) in hHO) that stabilizes the hydroperoxy intermediate . The dynamic stability of this H-bond network in HmuO is significantly greater than in hHO and may account for the slower catalytic rate in bacterial HO compared with mammalian HO.

J Biol Chem, 2003 Feb 21, 278(8), 5718 - 27 Epub 2002 Dec 04.
A specific bacterial aminoacylase cleaves odorant precursors secreted in the human axilla; Natsch A et al.; Human axillary odor is known to be formed upon the action of Corynebacteria sp . on odorless axilla secretions . The known axilla odor determinant 3-methyl-2-hexenoic acid was identified in hydrolyzed axilla secretions along with a chemically related compound, 3-hydroxy-3-methylhexanoic acid . The natural precursors of both these acids were purified from non-hydrolyzed axilla secretions . From liquid chromatography/mass spectrometry analysis, it appeared that the acids are covalently linked to a glutamine residue in fresh axilla secretions, and the corresponding conjugates were synthesized for confirmation . Bacterial isolates obtained from the human axilla and belonging to the Corynebacteria were found to release the acids from these odorless precursors in vitro . A Zn(2+)-dependent aminoacylase mediating this cleavage was purified from Corynebacterium striatum Ax20, and the corresponding gene agaA was cloned and heterologously expressed in Escherichia coli . The enzyme is highly specific for the glutamine residue but has a low specificity for the acyl part of the substrate . agaA is closely related to many genes coding for enzymes involved in the cleavage of N-terminal acyl and aryl substituents from amino acids . This is the first report of the structure elucidation of precursors for human body odorants and the isolation of the bacterial enzyme involved in their cleavage.

Appl Microbiol Biotechnol, 2002 Dec, 60(4), 437 - 41 Epub 2002 Nov 06.
3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum: the C-terminal domain is not essential for activity but is required for inhibition by L-serine; Peters-Wendisch P et al.; The serA gene of Corynebacterium glutamicum coding for 3-phosphoglycerate dehydrogenase (PGDH) was isolated and functionally characterized . It encodes a polypeptide of 530 aminoacyl residues (aa), which is substantially longer than the corresponding Escherichia coli polypeptide of 410 aa . The difference is largely due to an additional stretch of aa in the carboxy- (C)-terminal part of the polypeptide . Overexpression of serA in C . glutamicum results in a 16-fold increase in specific PGDH activity to 2.1 U/mg protein, with activity being inhibited by high concentrations of L-serine . A set of muteins that were progressively truncated at the C-terminal end was constructed . When overexpressed, mutein SerADelta197 showed a specific PGDH dehydrogenase activity of 1.3 U/mg protein, with the activity no longer being sensitive to L-serine . Gel filtration experiments showed that wild type PGDH is a homotetramer, whereas mutein SerADelta197 constitutes a dimer . Thus, the specific regulatory features of C . glutamicum PGDH are due to the C-terminal part of the polypeptide, which can be deleted with almost no effect on the catalytic activity of the enzyme.

Eur J Pharmacol, 2002 Dec 13, 457(1), 57 - 69
A role for histamine in cytokine modulation by the adenosine A(3) receptor agonist, 2-Cl-IB-MECA; Smith SR et al.; The effects of adenosine receptor agonists on cytokine production in vivo were investigated in mouse models of endotoxemia . Selective adenosine A(3) (2-chloro-N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide) (2-Cl-IB-MECA) and A(2A) (2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride) (CGS 21860) receptor agonists were found to modulate endotoxin-induced cytokine responses in mice sensitized to D-galactosamine or primed with Corynebacterium parvum . The adenosine receptor agonists had similar effects in these models of endotoxemia, suppressing the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-12 while enhancing that of interleukin-10 . However, 2-Cl-IB-MECA also caused a dramatic increase in circulating histamine levels shortly after its injection into mice . The cytokine modulatory activities of 2-Cl-IB-MECA were mimicked by the mast cell depleting compound 48/80 and both drugs only produced such effects at doses that caused an elevation in circulating histamine levels . Furthermore, the capacity of 2-Cl-IB-MECA to modulate cytokine responses was greatly diminished when the drug was administered to mast cell deficient (WBB6F-W/W(V)) mice . Together, these results strongly suggest a role for histamine in cytokine modulation by 2-Cl-IB-MECA . Cimetidine, a histamine H(2) receptor antagonist, did not reverse cytokine modulation by 2-Cl-IB-MECA and pyrilamine, a histamine H(1) receptor antagonist, prevented the increase in serum histamine that was induced by 2-Cl-IB-MECA . This effect of pyrilamine and other histamine H(1) receptor antagonists confounded attempts to determine a role for the histamine H(1) receptor in cytokine modulation by 2-Cl-IB-MECA . However, under some experimental conditions, pyrilamine appeared to antagonize the modulatory effects of the adenosine A(3) receptor agonist on cytokine responses . The apparent antagonism of pyrilamine was unrelated to its suppressive effects on histamine release and appeared to reflect activity at the level of the histamine H(1) receptor.

Diagn Microbiol Infect Dis, 2002 Oct, 44(2), 205 - 7
Corynebacterium macginleyi isolation from conjunctival swab in Italy; Giammanco GM et al.; Corynebacterium macginleyi was isolated from conjunctival swabs of a farmer suffering from purulent conjunctivitis . This species has only recently been reported in Switzerland and Germany to be exclusively isolated from ocular surfaces . This represents the first isolation of C . macginleyi in Italy indicating that its circulation is not geographically limited.

J Clin Microbiol, 2002 Dec, 40(12), 4713 - 9
Development of a real-time fluorescence PCR assay for rapid detection of the diphtheria toxin gene; Mothershed EA et al.; We developed and evaluated a real-time fluorescence PCR assay for detecting the A and B subunits of diphtheria toxin (tox) gene . When 23 toxigenic Corynebacterium diphtheriae strains, 9 nontoxigenic C . diphtheriae strains, and 44 strains representing the diversity of pathogens and normal respiratory flora were tested, this real-time PCR assay exhibited 100% sensitivity and specificity . It allowed for the detection of both subunits of the tox gene at 750 times greater sensitivity (2 CFU) than the standard PCR (1,500 CFU) . When used directly on specimens collected from patients with clinical diphtheria, one or both subunits of the tox gene were detected in 34 of 36 specimens by using the real-time PCR assay; only 9 specimens were found to be positive by standard PCR . Reamplification by standard PCR and DNA sequencing of the amplification product confirmed all real-time PCR tox-positive reactions . This real-time PCR format is a more sensitive and rapid alternative to standard PCR for detection of the tox gene in clinical material.

Appl Environ Microbiol, 2002 Dec, 68(12), 5843 - 59
Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria; Wittmann C et al.; A comprehensive approach of metabolite balancing, (13)C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum . The five strains examined (C . glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection . Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux . Strain optimization was accompanied by significant changes in intracellular flux distributions . The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield . Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes . The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants . In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization . Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor . The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C . glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions . Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that the observed differences were clearly strain specific.

J Biol Chem, 2003 Feb 7, 278(6), 4339 - 46 Epub 2002 Nov 20.
Purification of a cytochrome bc-aa3 supercomplex with quinol oxidase activity from Corynebacterium glutamicum . Identification of a fourth subunity of cytochrome aa3 oxidase and mutational analysis of diheme cytochrome c1; Niebisch A et al.; The aerobic respiratory chain of the Gram-positive Corynebacterium glutamicum involves a bc(1) complex with a diheme cytochrome c(1) and a cytochrome aa(3) oxidase but no additional c-type cytochromes . Here we show that the two enzymes form a supercomplex, because affinity chromatography of either strep-tagged cytochrome b (QcrB) or strep-tagged subunit I (CtaD) of cytochrome aa(3) always resulted in the copurification of the subunits of the bc(1) complex (QcrA, QcrB, QcrC) and the aa(3) complex (CtaD, CtaC, CtaE) . The isolated bc(1)-aa(3) supercomplexes had quinol oxidase activity, indicating functional electron transfer between cytochrome c(1) and the Cu(A) center of cytochrome aa(3) . Besides the known bc(1) and aa(3) subunits, few additional proteins were copurified, one of which (CtaF) was identified as a fourth subunit of cytochrome aa(3) . If either of the two CXXCH motifs for covalent heme attachment in cytochrome c(1) was changed to SXXSH, the resulting mutants showed severe growth defects, had no detectable c-type cytochrome, and their cytochrome b level was strongly reduced . This indicates that the attachment of both heme groups to apo-cytochrome c(1) is not only required for the activity but also for the assembly and/or stability of the bc(1) complex.

J Bacteriol, 2002 Dec, 184(24), 6882 - 92
Analysis of a DtxR-like metalloregulatory protein, MntR, from Corynebacterium diphtheriae that controls expression of an ABC metal transporter by an Mn(2+)-dependent mechanism; Schmitt MP; The DtxR protein is a global iron-dependent repressor in Corynebacterium diphtheriae that regulates transcription from multiple promoters . A search of the partially completed C . diphtheriae genome identified a gene, mntR, whose predicted product has significant homology with the DtxR repressor protein . The mntR gene is the terminal gene in a five-gene operon that also carries the mntABCD genes, whose predicted products are homologous to ABC metal transporters . Transcription of this genetic system, as measured by expression of an mntA-lacZ reporter fusion, is strongly repressed by Mn(2+) . The divalent metals Fe(2+), Cu(2+), and Zn(2+) did not repress expression of the mntA-lacZ construct . A mutation in the mntR gene abolished Mn(2+)-dependent repression of the mntA-lacZ fusion, demonstrating that MntR is essential for the Mn(2+)-dependent regulation of this promoter . Footprinting experiments showed that MntR protects from DNase I digestion an approximately 73-bp AT-rich region that includes the entire mntA promoter . This large region protected from DNase I suggests that as many as three MntR dimer pairs may bind to this region . Binding studies also revealed that DtxR failed to bind to the MntR binding site and that MntR exhibited weak and diffuse binding at the DtxR binding site at the tox promoter . A C . diphtheriae mntA mutant grew as well as the wild type in a low-Mn(2+) medium, which suggests that the mntABCD metal transporter is not required for growth in a low-Mn(2+) medium and that additional Mn(2+) transport systems may be present in C . diphtheriae . This study reports the characterization of MntR, a Mn(2+)-dependent repressor, and the second member of the family of DtxR-like metalloregulatory proteins to be identified in C . diphtheriae.

FEMS Microbiol Lett, 2002 Nov 19, 217(1), 103 - 7
Purification, characterization and identification of cysteine desulfhydrase of Corynebacterium glutamicum, and its relationship to cysteine production; Wada M et al.; We highly purified the enzyme having L-cysteine desulfhydrase activity from Corynebacterium glutamicum . According to its partial amino acid sequence, the enzyme was identified as the aecD gene product, a C-S lyase with alpha, beta-elimination activity {I . Rossol and A . Puhler (1992) J . Bacteriol . 174, 2968-2977} . To produce L-cysteine in C . glutamicum, the Escherichia coli-altered cysE gene encoding Met256Ile mutant serine acetyltransferase, which is desensitized to feedback inhibition by L-cysteine, was introduced into C . glutamicum . When the altered cysE gene was expressed in the aecD-disrupted strain, the transformants produced approximately 290 mg of L-cysteine plus L-cystine per liter from glucose . The produced amount of these amino acids was about two-fold higher than that of the wild-type strain.

FEMS Microbiol Lett, 2002 Nov 19, 217(1), 71 - 9
S-layer protein transport across the cell wall of Corynebacterium glutamicum: in vivo kinetics and energy requirements; Houssin C et al.; Corynebacteria are Gram-positive bacteria with a very peculiar cell envelope structure as it is constituted of an inner membrane and an outer membrane-like structure . Protein secretion in Corynebacterium glutamicum was studied in vivo, using the S-layer protein PS2 as a model . We show that different variants of PS2 protein are exported through the whole cell envelope with a half-life ranging between 2 and 4 min, by a two-step mechanism . The first step, which is over after about 1.5 min, is ATP- and proton motive force-dependent and may correspond to translocation across the inner membrane via the 'Sec' machinery . The second step, across the cell wall and the outer mycolate layer, is rapid but independent of energy sources . This very efficient secretion process across the mycolate layer raises the question of the existence in this layer of a specific machinery.

Clin Infect Dis, 2002 Dec 1, 35(11), 1434 - 40 Epub 2002 Nov 14.
Corynebacterium species isolated from patients with mastitis; Paviour S et al.; Corynebacteria were isolated from breast tissue, pus, or deep wound swabs of 24 women; the most common species isolated was the newly described Corynebacterium kroppenstedtii, followed by Corynebacterium amycolatum and Corynebacterium tuberculostearicum . Gram-positive bacilli were seen in samples sent for culture or in histological specimens for 12 women, and 9 of the 12 women from whom adequate histological specimens were obtained had conditions that met the criteria for granulomatous lobular mastitis, a chronic inflammatory disease of unknown etiology.

Curr Microbiol, 2003 Jan, 46(1), 1 - 5
Characterization of Cr(VI)-resistant bacteria isolated from chromium-contaminated soil by tannery activity; Viti C et al.; Bacterial strains, previously isolated from a chromium-polluted soil, were identified on the basis of Gram reaction and biochemical characteristics (Biolog system) . Moreover, chromate MICs, chromate reduction capability, multiple heavy metal tolerance, and antibiotic susceptibility were tested for each isolate . All strains but one were Gram-positive and resistant to high concentrations of chromate . The most Cr(VI)-resistant isolate (22m M) was identified as Corynebacterium hoagii . All Cr(VI)-resistant strains except the isolate ChrC20 were capable of catalyzing the reduction of Cr(VI) to Cr(III), a less toxic and less water-soluble form of chromium . The only isolate Cr(VI)-sensitive, attributed to the Pseudomonas genus, also exhibited Cr(VI)-reduction . Isolates were also screened for the presence of plasmid DNA . The strains ChrC20 and ChrB20 harbored one and two plasmids of high molecular mass, respectively . This approach permitted selection of some bacterial strains, which could be used for bioremediation of Cr(VI)-polluted environments.

FEMS Microbiol Lett, 2002 Oct 29, 216(1), 71 - 5
Characterization of a novel plasmid pXZ608 from Corynebacterium glutamicum; Lei C et al.; The complete nucleotide sequence of a novel cryptic plasmid pXZ608 from Corynebacterium glutamicum 227 was determined . pXZ608 was 5949 bp with six open reading frames (ORF1-6) . The predicted ORF1 gene product was homologous to replication proteins of rolling circle replication plasmids . The conserved single- and double-stranded origins of rolling circle replication were found, and interestingly, the two origins were both located on ORF1, which indicated that the Rep protein encoded by ORF1 could bind to its own gene region . Deletion analysis revealed that the minimal replicon was located on the 2.14-kb SacI-BstEII fragment.

Folia Microbiol (Praha), 2002, 47(4), 359 - 63
Biological activity of secondary metabolites produced by a strain of Pseudomonas fluorescens; Boruah HP et al.; Biological activity of secondary metabolites produced by a plant-growth-promoting Pseudomonas fluorescens was evaluated . The strain produced antibiotics phenazine (PHE), 2,4-diacetylphloroglucinol (PHL) and siderophore pyoverdin (PYO) in standard King's B and succinic acid media, respectively . After extraction, PYO was identified by comparing the UV-spectra and moss-green color development after 'diazotized sulfanilic acid' (DSA) spray in TLC . PHE and PHL were identified by comparing standard compounds on TLC and orange-color development immediately after DSA spray . In vitro antibiosis study of the metabolites revealed their antibacterial and antifungal activity against bacterial test organisms Corynebacterium sp., Mycobacterium phlei and M . smegmatis and test fungi Fusarium moniliforme, F . oxysporum, F . semitectum, F . solani and Rhizoctonia solani . A statistically significantly higher plant growth was recorded in siderophore-amended plantlets under gnotobiotic conditions whereas PHE and PHL did not show any plant-growth-promoting activity . These results support the importance of the secondary metabolites produced by the strain P . fluorescens in enhancing plant growth and in controlling fungal and bacterial pathogens.

Folia Microbiol (Praha), 2002, 47(4), 307 - 10
Atypical location of double-strand origin of replication (nic site) on the plasmid pGA1 from Corynebacterium glutamicum; Abrhamova Z et al.; The double-strand origin of replication (dso) of the rolling-circle-replicating (RC) plasmid pGA1 from Corynebacterium glutamicum was analyzed using the runoff DNA synthesis assay . The site- and strand-specific breakage of double-stranded plasmid DNA, representing the nic site of dso, was localized precisely within the sequence 5'-CTGG decreases AT-3' in the distal part of the pGA1 rep gene . This location of dso differs from the dso positions found on other RC plasmids and is in agreement with the classification of the plasmid pGA1 into a new group of RC plasmids.

An Med Interna, 2002 Sep, 19(9), 473 - 6
{Infections by Arcanobacterium haemolyticum: an emerging pathogen}; Puerto Alonso JL et al.; Arcanobacterium haemolyticum is a grampositive rod wich belonged, until a short time ago, to Corynebacterium genus, and recently classified in a new genus, with only one specie . Human is the main reservoir . It has been isolated from the skin and pharinx of healthy individuals, but also it is cause of infection, specially pharingitis, in children, and chronic cutaneous ulcus, in diabetic patients . Less frequently, it is cause of osteomyelitis, meningitis, pneumonia, abscess, endocarditis and sepsis . Diagnosis is difficult because its double quality: comensal and pathogen . There are not established guidelines for the treatment of these infections, although most of isolated strains are susceptibles to penicillin, erythromicin, clindamycin and tetracycline . High doses of penicillin, with or without gentamicin, it is recommended for the treatment of deep infections.

An Med Interna, 2002 Sep, 19(9), 463 - 5
{Pneumonia caused by Corynebacterium pseudodiphteriticum, an entity worth knowing}; Aspiroz Sancho C et al.; Respiratory infections are challenging for clinicians and new microbes or those considered previously as normal flora or less virulent forms seen responsible for some cases . Thus, the case reported here is a nosocomial pneumonia caused by Corynebacterium pseudodiphteriticum in a man suffering chronic obstructive pulmonary disease and resolved with cefotaxime . This microorganism is part of the oropharingeal bacterial flora and is therefore associated mainly with respiratory disease an less commonly with endocarditis, prostheses or wound infections . Susceptibility testing found uniform susceptibility to b-lactamases, aminoglycosides, rifampin and tetracycline . Susceptibility to ciprofloxacine is variable and resistance to macrolides (erythromycin and clindamycin) was frequent.

J Dairy Sci, 2002 Oct, 85(10), 2512 - 20
The effect of an intramammary teat seal on new intramammary infections; Berry EA et al.; As concern over the possible overuse of antibacterials increases, attention has focused on reduction of antibiotic usage and on nonantibiotic alternatives . A nonantibiotic intramammary teat sealant, Teat Seal (Cross Vetpharm Group Ltd., Tallaght, Dublin, Ireland), has been available in Ireland, in combination with an intramammary tube of cloxacillin . Teat Seal has been reformulated for use in cows with low cell counts as an alternative to antibiotic dry cow therapy at the end of lactation . The product is now marketed as Orbeseal (Pfizer Animal Health) . A comparison between this teat sealant and no treatment was made on new intramammary infections and clinical mastitis, on all cows within four herds, and on low cell count cows in three herds . No cases of clinical mastitis in the dry period were observed in cows treated with Teat Seal (n = 197), whereas a significant number (6 cows) were observed in the untreated cows (n = 204) . In all herds, significantly more new infections at calving were found in the untreated group (62 cows in the untreated group compared with 21 cows in the Teat Seal group) . In those quarters where infections were first detected at calving, the incidence of clinical mastitis was significantly greater in the untreated group . Quarters in both treatment groups that were infected at drying off with Corynebacterium spp . or coagulase-negative staphylococci were not protected against new infections and had an increased risk of new infection by Streptococcus uberis . The results will inform those restricting their use of antibiotic dry cow therapy in alternative management strategies and the additional risk of new intramammary infection.

J Clin Microbiol, 2002 Nov, 40(11), 4375 - 81
Characteristics of rare or recently described corynebacterium species recovered from human clinical material in Canada; Bernard KA et al.; Nineteen new Corynebacterium species or taxa described since 1995 have been associated with human disease . We report the characteristics of 72 strains identified as or most closely resembling 14 of these newer, medically relevant Corynebacterium species or taxa, as well as describe in brief an isolate of Corynebacterium bovis, a rare pathogen for humans . The bacteria studied in this report were nearly all derived from human clinical specimens and were identified by a polyphasic approach . Most were characterized by nearly full 16S rRNA gene sequence analysis . Some isolates were recovered from previously unreported sources and exhibited unusual phenotypes or represented the first isolates found outside Europe . Products of fermentation, with emphasis on the presence or absence of propionic acid, were also studied in order to provide an additional characteristic with which to differentiate among phenotypically similar species.

Appl Environ Microbiol, 2002 Nov, 68(11), 5422 - 8
Effect of pyruvate carboxylase overexpression on the physiology of Corynebacterium glutamicum; Koffas MA et al.; Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria . In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C . glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate . In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities . If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis . Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.

Vet Immunol Immunopathol, 2002 Nov, 90(1-2), 55 - 63
Efficacy of DNA vaccination by different routes of immunisation in sheep; De Rose R et al.; DNA vaccination, delivered through various routes, has been used extensively in laboratory animals . Few studies have focused on veterinary species and while results obtained in laboratory animals can often be extrapolated to veterinary species this is not always the case . In this study we have compared the effect of the route of immunisation with DNA on the induction of immune responses and protection of sheep to challenge with live Corynebacterium pseudotuberculosis . Intramuscular injection of plasmid DNA encoding an inactivated form of the phospholipase D (PLD) antigen linked to CTLA4-Ig resulted in the induction of a strong memory response and sterile immunity following challenge in 45% of the animals . In contrast, gene gun delivery or subcutaneous (SC) injection of the DNA vaccine induced comparatively poor responses and insignificant levels of protection . Thus, DNA vaccine efficacy in sheep is strongly influenced by the route of vaccination . Amongst intramuscular vaccinates, protected sheep had significantly elevated IgG2 responses compared to unprotected animals, while both subgroups had equivalent IgG1 levels . This suggests that the presence of IgG2 antibodies and hence a Th1-like response, induced by the DNA vaccine gave rise to protective immunity against C . pseudotuberculosis.

Crit Rev Oncol Hematol, 2002 Oct, 44(1), 81 - 102
Adjuvant therapy of malignant melanoma; Molife R et al.; High risk surgically resected melanoma is associated with a less than 50% 5-year survival . Adjuvant therapy is an appropriate treatment modality in this setting, and is more likely to be effective as the tumour burden here is small . Clinical observations of spontaneous tumour regressions and a highly variable rate of disease progression suggest a role of the immune system in the natural history of melanoma . Biological agents have therefore been the subjects of numerous adjuvant studies . Early, randomised controlled trials (RCTs) of Bacillus Calmette-Guerin (BCG), levamisole, Corynebacterium parvum, chemotherapy, isolated limb perfusion (ILP), radiotherapy, transfer factor (TF), megestrol acetate and vitamin A yielded largely negative results . Current trials focus on vaccines and the interferons . To date the latter is the only therapy to have shown a significant benefit in the prospective randomised controlled phase III setting . This report represents a systematic review of studies in adjuvant therapy in melanoma . Data from ongoing studies is awaited before a role for adjuvant agents in high risk melanoma is confirmed.

CLAO J, 2002 Oct, 28(4), 192 - 5
Microbiologic evaluation of frequent-replacement soft contact lenses; Iskeleli G et al.; PURPOSE: This article reports a microbiologic study of two kinds of monthly frequent-replacement daily wear soft contact lenses, with different amounts of water content, in asymptomatic contact lens wearers . METHOD: We studied 35 lenses of 18 patients who wear frequent-replacement soft contact lenses with a water content of 38% and 40 lenses of 20 patients using frequent-replacement contact lenses with a water content of 55% . The lenses worn by patients regularly for 1 month were removed from their eyes in a sterile manner on the 30th day and were studied microbiologically to isolate pathogenic agents . RESULTS: In the group of monthly frequent-replacement soft contact lenses with a water content of 38%, microorganisms were isolated at a rate of 91%; and in the group of monthly frequent-replacement soft contact lenses with a water content of 55%, microorganisms were isolated at a rate of 85% . When the two groups were compared, there was no statistically significant difference (P=0.31) . Although coagulase-negative staphylococci, Corynebacterium spp, and gram-negative rods were detected in both groups, Staphylococcus aureus, non-hemolytic streptococci, Neisseriae spp, and Penicillium spp also were isolated in the group with the higher water content . CONCLUSION: Bacteria spreading from the environment or from skin flora to the eyes showed more diversity in the group of frequent-replacement soft contact lenses with a high water content . Additionally, Penicillium spp also was isolated in this group . Therefore scrupulous attention to daily lens care is crucial for people who wear frequent-replacement soft contact lenses.

FEMS Microbiol Lett, 2002 Sep 24, 215(1), 115 - 9
Intracellular viability of toxigenic Corynebacterium diphtheriae strains in HEp-2 cells; Hirata R et al.; Corynebacterium diphtheriae, generally considered an extracellular coloniser, was evaluated for its ability to enter and survive within HEp-2 monolayers by gentamicin protection assay . Intracellular viability of HC01 strain, isolated from endocarditis, was more expressive (2.59%) than observed in 241 (0.21%) and CDC-E8392 (1.93%) strains . Electron microscopy of C . diphtheriae-infected HEp-2 cells revealed intracellular bacteria inside membrane-bound vacuoles . Bacterial internalisation was totally inhibited by 5 microM cytochalasin E and significantly inhibited by 100 microM genistein (P<0.05) . Therefore, C . diphtheriae presents the ability to survive within cultured epithelial cells and signalling cascade as well as actin polymerisation are required for entry of diphtheria bacilli into HEp-2 cells.

Plasmid, 2002 Sep, 48(2), 117 - 29
The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100; Tauch A et al.; We determined the complete nucleotide sequence of the 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum LP-6 which encodes streptomycin, spectinomycin, and tetracycline resistance . The antibiotic resistance determinant of pTET3 comprises an intI1-like gene, which was truncated by the insertion sequence IS6100, and the novel aminoglycoside adenyltransferase gene cassette aadA9 . The deduced AADA9 protein showed 61% identity and 71% similarity to AADA6 of integron In51 from Pseudomonas aeruginosa . In addition, pTET3 carries the novel repressor-regulated tetracycline resistance determinant Tet 33 which revealed amino acid sequence homology to group 1 tetracycline efflux systems . The highest level of similarity was observed to the tetracycline efflux protein TetA(Z) from the C . glutamicum plasmid pAG1 with 65% identical and 77% similar amino acids . Each antibiotic resistance region of pTET3 is flanked by identical copies of the widespread insertion sequence IS6100 initially identified in Mycobacterium fortuitum . Transposition assays with a cloned copy of IS6100 revealed that this element is transpositionally active in C . glutamicum . These data suggest a central role of IS6100 in the evolutionary history of pTET3 by mediating the cointegrative assembly of resistance gene-carrying DNA segments.

Scand J Infect Dis, 2002, 34(9), 699 - 700
Corynebacterium macginleyi isolated from urine in a patient with a permanent bladder catheter; Villanueva JL et al.; An 82-y-old male patient with a neurogenic bladder and vesical stones presented with a urinary tract infection caused by Corynebacterium macginleyi . This is the first case of isolation of C . macginleyi from a non-conjunctival specimen . The patient recovered fully with antimicrobial treatment.

Bioresour Technol, 2002 Dec, 85(3), 257 - 61
Towards efficient crude oil degradation by a mixed bacterial consortium; Rahman KS et al.; A laboratory study was undertaken to assess the optimal conditions for biodegradation of Bombay High (BH) crude oil . Among 130 oil degrading bacterial cultures isolated from oil contaminated soil samples, Micrococcus sp . GS2-22, Corynebacterium sp . GS5-66, Flavobacterium sp . DS5-73, Bacillus sp . DS6-86 and Pseudomonas sp . DS10-129 were selected for the study based on the efficiency of crude oil utilisation . A mixed bacterial consortium prepared using the above strains was also used . Individual bacterial cultures showed less growth and degradation than did the mixed bacterial consortium . At 1% crude oil concentration, the mixed bacterial consortium degraded a maximum of 78% of BH crude oil . This was followed by 66% by Pseudomonas sp . DS10-129, 59% by Bacillus sp . DS6-86, 49% by Micrococcus sp . GS2-22, 43% by Corynebacterium sp . GS5-66 and 41% by Flavobacterium sp . DS5-73 . The percentage of degradation by the mixed bacterial consortium decreased from 78% to 52% as the concentration of crude oil was increased from 1% to 10% . Temperature of 30 degrees C and pH 7.5 were found to be optima for maximum biodegradation.

Environ Technol, 2002 Sep, 23(9), 1033 - 42
Microorganism selection and performance in bioslurry reactors treating PAH-contaminated soil; Cassidy DP et al.; A continuous-flow reactor (CSTR) and a soil slurry-sequencing batch reactor (SS-SBR) were operated in 81 vessels for 200 days to treat a soil contaminated with polycyclic aromatic hydrocarbons (PAH) . Filtered slurry samples were used to quantify bulk biosurfactant concentrations and PAH emulsification . Concentrations of Corynebacterium aquaticum, Flavobacterium mizutaii, Mycobacterium gastri, Pseudomonas aeruginosa, and Pseudomonas putida were determined using fatty acid methyl ester (FAME) analysis . The CSTR and SS-SBR selected microbial consortia with markedly different surfactant-producing and PAH-degrading abilities . Biosurfactant levels in the SS-SBR reached 4 times the critical micelle concentration (CMC) that resulted in considerable emulsification of PAH . In contrast, CSTR operation resulted in nomeasurable biosurfactant production . Total PAH removal efficiency was 93% in the SS-SBR, compared with only 66% in the CSTR, and stripping of PAH was 3 times less in the SS-SBR . Reversing the mode of operation on day 100 caused a complete reversal in microbial consortia and in reactor performance by day 140 . These results show that bioslurry reactor operation can be manipulated to control overall reactor performance.

Biosci Biotechnol Biochem, 2002 Aug, 66(8), 1719 - 22
Determination of the absolute configurations of the anteiso acid moieties of glycoglycerolipid S365A isolated from Corynebacterium aquaticum; Akasaka K et al.; The absolute configurations of the two acid moieties, 12-methyltetradecanoate and 14-methylhexadecanoate, of glycoglycerolipid S365A isolated from Corynebacterium aquaticum were determined by an HPLC analysis after their conversion with the chiral fluorescent labeling reagents, (1S,2S)- and (1R,2R)-2-(2,3-anthracenedicarboximido)cyclohexanol . Both anteiso acids had the S configuration.

Popul Sci, 1981, (2), 137 - 48
Histological study of the draining lymph nodes after injection of antigen and adjuvant materials tried for fertility control; Nasr EM et al.; PIP: Potential adjuvants for use in immunizing women against beta hCG (beta-chain human chorionic gonadotropin) for contraception were tested in mice for their trapping effect: trapping refers to the sequestation of lymphocytes in the draining lymph node after immunization . The substances tested were Freund's complete adjuvant (FCA) as a positive control, Corynebacterium parvum, tetanus toxoid as a carrier protein for beta-hCG, hCG, beta-hcg-tetanus toxoid conjugate + C . parvum, and saline as a negative control . FCA is unsuitable for human use . Female mice were injected in the left food pad and the popliteal lymph nodes were studied histologically at 4 and 8 days . Tetanus toxoid induced enlargement and blast cell formation at 8 days . Fca caused enlargement and blast cell transformation at 4 days and marked enlargement and formation of germinal centers at 8 days . C . Parvum evoked a similar response to FCA, with slight enlargement of the right lymph nodes at Day 8 . hCG brought no response except for slight enlargement at 8 days . The combined beta-hCG-tetanus toxoid + C . parvum elicited the greatest response, enlargement, hypercellular paracortex, blast cell transformation at 4 days and numerous germinal centers at 8 days, with enlargement of the contralateral nodes also . Since, neither tetanus toxoid nor beta-hCG causes trapping, the use of C . parvum as an adjuvant is suggested to enhance women's immune response to beta-hCG .

Rev Med Vet (Toulouse), 1975 Nov, 126(11), 1371 - 88
{Abortifacient agents in the sow}; Berthelon M et al.; PIP: Abortion in sows may be complete, or much more often partial, since the average litter size is about 10 . This review describes the clinical and serological findings, mode of transmission and recommended treatment for the most common parasitic, fungal, mycotoxin, deficient, and toxic causes of abortion in sows . The most likely possibilities in France are brucellosis, leptospirosis, Aujeszky virus, mycotoxin, or dietary deficiencies . The bacterialtion in French sows are Brucella species, Leptospira species, E . coli, streptococci, staphylococci, Pseudomonas, Hemophilus, Corynebacterium pyogenes, salmonellae, Listeria monocytogenes, Mycobacteriumr fetus, Chlamydia and Mycoplasma bovi genitalium . Toxoplasma gondii are parasites known to cause abortion in sows . Mycotoxins from Fusarium species may contaminate feed . Noninfectious causes include poisoning from nitrates, nitrites, estrogens, or insecticides and deficiencies of Vitamins-A, -B, in abortion .

Acta Med Colomb, 1985 Sep-Oct, 10(5), 197 - 203
{Vaginitis due to Gardnerella vaginalis in a university medical service}; Diaz F et al.; PIP: Between August 1983 and June 1984, a total of 363 women were studied who had been referred to the medical service of the School of Bacteriology and Laboratory Clinic of the University of Antioquia, Medellin, Colombia, for obtaining vaginal cytology . They either had or did not have vaginal discharge . The smears were tested for Gardnerella vaginalis and Trichomonas vaginalis . The majority of the patients were in the third, fourth, and fifth decades of their lives: 51%, 24.5%, and 14%, respectively . 104 (28.7%) patients had discharge as ascertained both subjectively and objectively . 74 (20.4%) complained of discharge, but it could not be confirmed objectively; 38 (10.5%) did not have discharge by objective findings; and 147 (40.5%) neither complained of, nor were found to have, discharge . 223 (61.4%) women did not use contraceptives . 56 (15.4%) women used hormonal contraceptives and 45 (12.4%) used IUDs; 20 (5.5%) had undergone tubal ligation; 8 (2.2%) used spermicides and 10 (2.8%) had undergone hysterectomy; and 1 woman used various combinations of methods . 70 (21.8%) had vaginitis caused by Gardnerella, 28 (7.7%) had candidiasis, and 10 (2.8%) had trichomoniasis; 4 cases were associated with Gardnerella and Candida and 2 cases had mixed infection (both Candida and Trichomonas); and 240 (66.1%) patients did not show any kind of bacteria . The following variables were associated with Gardnerella vaginitis (p 0.0001) as opposed to other forms of vaginitis: Gram compatibility with Gardnerella; the pH of the vaginal secretion was 4.51 or higher; the homogeneous aspect of the discharge; the presence of cells and odor; the absence of lactobacilli and Corynebacteria; the positivity of Gardnerella culture; and the absence or low count of leukocytes (p 0.02) . 20 (25.3%) of the 79 patients with Gardnerella vaginitis, 3 (10.7%) of the 28 patients with candidiasis, and 2 (20%) of 10 patients with trichomoniasis neither had discharge nor could that be confirmed by speculum examination . On the other hand, 62 (25.8%) of the 240 patients without the etiologic agents of vaginitis did have discharge as ascertained objectively . Of these, 22 (35.5%) displayed congestion and erosion of the cervix .

NPN Med, 1985 Jan 1, 5(81), 19 - 24
{Cutaneous effects in hormonal contraception}; Thomas P et al.; PIP: Oral contraceptives (OCs) can affect the skin through their hormonal effects or through iatrogenic effects associated with their toxicity in certain individuals . They may also be beneficial in certain androgen-dependent dermatoses . Toxic effects of OCs are rare but potentially serious; they should be diagnosed early and require permanent termination of OC use . The clinical manifestations are variable and not specific to the medication . The most frequently reported manifestations are allergic vascularities which may lead to serious renal complications, fixed pigmented erythema, urticaria, which may have other etiologic factors, and lichenoid eruptions . Combined OCs, because of their estrogen content, may cause sensitivity to light in susceptible women . Other dermatoses can be initiated or aggravated by OCs without direct relation to their hormonal effects . OCs are therefore contraindicated if there is a personal or family history of porphyries or a personal history of systemic lupus erythematosus, erythema nouex, herpes gestationis, or malignant melanoma . Hormonal-related dermatological effects caused by either progestins or estrogens have become less frequent as dose levels have declined . Chloasma, either melasma or a poorly defined spotty pigmentation, accounts for 2/3 of cases of OC-related dermatoses . It is more common in women of Mediterranean background . 80% of affected OC users have a history of "mask of pregnancy", but the condition is also found in nulliparas . Exposure to sunlight is a factor . Women with a history of chloasma of pregnancy and dark coloring should not use OCs . Seborrhea is directly related to the androgen effect of OCs and is less likely to occur with 17 OH progesterone derivatives than with 19 norsteroid derivatives . The role of androgens in acne is well known, but 2 other factors are necessary: an anomaly in keratinization and proliferation of corynebacterium acnes, a saprophyte of the follicles . OCs do not necessarily need to be suspended during well-conducted acne treatment . Alopecia is rare but difficult to diagnose because of its psychological aspects . Androgenic alopecia is aggravated by progestins derived from 19 norsteroids . True hirsutism caused by an androgen-producing ovarian pathology is not related to OC use . Estrogens are incriminated in the etiology of telangiectasies, permanent dilatations of the arterioles . Once developed the condition does not regress and requires treatment with sclerosing agents, electrocoagulation, or laser . The various dermatological risk factors should be ruled out before prescription of an OC . Classic contraceptive pills are not commonly used in treatment of common acne because the strongly estrogenic climate required for therapeutic utility carries the risk of hypertriglyceridemia, thrombophlebitis, and possibly carcinogenesis . The recent development of pills containing the antiandrogen cyproterone acetate instead of a progestin in combination with ethinyl estradiol reduces androgenic effects in women . This pill may be useful in cases of severe acne, severe seborrhea, androgenic alopecia, or excessive facial hair .

J Bacteriol, 2002 Oct, 184(20), 5723 - 32
Construction and characterization of transposon insertion mutations in Corynebacterium diphtheriae that affect expression of the diphtheria toxin repressor (DtxR); Oram DM et al.; Transcription of the bacteriophage-borne diphtheria toxin gene tox is negatively regulated, in response to intracellular Fe(2+) concentration, by the chromosomally encoded diphtheria toxin repressor (DtxR) . Due to a scarcity of tools, genetic analysis of Corynebacterium diphtheriae has primarily relied on analysis of chemically induced and spontaneously occurring mutants and on the results of experiments with C . diphtheriae genes cloned in Escherichia coli or analyzed in vitro . We modified a Tn5-based mutagenesis technique for use with C . diphtheriae, and we used it to construct the first transposon insertion libraries in the chromosome of this gram-positive pathogen . We isolated two insertions that affected expression of DtxR, one 121 bp upstream of dtxR and the other within an essential region of the dtxR coding sequence, indicating for the first time that dtxR is a dispensable gene in C . diphtheriae . Both mutant strains secrete diphtheria toxin when grown in medium containing sufficient iron to repress secretion of diphtheria toxin by wild-type C . diphtheriae . The upstream insertion mutant still produces DtxR in decreased amounts and regulates siderophore secretion in response to iron in a manner similar to its wild-type parent . The mutant containing the transposon insertion within dtxR does not produce DtxR and overproduces siderophore in the presence of iron . Differences in the ability of the two mutant strains to survive oxidative stress also indicated that the upstream insertion retained slight DtxR activity, whereas the insertion within dtxR abolished DtxR activity . This is the first evidence that DtxR plays a role in protecting the cell from oxidative stress.

Mol Biol Rep, 2002, 29(1-2), 129 - 34
Carbon flux analysis in a pantothenate overproducing Corynebacterium glutamicum strain; Chassagnole C et al.; Carbon flux analysis during a pseudo-stationary phase of metabolite accumulation in a genetically engineered strain of Corynebacterium glutamicum, containing plasmids leading to over-expression of the ilvBNCD and panBC operons, has identified the basic metabolic constraints governing the potential of this bacterium to produce pantothenate . Carbon flux converging on pyruvate (75% of glucose uptake) is controlled by anabolic precursor requirements and NADPH demand provoking high carbon loss as CO2 via the pentose pathway . Virtually all the flux of pyruvate is directed into the branched pathway leading to both valine and pantothenate production, but flux towards valine is tenfold higher than that transformed to pantothenate, indicating that significant improvements will only be obtained if carbon flux at the ketoisovalerate branchpoint can be modulated.

Curr Microbiol, 2002 Nov, 45(5), 362 - 7
Efficient electrotransformation of corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1; Tauch A et al.; Efficient transformation of the human pathogen Corynebacterium diphtheriae was achieved with novel cloning vectors consisting of a mini-replicon from the cryptic C . glutamicum plasmid pGA1 as well as of the aph(3')-IIa or tetA(Z ) antibiotic resistance genes . Plasmid-containing transformants of C . diphtheriae were recovered at frequencies ranging from 1.3 x 10(5) to 4.8 x 10(6) colony forming units (cfu)/microg of plasmid DNA . Vector DNA was directly transferred from Escherichia coli into C . diphtheriae with frequencies up to 5.6 x 10(5) cfu/microg of plasmid DNA . On the basis of the pGA1 mini-replicon, an expression vector system was established for C . diphtheriae by means of the P(tac) promoter and the green fluorescent reporter protein . In addition, other commonly used vector systems from C . glutamicum, including the pBL1 and pHM1519 replicons, and the sacB conditionally lethal selection marker from Bacillus subtilis, were shown to be functional in C . diphtheriae . Thus, the ability to apply the standard methods of C . glutamicum recombinant DNA technology will greatly facilitate the functional analysis of the recently completed C . diphtheriae genome sequence.

Clin Infect Dis, 2002 Oct 1, 35(7), e72 - 7 Epub 2002 Sep 10.
Central venous catheter-related bacteremia due to Tsukamurella species in the immunocompromised host: a case series and review of the literature; Schwartz MA et al.; We report 6 cases of bacteremia due to Tsukamurella species, all of which were in immunosuppressed patients with indwelling central venous catheters (CVCs) . Fewer than 20 cases of serious illness due to these gram-positive bacilli have been reported in the medical literature; these cases have mostly been ascribed to the species Tsukamurella paurometabola . Tsukamurella species are frequently misidentified as Rhodococcus or Corynebacterium species . We used high-performance liquid chromatography to identify these organisms to the genus level and 16S ribosomal RNA gene sequencing and DNA-DNA dot blots for species identification . Three of our isolates were identified as Tsukamurella pulmonis, 1 was identified as Tsukamurella tyrosinosolvans, and 1 was identified as a unique species . One isolate was not maintained long enough for species identification . All patients were successfully treated with antimicrobial therapy and CVC removal . Infection with this organism should be considered in the immunosuppressed patient with an indwelling CVC and gram-positive bacilli in the blood.

Bioresour Technol, 2002 Nov, 85(2), 207 - 11
Optimisation of a culture medium containing fish silage for L-lysine production by Corynebacterium glutamicum; Coello N et al.; In a first step the effects of 10 components of a culture medium designed for L-lysine production were evaluated with a 2(10-6) factorial design . Among them, glucose, fish silage, and ammonium sulphate showed a significant effect . In a second step, an orthogonal-central composite experimental design and response surface methodology was performed with five from the 10 initial compounds . The determination coefficient (R2) of the fitted second-order model was 0.990 . L-lysine production with the optimised medium increased 2.6 times as compared with the original medium.

Immunology, 2002 Sep, 107(1), 93 - 101
Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases; Tambourgi DV et al.; We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induce complement (C)-dependent lysis of autologous erythrocytes by induction of the cleavage of cell surface glycophorins through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway of C . Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to those observed after spider envenomation . The aim of this study was to investigate if the bacterial PLD-induced haemolysis of human erythrocytes was C dependent and if cleavage of glycophorins occurred . We show here that haemolysis of both PLD- and P1-treated human erythrocytes was C dependent, but while PLD-mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathways . P1, but not PLD, induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins . In both cases, annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine to the cell surface . Our results suggest that C susceptibility induced by L . intermedia and C . pseudotuberculosis PLD is a result of exposure of phosphatidylserine, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.

Aust Vet J, 2002 Aug, 80(8), 494 - 6
Effect of the interval between shearing and dipping on the spread of Corynebacterium pseudotuberculosis infection in sheep; Paton MW et al.; OBJECTIVE: To determine whether the spread of Corynebacterium pseudotuberculosis infection to sheep in dips could be controlled by increasing the time between shearing and dipping . DESIGN: A controlled treatment trial where only the time between shearing and dipping was varied . ANIMALS AND PROCEDURE: One hundred and ninety-five sheep were found to be negative for C . pseudotuberculosis exposure by assay of CLA toxin antibody, were divided into four treatment groups . Each was shorn at either 0, 2, 4 or 8 weeks before dipping in a solution containing C . pseudotuberculosis . Blood samples were taken 6 weeks after dipping and sheep were slaughtered 12 weeks after dipping . A fifth smaller group of 14 sheep shorn 26 weeks before dipping, was also exposed to C . pseudotuberculosis and was slaughtered with the other sheep . RESULTS: The occurrence of caseous lymphadenitis abscesses did not differ between groups or with sheep shorn 26 weeks before dipping . The proportion of sheep that seroconverted to the C . pseudotuberculosis toxin and cell wall ELISA was larger in sheep dipped immediately after shearing than in sheep in the other groups . CONCLUSIONS: Delaying dipping until 8 weeks after shearing did not decrease the C . pseudotuberculosis infection rate due to dipping . Sheep dipped immediately after shearing developed higher concentrations of antibody to C . pseudotuberculosis than sheep when dipping occurred between 2 and 8 weeks and later after shearing.

Stomatologiia (Mosk), 2002, 81(3), 4 - 8
{Microbiological validation of the choice of basic plastic for removable dentures}; Aputiunov SD et al.; Adhesion of obligate and facultative anaerobic bacteria, favoring the development of oral inflammatory diseases, including the cariesogenic and periodontogenic bacteria and Candida albicans fungi, isolated from patients with periodontitis, to 13 basic materials used in removable denture making, was studied . The adhesion of all bacteria (Streptococcus sanguis, Prevotella melangogenica, Fusobacterium nucleatum, Corynebacterium xerosis) and fungi to hot polymerization basic materials was the maximum . The most perspective basic plastic for clinical use (preserving intact oral microbiocenosis and preventing stomatitis induced by denture wearing) are cold polymerization materials, such as Redont-03, Dentoplast Breden, Leocryl, and UHF polymerization materials Acron GC, AKR-MV, and Etakril-02.

J Fr Ophtalmol, 2002 Jun, 25(6), 599 - 603
{Therapeutic strategy in delayed postoperative endophtalmitis: a report on 15 cases}; Monnet D et al.; PURPOSE: To report the treatment strategies and visual acuity outcomes of chronic postoperative endophthalmitis . MATERIAL: and methods: The authors reviewed the records of 15 patients presenting 3 or more weeks after cataract surgery with intraocular inflammation and treated at Bicetre Hospital from 1992 to 1998 . Group I included 6 consecutive patients treated with vitrectomy and intravitreal antibiotic injection (vancomycin and cefazolin) . Group II included 9 consecutive patients treated with intravitreal antibiotic injection (vancomycin and ceftazidime) and irrigation of the capsular bag (vancomycin) . The minimum follow-up period was 1 year . RESULTS: In group I, 2 patients had recurrent inflammation . In these patients, the capsular bag and the intraocular implant were removed . In 1 patient there was culture-proven Corynebacterium and in 1 patient a Staphylococcus epidermidis was found . Final visual acuity was 20/40 or better in 5 patients and 20/100 in 1 patient . Visual acuity improved in all cases . In group II no recurrence was seen in the 12-20 months of follow-up . In 2 patients there was proven Staphylococcus epidermidis and in one patient Propionibacterium acnes was found . Final visual acuity was 20/40 or more in 3 patients, 20/100 or more in 4 patients and less than 20/200 in 2 patients . Visual acuity improved in 8 cases . CONCLUSIONS: Intravitreal antibiotic injection with vitrectomy and intravitreal antibiotic injection with antibiotic irrigation of the capsular bag are both effective in the treatment of delayed chronic postoperative endophthalmitis; however, with the second approach, there is minimal surgical trauma and the intraocular implant is retained.

Arch Latinoam Nutr, 2002 Mar, 52(1), 68 - 73
{Isolation of Gram-positive bacteria from raw milk with antimicrobial residues}; Faria Reyes J et al.; Two hundred samples of raw milk were collected at the receiving plants located in three areas of high milk production in Zulia state, Venezuela . The CTT test and trial disk were used in order to detect the presence of antimicrobials . The positive samples were inoculated in tripticase soy broth, human blood agar and manitol salt agar in order to isolate Gram-positive bacteria . The identification of species was performed through biochemical tests . It was found that 45 samples (22.5%) of analyzed milk contained antimicrobials, and bacterial growth was obtained in 35 of them . 100 strains were isolated namely: 44 Staphylococcus, 19 Streptococcus, 17 Enterococcus, 9 Bacillus, 4 Micrococcus, 4 Corynebacterium and 3 Lactococcus . The most frequently isolated specie was S . aureus, the main producing agent of bovine mastitis in Zulia state, a microorganism frequently associated in the country to food-borne intoxications, associated to cheese processed from raw milk . It is recommended to apply control programs for the use of antibiotics.

J Basic Microbiol, 2002, 42(4), 284 - 91
Occurrence of crude oil degrading bacteria in gasoline and diesel station soils; Rahman KS et al.; Microbial enumeration and identification were carried out on several oil contaminated soil samples collected from gasoline and diesel stations . Bacteria were the most dominant microbiota and were therefore classified to generic level . Eleven main genera were detected and Corynebacterium was the predominant genus in all the samples . Biochemical characterisation and substrate utilisation showed high percentage of lipolytic ability combined with high inorganic nitrogen utilisers . The ability of these cultures to degrade crude oil was tested individually and in mixed bacterial consortium at different temperatures and pH values . Maximum crude oil biodegradation of 78% was achieved using a bacterial consortium containing five cultures (Micrococcus sp . GS2-22, Corynebacterium sp . GS5-66, Flavobacterium sp . DS5-73, Bacillus sp . DS6-86 and Pseudomonas sp . DS10-129) with 1% crude oil at 30 degrees C and pH 7.5 . Such a consortium may be useful for bioaugmentation of oil contaminated environments.

J Biotechnol, 2002 Oct 9, 99(1), 79 - 91
The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains; Tauch A et al.; The potential of the alanine racemase gene alr from Corynebacterium glutamicum ATCC 13032 to substitute for antibiotic resistance determinants in cloning systems has been investigated . The alr gene was identified by a PCR technique and its nucleotide sequence was determined . The deduced protein revealed the highest amino acid sequence similarity to the Alr protein from Mycobacterium smegmatis with 45% identical and 58% similar amino acids . A defined alr deletion mutant of C . glutamicum displayed a strict dependence on the presence of D-alanine for growth on complex and minimal medium . The alr gene was placed on a novel C . glutamicum vector which is completely free of antibiotic resistance genes . In vivo complementation of the chromosomal alr deletion with alr-carrying vectors permitted growth of the mutant strain in the absence of external D-alanine and provided strong selective pressure to maintain the plasmid . The alr gene enabled the selection of C . glutamicum transformants with a similar efficiency as the tetracycline resistance gene tetA(33) . These data provided experimental evidence that the alr gene can be applied as an alternative selection marker to antibiotic resistance genes in industrial C . glutamicum strains . In an application example, the novel deltaalr host-alr(+) vector-system for C . glutamicum was used to overproduce the vitamin D-pantothenic acid .

Vet Res, 2002 Jul-Aug, 33(4), 335 - 57
Quarter milk somatic cell count in infected dairy cows: a meta-analysis; Djabri B et al.; The aim of this paper was to evaluate the effects associated with intramammary infection (IMI) by a bacterium or a group of bacteria (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, coliforms, Staphylococci other than S . aureus, and Corynebacterium bovis) on the somatic cell count (SCC) in quarter milk of dairy cows . Papers selected for analysis had to provide SCC values associated with the natural infection in quarters by different bacteria . Sampling for measurement of SCC and determination of the infection had to be done on the same day . Only papers published in English or in French after 1971 were considered . Twenty-one papers fulfilled the selection criteria . The animals sampled, the measurement techniques for SCC and the bacteriological identification, as well as the definition of the infection, all differed widely among the selected studies . The meta-analysis method was used to estimate both the mean SCC (arithmetic and geometric) value and the average increase on SCC of each type of infection . The geometric mean SCC in bacteriologically negative quarters was 68 000 c/mL . In case of IMI, the retained SCC was 357 000, 857 000, 547 000, 1 024 000, 1 151 000, 138 000 and 105 000 c/mL in quarters infected by Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, coliforms, staphylococci other than S . aureus and Corynebacterium bovis, respectively . The variation factors that could influence these SCC values and the bacteriological results are discussed.

Ukr Biokhim Zh, 2001 Nov-Dec, 73(6), 73 - 6
{Specificity of antibodies to diphtheria toxin subunits in children with various forms of diphtheria infections}; Romaniuk SI et al.; In order to study the specificity of serum antibodies to separate subunits of diphtheria toxin, SDS-electrophoresis of diphtheria toxin preliminary disintegrated on the subunits via trypsin treatment was performed, followed by immunoblotting assay . 86 blood serum samples of children with diphtheria carriers of toxigenic and non-toxigenic strains of Corynebacterium diphtheriae as well as children with other infectious diseases similar to diphtheria in their clinical manifestation, and healthy ones immunized with DTP-vaccine were tested . A special computer program was written and applied for results processing and assumption . The data obtained showed that there were particular differences in frequency of predominating the antibodies to one or another subunit of diphtheria toxin among various groups of the children . We consider that the different specificity of antibodies of sick children and children-carriers is capable to predetermine the different course of infectious process.

Fish Shellfish Immunol, 2002 May, 12(5), 371 - 85
Antibacterial activity in four marine crustacean decapods; Haug T et al.; A search for antibacterial activity in different body-parts of Pandalus borealis (northern shrimp), Pagurus bernhardus (hermit crab), Hyas araneus (spider crab) and Paralithodes camtschatica (king crab) was conducted . Dried samples were extracted with 60% (v/v) acetonitrile, containing 0.1% (v/v) trifluoroacetic acid, and further extracted and concentrated on C18 cartridges . Eluates from the solid phase extraction were tested for antibacterial, lysozyme and haemolytic activity . Antibacterial activity against Escherichia coli, Vibrio anguillarum, Corynebacterium glutamicum and Staphylococcus aureus was detected in extracts from several tissues in all species tested, but mainly in the haemolymph and haemocyte extracts . V . anguillarum and C . glutamicum were generally the most sensitive micro-organisms . In P . borealis and P . bernhardus most of the active fractions were not affected by proteinase K treatment, while in H . araneus and P . camtschatica most fractions were sensitive to proteinase K treatment, indicating antibacterial factors of proteinaceous nature . In P . bernhardus the active fractions were generally heat labile, whereas in H . araneus the activities were resistant to heat . Differences between active extracts regarding hydrophobicity and sensitivity for heat and proteinase K treatment indicate that several compounds are responsible for the antibacterial activities detected . Lysozyme-like activity could be detected in some fractions and haemolytic activity against human red blood cells could be detected in haemolymph/haemocyte and exoskeleton extracts from all species tested.

Med Dosw Mikrobiol, 2002, 54(1), 35 - 43
{Novelty of laboratory diagnosis for diphtheria}; Kuklinska D et al.; Selected elements of simplified, bacteriological diagnosis of diphtheria were presented . The procedure of Corynebacterium strains isolation from diphtheria suspected persons and performing of toxin testing of potentially toxigenic isolates: C . diphtheriae, C . ulcerans and C . pseudotuberculosis were shortened . The role of selective tellurite media was underlined but Loeffler medium was rejected . Columbia blood agar plate was utilized for preliminary culture . Biochemical tests and toxin testing were performed from this medium . Presented diphtheria diagnosis scheme may have practical application for the laboratory work in Poland.

Microbiology, 2002 Aug, 148(Pt 8), 2479 - 87
Plasmid-borne macrolide resistance in Micrococcus luteus; Liebl W et al.; A plasmid designated pMEC2 which confers resistance to erythromycin, other macrolides, and lincomycin was detected in Micrococcus luteus strain MAW843 isolated from human skin . Curing of this approximately 4.2 kb plasmid from the host organism resulted in erythromycin sensitivity of the strain . Introduction of pMEC2 into a different M . luteus strain conferred erythromycin resistance upon this strain . Macrolide resistance in M . luteus MAW843 was an inducible trait . Induction occurred at subinhibitory erythromycin concentrations of about 0.02-0.05 micro g ml(-1) . Erythromycin and oleandomycin were inducers, while spiramycin and tylosin exerted no significant inducer properties . With heterologous expression experiments in Corynebacterium glutamicum, using hybrid plasmid constructs and deletion derivatives thereof, it was possible to narrow down the location of the plasmid-borne erythromycin-resistance determinant to a region of about 1.8 kb of pMEC2 . Sequence analysis of the genetic determinant, designated erm(36), identified an ORF putatively encoding a 281-residue protein with similarity to 23S rRNA adenine N(6)-methyltransferases . erm(36) was most related (about 52-54% identity) to erythromycin-resistance proteins found in high-G+C Gram-positive bacteria, including the (opportunistic) pathogenic corynebacteria Corynebacterium jeikeium, C . striatum, C . diphtheriae and Propionibacterium acnes . This is believed to be the first report of a plasmid-borne, inducible antibiotic resistance in micrococci . The possible role of non-pathogenic, saprophytic micrococci bearing antibiotic-resistance genes in the spreading of these determinants is discussed.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(2), 169 - 173
High-level Expression and Characterization of the Fusion Protein Consisting of Diphtheria Toxin and Human Interleukin 6; Zhang XJ et al.; Diphtheria toxin is a single chain exotoxin of 535 amino acids, secreted from beta Corynebacteriophage diphtheriae . Eukaryotic cells, especially tumor cells are very sensitive to DT . Just one or two molecules of DT can kill a cell . On the surface of some tumor cells, such as human myeloma, heptoma, etc, IL-6 receptor has been demonstrated to be expressed at a very-high level . Selective cytotoxicity mediated by IL-6 receptor could be useful in the targeting therapy of these tumors . Based on this strategy, a hybrid protein consisting of DT and IL-6 was constructed, expressed and characterized . IL-6 cDNA was first modified for constructing the fusion protein of DT/IL-6 and the receptor binding domain of DTDNA was replaced by IL-6 cDNA to obtain the expression vector pdeltaDT/TL-6 . After induction by IPTG, the fusion protein was expressed successfully, accounting for 20% of the total bacterial protein . The results of SDS-PAGE and Western blot showed that there was a band of about 64 kD . After preliminary purification, the IL-6 receptor competitive binding test and the cytotoxic activity assay of the deltaDT/TL-6 showed that the fusion protein possessed significant cytotoxic activity to U266 cells; while the cells expressing IL-6 receptors at a medium-level was resistant it to a certain degree.

Rev Soc Bras Med Trop, 2002 Jul-Aug, 35(4), 311 - 3
{Cutaneous leishmaniotic ulcers with Corynebacterium diphtheriae}; Vera LA et al.; In a prospective study to evaluate the influence of secondary bacterial infection on the evaluation of cutaneous leishmaniasis, in Corte de Pedra (Bahia), we isolated Corynebacterium diphtheriae in 7 (8.3%) out of 84 patients with ulcers studied . Due to the small number of patients with the presence of the bacteria in the ulcer, we could not conclude whether Corynebacterium diphtheriae behaves only as a colonizer nor its influence on the healing of the leishmaniotic ulcer.

J Bacteriol, 2002 Sep, 184(17), 4846 - 56
Identification of a DtxR-regulated operon that is essential for siderophore-dependent iron uptake in Corynebacterium diphtheriae; Qian Y et al.; The diphtheria toxin repressor (DtxR) uses Fe(2+) as a corepressor and inhibits transcription from iron-regulated promoters (IRPs) in Corynebacterium diphtheriae . A new IRP, designated IRP6, was cloned from C . diphtheriae by a SELEX-like procedure . DtxR bound to IRP6 in vitro only in the presence of appropriate divalent metal ions, and repression of IRP6 by DtxR in an Escherichia coli system was iron dependent . The open reading frames (ORFs) downstream from IRP6 and previously described promoter IRP1 were found to encode proteins homologous to components of ATP-binding cassette (ABC) transport systems involved in high-affinity iron uptake in other bacteria . IRP1 and IRP6 were repressed under high-iron conditions in wild-type C . diphtheriae C7(beta), but they were expressed constitutively in C7(beta) mutant strains HC1, HC3, HC4, and HC5, which were shown previously to be defective in corynebactin-dependent iron uptake . A clone of the wild-type irp6 operon (pCM6ABC) complemented the constitutive corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a clone of the wild-type irp1 operon failed to complement any of these strains . Complementation by subclones of pCM6ABC demonstrated that mutant alleles of irp6A, irp6C, and irp6B were responsible for the phenotypes of HC1, HC4, and HC5, respectively . The irp6A allele in HC1 and the irp6B allele in HC5 encoded single amino acid substitutions in their predicted protein products, and the irp6C allele in HC4 caused premature chain termination of its predicted protein product . Strain HC3 was found to have a chain-terminating mutation in dtxR in addition to a missense mutation in its irp6B allele . These findings demonstrated that the irp6 operon in C . diphtheriae encodes a putative ABC transporter, that specific mutant alleles of irp6A, irp6B, and irp6C are associated with defects in corynebactin-dependent iron uptake, and that complementation of these mutant alleles restores repression of corynebactin production under high-iron growth conditions, most likely as a consequence of restoring siderophore-dependent iron uptake mediated by the irp6 operon.

Biosci Biotechnol Biochem, 2002 Jun, 66(6), 1337 - 44
Transcriptome analysis of acetate metabolism in Corynebacterium glutamicum using a newly developed metabolic array; Hayashi M et al.; Following the determination of the whole-genome sequence of Corynebacterium glutamicum, we have developed a DNA array to extensively investigate gene expression and regulation relevant to carbon metabolism . For this purpose, a total of 120 C . glutamicum genes, including those in central metabolism and amino acid biosyntheses, were amplified by PCR and printed onto glass slides . The resulting array, designated a "metabolic array", was used for hybridization with fluorescently labeled cDNA probes generated by reverse transcription from total RNA samples . As the first demonstration of transcriptome analysis in this industrially important microorganism, we applied the metabolic array to study differential transcription profiles between cells grown on glucose and on acetate as the sole carbon source . The changes in gene expression observed for the known acetate-regulated genes (aceA, aceB, pta, and ack) were well consistent with the literature data of northern analyses and enzyme assays, indicating the utility of the metabolic array in transcriptome analysis of C . glutamicum . In addition to the known responses, many previously unrecognized co-regulated genes were identified . For example, several TCA cycle genes, such as gltA, sdhA, sdhB, fumH, and mdh, and the gluconeogenic gene pck were up-regulated in the acetate medium . On the other hand, a few genes involved in glycolysis and the pentose phosphate pathway, as well as many amino acid biosynthetic genes, were down-regulated in acetate . Furthermore, two gap genes, gapA and gapB, were found to be inversely regulated, suggesting the presence of a new regulatory step for carbon metabolism between glycolysis and gluconeogenesis.

Biosci Biotechnol Biochem, 2002 Jun, 66(6), 1256 - 61
Cloning and expression of fructosyl-amino acid oxidase gene from Corynebacterium sp . 2-4-1 in Escherichia coli; Sakaue R et al.; The gene encoding the fructosyl-amino acid oxidase (fructosyl-alpha-L-amino acid: oxygen oxidoreductase (defructosylating); EC 1.5.3) of Corynebacterium sp . 2-4-1 was cloned and expressed in Escherichia coli . The gene consists of 1,116 nucleotides and encodes a protein of 372 amino acids with a predicted molecular mass of 39,042 . The open reading frame was confirmed as the gene of the fructosyl-amino acid oxidase by comparison with the N-terminal amino acid sequence of the purified fructosyl-amino acid oxidase from Corynebacterium sp . 2-4-1 . The sequence of the AMP-binding motif, GXGXXG, was found in the deduced N-terminal region . The amino acid sequence of the fructosyl-amino acid oxidase showed no similarity to that of fungal fructosyl-amino acid oxidases . In addition, substrate specificities of this fructosyl-amino acid oxidase were different from those of other fructosyl-amino acid oxidases . The fructosyl-amino acid oxidase of Corynebacterium sp . 2-4-1 is an enzyme that has unique substrate specificity and primary structure in comparison with fungal fructosyl-amino acid oxidases.

Res Microbiol, 2002 Jun, 153(5), 307 - 11
Differentiation of Corynebacterium amycolatum, C . minutissimum, C . striatum and related species by pyrolysis-gas-liquid chromatography with atomic emission detection; Voisin S et al.; We report here the application of pyrolysis-gas chromatography followed by atomic emission detection (AED) for the characterisation of Corynebacterium amycolatum and related species (i.e., C . striatum, C . minutissimum, C . xerosis and the recently described C . freneyi) . This phenotypic method, which analyses the whole chemical composition of bacteria, clearly separates C . amycolatum from other species . Moreover, this C . amycolatum group is subdivided into two distinct subgroups . We cannot differentiate the C . minutissimum strains from those of C . striatum . On the other hand, C . freneyi and C . xerosis are clearly distinct from the other species.

Postgrad Med J, 2002 May, 78(919), 290 - 1
An unusual case of infective endocarditis; Houghton T et al.; A rare case of Corynebacterium striatum endocarditis on a bioprosthetic aortic valve replacement, treated medically, is reported . The presentation was subacute, and initially endocarditis screening was negative . Because of the failure of symptoms to settle further screening was performed which confirmed the organism in several sets of blood cultures . This emphasises the importance of persistent screening for endocarditis if the history raises any suspicion of this potentially serious infection, especially in the presence of prosthetic valves.

Vet Microbiol, 2002 Sep 2, 88(3), 287 - 97
An interferon-gamma assay for diagnosis of Corynebacterium pseudotuberculosis infection in adult sheep from a research flock; Prescott JF et al.; The optimal method of control of caseous lymphadenitis of sheep caused by Corynebacterium pseudotuberculosis is eradication of infection by identification and removal of infected carrier animals . Current serological approaches to identification of infected sheep are generally hampered by low sensitivity and specificity of available tests . The objective of this study was to develop a whole blood assay for detection of C . pseudotuberculosis-infected sheep, based on detection of IFN-gamma response to whole cell C . pseudotuberculosis antigens, and to determine the reliability of the assay . A commercially available bovine interferon-gamma assay enzyme-linked immunosorbent assay was used and the test optimised using experimentally infected sheep . The assay was also tested on known CLA-negative sheep . Setting a IFN-gamma optical density cut-off at 0.100 as positive under the conditions used, the test detected C . pseudotuberculosis experimentally infected sheep over a 450-day period with a reliability of 95.7% . It identified known non-infected sheep with a reliability of 95.5% . Repeated vaccination of three uninfected sheep with a commercially available bacterin-toxoid vaccine did not interfere with the assay . The IFN-gamma response of sheep whole blood to C . pseudotuberculosis antigens offers promise for use in a test-and-removal approach to eradication of CLA in sheep.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1165 - 9
Corynebacterium appendicis sp . nov; Yassin AF et al.; A lipophilic, coryneform bacterium isolated from a human clinical specimen was characterized by phenotypic and molecular-taxonomic methods . Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and short-chain mycolic acids consistent with the genus Corynebacterium . The isolate could be distinguished from other members of the genus Corynebacterium by positive urease and catalase tests as well as its failure to produce acid from carbohydrates . Comparative 16S rRNA gene sequencing showed that this isolate constitutes a distinct subline within the genus Corynebacterium, displaying >3.0% sequence divergence from other known Corynebacterium species . Based on both phenotypic and phylogenetic evidence, it is proposed that this isolate be classified as a novel species, Corynebacterium appendicis sp . nov., represented by strain IMMIB R-3491T (= DSM 44531T = NRRL B-24151T).

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1127 - 31
Corynebacterium efficiens sp . nov., a glutamic-acid-producing species from soil and vegetables; Fudou R et al.; Three glutamic-acid-producing coryneform strains were isolated from soil and vegetable samples . Chemotaxonomic investigations indicated that these strains belonged to the genus Corynebacterium . Phylogenetic studies, based on 16S rDNA analysis, demonstrated that the three strains formed a distinct cluster within the genus Corynebacterium and that their nearest relatives were Corynebacterium glutamicum and Corynebacterium callunae, also known as glutamic-acid-producing species . The data from 16S rDNA sequence and DNA-DNA relatedness studies clearly indicated that the three isolates represented a new species within the genus Corynebacterium . All of the isolates could grow at 45 degrees C and produced acid from dextrin; these were the most significant characteristics differentiating the three isolates from their neighbours . On the basis of the data presented here, it is proposed that the three glutamic-acid-producing isolates together be classified as Corynebacterium efficiens sp . nov., the type strain of which is YS-314T (= AJ 12310T = JCM 11189T = DSM 44549T).

J Dairy Sci, 2002 Jun, 85(6), 1370 - 5
Microbiological quality and somatic cell count of ewe milk with special reference to staphylococci; Ariznabarreta A et al.; A total 1502 useful half udders of 762 Churra ewes from eight herds were aseptically sampled in midlactation to study both the bacteriological isolates and the SCC of milk . Corynebacteria, enterococci, micrococci, staphylococci, and streptococci represented 11.2, 2.9, 1.4, 78.9, and 3.1% of all isolates, respectively . Within staphylococci, novobiocin-sensitive species (71.1%) were much more frequently isolated than novobiocin-resistant ones (7.8%) . Staphylococcus epidermidis was the most prevalent species (53.2% of the isolates) . Log SCC of uninfected half udder milk was 4.86 . Isolates of novobiocin-resistant coagulase-negative staphylococci, micrococci, and corynebacteria were associated to low values of log SCC (4.85 to 5.20) . In contrast, infection by novobiocin-sensitive coagulase-negative staphylococci, streptococci, and enterococci organisms was related to a sharp inflammatory response with log SCC means between 5.92 and 6.32 . The species that showed the highest log SCC were Pasteurella haemolytica (7.62), Streptococcus agalactiae (7.28), and Staphylococcus aureus (6.68) . High prevalence of infections by novobiocin-sensitive staphylococci together with high SCC related to such infections show a relevant role of these organisms in ewe mastitis . Consequently, implementation of staphylococcal mastitis control programs would be of great interest in dairy ewe herds to improve microbiological and hygienic quality of milk.

Clin Infect Dis, 2002 Aug 15, 35(4), e40 - 2 Epub 2002 Jul 17.
Corynebacterium minutissimum bacteremia in an immunocompetent host with cellulitis; Granok AB et al.; Since its original description in 1961, Corynebacterium minutissimum, the causative agent of erythrasma, has rarely been associated with extracutaneous disease . We report a case of cellulitis and bacteremia due to C . minutissimum . We discuss the treatment of C . minutissimum infection and describe the clinical settings in which isolation of Corynebacterium species from blood cultures should be considered significant.

J Biotechnol, 2002 Sep 25, 98(2-3), 255 - 68
Genome-wide transcription profiling of Corynebacterium glutamicum after heat shock and during growth on acetate and glucose; Muffler A et al.; To monitor the global gene expression of Corynebacterium glutamicum we established two formats of DNA-arrays on nylon membranes . We produced an ordered DNA-array of PCR fragments from a shotgun library of C . glutamicum representing a threefold coverage of the genome . With this format we studied genome-wide transcriptional changes after heat shock . Sequence and subsequent BLAST analysis of PCR fragments with elevated expression after heat shock revealed PCR fragments harboring genes that encode several proteins of the heat shock family, proteins of the oxidative stress response and proteins with unknown function . DNA-arrays based on PCR fragments representing 2804 annotated ORFs of C . glutamicum were used to monitor the transcript levels during growth on acetate and glucose . We determined minimal detectable ratios and compared labeling approaches with random hexamers and ORF-specific primers . ORF-based DNA-array analysis with different labeling approaches showed similar results: e.g . increased mRNA levels of the pta-ack operon, aceA, aceB and genes encoding phosphoenolpyruvate carboxykinase and enzymes of the citric acid cycle during growth on acetate and elevated mRNA levels of some enzymes of the glycolytic pathway and lactate dehydrogenase upon growth on glucose . These results demonstrate that shotgun DNA-arrays and ORF-based DNA-arrays are appropriate tools to study physiology of microorganism.

Int J Med Microbiol, 2002 Jun, 292(1), 59 - 63
A fatal case of necrotizing sinusitis due to toxigenic Corynebacterium ulcerans; Wellinghausen N et al.; A 77-year-old farmer developed cough with sputum production, fever, bloody nasal discharge and a mass in his right maxillary sinus leading to necrotic ulceration of the sinus . Corynebacterium ulcerans, carrying the beta-phage for the diphtheria toxin and secreting the toxin, was detected microscopically and by culture from the sinusoidal and ulcer discharge . Despite immediate antimicrobial chemotherapy the patient died of pulmonary failure associated with the production of large amounts of very viscous sputum . Identification of the causative agent, pathophysiological aspects and risk factors of this unusal infection are discussed.

J Small Anim Pract, 2002 Jul, 43(7), 299 - 302
Urinary tract infection caused by Corynebacterium urealyticum in a dog; Suarez ML et al.; Urinary tract infection (UTI) caused by Corynebacterium urealyticum is a rarely recognised condition in veterinary medicine . This report describes a case in a 13-month-old dog which presented with a history of dysuria and haematuria . C urealyticum was identified as the cause of UTI . The clinical, radiological and ultrasonographic features and the results of urinalysis and urine bacteriological culture are described, as are the therapeutic challenges presented by this particular infection.

Nucleic Acids Res, 2002 Jul 15, 30(14), 3141 - 51
Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation; Vitreschak AG et al.; The riboflavin biosynthesis in bacteria was analyzed using comparative analysis of genes, operons and regulatory elements . A model for regulation based on formation of alternative RNA structures involving the RFN elements is suggested . In Gram-positive bacteria including actinomycetes, Thermotoga, Thermus and Deinococcus, the riboflavin metabolism and transport genes are predicted to be regulated by transcriptional attenuation, whereas in most Gram-negative bacteria, the riboflavin biosynthesis genes seem to be regulated on the level of translation initiation . Several new candidate riboflavin transporters were identified (impX in Desulfitobacterium halfniense and Fusobacterium nucleatum; pnuX in several actinomycetes, including some Corynebacterium species and Strepto myces coelicolor; rfnT in Rhizobiaceae) . Traces of a number of likely horizontal transfer events were found: the complete riboflavin operon with the upstream regulatory element was transferred to Haemophilus influenzae and Actinobacillus pleuropneumoniae from some Gram-positive bacterium; non-regulated riboflavin operon in Pyrococcus furiousus was likely transferred from Thermotoga; and the RFN element was inserted into the riboflavin operon of Pseudomonas aeruginosa from some other Pseudomonas species, where it had regulated the ribH2 gene.

Comp Immunol Microbiol Infect Dis, 2002 Jul, 25(4), 237 - 48
Distribution of rabies virus in infected mice, vaccinated and submitted to P . acnes as immunomodulator; Megid J et al.; The lethality and distribution of rabies virus were evaluated in swiss mice experimentally infected with street rabies virus, vaccinated and submitted to immunomodulation by P .acnes (formerly Corynebacterium parvum) . The animals were sacrificed at different times,when the different tissues were collected and submitted to fluorescent antibody test (FAT) and mouse inoculation test (MIT) . The group submitted to vaccination and P . acnes treatment presented a percentage of survival superior to that observed in infected mice only treated with P . acnes . Control infected animals had the lowest survival rates.The distribution of rabies virus in spleen of infected mice, vaccinated and submitted to P . acnes was superior to that verified in infected mice not treated with P.acnes . The increased survival correlated with the distribution of rabies virus in lymphoid tissues, could be interpreted as the consequence of P . acnes activity on macrophages . The results suggest the role of macrophages against rabies virus infection in mice and the importance of vaccination in the post expositive treatment of rabies.

J Vet Med B Infect Dis Vet Public Health, 2002 Jun, 49(5), 230 - 4
Effect of infectious status and parity on somatic cell count and California mastitis test in pampinta dairy ewes; Suarez VH et al.; The relationship between somatic cell counts (SCC) and California mastitis test (CMT) results according to the infectious status of mammary halves and parity of Pampinta dairy ewes was evaluated . Tests were associated to bacteriological analysis and classified into three groups: uninfected (negative culture), infected by minor pathogens and infected by major pathogens . Coagulase-negative Staphylococcus (32.4%), Micrococcus spp . (32.4%), Corynebacterium spp . (5.4%), and Bacillus spp . (1.4%) were the minor pathogens isolated, while Staphylococcus aureus (27%) and Escherichia coli (1.4%) were the major pathogens isolated . A good correlation was found between the CMT and SCC, which included inflammatory and epithelial cells (r = 0.64; P < 0.0001) . SCC averages for the CMT scores shown in parentheses were 223 576 (0); 245,248 (1); 397,778 (2); 1,159,109 (3) and 2,460,833 (4) cells/ml . The correlation between SCC and the infectious status of udder halves was 0.58 (P < 0.0001) . The relationship between SCC and CMT profiles and infectious status studied by a discriminant analysis showed, with an accuracy of 65%, three infectious status groups . SCC arithmetic means were 244,470 cells/ml for negative culture, 1,044,100 cells/ml for minor pathogens and 2,045,652 cells/ ml for major pathogens . With the exception of 1-year-old ewes, no significant differences were observed in SCC as affected by age or parity.

Infection, 2002 Jun, 30(3), 168 - 70
Laryngopharyngitis by Corynebacterium ulcerans; Kaufmann D et al.; A 71-year-old female patient was hospitalized with membranous laryngopharyngitis typical of classical diphtheria . A toxigenic strain of Corynebacterium ulcerans was isolated from the throat . The patient was treated for 6 days with amoxicillin-clavulanic acid and recovered without complications . This second reported case of diphtheric laryngopharyngitis caused by C . ulcerans in Switzerland is a reminder that C . ulcerans should be included as a possible agent in patients with classical diphtheria symptoms.

Vet Microbiol, 2002 Aug 2, 88(1), 75 - 83
Identification of Corynebacterium pseudotuberculosis isolates from sheep and goats by PCR; Cetinkaya B et al.; The present study was carried out to estimate the prevalence of caseous lymphadenitis (CL) in sheep and goats slaughtered at the local abattoir in Elazig province located in the east of Turkey, between September and December 2000 . A total of 2046 sheep and 2262 goat carcasses were examined during the study period and 118 abscessed lymph nodes, 89 from sheep and 29 from goats, were collected . Corynebacterium spp . strains were isolated from 81.4% of the abscesses, giving an overall prevalence of 2.2% . The prevalence was 3.5 and 1.1% in sheep and goats, respectively . PCR on DNA extracted from 96 suspicious isolates, using a pair of Corynebacterium pseudotuberculosis-specific primers, was positive for 93 . Although cross-reaction with C . ulcerans, a human/bovine species, was observed, the PCR assay used in this study may successfully be applied for the diagnosis of CL in goats and sheep as an alternative to conventional methods, owing to its advantages of specificity and speed.

FEMS Microbiol Lett, 2002 Jul 2, 212(2), 209 - 16
Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476; Srivastava P et al.; A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2 . Efficient expression of GST driven by inducible tac promoter of E . coli was observed in Corynebacterium acetoacidophilum . Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins . The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.

Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 205 - 10 Epub 2002 Apr 04.
Influence of threonine exporters on threonine production in Escherichia coli; Kruse D et al.; Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E . coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE . Both E . coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from {(14)C} L-threonine . The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.

J Infect . 2002 Apr;44(3):193.
Corynebacterium striatum first reported case of prosthetic valve endocarditis; de Arriba JJ et al.; We describe the case of a prosthetic valve endocarditis in a 72-year-old woman . Corynebacterium striatum was isolated in the blood samples . This organism has been described in a few cases of native valve endocarditis, but this is the first case reported of prosthetic valve endocarditis .

J Antimicrob Chemother, 2002 Jul, 50(1), 125 - 8
Penicillin tolerance amongst non-toxigenic Corynebacterium diphtheriae isolated from cases of pharyngitis; von Hunolstein C et al.; Twenty-four strains of non-toxigenic Corynebacterium diphtheriae biotype gravis from the throats of patients with pharyngitis/tonsillitis were assayed for susceptibility to penicillin and erythromycin using determination of MIC, MBC and time-kill curves . There were no differences between the MICs of penicillin for susceptible and tolerant strains . All but one strain had penicillin MBCs > or = 2 mg/L . Seventy-one per cent (17/24) of the strains were tolerant to penicillin . In contrast, all strains were susceptible to erythromycin (MIC < or = 0.016 mg/L) . These aspects should be considered when choosing the therapy for treating non-toxigenic C . diphtheriae pharyngitis/tonsillitis.

Appl Environ Microbiol, 2002 Jul, 68(7), 3321 - 7
Identification of glyA (encoding serine hydroxymethyltransferase) and its use together with the exporter ThrE to increase L-threonine accumulation by Corynebacterium glutamicum; Simic P et al.; L-threonine can be made by the amino acid-producing bacterium Corynebacterium glutamicum . However, in the course of this process, some of the L-threonine is degraded to glycine . We detected an aldole cleavage activity of L-threonine in crude extracts with an activity of 2.2 nmol min(-1) (mg of protein)(-1) . In order to discover the molecular reason for this activity, we cloned glyA, encoding serine hydroxymethyltransferase (SHMT) . By using affinity-tagged glyA, SHMT was isolated and its substrate specificity was determined . The aldole cleavage activity of purified SHMT with L-threonine as the substrate was 1.3 micromol min(-1) (mg of protein)(-1), which was 4% of that with L-serine as substrate . Reduction of SHMT activity in vivo was obtained by placing the essential glyA gene in the chromosome under the control of P(tac), making glyA expression isopropylthiogalactopyranoside dependent . In this way, the SHMT activity in an L-threonine producer was reduced to 8% of the initial activity, which led to a 41% reduction in glycine, while L-threonine was simultaneously increased by 49% . The intracellular availability of L-threonine to aldole cleavage was also reduced by overexpressing the L-threonine exporter thrE . In C . glutamicum DR-17, which overexpresses thrE, accumulation of 67 mM instead of 49 mM L-threonine was obtained . This shows that the potential for amino acid formation can be considerably improved by reducing its intracellular degradation and increasing its export.

Eur J Biochem, 2002 Jul, 269(13), 3142 - 9
Trehalose-based oligosaccharides isolated from the cytoplasm of Mycobacterium smegmatis . Relation to trehalose-based oligosaccharides attached to lipid; Ohta M et al.; A series of trehalose-based oligosaccharides were isolated from the cytoplasmic fraction of Mycobacterium smegmatis and purified by gel-filtration and paper chromatography and TLC . Their structures were determined by HPLC and GLC to determine sugar composition and ratios, MALDI-TOF MS to measure molecular mass, methylation analysis to determine linkages, (1)H-NMR to obtain anomeric configurations of glycosidic linkages, and exoglycosidase digestions followed by TLC to determine sequences and anomeric configurations of the monosaccharides . Six different oligosaccharides were identified all with trehalose as the basic structure and additional glucose or galactose residues attached in various linkages . One of these oligosaccharides is the disaccharide trehalose (Glcalpha1-1alphaGlc), which is present in substantial amounts in these cells and also in other mycobacteria . Two other oligosaccharides, the tetrasaccharides Glcalpha1-4Glcalpha1-1alphaGlc6-1alphaGal and Galalpha1-6Galalpha1-6Glcalpha1-1alphaGlc, have not previously been isolated from natural sources or synthesized chemically . The fourth oligosaccharide, Glcbeta1-6Glcbeta1-6Glcalpha1-1alphaGlc, has been isolated from corynebacteria, but not reported in other organisms . Two other oligosaccharides, Glcalpha1-4Glcalpha1-1alphaGlc, which has been synthesized chemically and isolated from insects but not previously reported in mycobacteria, and Glcbeta1-6Glcalpha1-1alphaGlc, which was previously isolated from Mycobacterium fortuitum and yeast, were also characterized . Another trisaccharide found in the cytosol has been partially characterized as arabinosyl-1-4trehalose, but neither the anomeric configuration nor the D or L configuration of the arabinose is known . In analogy with sucrose and its higher homologs, raffinose and stachyose, which may act as protective agents during maturation drying in plants, these trehalose homologs may also have a protective role in mycobacteria, perhaps during latency.

J Bacteriol, 2002 Jul, 184(14), 3947 - 56
Export of L-isoleucine from Corynebacterium glutamicum: a two-gene-encoded member of a new translocator family; Kennerknecht N et al.; Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes L-isoleucine in a process dependent on the proton motive force . In order to identify the system responsible for L-isoleucine export, we have used transposon mutagenesis to isolate mutants of C . glutamicum sensitive to the peptide isoleucyl-isoleucine . In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices . brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices . A mutant deleted of both genes no longer exports L-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate . BrnF and BrnE together are also required for the export of L-leucine and L-valine . BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids . Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export . Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon . Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in alpha-proteobacteria but not in other prokaryotes analyzed.

Pediatr Infect Dis J, 2002 Apr, 21(4), 350 - 2
Safety, tolerability and immunogenicity of low dose Haemophilus influenzae type b conjugated to the outer membrane protein complex of Neisseria meningitidis group B; Anderson EL et al.; Infants vaccinated with three doses followed by a booster of either 1.0 microg of Haemophilus influenzae type b conjugated to the outer membrane protein complex of Neisseria meningitidis group B or H . influenzae 10-microg oligosaccharide vaccine conjugated to 25 microg mutant toxin (CRM197) isolated from Corynebacterium diphtheriae had protective antipolyribosylribitol phosphate concentrations.

Clin Exp Metastasis, 2002, 19(3), 259 - 64
IFNgamma and TNFalpha account for a pro-clonogenic activity secreted by activated murine peritoneal macrophages; Calorini L et al.; In the present study, we found that murine peritoneal macrophages elicited by BCG or Listeria monocytogenes release into the media an activity capable of stimulating the lung colonization as well as the expression of MHC class I antigens in B16 melanoma cells . A similar activity has previously been found in media conditioned by Corynebacterium parvum-elicited macrophages . Analysis by gel filtration chromatography of media conditioned by Corynebacterium parvum-, BCG- or Listeria monocytogenes-elicited macrophages revealed that the material responsible for the pro-clonogenic activity concentrated in chromatographic fractions corresponding to molecular weights (25 to 52 kDa) which are characteristic of certain cytokines . Thus, we challenged the various macrophage-conditioned media with polyclonal antibodies against IFNgamma and TNFalpha, and found that the macrophage pro-clonogenic activity was completely abolished in the presence of anti-IFNgamma antibodies, but only partially inhibited by anti-TNFalpha antibodies . This finding suggests a cooperative participation of the two cytokines to the pro-clonogenic activity of the media conditioned by Corynebacterium parvum-, BCG- or Listeria monocytogenes-elicited macrophages.

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 1001 - 5
Corynebacterium aurimucosum sp . nov . and emended description of Corynebacterium minutissimum Collins and Jones (1983); Yassin AF et al.; Two coryneform bacteria isolated from human clinical specimens were characterized by phenotypic and molecular taxonomic methods . Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and short-chain mycolic acids consistent with the genus Corynebacterium sensu stricto . Comparative 16S rRNA gene sequence analysis showed that the two strains are genealogically highly related (99.8% sequence similarity) and constitute a new subline within the genus Corynebacterium, with Corynebacterium minutissimum as their nearest phylogenetic neighbours (98.8% sequence similarity) . However, DNA-DNA hybridization experiments demonstrated unambiguously that the isolates are genealogically distinct from Corynebacterium minutissimum (42% homology) . Biochemical testing indicated that the two isolates were hardly differentiated from Corynebacterium minutissimum . Based on both phenotypic and phylogenetic evidence it is proposed that these isolates be classified as a new species, Corynebacterium aurimucosum sp . nov . The type strain of Corynebacterium aurimucosum is represented by strain IMMIB D-1488T (= DSM 44532T = NRRL B-24143T).

Public Health Rep, 2001 Jul-Aug, 116(4), 362 - 8
Diphtheria and hearing loss; Schubert CR et al.; OBJECTIVE: To determine if infectious diseases usually experienced in childhood have an effect on hearing ability later in life . METHODS: The Epidemiology of Hearing Loss Study (N = 3,753) is a population-based study of age-related hearing loss in adults aged 48 to 92 years in Beaver Dam, Wisconsin . As part of this study, infectious disease history was obtained and hearing was tested using pure-tone audiometry . Hearing loss was defined as a pure-tone average of thresholds at 500 Hz, 1,000 Hz, 2,000 Hz, and 4,000 Hz greater than 25 decibels hearing level in either ear . RESULTS: After adjusting for confounders, only a history of diphtheria (n = 37) was associated with hearing loss (odds ratio {OR} 2.79; 95% confidence interval {CI} 1.05, 7.36) . There was no relationship between hearing loss and history of chickenpox, measles, mumps, pertussis, polio, rheumatic fever, rubella, or scarlet fever . Only two participants with a history of diphtheria and hearing loss reported having a hearing loss before age 20 . CONCLUSIONS: Diphtheria in childhood may have consequences for hearing that do not become apparent until later in life . A possible biological mechanism for a diphtheria effect on hearing ability exists: The toxin produced by the Corynebacterium diphtheriae bacteria can cause damage to cranial nerves and therefore may affect the auditory neural pathway . These data may have important implications for areas facing a resurgence of diphtheria cases.

Ukr Biokhim Zh, 2001 May-Jun, 73(3), 61 - 70
{Electrophoretic patterns of cell wall protein as a criterion for the identification and classification of Corynebacteria}; Mykhal's'kyi LO et al.; Electrophoretic patterns of cell wall protein of three industrial strains, that were used for production of lysin, and eight collection strains from the genus Corynevacterium were studied to analyze their similarity as well as to estimate an opportunity of using this parameter as an additional criterion for identification and classification of corynebacteria . Similarity coefficient of cell wall overall and main protein electrophoretic patterns were determined by a specially created computer program . Electrophoretic analysis showed that every specie had an individual protein profile . There were determined biopolymers common for the specie, genus and individual among the overall majors and minors . The obtained results showed, that the patterns of main proteins were more conservative and informative in comparison with those ones of overall proteins . The definition of similarity coefficient by the main protein patterns has correlated with the protein profile characteristics of every analyzed strain, and it managed to distribute them into the separate groups . The similarity coefficient of preparations by the main protein patterns allows to separate one specie or a strain from another, and that gives us a chance to claim that this parameter could be used as an additional criterion for differentiation and referring the corynebacteria to a certain taxonomic group.

J Ind Microbiol Biotechnol, 2002 Jun, 28(6), 338 - 43
Influence of glucose, fructose and sucrose as carbon sources on kinetics and stoichiometry of lysine production by Corynebacterium glutamicum; Kiefer P et al.; Batch cultivations of l-lysine-producing Corynebacterium glutamicum ATCC 21253 were carried out on the different carbon sources, glucose, sucrose and fructose . The time profiles of substrate and product concentrations were evaluated to compare kinetics and stoichiometry of lysine production . The lysine yield (mol C/mol C) on glucose was 8% higher than on sucrose and 30% higher than on fructose . The highest final biomass concentration of 5.0 g/l was obtained on glucose, whereas fructose and sucrose yielded 20% less biomass . Compared to glucose, fructose resulted in significantly higher respiration rates, a higher substrate uptake rate but a lower lysine production rate during the cultivation process . This was probably due to a higher tricarboxylic cycle activity combined with a lower activity of the pentose phosphate pathway . On sucrose, specific rates and yields differed significantly from those on fructose and glucose . Transport and metabolism of sucrose, therefore, are not a simple superposition of its building blocks, glucose and fructose.

J Ind Microbiol Biotechnol, 2002 Jun, 28(6), 333 - 7
Flexibility of the metabolism of Corynebacterium glutamicum 2262, a glutamic acid-producing bacterium, in response to temperature upshocks; Delaunay S et al.; In order to test the temperature sensitivity of glutamate production metabolism, several temperature shifts, from 33 to 37, 38, 39, 40 or 41 degrees C, were applied to the temperature-sensitive strain, Corynebacterium glutamicum 2262, cultivated in a 24-h fed-batch process . Whereas glucose was entirely dedicated to biomass synthesis when cells were grown at 33 degrees C, applying temperature upshocks, whatever their range, triggered a redistribution of the carbon utilisation between glutamate, biomass and lactate production . Although increasing the culture temperature from 33 to 37, 38, 39 or 40 degrees C resulted in final glutamate titers superior to 80 g/l, temperatures resulting in the best chanelling of the carbon flow towards glutamic acid synthesis were 39 and 40 degrees C . Moreover, this study showed that the higher the temperature, the slower the growth rate and the higher the lactate accumulation.

New Microbiol, 2002 Apr, 25(2), 187 - 93
Characterization of a coryneform isolate from fasciated mugwort (Artemisia vulgaris); Battikhi MN; The morphological and physiological features of coryneform isolated from fasciated mugwort (Artemisia vulgaris) display many morphological similarities with the plant pathogenic corynebacteria, but differ from Corynebacterium fascians in exhibiting motility albeit in only a small proportion of each cell population, and by its ability to hydrolyze asculin, its failure to produce urease and differences in pigmentation . The isolate appears to be related to Corynebacterium fascians in its ability to cause fasciation but physiologically and biochemically it resembles Cornyebacterium poinsettiae and C . flaccumfaciens, both of which were transferred to the genus Curtobacterium.

Rhinology, 2002 Mar, 40(1), 34 - 40
Infectious and neoplastic diseases of the sphenoid sinus--a report of 10 cases; Mra Z et al.; Sphenoid opacifications may be discovered during the radiological work up of patients presenting with fever, headache, or neurological changes . While most of these patients do not require surgical intervention, prompt assessment and management is nevertheless required . Ten patients who underwent sphenoidotomy for drainage or biopsy at Montefiore Hospital during a 4-year period from September 1995 through January 2000 are presented . Nine out of 10 patients had predisposing factors such as AIDS, diabetes, leukemia, and end-stage renal disease . The most common presentation was altered mental status . One patient rapidly developed cavernous sinus thrombosis . Microbiology of sphenoid cultures included various fungi, Mycobacterium avium intracellulare, coagulase negative Staphylococci, and Corynebacterium . Neoplastic processes included non-Hodgkin's lymphoma and sinonasal undifferentiated carcinoma . When evaluating hospitalized patients with sphenoid sinus disease, a thorough history and a bedside nasal endoscopy should be performed . Conservative management in the form of intravenous antibiotics and topical decongestion should always be the first line of treatment . Those patients with clinical or radiological evidence of disease extending beyond the confines of the sphenoid sinus require immediate surgical intervention.

Mol Microbiol, 2002 May, 44(4), 1109 - 22
Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis; Puech V et al.; Mycobacterium tuberculosis produces a series of major secreted proteins, the fibronectin-binding proteins (Fbps), also known as the antigen 85 complex, that are believed to play an essential role in the pathogenesis of tuberculosis through their mycoloyltransferase activity required for maintaining the integrity of the bacterial cell envelope . Four different fbp genes are found in the genome of M . tuberculosis, but the reason for the existence of these Fbps sharing the same substrate specificity in vitro in mycobacteria is unknown . We have shown previously that, in the heterologous host, Corynebacterium glutamicum, FbpA, FbpB and FbpC can all add mycoloyl residues to the cell wall arabinogalactan and that, in M . tuberculosis, the cell wall mycoloylation decreases by 40% when fbpC is knocked out . To investigate whether the remaining 60% mycoloylation came from the activity of FbpA and/or FbpB, fbpA- and fbpB-inactivated mutant strains were biochemically characterized and compared with the previously studied fbpC-disrupted mutant . Unexpectedly, both mutants produced normally mycoloylated cell walls . Overproduction of FbpA, FbpB or FbpC, but not FbpD, in the fbpC-inactivated mutant strain of M . tuberculosis restored both the cell wall-linked mycolate defect and the outer cell envelope permeability barrier property . These results are consistent with all three enzymes being involved in cell wall mycoloylation and FbpC playing a more critical role than the others or, alternatively, FbpC is able to compensate for FbpA and FbpB in ways that these enzymes cannot compensate for FbpC, pointing to a partial redundancy of Fbps . In sharp contrast, FbpD does not appear to be an active mycoloyltransferase enzyme, as it cannot complement the fbpC-inactivated mutant . Most importantly, application of Smith degradation to the cell walls of transformants demonstrated that the multiple Fbp enzymes are redundant rather than specific for the various arabinogalactan mycoloylation regions . Neither FbpA nor FbpB attaches mycoloyl residues to specific sites but, like FbpC, each enzyme transfers mycoloyl residues onto the four sites present in the arabinogalactan non-reducing end hexaarabinosides.

Drugs, 2002, 62(8), 1131 - 41
Management of cutaneous erythrasma; Holdiness MR; Corynebacterium minutissimum is the bacteria that leads to cutaneous eruptions of erythrasma and is the most common cause of interdigital foot infections . It is found mostly in occluded intertriginous areas such as the axillae, inframammary areas, interspaces of the toes, intergluteal and crural folds, and is more common in individuals with diabetes mellitus than other clinical patients . This organism can be isolated from a cutaneous site along with a concurrent dermatophyte or Candida albicans infection . The differential diagnosis of erythrasma includes psoriasis, dermatophytosis, candidiasis and intertrigo, and methods for differentiating include Wood's light examination and bacterial and mycological cultures . Erythromycin 250mg four times daily for 14 days is the treatment of choice and other antibacterials include tetracycline and chloramphenicol; however, the use of chloramphenicol is limited by bone marrow suppression potentially leading to neutropenia, agranulocytosis and aplastic anaemia . Further studies are needed but clarithromycin may be an additional drug for use in the future . Where there is therapeutic failure or intertriginous involvement, topical solutions such as clindamycin, Whitfield's ointment, sodium fusidate ointment and antibacterial soaps may be required for both treatment and prophylaxis . Limited studies on the efficacy of these medications exist, however, systemic erythromycin demonstrates cure rates as high as 100% . Compared with tetracyclines, systemic erythromycin has greater efficacy in patients with involvement of the axillae and groin, and similar efficacy for interdigital infections . Whitfield's ointment has equal efficacy to systemic erythromycin in the axillae and groin, but shows greater efficacy in the interdigital areas and is comparable with 2% sodium fusidate ointment for treatment of all areas . Adverse drug effects and potential drug interactions need to be considered . No cost-effectiveness data are available but there are limited data on cost-related treatment issues . A guideline is proposed for the detection, evaluation, treatment and prophylaxis of this cutaneous eruption.

Biochemistry, 2002 May 21, 41(20), 6226 - 36
Optimizing an artificial metabolic pathway: engineering the cofactor specificity of Corynebacterium 2,5-diketo-D-gluconic acid reductase for use in vitamin C biosynthesis; Banta S et al.; The strict cofactor specificity of many enzymes can potentially become a liability when these enzymes are to be employed in an artificial metabolic pathway . The preference for NADPH over NADH exhibited by the Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase may not be ideal for use in industrial scale vitamin C biosynthesis . We have previously reported making a number of site-directed mutations at five sites located in the cofactor-binding pocket that interact with the 2'-phosphate group of NADPH . These mutations conferred greater activity with NADH upon the Corynebacterium 2,5-DKG reductase {Banta, S., Swanson, B . A., Wu, S., Jarnagin, A., and Anderson, S . (2002) Protein Eng . 15, 131-140; (1)} . The best of these mutations have now been combined to see if further improvements can be obtained . In addition, several chimeric mutants have been produced that contain the same residues as are found in other members of the aldo-keto reductase superfamily that are naturally able to use NADH as a cofactor . The most active mutants obtained in this work were also combined with a previously reported substrate-binding pocket double mutant, F22Y/A272G . Mutant activity was assayed using activity-stained native polyacrylamide gels . Superior mutants were purified and subjected to a simplified kinetic analysis . The simplified kinetic analysis was extended for the most active mutants in order to obtain the kinetic parameters in the full-ordered bi bi rate equation in the absence of products, with both NADH and NADPH as cofactors . The best mutant 2,5-DKG reductase produced in this work was the F22Y/K232G/R238H/A272G quadruple mutant, which exhibits activity with NADH that is more than 2 orders of magnitude higher than that of the wild-type enzyme, and it retains a high level of activity with NADPH . This new 2,5-DKG reductase may be a valuable new catalyst for use in vitamin C biosynthesis.

Nature, 2002 May 9, 417(6885), 141 - 7
Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2); Bentley SD et al.; Streptomyces coelicolor is a representative of the group of soil-dwelling, filamentous bacteria responsible for producing most natural antibiotics used in human and veterinary medicine . Here we report the 8,667,507 base pair linear chromosome of this organism, containing the largest number of genes so far discovered in a bacterium . The 7,825 predicted genes include more than 20 clusters coding for known or predicted secondary metabolites . The genome contains an unprecedented proportion of regulatory genes, predominantly those likely to be involved in responses to external stimuli and stresses, and many duplicated gene sets that may represent 'tissue-specific' isoforms operating in different phases of colonial development, a unique situation for a bacterium . An ancient synteny was revealed between the central 'core' of the chromosome and the whole chromosome of pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae . The genome sequence will greatly increase our understanding of microbial life in the soil as well as aiding the generation of new drug candidates by genetic engineering.

Biosci Biotechnol Biochem, 2002 Feb, 66(2), 464 - 6
Kinetic resolutions of indan derivatives using bacteria; Tarui N et al.; Racemic indan derivatives have been resolved by the hydrolysis of amide bonds using Corynebacterium ammoniagenes IFO12612 to produce (S)-amine and (R)-amides . In the kinetic resolution of 1 (N-12-(6-methoxy-indan-1-yl)ethyl}acetamide), it was possible to run the reaction to 44% conversion on a 10-g scale, obtaining (S)-amine 4 ((S)-2-(6-methoxy-indan-1-yl)ethylamine) at >99% enantiomeric excess (ee) and (R)-1 at 98% ee.

Emerg Infect Dis, 2002 May, 8(5), 516 - 8
Molecular characterization of Corynebacterium diphtheriae isolates, Russia, 1957-1987; Skogen V et al.; In the 1990s, the Newly Independent and Baltic States of the former Soviet Union experienced the largest diphtheria outbreak since the 1960s; it was caused by Corynebacterium diphtheriae strains of a unique clonal group . To address its origin, we studied 47 clinical isolates from Russia and demonstrated that this clonal group was an integral part of the endemic reservoir that existed in Russia at least 5 years before the epidemic began.

Mol Microbiol, 2002 May, 44(3), 675 - 84
Charting and unzipping the surface layer of Corynebacterium glutamicum with the atomic force microscope; Scheuring S et al.; Bacterial surface layers (S-layers) are extracellular protein networks that act as molecular sieves and protect a large variety of archaea and bacteria from hostile environments . Atomic force microscopy (AFM) was used to asses the S-layer of Coryne-bacterium glutamicum formed of PS2 proteins that assemble into hexameric complexes within a hexagonal lattice . Native and trypsin-treated S-layers were studied . Using the AFM stylus as a nanodissector, native arrays that adsorbed to mica as double layers were separated . All surfaces of native and protease-digested S-layers were imaged at better than 1 nm lateral resolution . Difference maps of the topographies of native and proteolysed samples revealed the location of the cleaved C-terminal fragment and the sidedness of the S-layer . Because the corrugation depths determined from images of both sides span the total thickness of the S-layer, a three-dimensional reconstruction of the S-layer could be calculated . Lattice defects visualized at 1 nm resolution revealed the molecular boundaries of PS2 proteins . The combination of AFM imaging and single molecule force spectroscopy allowed the mechanical properties of the Corynebacterium glutamicum S-layer to be examined . The results provide a basis for understanding the amazing stability of this protective bacterial surface coat.

Eur J Biochem, 2002 May, 269(9), 2394 - 402
Chiral alcohol production by NADH-dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH; Itoh N et al.; Phenylacetaldehyde reductase (PAR) produced by styrene-assimilating Corynebacterium strain ST-10 was used to synthesize chiral alcohols . This enzyme with a broad substrate range reduced various prochiral aromatic ketones and beta-ketoesters to yield optically active secondary alcohols with an enantiomeric purity of more than 98% enantiomeric excess (e.e.) . The Escherichia coli recombinant cells which expressed the par gene could efficiently produce important pharmaceutical intermediates; (R)-2-chloro-1-(3-chlorophenyl)ethanol (28 mg.mL-1) from m-chlorophenacyl chloride, ethyl (R)-4-chloro-3-hydroxy butanoate) (28 mg.mL-1) from ethyl 4-chloro-3-oxobutanoate and (S)-N-tert-butoxycarbonyl(Boc)-3-pyrrolidinol from N-Boc-3-pyrrolidinone (51 mg.mL-1), with more than 86% yields . The high yields were due to the fact that PAR could concomitantly reproduce NADH in the presence of 3-7% (v/v) 2-propanol in the reaction mixture . This biocatalytic process provided one of the best asymmetric reductions ever reported.

J Cataract Refract Surg, 2002 May, 28(5), 826 - 33
Bacterial contamination of the anterior chamber during phacoemulsification cataract surgery; Leong JK et al.; PURPOSE: To determine the incidence of bacterial contamination of the anterior chamber after phacoemulsification cataract surgery with intraocular lens (IOL) implantation . SETTING: Department of Ophthalmology, Royal Prince Alfred Hospital, Sydney, Australia . METHODS: Ninety-eight consecutive eyes of 96 patients having phacoemulsification cataract surgery with IOL implantation were included in this prospective study . Two intraoperative anterior chamber aspirates were obtained from each patient, 1 taken at the start and the other at the conclusion of surgery . In addition, preoperative and postoperative conjunctival swabs were acquired . The 4 specimens were cultured using direct culturing techniques under aerobic and anaerobic conditions for 14 days . No preoperative antibiotics were used . RESULTS: The incidence of intraoperative anterior chamber contamination was 0% (95% confidence interval, 0%-3.7%) as all intraoperative anterior chamber samples proved culture negative . Sixty-five percent of the preoperative conjunctival swabs were positive for growth, with corynebacteria, coagulase-negative staphylococci, and Propionibacterium acnes being the most frequently cultured organisms . Sixteen percent of the postoperative conjunctival swabs were positive for growth, with corynebacteria and coagulase-negative staphylococci being the most common bacteria . One patient developed culture-positive postoperative endophthalmitis; using pulsed-field gel electrophoresis for further typing, the implicated Staphylococcus epidermidis was indistinguishable from that isolated from the patient's preoperative conjunctival swab . CONCLUSIONS: The bacterial contamination rate of the anterior chamber after phacoemulsification and IOL implantation was extremely low . Additional findings support the conjunctiva as being a primary source of bacteria causing postoperative endophthalmitis as well as the ability of povidone-iodine to reduce the conjunctival bacterial load.

J Bacteriol, 2002 May, 184(10), 2728 - 39
Identification of two prpDBC gene clusters in Corynebacterium glutamicum and their involvement in propionate degradation via the 2-methylcitrate cycle; Claes WA et al.; Genome sequencing revealed that the Corynebacterium glutamicum genome contained, besides gltA, two additional citrate synthase homologous genes (prpC) located in two different prpDBC gene clusters, which were designated prpD1B1C1 and prpD2B2C2 . The coding regions of the two gene clusters as well as the predicted gene products showed sequence identities of about 70 to 80% . Significant sequence similarities were found also to the prpBCDE operons of Escherichia coli and Salmonella enterica, which are known to encode enzymes of the propionate-degrading 2-methylcitrate pathway . Homologous and heterologous overexpression of the C . glutamicum prpC1 and prpC2 genes revealed that their gene products were active as citrate synthases and 2-methylcitrate synthases . Growth tests showed that C . glutamicum used propionate as a single or partial carbon source, although the beginning of the exponential growth phase was strongly delayed by propionate for up to 7 days . Compared to growth on acetate, the specific 2-methylcitrate synthase activity increased about 50-fold when propionate was provided as the sole carbon source, suggesting that in C . glutamicum the oxidation of propionate to pyruvate occurred via the 2-methylcitrate pathway . Additionally, two-dimensional gel electrophoresis experiments combined with mass spectrometry showed strong induction of the expression of the C . glutamicum prpD2B2C2 genes by propionate as an additional carbon source . Mutational analyses revealed that only the prpD2B2C2 genes were essential for the growth of C . glutamicum on propionate as a sole carbon source, while the function of the prpD1B1C1 genes remains obscure.

Appl Environ Microbiol, 2002 May, 68(5), 2246 - 50
Linking central metabolism with increased pathway flux: L-valine accumulation by Corynebacterium glutamicum; Radmacher E et al.; Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE, which encode dihydroxy acid dehydratase and transaminase B, respectively . These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete L-valine, to result in an accumulation of this amino acid to a concentration of 42 mM . Since L-valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate . The same genes, ilvBNCD, overexpressed in an ilvA deletion mutant which is unable to synthesize L-isoleucine increased the concentration of this amino acid to 58 mM . A further dramatic increase was obtained when panBC was deleted, making the resulting mutant auxotrophic for D-pantothenate . When the resulting strain, C . glutamicum 13032DeltailvADeltapanBC with ilvBNCD overexpressed, was grown under limiting conditions it accumulated 91 mM L-valine . This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the L-valine biosynthesis pathway.

FEMS Microbiol Lett, 2002 Mar 5, 208(2), 287 - 93
Nitrogen assimilation in Corynebacterium diphtheriae: pathways and regulatory cascades; Nolden L et al.; Genes encoding proteins for ammonium uptake, assimilation, and the nitrogen regulatory system in Corynebacterium diphtheriae were studied on basis of homology searches using Corynebacterium glutamicum genes as query sequences . Regulation of transcription of these genes in response to nitrogen starvation was analyzed by RNA hybridization experiments and knock-out mutants were generated to verify the function of distinct genes . In this communication, we were able to identify the key components of ammonium assimilation pathways and nitrogen regulation in C . diphtheriae . Moreover, we show in this study that molecular biology methods and vectors developed for C . glutamicum can be applied in C . diphtheriae . The results obtained strengthens the role of C . glutamicum as a model organism for mycolic acids-containing actinomycetes.

Appl Microbiol Biotechnol, 2002 Mar, 58(3), 265 - 74 Epub 2001 Dec 21.
Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications; Burkovski A et al.; Transport processes play a pivotal role in cellular metabolism, e.g . for the uptake of nutrients or the excretion of metabolic waste products . Moreover, they are also important in biotechnological processes such as the production of various amino acids by the use of microorganisms . The focus of this review is on bacterial amino acid transport systems, in particular those of Corynebacterium glutamicum and Escherichia coli, with respect to their function and biotechnological significance.

FEMS Microbiol Lett, 2002 Feb 19, 208(1), 41 - 5
Identification and role in virulence of putative iron acquisition genes from Corynebacterium pseudotuberculosis; Billington SJ et al.; Four genes, fagA, B, C and D, encoding products with 32-47% identity to proteins involved in bacterial iron uptake systems, were identified immediately downstream of the Corynebacterium pseudotuberculosis phospholipase D gene . beta-Galactosidase assays on a C . pseudotuberculosis strain carrying a fagA-lacZ fusion indicated that the putative fagABC operon was poorly expressed in iron-rich media . However, similar experiments in iron-limited media resulted in an approximately three-fold increase in beta-galactosidase activity, suggesting that this operon is regulated by iron in vitro . Although no defect in iron utilization could be determined for a C . pseudotuberculosis fagB(C) mutant in vitro, this mutant showed reduced virulence compared to wild-type in a goat model of caseous lymphadenitis . Thus, expression of the fag genes in the host appears to contribute to virulence.

Oral Microbiol Immunol, 2002 Apr, 17(2), 132 - 5
Bacteriology and antimicrobial susceptibility of gram-positive cocci isolated from pus specimens of orofacial odontogenic infections; Kuriyama T et al.; We recently reported the beta-lactamase production and antimicrobial susceptibility of anaerobic gram-negative rods isolated from pus specimens of 93 orofacial odontogenic infections . In this report, we determine the bacteriology and antimicrobial susceptibility of bacteria other than anaerobic gram-negative rods, mainly gram-positive cocci, isolated from the same specimens . Streptococcus constellatus and Peptostreptococcus micros were frequent isolates from all types of infection examined . Peptostreptococcus prevotii, Corynebacterium species, and Eubacterium species were recovered only from dentoalveolar infections, while Gemella morbillorum was found more frequently in periodontitis than in the other infections . beta-Lactamase-positive strains were detected only in staphylococci . Ampicillin, ampicillin/sulbactam, cefazolin, cefotaxime, imipenem, erythromycin, clindamycin and levofloxacin showed high susceptibility rates (> or = 77%) against viridans streptococci, Peptostreptococcus and Gemella . Minocycline showed a high MIC90 value against viridans streptococci (32 microg/ml), and metronidazole was effective against Peptostreptococcus and Gemella . These results provide useful information for the treatment of orofacial odontogenic infections.

J Investig Dermatol Symp Proc, 2001 Dec, 6(3), 170 - 4
Skin microflora and bacterial infections of the skin; Chiller K et al.; The skin is a milieu for controlled bacterial growth . Skin supports the growth of commensal bacteria, which protect the host from pathogenic bacteria . Environmental and local factors, host immunity, and organism adherence and virulence are intricately related to cutaneous infection . Resident gram-positive bacteria include Staphylococcus, Micrococcus, and Corynebacterium sp . Staphylococcus aureus and Streptococcus pyogenes are notoriously pathogenic in the skin . In order for bacteria to be pathogenic, they must be able to adhere to, grow on, and invade the host . Bacteria possess numerous virulence genes that allow for growth in these privileged niches . Epidermal infections caused by S . aureus and S . pyogenes include impetigo and ecthyma . Dermal infections consist of erysipelas, cellulitis, and necrotizing fasciitis . The pilosebaceous unit is involved in folliculitis, furunculosis, and carbunculosis . Moreover, S . aureus and S . pyogenes produce toxins that may elicit a superantigen response, causing massive release of cytokines . Staphylococcal scalded skin syndrome, toxic shock syndrome, and scarlet fever are all superantigen-mediated . Gram-negative organisms such as Pseudomonas aeruginosa, Pasteurella multocida, Capnocytophaga canimorsus, Bartonella sp., Klebsiella rhinoscleromatis, and Vibrio vulnificus are not typical resident skin microflora but may cause cutaneous infection.

Protein Eng, 2002 Feb, 15(2), 131 - 40
Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A; Banta S et al.; The NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme is a required component in some novel biosynthetic vitamin C production processes . This enzyme catalyzes the conversion of 2,5-DKG to 2-keto-L-gulonic acid, which is an immediate precursor to L-ascorbic acid . Forty unique site-directed mutations were made at five residues in the cofactor-binding pocket of 2,5-DKG reductase A in an attempt to improve its ability to use NADH as a cofactor . NADH is more stable, less expensive and more prevalent in the cell than is NADPH . To the best of our knowledge, this is the first focused attempt to alter the cofactor specificity of a member of the aldo-keto reductase superfamily by engineering improved activity with NADH into the enzyme . Activity of the mutants with NADH or NADPH was assayed using activity-stained native polyacrylamide gels . Eight of the mutants at three different sites were identified as having improved activity with NADH . These mutants were purified and subjected to a kinetic characterization with NADH as a cofactor . The best mutant obtained, R238H, produced an almost 7-fold improvement in catalysis with NADH compared with the wild-type enzyme . Surprisingly, most of this catalytic improvement appeared to be due to an improvement in the apparent kcat for the reaction rather than a large improvement in the affinity of the enzyme for NADH.

Comp Biochem Physiol A Mol Integr Physiol, 2001 Oct, 130(3), 437 - 60
Osmosensing and osmoregulatory compatible solute accumulation by bacteria; Wood JM et al.; Bacteria inhabit natural and artificial environments with diverse and fluctuating osmolalities, salinities and temperatures . Many maintain cytoplasmic hydration, growth and survival most effectively by accumulating kosmotropic organic solutes (compatible solutes) when medium osmolality is high or temperature is low (above freezing) . They release these solutes into their environment when the medium osmolality drops . Solutes accumulate either by synthesis or by transport from the extracellular medium . Responses to growth in high osmolality medium, including biosynthetic accumulation of trehalose, also protect Salmonella typhimurium from heat shock . Osmotically regulated transporters and mechanosensitive channels modulate cytoplasmic solute levels in Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Lactobacillus plantarum, Lactococcus lactis, Listeria monocytogenes and Salmonella typhimurium . Each organism harbours multiple osmoregulatory transporters with overlapping substrate specificities . Membrane proteins that can act as both osmosensors and osmoregulatory transporters have been identified (secondary transporters ProP of E . coli and BetP of C . glutamicum as well as ABC transporter OpuA of L . lactis) . The molecular bases for the modulation of gene expression and transport activity by temperature and medium osmolality are under intensive investigation with emphasis on the role of the membrane as an antenna for osmo- and/or thermosensors.

Folia Morphol (Warsz), 2002, 61(1), 31 - 5
Effect of unilateral, intraovarian infusions of bacteria on ovarian morphology in gilts; Jana B et al.; The aim of this study was to investigate whether unilateral, intraovarian infusions of bacteria might have induced morphological changes in the contralateral ovary . Eleven sexually matured gilts with controlled estrous cycle were used . The animals were randomly divided into two groups: I (Gr . I, treated; n = 4), and II (Gr . II, control; n = 7) . In Gr . I, 1 ml of bacterial suspension (10(3) colony forming units/ml of saline of Escherichia coli, Staphylococcus aureus and Corynebacterium pyogenes, in proportion 1:1:1) was infused into the hilus of one ovary from the 15th to the 19th day of the estrous cycle . At the same time, 1 ml of saline was infused into the hilus of the contralateral ovary and into both ovaries of the control gilts . On the 7th day of the next cycle, the ovaries were dissected out . There were no significant differences in the number of follicles and corpora lutea (CL) as well as in weight and size between the bacteria-infused, contralateral and control ovaries . The microscopic observations of the bacteria-infused ovaries revealed the presence of focal infiltrations of neutrophils in the softened stroma, especially around dilated blood vessels filled with erythrocytes . In the contralateral ovaries, the number of regularly distributed neutrophils in the softened stroma was greater than that found in the bacteria-treated ovaries . CL of the bacteria-infused ovaries had more numerous, dilated blood vessels than CL observed in the contralateral gonads . More neutrophils were found in CL of both ovaries in Gr . I as compared to those observed in Gr . II . In Gr . II, single neutrophils were found also in the stroma where the tip of the cannula was inserted . This study revealed that in gilts, unilateral, intraovarian administration of bacteria did not change the number of ovarian structures, the weight and size of the bacteria-infused and contralateral ovary, but induced inflammatory changes in both ovaries.

Kansenshogaku Zasshi, 2002 Feb, 76(2), 89 - 95
{Investigate of nasopharyngeal flora in highly aged patients}; Uno Y; To clarify the bacteriological interpretation of flora in the nasopharynx of highly aged patients (n = 107), healthy nasopharyngeal swabs were obtained from subjects of advanced age . Chief pathogenic bacteria isolated from highly aged persons were coagulase negative Staphylococcus (43 strains), Corynebacterium spp . (14 strains), methicillin-susceptible Staphylococcus aureus (beta-lactamase production and non-production) (16 strains), methicillin-resistant Staphylococcus aureus (beta-lactamase production) (6 strains) . Chief nonpathogenic bacteria isolated from highly aged persons were alpha-streptococcus (14 strains), Neisseria sp (3 strains) . Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis which are the chief bacteria isolated from children, demonstrated only 3 strains for each bacteria . In cases showing detection of multiple detected bacteria, common combinations were non-pathogenic bacteria and weakly pathogenic bacteria and enteric bacteria and pathogenic bacteria . The differences between nasopharyngeal flora of children and highly aged persons are suspected to be due to the differences in immunological and anatomical factors . We should actively examine these factors in highly aged subjects.

J Protein Chem, 2002 Jan, 21(1), 59 - 64
Cofactors in sarcosine oxidase from Corynebacterium sp . U-96; Mukouyama EB et al.; Sarcosine oxidase from Corynebacterium sp . U-96 is a heterotetrameric enzyme that was reported to contain 1 mol of covalently bound FAD and 1 mol of non-covalently-bound FAD . This work describes the result of reinvestigation of the cofactors in this enzyme . The enzyme was found to contain 1 mol of non-covalently-bound NAD+, 1 mol of non-covalently-bound FAD, and 1 mol of covalent FMN . The covalent FMN was identified by the mass and amino acid sequence analyses of the flavin peptide.

Res Microbiol, 2002 Mar, 153(2), 99 - 106
Study of Corynebacterium diphtheriae strains isolated in Romania, northwestern Russia and the Republic of Moldova; Damian M et al.; A selection of 167 Corynebacterium diphtheriae strains isolated in Romania, the Russian Federation and the Republic of Moldova were analysed by biotyping, phage typing, the toxin production test and by molecular techniques such as ribotyping, pulsed field gel electrophoresis and random amplified polymorphic DNA, in order to establish the epidemiological relatedness, genetic divergence and strain circulation within and between the bordering countries . Using a set of five digoxigenin-labeled oligonucleotides and BstEII digestion, 34 ribotypes were identified . The strains isolated in the epidemic areas (Russia and Moldova) were very closely related but different from those isolated in Romania . C1 and C5 were the main ribotypes identified in these areas . Neither ribotype was found in Romania, where the main circulating types were C3 and C7 . Field inversion gel electrophoresis was more discriminative than ribotyping and revealed 54 macrorestriction profiles after SfiI restriction . Both methods showed a significant homogeneity of the strains from epidemic areas and a large diversity among the Romanian strains . Random amplification was useful as an identification method for the epidemic strains, but not for the Romanian ones which displayed a large number of amplification profiles . The phenotypic methods associated with molecular typing techniques enabled distinguishing between strains, detecting the epidemic clone, and sustaining the absence of transmission across borders.

Actas Urol Esp, 2002 Jan, 26(1), 53 - 6
{Encrusted pyelitis . Lithiasic disease with infectious etiology}; Moreno Arcas P et al.; We report on two new cases of encrusted pielitis, a lithiasic disease of infectious ethiology--Corynebacterium of D group- . The clinic diagnostic is difficult and this disease develops in immunosuppressed patients, mainly in renal transplanted ones . One of our two cases is diagnosed in a patient with a transplanted kidney and the other one develops the disease within her native kidneys . We remark on the clinic features and therapeutic options.

Trop Anim Health Prod, 2002 Feb, 34(1), 19 - 25
Prevalence and aetiology of mastitis in cows from two major Ethiopian dairies; Workineh S et al.; The study was undertaken to determine the aetiology and prevalence of mastitis in hand-milked cows (n = 186) in two major Ethiopian dairies . The California Mastitis Test and culturing for bacteria revealed that 21.5% of the cows were clinically infected and 38.2% had subclinical mastitis . Most mastitis pathogens isolated from milk samples testing positive by the California Mastitis Test were Gram-positive cocci . Staphylococci constituted 57% of the isolates, of which the predominant cause of bovine mastitis was Staphylococcus aureus (40.5%) . Other mastitis pathogens isolated include streptococci (16.5%), coliforms (9%) and corynebacteria (5%) . Retrospective analysis of farm records indicated that mastitis was the second most important cause of culling and accounted for 27% of the cows removed from these two dairies.

Mol Microbiol, 2001 Dec, 42(5), 1281 - 95
Sensing nitrogen limitation in Corynebacterium glutamicum: the role of glnK and glnD; Nolden L et al.; A novel nitrogen control system regulating the transcription of genes expressed in response to nitrogen starvation in Corynebacterium glutamicum was identified by us recently . In this communication, we also show that the nitrogen regulation cascade in C . glutamicum functions by a new mechanism, although components highly similar to sensor and signal transmitter proteins of Escherichia coli are used, namely uridylyltransferase and a PII-type GlnK protein . The genes encoding these key components of the nitrogen regulation cascade, glnD and glnK, are organized in an operon together with amtB, which codes for an ammonium permease . Using a combination of site-directed mutagenesis, RNA hybridization experiments, reporter gene assays, transport measurements and non-denaturing gel electrophoresis followed by immunodetection, we showed that GlnK is essential for nitrogen control and that signal transduction is transmitted by uridylylation of this protein . As a consequence of the latter, a glnD deletion strain lacking uridylyltransferase is impaired in its response to nitrogen shortage . The glnD mutant revealed a decreased growth rate in the presence of limiting amounts of ammonium or urea; additionally, changes in its protein profile were observed, as shown by in vivo labelling and two-dimensional PAGE . In contrast to E . coli, expression of glnD is upregulated upon nitrogen limitation in C . glutamicum . This indicates that the glnD gene product is probably not the primary sensor of nitrogen status in C . glutamicum as shown for enterobacteria . In accordance with this hypothesis, we found a deregulated nitrogen control as a result of the overexpression of glnD . Furthermore, quantification of cytoplasmic amino acid pools excluded the possibility that a fall in glutamine concentration is perceived as the signal for nitrogen starvation by C . glutamicum, as is found in enterobacteria . Direct measurements of the intracellular ammonium pool indicated that the concentration of this compound might indicate the cellular nitrogen status . Deduced from glnK and glnD expression patterns and the genetic organization of these genes, this regulatory mechanism is also present in Corynebacterium diphtheriae, the causative agent of diphtheria.

J Immunol, 2002 Mar 15, 168(6), 2590 - 4
Cutting edge: identification of c-Rel-dependent and -independent pathways of IL-12 production during infectious and inflammatory stimuli; Mason N et al.; The production of IL-12 is required for immunity to many intracellular pathogens . Recent studies have shown that c-Rel, a member of the NF-kappaB family of transcription factors, is essential for LPS-induced IL-12p40 production by macrophages . In this study, we demonstrate that c-Rel is also required for IL-12p40 production by macrophages in response to Corynebacterium parvum, CpG oligodeoxynucleotides, anti-CD40 and low molecular weight hyaluronic acid . However, c-Rel(-/-) mice infected with Toxoplasma gondii produce comparable amounts of IL-12p40 to infected wild-type mice and have an IL-12-dependent mechanism of resistance to this infection . Furthermore, c-Rel was not required for IL-12p40 production by macrophages or dendritic cells in response to soluble Toxoplasma Ag, and neutrophils from c-Rel(-/-) mice contain normal amounts of preformed IL-12p40 . Together these studies reveal the presence of c-Rel-dependent pathways critical for IL-12p40 production in response to inflammatory stimuli and demonstrate a novel c-Rel-independent pathway of IL-12p40 production during toxoplasmosis.

J Biotechnol, 2002 Apr 25, 95(1), 25 - 38
Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: use of a cosmid and a bacterial artificial chromosome library; Tauch A et al.; The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library . High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization . Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled . Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C . glutamicum genome were represented by the cosmid library . To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C . glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping . The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C . glutamicum genome (3.28 Mb) . Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C . glutamicum . The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb . A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C . glutamicum.

Appl Microbiol Biotechnol, 2002 Feb, 58(2), 217 - 23
A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant; Ohnishi J et al.; Classical whole-cell mutagenesis has achieved great success in development of many industrial fermentation strains, but has the serious disadvantage of accumulation of uncharacterized secondary mutations that are detrimental to their performance . In the post-genomic era, a novel methodology which avoids this drawback presents itself . This "genome-based strain reconstruction" involves identifying mutations by comparative genomic analysis, defining mutations beneficial for production, and assembling them in a single wild-type background . Described herein is an initial challenge involving reconstruction of classically derived L-lysine-producing Corynebacterium glutamicum . Comparative genomic analysis for the relevant terminal pathways, the efflux step, and the anaplerotic reactions between the wild-type and production strains identified a Val-59-->Ala mutation in the homoserine dehydrogenase gene (hom), a Thr-311-->Ile mutation in the aspartokinase gene (lysC), and a Pro-458-->Ser mutation in the pyruvate carboxylase gene (pyc) . Introduction of the hom and lysC mutations into the wild-type strain by allelic replacement resulted in accumulation of 8 g and 55 g of L-lysine/l, respectively, indicating that both these specific mutations are relevant to production . The two mutations were then reconstituted in the wild-type genome, which led to a synergistic effect on production (75 g/l) . Further introduction of the pyc mutation resulted in an additional contribution and accumulation of 80 g/l after only 27 h . This high-speed fermentation achieved the highest productivity (3.0 g l(-1) h(-1)) so far reported for microbes producing L-lysine in fed-batch fermentation.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Sep-Oct, (5), 74 - 6
{Effect of lysogenic conversion of Corynebacterium diphtheriae strain on its iron assimilation and spectrum of cellular proteins}; Gavrilova NA et al.; Growth of the isogenic pair of C . diphtheriae strains, the nontoxigenic strain C7 (-) and the toxigenic strain C7 (beta), was studied under conditions of limited availability of the iron source . The growth of the toxigenic strain was shown to depend on the concentration of iron in the medium to a lesser degree than the nontoxigenic one . Lysogenic conversion results in the synthesis of additional iron-dependent proteins, absent in C . diphtheriae initial nontoxigenic strain C7 (-) . Special attention was paid to proteins with a mol . wt . 66 kD, synthesized by the toxigenic strain irrespective of the concentration of iron in the medium, while in the toxigenic strain these proteins were detected only under conditions of iron deficiency.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Sep-Oct, (5), 71 - 2
{Effect of medicinal plant extracts on the growth of microorganisms}; Baronets NG et al.; Extracts obtained from sweatweed and licorice roots, flax seeds, milfoil, bur-marigold, plantain, coltsfoot, nettle, Indian corn stigmas, laminaria produced a stimulating effect on the growth of Candida albicans test strain and Streptococcus pyogenes test strain Dick 1 . Sweatweed, licorice, Aerva lanata and violet extracts influenced the growth of Corynebacterium xerosis 1911, while sweatweed, violet, horse-tail, bur-marigold, camomile, plantain, and nettle extracts influenced the growth of shigellae . The stimulating effect could be supposedly produced by biologically active substances contained in medicinal plants (organic acids, alkaloids, carotinoids, vitamins, microelements) . Further studies aimed at the identification of substances producing the stimulating effect are planned.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Sep-Oct, (5), 12 - 5
{The microflora of ulzerous zone mucosa in patients with duodenal ulcer}; Chervinets VM et al.; Bacteriological study of the biopsies taken from gastric and duodenal mucosa of 10 healthy volunteers and 74 patients with duodenal ulcer, was carried out . In the gastroduodenal zone of healthy subjects microorganisms of 6 genera (Streptococcus, Candida, Staphylococcus, Bacillus, Helicobacter and Lactobacillus) were detected . H . pylori was isolated in 20% of cases only in biopsy specimens taken from the antral section of the stomach of healthy as monoculture or in combination with C . albicans . In patients with duodenal ulcer activation of opportunistic microflora was observed in the periulcerous zone . More often H . pylori occurred in associations with fungi of the genus Candida, streptococci, staphylococci, enterobacteria, Pseudomonas and other microorganisms (of more than 30 genera) . Quantitatively the dominating microorganisms (3.8-5.7 lg CFU/g) were H . pylori, fungi of the genus Candida, bacteria of the genera Streptococcus, Peptostreptococcus, Bacteroides, Gemella, Prevotella, Veillonella, Peptococcus, Bacillus, different species of opportunistic enterobacteria, as well as bacteria of the genera Staphylococcus, Micrococcus, Corynebacterium, Neisseria, Pseudomonas, etc . Opportunistic bacteria detected in the ulcerous zone, as a rule, expressed hemolytic, lecithinase, RNAase, caseinolytic, catalase and urease activity . Sonicated filtrates of such cultures produced a cytotoxic effect on cells HEp-2 . Ulcer is an infected wound that needs sanitation.

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