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