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Sample records for recombinant corynebacterium glutamicum

  1. Transcriptome analysis of Corynebacterium glutamicum in the process of recombinant protein expression in bioreactors

    PubMed Central

    Sun, Yang; Guo, Wenwen; Wang, Fen; Zhan, Chunjun; Yang, Yankun; Liu, Xiuxia; Bai, Zhonghu

    2017-01-01

    Corynebacterium glutamicum (C. glutamicum) is a favorable host cell for the production of recombinant proteins, such as important enzymes and pharmaceutical proteins, due to its excellent potential advantages. Herein, we sought to systematically explore the influence of recombinant protein expression on the transcription and metabolism of C. glutamicum. Two C. glutamicum strains, the wild-type strain and an engineered strain expressing enhanced green fluorescent protein (EGFP), were cultured in parallel in 5-L bioreactors to study the change in metabolism in the process of EGFP expression. The results revealed that EGFP expression had great effects on the growth and metabolism of C. glutamicum and contributed to metabolism-like anaerobic conditions as follows: glycolysis was enhanced, the TCA cycle was shunted, and Glu, Val, Met, lactate and acetate were accumulated to produce sufficient ATP for EGFP production and transfer. Many differentially expressed genes related to ribosomal protein, transcriptional regulators, and energy metabolism were found to be expressed in the presence of EGFP, laying the foundation for identifying genomic loci to change the flow of the host cell metabolism to improve the ability of expressing foreign proteins in C. glutamicum. PMID:28369109

  2. The impact of PHB accumulation on L-glutamate production by recombinant Corynebacterium glutamicum.

    PubMed

    Liu, Qian; Ouyang, Shao-Ping; Kim, Jonghyok; Chen, Guo-Qiang

    2007-11-01

    Corynebacterium glutamicum, a gram-positive soil bacterium, has been used extensively for the industrial production of l-glutamate and other amino acids. In this study, an Escherichia coli-C. glutamicum shuttle expression plasmid harboring polyhydroxybutyrate (PHB) synthesis genes, phbCAB from Ralstonia eutropha, was constructed under the Ptrc promoter. C. glutamicum harboring this plasmid accumulated 3-13% PHB with a weight average molecular mass of 125,400 and a polydispersity of 11.3 when grown on glucose. PHB synthesis related enzyme activities including beta-ketothiolase (PhbA), acetoacetyl-CoA reductase (PhbB) and PHB synthase (PhbC) were found to be constitutively produced independent of IPTG. l-Glutamate production increased 39-68% in two C. glutamicum strains harboring PHB synthesis genes compared with their parent strains in shake flask experiments. In fermentor studies, the recombinant produced approximately 23% more l-glutamate compared with that of the wild type, and yielded less intermediate metabolites or by-products including alpha-ketoglutarate, l-glutamine and lactate. These results suggested that the expression of phbCAB genes in C. glutamicum could help regulate glutamate production metabolism. This demonstrated that the expression of PHB synthesis genes has a positive effect on l-glutamate production in C. glutamicum.

  3. Biosynthesis of trans-4-hydroxyproline by recombinant strains of Corynebacterium glutamicum and Escherichia coli

    PubMed Central

    2014-01-01

    Background Trans-4-hydroxy-L-proline (trans-Hyp), one of the hydroxyproline (Hyp) isomers, is a useful chiral building block in the production of many pharmaceuticals. Although there are some natural biosynthetic pathways of trans-Hyp existing in microorganisms, the yield is still too low to be scaled up for industrial applications. Until now the production of trans-Hyp is mainly from the acid hydrolysis of collagen. Due to the increasing environmental concerns on those severe chemical processes and complicated downstream separation, it is essential to explore some environment-friendly processes such as constructing new recombinant strains to develop efficient process for trans-Hyp production. Result In this study, the genes of trans-proline 4-hydroxylase (trans-P4H) from diverse resources were cloned and expressed in Corynebacterium glutamicum and Escherichia coli, respectively. The trans-Hyp production by these recombinant strains was investigated. The results showed that all the genes from different resources had been expressed actively. Both the recombinant C. glutamicum and E. coli strains could produce trans-Hyp in the absence of proline and 2-oxoglutarate. Conclusions The whole cell microbial systems for trans-Hyp production have been successfully constructed by introducing trans-P4H into C. glutamicum and E. coli. Although the highest yield was obtained in recombinant E. coli, using recombinant C. glutamicum strains to produce trans-Hyp was a new attempt. PMID:24885047

  4. High-titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum.

    PubMed

    Cheng, Fangyu; Gong, Qianying; Yu, Huimin; Stephanopoulos, Gregory

    2016-03-01

    Hyaluronic acid (HA) plays important roles in human tissue system, thus it is highly desirable for various applications, such as in medical, clinic and cosmetic fields. The wild microbial producer of HA, streptococcus, was restricted by its potential pathogens, hence different recombinant hosts are being explored. In this work, we engineered Corynebacterium glutamicum, a GRAS (Generally Recognized as Safe) organism free of exotoxins and endotoxins to produce HA with high titer and satisfied Mw . The ssehasA gene encoding hyaluronan synthase (HasA) was artificially synthesized with codon preference of C. glutamicum. Other genes involved in the HA synthetic pathway were directly cloned from the C. glutamicum genome. The operon structures and constitutive or inducible promoters were particularly compared and the preferred environmental conditions were also optimized. Using glucose and corn syrup powder as carbon and nitrogen sources, batch cultures of the engineered C.glutamicum with operon ssehasA-hasB driven by Ptac promoter were performed in a 5 L fermentor. The maximal HA titer, productivity and yield reached 8.3 g/L, 0.24 g/L/h and 0.22 gHA/gGlucose, respectively; meanwhile the maximal Mw was 1.30 MDa. This work provides a safe and efficient novel producer of HA with huge industrial prospects.

  5. A New Strategy for Production of 5-Aminolevulinic Acid in Recombinant Corynebacterium glutamicum with High Yield

    PubMed Central

    Yang, Peng; Liu, Wenjing; Cheng, Xuelian; Wang, Jing; Qi, Qingsheng

    2016-01-01

    ABSTRACT 5-Aminolevulinic acid (ALA), a nonprotein amino acid involved in tetrapyrrole synthesis, has been widely applied in agriculture, medicine, and food production. Many engineered metabolic pathways have been constructed; however, the production yields are still low. In this study, several 5-aminolevulinic acid synthases (ALASs) from different sources were evaluated and compared with respect to their ALA production capacities in an engineered Corynebacterium glutamicum CgS1 strain that can accumulate succinyl-coenzyme A (CoA). A codon-optimized ALAS from Rhodobacter capsulatus SB1003 displayed the best potential. Recombinant strain CgS1/pEC-SB produced 7.6 g/liter ALA using a mineral salt medium in a fed-batch fermentation mode. Employing two-stage fermentation, 12.46 g/liter ALA was produced within 17 h, with a productivity of 0.73 g/liter/h, in recombinant C. glutamicum. Through overexpression of the heterologous nonspecific ALA exporter RhtA from Escherichia coli, the titer was further increased to 14.7 g/liter. This indicated that strain CgS1/pEC-SB-rhtA holds attractive industrial application potential for the future. IMPORTANCE In this study, a two-stage fermentation strategy was used for production of the value-added nonprotein amino acid 5-aminolevulinic acid from glucose and glycine in a generally recognized as safe (GRAS) host, Corynebacterium glutamicum. The ALA titer represented the highest in the literature, to our knowledge. This high production capacity, combined with the potential easy downstream processes, made the recombinant strain an attractive candidate for industrial use in the future. PMID:26921424

  6. Productivity of cyclohexanone oxidation of the recombinant Corynebacterium glutamicum expressing chnB of Acinetobacter calcoaceticus.

    PubMed

    Doo, Eun-Hee; Lee, Won-Heong; Seo, Hyo-Seel; Seo, Jin-Ho; Park, Jin-Byung

    2009-06-15

    The biocatalytic efficiency of recombinant Corynebacterium glutamicum expressing the chnB gene encoding cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871 was investigated. Optimization of an expression system and induction conditions enabled the recombinant biocatalyst to produce CHMO to a specific activity of ca. 0.5 U mg(-1) protein. Tight control of feeding of an energy source (i.e., glucose) and dissolved oxygen tension during fed-batch culture-based biotransformation allowed the cells to produce epsilon-caprolactone to a concentration of 16.0 g l(-1). The specific and volumetric productivity for cyclohexanone oxidation were 0.12 g g drycells(-1)h(-1) (17.5 U g(-1) of dry cells) and 2.3 g l(-1)h(-1) (330 U l(-1)), respectively. These values correspond to over 5.4- and 2.7-fold of recombinant Escherichia coli expressing the same gene under similar reaction conditions. It could be concluded that the recombinant C. glutamicum is a promising biocatalyst for Baeyer-Villiger oxidations.

  7. Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum.

    PubMed

    Yim, Sung Sun; Choi, Jae Woong; Lee, Roo Jin; Lee, Yong Jae; Lee, Se Hwa; Kim, So Young; Jeong, Ki Jun

    2016-01-01

    Corynebacterium glutamicum, which has been for long an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large-scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α-amylase, and camelid antibody fragment (VHH). For large-scale production, fed-batch cultivations were also conducted and high yields were successfully achieved--as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α-amylase), and 1.57 g/L (VHH)--in the extracellular medium. From the culture media, all model proteins could be simply purified by one-step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum.

  8. 3-Amino-4-hydroxybenzoic acid production from sweet sorghum juice by recombinant Corynebacterium glutamicum.

    PubMed

    Kawaguchi, Hideo; Sasaki, Kengo; Uematsu, Kouji; Tsuge, Yota; Teramura, Hiroshi; Okai, Naoko; Nakamura-Tsuruta, Sachiko; Katsuyama, Yohei; Sugai, Yoshinori; Ohnishi, Yasuo; Hirano, Ko; Sazuka, Takashi; Ogino, Chiaki; Kondo, Akihiko

    2015-12-01

    The production of the bioplastic precursor 3-amino-4-hydroxybenzoic acid (3,4-AHBA) from sweet sorghum juice, which contains amino acids and the fermentable sugars sucrose, glucose and fructose, was assessed to address the limitations of producing bio-based chemicals from renewable feedstocks. Recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI derived from Streptomyces griseus produced 3,4-AHBA from the sweet sorghum juice of cultivar SIL-05 at a final concentration (1.0 g l(-1)) that was 5-fold higher than that from pure sucrose. Fractionation of sweet sorghum juice by nanofiltration (NF) membrane separation (molecular weight cut-off 150) revealed that the NF-concentrated fraction, which contained the highest concentrations of amino acids, increased 3,4-AHBA production, whereas the NF-filtrated fraction inhibited 3,4-AHBA biosynthesis. Amino acid supplementation experiments revealed that leucine specifically enhanced 3,4-AHBA production by strain KT01. Taken together, these results suggest that sweet sorghum juice is a potentially suitable feedstock for 3,4-AHBA production by recombinant C. glutamicum.

  9. Production of L-ornithine from sucrose and molasses by recombinant Corynebacterium glutamicum.

    PubMed

    Zhang, Yuan-Yuan; Bu, Yi-Fan; Liu, Jian-Zhong

    2015-09-01

    Sucrose and molasses are attractive raw materials for industrial fermentation. Although Corynebacterium glutamicum shows sucrose-utilizing activity, sucrose or molasses is only a fraction of carbon source used in the fermentation medium in most works. An engineered C. glutamicum strain was constructed for producing L-ornithine with sucrose or molasses as a sole carbon source by transferring Mannheimia succiniciproducens β-fructofuranosidase gene (sacC). The engineered strain, C. glutamicum ΔAPE6937R42 (pEC-sacC), produced 22.0 g/L of L-ornithine with sucrose as the sole carbon source, which is on par with that obtained by the parent strain C. glutamicum ΔAPE6937R42 with glucose as the sole carbon. The resulting strain C. glutamicum ΔAPE6937R42 (pEC-sacC) produced 27.0 g/L of L-ornithine with molasses as the sole carbon source, which is higher than that obtained by the parent strain C. glutamicum ΔAPE6937R42 with glucose as the sole carbon. This strategy can be applied for developing sucrose- or molasses-utilizing industrial strains.

  10. Ethambutol-mediated cell wall modification in recombinant Corynebacterium glutamicum increases the biotransformation rates of cyclohexanone derivatives.

    PubMed

    Yun, Ji-Yeong; Lee, Jung-Eun; Yang, Kyung-Mi; Cho, Suekyung; Kim, Arim; Kwon, Yong-Uk; Kwon, Yong-Euk; Park, Jin-Byung

    2012-01-01

    The effects of structural modification of cell wall on the biotransformation capability by recombinant Corynebacterium glutamicum cells, expressing the chnB gene encoding cyclohexanone monooxygenase of Acinetobacter calcoaceticus NCIMB 9871, were investigated. Baeyer-Villiger oxygenation of 2-(2'-acetoxyethyl) cyclohexanone (MW 170 Da) into R-7-(2'-acetoxyethyl)-2-oxepanone was used as a model reaction. The whole-cell biotransformation followed Michaelis-Menten kinetics. The V (max) and K (S) values were estimated as 96.8 U g(-1) of dry cells and 0.98 mM, respectively. The V (max) was comparable with that of cyclohexanone oxygenation, whereas the K (S) was almost eightfold higher. The K (S) value of 2-(2'-acetoxyethyl) cyclohexanone oxygenation was reduced by ca. 30% via altering the cell envelop structure of C. glutamicum with ethambutol, which inhibits arabinosyl transferases involved in the biosynthesis of cell wall arabinogalactan and mycolate layers. The higher whole-cell biotransformation rate was also observed in the oxygenation of ethyl 2-cyclohexanone acetate upon ethambutol treatment of the recombinant C. glutamicum. Therefore, it was assumed that the biotransformation efficiency of C. glutamicum-based biocatalysts, with respect to medium- to large-sized lipophilic organic substrates (MW > ca. 170), can be enhanced by engineering their cell wall outer layers, which are known to function as a formidable barrier to lipophilic molecules.

  11. Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing Corynebacterium glutamicum.

    PubMed

    Gopinath, Vipin; Meiswinkel, Tobias M; Wendisch, Volker F; Nampoothiri, K Madhavan

    2011-12-01

    Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the L-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more L-glutamate and L-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM L-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM L-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.

  12. Putrescine production by engineered Corynebacterium glutamicum.

    PubMed

    Schneider, Jens; Wendisch, Volker F

    2010-10-01

    Here, we report the engineering of the industrially relevant Corynebacterium glutamicum for putrescine production. C. glutamicum grew well in the presence of up to 500 mM of putrescine. A reduction of the growth rate by 34% and of biomass formation by 39% was observed at 750 mM of putrescine. C. glutamicum was enabled to produce putrescine by heterologous expression of genes encoding enzymes of the arginine- and ornithine decarboxylase pathways from Escherichia coli. The results showed that the putrescine yield by recombinant C. glutamicum strains provided with the arginine-decarboxylase pathway was 40 times lower than the yield by strains provided with the ornithine decarboxylase pathway. The highest production efficiency was reached by overexpression of speC, encoding the ornithine decarboxylase from E. coli, in combination with chromosomal deletion of genes encoding the arginine repressor ArgR and the ornithine carbamoyltransferase ArgF. In shake-flask batch cultures this strain produced putrescine up to 6 g/L with a space time yield of 0.1 g/L/h. The overall product yield was about 24 mol% (0.12 g/g of glucose).

  13. Enzymatic synthesis of S-adenosylhomocysteine: immobilization of recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (ATCC 13032).

    PubMed

    Lozada-Ramírez, J D; Sánchez-Ferrer, A; García-Carmona, F

    2012-03-01

    Recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (CgSAHase) was covalently bound to Eupergit® C. The maximum yield of bound protein was 91% and the catalytic efficiency was 96.9%. When the kinetic results for the immobilized enzyme were compared with those for the soluble enzyme, no decrease in the catalytic efficiency of the former was detected. Both soluble and immobilized enzymes showed similar optimum pH and temperature ranges. The reuse of immobilized CgSAHase caused a loss of synthetic activity due to NAD(+) release, although the binding to the support was sufficiently strong for up to 5 cycles with 95% conversion efficiency. The immobilized enzyme was incubated every 3 cycles with 100 μM NAD(+) to recover the loss of activity after 5 cycles. This maintained the activity for another 50 cycles. The purification of S-adenosylhomocysteine (SAH) provided an overall yield of 76% and 98% purity as determined by HPLC and NMR analyses. The results indicate the suitability of immobilized CgSAHase for synthesizing SAH and other important S-nucleosidylhomocysteine.

  14. The Actinobacterium Corynebacterium glutamicum, an Industrial Workhorse.

    PubMed

    Lee, Joo-Young; Na, Yoon-Ah; Kim, Eungsoo; Lee, Heung-Shick; Kim, Pil

    2016-05-28

    Starting as a glutamate producer, Corynebacterium glutamicum has played a variety of roles in the industrial production of amino acids, one of the most important areas of white biotechnology. From shortly after its genome information became available, C. glutamicum has been applied in various production processes for value-added chemicals, fuels, and polymers, as a key organism in industrial biotechnology alongside the surprising progress in systems biology and metabolic engineering. In addition, recent studies have suggested another potential for C. glutamicum as a synthetic biology platform chassis that could move the new era of industrial microbial biotechnology beyond the classical field. Here, we review the recent progress and perspectives in relation to C. glutamicum, which demonstrate it as one of the most promising and valuable workhorses in the field of industrial biotechnology.

  15. Production of the amino acids l-glutamate, l-lysine, l-ornithine and l-arginine from arabinose by recombinant Corynebacterium glutamicum.

    PubMed

    Schneider, Jens; Niermann, Karin; Wendisch, Volker F

    2011-07-10

    Amino acid production processes with Corynebacterium glutamicum are based on media containing glucose from starch hydrolysis or fructose and sucrose as present in molasses. Simultaneous utilization of various carbon sources, including glucose, fructose and sucrose, in blends is a typical characteristic of this bacterium. The renewable non-food carbon source arabinose, which is present in hemicellulosic hydrolysates, cannot be utilized by most C. glutamicum strains. Heterologous expression of the araBAD operon from Escherichia coli in the wild-type and in an l-lysine producing strain of C. glutamicum was shown to enable production of l-glutamate and l-lysine, respectively, from arabinose as sole carbon source. l-Ornithine and l-arginine producing strains were constructed and shown to produce l-ornithine and l-arginine from arabinose when araBAD from E. coli was expressed. Moreover, the recombinant strains produced l-glutamate, l-lysine, l-ornithine and l-arginine respectively, from arabinose also when glucose-arabinose blends were used as carbon sources.

  16. Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi).

    PubMed

    Cleto, Sara; Jensen, Jaide Vk; Wendisch, Volker F; Lu, Timothy K

    2016-05-20

    Corynebacterium glutamicum is an important organism for the industrial production of amino acids. Metabolic pathways in this organism are usually engineered by conventional methods such as homologous recombination, which depends on rare double-crossover events. To facilitate the mapping of gene expression levels to metabolic outputs, we applied CRISPR interference (CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in C. glutamicum. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of pgi and pck up to 98%, and of pyk up to 97%, resulting in titer enhancement ratios of l-lysine and l-glutamate production comparable to levels achieved by gene deletion. This approach for C. glutamicum metabolic engineering, which only requires 3 days, indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection.

  17. Glycerol-3-phosphatase of Corynebacterium glutamicum.

    PubMed

    Lindner, Steffen N; Meiswinkel, Tobias M; Panhorst, Maren; Youn, Jung-Won; Wiefel, Lars; Wendisch, Volker F

    2012-06-15

    Formation of glycerol as by-product of amino acid production by Corynebacterium glutamicum has been observed under certain conditions, but the enzyme(s) involved in its synthesis from glycerol-3-phosphate were not known. It was shown here that cg1700 encodes an enzyme active as a glycerol-3-phosphatase (GPP) hydrolyzing glycerol-3-phosphate to inorganic phosphate and glycerol. GPP was found to be active as a homodimer. The enzyme preferred conditions of neutral pH and requires Mg²⁺ or Mn²⁺ for its activity. GPP dephosphorylated both L- and D-glycerol-3-phosphate with a preference for the D-enantiomer. The maximal activity of GPP was estimated to be 31.1 and 1.7 U mg⁻¹ with K(M) values of 3.8 and 2.9 mM for DL- and L-glycerol-3-phosphate, respectively. For physiological analysis a gpp deletion mutant was constructed and shown to lack the ability to produce detectable glycerol concentrations. Vice versa, gpp overexpression increased glycerol accumulation during growth in fructose minimal medium. It has been demonstrated previously that intracellular accumulation of glycerol-3-phosphate is growth inhibitory as shown for a recombinant C. glutamicum strain overproducing glycerokinase and glycerol facilitator genes from E. coli in media containing glycerol. In this strain, overexpression of gpp restored growth in the presence of glycerol as intracellular glycerol-3-phosphate concentrations were reduced to wild-type levels. In C. glutamicum wild type, GPP was shown to be involved in utilization of DL-glycerol-3-phosphate as source of phosphorus, since growth with DL-glycerol-3-phosphate as sole phosphorus source was reduced in the gpp deletion strain whereas it was accelerated upon gpp overexpression. As GPP homologues were found to be encoded in the genomes of many other bacteria, the gpp homologues of Escherichia coli (b2293) and Bacillus subtilis (BSU09240, BSU34970) as well as gpp1 from the plant Arabidosis thaliana were overexpressed in E. coli MG1655 and

  18. D-Allulose Production from D-Fructose by Permeabilized Recombinant Cells of Corynebacterium glutamicum Cells Expressing D-Allulose 3-Epimerase Flavonifractor plautii

    PubMed Central

    Park, Chul-Soon; Kim, Taeyong; Hong, Seung-Hye; Shin, Kyung-Chul; Kim, Kyoung-Rok; Oh, Deok-Kun

    2016-01-01

    A d-allulose 3-epimerase from Flavonifractor plautii was cloned and expressed in Escherichia coli and Corynebacterium glutamicum. The maximum activity of the enzyme purified from recombinant E. coli cells was observed at pH 7.0, 65°C, and 1 mM Co2+ with a half-life of 40 min at 65°C, Km of 162 mM, and kcat of 25280 1/s. For increased d-allulose production, recombinant C. glutamicum cells were permeabilized via combined treatments with 20 mg/L penicillin and 10% (v/v) toluene. Under optimized conditions, 10 g/L permeabilized cells produced 235 g/L d-allulose from 750 g/L d-fructose after 40 min, with a conversion rate of 31% (w/w) and volumetric productivity of 353 g/L/h, which were 1.4- and 2.1-fold higher than those obtained for nonpermeabilized cells, respectively. PMID:27467527

  19. Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum

    PubMed Central

    2012-01-01

    Background Corynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin. Results Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 ɛ-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum ΔcrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum ΔcrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 ± 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum ΔcrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 ± 0.3 mg/g CDW were obtained. Conclusion C. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions

  20. Synthetic promoter libraries for Corynebacterium glutamicum.

    PubMed

    Rytter, Jakob Vang; Helmark, Søren; Chen, Jun; Lezyk, Mateusz Jakub; Solem, Christian; Jensen, Peter Ruhdal

    2014-03-01

    The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in β-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7-59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms.

  1. Functional Characterization of Corynebacterium glutamicum Mycothiol S-Conjugate Amidase

    PubMed Central

    Si, Meiru; Long, Mingxiu; Chaudhry, Muhammad Tausif; Xu, Yixiang; Zhang, Pan; Zhang, Lei; Shen, Xihui

    2014-01-01

    The present study focuses on the genetic and biochemical characterization of mycothiol S-conjugate amidase (Mca) of Corynebacterium glutamicum. Recombinant C. glutamicum Mca was heterologously expressed in Escherichia coli and purified to apparent homogeneity. The molecular weight of native Mca protein determined by gel filtration chromatography was 35 kDa, indicating that Mca exists as monomers in the purification condition. Mca showed amidase activity with mycothiol S-conjugate of monobromobimane (MSmB) in vivo while mca mutant lost the ability to cleave MSmB. In addition, Mca showed limited deacetylase activity with N-acetyl-D-glucosamine (GlcNAc) as substrate. Optimum pH for amidase activity was between 7.5 and 8.5, while the highest activity in the presence of Zn2+ confirmed Mca as a zinc metalloprotein. Amino acid residues conserved among Mca family members were located in C. glutamicum Mca and site-directed mutagenesis of these residues indicated that Asp14, Tyr137, His139 and Asp141 were important for activity. The mca deletion mutant showed decreased resistance to antibiotics, alkylating agents, oxidants and heavy metals, and these sensitive phenotypes were recovered in the complementary strain to a great extent. The physiological roles of Mca in resistance to various toxins were further supported by the induced expression of Mca in C. glutamicum under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. PMID:25514023

  2. 3-Methyl-1-butanol Biosynthesis in an Engineered Corynebacterium glutamicum.

    PubMed

    Xiao, Shiyuan; Xu, Jingliang; Chen, Xiaoyan; Li, Xiekun; Zhang, Yu; Yuan, Zhenhong

    2016-05-01

    Biofuel offers a promising solution to the adverse environmental problems and depletion in reserves of fossil fuels. Higher alcohols including 3-methyl-1-butanol were paid much more attention as fuel substitute in recent years, due to its similar properties to gasoline. In the present work, 3-methyl-1-butanol production in engineered Corynebacterium glutamicum was studied. α-Ketoisovalerate decarboxylase gene (kivd) from Lactococcus lactis combined with alcohol dehydrogenase gene (adh2, adhA, and adh3) from three organisms were overexpressed in C. glutamicum. Enzymatic assay and alcohol production results showed that adh3 from Zymomonas mobilis was the optimum candidate for 3-methyl-1-butanol production in C. glutamicum. The recombinant with kivd and adh3 could produce 0.182 g/L of 3-methyl-1-butanol and 0.144 g/L of isobutanol after 12 h of incubation. Further inactivation of the E1 subunit of pyruvate dehydrogenase complex gene (aceE) and lactic dehydrogenase gene (ldh) in the above C. glutamicum strain would improve the 3-Methyl-1-butanol titer to 0.497 g/L after 12 h of incubation.

  3. Corynebacterium glutamicum promoters: a practical approach

    PubMed Central

    Pátek, Miroslav; Holátko, Jiří; Busche, Tobias; Kalinowski, Jörn; Nešvera, Jan

    2013-01-01

    Summary Transcription initiation is the key step in gene expression in bacteria, and it is therefore studied for both theoretical and practical reasons. Promoters, the traffic lights of transcription initiation, are used as construction elements in biotechnological efforts to coordinate ‘green waves’ in the metabolic pathways leading to the desired metabolites. Detailed analyses of Corynebacterium glutamicum promoters have already provided large amounts of data on their structures, regulatory mechanisms and practical capabilities in metabolic engineering. In this minireview the main aspects of promoter studies, the methods developed for their analysis and their practical use in C. glutamicum are discussed. These include definitions of the consensus sequences of the distinct promoter classes, promoter localization and characterization, activity measurements, the functions of transcriptional regulators and examples of practical uses of constitutive, inducible and modified promoters in biotechnology. The implications of the introduction of novel techniques, such as in vitro transcription and RNA sequencing, to C. glutamicum promoter studies are outlined. PMID:23305350

  4. Enhancement of γ-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis.

    PubMed

    Shi, Feng; Jiang, Junjun; Li, Yongfu; Li, Youxin; Xie, Yilong

    2013-11-01

    γ-Aminobutyric acid (GABA), a non-protein amino acid, is a bioactive component in the food, feed and pharmaceutical fields. To establish an effective single-step production system for GABA, a recombinant Corynebacterium glutamicum strain co-expressing two glutamate decarboxylase (GAD) genes (gadB1 and gadB2) derived from Lactobacillus brevis Lb85 was constructed. Compared with the GABA production of the gadB1 or gadB2 single-expressing strains, GABA production by the gadB1-gadB2 co-expressing strain increased more than twofold. By optimising urea supplementation, the total production of L-glutamate and GABA increased from 22.57 ± 1.24 to 30.18 ± 1.33 g L⁻¹, and GABA production increased from 4.02 ± 0.95 to 18.66 ± 2.11 g L⁻¹ after 84-h cultivation. Under optimal urea supplementation, L-glutamate continued to be consumed, GABA continued to accumulate after 36 h of fermentation, and the pH level fluctuated. GABA production increased to a maximum level of 27.13 ± 0.54 g L⁻¹ after 120-h flask cultivation and 26.32 g L⁻¹ after 60-h fed-batch fermentation. The conversion ratio of L-glutamate to GABA reached 0.60-0.74 mol mol⁻¹. By co-expressing gadB1 and gadB2 and optimising the urea addition method, C. glutamicum was genetically improved for de novo biosynthesis of GABA from its own accumulated L-glutamate.

  5. Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

    NASA Astrophysics Data System (ADS)

    Wittmann, Christoph

    The Gram-positive soil bacterium Corynebacterium glutamicum was discovered as a natural overproducer of glutamate about 50 years ago. Linked to the steadily increasing economical importance of this microorganism for production of glutamate and other amino acids, the quest for efficient production strains has been an intense area of research during the past few decades. Efficient production strains were created by applying classical mutagenesis and selection and especially metabolic engineering strategies with the advent of recombinant DNA technology. Hereby experimental and computational approaches have provided fascinating insights into the metabolism of this microorganism and directed strain engineering. Today, C. glutamicum is applied to the industrial production of more than 2 million tons of amino acids per year. The huge achievements in recent years, including the sequencing of the complete genome and efficient post genomic approaches, now provide the basis for a new, fascinating era of research - analysis of metabolic and regulatory properties of C. glutamicum on a global scale towards novel and superior bioprocesses.

  6. Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi)

    PubMed Central

    2016-01-01

    Corynebacterium glutamicum is an important organism for the industrial production of amino acids. Metabolic pathways in this organism are usually engineered by conventional methods such as homologous recombination, which depends on rare double-crossover events. To facilitate the mapping of gene expression levels to metabolic outputs, we applied CRISPR interference (CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in C. glutamicum. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of pgi and pck up to 98%, and of pyk up to 97%, resulting in titer enhancement ratios of l-lysine and l-glutamate production comparable to levels achieved by gene deletion. This approach for C. glutamicum metabolic engineering, which only requires 3 days, indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection. PMID:26829286

  7. Silencing of cryptic prophages in Corynebacterium glutamicum

    PubMed Central

    Pfeifer, Eugen; Hünnefeld, Max; Popa, Ovidiu; Polen, Tino; Kohlheyer, Dietrich; Baumgart, Meike; Frunzke, Julia

    2016-01-01

    DNA of viral origin represents a ubiquitous element of bacterial genomes. Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity. In this study, we describe the nucleoid-associated protein CgpS, which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum. CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3. Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq) revealed its association with AT-rich DNA elements, including the entire CGP3 prophage region (187 kbp), as well as several other elements acquired by horizontal gene transfer. Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage. In contrast, a strain lacking the CGP3 prophage was not affected and displayed stable growth. In a bioinformatics approach, cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes. Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure, supporting an ancient function of these xenogeneic silencers in phage-host interaction. PMID:27492287

  8. Silencing of cryptic prophages in Corynebacterium glutamicum.

    PubMed

    Pfeifer, Eugen; Hünnefeld, Max; Popa, Ovidiu; Polen, Tino; Kohlheyer, Dietrich; Baumgart, Meike; Frunzke, Julia

    2016-12-01

    DNA of viral origin represents a ubiquitous element of bacterial genomes. Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity. In this study, we describe the nucleoid-associated protein CgpS, which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3. Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq) revealed its association with AT-rich DNA elements, including the entire CGP3 prophage region (187 kbp), as well as several other elements acquired by horizontal gene transfer. Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage. In contrast, a strain lacking the CGP3 prophage was not affected and displayed stable growth. In a bioinformatics approach, cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes. Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure, supporting an ancient function of these xenogeneic silencers in phage-host interaction.

  9. Metabolic engineering of Corynebacterium glutamicum for methanol metabolism.

    PubMed

    Witthoff, Sabrina; Schmitz, Katja; Niedenführ, Sebastian; Nöh, Katharina; Noack, Stephan; Bott, Michael; Marienhagen, Jan

    2015-03-01

    Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [(13)C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum Δald ΔadhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO2, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.

  10. Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

    PubMed Central

    Witthoff, Sabrina; Schmitz, Katja; Niedenführ, Sebastian; Nöh, Katharina; Noack, Stephan

    2015-01-01

    Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum Δald ΔadhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO2, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate. PMID:25595770

  11. Production of the Marine Carotenoid Astaxanthin by Metabolically Engineered Corynebacterium glutamicum.

    PubMed

    Henke, Nadja A; Heider, Sabine A E; Peters-Wendisch, Petra; Wendisch, Volker F

    2016-06-30

    Astaxanthin, a red C40 carotenoid, is one of the most abundant marine carotenoids. It is currently used as a food and feed additive in a hundred-ton scale and is furthermore an attractive component for pharmaceutical and cosmetic applications with antioxidant activities. Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin, is an industrially relevant microorganism used in the million-ton amino acid production. In this work, engineering of a genome-reduced C. glutamicum with optimized precursor supply for astaxanthin production is described. This involved expression of heterologous genes encoding for lycopene cyclase CrtY, β-carotene ketolase CrtW, and hydroxylase CrtZ. For balanced expression of crtW and crtZ their translation initiation rates were varied in a systematic approach using different ribosome binding sites, spacing, and translational start codons. Furthermore, β-carotene ketolases and hydroxylases from different marine bacteria were tested with regard to efficient astaxanthin production in C. glutamicum. In shaking flasks, the C. glutamicum strains developed here overproduced astaxanthin with volumetric productivities up to 0.4 mg·L(-1)·h(-1) which are competitive with current algae-based production. Since C. glutamicum can grow to high cell densities of up to 100 g cell dry weight (CDW)·L(-1), the recombinant strains developed here are a starting point for astaxanthin production by C. glutamicum.

  12. Production of the Marine Carotenoid Astaxanthin by Metabolically Engineered Corynebacterium glutamicum

    PubMed Central

    Henke, Nadja A.; Heider, Sabine A. E.; Peters-Wendisch, Petra; Wendisch, Volker F.

    2016-01-01

    Astaxanthin, a red C40 carotenoid, is one of the most abundant marine carotenoids. It is currently used as a food and feed additive in a hundred-ton scale and is furthermore an attractive component for pharmaceutical and cosmetic applications with antioxidant activities. Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin, is an industrially relevant microorganism used in the million-ton amino acid production. In this work, engineering of a genome-reduced C. glutamicum with optimized precursor supply for astaxanthin production is described. This involved expression of heterologous genes encoding for lycopene cyclase CrtY, β-carotene ketolase CrtW, and hydroxylase CrtZ. For balanced expression of crtW and crtZ their translation initiation rates were varied in a systematic approach using different ribosome binding sites, spacing, and translational start codons. Furthermore, β-carotene ketolases and hydroxylases from different marine bacteria were tested with regard to efficient astaxanthin production in C. glutamicum. In shaking flasks, the C. glutamicum strains developed here overproduced astaxanthin with volumetric productivities up to 0.4 mg·L−1·h−1 which are competitive with current algae-based production. Since C. glutamicum can grow to high cell densities of up to 100 g cell dry weight (CDW)·L−1, the recombinant strains developed here are a starting point for astaxanthin production by C. glutamicum. PMID:27376307

  13. Ohr Protects Corynebacterium glutamicum against Organic Hydroperoxide Induced Oxidative Stress

    PubMed Central

    Xiao, Xiao; Guan, Jingyuan; Zhang, Yaoling; Ding, Wei; Chaudhry, Muhammad Tausif; Wang, Yao; Shen, Xihui

    2015-01-01

    Ohr, a bacterial protein encoded by the Organic Hydroperoxide Resistance (ohr) gene, plays a critical role in resistance to organic hydroperoxides. In the present study, we show that the Cys-based thiol-dependent Ohr of Corynebacterium glutamicum decomposes organic hydroperoxides more efficiently than hydrogen peroxide. Replacement of either of the two Cys residues of Ohr by a Ser residue resulted in drastic loss of activity. The electron donors supporting regeneration of the peroxidase activity of the oxidized Ohr of C. glutamicum were principally lipoylated proteins (LpdA and Lpd/SucB). A Δohr mutant exhibited significantly decreased resistance to organic hydroperoxides and marked accumulation of reactive oxygen species (ROS) in vivo; protein carbonylation was also enhanced notably. The resistance to hydrogen peroxide also decreased, but protein carbonylation did not rise to any great extent. Together, the results unequivocally show that Ohr is essential for mediation of organic hydroperoxide resistance by C. glutamicum. PMID:26121694

  14. High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production.

    PubMed

    Kang, Mi-Suk; Han, Sang-Soo; Kim, Mi-Young; Kim, Bu-Youn; Huh, Jong-Pil; Kim, Hak-Sung; Lee, Jin-Ho

    2014-05-01

    The nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase (NHase), converting acrylonitrile into acrylamide, was cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. The specific enzyme activity in recombinant C. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (DCW). To overexpress the NHase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (TIR) of nhhB. Of them, pNBM4 with seven mutations showed the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reached 53.4 g DCW/L within 18 h, and the specific and total enzyme activities were estimated to be 37.3 μmol/min/mg DCW and 1,992 μmol/min/mL, respectively. The use of recombinant Corynebacterium cells for the production of acrylamide from acrylonitrile resulted in a conversion yield of 93 % and a final acrylamide concentration of 42.5 % within 6 h when the total amount of fed acrylonitrile was 456 g.

  15. Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum.

    PubMed

    Ding, Wei; Si, Meiru; Zhang, Weipeng; Zhang, Yaoling; Chen, Can; Zhang, Lei; Lu, Zhiqiang; Chen, Shaolin; Shen, Xihui

    2015-01-27

    Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51 kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2 kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30°C, and interestingly, it could utilize NAD(+) and NADP(+) as coenzymes with similar efficiency and showed no obvious difference toward NAD(+) and NADP(+). In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum.

  16. Engineering biotin prototrophic Corynebacterium glutamicum strains for amino acid, diamine and carotenoid production.

    PubMed

    Peters-Wendisch, P; Götker, S; Heider, S A E; Komati Reddy, G; Nguyen, A Q; Stansen, K C; Wendisch, V F

    2014-12-20

    The Gram-positive Corynebacterium glutamicum is auxotrophic for biotin. Besides the biotin uptake system BioYMN and the transcriptional regulator BioQ, this bacterium possesses functional enzymes for the last three reactions of biotin synthesis starting from pimeloyl-CoA. Heterologous expression of bioF from the Gram-negative Escherichia coli enabled biotin synthesis from pimelic acid added to the medium, but expression of bioF together with bioC and bioH from E. coli did not entail biotin prototrophy. Heterologous expression of bioWAFDBI from Bacillus subtilis encoding another biotin synthesis pathway in C. glutamicum allowed for growth in biotin-depleted media. Stable growth of the recombinant was observed without biotin addition for eight transfers to biotin-depleted medium while the empty vector control stopped growth after the first transfer. Expression of bioWAFDBI from B. subtilis in C. glutamicum strains overproducing the amino acids l-lysine and l-arginine, the diamine putrescine, and the carotenoid lycopene, respectively, enabled formation of these products under biotin-depleted conditions. Thus, biotin-prototrophic growth and production by recombinant C. glutamicum were achieved.

  17. Effect of Corynebacterium glutamicum on Livestock Material Burial Treatment.

    PubMed

    Kim, Bit-Na; Cho, Ho-Seong; Cha, Yougin; Park, Joon-Kyu; Kim, Geonha; Kim, Yang-Hoon; Min, Jiho

    2016-08-28

    In recent years, foot-and-mouth disease has occurred in all parts of the world. The animals with the disease are buried in the ground; therefore, their concentration could affect ground or groundwater. Moreover, the complete degradation of carcasses is not a certainty, and their disposal is important to prevent humans, livestock, and the environment from being affected with the disease. The treatment of Corynebacterium glutamicum is a feasible method to reduce the risk of carcass decomposition affecting humans or the environment. Therefore, this study aimed to investigate the effect of C. glutamicum on the soil environment with a carcass. The composition of amino acids in the soil treated with C. glutamicum was generally higher than those in the untreated soil. Moreover, the plant root in the soil samples treated with C. glutamicum had 84.0% amino acids relative to the standard value and was similar to that of the control. The results of this study suggest the possibility to reduce the toxicity of a grave land containing animals with this disease.

  18. Histidine biosynthesis, its regulation and biotechnological application in Corynebacterium glutamicum.

    PubMed

    Kulis-Horn, Robert K; Persicke, Marcus; Kalinowski, Jörn

    2014-01-01

    l-Histidine biosynthesis is an ancient metabolic pathway present in bacteria, archaea, lower eukaryotes, and plants. For decades l-histidine biosynthesis has been studied mainly in Escherichia coli and Salmonella typhimurium, revealing fundamental regulatory processes in bacteria. Furthermore, in the last 15 years this pathway has been also investigated intensively in the industrial amino acid-producing bacterium Corynebacterium glutamicum, revealing similarities to E. coli and S. typhimurium, as well as differences. This review summarizes the current knowledge of l-histidine biosynthesis in C. glutamicum. The genes involved and corresponding enzymes are described, in particular focusing on the imidazoleglycerol-phosphate synthase (HisFH) and the histidinol-phosphate phosphatase (HisN). The transcriptional organization of his genes in C. glutamicum is also reported, including the four histidine operons and their promoters. Knowledge of transcriptional regulation during stringent response and by histidine itself is summarized and a translational regulation mechanism is discussed, as well as clues about a histidine transport system. Finally, we discuss the potential of using this knowledge to create or improve C. glutamicum strains for the industrial l-histidine production.

  19. Formation of volutin granules in Corynebacterium glutamicum.

    PubMed

    Pallerla, Srinivas Reddy; Knebel, Sandra; Polen, Tino; Klauth, Peter; Hollender, Juliane; Wendisch, Volker F; Schoberth, Siegfried M

    2005-02-01

    Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted.

  20. Analysis of Genes Involved in Arsenic Resistance in Corynebacterium glutamicum ATCC 13032†

    PubMed Central

    Ordóñez, Efrén; Letek, Michal; Valbuena, Noelia; Gil, José A.; Mateos, Luis M.

    2005-01-01

    Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons (ars1 and ars2) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum. The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC); operon ars1 contains an additional arsenate reductase gene (arsC1′) located immediately downstream from arsC1. Additional arsenite permease and arsenate reductase genes (arsB3 and arsC4) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1, arsB1, arsC1, arsC1′, arsR2, arsB2, and arsC2 being inducible by arsenite. PMID:16204540

  1. Complete genome sequence of Corynebacterium glutamicum CP, a Chinese l-leucine producing strain.

    PubMed

    Gui, Yongli; Ma, Yuechao; Xu, Qingyang; Zhang, Chenglin; Xie, Xixian; Chen, Ning

    2016-02-20

    Here, we report the complete genome sequence of Corynebacterium glutamicum CP, an industrial l-leucine producing strain in China. The whole genome consists of a circular chromosome and a plasmid. The comparative genomics analysis shows that there are many mutations in the key enzyme coding genes relevant to l-leucine biosynthesis compared to C. glutamicum ATCC 13032.

  2. Accelerated pentose utilization by Corynebacterium glutamicum for accelerated production of lysine, glutamate, ornithine and putrescine

    PubMed Central

    Meiswinkel, Tobias M; Gopinath, Vipin; Lindner, Steffen N; Nampoothiri, K Madhavan; Wendisch, Volker F

    2013-01-01

    Summary Because of their abundance in hemicellulosic wastes arabinose and xylose are an interesting source of carbon for biotechnological production processes. Previous studies have engineered several Corynebacterium glutamicum strains for the utilization of arabinose and xylose, however, with inefficient xylose utilization capabilities. To improve xylose utilization, different xylose isomerase genes were tested in C. glutamicum. The gene originating from Xanthomonas campestris was shown to have the highest effect, resulting in growth rates of 0.14 h−1, followed by genes from Bacillus subtilis, Mycobacterium smegmatis and Escherichia coli. To further increase xylose utilization different xylulokinase genes were expressed combined with X. campestris xylose isomerase gene. All combinations further increased growth rates of the recombinant strains up to 0.20 h−1 and moreover increased biomass yields. The gene combination of X. campestris xylose isomerase and C. glutamicum xylulokinase was the fastest growing on xylose and compared with the previously described strain solely expressing E. coli xylose isomerase gene delivered a doubled growth rate. Productivity of the amino acids glutamate, lysine and ornithine, as well as the diamine putrescine was increased as well as final titres except for lysine where titres remained unchanged. Also productivity in medium containing rice straw hydrolysate as carbon source was increased. Funding Information No funding information provided. PMID:23164409

  3. Systems metabolic engineering of Corynebacterium glutamicum for production of the chemical chaperone ectoine

    PubMed Central

    2013-01-01

    Background The stabilizing and function-preserving effects of ectoines have attracted considerable biotechnological interest up to industrial scale processes for their production. These rely on the release of ectoines from high-salinity-cultivated microbial producer cells upon an osmotic down-shock in rather complex processor configurations. There is growing interest in uncoupling the production of ectoines from the typical conditions required for their synthesis, and instead design strains that naturally release ectoines into the medium without the need for osmotic changes, since the use of high-salinity media in the fermentation process imposes notable constraints on the costs, design, and durability of fermenter systems. Results Here, we used a Corynebacterium glutamicum strain as a cellular chassis to establish a microbial cell factory for the biotechnological production of ectoines. The implementation of a mutant aspartokinase enzyme ensured efficient supply of L-aspartate-beta-semialdehyde, the precursor for ectoine biosynthesis. We further engineered the genome of the basic C. glutamicum strain by integrating a codon-optimized synthetic ectABCD gene cluster under expressional control of the strong and constitutive C. glutamicum tuf promoter. The resulting recombinant strain produced ectoine and excreted it into the medium; however, lysine was still found as a by-product. Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported. Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L-1 day-1 under growth conditions that did not rely on the use of high-salinity media. Conclusions The present study describes the construction of a stable microbial cell factory for recombinant production of ectoine. We successfully applied metabolic engineering strategies to optimize its synthetic production in the

  4. Metabolic engineering of Corynebacterium glutamicum to produce GDP-L-fucose from glucose and mannose.

    PubMed

    Chin, Young-Wook; Park, Jin-Byung; Park, Yong-Cheol; Kim, Kyoung Heon; Seo, Jin-Ho

    2013-06-01

    Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5'-diphosphate (GDP)-L-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-L-fucose from GDP-D-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-L-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-D-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-L-fucose at the specific rate of 0.11 mg g cell(-1) h(-1). The specific GDP-L-fucose content reached 5.5 mg g cell(-1), which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity.

  5. Ornithine cyclodeaminase-based proline production by Corynebacterium glutamicum

    PubMed Central

    2013-01-01

    Background The soil bacterium Corynebacterium glutamicum, best known for its glutamate producing ability, is suitable as a producer of a variety of bioproducts. Glutamate is the precursor of the amino acid proline. Proline biosynthesis typically involves three enzymes and a spontaneous cyclisation reaction. Alternatively, proline can be synthesised from ornithine, an intermediate of arginine biosynthesis. The direct conversion of ornithine to proline is catalysed by ornithine cyclodeaminase. An ornithine overproducing platform strain with deletions of argR and argF (ORN1) has been employed for production of derived compounds such as putrescine. By heterologous expression of ocd this platform strain can be engineered further for proline production. Results Plasmid-based expression of ocd encoding the putative ornithine cyclodeaminase of C. glutamicum did not result in detectable proline accumulation in the culture medium. However, plasmid-based expression of ocd from Pseudomonas putida resulted in proline production with yields up to 0.31 ± 0.01 g proline/g glucose. Overexpression of the gene encoding a feedback-alleviated N-acetylglutamate kinase further increased proline production to 0.36 ± 0.01 g/g. In addition, feedback-alleviation of N-acetylglutamate kinase entailed growth-coupled production of proline and reduced the accumulation of by-products in the culture medium. Conclusions The product spectrum of the platform strain C. glutamicum ORN1 was expanded to include the amino acid L-proline. Upon further development of the ornithine overproducing platform strain, industrial production of amino acids of the glutamate family and derived bioproducts such as diamines might become within reach. PMID:23806148

  6. Anaerobic Growth of Corynebacterium glutamicum via Mixed-Acid Fermentation

    PubMed Central

    Michel, Andrea; Koch-Koerfges, Abigail; Krumbach, Karin; Brocker, Melanie

    2015-01-01

    Corynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to l-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD600) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO2 is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions. PMID:26276118

  7. Bio-based production of organic acids with Corynebacterium glutamicum.

    PubMed

    Wieschalka, Stefan; Blombach, Bastian; Bott, Michael; Eikmanns, Bernhard J

    2013-03-01

    The shortage of oil resources, the steadily rising oil prices and the impact of its use on the environment evokes an increasing political, industrial and technical interest for development of safe and efficient processes for the production of chemicals from renewable biomass. Thus, microbial fermentation of renewable feedstocks found its way in white biotechnology, complementing more and more traditional crude oil-based chemical processes. Rational strain design of appropriate microorganisms has become possible due to steadily increasing knowledge on metabolism and pathway regulation of industrially relevant organisms and, aside from process engineering and optimization, has an outstanding impact on improving the performance of such hosts. Corynebacterium glutamicum is well known as workhorse for the industrial production of numerous amino acids. However, recent studies also explored the usefulness of this organism for the production of several organic acids and great efforts have been made for improvement of the performance. This review summarizes the current knowledge and recent achievements on metabolic engineering approaches to tailor C. glutamicum for the bio-based production of organic acids. We focus here on the fermentative production of pyruvate, L- and D-lactate, 2-ketoisovalerate, 2-ketoglutarate, and succinate. These organic acids represent a class of compounds with manifold application ranges, e.g. in pharmaceutical and cosmetics industry, as food additives, and economically very interesting, as precursors for a variety of bulk chemicals and commercially important polymers.

  8. Purification and characterization of fumarase from Corynebacterium glutamicum.

    PubMed

    Genda, Tomoko; Watabe, Shoji; Ozaki, Hachiro

    2006-05-01

    Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K(eq)) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K(eq)=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.

  9. Metabolic engineering of Corynebacterium glutamicum for L-arginine production.

    PubMed

    Park, Seok Hyun; Kim, Hyun Uk; Kim, Tae Yong; Park, Jun Seok; Kim, Suok-Su; Lee, Sang Yup

    2014-08-05

    L-arginine is an important amino acid for diverse industrial and health product applications. Here we report the development of metabolically engineered Corynebacterium glutamicum ATCC 21831 for the production of L-arginine. Random mutagenesis is first performed to increase the tolerance of C. glutamicum to L-arginine analogues, followed by systems metabolic engineering for further strain improvement, involving removal of regulatory repressors of arginine operon, optimization of NADPH level, disruption of L-glutamate exporter to increase L-arginine precursor and flux optimization of rate-limiting L-arginine biosynthetic reactions. Fed-batch fermentation of the final strain in 5 l and large-scale 1,500 l bioreactors allows production of 92.5 and 81.2 g l(-1) of L-arginine with the yields of 0.40 and 0.35 g L-arginine per gram carbon source (glucose plus sucrose), respectively. The systems metabolic engineering strategy described here will be useful for engineering Corynebacteria strains for the industrial production of L-arginine and related products.

  10. Structural basis for cytokinin production by LOG from Corynebacterium glutamicum

    PubMed Central

    Seo, Hogyun; Kim, Sangwoo; Sagong, Hye-Young; Son, Hyeoncheol Francis; Jin, Kyeong Sik; Kim, Il-Kwon; Kim, Kyung-Jin

    2016-01-01

    “Lonely guy” (LOG) has been identified as a cytokinin-producing enzyme in plants and plant-interacting fungi. The gene product of Cg2612 from the soil-dwelling bacterium Corynebacterium glutamicum was annotated as an LDC. However, the facts that C. glutamicum lacks an LDC and Cg2612 has high amino acid similarity with LOG proteins suggest that Cg2612 is possibly an LOG protein. To investigate the function of Cg2612, we determined its crystal structure at a resolution of 2.3 Å. Cg2612 functions as a dimer and shows an overall structure similar to other known LOGs, such as LOGs from Arabidopsis thaliana (AtLOG), Claviceps purpurea (CpLOG), and Mycobacterium marinum (MmLOG). Cg2612 also contains a “PGGXGTXXE” motif that contributes to the formation of an active site similar to other LOGs. Moreover, biochemical studies on Cg2612 revealed that the protein has phosphoribohydrolase activity but not LDC activity. Based on these structural and biochemical studies, we propose that Cg2612 is not an LDC family enzyme, but instead belongs to the LOG family. In addition, the prenyl-binding site of Cg2612 (CgLOG) comprised residues identical to those seen in AtLOG and CpLOG, albeit dissimilar to those in MmLOG. The work provides structural and functional implications for LOG-like proteins from other microorganisms. PMID:27507425

  11. Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum.

    PubMed Central

    Farwick, M; Siewe, R M; Krämer, R

    1995-01-01

    Osmoregulatory uptake of glycine betaine in whole cells of Corynebacterium glutamicum ATCC 13032 (wild type) was studied. The cells actively take up glycine betaine when they are osmotically shocked. The total accumulation and uptake rate were dependent on the osmotic strength of the medium. Kinetic analysis revealed a high-affinity transport system (Km, 8.6 +/- 0.4 microM) with high maximum velocity (110 nmol.min-1.mg [dry weight]-1). Glycine betaine functioned as a compatible solute when added to the medium and allowed growth at an otherwise inhibitory osmotic strength of 1.5 M NaCl. Proline and ectoine could also be used as osmoprotectants. Glycine betaine is neither synthesized nor metabolized by C. glutamicum. The glycine betaine transport system is constitutively expressed at a basal level of activity. It can be induced up to eightfold by osmotic stress and is strongly regulated at the level of activity. The transport system is highly specific and has its pH optimum in the slightly alkaline range at about pH 8. The uptake of the zwitterionic glycine betaine is mediated by a secondary symport system coupled to cotransport of at least two Na+ ions. It is thus driven both by the membrane potential and the Na+ gradient. An extremely high accumulation (internal/external) ratio of up to 4 x 10(6) was measured, which represents the highest accumulation ratio observed for any transport system. PMID:7642496

  12. Structural basis for cytokinin production by LOG from Corynebacterium glutamicum.

    PubMed

    Seo, Hogyun; Kim, Sangwoo; Sagong, Hye-Young; Son, Hyeoncheol Francis; Jin, Kyeong Sik; Kim, Il-Kwon; Kim, Kyung-Jin

    2016-08-10

    "Lonely guy" (LOG) has been identified as a cytokinin-producing enzyme in plants and plant-interacting fungi. The gene product of Cg2612 from the soil-dwelling bacterium Corynebacterium glutamicum was annotated as an LDC. However, the facts that C. glutamicum lacks an LDC and Cg2612 has high amino acid similarity with LOG proteins suggest that Cg2612 is possibly an LOG protein. To investigate the function of Cg2612, we determined its crystal structure at a resolution of 2.3 Å. Cg2612 functions as a dimer and shows an overall structure similar to other known LOGs, such as LOGs from Arabidopsis thaliana (AtLOG), Claviceps purpurea (CpLOG), and Mycobacterium marinum (MmLOG). Cg2612 also contains a "PGGXGTXXE" motif that contributes to the formation of an active site similar to other LOGs. Moreover, biochemical studies on Cg2612 revealed that the protein has phosphoribohydrolase activity but not LDC activity. Based on these structural and biochemical studies, we propose that Cg2612 is not an LDC family enzyme, but instead belongs to the LOG family. In addition, the prenyl-binding site of Cg2612 (CgLOG) comprised residues identical to those seen in AtLOG and CpLOG, albeit dissimilar to those in MmLOG. The work provides structural and functional implications for LOG-like proteins from other microorganisms.

  13. Elimination of polyamine N-acetylation and regulatory engineering improved putrescine production by Corynebacterium glutamicum.

    PubMed

    Nguyen, Anh Q D; Schneider, Jens; Wendisch, Volker F

    2015-05-10

    Corynebacterium glutamicum has been engineered for production of the polyamide monomer putrescine or 1,4-diaminobutane. Here, N-acetylputrescine was shown to be a significant by-product of putrescine production by recombinant putrescine producing C. glutamicum strains. A systematic gene deletion approach of 18 (putative) N-acetyltransferase genes revealed that the cg1722 gene product was responsible for putrescine acetylation. The encoded enzyme was purified and characterized as polyamine N-acetyltransferase. The enzyme accepted acetyl-CoA and propionyl-CoA as donors for acetylation of putrescine and other diamines as acceptors, but showed highest catalytic efficiency with the triamine spermidine and the tetraamine spermine and, hence, was named SnaA. Upon deletion of snaA in the putrescine producing strain PUT21, no acteylputrescine accumulated, but about 41% more putrescine as compared to the parent strain. Moreover, a transcriptome approach identified increased expression of the cgmAR operon encoding a putative permease and a transcriptional TetR-family repressor upon induction of putrescine production in C. glutamicum PUT21. CgmR is known to bind to cgmO upstream of cgmAR and gel mobility shift experiments with purified CgmR revealed that putrescine and other diamines perturbed CgmR-cgmO complex formation, but not migration of free cgmO DNA. Deletion of the repressor gene cgmR resulted in expression changes of a number of genes and increased putrescine production of C. glutamicum PUT21 by 19% as compared to the parent strain. Overexpression of the putative transport gene cgmA increased putrescine production by 24% as compared to the control strain. However, cgmA overexpression in PUT21ΔsnaA did not further improve putrescine production, hence, the beneficial effects of both targets were not synergistic at the highest described yield of 0.21 g g(-1).

  14. Glutamate Fermentation-2: Mechanism of L-Glutamate Overproduction in Corynebacterium glutamicum.

    PubMed

    Hirasawa, Takashi; Wachi, Masaaki

    2016-12-03

    The nonpathogenic coryneform bacterium, Corynebacterium glutamicum, was isolated as an L-glutamate-overproducing microorganism by Japanese researchers and is currently utilized in various amino acid fermentation processes. L-Glutamate production by C. glutamicum is induced by limitation of biotin and addition of fatty acid ester surfactants and β-lactam antibiotics. These treatments affect the cell surface structures of C. glutamicum. After the discovery of C. glutamicum, many researchers have investigated the underlying mechanism of L-glutamate overproduction with respect to the cell surface structures of this organism. Furthermore, metabolic regulation during L-glutamate overproduction by C. glutamicum, particularly, the relationship between central carbon metabolism and L-glutamate biosynthesis, has been investigated. Recently, the role of a mechanosensitive channel protein in L-glutamate overproduction has been reported. In this chapter, mechanisms of L-glutamate overproduction by C. glutamicum have been reviewed.

  15. Bio-based production of organic acids with Corynebacterium glutamicum

    PubMed Central

    Wieschalka, Stefan; Blombach, Bastian; Bott, Michael; Eikmanns, Bernhard J

    2013-01-01

    The shortage of oil resources, the steadily rising oil prices and the impact of its use on the environment evokes an increasing political, industrial and technical interest for development of safe and efficient processes for the production of chemicals from renewable biomass. Thus, microbial fermentation of renewable feedstocks found its way in white biotechnology, complementing more and more traditional crude oil-based chemical processes. Rational strain design of appropriate microorganisms has become possible due to steadily increasing knowledge on metabolism and pathway regulation of industrially relevant organisms and, aside from process engineering and optimization, has an outstanding impact on improving the performance of such hosts. Corynebacterium glutamicum is well known as workhorse for the industrial production of numerous amino acids. However, recent studies also explored the usefulness of this organism for the production of several organic acids and great efforts have been made for improvement of the performance. This review summarizes the current knowledge and recent achievements on metabolic engineering approaches to tailor C. glutamicum for the bio-based production of organic acids. We focus here on the fermentative production of pyruvate, l-and d-lactate, 2-ketoisovalerate, 2-ketoglutarate, and succinate. These organic acids represent a class of compounds with manifold application ranges, e.g. in pharmaceutical and cosmetics industry, as food additives, and economically very interesting, as precursors for a variety of bulk chemicals and commercially important polymers. Funding Information Work in the laboratories of the authors was supported by the Fachagentur Nachwachsende Rohstoffe (FNR) of the Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV; FNR Grants 220-095-08A and 220-095-08D; Bio-ProChemBB project, ERA-IB programme), by the Deutsche Bundesstiftung Umwelt (DBU Grant AZ13040/05) and the Evonik Degussa AG. PMID

  16. Crude glycerol-based production of amino acids and putrescine by Corynebacterium glutamicum.

    PubMed

    Meiswinkel, Tobias M; Rittmann, Doris; Lindner, Steffen N; Wendisch, Volker F

    2013-10-01

    Corynebacterium glutamicum possesses genes for glycerol kinase and glycerol-3-phosphate dehydrogenase that were shown to support slow growth with glycerol only when overexpressed from a plasmid. Pure glycerol and crude glycerol from biodiesel factories were tested for growth of recombinant strains expressing glpF, glpK and glpD from Escherichia coli. Some, but not all crude glycerol lots served as good carbon sources. Although the inhibitory compound(s) present in these crude glycerol lots remained unknown, the addition of substoichiometric glucose concentrations (below 10% by weight) enabled the utilization of some of the inhibitory crude glycerol lots. Besides growth, production of the amino acids L-glutamate, L-lysine, L-ornithine and L-arginine as well as of the diamine putrescine based on crude glycerol qualities from biodiesel factories was demonstrated.

  17. Updates on industrial production of amino acids using Corynebacterium glutamicum.

    PubMed

    Wendisch, Volker F; Jorge, João M P; Pérez-García, Fernando; Sgobba, Elvira

    2016-06-01

    L-Amino acids find various applications in biotechnology. L-Glutamic acid and its salts are used as flavor enhancers. Other L-amino acids are used as food or feed additives, in parenteral nutrition or as building blocks for the chemical and pharmaceutical industries. L-amino acids are synthesized from precursors of central carbon metabolism. Based on the knowledge of the biochemical pathways microbial fermentation processes of food, feed and pharma amino acids have been developed. Production strains of Corynebacterium glutamicum, which has been used safely for more than 50 years in food biotechnology, and Escherichia coli are constantly improved using metabolic engineering approaches. Research towards new processes is ongoing. Fermentative production of L-amino acids in the million-ton-scale has shaped modern biotechnology and its markets continue to grow steadily. This review focusses on recent achievements in strain development for amino acid production including the use of CRISPRi/dCas9, genome-reduced strains, biosensors and synthetic pathways to enable utilization of alternative carbon sources.

  18. Proteomics of FACS-sorted heterogeneous Corynebacterium glutamicum populations.

    PubMed

    Harst, Andreas; Albaum, Stefan P; Bojarzyn, Tanja; Trötschel, Christian; Poetsch, Ansgar

    2017-03-18

    The metabolic status of individual cells in microbial cultures can differ, being relevant for biotechnology, environmental and medical microbiology. However, it is hardly understood in molecular detail due to limitations of current analytical tools. Here, we demonstrate that FACS in combination with proteomics can be used to sort and analyze cell populations based on their metabolic state. A previously established GFP reporter system was used to detect and sort single Corynebacterium glutamicum cells based on the concentration of branched chain amino acids (BCAA) using FACS. A proteomics workflow optimized for small cell numbers was used to quantitatively compare proteomes of a ΔaceE mutant, lacking functional pyruvate dehydrogenase (PD), and the wild type. About 800 proteins could be quantified from 1,000,000 cells. In the ΔaceE mutant BCAA production was coordinated with upregulation of the glyoxylate cycle and TCA cycle to counter the lack of acetyl CoA resulting from the deletion of aceE.

  19. Recent progress in development of synthetic biology platforms and metabolic engineering of Corynebacterium glutamicum.

    PubMed

    Woo, Han Min; Park, Jin-Byung

    2014-06-20

    The paradigm of synthetic biology has been evolving, along with relevant engineering, to achieve designed bio-systems. Synthetic biology has reached the point where it is possible to develop microbial strains to produce desired chemicals. Recent advances in this field have promoted metabolic engineering of Corynebacterium glutamicum as an amino-acid producer for use in intelligent microbial-cell factories. Here, we review recent advances that address C. glutamicum as a potential model organism for synthetic biology, and evaluate their industrial applications. Finally, we highlight the perspective of developing C. glutamicum as a step toward advanced microbial-cell factories that could produce valuable chemicals from renewable resources.

  20. Optimization of the IPP Precursor Supply for the Production of Lycopene, Decaprenoxanthin and Astaxanthin by Corynebacterium glutamicum

    PubMed Central

    Heider, Sabine A. E.; Wolf, Natalie; Hofemeier, Arne; Peters-Wendisch, Petra; Wendisch, Volker F.

    2014-01-01

    The biotechnologically relevant bacterium Corynebacterium glutamicum, currently used for the million ton-scale production of amino acids for the food and feed industries, is pigmented due to synthesis of the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides. The precursors of carotenoid biosynthesis, isopenthenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate, are synthesized in this organism via the methylerythritol phosphate (MEP) or non-mevalonate pathway. Terminal pathway engineering in recombinant C. glutamicum permitted the production of various non-native C50 and C40 carotenoids. Here, the role of engineering isoprenoid precursor supply for lycopene production by C. glutamicum was characterized. Overexpression of dxs encoding the enzyme that catalyzes the first committed step of the MEP-pathway by chromosomal promoter exchange in a prophage-cured, genome-reduced C. glutamicum strain improved lycopene formation. Similarly, an increased IPP supply was achieved by chromosomal integration of two artificial operons comprising MEP pathway genes under the control of a constitutive promoter. Combined overexpression of dxs and the other six MEP pathways genes in C. glutamicum strain LYC3-MEP was not synergistic with respect to improving lycopene accumulation. Based on C. glutamicum strain LYC3-MEP, astaxanthin could be produced in the milligrams per gram cell dry weight range when the endogenous genes crtE, crtB, and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were coexpressed with the genes for lycopene cyclase and β-carotene hydroxylase from Pantoea ananatis and carotene C(4) oxygenase from Brevundimonas aurantiaca. PMID:25191655

  1. Overexpression of Mycothiol Disulfide Reductase Enhances Corynebacterium glutamicum Robustness by Modulating Cellular Redox Homeostasis and Antioxidant Proteins under Oxidative Stress

    PubMed Central

    Si, Meiru; Zhao, Chao; Zhang, Bing; Wei, Dawei; Chen, Keqi; Yang, Xu; Xiao, He; Shen, Xihui

    2016-01-01

    Mycothiol (MSH) is the dominant low-molecular-weight thiol (LMWT) unique to high-(G+C)-content Gram-positive Actinobacteria, such as Corynebacterium glutamicum, and is oxidised into its disulfide form mycothiol disulfide (MSSM) under oxidative conditions. Mycothiol disulfide reductase (Mtr), an NADPH-dependent enzyme, reduces MSSM to MSH, thus maintaining intracellular redox homeostasis. In this study, a recombinant plasmid was constructed to overexpress Mtr in C. glutamicum using the expression vector pXMJ19-His6. Mtr-overexpressing C. glutamicum cells showed increased tolerance to ROS induced by oxidants, bactericidal antibiotics, alkylating agents, and heavy metals. The physiological roles of Mtr in resistance to oxidative stresses were corroborated by decreased ROS levels, reduced carbonylation damage, decreased loss of reduced protein thiols, and a massive increase in the levels of reversible protein thiols in Mtr-overexpressing cells exposed to stressful conditions. Moreover, overexpression of Mtr caused a marked increase in the ratio of reduced to oxidised mycothiol (MSH:MSSM), and significantly enhanced the activities of a variety of antioxidant enzymes, including mycothiol peroxidase (MPx), mycoredoxin 1 (Mrx1), thioredoxin 1 (Trx1), and methionine sulfoxide reductase A (MsrA). Taken together, these results indicate that the Mtr protein functions in C. glutamicum by protecting cells against oxidative stress. PMID:27383057

  2. Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

    PubMed Central

    Padilla, Leandro; Krämer, Reinhard; Stephanopoulos, Gregory; Agosin, Eduardo

    2004-01-01

    Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum. PMID:14711665

  3. Degradation and assimilation of aromatic compounds by Corynebacterium glutamicum: another potential for applications for this bacterium?

    PubMed

    Shen, Xi-Hui; Zhou, Ning-Yi; Liu, Shuang-Jiang

    2012-07-01

    With the implementation of the well-established molecular tools and systems biology techniques, new knowledge on aromatic degradation and assimilation by Corynebacterium glutamicum has been emerging. This review summarizes recent findings on degradation of aromatic compounds by C. glutamicum. Among these findings, the mycothiol-dependent gentisate pathway was firstly discovered in C. glutamicum. Other important knowledge derived from C. glutamicum would be the discovery of linkages among aromatic degradation and primary metabolisms such as gluconeogenesis and central carbon metabolism. Various transporters in C. glutamicum have also been identified, and they play an essential role in microbial assimilation of aromatic compounds. Regulation on aromatic degradation occurs mainly at transcription level via pathway-specific regulators, but global regulator(s) is presumably involved in the regulation. It is concluded that C. glutamicum is a very useful model organism to disclose new knowledge of biochemistry, physiology, and genetics of the catabolism of aromatic compounds in high GC content Gram-positive bacteria, and that the new physiological properties of aromatic degradation and assimilation are potentially important for industrial applications of C. glutamicum.

  4. Genetic and biochemical identification of the chorismate mutase from Corynebacterium glutamicum.

    PubMed

    Li, Pan-Pan; Liu, Ya-Jun; Liu, Shuang-Jiang

    2009-10-01

    Chorismate mutase (CM) catalyses the rearrangement of chorismate to prephenate and is also the first and the key enzyme that diverges the shikimate pathway to either tryptophan (Trp) or phenylalanine (Phe) and tyrosine (Tyr). Corynebacterium glutamicum is one of the most important amino acid producers for the fermentation industry and has been widely investigated. However, the gene(s) encoding CM has not been experimentally identified in C. glutamicum. In this study, the ncgl0819 gene, which was annotated as 'conserved hypothetical protein' in the C. glutamicum genome, was genetically characterized to be essential for growth in minimal medium, and a mutant deleted of ncgl0819 was a Phe and Tyr auxotroph. Genetic cloning and expression of ncgl0819 in Escherichia coli resulted in the formation of a new protein (NCgl0819) having CM activity. It was concluded that ncgl0819 encoded the CM of C. glutamicum (CM0819). CM0819 was demonstrated to be a homodimer and is a new member of the monofunctional CMs of the AroQ structural class. The CM0819 activity was not affected by Phe, Tyr or Trp. Two 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthases (DS0950 and DS2098, formerly NCgl0950 and NCgl2098) had been previously identified from C. glutamicum. CM0819 significantly stimulated DAHP synthase (DS2098) activity. Physical interaction between CM0819 and DS2098 was observed. When CM0819 was present, DS2098 activity was subject to allosteric inhibition by chorismate and prephenate. Conserved hypothetical proteins homologous to CM0819 were identified in all known Corynebacterium genomes, suggesting a universal occurrence of CM0819-like CMs in the genus Corynebacterium.

  5. Global transcriptomic analysis of the response of Corynebacterium glutamicum to ferulic acid.

    PubMed

    Chen, Can; Pan, Junfeng; Yang, Xiaobing; Xiao, He; Zhang, Yaoling; Si, Meiru; Shen, Xihui; Wang, Yao

    2017-03-01

    Corynebacterium glutamicum can survive by using ferulic acid as the sole carbon source. In this study, we assessed the response of C. glutamicum to ferulic acid stress by means of a global transcriptional response analysis. The transcriptional data showed that several genes involved in degradation of ferulic acid were affected. Moreover, several genes related to the stress response; protein protection or degradation and DNA repair; replication, transcription and translation; and the cell envelope were differentially expressed. Deletion of the katA or sigE gene in C. glutamicum resulted in a decrease in cell viability under ferulic acid stress. These insights will facilitate further engineering of model industrial strains, with enhanced tolerance to ferulic acid to enable easy production of biofuels from lignocellulose.

  6. [Construction and structural analysis of integrated cellular network of Corynebacterium glutamicum].

    PubMed

    Jiang, Jinguo; Song, Lifu; Zheng, Ping; Jia, Shiru; Sun, Jibin

    2012-05-01

    Corynebacterium glutamicum is one of the most important traditional industrial microorganisms and receiving more and more attention towards a novel cellular factory due to the recently rapid development in genomics and genetic operation toolboxes for Corynebacterium. However, compared to other model organisms such as Escherichia coli, there were few studies on its metabolic regulation, especially a genome-scale integrated cellular network model currently missing for Corynebacterium, which hindered the systematic study of Corynebacterium glutamicum and large-scale rational design and optimization for strains. Here, by gathering relevant information from a number of public databases, we successfully constructed an integrated cellular network, which was composed of 1384 reactions, 1276 metabolites, 88 transcriptional factors and 999 pairs of transcriptional regulatory relationships. The transcriptional regulatory sub-network could be arranged into five layers and the metabolic sub-network presented a clear bow-tie structure. We proposed a new method to extract complex metabolic and regulatory sub-network for product-orientated study taking lysine biosynthesis as an example. The metabolic and regulatory sub-network extracted by our method was more close to the real functional network than the simplex biochemical pathways. The results would be greatly helpful for understanding the high-yielding biomechanism for amino acids and the re-design of the industrial strains.

  7. RND transporters protect Corynebacterium glutamicum from antibiotics by assembling the outer membrane.

    PubMed

    Yang, Liang; Lu, Shuo; Belardinelli, Juan; Huc-Claustre, Emilie; Jones, Victoria; Jackson, Mary; Zgurskaya, Helen I

    2014-08-01

    Corynebacterium-Mycobacterium-Nocardia (CMN) group are the causative agents of a broad spectrum of diseases in humans. A distinctive feature of these Gram-positive bacteria is the presence of an outer membrane of unique structure and composition. Recently, resistance-nodulation-division (RND) transporters (nicknamed MmpLs, Mycobacterial membrane protein Large) have emerged as major contributors to the biogenesis of the outer membranes in mycobacteria and as promising drug targets. In this study, we investigated the role of RND transporters in the physiology of Corynebacterium glutamicum and analyzed properties of these proteins. Our results show that in contrast to Gram-negative species, in which RND transporters actively extrude antibiotics from cells, in C. glutamicum and relatives these transporters protect cells from antibiotics by playing essential roles in the biogenesis of the low-permeability barrier of the outer membrane. Conditional C. glutamicum mutants lacking RND proteins and with the controlled expression of either NCgl2769 (CmpL1) or NCgl0228 (CmpL4) are hypersusceptible to multiple antibiotics, have growth deficiencies in minimal medium and accumulate intracellularly trehalose monocorynomycolates, free corynomycolates, and the previously uncharacterized corynomycolate-containing lipid. Our results also suggest that similar to other RND transporters, Corynebacterial membrane proteins Large (CmpLs) functions are dependent on a proton-motive force.

  8. Transcriptome and Multivariable Data Analysis of Corynebacterium glutamicum under Different Dissolved Oxygen Conditions in Bioreactors

    PubMed Central

    Sun, Yang; Guo, Wenwen; Wang, Fen; Peng, Feng; Yang, Yankun; Dai, Xiaofeng; Liu, Xiuxia; Bai, Zhonghu

    2016-01-01

    Dissolved oxygen (DO) is an important factor in the fermentation process of Corynebacterium glutamicum, which is a widely used aerobic microbe in bio-industry. Herein, we described RNA-seq for C. glutamicum under different DO levels (50%, 30% and 0%) in 5 L bioreactors. Multivariate data analysis (MVDA) models were used to analyze the RNA-seq and metabolism data to investigate the global effect of DO on the transcriptional distinction of the substance and energy metabolism of C. glutamicum. The results showed that there were 39 and 236 differentially expressed genes (DEGs) under the 50% and 0% DO conditions, respectively, compared to the 30% DO condition. Key genes and pathways affected by DO were analyzed, and the result of the MVDA and RNA-seq revealed that different DO levels in the fermenter had large effects on the substance and energy metabolism and cellular redox balance of C. glutamicum. At low DO, the glycolysis pathway was up-regulated, and TCA was shunted by the up-regulation of the glyoxylate pathway and over-production of amino acids, including valine, cysteine and arginine. Due to the lack of electron-acceptor oxygen, 7 genes related to the electron transfer chain were changed, causing changes in the intracellular ATP content at 0% and 30% DO. The metabolic flux was changed to rebalance the cellular redox. This study applied deep sequencing to identify a wealth of genes and pathways that changed under different DO conditions and provided an overall comprehensive view of the metabolism of C. glutamicum. The results provide potential ways to improve the oxygen tolerance of C. glutamicum and to modify the metabolic flux for amino acid production and heterologous protein expression. PMID:27907077

  9. Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction.

    PubMed

    Mizuno, Yuta; Nagano-Shoji, Megumi; Kubo, Shosei; Kawamura, Yumi; Yoshida, Ayako; Kawasaki, Hisashi; Nishiyama, Makoto; Yoshida, Minoru; Kosono, Saori

    2016-02-01

    The bacterium Corynebacterium glutamicum is utilized during industrial fermentation to produce amino acids such as L-glutamate. During L-glutamate fermentation, C. glutamicum changes the flux of central carbon metabolism to favor L-glutamate production, but the molecular mechanisms that explain these flux changes remain largely unknown. Here, we found that the profiles of two major lysine acyl modifications were significantly altered upon glutamate overproduction in C. glutamicum; acetylation decreased, whereas succinylation increased. A label-free semi-quantitative proteomic analysis identified 604 acetylated proteins with 1328 unique acetylation sites and 288 succinylated proteins with 651 unique succinylation sites. Acetylation and succinylation targeted enzymes in central carbon metabolic pathways that are directly related to glutamate production, including the 2-oxoglutarate dehydrogenase complex (ODHC), a key enzyme regulating glutamate overproduction. Structural mapping revealed that several critical lysine residues in the ODHC components were susceptible to acetylation and succinylation. Furthermore, induction of glutamate production was associated with changes in the extent of acetylation and succinylation of lysine, suggesting that these modifications may affect the activity of enzymes involved in glutamate production. Deletion of phosphotransacetylase decreased the extent of protein acetylation in nonproducing condition, suggesting that acetyl phosphate-dependent acetylation is active in C. glutamicum. However, no effect was observed on the profiles of acetylation and succinylation in glutamate-producing condition upon disruption of acetyl phosphate metabolism or deacetylase homologs. It was considered likely that the reduced acetylation in glutamate-producing condition may reflect metabolic states where the flux through acid-producing pathways is very low, and substrates for acetylation do not accumulate in the cell. Succinylation would occur more

  10. Metabolic engineering of Corynebacterium glutamicum aimed at alternative carbon sources and new products

    PubMed Central

    Zahoor, Ahmed; Lindner, Steffen N.; Wendisch, Volker F.

    2012-01-01

    Corynebacterium glutamicum is well known as the amino acid-producing workhorse of fermentation industry, being used for multi-million-ton scale production of glutamate and lysine for more than 60 years. However, it is only recently that extensive research has focused on engineering it beyond the scope of amino acids. Meanwhile, a variety of corynebacterial strains allows access to alternative carbon sources and/or allows production of a wide range of industrially relevant compounds. Some of these efforts set new standards in terms of titers and productivities achieved whereas others represent a proof-of-principle. These achievements manifest the position of C. glutamicum as an important industrial microorganism with capabilities far beyond the traditional amino acid production. In this review we focus on the state of the art of metabolic engineering of C. glutamicum for utilization of alternative carbon sources, (e.g. coming from wastes and unprocessed sources), and construction of C. glutamicum strains for production of new products such as diamines, organic acids and alcohols PMID:24688664

  11. In vitro functional characterization of the Na+/H+ antiporters in Corynebacterium glutamicum.

    PubMed

    Xu, Ning; Wang, Lei; Cheng, Haijiao; Liu, Qingdai; Liu, Jun; Ma, Yanhe

    2016-02-01

    Corynebacterium glutamicum, typically used as industrial workhorse for amino acid production, is a moderately salt-alkali-tolerant microorganism with optimal growth at pH 7-9. However, little is known about the mechanisms of salt-alkali tolerance in C. glutamicum. Here, the catalytic capacity of three putative Na(+)/H(+) antiporters from C. glutamicum (designated as Cg-Mrp1, Cg-Mrp2 and Cg-NhaP) were characterized in an antiporter-deficient Escherichia coli KNabc strain. Only Cg-Mrp1 was able to effectively complement the Na(+)-sensitive of E. coli KNabc. Cg-Mrp1 exhibited obvious Na(+)(Li(+))/H(+) antiport activities with low apparent Km values of 1.08 mM and 1.41 mM for Na(+) and Li(+), respectively. The Na(+)/H(+) antiport activity of Cg-Mrp1 was optimal in the alkaline pH range. All three antiporters showed detectable K(+)/H(+) antiport activitiy. Cg-NhaP also exhibited Na(+)(Li(+),Rb(+))/H(+) antiport activities but at lower levels of activity. Interestingly, overexpression of Cg-Mrp2 exhibited clear Na(+)(K(+))/H(+) antiport activities. These results suggest that C. glutamicum Na(+)(K(+))/H(+) antiporters may have overlapping roles in coping with salt-alkali and perhaps high-osmolarity stress.

  12. Inactivation of the phosphoglucomutase gene pgm in Corynebacterium glutamicum affects cell shape and glycogen metabolism

    PubMed Central

    Seibold, Gerd M.; Eikmanns, Bernhard J.

    2013-01-01

    In Corynebacterium glutamicum formation of glc-1-P (α-glucose-1-phosphate) from glc-6-P (glucose-6-phosphate) by α-Pgm (phosphoglucomutase) is supposed to be crucial for synthesis of glycogen and the cell wall precursors trehalose and rhamnose. Furthermore, Pgm is probably necessary for glycogen degradation and maltose utilization as glucan phosphorylases of both pathways form glc-1-P. We here show that C. glutamicum possesses at least two Pgm isoenzymes, the cg2800 (pgm) encoded enzyme contributing most to total Pgm activity. By inactivation of pgm we created C. glutamicum IMpgm showing only about 12% Pgm activity when compared to the parental strain. We characterized both strains during cultivation with either glucose or maltose as substrate and observed that (i) the glc-1-P content in the WT (wild-type) and the mutant remained constant independent of the carbon source used, (ii) the glycogen levels in the pgm mutant were lower during growth on glucose and higher during growth on maltose, and (iii) the morphology of the mutant was altered with maltose as a substrate. We conclude that C. glutamicum employs glycogen as carbon capacitor to perform glc-1-P homeostasis in the exponential growth phase and is therefore able to counteract limited Pgm activity for both anabolic and catabolic metabolic pathways. PMID:23863124

  13. Metabolic engineering of Corynebacterium glutamicum aimed at alternative carbon sources and new products.

    PubMed

    Zahoor, Ahmed; Lindner, Steffen N; Wendisch, Volker F

    2012-01-01

    Corynebacterium glutamicum is well known as the amino acid-producing workhorse of fermentation industry, being used for multi-million-ton scale production of glutamate and lysine for more than 60 years. However, it is only recently that extensive research has focused on engineering it beyond the scope of amino acids. Meanwhile, a variety of corynebacterial strains allows access to alternative carbon sources and/or allows production of a wide range of industrially relevant compounds. Some of these efforts set new standards in terms of titers and productivities achieved whereas others represent a proof-of-principle. These achievements manifest the position of C. glutamicum as an important industrial microorganism with capabilities far beyond the traditional amino acid production. In this review we focus on the state of the art of metabolic engineering of C. glutamicum for utilization of alternative carbon sources, (e.g. coming from wastes and unprocessed sources), and construction of C. glutamicum strains for production of new products such as diamines, organic acids and alcohols.

  14. Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum.

    PubMed

    Takeno, Seiki; Ohnishi, Junko; Komatsu, Tomoha; Masaki, Tatsuya; Sen, Kikuo; Ikeda, Masato

    2007-07-01

    Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O(2)), while no visible colonies were formed in the absence of O(2). However, in the presence of nitrate (NO3-), the organism exhibited limited growth anaerobically with production of nitrite (NO2-), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using L-lysine- and L-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.

  15. Global Transcriptomic Analysis of the Response of Corynebacterium glutamicum to Vanillin

    PubMed Central

    Chen, Can; Pan, Junfeng; Yang, Xiaobing; Guo, Chenghao; Ding, Wei; Si, Meiru; Zhang, Yi; Shen, Xihui; Wang, Yao

    2016-01-01

    Lignocellulosic biomass is an abundant and renewable resource for biofuels and bio-based chemicals. Vanillin is one of the major phenolic inhibitors in biomass production using lignocellulose. To assess the response of Corynebacterium glutamicum to vanillin stress, we performed a global transcriptional response analysis. The transcriptional data showed that the vanillin stress not only affected the genes involved in degradation of vanillin, but also differentially regulated several genes related to the stress response, ribosome/translation, protein secretion, and the cell envelope. Moreover, deletion of the sigH or msrA gene in C. glutamicum resulted in a decrease in cell viability under vanillin stress. These insights will promote further engineering of model industrial strains, with enhanced tolerance or degradation ability to vanillin to enable suitable production of biofuels and bio-based chemicals from lignocellulosic biomass. PMID:27760214

  16. Global Transcriptomic Analysis of the Response of Corynebacterium glutamicum to Vanillin.

    PubMed

    Chen, Can; Pan, Junfeng; Yang, Xiaobing; Guo, Chenghao; Ding, Wei; Si, Meiru; Zhang, Yi; Shen, Xihui; Wang, Yao

    2016-01-01

    Lignocellulosic biomass is an abundant and renewable resource for biofuels and bio-based chemicals. Vanillin is one of the major phenolic inhibitors in biomass production using lignocellulose. To assess the response of Corynebacterium glutamicum to vanillin stress, we performed a global transcriptional response analysis. The transcriptional data showed that the vanillin stress not only affected the genes involved in degradation of vanillin, but also differentially regulated several genes related to the stress response, ribosome/translation, protein secretion, and the cell envelope. Moreover, deletion of the sigH or msrA gene in C. glutamicum resulted in a decrease in cell viability under vanillin stress. These insights will promote further engineering of model industrial strains, with enhanced tolerance or degradation ability to vanillin to enable suitable production of biofuels and bio-based chemicals from lignocellulosic biomass.

  17. A Brevibacterium linens pRBL1 replicon functional in Corynebacterium glutamicum.

    PubMed

    Ankri, S; Bouvier, I; Reyes, O; Predali, F; Leblon, G

    1996-07-01

    Brevibacterium linens RBL strain cryptic plasmid pRBL1 (8.0 kb) is described. A region involved in pRBL1 autonomous replication in Corynebacterium glutamicum was identified by insertion and deletion mapping and partially sequenced. This region encodes for a hypothetical 310-amino acid (aa) protein closely related to the replicases of plasmids pXZ10142 (C. glutamicum) and pAL5000 (Mycobacterium fortuitum). The 310-aa protein also shows significant homology to proteins of pColE5-099 (Shigella sonnei) and pJD1 (Neisseria gonorrhoea). At least one of these proteins, the Rep protein of pColE5-099, is known to be involved in theta replication.

  18. A giant market and a powerful metabolism: L-lysine provided by Corynebacterium glutamicum.

    PubMed

    Eggeling, Lothar; Bott, Michael

    2015-04-01

    L-lysine is made in an exceptional large quantity of currently 2,200,000 tons/year and belongs therefore to one of the leading biotechnological products. Production is done almost exclusively with mutants of Corynebacterium glutamicum. The increasing L-lysine market forces companies to improve the production process fostering also a deeper understanding of the microbial physiology of C. glutamicum. Current major challenges are the identification of ancillary mutations not intuitively related with product increase. This review gives insights on how cellular characteristics enable to push the carbon flux in metabolism towards its theoretical maximum, and this example may also serve as a guide to achieve and increase the formation of other products of interest in microbial biotechnology.

  19. Impact of Heterologous Expression of Escherichia coli UDP-Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum

    PubMed Central

    Padilla, Leandro; Morbach, Susanne; Krämer, Reinhard; Agosin, Eduardo

    2004-01-01

    Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway. PMID:15240254

  20. The pyruvate dehydrogenase complex of Corynebacterium glutamicum: an attractive target for metabolic engineering.

    PubMed

    Eikmanns, Bernhard J; Blombach, Bastian

    2014-12-20

    The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative thiamine pyrophosphate-dependent decarboxylation of pyruvate to acetyl-CoA and CO2. Since pyruvate is a key metabolite of the central metabolism and also the precursor for several relevant biotechnological products, metabolic engineering of this multienzyme complex is a promising strategy to improve microbial production processes. This review summarizes the current knowledge and achievements on metabolic engineering approaches to tailor the PDHC of Corynebacterium glutamicum for the bio-based production of l-valine, 2-ketosiovalerate, pyruvate, succinate and isobutanol and to improve l-lysine production.

  1. Biotin protein ligase from Corynebacterium glutamicum: role for growth and L: -lysine production.

    PubMed

    Peters-Wendisch, P; Stansen, K C; Götker, S; Wendisch, V F

    2012-03-01

    Corynebacterium glutamicum is a biotin auxotrophic Gram-positive bacterium that is used for large-scale production of amino acids, especially of L-glutamate and L-lysine. It is known that biotin limitation triggers L-glutamate production and that L-lysine production can be increased by enhancing the activity of pyruvate carboxylase, one of two biotin-dependent proteins of C. glutamicum. The gene cg0814 (accession number YP_225000) has been annotated to code for putative biotin protein ligase BirA, but the protein has not yet been characterized. A discontinuous enzyme assay of biotin protein ligase activity was established using a 105aa peptide corresponding to the carboxyterminus of the biotin carboxylase/biotin carboxyl carrier protein subunit AccBC of the acetyl CoA carboxylase from C. glutamicum as acceptor substrate. Biotinylation of this biotin acceptor peptide was revealed with crude extracts of a strain overexpressing the birA gene and was shown to be ATP dependent. Thus, birA from C. glutamicum codes for a functional biotin protein ligase (EC 6.3.4.15). The gene birA from C. glutamicum was overexpressed and the transcriptome was compared with the control strain revealing no significant gene expression changes of the bio-genes. However, biotin protein ligase overproduction increased the level of the biotin-containing protein pyruvate carboxylase and entailed a significant growth advantage in glucose minimal medium. Moreover, birA overexpression resulted in a twofold higher L-lysine yield on glucose as compared with the control strain.

  2. Crystallization and initial crystallographic characterization of the Corynebacterium glutamicum nitrilotriacetate monooxygenase component A

    SciTech Connect

    Kim, Kyung-Jin; Kim, Sujin; Lee, Sujin; Kang, Beom Sik; Lee, Heung-Soo; Oh, Tae-Kwang; Kim, Myung Hee

    2006-11-01

    The Corynebacterium glutamicum NTA monooxygenase component A protein, which plays the central role in NTA biodegradation, was crystallized. The initial X-ray crystallographic characterization is reported. Safety and environmental concerns have recently dictated the proper disposal of nitrilotriacetate (NTA). Biodegradation of NTA is initiated by NTA monooxygenase, which is composed of two proteins: component A and component B. The NTA monooxygenase component A protein from Corynebacterium glutamicum was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate as the precipitant. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 111.04, b = 98.51, c = 171.61 Å, β = 101.94°. The asymmetric unit consists of four molecules, corresponding to a packing density of 2.3 Å{sup 3} Da{sup −1}. The structure was solved by molecular replacement. Structure refinement is in progress.

  3. Metabolic engineering of Corynebacterium glutamicum for the production of L-ornithine.

    PubMed

    Kim, Seo Yun; Lee, Joungmin; Lee, Sang Yup

    2015-02-01

    L-ornithine is a non-essential amino acid for various industrial applications in food industry. In this study, Corynebacterium glutamicum ATCC 13032 was metabolically engineered for the production of L-ornithine. First, the proB and argF genes were deleted to block the competitive branch pathway and to block the conversion of L-ornithine to citrulline, respectively. In addition, the argR gene encoding the regulatory repressor of the L-arginine operon was also deleted. The resulting strain produced 230 mg/L of L-ornithine from glucose in flask culture. This base strain was further engineered by the plasmid-based overexpression of the argCJBD genes from C. glutamicum ATCC 21831, which resulted in the production of 7.19 g/L of L-ornithine. To enrich the NADPH pool, the carbon flux was redirected towards the pentose phosphate pathway by changing the start codons of the pgi and zwf genes and replacing the native promoter of the tkt operon with the strong sod promoter. Fed-batch cultivation of this final strain YW06 (pSY223) allowed production of 51.5 g/L of L-ornithine in 40 h with the overall productivity of 1.29 g/L/h. The results obtained in this study demonstrate the possibility of efficiently producing L-ornithine by metabolically engineered C. glutamicum.

  4. Chromosome segregation impacts on cell growth and division site selection in Corynebacterium glutamicum.

    PubMed

    Donovan, Catriona; Schauss, Astrid; Krämer, Reinhard; Bramkamp, Marc

    2013-01-01

    Spatial and temporal regulation of bacterial cell division is imperative for the production of viable offspring. In many rod-shaped bacteria, regulatory systems such as the Min system and nucleoid occlusion ensure the high fidelity of midcell divisome positioning. However, regulation of division site selection in bacteria lacking recognizable Min and nucleoid occlusion remains less well understood. Here, we describe one such rod-shaped organism, Corynebacterium glutamicum, which does not always place the division septum precisely at midcell. Here we now show at single cell level that cell growth and division site selection are spatially and temporally regulated by chromosome segregation. Mutants defective in chromosome segregation have more variable cell growth and aberrant placement of the division site. In these mutants, division septa constrict over and often guillotine the nucleoid, leading to nonviable, DNA-free cells. Our results suggest that chromosome segregation or some nucleoid associated factor influences growth and division site selection in C. glutamicum. Understanding growth and regulation of C. glutamicum cells will also be of importance to develop strains for industrial production of biomolecules, such as amino acids.

  5. Chromosome Segregation Impacts on Cell Growth and Division Site Selection in Corynebacterium glutamicum

    PubMed Central

    Donovan, Catriona; Schauss, Astrid; Krämer, Reinhard; Bramkamp, Marc

    2013-01-01

    Spatial and temporal regulation of bacterial cell division is imperative for the production of viable offspring. In many rod-shaped bacteria, regulatory systems such as the Min system and nucleoid occlusion ensure the high fidelity of midcell divisome positioning. However, regulation of division site selection in bacteria lacking recognizable Min and nucleoid occlusion remains less well understood. Here, we describe one such rod-shaped organism, Corynebacterium glutamicum, which does not always place the division septum precisely at midcell. Here we now show at single cell level that cell growth and division site selection are spatially and temporally regulated by chromosome segregation. Mutants defective in chromosome segregation have more variable cell growth and aberrant placement of the division site. In these mutants, division septa constrict over and often guillotine the nucleoid, leading to nonviable, DNA-free cells. Our results suggest that chromosome segregation or some nucleoid associated factor influences growth and division site selection in C. glutamicum. Understanding growth and regulation of C. glutamicum cells will also be of importance to develop strains for industrial production of biomolecules, such as amino acids. PMID:23405112

  6. FudC, a protein primarily responsible for furfural detoxification in Corynebacterium glutamicum.

    PubMed

    Tsuge, Yota; Kudou, Motonori; Kawaguchi, Hideo; Ishii, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

    2016-03-01

    Lignocellulosic hydrolysates contain compounds that inhibit microbial growth and fermentation, thereby decreasing the productivity of biofuel and biochemical production. In particular, the heterocyclic aldehyde furfural is one of the most toxic compounds found in these hydrolysates. We previously demonstrated that Corynebacterium glutamicum converts furfural into the less toxic compounds furfuryl alcohol and 2-furoic acid. To date, however, the genes involved in these oxidation and reduction reactions have not been identified in the C. glutamicum genome. Here, we show that Cgl0331 (designated FudC) is mainly responsible for the reduction of furfural into furfuryl alcohol in C. glutamicum. Deletion of the gene encoding FudC markedly diminished the in vivo reduction of furfural to furfuryl alcohol. Purified His-tagged FudC protein from Escherichia coli was also shown to convert furfural into furfuryl alcohol in an in vitro reaction utilizing NADPH, but not NADH, as a cofactor. Kinetic measurements demonstrated that FudC has a high affinity for furfural but has a narrow substrate range for other aldehydes compared to the protein responsible for furfural reduction in E. coli.

  7. Molecular mechanisms and metabolic engineering of glutamate overproduction in Corynebacterium glutamicum.

    PubMed

    Hirasawa, Takashi; Kim, Jongpill; Shirai, Tomokazu; Furusawa, Chikara; Shimizu, Hiroshi

    2012-01-01

    Glutamate is a commercially important chemical. It is used as a flavor enhancer and is a major raw material for producing industrially useful chemicals. A coryneform bacterium, Corynebacterium glutamicum, was isolated in 1956 by Japanese researchers as a glutamate-overproducing bacterium and since then, remarkable progress in glutamate production has been made using this microorganism. Currently, the global market for glutamate is over 2.5 million tons per year. Glutamate overproduction by C. glutamicum is induced by specific treatments-biotin limitation, addition of fatty acid ester surfactants such as Tween 40, and addition of β-lactam antibiotics such as penicillin. Molecular biology and metabolic engineering studies on glutamate overproduction have revealed that metabolic flow is significantly altered by these treatments. These studies have also provided insight into the molecular mechanisms underlying these changes. In this chapter, we review our current understanding of the molecular mechanisms of glutamate overproduction in C. glutamicum, and we discuss the advances made by metabolic engineering of this microorganism.

  8. IdsA is the major geranylgeranyl pyrophosphate synthase involved in carotenogenesis in Corynebacterium glutamicum.

    PubMed

    Heider, Sabine A E; Peters-Wendisch, Petra; Beekwilder, Jules; Wendisch, Volker F

    2014-11-01

    Corynebacterium glutamicum, a yellow-pigmented soil bacterium that synthesizes the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides, has been engineered for the production of various carotenoids. CrtE was assumed to be the major geranylgeranyl pyrophosphate (GGPP) synthase in carotenogenesis; however, deletion of crtE did not abrogate carotenoid synthesis. In silico analysis of the repertoire of prenyltransferases encoded by the C. glutamicum genome revealed two candidate GGPPS genes (idsA and ispB). The absence of pigmentation of an idsA deletion mutant and complementation experiments with a double deletion mutant lacking both idsA and crtE showed that IdsA is the major GGPPS of C. glutamicum and that crtE overexpression compensated for the lack of IdsA, whereas plasmid-borne overexpression of ispB did not. Purified His-tagged CrtE was active as a homodimer, whereas the active form of IdsA was homotetrameric. Both enzymes catalyzed prenyl transfer with isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesylphosphate (FPP) as substrates. IdsA showed the highest catalytic efficiency with dimethylallyl pyrophosphate and IPP, whereas the catalytic efficiency of CrtE was highest with geranyl pyrophosphate and IPP. Finally, application of prenyltransferase overexpression revealed that combined overexpression of idsA and the IPP isomerase gene idi in the absence of crtE led to the highest decaprenoxanthin titer reported to date.

  9. Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis.

    PubMed

    Küberl, Andreas; Fränzel, Benjamin; Eggeling, Lothar; Polen, Tino; Wolters, Dirk Andreas; Bott, Michael

    2014-06-01

    In a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome-lacking non-pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni(2+)-chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI-TOF-MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins.

  10. Characterization of citrate utilization in Corynebacterium glutamicum by transcriptome and proteome analysis.

    PubMed

    Polen, Tino; Schluesener, Daniela; Poetsch, Ansgar; Bott, Michael; Wendisch, Volker F

    2007-08-01

    Corynebacterium glutamicum grows aerobically on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. To characterize the citrate utilization in C. glutamicum on a genomewide scale, a comparative analysis was carried out by combining transcriptome and proteome analysis. In cells grown on citrate, transcriptome analysis revealed highest expression changes for two different citrate-uptake systems encoded by citM and tctCBA, whereas genes encoding uptake systems for the glucose- (ptsG), sucrose- (ptsS) and fructose- (ptsF) specific PTS components and permeases for gluconate (gntP) and glutamate (gluC) displayed decreased mRNA levels in citrate-grown cells. This pattern was also observed when cells grown in Luria-Bertani (LB) medium plus citrate were compared with cells grown in LB medium, indicating some kind of catabolite repression. Genes encoding enzymes of the tricarboxylic acid cycle (aconitase, succinyl-CoA synthetase, succinate dehydrogenase and fumarase), malic enzyme, PEP carboxykinase, gluconeogenic glyceraldehyde-3-phosphate dehydrogenase and ATP synthase displayed increased expression in cells grown on citrate. Accordingly, proteome analysis revealed elevated protein levels of these enzymes and showed a good correlation with the mRNA levels. In conclusion, this study revealed the citrate stimulon in C. glutamicum and the regulated central metabolic genes when grown on citrate.

  11. Succinate production from CO₂-grown microalgal biomass as carbon source using engineered Corynebacterium glutamicum through consolidated bioprocessing.

    PubMed

    Lee, Jungseok; Sim, Sang Jun; Bott, Michael; Um, Youngsoon; Oh, Min-Kyu; Woo, Han Min

    2014-07-24

    The potential for production of chemicals from microalgal biomass has been considered as an alternative route for CO₂ mitigation and establishment of biorefineries. This study presents the development of consolidated bioprocessing for succinate production from microalgal biomass using engineered Corynebacterium glutamicum. Starch-degrading and succinate-producing C. glutamicum strains produced succinate (0.16 g succinate/g total carbon source) from a mixture of starch and glucose as a model microalgal biomass. Subsequently, the engineered C. glutamicum strains were able to produce succinate (0.28 g succinate/g of total sugars including starch) from pretreated microalgal biomass of CO₂-grown Chlamydomonas reinhardtii. For the first time, this work shows succinate production from CO₂ via sequential fermentations of CO₂-grown microalgae and engineered C. glutamicum. Therefore, consolidated bioprocessing based on microalgal biomass could be useful to promote variety of biorefineries.

  12. Identification of the phd gene cluster responsible for phenylpropanoid utilization in Corynebacterium glutamicum.

    PubMed

    Kallscheuer, Nicolai; Vogt, Michael; Kappelmann, Jannick; Krumbach, Karin; Noack, Stephan; Bott, Michael; Marienhagen, Jan

    2016-02-01

    Phenylpropanoids as abundant, lignin-derived compounds represent sustainable feedstocks for biotechnological production processes. We found that the biotechnologically important soil bacterium Corynebacterium glutamicum is able to grow on phenylpropanoids such as p-coumaric acid, ferulic acid, caffeic acid, and 3-(4-hydroxyphenyl)propionic acid as sole carbon and energy sources. Global gene expression analyses identified a gene cluster (cg0340-cg0341 and cg0344-cg0347), which showed increased transcription levels in response to phenylpropanoids. The gene cg0340 (designated phdT) encodes for a putative transporter protein, whereas cg0341 and cg0344-cg0347 (phdA-E) encode enzymes involved in the β-oxidation of phenylpropanoids. The phd gene cluster is transcriptionally controlled by a MarR-type repressor encoded by cg0343 (phdR). Cultivation experiments conducted with C. glutamicum strains carrying single-gene deletions showed that loss of phdA, phdB, phdC, or phdE abolished growth of C. glutamicum with all phenylpropanoid substrates tested. The deletion of phdD (encoding for putative acyl-CoA dehydrogenase) additionally abolished growth with the α,β-saturated phenylpropanoid 3-(4-hydroxyphenyl)propionic acid. However, the observed growth defect of all constructed single-gene deletion strains could be abolished through plasmid-borne expression of the respective genes. These results and the intracellular accumulation of pathway intermediates determined via LC-ESI-MS/MS in single-gene deletion mutants showed that the phd gene cluster encodes for a CoA-dependent, β-oxidative deacetylation pathway, which is essential for the utilization of phenylpropanoids in C. glutamicum.

  13. Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization.

    PubMed

    Kang, Min-Kyoung; Lee, Jungseok; Um, Youngsoon; Lee, Taek Soon; Bott, Michael; Park, Si Jae; Woo, Han Min

    2014-07-01

    Currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as Escherichia coli and Saccharomyces cerevisiae. In order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called CoryneBrick, for gene expression in Corynebacterium glutamicum as a set of E. coli-C. glutamicum shuttle vectors whose elements are interchangeable with BglBrick standard parts. C. glutamicum is an established industrial microorganism for the production of amino acids, proteins, and commercially promising chemicals. Using the CoryneBrick vectors, we showed various time-dependent expression profiles of a red fluorescent protein. This CoryneBrick platform was also applicable for two-plasmid expression systems with a conventional C. glutamicum expression vector. In order to demonstrate the practical application of the CoryneBrick vectors, we successfully reconstructed the xylose utilization pathway in the xylose-negative C. glutamicum wild type by fast BglBrick cloning methods using multiple genes encoding for xylose isomerase and xylulose kinase, resulting in a growth rate of 0.11 ± 0.004 h(-1) and a xylose uptake rate of 3.35 mmol/gDW/h when 1 % xylose was used as sole carbon source. Thus, CoryneBrick vectors were shown to be useful engineering tools in order to exploit Corynebacterium as a synthetic platform for the production of chemicals by controllable expression of the genes of interest.

  14. Engineering of Corynebacterium glutamicum for growth and L-lysine and lycopene production from N-acetyl-glucosamine.

    PubMed

    Matano, Christian; Uhde, Andreas; Youn, Jung-Won; Maeda, Tomoya; Clermont, Lina; Marin, Kay; Krämer, Reinhard; Wendisch, Volker F; Seibold, Gerd M

    2014-06-01

    Sustainable supply of feedstock has become a key issue in process development in microbial biotechnology. The workhorse of industrial amino acid production Corynebacterium glutamicum has been engineered towards utilization of alternative carbon sources. Utilization of the chitin-derived aminosugar N-acetyl-glucosamine (GlcNAc) for both cultivation and production with C. glutamicum has hitherto not been investigated. Albeit this organism harbors the enzymes N-acetylglucosamine-6-phosphatedeacetylase and glucosamine-6P deaminase of GlcNAc metabolism (encoded by nagA and nagB, respectively) growth of C. glutamicum with GlcNAc as substrate was not observed. This was attributed to the lack of a functional system for GlcNAc uptake. Of the 17 type strains of the genus Corynebacterium tested here for their ability to grow with GlcNAc, only Corynebacterium glycinophilum DSM45794 was able to utilize this substrate. Complementation studies with a GlcNAc-uptake deficient Escherichia coli strain revealed that C. glycinophilum possesses a nagE-encoded EII permease for GlcNAc uptake. Heterologous expression of the C. glycinophilum nagE in C. glutamicum indeed enabled uptake of GlcNAc. For efficient GlcNac utilization in C. glutamicum, improved expression of nagE with concurrent overexpression of the endogenous nagA and nagB genes was found to be necessary. Based on this strategy, C. glutamicum strains for the efficient production of the amino acid L-lysine as well as the carotenoid lycopene from GlcNAc as sole substrate were constructed.

  15. Process inhomogeneity leads to rapid side product turnover in cultivation of Corynebacterium glutamicum

    PubMed Central

    2014-01-01

    Background Corynebacterium glutamicum has large scale industrial applications in the production of amino acids and the potential to serve as a platform organism for new products. This means the demand for industrial process development is likely to increase. However, large scale cultivation conditions differ from laboratory bioreactors, mostly due to the formation of concentration gradients at the industrial scale. This leads to an oscillating supply of oxygen and nutrients for microorganisms with uncertain impact on metabolism. Scale-down bioreactors can be applied to study robustness and physiological reactions to oscillating conditions at a laboratory scale. Results In this study, C. glutamicum ATCC13032 was cultivated by glucose limited fed-batch cultivation in a two-compartment bioreactor consisting of an aerobic stirred tank and a connected non-aerated plug flow reactor with optional feeding. Continuous flow through both compartments generated oscillating profiles with estimated residence times of 45 and 87 seconds in the non-aerated plug flow compartment. Oscillation of oxygen supply conditions at substrate excess and oscillation of both substrate and dissolved oxygen concentration were compared to homogeneous reference cultivations. The dynamic metabolic response of cells within the anaerobic plug flow compartment was monitored throughout the processes, detecting high turnover of substrate into metabolic side products and acidification within oxygen depleted zones. It was shown that anaerobic secretion of lactate into the extracellular culture broth, with subsequent reabsorption in the aerobic glucose-limited environment, leads to mixed-substrate growth in fed-batch processes. Apart from this, the oscillations had only a minor impact on growth and intracellular metabolite characteristics. Conclusions Carbon metabolism of C. glutamicum changes at oscillating oxygen supply conditions, leading to a futile cycle over extracellular side products and back into

  16. Aerobic production of succinate from arabinose by metabolically engineered Corynebacterium glutamicum.

    PubMed

    Chen, Tao; Zhu, Nianqing; Xia, Huihua

    2014-01-01

    Arabinose is considered as an ideal feedstock for the microbial production of value-added chemicals due to its abundance in hemicellulosic wastes. In this study, the araBAD operon from Escherichia coli was introduced into succinate-producing Corynebacterium glutamicum, which enabled aerobic production of succinate using arabinose as sole carbon source. The engineered strain ZX1 (pXaraBAD, pEacsAgltA) produced 74.4 mM succinate with a yield of 0.58 mol (mol arabinose)(-1), which represented 69.9% of the theoretically maximal yield. Moreover, this strain produced 110.2 mM succinate using combined substrates of glucose and arabinose. To date, this is the highest succinate production under aerobic conditions in minimal medium.

  17. Impact of pulsed electric fields on Corynebacterium glutamicum cell membrane permeabilization.

    PubMed

    Tryfona, Theodora; Bustard, Mark T

    2008-04-01

    The permeability barrier of the microbial cell envelope for substrates and products often causes very low reaction rates of whole cells. Therefore, it is of interest to develop an effective method to reduce this permeability barrier in order to increase product yields. Utilisation of pulse electric fields may improve amino acid release from Corynebacterium glutamicum by up to several orders of magnitude. In particular pulsed electric fields may change the cell/membrane's dielectric properties and induce the release of intracellular metabolites. In this study the parameters for successful electropermeabilization were determined and the viabilities of treated cells were examined. We also found that pulse treated cells not only maintained their viabilities but also their ability to reproduce, post-pulse treatment. Since electropermeabilized cells could maintain both their viabilities and ability to reproduce, we believe that this preliminary data may contribute to the optimization of fermentative production of amino acids and bioprocess enhancement through electropermeabilization and may be beneficial to industrial bioprocesses.

  18. PorA Represents the Major Cell Wall Channel of the Gram-Positive Bacterium Corynebacterium glutamicum

    PubMed Central

    Costa-Riu, Noelia; Burkovski, Andreas; Krämer, Reinhard; Benz, Roland

    2003-01-01

    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 13032ΔporA. 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. PMID:12896997

  19. Formation of xylitol and xylitol-5-phosphate and its impact on growth of d-xylose-utilizing Corynebacterium glutamicum strains.

    PubMed

    Radek, Andreas; Müller, Moritz-Fabian; Gätgens, Jochem; Eggeling, Lothar; Krumbach, Karin; Marienhagen, Jan; Noack, Stephan

    2016-08-10

    Wild-type Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the lignocellulosic pentose d-xylose for cell growth. Therefore, two different engineering approaches have been pursued resulting in platform strains harbouring a functional version of either the Isomerase (ISO) or the Weimberg (WMB) pathway for d-xylose assimilation. In a previous study we found for C. glutamicum WMB by-product formation of xylitol during growth on d-xylose and speculated that the observed lower growth rates are due to the growth inhibiting effect of this compound. Based on a detailed phenotyping of the ISO, WMB and the wild-type strain of C. glutamicum, we here show that this organism has a natural capability to synthesize xylitol from d-xylose under aerobic cultivation conditions. We furthermore observed the intracellular accumulation of xylitol-5-phosphate as a result of the intracellular phosphorylation of xylitol, which was particularly pronounced in the C. glutamicum ISO strain. Interestingly, low amounts of supplemented xylitol strongly inhibit growth of this strain on d-xylose, d-glucose and d-arabitol. These findings demonstrate that xylitol is a suitable substrate of the endogenous xylulokinase (XK, encoded by xylB) and its overexpression in the ISO strain leads to a significant phosphorylation of xylitol in C. glutamicum. Therefore, in order to circumvent cytotoxicity by xylitol-5-phosphate, the WMB pathway represents an interesting alternative route for engineering C. glutamicum towards efficient d-xylose utilization.

  20. Fermentative Production of the Diamine Putrescine: System Metabolic Engineering of Corynebacterium Glutamicum

    PubMed Central

    Nguyen, Anh Q. D.; Schneider, Jens; Reddy, Gajendar Komati; Wendisch, Volker F.

    2015-01-01

    Corynebacterium glutamicum shows great potential for the production of the glutamate-derived diamine putrescine, a monomeric compound of polyamides. A genome-scale stoichiometric model of a C. glutamicum strain with reduced ornithine transcarbamoylase activity, derepressed arginine biosynthesis, and an anabolic plasmid-addiction system for heterologous expression of E. coli ornithine decarboxylase gene speC was investigated by flux balance analysis with respect to its putrescine production potential. Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering. Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%. Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant. The resulting C. glutamicum strain NA6 obtained by systems metabolic engineering accumulated two fold more putrescine than the base strain, i.e., 58.1 ± 0.2 mM, and showed a specific productivity of 0.045 g·g−1·h−1 and a yield on glucose of 0.26 g·g−1. PMID:25919117

  1. C1 Metabolism in Corynebacterium glutamicum: an Endogenous Pathway for Oxidation of Methanol to Carbon Dioxide

    PubMed Central

    Witthoff, Sabrina; Mühlroth, Alice

    2013-01-01

    Methanol is considered an interesting carbon source in “bio-based” microbial production processes. Since Corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies of the response of this organism to methanol. The C. glutamicum wild type was able to convert 13C-labeled methanol to 13CO2. Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be upregulated, indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol dehydrogenase gene adhA showed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde. Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The Δald ΔadhE and Δald ΔmshC deletion mutants were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO2 was still possible. The oxidation of formate to CO2 is catalyzed by the formate dehydrogenase FdhF, recently identified by us. Similar to the case with ethanol, methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for expression of adhA and ald. In conclusion, we were able to show that C. glutamicum possesses an endogenous pathway for methanol oxidation to CO2 and to identify the enzymes and a transcriptional regulator involved in this pathway. PMID:24014532

  2. Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production.

    PubMed

    Lai, Shujuan; Zhang, Yun; Liu, Shuwen; Liang, Yong; Shang, Xiuling; Chai, Xin; Wen, Tingyi

    2012-04-01

    L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDH(r)). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached (14.22 ± 1.41) μmol g(CDM) (-1), which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDH(r) improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C(1) units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for L-serine production in C. glutamicum.

  3. C1 metabolism in Corynebacterium glutamicum: an endogenous pathway for oxidation of methanol to carbon dioxide.

    PubMed

    Witthoff, Sabrina; Mühlroth, Alice; Marienhagen, Jan; Bott, Michael

    2013-11-01

    Methanol is considered an interesting carbon source in "bio-based" microbial production processes. Since Corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies of the response of this organism to methanol. The C. glutamicum wild type was able to convert (13)C-labeled methanol to (13)CO2. Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be upregulated, indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol dehydrogenase gene adhA showed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde. Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The Δald ΔadhE and Δald ΔmshC deletion mutants were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO2 was still possible. The oxidation of formate to CO2 is catalyzed by the formate dehydrogenase FdhF, recently identified by us. Similar to the case with ethanol, methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for expression of adhA and ald. In conclusion, we were able to show that C. glutamicum possesses an endogenous pathway for methanol oxidation to CO2 and to identify the enzymes and a transcriptional regulator involved in this pathway.

  4. A proteomic study of Corynebacterium glutamicum AAA+ protease FtsH

    PubMed Central

    Lüdke, Alja; Krämer, Reinhard; Burkovski, Andreas; Schluesener, Daniela; Poetsch, Ansgar

    2007-01-01

    Background The influence of the membrane-bound AAA+ protease FtsH on membrane and cytoplasmic proteins of Corynebacterium glutamicum was investigated in this study. For the analysis of the membrane fraction, anion exchange chromatography was combined with SDS-PAGE, while the cytoplasmic protein fraction was studied by conventional two-dimensional gel electrophoresis. Results In contrast to the situation in other bacteria, deletion of C. glutamicum ftsH has no significant effect on growth in standard minimal medium or response to heat or osmotic stress. On the proteome level, deletion of the ftsH gene resulted in a strong increase of ten cytoplasmic and membrane proteins, namely biotin carboxylase/biotin carboxyl carrier protein (accBC), glyceraldehyde-3-phosphate dehydrogenase (gap), homocysteine methyltransferase (metE), malate synthase (aceB), isocitrate lyase (aceA), a conserved hypothetical protein (NCgl1985), succinate dehydrogenase A (sdhA), succinate dehydrogenase B (sdhB), succinate dehydrogenase CD (sdhCD), and glutamate binding protein (gluB), while 38 cytoplasmic and membrane-associated proteins showed a decreased abundance. The decreasing amount of succinate dehydrogenase A (sdhA) in the cytoplasmic fraction of the ftsH mutant compared to the wild type and its increasing abundance in the membrane fraction indicates that FtsH might be involved in the cleavage of a membrane anchor of this membrane-associated protein and by this changes its localization. Conclusion The data obtained hint to an involvement of C. glutamicum FtsH protease mainly in regulation of energy and carbon metabolism, while the protease is not involved in stress response, as found in other bacteria. PMID:17254330

  5. Membrane alteration is necessary but not sufficient for effective glutamate secretion in Corynebacterium glutamicum.

    PubMed Central

    Hoischen, C; Krämer, R

    1990-01-01

    We showed recently that secretion of glutamate in biotin-limited cells of Corynebacterium glutamicum is mediated by carrier systems in the plasma membrane (C. Hoischen and R. Krämer, Arch. Microbiol. 151:342-347, 1989). In view of the generally accepted hypothesis that glutamate efflux is directly caused by alterations of the membrane, it was necessary to examine the kind of correlation between changes in lipid content and composition of the bacterial membrane and glutamate secretion activity. Two new experimental approaches were used. (i) Changes in lipid content and composition were analyzed in glutamate-producing cells which were forced to switch to nonproducers by addition of biotin in a short-term fermentation. (ii) The time courses of both the fatty acid or phospholipid composition and the efflux activity were analyzed within the first minutes of the switch from high to low secretion activity. The following results were obtained. (i) The time course of the change in fatty acid or phospholipid content and composition was not related to the change in secretion behavior. (ii) There was no specific fatty acid or phospholipid compound which regulated glutamate efflux. (iii) High efflux activity could only be induced when the total lipid content of the membrane was reduced. (iv) Although consistently correlated to high secretion activity, membrane alteration was never a sufficient prerequisite for glutamate efflux in C. glutamicum. PMID:1971623

  6. Metabolic engineering of Corynebacterium glutamicum for the de novo production of ethylene glycol from glucose.

    PubMed

    Chen, Zhen; Huang, Jinhai; Wu, Yao; Liu, Dehua

    2016-01-01

    Development of sustainable biological process for the production of bulk chemicals from renewable feedstock is an important goal of white biotechnology. Ethylene glycol (EG) is a large-volume commodity chemical with an annual production of over 20 million tons, and it is currently produced exclusively by petrochemical route. Herein, we report a novel biosynthetic route to produce EG from glucose by the extension of serine synthesis pathway of Corynebacterium glutamicum. The EG synthesis is achieved by the reduction of glycoaldehyde derived from serine. The transformation of serine to glycoaldehyde is catalyzed either by the sequential enzymatic deamination and decarboxylation or by the enzymatic decarboxylation and oxidation. We screened the corresponding enzymes and optimized the production strain by combinatorial optimization and metabolic engineering. The best engineered C. glutamicum strain is able to accumulate 3.5 g/L of EG with the yield of 0.25 mol/mol glucose in batch cultivation. This study lays the basis for developing an efficient biological process for EG production.

  7. Production of L-lysine on different silage juices using genetically engineered Corynebacterium glutamicum.

    PubMed

    Neuner, Andreas; Wagner, Ines; Sieker, Tim; Ulber, Roland; Schneider, Konstantin; Peifer, Susanne; Heinzle, Elmar

    2013-01-20

    Corynebacterium glutamicum, the best established industrial producer organism for lysine was genetically modified to allow the production of lysine on grass and corn silages. The resulting strain C. glutamicum lysC(fbr)dld(Psod)pyc(Psod)malE(Psod)fbp(Psod)gapX(Psod) was based on earlier work (Neuner and Heinzle, 2011). That mutant carries a point mutation in the aspartokinase (lysC) regulatory subunit gene as well as overexpression of D-lactate dehydrogenase (dld), pyruvate carboxylase (pyc) and malic enzyme (malE) using the strong Psod promoter. Here, we additionally overexpressed fructose 1,6-bisphosphatase (fbp) and glyceraldehyde 3-phosphate dehydrogenase (gapX) using the same promoter. The resulting strain grew readily on grass and corn silages with a specific growth rate of 0.35 h⁻¹ and lysine carbon yields of approximately 90 C-mmol (C-mol)⁻¹. Lysine yields were hardly affected by oxygen limitation whereas linear growth was observed under oxygen limiting conditions. Overall, this strain seems very robust with respect to the composition of silage utilizing all quantified low molecular weight substrates, e.g. lactate, glucose, fructose, maltose, quinate, fumarate, glutamate, leucine, isoleucine and alanine.

  8. Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum.

    PubMed

    Vogt, Michael; Brüsseler, Christian; Ooyen, Jan van; Bott, Michael; Marienhagen, Jan

    2016-11-01

    The pentanol isomers 2-methyl-1-butanol and 3-methyl-1-butanol represent commercially interesting alcohols due to their potential application as biofuels. For a sustainable microbial production of these compounds, Corynebacterium glutamicum was engineered for producing 2-methyl-1-butanol and 3-methyl-1-butanol via the Ehrlich pathway from 2-keto-3-methylvalerate and 2-ketoisocaproate, respectively. In addition to an already available 2-ketoisocaproate producer, a 2-keto-3-methylvalerate accumulating C. glutamicum strain was also constructed. For this purpose, we reduced the activity of the branched-chain amino acid transaminase in an available C. glutamicuml-isoleucine producer (K2P55) via a start codon exchange in the ilvE gene enabling accumulation of up to 3.67g/l 2-keto-3-methylvalerate. Subsequently, nine strains expressing different gene combinations for three 2-keto acid decarboxylases and three alcohol dehydrogenases were constructed and characterized. The best strains accumulated 0.37g/l 2-methyl-1-butanol and 2.76g/l 3-methyl-1-butanol in defined medium within 48h under oxygen deprivation conditions, making these strains ideal candidates for additional strain and process optimization.

  9. tRNA-dependent alanylation of diacylglycerol and phosphatidylglycerol in Corynebacterium glutamicum.

    PubMed

    Smith, Angela M; Harrison, Jesse S; Grube, Christopher D; Sheppe, Austin E F; Sahara, Nahoko; Ishii, Ryohei; Nureki, Osamu; Roy, Hervé

    2015-11-01

    Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are membrane proteins that utilize aminoacylated tRNAs to modify membrane lipids with amino acids. Aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane and enables bacteria to adapt to changes in environmental conditions. aaPGSs utilize alanine, lysine and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (PG) and cardiolipin. Here we identify a new pathway for lipid aminoacylation, conserved in many Actinobacteria, which results in formation of Ala-PG and a novel alanylated lipid, Alanyl-diacylglycerol (Ala-DAG). Ala-DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, whereas formation of Ala-PG requires the same enzyme acting in concert with a putative esterase encoded upstream. The presence of alanylated lipids is sufficient to enhance the bacterial fitness of C. glutamicum cultured in the presence of certain antimicrobial agents, and elucidation of this system expands the known repertoire of membrane lipids acting as substrates for amino acid modification in bacterial cells.

  10. Expression, crystallization and preliminary crystallographic study of GluB from Corynebacterium glutamicum

    PubMed Central

    Liu, Qingbo; Li, Defeng; Hu, Yonglin; Wang, Da-Cheng

    2013-01-01

    GluB is a substrate-binding protein (SBP) which participates in the uptake of glutamic acid in Corynebacterium glutamicum, a Gram-positive bacterium. It is part of an ATP-binding cassette (ABC) transporter system. Together with the transmembrane proteins GluC and GluD and the cytoplasmic protein GluA, which couples the hydrolysis of ATP to the translocation of glutamate, they form a highly active glutamate-uptake system. As part of efforts to study the amino-acid metabolism, especially the metabolism of glutamic acid by C. glutamicum, a bacterium that is widely used in the industrial production of glutamic acid, the GluB protein was expressed, purified and crystallized, an X-ray diffraction data set was collected to a resolution of 1.9 Å and preliminary crystallographic analysis was performed. The crystal belonged to space group P3121 or P3221, with unit-cell parameters a = b = 82.50, c = 72.69 Å. PMID:23722846

  11. Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum

    PubMed Central

    Koffas, Mattheos A. G.; Jung, Gyoo Yeol; Aon, Juan C.; Stephanopoulos, Gregory

    2002-01-01

    Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology. PMID:12406733

  12. Transcriptomic analysis for elucidating the physiological effects of 5-aminolevulinic acid accumulation on Corynebacterium glutamicum.

    PubMed

    Yu, Xiaoli; Jin, Haiying; Cheng, Xuelian; Wang, Qian; Qi, Qingsheng

    2016-11-01

    5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthetic pathway, attracts close attention among researchers because of its potential applications to cancer treatment and agriculture. Overexpression of heterologous hemA and hemL, which encode glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase, respectively, in Corynebacterium glutamicum produces ALA, although whether ALA accumulation causes unintended effects on the host is unknown. Here we used an integrated systems approach to compare global transcriptional changes induced by the expression of hemA and hemL. Metabolic pathway such as glycolysis was inhibited, but tricarboxylic acid cycle, pentose phosphate pathway, and respiratory metabolism were stimulated. Moreover, the transcriptional levels of certain genes involved in heme biosynthesis were up-regulated, and the data implicate the two-component system (TCS) HrrSA was involved in the regulation of heme synthesis. With these understandings, it is proposed that ALA accumulation stimulates heme synthesis pathway and respiratory metabolism. Our study illuminates the physiological effects of overexpressing hemA and hemL on the phenotype of C. glutamicum and contributes important insights into the regulatory mechanisms of the heme biosynthetic pathways.

  13. tRNA-dependent alanylation of diacylglycerol and phosphatidylglycerol in Corynebacterium glutamicum

    PubMed Central

    Smith, Angela M.; Harrison, Jesse S.; Grube, Christopher D.; Sheppe, Austin E.F.; Sahara, Nahoko; Ishii, Ryohei; Nureki, Osamu; Roy, Hervé

    2015-01-01

    Summary Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are membrane proteins that utilize aminoacylated tRNAs to modify membrane lipids with amino acids. Aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane, and enables bacteria to adapt to changes in environmental conditions. aaPGSs utilize alanine, lysine, and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (PG) and cardiolipin. Here we identify a new pathway for lipid aminoacylation, conserved in many Actinobacteria, which results in formation of Ala-PG and a novel alanylated lipid, Ala-diacylglycerol (Ala-DAG). Ala-DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, while formation of Ala-PG requires the same enzyme acting in concert with a putative esterase encoded upstream. The presence of alanylated lipids is sufficient to enhance the bacterial fitness of C. glutamicum cultured in the presence of certain antimicrobial agents, and elucidation of this system expands the known repertoire of membrane lipids acting as substrates for amino acid modification in bacterial cells. PMID:26235234

  14. The small 6C RNA of Corynebacterium glutamicum is involved in the SOS response.

    PubMed

    Pahlke, Jennifer; Dostálová, Hana; Holátko, Jiří; Degner, Ursula; Bott, Michael; Pátek, Miroslav; Polen, Tino

    2016-09-01

    The 6C RNA family is a class of small RNAs highly conserved in Actinobacteria, including the genera Mycobacterium, Streptomyces and Corynebacterium whose physiological function has not yet been elucidated. We found that strong transcription of the cgb_03605 gene, which encodes 6C RNA in C. glutamicum, was driven by the SigA- and SigB-dependent promoter Pcgb_03605. 6C RNA was detected at high level during exponential growth phase (180 to 240 molcules per cell) which even increased at the entry of the stationary phase. 6C RNA level did not decrease within 240 min after transcription had been stopped with rifampicin, which suggests high 6C RNA stability. The expression of cgb_03605 further increased approximately twofold in the presence of DNA-damaging mitomycin C (MMC) and nearly threefold in the absence of LexA. Deletion of the 6C RNA gene cgb_03605 resulted in a higher sensitivity of C. glutamicum toward MMC and UV radiation. These results indicate that 6C RNA is involved in the DNA damage response. Both 6C RNA level-dependent pausing of cell growth and branched cell morphology in response to MMC suggest that 6C RNA may also be involved in a control of cell division.

  15. Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum.

    PubMed

    Mahr, Regina; Gätgens, Cornelia; Gätgens, Jochem; Polen, Tino; Kalinowski, Jörn; Frunzke, Julia

    2015-11-01

    Adaptive laboratory evolution has proven a valuable strategy for metabolic engineering. Here, we established an experimental evolution approach for improving microbial metabolite production by imposing an artificial selective pressure on the fluorescent output of a biosensor using fluorescence-activated cell sorting. Cells showing the highest fluorescent output were iteratively isolated and (re-)cultivated. The L-valine producer Corynebacterium glutamicum ΔaceE was equipped with an L-valine-responsive sensor based on the transcriptional regulator Lrp of C. glutamicum. Evolved strains featured a significantly higher growth rate, increased L-valine titers (~25%) and a 3-4-fold reduction of by-product formation. Genome sequencing resulted in the identification of a loss-of-function mutation (UreD-E188*) in the gene ureD (urease accessory protein), which was shown to increase L-valine production by up to 100%. Furthermore, decreased L-alanine formation was attributed to a mutation in the global regulator GlxR. These results emphasize biosensor-driven evolution as a straightforward approach to improve growth and productivity of microbial production strains.

  16. Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum

    PubMed Central

    Shen, Xi-Hui; Jiang, Cheng-Ying; Huang, Yan; Liu, Zhi-Pei; Liu, Shuang-Jiang

    2005-01-01

    Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Δncg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified. PMID:16000747

  17. Recent advances in the metabolic engineering of Corynebacterium glutamicum for the production of lactate and succinate from renewable resources.

    PubMed

    Tsuge, Yota; Hasunuma, Tomohisa; Kondo, Akihiko

    2015-03-01

    Recent increasing attention to environmental issues and the shortage of oil resources have spurred political and industrial interest in the development of environmental friendly and cost-effective processes for the production of bio-based chemicals from renewable resources. Thus, microbial production of commercially important chemicals is viewed as a desirable way to replace current petrochemical production. Corynebacterium glutamicum, a Gram-positive soil bacterium, is one of the most important industrial microorganisms as a platform for the production of various amino acids. Recent research has explored the use of C. glutamicum as a potential cell factory for producing organic acids such as lactate and succinate, both of which are commercially important bulk chemicals. Here, we summarize current understanding in this field and recent metabolic engineering efforts to develop C. glutamicum strains that efficiently produce L- and D-lactate, and succinate from renewable resources.

  18. Corynebacterium glutamicum harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate.

    PubMed

    Witthoff, Sabrina; Eggeling, Lothar; Bott, Michael; Polen, Tino

    2012-09-01

    Here, we show that Corynebacterium glutamicum ATCC 13032 co-metabolizes formate when it is grown with glucose as the carbon and energy source. CO(2) measurements during bioreactor cultivation and use of (13)C-labelled formate demonstrated that formate is almost completely oxidized to CO(2). The deletion of fdhF (cg0618), annotated as formate dehydrogenase (FDH) and located in a cluster of genes conserved in the family Corynebacteriaceae, prevented formate utilization. Similarly, deletion of fdhD (cg0616) resulted in the inability to metabolize formate and deletion of cg0617 markedly reduced formate utilization. These results illustrated that all three gene products are required for FDH activity. Growth studies with molybdate and tungstate indicated that the FDH from C. glutamicum ATCC 13032 is a molybdenum-dependent enzyme. The presence of 100 mM formate caused a 25 % lowered growth rate during cultivation of C. glutamicum ATCC 13032 wild-type in glucose minimal medium. This inhibitory effect was increased in the strains lacking FDH activity. Our data demonstrate that C. glutamicum ATCC 13032 possesses an FDH with a currently unknown electron acceptor. The presence of the FDH might help the soil bacterium C. glutamicum ATCC 13032 to alleviate growth retardation caused by formate, which is ubiquitously present in the environment.

  19. Efficient hydroxyproline production from glucose in minimal media by Corynebacterium glutamicum.

    PubMed

    Falcioni, Francesco; Bühler, Bruno; Schmid, Andreas

    2015-02-01

    The efficient coupling of biotransformation steps to an existing fermentation pathway is an interesting strategy to expand the product portfolio of Corynebacterium glutamicum as whole-cell biocatalyst. This is especially challenging if the biotransformation step comprises a direct link to central metabolism, as in the case of α-ketoglutarate-dependent oxygenase catalysis. Aiming at trans-4-hydroxy-L-proline (Hyp) production from glucose in a minimal medium, the proline-4-hydroxylase gene from Dactylosporangium sp. strain RH1 was introduced into a proline-producing, isoleucine-bradytroph C. glutamicum strain. The production of proline was found to be induced by isoleucine limitation. Proline and Hyp production were found to depend differently on isoleucine limitation. Severe isoleucine limitation was shown to result in proline accumulation and low hydroxylation rates both in batch and continuous cultivation set-ups. The investigation of different steady states with various glucose/isoleucine molar ratios revealed that optimal conditions for Hyp production are met around a molar ratio of 46:1, where isoleucine limitation is sufficient to trigger proline production but the hydroxylation rate is high enough to convert the majority of formed proline to Hyp. A high cell-density fed-batch set-up was designed, capable of producing 7.1 g L(-1) of Hyp from glucose in 23 h with 98.5% conversion of proline to Hyp. Reaction engineering, specifically the fine-tuning of the glucose/isoleucine concentration ratio, enabled control of the fermentation profile and thus the accumulation of the desired product Hyp from glucose in minimal and defined media.

  20. Enhancing pentose phosphate pathway in Corynebacterium glutamicum to improve l-isoleucine production.

    PubMed

    Ma, Wenjian; Wang, Jianli; Li, Ye; Hu, Xiaoqing; Shi, Feng; Wang, Xiaoyuan

    2016-11-01

    Three genes, gnd, pgl, and fbp, relevant to the pentose phosphate pathway (PPP) were overexpressed in Corynebacterium glutamicum IWJ001, leading to increase of l-isoleucine production. The transcriptional levels of gnd, pgl, and fbp significantly increased in IWJ001/pDXW-8-gnd-fbp-pgl. Compared with the control strain IWJ001/pDXW-8, intracellular NADPH/NADP(+) ratios in IWJ001/pDXW-8-gnd and IWJ001/pDXW-8-gnd-fbp cells grown for 36 H increased threefold and fourfold, respectively, indicating that overexpression of gnd and fbp redirected the carbon flux to PPP. Intracellular NADPH/NADP(+) ratio in IWJ001/pDXW-8-gnd-fbp-pgl grown for 36 H was similar to IWJ001/pDXW-8, suggesting that the NADPH produced by PPP could be quickly consumed for l-isoleucine production. 10.9 and 28.96 g/L of l-isoleucine was produced in IWJ001/pDXW-8-gnd-fbp-pgl in shake flask cultivation and fed-batch fermentation, respectively. In addition, IWJ001/pDXW-8-gnd-fbp-pgl grew fast, its dry cell weight reached 49 g/L after 48 H, whereas the start strain IWJ001/pDXW-8 reached only 40 g/L. After 96 H fermentation, l-isoleucine yield on glucose in IWJ001/pDXW-8-gnd-fbp-pgl reached 0.138 g/g. The results demonstrate that carbon flux redirection to PPP is an efficient approach to enhance l-isoleucine production in C. glutamicum.

  1. Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction.

    PubMed

    Vogt, Michael; Haas, Sabine; Klaffl, Simon; Polen, Tino; Eggeling, Lothar; van Ooyen, Jan; Bott, Michael

    2014-03-01

    Using metabolic engineering, an efficient L-leucine production strain of Corynebacterium glutamicum was developed. In the wild type of C. glutamicum, the leuA-encoded 2-isopropylmalate synthase (IPMS) is inhibited by low L-leucine concentrations with a K(i) of 0.4 mM. We identified a feedback-resistant IMPS variant, which carries two amino acid exchanges (R529H, G532D). The corresponding leuA(fbr) gene devoid of the attenuator region and under control of a strong promoter was integrated in one, two or three copies into the genome and combined with additional genomic modifications aimed at increasing L-leucine production. These modifications involved (i) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (ii) deletion of the gene encoding the transcriptional regulator IolR to increase glucose uptake, (iii) reduction of citrate synthase activity to increase precursor supply, and (iv) introduction of a gene encoding a feedback-resistant acetohydroxyacid synthase. The production performance of the resulting strains was characterized in bioreactor cultivations. Under fed-batch conditions, the best producer strain accumulated L-leucine to levels exceeding the solubility limit of about 24 g/l. The molar product yield was 0.30 mol L-leucine per mol glucose and the volumetric productivity was 4.3 mmol l⁻¹ h⁻¹. These values were obtained in a defined minimal medium with a prototrophic and plasmid-free strain, making this process highly interesting for industrial application.

  2. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    PubMed

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum.

  3. Thermal and Solvent Stress Cross-Tolerance Conferred to Corynebacterium glutamicum by Adaptive Laboratory Evolution

    PubMed Central

    Oide, Shinichi; Gunji, Wataru; Moteki, Yasuhiro; Yamamoto, Shogo; Suda, Masako; Jojima, Toru; Yukawa, Hideaki

    2015-01-01

    Reinforcing microbial thermotolerance is a strategy to enable fermentation with flexible temperature settings and thereby to save cooling costs. Here, we report on adaptive laboratory evolution (ALE) of the amino acid-producing bacterium Corynebacterium glutamicum under thermal stress. After 65 days of serial passage of the transgenic strain GLY3, in which the glycolytic pathway is optimized for alanine production under oxygen deprivation, three strains adapted to supraoptimal temperatures were isolated, and all the mutations they acquired were identified by whole-genome resequencing. Of the 21 mutations common to the three strains, one large deletion and two missense mutations were found to promote growth of the parental strain under thermal stress. Additive effects on thermotolerance were observed among these mutations, and the combination of the deletion with the missense mutation on otsA, encoding a trehalose-6-phosphate synthase, allowed the parental strain to overcome the upper limit of growth temperature. Surprisingly, the three evolved strains acquired cross-tolerance for isobutanol, which turned out to be partly attributable to the genomic deletion associated with the enhanced thermotolerance. The deletion involved loss of two transgenes, pfk and pyk, encoding the glycolytic enzymes, in addition to six native genes, and elimination of the transgenes, but not the native genes, was shown to account for the positive effects on thermal and solvent stress tolerance, implying a link between energy-producing metabolism and bacterial stress tolerance. Overall, the present study provides evidence that ALE can be a powerful tool to refine the phenotype of C. glutamicum and to investigate the molecular bases of stress tolerance. PMID:25595768

  4. Towards methionine overproduction in Corynebacterium glutamicum--methanethiol and dimethyldisulfide as reduced sulfur sources.

    PubMed

    Bolten, Christoph J; Schröder, Hartwig; Dickschat, Jeroen; Wittmann, Christoph

    2010-08-01

    In the present work, methanethiol and dimethyldisulfide were investigated as sulfur source for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis has predicted a high methionine yield for these reduced compounds provided that they can be utilized. Wild type cells were able to grow on methanethiol and on dimethyldisulfide as sole sulfur source, respectively. Isotope labeling studies with mutant strains exhibiting targeted modification of methionine biosynthesis gave detailed insight into the underlying pathways involved in assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as entire molecule, adding the terminal S-CH3 group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as enzyme catalyzing this reaction. Deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur source. Plasmid based overexpression of metY in the delta metY background restored the capability to grow on methanethiol or dimethyldisulfide as sole sulfur source. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased two-fold in the engineered strain. This positive effect was limited by depletion of the metY substrate O-acetylhomoserine, requesting for further metabolic engineering targets towards competitive production strains.

  5. Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number.

    PubMed Central

    Nesvera, J; Pátek, M; Hochmannová, J; Abrhámová, Z; Becvárová, V; Jelínkova, M; Vohradský, J

    1997-01-01

    The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode. PMID:9045809

  6. Transcriptional Regulation of the β-Type Carbonic Anhydrase Gene bca by RamA in Corynebacterium glutamicum.

    PubMed

    Shah, Adnan; Eikmanns, Bernhard J

    2016-01-01

    Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide "A" of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an "A" residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene.

  7. Characterization of a Unique Pathway for 4-Cresol Catabolism Initiated by Phosphorylation in Corynebacterium glutamicum*

    PubMed Central

    Du, Lei; Ma, Li; Qi, Feifei; Zheng, Xianliang; Jiang, Chengying; Li, Ailei; Wan, Xiaobo; Liu, Shuang-Jiang; Li, Shengying

    2016-01-01

    4-Cresol is not only a significant synthetic intermediate for production of many aromatic chemicals, but also a priority environmental pollutant because of its toxicity to higher organisms. In our previous studies, a gene cluster implicated to be involved in 4-cresol catabolism, creCDEFGHIR, was identified in Corynebacterium glutamicum and partially characterized in vivo. In this work, we report on the discovery of a novel 4-cresol biodegradation pathway that employs phosphorylated intermediates. This unique pathway initiates with the phosphorylation of the hydroxyl group of 4-cresol, which is catalyzed by a novel 4-methylbenzyl phosphate synthase, CreHI. Next, a unique class I P450 system, CreJEF, specifically recognizes phosphorylated intermediates and successively oxidizes the aromatic methyl group into carboxylic acid functionality via alcohol and aldehyde intermediates. Moreover, CreD (phosphohydrolase), CreC (alcohol dehydrogenase), and CreG (aldehyde dehydrogenase) were also found to be required for efficient oxidative transformations in this pathway. Steady-state kinetic parameters (Km and kcat) for each catabolic step were determined, and these results suggest that kinetic controls serve a key role in directing the metabolic flux to the most energy effective route. PMID:26817843

  8. Structural Insights into a Novel Class of Aspartate Aminotransferase from Corynebacterium glutamicum

    PubMed Central

    Son, Hyeoncheol Francis; Kim, Kyung-Jin

    2016-01-01

    Aspartate aminotransferase from Corynebacterium glutamicum (CgAspAT) is a PLP-dependent enzyme that catalyzes the production of L-aspartate and α-ketoglutarate from L-glutamate and oxaloacetate in L-lysine biosynthesis. In order to understand the molecular mechanism of CgAspAT and compare it with those of other aspartate aminotransferases (AspATs) from the aminotransferase class I, we determined the crystal structure of CgAspAT. CgAspAT functions as a dimer, and the CgAspAT monomer consists of two domains, the core domain and the auxiliary domain. The PLP cofactor is found to be bound to CgAspAT and stabilized through unique residues. In our current structure, a citrate molecule is bound at the active site of one molecule and mimics binding of the glutamate substrate. The residues involved in binding of the PLP cofactor and the glutamate substrate were confirmed by site-directed mutagenesis. Interestingly, compared with other AspATs from aminotransferase subgroup Ia and Ib, CgAspAT exhibited unique binding sites for both cofactor and substrate; moreover, it was found to have unusual structural features in the auxiliary domain. Based on these structural differences, we propose that CgAspAT does not belong to either subgroup Ia or Ib, and can be categorized into a subgroup Ic. The phylogenetic tree and RMSD analysis also indicates that CgAspAT is located in an independent AspAT subgroup. PMID:27355211

  9. Dihydroxyacetone production in an engineered Escherichia coli through expression of Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase.

    PubMed

    Jain, Vishist Kumar; Tear, Crystal Jing Ying; Lim, Chan Yuen

    2016-05-01

    Dihydroxyacetone (DHA) has several industrial applications such as a tanning agent in tanning lotions in the cosmetic industry; its production via microbial fermentation would present a more sustainable option for the future. Here we genetically engineered Escherichia coli (E. coli) for DHA production from glucose. Deletion of E. coli triose phosphate isomerase (tpiA) gene was carried out to accumulate dihydroxyacetone phosphate (DHAP), for use as the main intermediate or precursor for DHA production. The accumulated DHAP was then converted to DHA through the heterologous expression of Corynebacterium glutamicum DHAP dephosphorylase (cghdpA) gene. To conserve DHAP exclusively for DHA production we removed methylglyoxal synthase (mgsA) gene in the ΔtpiA strain. This drastically improved DHA production from 0.83g/l (0.06g DHA/g glucose) in the ΔtpiA strain bearing cghdpA to 5.84g/l (0.41g DHA/g glucose) in the ΔtpiAΔmgsA double mutant containing the same gene. To limit the conversion of intracellular DHA to glycerol, glycerol dehydrogenase (gldA) gene was further knocked out resulting in a ΔtpiAΔmgsAΔgldA triple mutant. This triple mutant expressing the cghdpA gene produced 6.60g/l of DHA at 87% of the maximum theoretical yield. In summary, we demonstrated an efficient system for DHA production in genetically engineered E. coli strain.

  10. Characterization of a Unique Pathway for 4-Cresol Catabolism Initiated by Phosphorylation in Corynebacterium glutamicum.

    PubMed

    Du, Lei; Ma, Li; Qi, Feifei; Zheng, Xianliang; Jiang, Chengying; Li, Ailei; Wan, Xiaobo; Liu, Shuang-Jiang; Li, Shengying

    2016-03-18

    4-Cresol is not only a significant synthetic intermediate for production of many aromatic chemicals, but also a priority environmental pollutant because of its toxicity to higher organisms. In our previous studies, a gene cluster implicated to be involved in 4-cresol catabolism, creCDEFGHIR, was identified in Corynebacterium glutamicum and partially characterized in vivo. In this work, we report on the discovery of a novel 4-cresol biodegradation pathway that employs phosphorylated intermediates. This unique pathway initiates with the phosphorylation of the hydroxyl group of 4-cresol, which is catalyzed by a novel 4-methylbenzyl phosphate synthase, CreHI. Next, a unique class I P450 system, CreJEF, specifically recognizes phosphorylated intermediates and successively oxidizes the aromatic methyl group into carboxylic acid functionality via alcohol and aldehyde intermediates. Moreover, CreD (phosphohydrolase), CreC (alcohol dehydrogenase), and CreG (aldehyde dehydrogenase) were also found to be required for efficient oxidative transformations in this pathway. Steady-state kinetic parameters (Km and kcat) for each catabolic step were determined, and these results suggest that kinetic controls serve a key role in directing the metabolic flux to the most energy effective route.

  11. Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of L-threonine.

    PubMed

    Dong, Xunyan; Quinn, Peter J; Wang, Xiaoyuan

    2011-01-01

    L-threonine is an essential amino acid for mammals and as such has a wide and expanding application in industry with a fast growing market demand. The major method of production of l-threonine is microbial fermentation. To optimize L-threonine production the fundamental solution is to develop robust microbial strains with high productivity and stability. Metabolic engineering provides an effective alternative to the random mutation for strain development. In this review, the updated information on genetics and molecular mechanisms for regulation of L-threonine pathways in Escherichia coli and Corynebacterium glutamicum are summarized, including L-threonine biosynthesis, intracellular consumption and trans-membrane export. Upon such knowledge, genetically defined L-threonine producing strains have been successfully constructed, some of which have already achieved the productivity of industrial producing strains. Furthermore, strategies for strain construction are proposed and potential problems are identified and discussed. Finally, the outlook for future strategies to construct industrially advantageous strains with respect to recent advances in biology has been considered.

  12. Characterization of lysine acetylation of a phosphoenolpyruvate carboxylase involved in glutamate overproduction in Corynebacterium glutamicum.

    PubMed

    Nagano-Shoji, Megumi; Hamamoto, Yuma; Mizuno, Yuta; Yamada, Ayuka; Kikuchi, Masaki; Shirouzu, Mikako; Umehara, Takashi; Yoshida, Minoru; Nishiyama, Makoto; Kosono, Saori

    2017-03-03

    Protein Nε-acylation is emerging as a ubiquitous post-translational modification. In Corynebacterium glutamicum, which is utilized for industrial production of L-glutamate, the levels of protein acetylation and succinylation change drastically under the conditions that induce glutamate overproduction. Here, we characterized the acylation of phosphoenolpyruvate carboxylase (PEPC), an anaplerotic enzyme that supplies oxaloacetate for glutamate overproduction. We showed that acetylation of PEPC at lysine 653 decreased enzymatic activity, leading to reduced glutamate production. An acetylation-mimic (KQ) mutant of K653 showed severely reduced glutamate production, while the corresponding KR mutant showed normal production levels. Using an acetyllysine-incorporated PEPC protein, we verified that K653-acetylation negatively regulates PEPC activity. In addition, NCgl0616, a sirtuin-type deacetylase, deacetylated K653-acetylated PEPC in vitro. Interestingly, the specific activity of PEPC was increased during glutamate overproduction, which was blocked by the K653R mutation or deletion of sirtuin-type deacetylase homologues. These findings suggested that deacetylation of K653 by NCgl0616 likely plays a role in the activation of PEPC, which maintains carbon flux under glutamate-producing conditions. PEPC deletion increased protein acetylation levels in cells under glutamate-producing conditions, supporting our hypothesis that PEPC is responsible for a large carbon flux change under glutamate-producing conditions. This article is protected by copyright. All rights reserved.

  13. Functional analysis of all aminotransferase proteins inferred from the genome sequence of Corynebacterium glutamicum.

    PubMed

    Marienhagen, Jan; Kennerknecht, Nicole; Sahm, Hermann; Eggeling, Lothar

    2005-11-01

    Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5'-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with L-glutamate, L-aspartate, and L-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) L-glutamate > 2-aminobutyrate > L-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes L-alanine to aminate 2-oxo-isovalerate, the L-valine precursor, and 2-oxo-butyrate. A second AT active with the L-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for L-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far.

  14. Metabolic quenching of Corynebacterium glutamicum: efficiency of methods and impact of cold shock.

    PubMed

    Wellerdiek, Max; Winterhoff, Dajana; Reule, Waldemar; Brandner, Jürgen; Oldiges, Marco

    2009-08-01

    Representative and valid cytoplasmic concentrations are essential for ensuring the significance of results in the field of metabolome analysis. One of the most crucial points in this respect is the sampling itself. A rapid and sudden stopping of the metabolism on a timescale that is much faster than the conversion rates of investigated metabolites is worthwhile. This can be achieved by applying of cold methanol quenching combined with reproducible, fast, and automated sampling. Unfortunately, quenching the metabolism by a sharp temperature shift leads to what is known as cold shock or the cell-leakage effect. In the present work, we applied a microstructure heat exchanger to analyze the cold shock effect using Corynebacterium glutamicum as a model microorganism. Using this apparatus together with a silicon pipe, it was possible to assay the leakage effect on a timescale starting at 1 s after cooling cell suspension. The high turnover rates not only require a rapid quenching technique, but also the correct application. Moreover, we succeeded in showing that even when the required appropriate setup of methanol quenching is not used, the metabolism is not stopped within the required timescale. By applying robust techniques like rapid sampling in combination with reproducible sample processing, we ensured fast and reliable metabolic inactivation during all steps.

  15. Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones.

    PubMed

    Kallscheuer, Nicolai; Vogt, Michael; Stenzel, Anton; Gätgens, Jochem; Bott, Michael; Marienhagen, Jan

    2016-11-01

    Corynebacterium glutamicum is an important organism in industrial biotechnology for the microbial production of bulk chemicals, in particular amino acids. However, until now activity of a complex catabolic network for the degradation of aromatic compounds averted application of C. glutamicum as production host for aromatic compounds of pharmaceutical or biotechnological interest. In the course of the construction of a suitable C. glutamicum platform strain for plant polyphenol production, four gene clusters comprising 21 genes involved in the catabolism of aromatic compounds were deleted. Expression of plant-derived and codon-optimized genes coding for a chalcone synthase (CHS) and a chalcone isomerase (CHI) in this strain background enabled formation of 35mg/L naringenin and 37mg/L eriodictyol from the supplemented phenylpropanoids p-coumaric acid and caffeic acid, respectively. Furthermore, expression of genes coding for a 4-coumarate: CoA-ligase (4CL) and a stilbene synthase (STS) led to the production of the stilbenes pinosylvin, resveratrol and piceatannol starting from supplemented phenylpropanoids cinnamic acid, p-coumaric acid and caffeic acid, respectively. Stilbene concentrations of up to 158mg/L could be achieved. Additional engineering of the amino acid metabolism for an optimal connection to the synthetic plant polyphenol pathways enabled resveratrol production directly from glucose. The construction of these C. glutamicum platform strains for the synthesis of plant polyphenols opens the door towards the microbial production of high-value aromatic compounds from cheap carbon sources with this microorganism.

  16. Economically enhanced succinic acid fermentation from cassava bagasse hydrolysate using Corynebacterium glutamicum immobilized in porous polyurethane filler.

    PubMed

    Shi, Xinchi; Chen, Yong; Ren, Hengfei; Liu, Dong; Zhao, Ting; Zhao, Nan; Ying, Hanjie

    2014-12-01

    An immobilized fermentation system, using cassava bagasse hydrolysate (CBH) and mixed alkalis, was developed to achieve economical succinic acid production by Corynebacterium glutamicum. The C. glutamicum strains were immobilized in porous polyurethane filler (PPF). CBH was used efficiently as a carbon source instead of more expensive glucose. Moreover, as a novel method for regulating pH, the easily decomposing NaHCO3 was replaced by mixed alkalis (NaOH and Mg(OH)2) for succinic acid production by C. glutamicum. Using CBH and mixed alkalis in the immobilized batch fermentation system, succinic acid productivity of 0.42gL(-1)h(-1) was obtained from 35gL(-1) glucose of CBH, which is similar to that obtained with conventional free-cell fermentation with glucose and NaHCO3. In repeated batch fermentation, an average of 22.5gL(-1) succinic acid could be obtained from each batch, which demonstrated the enhanced stability of the immobilized C. glutamicum cells.

  17. Lactate production as representative of the fermentation potential of Corynebacterium glutamicum 2262 in a one-step process.

    PubMed

    Khuat, Hoang Bao Truc; Kaboré, Abdoul Karim; Olmos, Eric; Fick, Michel; Boudrant, Joseph; Goergen, Jean-Louis; Delaunay, Stéphane; Guedon, Emmanuel

    2014-01-01

    The fermentative properties of thermo-sensitive strain Corynebacterium glutamicum 2262 were investigated in processes coupling aerobic cell growth and the anaerobic fermentation phase. In particular, the influence of two modes of fermentation on the production of lactate, the fermentation product model, was studied. In both processes, lactate was produced in significant amount, 27 g/L in batch culture, and up to 55.8 g/L in fed-batch culture, but the specific production rate in the fed-batch culture was four times lower than that in the batch culture. Compared to other investigated fermentation processes, our strategy resulted in the highest yield of lactic acid from biomass. Lactate production by C. glutamicum 2262 thus revealed the capability of the strain to produce various fermentation products from pyruvate.

  18. Corynebacterium glutamicum ggtB encodes a functional γ-glutamyl transpeptidase with γ-glutamyl dipeptide synthetic and hydrolytic activity.

    PubMed

    Walter, Frederik; Grenz, Sebastian; Ortseifen, Vera; Persicke, Marcus; Kalinowski, Jörn

    2016-08-20

    In this work the role of γ-glutamyl transpeptidase in the metabolism of γ-glutamyl dipeptides produced by Corynebacterium glutamicum ATCC 13032 was studied. The enzyme is encoded by the gene ggtB (cg1090) and synthesized as a 657 amino acids long preprotein. Gamma-glutamyl transpeptidase activity was found to be associated with intact cells of C. glutamicum and was abolished upon deletion of ggtB. Bioinformatic analysis indicated that the enzyme is a lipoprotein and is attached to the outer side of the cytoplasmic membrane. Biochemical parameters of recombinant GgtB were determined using the chromogenic substrate γ-glutamyl-p-nitroanilide. Highest activity of the enzyme was measured in sodium bicarbonate buffer at pH 9.6 and 45°C. The KM value was 123μM. GgtB catalyzed the concentration-dependent synthesis and hydrolysis of γ-glutamyl dipeptides and showed strong glutaminase activity. The intracellular concentrations of five γ-glutamyl dipeptides (γ-Glu-Glu, γ-Glu-Gln, γ-Glu-Val, γ-Glu-Leu, γ-Glu-Met) were determined by HPLC-MS and ranged from 0.15 to 0.4mg/g CDW after exponential growth in minimal media. Although deletion and overexpression of ggtB had significant effects on intracellular dipeptide concentrations, it was neither essential for biosynthesis nor catabolism of these dipeptides in vivo.

  19. Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum

    PubMed Central

    Srisimarat, Wiraya; Kaulpiboon, Jarunee; Krusong, Kuakarun; Zimmermann, Wolfgang

    2012-01-01

    Corynebacterium glutamicum amylomaltase (CgAM) catalyzes the formation of large-ring cyclodextrins (LR-CDs) with a degree of polymerization of 19 and higher. The cloned CgAM gene was ligated into the pET-17b vector and used to transform Escherichia coli BL21(DE3). Site-directed mutagenesis of Tyr-172 in CgAM to alanine (Y172A) was performed to determine its role in the control of LR-CD production. Both the recombinant wild-type (WT) and Y172A enzymes were purified to apparent homogeneity and characterized. The Y172A enzyme exhibited lower disproportionation, cyclization, and hydrolysis activities than the WT. The kcat/Km of the disproportionation reaction of the Y172A enzyme was 2.8-fold lower than that of the WT enzyme. The LR-CD product profile from enzyme catalysis depended on the incubation time and the enzyme concentration. Interestingly, the Y172A enzyme showed a product pattern different from that of the WT CgAM at a long incubation time. The principal LR-CD products of the Y172A mutated enzyme were a cycloamylose mixture with a degree of polymerization of 28 or 29 (CD28 or CD29), while the principal LR-CD product of the WT enzyme was CD25 at 0.05 U of amylomaltase. These results suggest that Tyr-172 plays an important role in determining the LR-CD product profile of this novel CgAM. PMID:22865069

  20. Enhancement of biomolecule transport by electroporation: a review of theory and practical application to transformation of Corynebacterium glutamicum.

    PubMed

    Tryfona, Theodora; Bustard, Mark T

    2006-02-20

    Selective and reversible permeabilization of the cell wall permeability barrier is the focus for many biotechnological applications. In this article, the basic principles for reversible membrane permeabilization, based on biological, chemical, and physical methods are reviewed. Emphasis is given to electroporation (electropermeabilization) which tends to be the most popular method for membrane permeabilization and for introduction of foreign molecules into the cells. The applications of this method in industrial processes as well as the critical factors and parameters which affect the success of this approach are discussed. The different strategies developed throughout the years for increased transformation efficiencies of the industrially important amino acid-overproducing bacterium Corynebacterium glutamicum, are also summarized.

  1. Reengineering of a Corynebacterium glutamicum l-Arginine and l-Citrulline Producer▿

    PubMed Central

    Ikeda, Masato; Mitsuhashi, Satoshi; Tanaka, Kenji; Hayashi, Mikiro

    2009-01-01

    Toward the creation of a robust and efficient producer of l-arginine and l-citrulline (arginine/citrulline), we have performed reengineering of a Corynebacterium glutamicum strain by using genetic information of three classical producers. Sequence analysis of their arg operons identified three point mutations (argR123, argG92up, and argG45) in one producer and one point mutation (argB26 or argB31) in each of the other two producers. Reconstitution of the former three mutations or of each argB mutation on a wild-type genome led to no production. Combined introduction of argB26 or argB31 with argR123 into a wild type gave rise to arginine/citrulline production. When argR123 was replaced by an argR-deleted mutation (ΔargR), the production was further increased. The best mutation set, ΔargR and argB26, was used to screen for the highest productivity in the backgrounds of different wild-type strains of C. glutamicum. This yielded a robust producer, RB, but the production was still one-third of that of the best classical producer. Transcriptome analysis revealed that the arg operon of the classical producer was much more highly upregulated than that of strain RB. Introduction of leuC456, a mutation derived from a classical l-lysine producer and provoking global induction of the amino acid biosynthesis genes, including the arg operon, into strain RB led to increased production but incurred retarded fermentation. On the other hand, replacement of the chromosomal argB by heterologous Escherichia coli argB, natively insensitive to arginine, caused a threefold-increased production without retardation, revealing that the limitation in strain RB was the activity of the argB product. To overcome this, in addition to argB26, the argB31 mutation was introduced into strain RB, which caused higher deregulation of the enzyme and resulted in dramatically increased production, like the strain with E. coli argB. This reconstructed strain displayed an enhanced performance, thus

  2. Transcriptional response of Corynebacterium glutamicum ATCC 13032 to hydrogen peroxide stress and characterization of the OxyR regulon.

    PubMed

    Milse, Johanna; Petri, Kathrin; Rückert, Christian; Kalinowski, Jörn

    2014-11-20

    The aerobic soil bacterium Corynebacterium glutamicum ATCC 13032 has a remarkable natural resistance to hydrogen peroxide. A major player in hydrogen peroxide defense is the LysR type transcriptional regulator OxyR, homologs of which are present in a wide range of bacteria. In this study, the global transcriptional response of C. glutamicum to oxidative stress induced by hydrogen peroxide was examined using whole genome DNA microarrays, demonstrating the dynamic reaction of the regulatory networks. Deletion of oxyR resulted in an increased resistance of the C. glutamicum mutant to hydrogen peroxide. By performing DNA microarray hybridizations and RT-qPCR, differentially expressed genes were detected in the mutant. The direct control by OxyR was verified by electrophoretic mobility shift assays for 12 target regions. The results demonstrated that OxyR in C. glutamicum acts as a transcriptional repressor under non-stress conditions for a total of 23 genes. The regulated genes encode proteins related to oxidative stress response (e.g. katA), iron homeostasis (e.g. dps) and sulfur metabolism (e.g. suf cluster). Besides the regulator of the suf cluster, SufR, OxyR regulated the gene cg1695 encoding a putative transcriptional regulator, indicating the role of OxyR as a master regulator in defense against oxidative stress. Using a modified DNase footprint approach, the OxyR-binding sites in five target promoter regions, katA, cydA, hemH, dps and cg1292, were localized and in each upstream region at least two overlapping binding sites were found. The DNA regions protected by the OxyR protein are about 56bp in length and do not have evident sequence similarities. Still, by giving an insight in the H2O2 stimulon and extending the OxyR regulon this study considerably contributes to the understanding of the response of C. glutamicum to hydrogen peroxide-mediated oxidative stress.

  3. Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes

    PubMed Central

    Vogt, Michael; Haas, Sabine; Polen, Tino; van Ooyen, Jan; Bott, Michael

    2015-01-01

    2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l−1) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l−1 h−1. Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol. PMID:25488800

  4. Identification and functional analysis of the gene cluster for L-arabinose utilization in Corynebacterium glutamicum.

    PubMed

    Kawaguchi, Hideo; Sasaki, Miho; Vertès, Alain A; Inui, Masayuki; Yukawa, Hideaki

    2009-06-01

    Corynebacterium glutamicum ATCC 31831 grew on l-arabinose as the sole carbon source at a specific growth rate that was twice that on d-glucose. The gene cluster responsible for l-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three l-arabinose-catabolizing genes, araA (encoding l-arabinose isomerase), araB (l-ribulokinase), and araD (l-ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (LacI-type transcriptional regulator), araE (l-arabinose transporter), and galM (putative aldose 1-epimerase), were present in the opposite direction. Inactivation of the araA, araB, or araD gene eliminated growth on l-arabinose, and each of the gene products was functionally homologous to its Escherichia coli counterpart. Moreover, compared to the wild-type strain, an araE disruptant exhibited a >80% decrease in the growth rate at a lower concentration of l-arabinose (3.6 g liter(-1)) but not at a higher concentration of l-arabinose (40 g liter(-1)). The expression of the araBDA operon and the araE gene was l-arabinose inducible and negatively regulated by the transcriptional regulator AraR. Disruption of araR eliminated the repression in the absence of l-arabinose. Expression of the regulon was not repressed by d-glucose, and simultaneous utilization of l-arabinose and d-glucose was observed in aerobically growing wild-type and araR deletion mutant cells. The regulatory mechanism of the l-arabinose regulon is, therefore, distinct from the carbon catabolite repression mechanism in other bacteria.

  5. Engineering of Corynebacterium glutamicum for xylitol production from lignocellulosic pentose sugars.

    PubMed

    Dhar, Kiran S; Wendisch, Volker F; Nampoothiri, Kesavan Madhavan

    2016-07-20

    Xylitol is a non-fermentable sugar alcohol used as sweetener. Corynebacterium glutamicum ATCC13032 was metabolically engineered for xylitol production from the lignocellulosic pentose sugars xylose and arabinose. Direct conversion of xylose to xylitol was achieved through the heterologous expression of NAD(P)H-dependent xylose reductase (xr) gene from Rhodotorula mucilaginosa. Xylitol synthesis from arabinose was attained through polycistronic expression of l-arabinose isomerase (araA), d-psicose 3 epimerase (dpe) and l-xylulose reductase (lxr) genes from Escherichia coli, Agrobacterium tumefaciens and Mycobacterium smegmatis, respectively. Expression of xr and the synthetic araA-dpe-lxr operon under the control of IPTG-inducible Ptac promoter enabled production of xylitol from both xylose and arabinose in the mineral (CGXII) medium with glucose as carbon source. Additional expression of a pentose transporter (araTF) gene enhanced xylitol production by about four-fold compared to the parent strain. The constructed strain Cg-ax3 produced 6.7±0.4g/L of xylitol in batch fermentations and 31±0.5g/L of xylitol in fed-batch fermentations with a specific productivity of 0.28±0.05g/g cdw/h. The strain Cg-ax3 was also validated for xylitol production from pentose rich, acid pre-treated liquor of sorghum stover (SAPL) and the results were comparable in both SAPL (27±0.3g/L) and mineral medium (31±0.5g/L).

  6. Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence of Corynebacterium glutamicum

    PubMed Central

    Marienhagen, Jan; Kennerknecht, Nicole; Sahm, Hermann; Eggeling, Lothar

    2005-01-01

    Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5′-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with l-glutamate, l-aspartate, and l-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) l-glutamate > 2-aminobutyrate > l-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes l-alanine to aminate 2-oxo-isovalerate, the l-valine precursor, and 2-oxo-butyrate. A second AT active with the l-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for l-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far. PMID:16267288

  7. Chill activation of compatible solute transporters in Corynebacterium glutamicum at the level of transport activity.

    PubMed

    Ozcan, Nuran; Krämer, Reinhard; Morbach, Susanne

    2005-07-01

    The gram-positive soil bacterium Corynebacterium glutamicum harbors four osmoregulated secondary uptake systems for compatible solutes, BetP, EctP, LcoP, and ProP. When reconstituted in proteoliposomes, BetP was shown to sense hyperosmotic conditions via the increase in luminal K(+) and to respond by instant activation. To study further putative ways of stimulus perception and signal transduction, we have investigated the responses of EctP, LcoP, and BetP, all belonging to the betaine-carnitine-choline transporter family, to chill stress at the level of activity. When fully activated by hyperosmotic stress, they showed the expected increase of activity at increasing temperature. In the absence of osmotic stress, EctP was not activated by chill and LcoP to only a very low extent, whereas BetP was significantly stimulated at low temperature. BetP was maximally activated at 10 degrees C, reaching the same transport rate as that observed under hyperosmotic conditions at this temperature. A role of cytoplasmic K(+) in chill-dependent activation of BetP was ruled out, since (i) the cytoplasmic K(+) concentration did not change significantly at lower temperatures and (ii) a mutant BetP lacking the C-terminal 25 amino acids, which was previously shown to have lost the ability to be activated by luminal K(+), was fully competent in chill sensing. When heterologously expressed in Escherichia coli, BetP did not respond to chill stress. This may indicate that the membrane in which BetP is inserted plays an important role in chill activation and thus in signal transduction by BetP, different from the previously established K(+)-mediated process.

  8. Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes.

    PubMed

    Vogt, Michael; Haas, Sabine; Polen, Tino; van Ooyen, Jan; Bott, Michael

    2015-03-01

    2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l(-1)) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l(-1)  h(-1). Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol.

  9. Cutting the Gordian Knot: Identifiability of anaplerotic reactions in Corynebacterium glutamicum by means of (13) C-metabolic flux analysis.

    PubMed

    Kappelmann, Jannick; Wiechert, Wolfgang; Noack, Stephan

    2016-03-01

    Corynebacterium glutamicum is the major workhorse for the microbial production of several amino and organic acids. As long as these derive from tricarboxylic acid cycle intermediates, the activity of anaplerotic reactions is pivotal for a high biosynthetic yield. To determine single anaplerotic activities (13) C-Metabolic Flux Analysis ((13) C-MFA) has been extensively used for C. glutamicum, however with different network topologies, inconsistent or poorly determined anaplerotic reaction rates. Therefore, in this study we set out to investigate whether a focused isotopomer model of the anaplerotic node can at all admit a unique solution for all fluxes. By analyzing different scenarios of active anaplerotic reactions, we show in full generality that for C. glutamicum only certain anaplerotic deletion mutants allow to uniquely determine the anaplerotic fluxes from (13) C-isotopomer data. We stress that the result of this analysis for different assumptions on active enzymes is directly transferable to other compartment-free organisms. Our results demonstrate that there exist biologically relevant metabolic network topologies for which the flux distribution cannot be inferred by classical (13) C-MFA.

  10. Activity of exporters of Escherichia coli in Corynebacterium glutamicum, and their use to increase L-threonine production.

    PubMed

    Diesveld, Ramon; Tietze, Nadine; Fürst, Oliver; Reth, Alexander; Bathe, Brigitte; Sahm, Hermann; Eggeling, Lothar

    2009-01-01

    L-Threonine is an important biotechnological product and Corynebacterium glutamicum is able to synthesize and accumulate this amino acid to high intracellular levels. We here use four exporters of Escherichia coli and show that three of them operate in C. glutamicum, with RhtA and RhtC being the most effective. Whereas RhtA was unspecific, resulting in L-homoserine together with L-threonine excretion, this was not the case with RhtC. Expression of rhtC reduced the intracellular L-threonine concentration from 140 to 11 mM and resulted in maximal excretion rates of 11.2 nmol min(-1) mg(-1) as compared to 2.3 nmol min(-1) mg(-1) obtained without rhtC expression. In combination with an ilvA mutation generated and introduced into the chromosome, an accumulation of up to 54 mM L-threonine was achieved as compared to 21 mM obtained with the ancestor strain. This shows that expression of rhtC is the pivotal point for industrial relevant L-threonine production with C. glutamicum, and might encourage in general the use of heterologous exporters in the field of white biotechnology to make full use of biosynthesis pathways.

  11. Direct production of organic acids from starch by cell surface-engineered Corynebacterium glutamicum in anaerobic conditions

    PubMed Central

    2013-01-01

    We produced organic acids, including lactate and succinate, directly from soluble starch under anaerobic conditions using high cell-density cultures of Corynebacterium glutamicum displaying α-amylase (AmyA) from Streptococcus bovis 148 on the cell surface. Notably, reactions performed under anaerobic conditions at 35 and 40°C, which are higher than the optimal growth temperature of 30°C, showed 32% and 19%, respectively, higher productivity of the organic acids lactate, succinate, and acetate compared to that at 30°C. However, α-amylase was not stably anchored and released into the medium from the cell surface during reactions at these higher temperatures, as demonstrated by the 61% and 85% decreases in activity, respectively, from baseline, compared to the only 8% decrease at 30°C. The AmyA-displaying C. glutamicum cells retained their starch-degrading capacity during five 10 h reaction cycles at 30°C, producing 107.8 g/l of total organic acids, including 88.9 g/l lactate and 14.0 g/l succinate. The applicability of cell surface-engineering technology for the production of organic acids from biomass by high cell-density cultures of C. glutamicum under anaerobic conditions was demonstrated. PMID:24342107

  12. A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum

    PubMed Central

    2013-01-01

    Background Arginine biosynthesis in Corynebacterium glutamicum consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by N-acetylglutamate synthase (NAGS). There are different kinds of known NAGSs, for example, “classical” ArgA, bifunctional ArgJ, ArgO, and S-NAGS. However, since C. glutamicum possesses a monofunctional ArgJ, which catalyses only the fifth step of the arginine biosynthesis pathway, glutamate must be acetylated by an as of yet unknown NAGS gene. Results Arginine biosynthesis was investigated by metabolome profiling using defined gene deletion mutants that were expected to accumulate corresponding intracellular metabolites. HPLC-ESI-qTOF analyses gave detailed insights into arginine metabolism by detecting six out of seven intermediates of arginine biosynthesis. Accumulation of N-acetylglutamate in all mutants was a further confirmation of the unknown NAGS activity. To elucidate the identity of this gene, a genomic library of C. glutamicum was created and used to complement an Escherichia coli ΔargA mutant. The plasmid identified, which allowed functional complementation, contained part of gene cg3035, which contains an acetyltransferase domain in its amino acid sequence. Deletion of cg3035 in the C. glutamicum genome led to a partial auxotrophy for arginine. Heterologous overexpression of the entire cg3035 gene verified its ability to complement the E. coli ΔargA mutant in vivo and homologous overexpression led to a significantly higher intracellular N-acetylglutamate pool. Enzyme assays confirmed the N-acetylglutamate synthase activity of Cg3035 in vitro. However, the amino acid sequence of Cg3035 revealed no similarities to members of known NAGS gene families. Conclusions The N-acetylglutamate synthase Cg3035 is able to catalyse the first step of arginine biosynthesis in C. glutamicum. It represents a novel class of NAGS genes apparently present only in bacteria of the suborder Corynebacterineae, comprising

  13. Development and application of an arabinose-inducible expression system by facilitating inducer uptake in Corynebacterium glutamicum.

    PubMed

    Zhang, Yun; Shang, Xiuling; Lai, Shujuan; Zhang, Guoqiang; Liang, Yong; Wen, Tingyi

    2012-08-01

    Corynebacterium glutamicum is currently used for the industrial production of a variety of biological materials. Many available inducible expression systems in this species use lac-derived promoters from Escherichia coli that exhibit much lower levels of inducible expression and leaky basal expression. We developed an arabinose-inducible expression system that contains the L-arabinose regulator AraC, the P(BAD) promoter from the araBAD operon, and the L-arabinose transporter AraE, all of which are derived from E. coli. The level of inducible P(BAD)-based expression could be modulated over a wide concentration range from 0.001 to 0.4% L-arabinose. This system tightly controlled the expression of the uracil phosphoribosyltransferase without leaky expression. When the gene encoding green fluorescent protein (GFP) was under the control of P(BAD) promoter, flow cytometry analysis showed that GFP was expressed in a highly homogeneous profile throughout the cell population. In contrast to the case in E. coli, P(BAD) induction was not significantly affected in the presence of different carbon sources in C. glutamicum, which makes it useful in fermentation applications. We used this system to regulate the expression of the odhI gene from C. glutamicum, which encodes an inhibitor of α-oxoglutarate dehydrogenase, resulting in high levels of glutamate production (up to 13.7 mM) under biotin nonlimiting conditions. This system provides an efficient tool available for molecular biology and metabolic engineering of C. glutamicum.

  14. Identification of AcnR, a TetR-type repressor of the aconitase gene acn in Corynebacterium glutamicum.

    PubMed

    Krug, Andreas; Wendisch, Volker F; Bott, Michael

    2005-01-07

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

  15. Biosynthesis of l-Sorbose and l-Psicose Based on C-C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain.

    PubMed

    Yang, Jiangang; Li, Jitao; Men, Yan; Zhu, Yueming; Zhang, Ying; Sun, Yuanxia; Ma, Yanhe

    2015-07-01

    The property of loose stereochemical control at aldol products from aldolases helped to synthesize multiple polyhydroxylated compounds with nonnatural stereoconfiguration. In this study, we discovered for the first time that some fructose 1,6-diphosphate aldolases (FruA) and tagatose 1,6-diphosphate (TagA) aldolases lost their strict stereoselectivity when using l-glyceraldehyde and synthesized not only l-sorbose but also a high proportion of l-psicose. Among the aldolases tested, TagA from Bacillus licheniformis (BGatY) showed the highest enzyme activity with l-glyceraldehyde. Subsequently, a "one-pot" reaction based on BGatY and fructose-1-phosphatase (YqaB) generated 378 mg/liter l-psicose and 199 mg/liter l-sorbose from dihydroxyacetone-phosphate (DHAP) and l-glyceraldehyde. Because of the high cost and instability of DHAP, a microbial fermentation strategy was used further to produce l-sorbose/l-psicose from glucose and l-glyceraldehyde, in which DHAP was obtained from glucose through the glycolytic pathway, and some recombination pathways based on FruA or TagA and YqaB were constructed in Escherichia coli and Corynebacterium glutamicum strains. After evaluation of different host cells and combinations of FruA or TagA with YqaB and optimization of gene expression, recombinant C. glutamicum strain WT(pXFTY) was selected and produced 2.53 g/liter total ketoses, with a yield of 0.50 g/g l-glyceraldehyde. Moreover, deletion of gene cgl0331, encoding the Zn-dependent alcohol dehydrogenase in C. glutamicum, was confirmed for the first time to significantly decrease conversion of l-glyceraldehyde to glycerol and to increase yield of target products. Finally, fed-batch culture of strain SY14(pXFTY) produced 3.5 g/liter l-sorbose and 2.3 g/liter l-psicose, with a yield of 0.61 g/g l-glyceraldehyde. This microbial fermentation strategy also could be applied to efficiently synthesize other l-sugars.

  16. Biosynthesis of l-Sorbose and l-Psicose Based on C—C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain

    PubMed Central

    Yang, Jiangang; Li, Jitao; Men, Yan; Zhu, Yueming; Zhang, Ying; Ma, Yanhe

    2015-01-01

    The property of loose stereochemical control at aldol products from aldolases helped to synthesize multiple polyhydroxylated compounds with nonnatural stereoconfiguration. In this study, we discovered for the first time that some fructose 1,6-diphosphate aldolases (FruA) and tagatose 1,6-diphosphate (TagA) aldolases lost their strict stereoselectivity when using l-glyceraldehyde and synthesized not only l-sorbose but also a high proportion of l-psicose. Among the aldolases tested, TagA from Bacillus licheniformis (BGatY) showed the highest enzyme activity with l-glyceraldehyde. Subsequently, a “one-pot” reaction based on BGatY and fructose-1-phosphatase (YqaB) generated 378 mg/liter l-psicose and 199 mg/liter l-sorbose from dihydroxyacetone-phosphate (DHAP) and l-glyceraldehyde. Because of the high cost and instability of DHAP, a microbial fermentation strategy was used further to produce l-sorbose/l-psicose from glucose and l-glyceraldehyde, in which DHAP was obtained from glucose through the glycolytic pathway, and some recombination pathways based on FruA or TagA and YqaB were constructed in Escherichia coli and Corynebacterium glutamicum strains. After evaluation of different host cells and combinations of FruA or TagA with YqaB and optimization of gene expression, recombinant C. glutamicum strain WT(pXFTY) was selected and produced 2.53 g/liter total ketoses, with a yield of 0.50 g/g l-glyceraldehyde. Moreover, deletion of gene cgl0331, encoding the Zn-dependent alcohol dehydrogenase in C. glutamicum, was confirmed for the first time to significantly decrease conversion of l-glyceraldehyde to glycerol and to increase yield of target products. Finally, fed-batch culture of strain SY14(pXFTY) produced 3.5 g/liter l-sorbose and 2.3 g/liter l-psicose, with a yield of 0.61 g/g l-glyceraldehyde. This microbial fermentation strategy also could be applied to efficiently synthesize other l-sugars. PMID:25888171

  17. Identification of a HAD superfamily phosphatase, HdpA, involved in 1,3-dihydroxyacetone production during sugar catabolism in Corynebacterium glutamicum.

    PubMed

    Jojima, Toru; Igari, Takafumi; Gunji, Wataru; Suda, Masako; Inui, Masayuki; Yukawa, Hideaki

    2012-11-30

    Corynebacterium glutamicum produces 1,3-dihydroxyacetone (DHA) as metabolite of sugar catabolism but the responsible enzyme is yet to be identified. Using a transposon mutant library, the gene hdpA (cgR_2128) was shown to encode a haloacid dehalogenase superfamily member that catalyzes dephosphorylation of dihydroxyacetone phosphate to produce DHA. Inactivation of hdpA led to a drastic decrease in DHA production from each of glucose, fructose, and sucrose, indicating that HdpA is the main enzyme responsible for DHA production from sugars in C. glutamicum. Confirmation of DHA production via dihydroxyacetone phosphatase finally confirms a long-speculated route through which bacteria produce DHA.

  18. Modeling and optimization of glutamic acid production using mixed culture of Corynebacterium glutamicum NCIM2168 and Pseudomonas reptilivora NCIM2598.

    PubMed

    Kumar, Rajaram Shyam; Moorthy, Innasi Muthu Ganesh; Baskar, Rajoo

    2013-01-01

    In this study, a hybrid system of response surface methodology followed by genetic algorithm has been adopted to optimize the production medium for L-glutamic acid fermentation with mixed cultures of Corynebacterium glutamicum and Pseudomonas reptilovora. The optimal combination of media components for maximal production of L-glutamic acid was found to be 49.99 g L(-1) of glucose, 10 g L(-1) of urea, 18.06% (v/v) of salt solution, and 4.99% (v/v) of inoculum size. The experimental glutamic acid yield at optimum condition was 19.69 g L(-1), which coincided well to the value predicted by the model (19.61 g L(-1)). Using this methodology, a nonlinear regression model was developed for the glutamic acid production. The model was validated statistically and the determination coefficient (R (2)) was found to be 0.99.

  19. Purification, crystallization and preliminary X-ray diffraction studies of the arsenic repressor ArsR from Corynebacterium glutamicum

    PubMed Central

    Santha, Sangilimadan; Pandaranayaka, Eswari P. J.; Rosen, Barry P.; Thiyagarajan, Saravanamuthu

    2011-01-01

    ArsR is a member of the SmtB/ArsR family of metalloregulatory proteins that regulate prokaryotic arsenic-resistance operons. Here, the crystallization and preliminary X-ray diffraction studies of a cysteine-free derivative of ArsR from Corynebacterium glutamicum (CgArsR-C15/16/55S) are reported. CgArsR-C15/16/55S was expressed, purified, crystallized and X-ray diffraction data were collected to 1.86 Å resolution. The protein crystallized in a tetragonal space group (P4), with unit-cell parameters a = b = 41.84, c = 99.47 Å. PMID:22139180

  20. In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by 15N Nuclear Magnetic Resonance

    PubMed Central

    Tesch, M.; de Graaf, A. A.; Sahm, H.

    1999-01-01

    Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)–glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of 15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicum strains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4+ was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type. PMID:10049869

  1. Engineering of Corynebacterium glutamicum with an NADPH-Generating Glycolytic Pathway for l-Lysine Production ▿

    PubMed Central

    Takeno, Seiki; Murata, Ryosuke; Kobayashi, Ryosuke; Mitsuhashi, Satoshi; Ikeda, Masato

    2010-01-01

    A sufficient supply of NADPH is a critical factor in l-lysine production by Corynebacterium glutamicum. Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP+ to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant was engineered into an l-lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an l-lysine producer was evaluated. The amounts of l-lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ∼70% on glucose, ∼120% on fructose, and ∼100% on sucrose, indicating that the increased l-lysine production was attributed to GapN. These results demonstrate effective l-lysine production by C. glutamicum with an additional source of NADPH during glycolysis. PMID:20851994

  2. Investigation of phosphorylation status of OdhI protein during penicillin- and Tween 40-triggered glutamate overproduction by Corynebacterium glutamicum.

    PubMed

    Kim, Jongpill; Hirasawa, Takashi; Saito, Masaki; Furusawa, Chikara; Shimizu, Hiroshi

    2011-07-01

    Glutamate overproduction by Corynebacterium glutamicum is triggered by treatment with penicillin or Tween 40 and is accompanied by a decrease in 2-oxoglutarate dehydrogenase complex (ODHC) activity. We have reported that de novo synthesis of OdhI, which inhibits ODHC activity by interacting specifically with the E1o subunit of ODHC (OdhA), is induced by penicillin, and that odhI overexpression induces glutamate overproduction in the absence of any triggers for glutamate overproduction. In this study, to determine the function of OdhI in glutamate overproduction by C. glutamicum, changes in OdhI levels and phosphorylation status during penicillin- and Tween 40-induced glutamate overproduction were examined by western blot. The synthesis of both unphosphorylated and phosphorylated OdhI was increased by addition of Tween 40 or penicillin and the levels of unphosphorylated OdhI, which can inhibit ODHC activity, was significantly higher than those of phosphorylated OdhI, which is unable to inhibit ODHC activity. Meanwhile, the OdhA levels were maintained throughout the culture. These results indicate that OdhI synthesis is induced by additions of penicillin and Tween 40 and most synthesized OdhI is unphosphorylated, resulting in the decrease in ODHC activity and glutamate overproduction. Similarly, in the odhI-overexpressing strain, both unphosphorylated and phosphorylated OdhI were synthesized, while the levels of OdhA were nearly constant throughout culture. Our results suggest that high level of unphosphorylated OdhI regulates glutamate overproduction by C. glutamicum.

  3. Regulation of the malic enzyme gene malE by the transcriptional regulator MalR in Corynebacterium glutamicum.

    PubMed

    Krause, Jens P; Polen, Tino; Youn, Jung-Won; Emer, Denise; Eikmanns, Bernhard J; Wendisch, Volker F

    2012-06-15

    Corynebacterium glutamicum is a Gram-positive nonpathogenic bacterium that is used for the biotechnological production of amino acids. Here, we investigated the transcriptional control of the malE gene encoding malic enzyme (MalE) in C. glutamicum ATCC 13032, which is known to involve the nitrogen regulator AmtR. Gel shift experiments using purified regulators RamA and RamB revealed binding of these regulators to the malE promoter. In DNA-affinity purification experiments a hitherto uncharacterized transcriptional regulator belonging to the MarR family was found to bind to malE promoter DNA and was designated as MalR. C. glutamicum cells overexpressing malR showed reduced MalE activities in LB medium or in minimal media with acetate, glucose, pyruvate or citrate. Deletion of malR positively affected MalE activities during growth in LB medium and minimal media with pyruvate, glucose or the TCA cycle dicarboxylates l-malate, succinate and fumarate. Transcriptional fusion analysis revealed elevated malE promoter activity in the malR deletion mutant during growth in pyruvate minimal medium suggesting that MalR acts as a repressor of malE. Purified MalR bound malE promoter DNA in gel shift experiments. Two MalR binding sites were identified in the malE promoter by mutational analysis. Thus, MalR contributes to the complex transcriptional control of malE which also involves RamA, RamB and AmtR.

  4. Physiological adaptation of Corynebacterium glutamicum to benzoate as alternative carbon source - a membrane proteome-centric view.

    PubMed

    Haussmann, Ute; Qi, Su-Wei; Wolters, Dirk; Rögner, Matthias; Liu, Shuang-Jiang; Poetsch, Ansgar

    2009-07-01

    The ability of microorganisms to assimilate aromatic substances as alternative carbon sources is the basis of biodegradation of natural as well as industrial aromatic compounds. In this study, Corynebacterium glutamicum was grown on benzoate as sole carbon and energy source. To extend the scarce knowledge about physiological adaptation processes occurring in this cell compartment, the membrane proteome was investigated under quantitative and qualitative aspects by applying shotgun proteomics to reach a comprehensive survey. Membrane proteins were relatively quantified using an internal standard metabolically labeled with (15)N. Altogether, 40 proteins were found to change their abundance during growth on benzoate in comparison to glucose. A global adaptation was observed in the membrane of benzoate-grown cells, characterized by increased abundance of proteins of the respiratory chain, by a starvation response, and by changes in sulfur metabolism involving the regulator McbR. Additional to the relative quantification, stable isotope-labeled synthetic peptides were used for the absolute quantification of the two benzoate transporters of C. glutamicum, BenK and BenE. It was found that both transporters were expressed during growth on benzoate, suggesting that both contribute substantially to benzoate uptake.

  5. The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum.

    PubMed

    Nakayama, Yoshitaka; Becker, Michael; Ebrahimian, Haleh; Konishi, Tomoyuki; Kawasaki, Hisashi; Krämer, Reinhard; Martinac, Boris

    2016-01-01

    The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes.

  6. Cofactor recycling for co-production of 1,3-propanediol and glutamate by metabolically engineered Corynebacterium glutamicum.

    PubMed

    Huang, Jinhai; Wu, Yao; Wu, Wenjun; Zhang, Ye; Liu, Dehua; Chen, Zhen

    2017-02-08

    Production of 1,3-propanediol (1,3-PDO) from glycerol is a promising route toward glycerol biorefinery. However, the yield of 1,3-PDO is limited due to the requirement of NADH regeneration via glycerol oxidation process, which generates large amounts of undesired byproducts. Glutamate fermentation by Corynebacterium glutamicum is an important oxidation process generating excess NADH. In this study, we proposed a novel strategy to couple the process of 1,3-PDO synthesis with glutamate production for cofactor regeneration. With the optimization of 1,3-PDO synthesis route, C. glutamicum can efficiently convert glycerol into 1,3-PDO with the yield of ~ 1.0 mol/mol glycerol. Co-production of 1,3-PDO and glutamate was also achieved which increased the yield of glutamate by 18% as compared to the control. Since 1,3-PDO and glutamate can be easily separated in downstream process, this study provides a potential green route for coupled production of 1,3-PDO and glutamate to enhance the economic viability of biorefinery process.

  7. Modular pathway engineering of Corynebacterium glutamicum for production of the glutamate-derived compounds ornithine, proline, putrescine, citrulline, and arginine.

    PubMed

    Jensen, Jaide V K; Eberhardt, Dorit; Wendisch, Volker F

    2015-11-20

    The glutamate-derived bioproducts ornithine, citrulline, proline, putrescine, and arginine have applications in the food and feed, cosmetic, pharmaceutical, and chemical industries. Corynebacterium glutamicum is not only an excellent producer of glutamate but also of glutamate-derived products. Here, engineering targets beneficial for ornithine production were identified and the advantage of rationally constructing a platform strain for the production of the amino acids citrulline, proline, and arginine, and the diamine putrescine was demonstrated. Feedback alleviation of N-acetylglutamate kinase, tuning of the promoter of glutamate dehydrogenase gene gdh, lowering expression of phosphoglucoisomerase gene pgi, along with the introduction of a second copy of the arginine biosynthesis operon argCJB(A49V,M54V)D into the chromosome resulted in a C. glutamicum strain producing ornithine with a yield of 0.52 g ornithine per g glucose, an increase of 71% as compared to the parental ΔargFRG strain. Strains capable of producing 0.41 g citrulline per g glucose, 0.29 g proline per g glucose, 0.30 g arginine per g glucose, and 0.17 g putrescine per g glucose were derived from the ornithine-producing platform strain by plasmid-based overexpression of appropriate pathway modules with one to three genes.

  8. Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate.

    PubMed

    Leßmeier, Lennart; Pfeifenschneider, Johannes; Carnicer, Marc; Heux, Stephanie; Portais, Jean-Charles; Wendisch, Volker F

    2015-12-01

    Methanol, a one-carbon compound, can be utilized by a variety of bacteria and other organisms as carbon and energy source and is regarded as a promising substrate for biotechnological production. In this study, a strain of non-methylotrophic Corynebacterium glutamicum, which was able to produce the polyamide building block cadaverine as non-native product, was engineered for co-utilization of methanol. Expression of the gene encoding NAD+-dependent methanol dehydrogenase (Mdh) from the natural methylotroph Bacillus methanolicus increased methanol oxidation. Deletion of the endogenous aldehyde dehydrogenase genes ald and fadH prevented methanol oxidation to carbon dioxide and formaldehyde detoxification via the linear formaldehyde dissimilation pathway. Heterologous expression of genes for the key enzymes hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase of the ribulose monophosphate (RuMP) pathway in this strain restored growth in the presence of methanol or formaldehyde, which suggested efficient formaldehyde detoxification involving RuMP key enzymes. While growth with methanol as sole carbon source was not observed, the fate of 13C-methanol added as co-substrate to sugars was followed and the isotopologue distribution indicated incorporation into central metabolites and in vivo activity of the RuMP pathway. In addition, 13C-label from methanol was traced to the secreted product cadaverine. Thus, this synthetic biology approach led to a C. glutamicum strain that converted the non-natural carbon substrate methanol at least partially to the non-native product cadaverine.

  9. Gene expression profiling of Corynebacterium glutamicum during Anaerobic nitrate respiration: induction of the SOS response for cell survival.

    PubMed

    Nishimura, Taku; Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2011-03-01

    The gene expression profile of Corynebacterium glutamicum under anaerobic nitrate respiration revealed marked differences in the expression levels of a number of genes involved in a variety of cellular functions, including carbon metabolism and respiratory electron transport chain, compared to the profile under aerobic conditions using DNA microarrays. Many SOS genes were upregulated by the shift from aerobic to anaerobic nitrate respiration. An elongated cell morphology, similar to that induced by the DivS-mediated suppression of cell division upon cell exposure to the DNA-damaging reagent mitomycin C, was observed in cells subjected to anaerobic nitrate respiration. None of these transcriptional and morphological differences were observed in a recA mutant strain lacking a functional RecA regulator of the SOS response. The recA mutant cells additionally showed significantly reduced viability compared to wild-type cells similarly grown under anaerobic nitrate respiration. These results suggest a role for the RecA-mediated SOS response in the ability of cells to survive any DNA damage that may result from anaerobic nitrate respiration in C. glutamicum.

  10. Cofactor recycling for co-production of 1,3-propanediol and glutamate by metabolically engineered Corynebacterium glutamicum

    PubMed Central

    Huang, Jinhai; Wu, Yao; Wu, Wenjun; Zhang, Ye; Liu, Dehua; Chen, Zhen

    2017-01-01

    Production of 1,3-propanediol (1,3-PDO) from glycerol is a promising route toward glycerol biorefinery. However, the yield of 1,3-PDO is limited due to the requirement of NADH regeneration via glycerol oxidation process, which generates large amounts of undesired byproducts. Glutamate fermentation by Corynebacterium glutamicum is an important oxidation process generating excess NADH. In this study, we proposed a novel strategy to couple the process of 1,3-PDO synthesis with glutamate production for cofactor regeneration. With the optimization of 1,3-PDO synthesis route, C. glutamicum can efficiently convert glycerol into 1,3-PDO with the yield of ~ 1.0 mol/mol glycerol. Co-production of 1,3-PDO and glutamate was also achieved which increased the yield of glutamate by 18% as compared to the control. Since 1,3-PDO and glutamate can be easily separated in downstream process, this study provides a potential green route for coupled production of 1,3-PDO and glutamate to enhance the economic viability of biorefinery process. PMID:28176878

  11. Modular Optimization of a Hemicellulose-Utilizing Pathway in Corynebacterium glutamicum for Consolidated Bioprocessing of Hemicellulosic Biomass.

    PubMed

    Yim, Sung Sun; Choi, Jae Woong; Lee, Se Hwa; Jeong, Ki Jun

    2016-04-15

    Hemicellulose, which is the second most abundant polysaccharide in nature after cellulose, has the potential to become a major feedstock for microbial fermentation to produce various biofuels and chemicals. To utilize hemicellulose economically, it is necessary to develop a consolidated bioprocess (CBP), in which all processes from biomass degradation to the production of target products occur in a single bioreactor. Here, we report a modularly engineered Corynebacterium glutamicum strain suitable for CBP using hemicellulosic biomass (xylan) as a feedstock. The hemicellulose-utilizing pathway was divided into three distinct modules, and each module was separately optimized. In the module for xylose utilization, the expression level of the xylose isomerase (xylA) and xylulokinase (xylB) genes was optimized with synthetic promoters of different strengths. Then, the module for xylose transport was engineered with combinatorial sets of synthetic promoters and heterologous transporters to achieve the fastest cell growth rate on xylose (0.372 h(-1)). Next, the module for the enzymatic degradation of xylan to xylose was also engineered with different combinations of promoters and signal peptides to efficiently secrete both endoxylanase and xylosidase into the extracellular medium. Finally, each optimized module was integrated into a single plasmid to construct a highly efficient xylan-utilizing pathway. Subsequently, the direct production of lysine from xylan was successfully demonstrated with the engineered pathway. To the best of our knowledge, this is the first report of the development of a consolidated bioprocessing C. glutamicum strain for hemicellulosic biomass.

  12. Maltose uptake by the novel ABC transport system MusEFGK2I causes increased expression of ptsG in Corynebacterium glutamicum.

    PubMed

    Henrich, Alexander; Kuhlmann, Nora; Eck, Alexander W; Krämer, Reinhard; Seibold, Gerd M

    2013-06-01

    The Gram-positive Corynebacterium glutamicum efficiently metabolizes maltose by a pathway involving maltodextrin and glucose formation by 4-α-glucanotransferase, glucose phosphorylation by glucose kinases, and maltodextrin degradation via maltodextrin phosphorylase and α-phosphoglucomutase. However, maltose uptake in C. glutamicum has not been investigated. Interestingly, the presence of maltose in the medium causes increased expression of ptsG in C. glutamicum by an unknown mechanism, although the ptsG-encoded glucose-specific EII permease of the phosphotransferase system itself is not required for maltose utilization. We identified the maltose uptake system as an ABC transporter encoded by musK (cg2708; ATPase subunit), musE (cg2705; substrate binding protein), musF (cg2704; permease), and musG (cg2703; permease) by combination of data obtained from characterization of maltose uptake and reanalyses of transcriptome data. Deletion of the mus gene cluster in C. glutamicum Δmus abolished maltose uptake and utilization. Northern blotting and reverse transcription-PCR experiments revealed that musK and musE are transcribed monocistronically, whereas musF and musG are part of an operon together with cg2701 (musI), which encodes a membrane protein of unknown function with no homologies to characterized proteins. Characterization of growth and [(14)C]maltose uptake in the musI insertion strain C. glutamicum IMcg2701 showed that musI encodes a novel essential component of the maltose ABC transporter of C. glutamicum. Finally, ptsG expression during cultivation on different carbon sources was analyzed in the maltose uptake-deficient strain C. glutamicum Δmus. Indeed, maltose uptake by the novel ABC transport system MusEFGK2I is required for the positive effect of maltose on ptsG expression in C. glutamicum.

  13. The ncgl1108 (PheP (Cg)) gene encodes a new L-Phe transporter in Corynebacterium glutamicum.

    PubMed

    Zhao, Zhi; Ding, Jiu-Yuan; Li, Tang; Zhou, Ning-Yi; Liu, Shuang-Jiang

    2011-06-01

    Corynebacterium glutamicum played a central role in the establishment of fermentative production of amino acids, and it is a model for genetic and physiological studies. The general aromatic amino acid transporter, AroP(Cg), was the sole functionally identified aromatic amino acid transporter from C. glutamicum. In this study, the ncgl1108 (named as pheP (Cg), which is located upstream of the genetic cluster (ncgl1110 ~ ncgl1113) for resorcinol catabolism, was identified as a new L-Phe specific transporter from C. glutamicum RES167. The disruption of pheP (Cg) resulted in RES167∆ncgl1108, and this mutant showed decreased growth on L-Phe (as nitrogen source) but not on L-Tyr or L-Trp. Uptake assays with unlabeled and (14)C-labeled L-Phe and L-Tyr indicated that the mutants RES167∆ncgl1108 showed significant reduction in L-Phe uptake than RES167. Expression of pheP (Cg) in RES167∆ncgl1108/pGXKZ1 or RES167∆(ncgl1108-aroP (Cg))/pGXKZ1 restored their ability to uptake for L-Phe and growth on L-Phe. The uptake of L-Phe was not inhibited by nine amino acids but by L-Tyr. The K (m) and V (max) values of RES167∆(ncgl1108-aroP (Cg))/pGXKZ1 for L-Phe were determined to be 10.4 ± 1.5 μM and 1.2 ± 0.1 nmol min(-1) (mg DW)(-1), respectively, which are different from K (m) and V (max) values of RES167∆(ncgl1108-aroP (Cg)) for L-Phe [4.0 ± 0.4 μM and 0.6 ± 0.1 nmol min(-1) (mg DW)(-1)]. In conclusion, this PheP(Cg) is a new L-Phe transporter in C. glutamicum.

  14. Improving putrescine production by Corynebacterium glutamicum by fine-tuning ornithine transcarbamoylase activity using a plasmid addiction system.

    PubMed

    Schneider, Jens; Eberhardt, Dorit; Wendisch, Volker F

    2012-07-01

    Corynebacterium glutamicum shows a great potential for the production of the polyamide monomer putrescine (1,4-diaminobutane). Previously, we constructed the putrescine-producing strain PUT1 by deletion of argF, the gene for ornithine transcarbamoylase (OTC), and argR, encoding the L-arginine repressor, combined with heterologous expression of the Escherichia coli gene for L-ornithine decarboxylase SpeC. As a consequence of argF deletion, this strain requires supplementation of L-arginine and shows growth-decoupled putrescine production. To avoid costly supplementation with L-arginine and the strong feedback inhibition of the key enzyme N-acetylglutamate kinase (ArgB) by L-arginine, a plasmid addiction system for low-level argF expression was developed. By fine-tuning argF expression through modifications of the promoter, the translational start codon and/or the ribosome binding site, high productivity and titer could be obtained. OTC activity varied almost thousandfold between 960 and 1 mU mg⁻¹ resulting in putrescine yields on glucose from less than 0.001 up to 0.26 g g⁻¹, the highest yield in bacteria reported to date. The most promising strain, designated PUT21, was characterized comprehensively. PUT21 strain grew with a rate of 0.19 h⁻¹ in mineral salt medium without the need for L-arginine supplementation and produced putrescine with a yield of 0.16 g g⁻¹ glucose at a volumetric productivity of 0.57 g L⁻¹ h⁻¹ and a specific productivity of 0.042 g g⁻¹ h⁻¹. The carbon balance suggested that no major unidentified by-product was produced. Compared to the first-generation strain PUT1, the putrescine yield observed with PUT21 was increased by 60%. In fed-batch cultivation with C. glutamicum PUT21, a putrescine titer of 19 g L⁻¹ at a volumetric productivity of 0.55 g L⁻¹ h⁻¹ and a yield of 0.16 g g⁻¹ glucose could be achieved. Moreover, while plasmid segregation of the initial strain required antibiotic selection

  15. Production of L-glutamic Acid with Corynebacterium glutamicum (NCIM 2168) and Pseudomonas reptilivora (NCIM 2598): A Study on Immobilization and Reusability

    PubMed Central

    Shyamkumar, Rajaram; Moorthy, Innasi Muthu Ganesh; Ponmurugan, Karuppiah; Baskar, Rajoo

    2014-01-01

    Background L-glutamic acid is one of the major amino acids that is present in a wide variety of foods. It is mainly used as a food additive and flavor enhancer in the form of sodium salt. Corynebacterium glutamicum (C. glutamicum) is one of the major organisms widely used for glutamic acid production. Methods The study was dealing with immobilization of C. glutamicum and mixed culture of C. glutamicum and Pseudomonas reptilivora (P. reptilivora) for L-glutamic acid production using submerged fermentation. 2, 3 and 5% sodium alginate concentrations were used for production and reusability of immobilized cells for 5 more trials. Results The results revealed that 2% sodium alginate concentration produced the highest yield (13.026±0.247 g/l by C. glutamicum and 16.026±0.475 g/l by mixed immobilized culture). Moreover, reusability of immobilized cells was evaluated in 2% concentration with 5 more trials. However, when the number of cycles increased, the production of L-glutamic acid decreased. Conclusion Production of glutamic acid using optimized medium minimizes the time needed for designing the medium composition. It also minimizes external contamination. Glutamic acid production gradually decreased due to multiple uses of beads and consequently it reduces the shelf life. PMID:25215180

  16. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect L-Lysine Production in Corynebacterium glutamicum.

    PubMed

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-03-09

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in L-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport--NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885--were also expressed at significantly higher levels in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, L-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.

  17. Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.

    PubMed

    Yamamoto, Shogo; Gunji, Wataru; Suzuki, Hiroaki; Toda, Hiroshi; Suda, Masako; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

    2012-06-01

    We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159-165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD(+) ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.

  18. Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σH in Corynebacterium glutamicum

    PubMed Central

    Toyoda, Koichi; Teramoto, Haruhiko; Yukawa, Hideaki

    2014-01-01

    The extracytoplasmic function sigma factor σH is responsible for the heat and oxidative stress response in Corynebacterium glutamicum. Due to the hierarchical nature of the regulatory network, previous transcriptome analyses have not been able to discriminate between direct and indirect targets of σH. Here, we determined the direct genome-wide targets of σH using chromatin immunoprecipitation with microarray technology (ChIP-chip) for analysis of a deletion mutant of rshA, encoding an anti-σ factor of σH. Seventy-five σH-dependent promoters, including 39 new ones, were identified. σH-dependent, heat-inducible transcripts for several of the new targets, including ilvD encoding a labile Fe-S cluster enzyme, dihydroxy-acid dehydratase, were detected, and their 5′ ends were mapped to the σH-dependent promoters identified. Interestingly, functional internal σH-dependent promoters were found in operon-like gene clusters involved in the pentose phosphate pathway, riboflavin biosynthesis, and Zn uptake. Accordingly, deletion of rshA resulted in hyperproduction of riboflavin and affected expression of Zn-responsive genes, possibly through intracellular Zn overload, indicating new physiological roles of σH. Furthermore, sigA encoding the primary σ factor was identified as a new target of σH. Reporter assays demonstrated that the σH-dependent promoter upstream of sigA was highly heat inducible but much weaker than the known σA-dependent one. Our ChIP-chip analysis also detected the σH-dependent promoters upstream of rshA within the sigH-rshA operon and of sigB encoding a group 2 σ factor, supporting the previous findings of their σH-dependent expression. Taken together, these results reveal an additional layer of the sigma factor regulatory network in C. glutamicum. PMID:25404703

  19. SpiE interacts with Corynebacterium glutamicum WhcE and is involved in heat and oxidative stress responses.

    PubMed

    Park, Jung Chul; Park, Joon-Song; Kim, Younhee; Kim, Pil; Kim, Eung Soo; Lee, Heung-Shick

    2016-05-01

    The gene whcE in Corynebacterium glutamicum positively responds to oxidative and heat stress. To search for proteins that interact with WhcE, we employed a two-hybrid system with WhcE as the bait. Sequencing analysis of the isolated clones revealed peptide sequences, one of which showed high sequence identity to a hydrophobe/amphiphile efflux-1 family transporter encoded by NCgl1497. The interaction of the NCgl1497-encoded protein with WhcE in vivo was verified using reporter gene expression by real-time quantitative PCR (RT-qPCR). The WhcE protein strongly interacted with the NCgl1497-encoded protein in the presence of oxidative and heat stress. Furthermore, purified WhcE and NCgl1497-encoded proteins interacted in vitro, especially in the presence of the oxidant diamide, and the protein-protein interaction was disrupted in the presence of the reductant dithiothreitol. In addition, the transcription of NCgl1497 was activated approximately twofold in diamide- or heat-treated cells. To elucidate the function of the NCgl497 gene, an NCgl1497-deleted mutant strain was constructed. The mutant showed decreased viability in the presence of diamide and heat stress. The mutant strain also exhibited reduced transcription of the thioredoxin reductase gene, which is known to be regulated by whcE. Based on the results, NCgl1497 was named spiE (stress protein interacting with WhcE). Collectively, our data suggest that spiE is involved in the whcE-mediated oxidative stress response pathway of C. glutamicum.

  20. From zero to hero - production of bio-based nylon from renewable resources using engineered Corynebacterium glutamicum.

    PubMed

    Kind, Stefanie; Neubauer, Steffi; Becker, Judith; Yamamoto, Motonori; Völkert, Martin; Abendroth, Gregory von; Zelder, Oskar; Wittmann, Christoph

    2014-09-01

    Polyamides are important industrial polymers. Currently, they are produced exclusively from petrochemical monomers. Herein, we report the production of a novel bio-nylon, PA5.10 through an integration of biological and chemical approaches. First, systems metabolic engineering of Corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. In this way, a hyper-producer, with a high diaminopentane yield of 41% in shake flask culture, was generated. Subsequent fed-batch production of C. glutamicum DAP-16 allowed a molar yield of 50%, a productivity of 2.2gL(-1)h(-1), and a final titer of 88gL(-1). The streamlined producer accumulated diaminopentane without generating any by-products. Solvent extraction from alkalized broth and two-step distillation provided highly pure diaminopentane (99.8%), which was then directly accessible for poly-condensation. Chemical polymerization with sebacic acid, a ten-carbon dicarboxylic acid derived from castor plant oil, yielded the bio-nylon, PA5.10. In pure form and reinforced with glass fibers, the novel 100% bio-polyamide achieved an excellent melting temperature and the mechanical strength of the well-established petrochemical polymers, PA6 and PA6.6. It even outperformed the oil-based products in terms of having a 6% lower density. It thus holds high promise for applications in energy-friendly transportation. The demonstration of a novel route for generation of bio-based nylon from renewable sources opens the way to production of sustainable bio-polymers with enhanced material properties and represents a milestone in industrial production.

  1. Identification of two prpDBC gene clusters in Corynebacterium glutamicum and their involvement in propionate degradation via the 2-methylcitrate cycle.

    PubMed

    Claes, Wilfried A; Pühler, Alfred; Kalinowski, Jörn

    2002-05-01

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

  2. Reducing lactate secretion by ldhA Deletion in L-glutamate- producing strain Corynebacterium glutamicum GDK-9

    PubMed Central

    Zhang, Dalong; Guan, Dan; Liang, Jingbo; Guo, Chunqian; Xie, Xixian; Zhang, Chenglin; Xu, Qingyang; Chen, Ning

    2014-01-01

    L-lactate is one of main byproducts excreted in to the fermentation medium. To improve L-glutamate production and reduce L-lactate accumulation, L-lactate dehydrogenase-encoding gene ldhA was knocked out from L-glutamate producing strain Corynebacterium glutamicum GDK-9, designated GDK-9ΔldhA. GDK-9ΔldhA produced approximately 10.1% more L-glutamate than the GDK-9, and yielded lower levels of such by-products as α-ketoglutarate, L-lactate and L-alanine. Since dissolved oxygen (DO) is one of main factors affecting L-lactate formation during L-glutamate fermentation, we investigated the effect of ldhA deletion from GDK-9 under different DO conditions. Under both oxygen-deficient and high oxygen conditions, L-glutamate production by GDK-9ΔldhA was not higher than that of the GDK-9. However, under micro-aerobic conditions, GDK-9ΔldhA exhibited 11.61% higher L-glutamate and 58.50% lower L-alanine production than GDK-9. Taken together, it is demonstrated that deletion of ldhA can enhance L-glutamate production and lower the unwanted by-products concentration, especially under micro-aerobic conditions. PMID:25763057

  3. Mutations of the Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel homolog, induce L-glutamic acid production.

    PubMed

    Nakamura, Jun; Hirano, Seiko; Ito, Hisao; Wachi, Masaaki

    2007-07-01

    Corynebacterium glutamicum is a biotin auxotroph that secretes L-glutamic acid in response to biotin limitation; this process is employed in industrial L-glutamic acid production. Fatty acid ester surfactants and penicillin also induce L-glutamic acid secretion, even in the presence of biotin. However, the mechanism of L-glutamic acid secretion remains unclear. It was recently reported that disruption of odhA, encoding a subunit of the 2-oxoglutarate dehydrogenase complex, resulted in L-glutamic acid secretion without induction. In this study, we analyzed odhA disruptants and found that those which exhibited constitutive L-glutamic acid secretion carried additional mutations in the NCgl1221 gene, which encodes a mechanosensitive channel homolog. These NCgl1221 gene mutations lead to constitutive L-glutamic acid secretion even in the absence of odhA disruption and also render cells resistant to an L-glutamic acid analog, 4-fluoroglutamic acid. Disruption of the NCgl1221 gene essentially abolishes L-glutamic acid secretion, causing an increase in the intracellular L-glutamic acid pool under biotin-limiting conditions, while amplification of the wild-type NCgl1221 gene increased L-glutamate secretion, although only in response to induction. These results suggest that the NCgl1221 gene encodes an L-glutamic acid exporter. We propose that treatments that induce L-glutamic acid secretion alter membrane tension and trigger a structural transformation of the NCgl1221 protein, enabling it to export L-glutamic acid.

  4. Interaction between DAHP synthase and chorismate mutase endows new regulation on DAHP synthase activity in Corynebacterium glutamicum.

    PubMed

    Li, Pan-Pan; Li, De-Feng; Liu, Di; Liu, Yi-Ming; Liu, Chang; Liu, Shuang-Jiang

    2013-12-01

    Previous research on Corynebacterium glutamicum revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DSCg, formerly DS2098) interacts with chorismate mutase (CMCg, formerly CM0819). In this study, we investigated the interaction by means of structure-guided mutation and enzymatic assays. Our results show that the interaction imparted a new mechanism for regulation of DAHP activity: In the absence of CMCg, DSCg activity was not regulated by prephenate, whereas in the presence of CMCg, prephenate markedly inhibited DSCg activity. Prephenate competed with the substrate phosphoenolpyruvate, and the inhibition constant (K i) was determined to be 0.945 mM. Modeling based on the structure of the complex formed between DAHP synthase and chorismate mutase of Mycobacterium tuberculosis predicted the interaction surfaces of the putative DSCg-CMCg complex. The amino acid residues and structural domains that contributed to the interaction surfaces were experimentally identified to be the (212)SPAGARYE(219) sequence of DSCg and the (60)SGGTR(64) loop and C-terminus ((97)RGKLG(101)) of CMCg.

  5. Metabolic Engineering of Corynebacterium glutamicum for Trehalose Overproduction: Role of the TreYZ Trehalose Biosynthetic Pathway

    PubMed Central

    Carpinelli, Jorge; Krämer, Reinhard; Agosin, Eduardo

    2006-01-01

    Trehalose has many potential applications in biotechnology and the food industry due to its protective effect against environmental stress. Our work explores microbiological production methods based on the capacity of Corynebacterium glutamicum to excrete trehalose. We address here raising trehalose productivity through homologous overexpression of maltooligosyltrehalose synthase and the maltooligosyltrehalose trehalohydrolase genes. In addition, heterologous expression of the UDP-glucose pyrophosphorylase gene from Escherichia coli improved the supply of glycogen. Gene expression effects were tested on enzymatic activities and intracellular glycogen content, as well as on accumulated and excreted trehalose. Overexpression of the treY gene and the treY/treZ synthetic operon significantly increased maltooligosyltrehalose synthase activity, the rate-limiting step, and improved the specific productivity and the final titer of trehalose. Furthermore, a strong decrease was noted in glycogen accumulation. Expression of galU/treY and galU/treYZ synthetic operons showed a partial recovery in the intracellular glycogen levels and a significant improvement in both intra- and extracellular trehalose content. PMID:16517642

  6. Structural basis for redox sensitivity in Corynebacterium glutamicum diaminopimelate epimerase: an enzyme involved in l-lysine biosynthesis

    PubMed Central

    Sagong, Hye-Young; Kim, Kyung-Jin

    2017-01-01

    Diaminopimelate epimerase (DapF) is one of the crucial enzymes involved in l-lysine biosynthesis, where it converts l,l-diaminopimelate (l,l-DAP) into d,l-DAP. DapF is also considered as an attractive target for the development of antibacterial drugs. Here, we report the crystal structure of DapF from Corynebacterium glutamicum (CgDapF). Structures of CgDapF obtained under both oxidized and reduced conditions reveal that the function of CgDapF is regulated by redox-switch modulation via reversible disulfide bond formation between two catalytic cysteine residues. Under oxidized condition, two catalytic cysteine residues form a disulfide bond; these same cysteine residues exist in reduced form under reduced condition. Disulfide bond formation also induces a subsequent structural change in the dynamic catalytic loop at the active site, which results in open/closed conformational change at the active site. We also determined the crystal structure of CgDapF in complex with its product d,l-DAP, and elucidated how the enzyme recognizes its substrate l,l-DAP as a substrate. Moreover, the structure in complex with the d,l-DAP product reveals that CgDapF undergoes a large open/closed domain movement upon substrate binding, resulting in a completely buried active site with the substrate bound. PMID:28176858

  7. Promiscuous activity of (S,S)-butanediol dehydrogenase is responsible for glycerol production from 1,3-dihydroxyacetone in Corynebacterium glutamicum under oxygen-deprived conditions.

    PubMed

    Jojima, Toru; Igari, Takafumi; Moteki, Yasuhiro; Suda, Masako; Yukawa, Hideaki; Inui, Masayuki

    2015-02-01

    Corynebacterium glutamicum can consume glucose to excrete glycerol under oxygen deprivation. Although glycerol synthesis from 1,3-dihydroxyacetone (DHA) has been speculated, no direct evidence has yet been provided in C. glutamicum. Enzymatic and genetic investigations here indicate that the glycerol is largely produced from DHA and, unexpectedly, the reaction is catalyzed by (S,S)-butanediol dehydrogenase (ButA) that inherently catalyzes the interconversion between S-acetoin and (S,S)-2,3-butanediol. Consequently, the following pathway for glycerol biosynthesis in the bacterium emerges: dihydroxyacetone phosphate is dephosphorylated by HdpA to DHA, which is subsequently reduced to glycerol by ButA. This study emphasizes the importance of promiscuous activity of the enzyme in vivo.

  8. Citrate utilization by Corynebacterium glutamicum is controlled by the CitAB two-component system through positive regulation of the citrate transport genes citH and tctCBA.

    PubMed

    Brocker, Melanie; Schaffer, Steffen; Mack, Christina; Bott, Michael

    2009-06-01

    In this work, the molecular basis of aerobic citrate utilization by the gram-positive bacterium Corynebacterium glutamicum was studied. Genome analysis revealed the presence of two putative citrate transport systems. The permease encoded by citH belongs to the citrate-Mg(2+):H(+)/citrate-Ca(2+):H(+) symporter family, whereas the permease encoded by the tctCBA operon is a member of the tripartite tricarboxylate transporter family. The expression of citH or tctCBA in Escherichia coli enabled this species to utilize citrate aerobically, indicating that both CitH and TctABC are functional citrate transporters. Growth tests with the recombinant E. coli strains indicated that CitH is active with Ca(2+) or Sr(2+) but not with Mg(2+) and that TctABC is active with Ca(2+) or Mg(2+) but not with Sr(2+). We could subsequently show that, with 50 mM citrate as the sole carbon and energy source, the C. glutamicum wild type grew best when the minimal medium was supplemented with CaCl(2) but that MgCl(2) and SrCl(2) also supported growth. Each of the two transporters alone was sufficient for growth on citrate. The expression of citH and tctCBA was activated by citrate in the growth medium, independent of the presence or absence of glucose. This activation was dependent on the two-component signal transduction system CitAB, composed of the sensor kinase CitA and the response regulator CitB. CitAB belongs to the CitAB/DcuSR family of two-component systems, whose members control the expression of genes that are involved in the transport and catabolism of tricarboxylates or dicarboxylates. C. glutamicum CitAB is the first member of this family studied in Actinobacteria.

  9. Transcription of Sialic Acid Catabolism Genes in Corynebacterium glutamicum Is Subject to Catabolite Repression and Control by the Transcriptional Repressor NanR

    PubMed Central

    Uhde, Andreas; Brühl, Natalie; Goldbeck, Oliver; Matano, Christian; Gurow, Oksana; Rückert, Christian; Marin, Kay; Wendisch, Volker F.; Krämer, Reinhard

    2016-01-01

    ABSTRACT Corynebacterium glutamicum metabolizes sialic acid (Neu5Ac) to fructose-6-phosphate (fructose-6P) via the consecutive activity of the sialic acid importer SiaEFGI, N-acetylneuraminic acid lyase (NanA), N-acetylmannosamine kinase (NanK), N-acetylmannosamine-6P epimerase (NanE), N-acetylglucosamine-6P deacetylase (NagA), and glucosamine-6P deaminase (NagB). Within the cluster of the three operons nagAB, nanAKE, and siaEFGI for Neu5Ac utilization a fourth operon is present, which comprises cg2936, encoding a GntR-type transcriptional regulator, here named NanR. Microarray studies and reporter gene assays showed that nagAB, nanAKE, siaEFGI, and nanR are repressed in wild-type (WT) C. glutamicum but highly induced in a ΔnanR C. glutamicum mutant. Purified NanR was found to specifically bind to the nucleotide motifs A[AC]G[CT][AC]TGATGTC[AT][TG]ATGT[AC]TA located within the nagA-nanA and nanR-sialA intergenic regions. Binding of NanR to promoter regions was abolished in the presence of the Neu5Ac metabolism intermediates GlcNAc-6P and N-acetylmannosamine-6-phosphate (ManNAc-6P). We observed consecutive utilization of glucose and Neu5Ac as well as fructose and Neu5Ac by WT C. glutamicum, whereas the deletion mutant C. glutamicum ΔnanR simultaneously consumed these sugars. Increased reporter gene activities for nagAB, nanAKE, and nanR were observed in cultivations of WT C. glutamicum with Neu5Ac as the sole substrate compared to cultivations when fructose was present. Taken together, our findings show that Neu5Ac metabolism in C. glutamicum is subject to catabolite repression, which involves control by the repressor NanR. IMPORTANCE Neu5Ac utilization is currently regarded as a common trait of both pathogenic and commensal bacteria. Interestingly, the nonpathogenic soil bacterium C. glutamicum efficiently utilizes Neu5Ac as a substrate for growth. Expression of genes for Neu5Ac utilization in C. glutamicum is here shown to depend on the transcriptional regulator

  10. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    PubMed Central

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-01-01

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport—NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885—were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production. PMID:27005618

  11. High-throughput screening of a Corynebacterium glutamicum mutant library on genomic and metabolic level.

    PubMed

    Reimer, Lorenz C; Spura, Jana; Schmidt-Hohagen, Kerstin; Schomburg, Dietmar

    2014-01-01

    Due to impressive achievements in genomic research, the number of genome sequences has risen quickly, followed by an increasing number of genes with unknown or hypothetical function. This strongly calls for development of high-throughput methods in the fields of transcriptomics, proteomics and metabolomics. Of these platforms, metabolic profiling has the strongest correlation with the phenotype. We previously published a high-throughput metabolic profiling method for C. glutamicum as well as the automatic GC/MS processing software MetaboliteDetector. Here, we added a high-throughput transposon insertion determination for our C. glutamicum mutant library. The combination of these methods allows the parallel analysis of genotype/phenotype correlations for a large number of mutants. In a pilot project we analyzed the insertion points of 722 transposon mutants and found that 36% of the affected genes have unknown functions. This underlines the need for further information gathered by high-throughput techniques. We therefore measured the metabolic profiles of 258 randomly chosen mutants. The MetaboliteDetector software processed this large amount of GC/MS data within a few hours with a low relative error of 11.5% for technical replicates. Pairwise correlation analysis of metabolites over all genotypes showed dependencies of known and unknown metabolites. For a first insight into this large data set, a screening for interesting mutants was done by a pattern search, focusing on mutants with changes in specific pathways. We show that our transposon mutant library is not biased with respect to insertion points. A comparison of the results for specific mutants with previously published metabolic results on a deletion mutant of the same gene confirmed the concept of high-throughput metabolic profiling. Altogether the described method could be applied to whole mutant libraries and thereby help to gain comprehensive information about genes with unknown, hypothetical and known

  12. Effect of biotin on transcription levels of key enzymes and glutamate efflux in glutamate fermentation by Corynebacterium glutamicum.

    PubMed

    Cao, Yan; Duan, Zuoying; Shi, Zhongping

    2014-02-01

    Biotin is an important factor affecting the performance of glutamate fermentation by biotin auxotrophic Corynebacterium glutamicum and glutamate is over-produced only when initial biotin content is controlled at suitable levels or initial biotin is excessive but with Tween 40 addition during fermentation. The transcription levels of key enzymes at pyruvate, isocitrate and α-ketoglutarate metabolic nodes, as well as transport protein (TP) of glutamate were investigated under the conditions of varied biotin contents and Tween 40 supplementation. When biotin was insufficient, the genes encoding key enzymes and TP were down-regulated in the early production phase, in particular, the transcription level of isocitrate dehydrogenase (ICDH) which was only 2% of that of control. Although the cells' morphology transformation and TP level were not affected, low transcription level of ICDH led to lower final glutamate concentration (64 g/L). When biotin was excessive, the transcription levels of key enzymes were at comparable levels as those of control with ICDH as an exception, which was only 3-22% of control level throughout production phase. In this case, little intracellular glutamate accumulation (1.5 mg/g DCW) and impermeable membrane resulted in non glutamate secretion into broth, even though the quantity of TP was more than 10-folds of control level. Addition of Tween 40 when biotin was excessive stimulated the expression of all key enzymes and TP, intracellular glutamate content was much higher (10-12 mg/g DCW), and final glutamate concentration reached control level (75-80 g/L). Hence, the membrane alteration and TP were indispensable in glutamate secretion. Biotin and Tween 40 influenced the expression level of ICDH and glutamate efflux, thereby influencing glutamate production.

  13. Toward homosuccinate fermentation: metabolic engineering of Corynebacterium glutamicum for anaerobic production of succinate from glucose and formate.

    PubMed

    Litsanov, Boris; Brocker, Melanie; Bott, Michael

    2012-05-01

    Previous studies have demonstrated the capability of Corynebacterium glutamicum for anaerobic succinate production from glucose under nongrowing conditions. In this work, we have addressed two shortfalls of this process, the formation of significant amounts of by-products and the limitation of the yield by the redox balance. To eliminate acetate formation, a derivative of the type strain ATCC 13032 (strain BOL-1), which lacked all known pathways for acetate and lactate synthesis (Δcat Δpqo Δpta-ackA ΔldhA), was constructed. Chromosomal integration of the pyruvate carboxylase gene pyc(P458S) into BOL-1 resulted in strain BOL-2, which catalyzed fast succinate production from glucose with a yield of 1 mol/mol and showed only little acetate formation. In order to provide additional reducing equivalents derived from the cosubstrate formate, the fdh gene from Mycobacterium vaccae, coding for an NAD(+)-coupled formate dehydrogenase (FDH), was chromosomally integrated into BOL-2, leading to strain BOL-3. In an anaerobic batch process with strain BOL-3, a 20% higher succinate yield from glucose was obtained in the presence of formate. A temporary metabolic blockage of strain BOL-3 was prevented by plasmid-borne overexpression of the glyceraldehyde 3-phosphate dehydrogenase gene gapA. In an anaerobic fed-batch process with glucose and formate, strain BOL-3/pAN6-gap accumulated 1,134 mM succinate in 53 h with an average succinate production rate of 1.59 mmol per g cells (dry weight) (cdw) per h. The succinate yield of 1.67 mol/mol glucose is one of the highest currently described for anaerobic succinate producers and was accompanied by a very low level of by-products (0.10 mol/mol glucose).

  14. Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example

    PubMed Central

    Taniguchi, Hironori; Wendisch, Volker F.

    2015-01-01

    Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing each annotated sigma factor gene in Corynebacterium glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. High performance liquid chromatography analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes related to the pentose phosphate pathway and for enzymes dependent on flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production. PMID:26257719

  15. Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example.

    PubMed

    Taniguchi, Hironori; Wendisch, Volker F

    2015-01-01

    Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing each annotated sigma factor gene in Corynebacterium glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. High performance liquid chromatography analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes related to the pentose phosphate pathway and for enzymes dependent on flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production.

  16. Improvement of the Redox Balance Increases l-Valine Production by Corynebacterium glutamicum under Oxygen Deprivation Conditions

    PubMed Central

    Hasegawa, Satoshi; Uematsu, Kimio; Natsuma, Yumi; Suda, Masako; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki

    2012-01-01

    Production of l-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the l-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall l-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of l-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for l-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD+ ratio significantly decreased, and glucose consumption and l-valine production drastically improved. Moreover, l-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD+ ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for l-valine production under oxygen deprivation conditions. The l-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM l-valine after 24 h with a yield of 0.63 mol mol of glucose−1, and the l-valine productivity reached 1,940 mM after 48 h. PMID:22138982

  17. Corynebacterium glutamicum ATP-phosphoribosyl transferases suitable for L-histidine production--Strategies for the elimination of feedback inhibition.

    PubMed

    Kulis-Horn, Robert K; Persicke, Marcus; Kalinowski, Jörn

    2015-07-20

    L-Histidine biosynthesis in Corynebacterium glutamicum is mainly regulated by L-histidine feedback inhibition of the ATP-phosphoribosyltransferase HisG that catalyzes the first step of the pathway. The elimination of this feedback inhibition is the first and most important step in the development of an L-histidine production strain. For this purpose, a combined approach of random mutagenesis and rational enzyme redesign was performed. Mutants spontaneously resistant to the toxic L-histidine analog β-(2-thiazolyl)-DL-alanine (2-TA) revealed novel and unpredicted mutations in the C-terminal regulatory domain of HisG resulting in increased feedback resistance. Moreover, deletion of the entire C-terminal regulatory domain in combination with the gain of function mutation S143F in the catalytic domain resulted in a HisG variant that is still highly active even at L-histidine concentrations close to the solubility limit. Notably, the S143F mutation on its own provokes feedback deregulation, revealing for the first time an amino acid residue in the catalytic domain of HisG that is involved in the feedback regulatory mechanism. In addition, we investigated the effect of hisG mutations for L-histidine production on different levels. This comprised the analysis of different expression systems, including plasmid- and chromosome-based overexpression, as well as the importance of codon choice for HisG mutations. The combination of domain deletions, single amino acid exchanges, codon choice, and chromosome-based overexpression resulted in production strains accumulating around 0.5 g l(-1) L-histidine, demonstrating the added value of the different approaches.

  18. Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum.

    PubMed

    Issa, Hanane; Huc-Claustre, Emilie; Reddad, Thamila; Bonadé Bottino, Nolwenn; Tropis, Maryelle; Houssin, Christine; Daffé, Mamadou; Bayan, Nicolas; Dautin, Nathalie

    2017-01-01

    Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated.

  19. Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum

    PubMed Central

    Issa, Hanane; Huc-Claustre, Emilie; Reddad, Thamila; Bonadé Bottino, Nolwenn; Tropis, Maryelle; Houssin, Christine; Daffé, Mamadou; Bayan, Nicolas

    2017-01-01

    Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated. PMID:28199365

  20. Construction of a prophage-free variant of Corynebacterium glutamicum ATCC 13032 for use as a platform strain for basic research and industrial biotechnology.

    PubMed

    Baumgart, Meike; Unthan, Simon; Rückert, Christian; Sivalingam, Jasintha; Grünberger, Alexander; Kalinowski, Jörn; Bott, Michael; Noack, Stephan; Frunzke, Julia

    2013-10-01

    The activity of bacteriophages and phage-related mobile elements is a major source for genome rearrangements and genetic instability of their bacterial hosts. The genome of the industrial amino acid producer Corynebacterium glutamicum ATCC 13032 contains three prophages (CGP1, CGP2, and CGP3) of so far unknown functionality. Several phage genes are regularly expressed, and the large prophage CGP3 (∼190 kbp) has recently been shown to be induced under certain stress conditions. Here, we present the construction of MB001, a prophage-free variant of C. glutamicum ATCC 13032 with a 6% reduced genome. This strain does not show any unfavorable properties during extensive phenotypic characterization under various standard and stress conditions. As expected, we observed improved growth and fitness of MB001 under SOS-response-inducing conditions that trigger CGP3 induction in the wild-type strain. Further studies revealed that MB001 has a significantly increased transformation efficiency and produced about 30% more of the heterologous model protein enhanced yellow fluorescent protein (eYFP), presumably as a consequence of an increased plasmid copy number. These effects were attributed to the loss of the restriction-modification system (cg1996-cg1998) located within CGP3. The deletion of the prophages without any negative effect results in a novel platform strain for metabolic engineering and represents a useful step toward the construction of a C. glutamicum chassis genome of strain ATCC 13032 for biotechnological applications and synthetic biology.

  1. Gluconate as suitable potential reduction supplier in Corynebacterium glutamicum: cloning and expression of gntP and gntK in Escherichia coli.

    PubMed

    Porco, Antonietta; Gamero, Elida E; Mylonás, Elena; Istúriz, Tomás

    2008-01-01

    Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral medium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constitutively expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli M1-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.

  2. Methionine uptake in Corynebacterium glutamicum by MetQNI and by MetPS, a novel methionine and alanine importer of the NSS neurotransmitter transporter family.

    PubMed

    Trötschel, Christian; Follmann, Martin; Nettekoven, Jeannine A; Mohrbach, Tobias; Forrest, Lucy R; Burkovski, Andreas; Marin, Kay; Krämer, Reinhard

    2008-12-02

    The soil bacterium Corynebacterium glutamicum is a model organism in amino acid biotechnology. Here we present the identification of two different L-methionine uptake systems including the first characterization of a bacterial secondary methionine carrier. The primary carrier MetQNI is a high affinity ABC-type transporter specific for l-methionine. Its expression is under the control of the transcription factor McbR, the global regulator of sulfur metabolism in C. glutamicum. Besides MetQNI, a novel secondary methionine uptake system of the NSS (neurotransmitter:sodium symporter) family was identified and named MetP. The MetP system is characterized by a lower affinity for methionine and uses Na(+) ions for energetic coupling. It is also the main alanine transporter in C. glutamicum and is expressed constitutively. These observations are consistent with models of methionine, alanine, and leucine bound to MetP, derived from the X-ray crystal structure of the LeuT transporter from Aquifex aeolicus. Complementation studies show that MetP consists of two components, a large subunit with 12 predicted transmembrane segments and, surprisingly, an additional subunit with one predicted transmembrane segment only. Thus, this new member of the NSS transporter family adds a novel feature to this class of carriers, namely, the functional dependence on an additional small subunit.

  3. Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis.

    PubMed

    Neshat, Armin; Mentz, Almut; Rückert, Christian; Kalinowski, Jörn

    2014-11-20

    The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts.

  4. Attenuating l-lysine production by deletion of ddh and lysE and their effect on l-threonine and l-isoleucine production in Corynebacterium glutamicum.

    PubMed

    Dong, Xunyan; Zhao, Yue; Hu, Jinyu; Li, Ye; Wang, Xiaoyuan

    2016-11-01

    The fermentative production of l-threonine and l-isoleucine with Corynebacterium glutamicum is usually accompanied by the by-production of l-lysine, which shares partial biosynthesis pathway with l-threonine and l-isoleucine. Since the direct precursor for l-lysine synthesis, diaminopimelate, is a component of peptidoglycan and thus essential for cell wall synthesis, reducing l-lysine by-production could be troublesome. Here, a basal strain with eliminated l-lysine production was constructed from the wild type C. glutamicum ATCC13869 by deleting the chromosomal ddh and lysE. Furthermore, the basal strain as well as the ddh single mutant strain was engineered for l-threonine production by over-expressing lysC1, hom1 and thrB, and for l-isoleucine production by over-expressing lysC1, hom1, thrB and ilvA1. Fermentation experiments with the engineered strains showed that (i) deletion of ddh improved l-threonine production by 17%, and additional deletion of lysE further improved l-threonine production by 28%; (ii) deletion of ddh improved l-isoleucine production by 8% and improved cell growth by 21%, whereas additional deletion of lysE had no further influence on both l-isoleucine production and cell growth; (iii) l-lysine by-production was reduced by 95% and 86% in l-threonine and l-isoleucine production, respectively, by deletion of ddh and lysE. This is the first report on improving l-threonine and l-isoleucine production by deleting ddh and lysE in C. glutamicum. The results demonstrate deletion of ddh and lysE as an effective strategy to reduce l-lysine by-production without surrendering the cell growth of C. glutamicum.

  5. Single-Domain Peptidyl-Prolyl cis/trans Isomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures

    PubMed Central

    van Ooyen, Jan

    2015-01-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM l-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes in C. glutamicum ΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent. PMID:26341203

  6. Single-Domain Peptidyl-Prolyl cis/trans Isomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures.

    PubMed

    Kallscheuer, Nicolai; Bott, Michael; van Ooyen, Jan; Polen, Tino

    2015-11-01

    Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM l-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes in C. glutamicum ΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent.

  7. The Two-Component Signal Transduction System CopRS of Corynebacterium glutamicum Is Required for Adaptation to Copper-Excess Stress

    PubMed Central

    Schelder, Stephanie; Zaade, Daniela; Litsanov, Boris; Bott, Michael; Brocker, Melanie

    2011-01-01

    Copper is an essential cofactor for many enzymes but at high concentrations it is toxic for the cell. Copper ion concentrations ≥50 µM inhibited growth of Corynebacterium glutamicum. The transcriptional response to 20 µM Cu2+ was studied using DNA microarrays and revealed 20 genes that showed a ≥ 3-fold increased mRNA level, including cg3281-cg3289. Several genes in this genomic region code for proteins presumably involved in the adaption to copper-induced stress, e. g. a multicopper oxidase (CopO) and a copper-transport ATPase (CopB). In addition, this region includes the copRS genes (previously named cgtRS9) which encode a two-component signal transduction system composed of the histidine kinase CopS and the response regulator CopR. Deletion of the copRS genes increased the sensitivity of C. glutamicum towards copper ions, but not to other heavy metal ions. Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified. Evidence was obtained that CopR binds only to the intergenic region between cg3285 (copR) and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3281 and cg3286-cg3289. Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress. PMID:21799779

  8. Rapid Electron Transfer within the III-IV Supercomplex in Corynebacterium glutamicum

    PubMed Central

    Graf, Simone; Fedotovskaya, Olga; Kao, Wei-Chun; Hunte, Carola; Ädelroth, Pia; Bott, Michael; von Ballmoos, Christoph; Brzezinski, Peter

    2016-01-01

    Complex III in C. glutamicum has an unusual di-heme cyt. c1 and it co-purifies with complex IV in a supercomplex. Here, we investigated the kinetics of electron transfer within this supercomplex and in the cyt. aa3 alone (cyt. bc1 was removed genetically). In the reaction of the reduced cyt. aa3 with O2, we identified the same sequence of events as with other A-type oxidases. However, even though this reaction is associated with proton uptake, no pH dependence was observed in the kinetics. For the cyt. bc1-cyt. aa3 supercomplex, we observed that electrons from the c-hemes were transferred to CuA with time constants 0.1–1 ms. The b-hemes were oxidized with a time constant of 6.5 ms, indicating that this electron transfer is rate-limiting for the overall quinol oxidation/O2 reduction activity (~210 e−/s). Furthermore, electron transfer from externally added cyt. c to cyt. aa3 was significantly faster upon removal of cyt. bc1 from the supercomplex, suggesting that one of the c-hemes occupies a position near CuA. In conclusion, isolation of the III-IV-supercomplex allowed us to investigate the kinetics of electron transfer from the b-hemes, via the di-heme cyt. c1 and heme a to the heme a3-CuB catalytic site of cyt. aa3. PMID:27682138

  9. Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

    PubMed Central

    Buchholz, Jens; Schwentner, Andreas; Brunnenkan, Britta; Gabris, Christina; Grimm, Simon; Gerstmeir, Robert; Takors, Ralf; Eikmanns, Bernhard J.

    2013-01-01

    Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a YP/S of 0.24 mol per mol of glucose and a QP of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products. PMID:23835179

  10. Polynucleotide Phosphorylase, RNase E/G, and YbeY Are Involved in the Maturation of 4.5S RNA in Corynebacterium glutamicum.

    PubMed

    Maeda, Tomoya; Tanaka, Yuya; Wachi, Masaaki; Inui, Masayuki

    2017-03-01

    Corynebacterium glutamicum has been applied for the industrial production of various metabolites, such as amino acids. To understand the biosynthesis of the membrane protein in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP is found in all three domains of life and plays an important role in the membrane insertion of proteins. SRP RNA is initially transcribed as precursor molecules; however, relatively little is known about its maturation. In C. glutamicum, SRP consists of the Ffh protein and 4.5S RNA lacking an Alu domain. In this study, we found that 3'-to-5' exoribonuclease, polynucleotide phosphorylase (PNPase), and two endo-type RNases, RNase E/G and YbeY, are involved in the 3' maturation of 4.5S RNA in C. glutamicum The mature form of 4.5S RNA was inefficiently formed in ΔrneG Δpnp mutant cells, suggesting the existence of an alternative pathway for the 3' maturation of 4.5S RNA. Primer extension analysis also revealed that the 5' mature end of 4.5S RNA corresponds to that of the transcriptional start site. Immunoprecipitated Ffh protein contained immature 4.5S RNA in Δpnp, ΔrneG, and ΔybeY mutants, suggesting that 4.5S RNA precursors can interact with Ffh. These results imply that the maturation of 4.5S RNA can be performed in the 4.5S RNA-Ffh complex.IMPORTANCE Overproduction of a membrane protein, such as a transporter, is useful for engineering of strains of Corynebacterium glutamicum, which is a workhorse of amino acid production. To understand membrane protein biogenesis in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP contains the Ffh protein and SRP RNA and plays an important role in the membrane insertion of proteins. Although SRP RNA is highly conserved among the three domains of life, relatively little is known about its maturation. We show that PNPase, RNase E/G, and YbeY are involved in the 3' maturation of the SRP RNA (4.5S RNA) in this

  11. Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

    PubMed Central

    Mormann, Sascha; Lömker, Alexander; Rückert, Christian; Gaigalat, Lars; Tauch, Andreas; Pühler, Alfred; Kalinowski, Jörn

    2006-01-01

    Background Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. Results A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L

  12. A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level

    PubMed Central

    Kortmann, Maike; Kuhl, Vanessa; Klaffl, Simon; Bott, Michael

    2015-01-01

    Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-d-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg−1. It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum. PMID:25488698

  13. A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level.

    PubMed

    Kortmann, Maike; Kuhl, Vanessa; Klaffl, Simon; Bott, Michael

    2015-03-01

    Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-D-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg(-1). It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum.

  14. Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays.

    PubMed

    Jochmann, Nina; Kurze, Anna-Katharina; Czaja, Lisa F; Brinkrolf, Karina; Brune, Iris; Hüser, Andrea T; Hansmeier, Nicole; Pühler, Alfred; Borovok, Ilya; Tauch, Andreas

    2009-05-01

    The lexA gene of Corynebacterium glutamicum ATCC 13032 was deleted to create the mutant strain C. glutamicum NJ2114, which has an elongated cell morphology and an increased doubling time. To characterize the SOS regulon in C. glutamicum, the transcriptomes of NJ2114 and a DNA-damage-induced wild-type strain were compared with that of a wild-type control using DNA microarray hybridization. The expression data were combined with bioinformatic pattern searches for LexA binding sites, leading to the detection of 46 potential SOS boxes located upstream of differentially expressed transcription units. Binding of a hexahistidyl-tagged LexA protein to 40 double-stranded oligonucleotides containing the potential SOS boxes was demonstrated in vitro by DNA band shift assays. It turned out that LexA binds not only to SOS boxes in the promoter-operator region of upregulated genes, but also to SOS boxes detected upstream of downregulated genes. These results demonstrated that LexA controls directly the expression of at least 48 SOS genes organized in 36 transcription units. The deduced genes encode a variety of physiological functions, many of them involved in DNA repair and survival after DNA damage, but nearly half of them have hitherto unknown functions. Alignment of the LexA binding sites allowed the corynebacterial SOS box consensus sequence TcGAA(a/c)AnnTGTtCGA to be deduced. Furthermore, the common intergenic region of lexA and the differentially expressed divS-nrdR operon, encoding a cell division suppressor and a regulator of deoxyribonucleotide biosynthesis, was characterized in detail. Promoter mapping revealed differences in divS-nrdR expression during SOS response and normal growth conditions. One of the four LexA binding sites detected in the intergenic region is involved in regulating divS-nrdR transcription, whereas the other sites are apparently used for negative autoregulation of lexA expression.

  15. NdnR is an NAD-responsive transcriptional repressor of the ndnR operon involved in NAD de novo biosynthesis in Corynebacterium glutamicum.

    PubMed

    Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2012-04-01

    The Corynebacterium glutamicum ndnR gene, which is chromosomally located in a gene cluster involved in NAD de novo biosynthesis, negatively regulates expression of the cluster genes, i.e. nadA, nadC, nadS and ndnR itself. Although ndnR encodes a member of the recently identified NrtR family of transcriptional regulators, whether or not the NdnR protein directly regulates these NAD biosynthesis genes remains to be verified. Here, two NdnR binding sites in the promoter region of the ndnR-nadA-nadC-nadS operon in C. glutamicum were confirmed by in vitro DNA binding assay and analysis of in vivo expression of the chromosomally integrated ndnR promoter-lacZ reporter fusion. Electrophoretic mobility shift assay revealed that the NdnR protein binds to the 5'-upstream region of ndnR, and that the binding is significantly enhanced by NAD. Mutation in two 21 bp NdnR binding motifs in the ndnR promoter region inhibited the binding of NdnR in vitro. The mutation also enhanced the promoter activity in cells cultured in the presence of nicotinate, which is utilized in NAD biosynthesis, resulting in the loss of the repression in response to an exogenous NAD precursor; this is consistent with the effect of deletion of ndnR reported in our previous study. These results indicate that NAD acts as a co-repressor for the NdnR protein that directly regulates the ndnR operon involved in NAD de novo biosynthesis; the NAD-NdnR regulatory system likely plays an important role in the control of NAD homeostasis in C. glutamicum.

  16. Metabolic profile of 1,5-diaminopentane producing Corynebacterium glutamicum under scale-down conditions: Blueprint for robustness to bioreactor inhomogeneities.

    PubMed

    Limberg, Michael H; Schulte, Julia; Aryani, Tita; Mahr, Regina; Baumgart, Meike; Bott, Michael; Wiechert, Wolfgang; Oldiges, Marco

    2017-03-01

    Performance losses during scale-up are described since decades, but are still one of the major obstacles for industrial bioprocess development. Consequently, robustness to inhomogeneous cultivation environments is an important quality of industrial production organisms. Especially, Corynebacterium glutamicum was proven to have an outstanding resistance against rapid changes of oxygen and substrate availability as occurring in industrial scale bioreactors. This study focuses on the identification of metabolic key mechanisms for this robustness to get a deeper insight and provide future targets for process orientated strain development. A 1,5-diaminopentane producing C. glutamicum strain was cultivated in a two compartment scale-down device to create short-term environmental changes simulating industrial scale cultivation conditions. Using multi omics based methods, it is shown, that central metabolism is flexibly rearranged under short-term oxygen depletion and carbon source excess to overcome shortage in NAD(+) recycling. In order to balance the redox state, key enzymes for the non-oxygen dependent fermentative NAD(+) regeneration were significantly up-regulated while parts of non-essential pathways were down-regulated. The transfer of the cells back into the well aerated zones with low substrate concentration triggers an additional upregulation of genes for the re-assimilation of previously formed side products, showing L-lactate forming and utilizing reactions being active at the same time. Especially L-lactate as reversible and flexible external buffer for carbon and redox equivalents puts C. glutamicum in a robust position to deal with inhomogeneity in large scale processes. Biotechnol. Bioeng. 2017;114: 560-575. © 2016 Wiley Periodicals, Inc.

  17. Phosphatase activity of the histidine kinases ensures pathway specificity of the ChrSA and HrrSA two-component systems in Corynebacterium glutamicum.

    PubMed

    Hentschel, Eva; Mack, Christina; Gätgens, Cornelia; Bott, Michael; Brocker, Melanie; Frunzke, Julia

    2014-06-01

    The majority of bacterial genomes encode a high number of two-component systems controlling gene expression in response to a variety of different stimuli. The Gram-positive soil bacterium Corynebacterium glutamicum contains two homologous two-component systems (TCS) involved in the haem-dependent regulation of gene expression. Whereas the HrrSA system is crucial for utilization of haem as an alternative iron source, ChrSA is required to cope with high toxic haem levels. In this study, we analysed the interaction of HrrSA and ChrSA in C. glutamicum. Growth of TCS mutant strains, in vitro phosphorylation assays and promoter assays of P(hrtBA) and P(hmuO) fused to eyfp revealed cross-talk between both systems. Our studies further indicated that both kinases exhibit a dual function as kinase and phosphatase. Mutation of the conserved glutamine residue in the putative phosphatase motif DxxxQ of HrrS and ChrS resulted in a significantly increased activity of their respective target promoters (P(hmuO) and P(hrtBA) respectively). Remarkably, phosphatase activity of both kinases was shown to be specific only for their cognate response regulators. Altogether our data suggest the phosphatase activity of HrrS and ChrS as key mechanism to ensure pathway specificity and insulation of these two homologous systems.

  18. Efficient production of α-ketoglutarate in the gdh deleted Corynebacterium glutamicum by novel double-phase pH and biotin control strategy.

    PubMed

    Li, Yanjun; Sun, Lanchao; Feng, Jia; Wu, Ruifang; Xu, Qingyang; Zhang, Chenglin; Chen, Ning; Xie, Xixian

    2016-06-01

    Production of L-glutamate using a biotin-deficient strain of Corynebacterium glutamicum has a long history. The process is achieved by controlling biotin at suboptimal dose in the initial fermentation medium, meanwhile feeding NH4OH to adjust pH so that α-ketoglutarate (α-KG) can be converted to L-glutamate. In this study, we deleted glutamate dehydrogenase (gdh1 and gdh2) of C. glutamicum GKG-047, an L-glutamate overproducing strain, to produce α-KG that is the direct precursor of L-glutamate. Based on the method of L-glutamate fermentation, we developed a novel double-phase pH and biotin control strategy for α-KG production. Specifically, NH4OH was added to adjust the pH at the bacterial growth stage and NaOH was used when the cells began to produce acid; besides adding an appropriate amount of biotin in the initial medium, certain amount of additional biotin was supplemented at the middle stage of fermentation to maintain a high cell viability and promote the carbon fixation to the flux of α-KG production. Under this control strategy, 45.6 g/L α-KG accumulated after 30-h fermentation in a 7.5-L fermentor and the productivity and yield achieved were 1.52 g/L/h and 0.42 g/g, respectively.

  19. Roles of export genes cgmA and lysE for the production of L-arginine and L-citrulline by Corynebacterium glutamicum.

    PubMed

    Lubitz, Dorit; Jorge, João M P; Pérez-García, Fernando; Taniguchi, Hironori; Wendisch, Volker F

    2016-10-01

    L-arginine is a semi-essential amino acid with application in cosmetic, pharmaceutical, and food industries. Metabolic engineering strategies have been applied for overproduction of L-arginine by Corynebacterium glutamicum. LysE was the only known L-arginine exporter of this bacterium. However, an L-arginine-producing strain carrying a deletion of lysE still accumulated about 10 mM L-arginine in the growth medium. Overexpression of the putative putrescine and cadaverine export permease gene cgmA was shown to compensate for the lack of lysE with regard to L-arginine export. Moreover, plasmid-borne overexpression of cgmA rescued the toxic effect caused by feeding of the dipeptide Arg-Ala to lysE-deficient C. glutamicum and argO-deficient Escherichia coli strains. Deletion of the repressor gene cgmR improved L-arginine titers by 5 %. Production of L-lysine and L-citrulline was not affected by cgmA overexpression. Taken together, CgmA may function as an export system not only for the diamine putrescine and cadaverine but also for L-arginine. The major export system for L-lysine and L-arginine LysE may also play a role in L-citrulline export since production of L-citrulline was reduced when lysE was deleted and improved by 45 % when lysE was overproduced.

  20. Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

    PubMed Central

    Komati Reddy, Gajendar; Lindner, Steffen N.

    2015-01-01

    Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154–7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH

  1. Expression analysis of the csp-like genes from Corynebacterium glutamicum encoding homologs of the Escherichia coli major cold-shock protein cspA.

    PubMed

    Kim, Wan-Soo; Park, Soo-Dong; Lee, Seok-Myung; Kim, Younhee; Kim, Pil; Lee, Heung-Shick

    2007-08-01

    Three csp-like genes were identified in the Corynebacterium glutamicum genome and designated cspA, cspB, and cspA2. The genes cspA and cspA2 encode proteins, comprising of 67 amino acid residues, respectively. They share 83% identity with each other. Identity of those proteins with Escherichia coli Csp proteins was near 50%. The cspB gene encodes a protein composed of 127 amino acids, which has 40% and 35% sequence identity with CspA and CspA2, respectively, especially at its N-terminal region. Analysis of the gene expression profiles was done using transcriptional cat fusion, which identified not only active expression of the three genes at the physiological growth temperature of 30 degrees C but also growth phase-dependent expression with the highest activity at late log phase. The promoters of cspA and cspA2 were more active than that of cspB. The expression of the two genes increased by 30% after a temperature downshift to 15 degrees C, and such stimulation was more evident in the late growth phase. In addition, the cspA gene appeared to show DNA-binding activity in vivo, and the activity increased at lower temperatures. Interestingly, the presence of cspA in multicopy hindered the growth of the host C. glutamicum cells at 20 degrees C, but not at 30 degrees C. Altogether, these data suggest that cspA, cspB, and cspA2 perform functions related to cold shock as well as normal cellular physiology. Moreover, CspA and its ortholog CspA2 may perform additional functions as a transcriptional regulator.

  2. Increased glucose utilization and cell growth of Corynebacterium glutamicum by modifying the glucose-specific phosphotransferase system (PTS(Glc)) genes.

    PubMed

    Xu, Jianzhong; Zhang, Junlan; Liu, Dongdong; Zhang, Weiguo

    2016-12-01

    The phosphoenolpyruvate:glucose phosphotransferase system (PTS(Glc)) is the major pathway of glucose uptake in Corynebacterium glutamicum. This study investigated glucose consumption rate, cell growth, and metabolite changes resulting from modification of PTS(Glc). The classical l-lysine producer C. glutamicum XQ-8 exhibited low glucose consumption, cell growth, and l-lysine production rates, whereas these parameters were significantly increased during cultivating on glucose plus maltose, through inactivation of SugR, or by overexpression of PTS(Glc) genes. XQ-8sugR::cat/pDXW-8-ptsI exhibited the highest increase in glucose consumption, growth rate, and l-lysine production, followed by XQ-8sugR::cat/pDXW-8-ptsG. However, overexpression of ptsH had little effect on the above-mentioned factors. Although co-overexpression of ptsGHI led to the highest glucose consumption, growth rate, and final l-lysine production; the l-lysine production rate was lower than that of XQ-8sugR::cat/pDXW-8-ptsIH. In fed-batch fermentation, XQ-8sugR::cat/pDXW-8-ptsIH had a higher growth rate of 0.54 h(-1) to a dry cell mass of 66 g·L(-1) after 16 h, and had a higher l-lysine production rate of 159.2 g·L(-1) after 36 h. These results indicate that modification of the sugar transport systems improves amino acid production, especially for mutants obtained by repeated physical and (or) chemical mutagenesis. However, modification of these systems needs to be performed on a case-by-case basis.

  3. Comparative 13C metabolic flux analysis of pyruvate dehydrogenase complex-deficient, L-valine-producing Corynebacterium glutamicum.

    PubMed

    Bartek, Tobias; Blombach, Bastian; Lang, Siegmund; Eikmanns, Bernhard J; Wiechert, Wolfgang; Oldiges, Marco; Nöh, Katharina; Noack, Stephan

    2011-09-01

    L-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by (13)C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an L-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated for L-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP.

  4. Enzyme-substrate complexes of the quinate/shikimate dehydrogenase from Corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination.

    PubMed

    Höppner, Astrid; Schomburg, Dietmar; Niefind, Karsten

    2013-11-01

    Quinate dehydrogenase (QDH) catalyzes the reversible oxidation of quinate to 3-dehydroquinate by nicotineamide adenine dinucleotide (NADH) and is involved in the catabolic quinate metabolism required for the degradation of lignin. The enzyme is a member of the family of shikimate/quinate dehydrogenases (SDH/QDH) occurring in bacteria and plants. We characterized the dual-substrate quinate/shikimate dehydrogenase (QSDH) from Corynebacterium glutamicum (CglQSDH) kinetically and revealed a clear substrate preference of CglQSDH for quinate compared with shikimate both at the pH optimum and in a physiological pH range, which is a remarkable contrast to closely related SDH/QDH enzymes. With respect to the cosubstrate, CglQSDH is strictly NAD(H) dependent. These substrate and cosubstrate profiles correlate well with the details of three atomic resolution crystal structures of CglQSDH in different functional states we report here: with bound NAD+ (binary complex) and as ternary complexes with NADH plus either shikimate or quinate. The CglQSDH-NADH-quinate structure is the first complex structure of any member of the SDH/QDH family with quinate. Based on this novel structural information and systematic sequence and structure comparisons with closely related enzymes, we can explain the strict NAD(H) dependency of CglQSDH as well as its discrimination between shikimate and quinate.

  5. The Corynebacterium glutamicum gene pmt encoding a glycosyltransferase related to eukaryotic protein-O-mannosyltransferases is essential for glycosylation of the resuscitation promoting factor (Rpf2) and other secreted proteins.

    PubMed

    Mahne, Martina; Tauch, Andreas; Pühler, Alfred; Kalinowski, Jörn

    2006-06-01

    Two-dimensional gel electrophoresis and immunoassays revealed several proteins of the secretory subproteome of Corynebacterium glutamicum to be glycosylated. By genome-wide searches for genes involved in glycosylation, the C. glutamicum gene cg1014 was found to exhibit significant similarity to eukaryotic protein-O-mannosyltransferases (PMTs) and to a recently identified orthologue of Mycobacterium tuberculosis, Rv1002c, which is responsible for protein-O-mannosylation. The putative membrane protein Cg1014 showed the same predicted transmembrane topology as Saccharomyces cerevisiae PMT1 and M. tuberculosis Rv1002c along with conserved amino acid residues responsible for catalytic activity. Deletion of the C. glutamicum pmt gene (cg1014) caused a complete loss of glycosylation of secreted proteins including the resuscitation promoting factor 2 (Rpf2), which is involved in intercellular communication and growth stimulation of C. glutamicum. Because the gene pmt as well as rpf genes are present in the genomes of all actinobacteria sequenced so far, this work provides new insights into bacterial protein glycosylation and new opportunities to elucidate the molecular mechanisms of Rpf activity in pathogenic growth and infection.

  6. The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Corynebacterium glutamicum

    PubMed Central

    Gaigalat, Lars; Schlüter, Jan-Philip; Hartmann, Michelle; Mormann, Sascha; Tauch, Andreas; Pühler, Alfred; Kalinowski, Jörn

    2007-01-01

    Background The major uptake system responsible for the transport of fructose, glucose, and sucrose in Corynebacterium glutamicum ATCC 13032 is the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The genes encoding PTS components, namely ptsI, ptsH, and ptsF belong to the fructose-PTS gene cluster, whereas ptsG and ptsS are located in two separate regions of the C. glutamicum genome. Due to the localization within and adjacent to the fructose-PTS gene cluster, two genes coding for DeoR-type transcriptional regulators, cg2118 and sugR, are putative candidates involved in the transcriptional regulation of the fructose-PTS cluster genes. Results Four transcripts of the extended fructose-PTS gene cluster that comprise the genes sugR-cg2116, ptsI, cg2118-fruK-ptsF, and ptsH, respectively, were characterized. In addition, it was shown that transcription of the fructose-PTS gene cluster is enhanced during growth on glucose or fructose when compared to acetate. Subsequently, the two genes sugR and cg2118 encoding for DeoR-type regulators were mutated and PTS gene transcription was found to be strongly enhanced in the presence of acetate only in the sugR deletion mutant. The SugR regulon was further characterized by microarray hybridizations using the sugR mutant and its parental strain, revealing that also the PTS genes ptsG and ptsS belong to this regulon. Binding of purified SugR repressor protein to a 21 bp sequence identified the SugR binding site as an AC-rich motif. The two experimentally identified SugR binding sites in the fructose-PTS gene cluster are located within or downstream of the mapped promoters, typical for transcriptional repressors. Effector studies using electrophoretic mobility shift assays (EMSA) revealed the fructose PTS-specific metabolite fructose-1-phosphate (F-1-P) as a highly efficient, negative effector of the SugR repressor, acting in the micromolar range. Beside F-1-P, other sugar-phosphates like fructose-1,6-bisphosphate (F-1,6-P

  7. Characterization of myo-inositol utilization by Corynebacterium glutamicum: the stimulon, identification of transporters, and influence on L-lysine formation.

    PubMed

    Krings, Eva; Krumbach, Karin; Bathe, Brigitte; Kelle, Ralf; Wendisch, Volker F; Sahm, Hermann; Eggeling, Lothar

    2006-12-01

    Although numerous bacteria possess genes annotated iol in their genomes, there have been very few studies on the possibly associated myo-inositol metabolism and its significance for the cell. We found that Corynebacterium glutamicum utilizes myo-inositol as a carbon and energy source, enabling proliferation with a high maximum rate of 0.35 h-1. Whole-genome DNA microarray analysis revealed that 31 genes respond to myo-inositol utilization, with 21 of them being localized in two clusters of >14 kb. A set of genomic mutations and functional studies yielded the result that some genes in the two clusters are redundant, and only cluster I is necessary for catabolizing the polyol. There are three genes which encode carriers belonging to the major facilitator superfamily and which exhibit a >12-fold increased mRNA level on myo-inositol. As revealed by mutant characterizations, one carrier is not involved in myo-inositol uptake whereas the other two are active and can completely replace each other with apparent Kms for myo-inositol as a substrate of 0.20 mM and 0.45 mM, respectively. Interestingly, upon utilization of myo-inositol, the L-lysine yield is 0.10 mol/mol, as opposed to 0.30 mol/mol, with glucose as the substrate. This is probably not only due to myo-inositol metabolism alone since a mixture of 187 mM glucose and 17 mM myo-inositol, where the polyol only contributes 8% of the total carbon, reduced the L-lysine yield by 29%. Moreover, genome comparisons with other bacteria highlight the core genes required for growth on myo-inositol, whose metabolism is still weakly defined.

  8. Translation efficiency of antiterminator proteins is a determinant for the difference in glucose repression of two β-glucoside phosphotransferase system gene clusters in Corynebacterium glutamicum R.

    PubMed

    Tanaka, Yuya; Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2011-01-01

    Corynebacterium glutamicum R has two β-glucoside phosphoenolpyruvate, carbohydrate phosphotransferase systems (PTS) encoded by bglF and bglF2 located in the respective clusters, bglF-bglA-bglG and bglF2-bglA2-bglG2. Previously, we reported that whereas β-glucoside-dependent induction of bglF is strongly repressed by glucose, glucose repression of bglF2 is very weak. Here, we reveal the mechanism behind the different effects of glucose on the two bgl genes. Deletion of the ribonucleic antiterminator sequence and transcriptional terminator located upstream of the translation initiation codon of bglF markedly relieved the glucose repression of a bglF-lacZ fusion, indicating that glucose affects the antitermination mechanism that is responsible for the β-glucoside-dependent induction of the bglF cluster. The glucose repression of bglF mRNA was also relieved by introducing a multicopy plasmid carrying the bglG gene encoding an antiterminator of the bglF cluster. Moreover, replacement of the GUG translation initiation codon of bglG with AUG was effective in relieving the glucose repression of bglF and bglG. Inversely, expression of bglF2 and bglG2 was subject to strict glucose repression in a mutant strain in which the AUG translation initiation codon of bglG2 encoding antiterminator of the bglF2 cluster was replaced with GUG. These results suggest that the translation initiation efficiency of the antiterminator proteins, at least in part, determines whether the target genes are subject to glucose repression. We also found that bglF expression was induced by glucose in the BglG-overexpressing strains, which may be explained by the ability of BglF to transport glucose.

  9. AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

    PubMed Central

    Kuge, Takayuki; Teramoto, Haruhiko

    2015-01-01

    ABSTRACT In Corynebacterium glutamicum ATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression of l-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDA and galM-araR) and two (BSE1 and BSE2) upstream of araE. l-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSB mutation resulted in derepression of both araBDA and galM-araR operons. The effects of BSE1 and/or BSE2 mutation on araE expression revealed that the two sites independently function as the cis elements, but BSE1 plays the primary role. However, AraR was shown to bind to these sites with almost the same affinity in vitro. Taken together, the expression of araBDA and araE is strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the −10 or −35 region of the galM-araR and araE promoters is less effective in repression. Furthermore, downregulation of araBDA and araE dependent on l-arabinose catabolism observed in the BSB mutant and the AraR-independent araR promoter identified within galM-araR add complexity to regulation of the AraR regulon derepressed by l-arabinose. IMPORTANCE Corynebacterium glutamicum has a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. Most C. glutamicum strains are unable to use a pentose sugar l-arabinose as a carbon source. However, genes for l-arabinose utilization and its regulation have been recently identified in C. glutamicum ATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the

  10. Intraspecific diversity of Brevibacterium linens, Corynebacterium glutamicum and Rhodococcus erythropolis based on partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy.

    PubMed

    Oberreuter, Helene; Charzinski, Joachim; Scherer, Siegfried

    2002-05-01

    The intraspecific diversity of 31 strains of Brevibacterium linens, 27 strains of Corynebacterium glutamicum and 29 strains of Rhodococcus erythropolis was determined by partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy. As a prerequisite for the analyses, 27 strains derived from culture collections which had carried invalid or wrong species designations were reclassified in accordance with polyphasic taxonomical data. FT-IR spectroscopy proved to be a rapid and reliable method for screening for similar isolates and for identifying these actinomycetes at the species level. Two main conclusions emerged from the analyses. (1) Comparison of intraspecific 16S rDNA similarities suggested that R. erythropolis strains have a very low diversity, B. linens displays high diversity and C. glutamicum occupies an intermediate position. (2) No correlation of FT-IR spectral similarity and 16S rDNA sequence similarity below the species level (i.e. between strains of one species) was observed. Therefore, diversification of 16S rDNA sequences and microevolutionary change of the cellular components detected by FT-IR spectroscopy appear to be de-coupled.

  11. The dual transcriptional regulator CysR in Corynebacterium glutamicum ATCC 13032 controls a subset of genes of the McbR regulon in response to the availability of sulphide acceptor molecules

    PubMed Central

    Rückert, Christian; Milse, Johanna; Albersmeier, Andreas; Koch, Daniel J; Pühler, Alfred; Kalinowski, Jörn

    2008-01-01

    Background Regulation of sulphur metabolism in Corynebacterium glutamicum ATCC 13032 has been studied intensively in the last few years, due to its industrial as well as scientific importance. Previously, the gene cg0156 was shown to belong to the regulon of McbR, a global transcriptional repressor of sulphur metabolism in C. glutamicum. This gene encodes a putative ROK-type regulator, a paralogue of the activator of sulphonate utilisation, SsuR. Therefore, it is an interesting candidate for study to further the understanding of the regulation of sulphur metabolism in C. glutamicum. Results Deletion of cg0156, now designated cysR, results in the inability of the mutant to utilise sulphate and aliphatic sulphonates. DNA microarray hybridisations revealed 49 genes with significantly increased and 48 with decreased transcript levels in presence of the native CysR compared to a cysR deletion mutant. Among the genes positively controlled by CysR were the gene cluster involved in sulphate reduction, fpr2 cysIXHDNYZ, and ssuR. Gel retardation experiments demonstrated that binding of CysR to DNA depends in vitro on the presence of either O-acetyl-L-serine or O-acetyl-L-homoserine. Mapping of the transcription start points of five transcription units helped to identify a 10 bp inverted repeat as the possible CysR binding site. Subsequent in vivo tests proved this motif to be necessary for CysR-dependent transcriptional regulation. Conclusion CysR acts as the functional analogue of the unrelated LysR-type regulator CysB from Escherichia coli, controlling sulphide production in response to acceptor availability. In both bacteria, gene duplication events seem to have taken place which resulted in the evolution of dedicated regulators for the control of sulphonate utilisation. The striking convergent evolution of network topology indicates the strong selective pressure to control the metabolism of the essential but often toxic sulphur-containing (bio-)molecules. PMID:18854009

  12. Transcriptional control of the F0F1-ATP synthase operon of Corynebacterium glutamicum: SigmaH factor binds to its promoter and regulates its expression at different pH values

    PubMed Central

    Barriuso-Iglesias, Mónica; Barreiro, Carlos; Sola-Landa, Alberto; Martín, Juan F

    2013-01-01

    Corynebacterium glutamicum used in the amino acid fermentation industries is an alkaliphilic microorganism. Its F0F1-ATPase operon (atpBEFHAGDC) is expressed optimally at pH 9.0 forming a polycistronic (7.5 kb) and a monocistronic (1.2 kb) transcripts both starting upstream of the atpB gene. Expression of this operon is controlled by the SigmaH factor. The sigmaH gene (sigH) was cloned and shown to be co-transcribed with a small gene, cg0877, encoding a putative anti-sigma factor. A mutant deleted in the sigH gene expressed the atpBEFHAGDC operon optimally at pH 7.0 at difference of the wild-type strain (optimal expression at pH 9.0). These results suggested that the SigmaH factor is involved in pH control of expression of the F0F1 ATPase operon. The SigmaH protein was expressed in Escherichia coli fused to the GST (glutathione-S-transferase) and purified to homogeneity by affinity chromatography on a GSTrap HP column. The fused protein was identified by immunodetection with anti-GST antibodies. DNA-binding studies by electrophoretic mobility shift assays showed that the SigH protein binds to a region of the atpB promoter containing the sigmaH recognition sequence (−35)TTGGAT…18nt…GTTA(−10). SigmaH plays an important role in the cascade of control of pH stress in Corynebacterium. PMID:23298179

  13. Transcriptional control of the F0F1-ATP synthase operon of Corynebacterium glutamicum: SigmaH factor binds to its promoter and regulates its expression at different pH values.

    PubMed

    Barriuso-Iglesias, Mónica; Barreiro, Carlos; Sola-Landa, Alberto; Martín, Juan F

    2013-03-01

    Corynebacterium glutamicum used in the amino acid fermentation industries is an alkaliphilic microorganism. Its F(0)F(1)-ATPase operon (atpBEFHAGDC) is expressed optimally at pH 9.0 forming a polycistronic (7.5 kb) and a monocistronic (1.2 kb) transcripts both starting upstream of the atpB gene. Expression of this operon is controlled by the SigmaH factor. The sigmaH gene (sigH) was cloned and shown to be co-transcribed with a small gene, cg0877, encoding a putative anti-sigma factor. A mutant deleted in the sigH gene expressed the atpBEFHAGDC operon optimally at pH 7.0 at difference of the wild-type strain (optimal expression at pH 9.0). These results suggested that the SigmaH factor is involved in pH control of expression of the F(0) F(1) ATPase operon. The SigmaH protein was expressed in Escherichia coli fused to the GST (glutathione-S-transferase) and purified to homogeneity by affinity chromatography on a GSTrap HP column. The fused protein was identified by immunodetection with anti-GST antibodies. DNA-binding studies by electrophoretic mobility shift assays showed that the SigH protein binds to a region of the atpB promoter containing the sigmaH recognition sequence (-35)TTGGAT…18nt…GTTA(-10). SigmaH plays an important role in the cascade of control of pH stress in Corynebacterium.

  14. Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 composite: A novel biosorbent for removal of As(III) and As(V) ions

    NASA Astrophysics Data System (ADS)

    Podder, M. S.; Majumder, C. B.

    2016-11-01

    The optimization of biosorption/bioaccumulation process of both As(III) and As(V) has been investigated by using the biosorbent; biofilm of Corynebacterium glutamicum MTCC 2745 supported on granular activated carbon/MnFe2O4 composite (MGAC). The presence of functional groups on the cell wall surface of the biomass that may interact with the metal ions was proved by FT-IR. To determine the most appropriate correlation for the equilibrium curves employing the procedure of the non-linear regression for curve fitting analysis, isotherm studies were performed for As(III) and As(V) using 30 isotherm models. The pattern of biosorption/bioaccumulation fitted well with Vieth-Sladek isotherm model for As(III) and Brouers-Sotolongo and Fritz-Schlunder-V isotherm models for As(V). The maximum biosorption/bioaccumulation capacity estimated using Langmuir model were 2584.668 mg/g for As(III) and 2651.675 mg/g for As(V) at 30 °C temperature and 220 min contact time. The results showed that As(III) and As(V) removal was strongly pH-dependent with an optimum pH value of 7.0. D-R isotherm studies specified that ion exchange might play a prominent role.

  15. Application of DEN refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum.

    PubMed

    Brunger, Axel T; Das, Debanu; Deacon, Ashley M; Grant, Joanna; Terwilliger, Thomas C; Read, Randy J; Adams, Paul D; Levitt, Michael; Schröder, Gunnar F

    2012-04-01

    Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.

  16. Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 composite: A novel biosorbent for removal of As(III) and As(V) ions.

    PubMed

    Podder, M S; Majumder, C B

    2016-11-05

    The optimization of biosorption/bioaccumulation process of both As(III) and As(V) has been investigated by using the biosorbent; biofilm of Corynebacterium glutamicum MTCC 2745 supported on granular activated carbon/MnFe2O4 composite (MGAC). The presence of functional groups on the cell wall surface of the biomass that may interact with the metal ions was proved by FT-IR. To determine the most appropriate correlation for the equilibrium curves employing the procedure of the non-linear regression for curve fitting analysis, isotherm studies were performed for As(III) and As(V) using 30 isotherm models. The pattern of biosorption/bioaccumulation fitted well with Vieth-Sladek isotherm model for As(III) and Brouers-Sotolongo and Fritz-Schlunder-V isotherm models for As(V). The maximum biosorption/bioaccumulation capacity estimated using Langmuir model were 2584.668mg/g for As(III) and 2651.675mg/g for As(V) at 30°C temperature and 220min contact time. The results showed that As(III) and As(V) removal was strongly pH-dependent with an optimum pH value of 7.0. D-R isotherm studies specified that ion exchange might play a prominent role.

  17. Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis

    PubMed Central

    2009-01-01

    Background The maintenance of internal pH in bacterial cells is challenged by natural stress conditions, during host infection or in biotechnological production processes. Comprehensive transcriptomic and proteomic analyses has been conducted in several bacterial model systems, yet questions remain as to the mechanisms of pH homeostasis. Results Here we present the comprehensive analysis of pH homeostasis in C. glutamicum, a bacterium of industrial importance. At pH values between 6 and 9 effective maintenance of the internal pH at 7.5 ± 0.5 pH units was found. By DNA microarray analyses differential mRNA patterns were identified. The expression profiles were validated and extended by 1D-LC-ESI-MS/MS based quantification of soluble and membrane proteins. Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found. The functional analysis revealed for the first time the occurrence of oxidative stress in C. glutamicum cells at neutral and low pH conditions accompanied by activation of the iron starvation response. Intracellular metabolite pool analysis unraveled inhibition of the TCA and other pathways at low pH. Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions. Conclusions Novel limitations for C. glutamicum at non-optimal pH values were identified by a comprehensive analysis on the level of the transcriptome, proteome, and metabolome indicating a functional link between pH acclimatization, oxidative stress, iron homeostasis, and metabolic alterations. The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense. PMID:20025733

  18. The flexible feedstock concept in Industrial Biotechnology: Metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources.

    PubMed

    Wendisch, Volker F; Brito, Luciana Fernandes; Gil Lopez, Marina; Hennig, Guido; Pfeifenschneider, Johannes; Sgobba, Elvira; Veldmann, Kareen H

    2016-09-20

    Most biotechnological processes are based on glucose that is either present in molasses or generated from starch by enzymatic hydrolysis. At the very high, million-ton scale production volumes, for instance for fermentative production of the biofuel ethanol or of commodity chemicals such as organic acids and amino acids, competing uses of carbon sources e.g. in human and animal nutrition have to be taken into account. Thus, the biotechnological production hosts E. coli, C. glutamicum, pseudomonads, bacilli and Baker's yeast used in these large scale processes have been engineered for efficient utilization of alternative carbon sources. This flexible feedstock concept is central to the use of non-glucose second and third generation feedstocks in the emerging bioeconomy. The metabolic engineering efforts to broaden the substrate scope of E. coli, C. glutamicum, pseudomonads, B. subtilis and yeasts to include non-native carbon sources will be reviewed. Strategies to enable simultaneous consumption of mixtures of native and non-native carbon sources present in biomass hydrolysates will be summarized and a perspective on how to further increase feedstock flexibility for the realization of biorefinery processes will be given.

  19. The LacI-Type transcriptional regulator AraR acts as an L-arabinose-responsive repressor of L-arabinose utilization genes in Corynebacterium glutamicum ATCC 31831.

    PubMed

    Kuge, Takayuki; Teramoto, Haruhiko; Yukawa, Hideaki; Inui, Masayuki

    2014-06-01

    The Corynebacterium glutamicum ATCC 31831 araBDA operon consists of three l-arabinose catabolic genes, upstream of which the galM, araR, and araE genes are located in opposite orientation. araR encodes a LacI-type transcriptional regulator that negatively regulates the l-arabinose-inducible expression of araBDA and araE (encoding an l-arabinose transporter), through a mechanism that has yet to be identified. Here we show that the AraR protein binds in vitro to three sites: one upstream of araBDA and two upstream of araE. We verify that a 16-bp consensus palindromic sequence is essential for binding of AraR, using a series of mutations introduced upstream of araB in electrophoretic mobility shift assays. Moreover, the DNA-binding activity of AraR is reduced by l-arabinose. We employ quantitative reverse transcription-PCR (qRT-PCR) analyses using various mutant strains deficient in l-arabinose utilization genes to demonstrate that the prominent upregulation of araBDA and araE within 5 min of l-arabinose supplementation is dependent on the uptake but independent of the catabolism of l-arabinose. Similar expression patterns, together with the upregulation by araR disruption without l-arabinose, are evident with the apparent galM-araR operon, although attendant changes in expression levels are much smaller than those realized with the expression of araBDA and araE. The AraR-binding site upstream of araB overlaps the -10 region of the divergent galM promoter. These observations indicate that AraR acts as a transcriptional repressor of araBDA, araE, and galM-araR and that l-arabinose acts as an intracellular negative effector of the AraR-dependent regulation.

  20. Multilocus sequence typing identifies evidence for recombination and two distinct lineages of Corynebacterium diphtheriae.

    PubMed

    Bolt, Frances; Cassiday, Pamela; Tondella, Maria Lucia; Dezoysa, Aruni; Efstratiou, Androulla; Sing, Andreas; Zasada, Aleksandra; Bernard, Kathryn; Guiso, Nicole; Badell, Edgar; Rosso, Marie-Laure; Baldwin, Adam; Dowson, Christopher

    2010-11-01

    We describe the development of a multilocus sequence typing (MLST) scheme for Corynebacterium diphtheriae, the causative agent of the potentially fatal upper respiratory disease diphtheria. Global changes in diphtheria epidemiology are highlighted by the recent epidemic in the former Soviet Union (FSU) and also by the emergence of nontoxigenic strains causing atypical disease. Although numerous techniques have been developed to characterize C. diphtheriae, their use is hindered by limited portability and, in some instances, poor reproducibility. One hundred fifty isolates from 18 countries and encompassing a period of 50 years were analyzed by multilocus sequence typing (MLST). Strain discrimination was in accordance with previous ribotyping data, and clonal complexes associated with disease outbreaks were clearly identified by MLST. The data produced are portable, reproducible, and unambiguous. The MLST scheme described provides a valuable tool for monitoring and characterizing endemic and epidemic C. diphtheriae strains. Furthermore, multilocus sequence analysis of the nucleotide data reveals two distinct lineages within the population of C. diphtheriae examined, one of which is composed exclusively of biotype belfanti isolates and the other of multiple biotypes.

  1. Engineering of a Glycerol Utilization Pathway for Amino Acid Production by Corynebacterium glutamicum▿

    PubMed Central

    Rittmann, Doris; Lindner, Steffen N.; Wendisch, Volker F.

    2008-01-01

    The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures. PMID:18757581

  2. Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae.

    PubMed

    Wesener, Darryl A; Levengood, Matthew R; Kiessling, Laura L

    2017-02-17

    The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

  3. Reduced Folate Supply as a Key to Enhanced l-Serine Production by Corynebacterium glutamicum▿

    PubMed Central

    Stolz, Michael; Peters-Wendisch, Petra; Etterich, Helga; Gerharz, Tanja; Faurie, Robert; Sahm, Hermann; Fersterra, Holger; Eggeling, Lothar

    2007-01-01

    The amino acid l-serine is required for pharmaceutical purposes, and the availability of a sugar-based microbial process for its production is desirable. However, a number of intracellular utilization routes prevent overproduction of l-serine, with the essential serine hydroxymethyltransferase (SHMT) (glyA) probably occupying a key position. We found that constructs of Corynebacterium glutamicum strains where chromosomal glyA expression is dependent on Ptac and lacIQ are unstable, acquiring mutations in lacIQ, for instance. To overcome the inconvenient glyA expression control, we instead considered controlling SHMT activity by the availability of 5,6,7,8-tetrahydrofolate (THF). The pabAB and pabC genes of THF synthesis were identified and deleted in C. glutamicum, and the resulting strains were shown to require folate or 4-aminobenzoate for growth. Whereas the C. glutamicum ΔsdaA strain (pserACB) accumulates only traces of l-serine, with the C. glutamicum ΔpabABCΔsdaA strain (pserACB), l-serine accumulation and growth responded in a dose-dependent manner to an external folate supply. At 0.1 mM folate, 81 mM l-serine accumulated. In a 20-liter controlled fed-batch culture, a 345 mM l-serine accumulation was achieved. Thus, an efficient and highly competitive process for microbial l-serine production is available. PMID:17142381

  4. Characterization of OxyR as a negative transcriptional regulator that represses catalase production in Corynebacterium diphtheriae.

    PubMed

    Kim, Ju-Sim; Holmes, Randall K

    2012-01-01

    Corynebacterium diphtheriae and Corynebacterium glutamicum each have one gene (cat) encoding catalase. In-frame Δcat mutants of C. diphtheriae and C. glutamicum were hyper-sensitive to growth inhibition and killing by H(2)O(2). In C. diphtheriae C7(β), both catalase activity and cat transcription decreased ~2-fold during transition from exponential growth to early stationary phase. Prototypic OxyR in Escherichia coli senses oxidative stress and it activates katG transcription and catalase production in response to H(2)O(2). In contrast, exposure of C. diphtheriae C7(β) to H(2)O(2) did not stimulate transcription of cat. OxyR from C. diphtheriae and C. glutamicum have 52% similarity with E. coli OxyR and contain homologs of the two cysteine residues involved in H(2)O(2) sensing by E. coli OxyR. In-frame ΔoxyR deletion mutants of C. diphtheriae C7(β), C. diphtheriae NCTC13129, and C. glutamicum were much more resistant than their parental wild type strains to growth inhibition by H(2)O(2). In the C. diphtheriae C7(β) ΔoxyR mutant, cat transcripts were about 8-fold more abundant and catalase activity was about 20-fold greater than in the C7(β) wild type strain. The oxyR gene from C. diphtheriae or C. glutamicum, but not from E. coli, complemented the defect in ΔoxyR mutants of C. diphtheriae and C. glutamicum and decreased their H(2)O(2) resistance to the level of their parental strains. Gel-mobility shift, DNaseI footprint, and primer extension assays showed that purified OxyR from C. diphtheriae C7(β) bound, in the presence or absence of DTT, to a sequence in the cat promoter region that extends from nucleotide position -55 to -10 with respect to the +1 nucleotide in the cat ORF. These results demonstrate that OxyR from C. diphtheriae or C. glutamicum functions as a transcriptional repressor of the cat gene by a mechanism that is independent of oxidative stress induced by H(2)O(2).

  5. Structural characteristics of the Corynebacterium lilium bacteriophage CL31.

    PubMed Central

    Trautwetter, A; Blanco, C; Sicard, A M

    1987-01-01

    Bacteriophage CL31 was isolated on a Corynebacterium lilium strain. Out of 30 strains tested, only CL31 was able to form plaques on Corynebacterium glutamicum ATCC 13287, Brevibacterium lactofermentum ATCC 21086, and Arthrobacter sp. strain SI55, but at a very low frequency. This phage belongs to group B of Bradley's classification (D. E. Bradley, Bacteriol. Rev. 31:230-314; 1967). Its head is 53 nm in diameter, and its tail is 396 nm in length. The phage capsid contains three major proteins, of 12.5, 29.0, and 37.0 kilodaltons, and five minor ones (23.9, 26.0, 27.0, 40.0, and 55.4 kilodaltons). CL31 DNA is a linear molecule of 48 kilobases with cohesive ends. Restriction mapping was performed for endonucleases BglII, EcoRI, SalI, and KpnI. The expression of CL31 genes in Escherichia coli was studied by the maxicell technique; 12 different proteins were detected. Images PMID:3033280

  6. Assessment of robustness against dissolved oxygen/substrate oscillations for C. glutamicum DM1933 in two-compartment bioreactor.

    PubMed

    Käß, Friedrich; Hariskos, Ioanna; Michel, Andrea; Brandt, Hans-Jürgen; Spann, Robert; Junne, Stefan; Wiechert, Wolfgang; Neubauer, Peter; Oldiges, Marco

    2014-06-01

    Corynebacterium glutamicum is an important organism for industrial biotechnology; particularly, in amino acid production (e.g. L-lysine). Production scales often reach reactor working volumes of several hundred cubic meters, which triggers inhomogeneous distribution of substrates and dissolved gasses due to increasing mixing times. Individual cells which follow the flow profile through the reactor are experiencing oscillating microenvironments. Oscillations can have an influence on the process performance, which is a subject of scale-down experiments. In this work, L-lysine-producing C. glutamicum DM1933 was assessed for its robustness against continuous dissolved oxygen and substrate supply oscillation in two-compartment scale-down bioreactors. Aerobic, substrate-limited stirred tank and non-aerated, substrate-excess plug flow compartments were applied for oscillation. Inhomogeneity of substrate and oxygen supply was observed to cause rapid side product turnover, redistribution of oxygen uptake from oxygen limited into fully aerobic zones, and intermediate medium acidification. However, process inhomogeneity did not impair productivity or growth at plug flow residence times of several minutes. In a focused analysis of proteome, metabolome, transcriptome, and other physiological parameters, no changes were identified in response to process inhomogeneity. In conclusion, fed-batch processes with C. glutamicum DM1933 possess remarkable robustness against oxygen and substrate supply oscillation, which is a unique property in the field of published scale-down studies. Microbial physiology of C. glutamicum appears to be ideally adapted to both homogeneous and inhomogeneous conditions. This ensures exceptional suitability for cultivation at increased mixing times, which is suggested to constitute an important basis for the long-lasting success in large scale bioprocess application.

  7. The Corynebacterium xerosis composite transposon Tn5432 consists of two identical insertion sequences, designated IS1249, flanking the erythromycin resistance gene ermCX.

    PubMed

    Tauch, A; Kassing, F; Kalinowski, J; Pühler, A

    1995-09-01

    Analysis of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium xerosis M82B revealed that the erythromycin resistance gene, ermCX, is located on a 4524-bp composite transposable element, Tn5432. The ends of Tn5432 are identical, direct repeats of an insertion sequence, designated IS1249, encoding a putative transposase of the IS256 family. IS1249 consists of 1385 bp with 45/42 imperfect terminal inverted repeats. The nucleotide sequence of the 1754-bp Tn5432 central region is 99% identical to the previously sequenced erythromycin resistance region of the Corynebacterium diphtheriae plasmid pNG2. It encodes the erythromycin resistance gene, ermCX, and an ORF homologous to the amino-terminal end of the transposase of IS31831 from Corynebacterium glutamicum. Transposons with regions flanking the insertion sites were recovered from the C. glutamicum chromosome by a plasmid rescue technique. Insertion of Tn5432 created 8-bp target site duplications. A Tn5432-induced isoleucine/valine-auxotrophic mutant was found to carry the transposon in the 5' region of the ilvBNC cluster; in pTP10 the transposon is inserted in a region similar to replication and partitioning functions of the Enterococcus faecalis plasmid pAD1 and the Agrobacterium tumefaciens plasmid pTAR.

  8. Isolation of insertion elements from gram-positive Brevibacterium, Corynebacterium and Rhodococcus strains using the Bacillus subtilis sacB gene as a positive selection marker.

    PubMed

    Jäger, W; Schäfer, A; Kalinowski, J; Pühler, A

    1995-02-01

    The sacB gene of Bacillus subtilis was successfully applied in various Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus strains for the isolation of transposable elements. Three different insertion sequence (IS) elements entrapped in sacB were isolated. The IS elements IS-Bl and IS-Cg isolated from Brevibacterium lactofermentum and Corynebacterium glutamicum, respectively, were found to be similar in size (1.45 kb) and generated target duplications of 8 bp. Their inverted repeats showed homology. In contrast, the IS element IS-Rf isolated from Rhodococcus fascians was only 1.3 kb long and generated a 3-bp target duplication. IS-Cg and IS-Rf were not restricted to their original host strains, and we also found strains harbouring more than one element.

  9. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production

    PubMed Central

    Man, Zaiwei; Xu, Meijuan; Rao, Zhiming; Guo, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong

    2016-01-01

    L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L−1 L-arginine with yield up to 0.431 g L-arginine g−1 glucose in fed-batch fermentation. PMID:27338253

  10. Corynebacterium mooreparkense, a later heterotypic synonym of Corynebacterium variabile.

    PubMed

    Gelsomino, Roberto; Vancanneyt, Marc; Snauwaert, Cindy; Vandemeulebroecke, Katrien; Hoste, Bart; Cogan, Timothy M; Swings, Jean

    2005-05-01

    Strains of a Gram-positive bacterium were isolated from the Irish smear-ripened cheese Gubbeen, and assigned to a new species, Corynebacterium mooreparkense, in 2001. During a further study on the same cheese, no additional isolates from this species could be found. Instead, multiple isolates of its nearest phylogenetic neighbour, Corynebacterium variabile, were found. A first screening with rep-PCR and SDS-PAGE pointed to a similarity between C. mooreparkense and C. variabile. Following this peculiar result, attempts were made to collect all type strains deposited at different culture collections and all strains described by Brennan et al. [Int J Syst Evol Microbiol (2001) 51, 843-852]. Subsequently, 16S rRNA gene sequencing and DNA-DNA hybridizations were performed. All C. mooreparkense strains had a 16S rRNA gene sequence similarity of at least 99.5 % with C. variabile and the DNA-DNA relatedness was 95 %. On the basis of these results, it is concluded that C. mooreparkense is a later heterotypic synonym of C. variabile.

  11. Toxigenic Corynebacterium ulcerans in human and non-toxigenic Corynebacterium diphtheriae in cat.

    PubMed

    Detemmerman, L; Rousseaux, D; Efstratiou, A; Schirvel, C; Emmerechts, K; Wybo, I; Soetens, O; Piérard, D

    2013-10-01

    Corynebacterium diphtheriae and Corynebacterium ulcerans are rarely isolated from clinical samples in Belgium. A case of toxigenic C. ulcerans in a woman is described, which confirms that this pathogen is still present. During investigation of the patient's cats, only a non-toxigenic toxin-bearing C. diphtheriae strain was detected.

  12. Characterization of Corynebacterium species in macaques

    PubMed Central

    Venezia, Jaime; Cassiday, Pamela K.; Marini, Robert P.; Shen, Zeli; Buckley, Ellen M.; Peters, Yaicha; Taylor, Nancy; Dewhirst, Floyd E.; Tondella, Maria L.

    2012-01-01

    Bacteria of the genus Corynebacterium are important primary and opportunistic pathogens. Many are zoonotic agents. In this report, phenotypic (API Coryne analysis), genetic (rpoB and 16S rRNA gene sequencing), and physical methods (MS) were used to distinguish the closely related diphtheroid species Corynebacterium ulcerans and Corynebacterium pseudotuberculosis, and to definitively diagnose Corynebacterium renale from cephalic implants of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques used in cognitive neuroscience research. Throat and cephalic implant cultures yielded 85 isolates from 43 macaques. Identification by API Coryne yielded C. ulcerans (n = 74), Corynebacterium pseudotuberculosis (n = 2), C. renale or most closely related to C. renale (n = 3), and commensals and opportunists (n = 6). The two isolates identified as C. pseudotuberculosis by API Coryne required genetic and MS analysis for accurate characterization as C. ulcerans. Of three isolates identified as C. renale by 16S rRNA gene sequencing, only one could be confirmed as such by API Coryne, rpoB gene sequencing and MS. This study emphasizes the importance of adjunct methods in identification of coryneforms and is the first isolation of C. renale from cephalic implants in macaques. PMID:22723254

  13. Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov., isolated from the surface of a smear-ripened cheese.

    PubMed

    Brennan, N M; Brown, R; Goodfellow, M; Ward, A C; Beresford, T P; Simpson, P J; Fox, P F; Cogan, T M

    2001-05-01

    Ten isolates each of two different bacterial species isolated from the surface of a smear-ripened cheese were found to exhibit many characteristics of the genus Corynebacterium. The isolates were Gram-positive, catalase-positive, non-spore-forming rods that did not undergo a rod/coccus transformation when grown on complex media. Chemotaxonomic investigation revealed that the strains belonged unambiguously to the genus Corynebacterium. Their cell walls contained arabinose, galactose and short-chain mycolic acids (C22 to C36) and their peptidoglycan contained meso-diaminopimelic acid. The G+C content of the DNA was 51-60 mol%. MK-9 (H2) was the principal menaquinone. The 16S rDNA sequences of four isolates of each bacterium were determined and aligned with those of other members of the coryneform group. Phylogenetic analysis showed that the strains represented two new sublines within the genus Corynebacterium; Corynebacterium variabile and Corynebacterium ammoniagenes were their nearest known phylogenetic neighbours. Corynebacterium variabile and Corynebacterium ammoniagenes showed the highest levels of sequence homology with the isolates; however, DNA-DNA hydridization studies indicated that the Corynebacterium strains isolated from the cheese smear did not belong to either Corynebacterium variabile or Corynebacterium ammoniagenes (26 and 46% chromosomal similarity, respectively). On the basis of the phylogenetic and phenotypic distinctiveness of the unknown isolates, it is proposed that the bacteria be classified as two new Corynebacterium species, for which the names Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov. are proposed. Type strains have been deposited in culture collections as Corynebacterium mooreparkense LMG S-19265T (= NCIMB 30131T) and Corynebacterium casei LMG S-19264T (= NCIMB 30130T).

  14. A fatal case of urosepsis due to Corynebacterium riegelii.

    PubMed

    Aygun, Gokhan; Midilli, Kenan; Cilingir, Hatice; Yilmaz, Mesut; Kutukcu, Aysegul; Eker, Engin

    2013-01-01

    Corynebacterium species other than Corynebacterium diphtheriae rarely cause infections in human but rather reside in flora, however they have been reported to cause opportunistic infections in both immunocompromised and immunecompetent patients. Here we report for the first time a case of an elderly female patient presenting with a fatal urosepsis caused by a recently defined pathogen, Corynebacterium riegelii, identified on second day after patient hospitalization leading to a progressive worsening and death of the patient on 6th day.

  15. Corynebacterium ulcerans, an emerging human pathogen.

    PubMed

    Hacker, Elena; Antunes, Camila A; Mattos-Guaraldi, Ana L; Burkovski, Andreas; Tauch, Andreas

    2016-09-01

    While formerly known infections of Corynebacterium ulcerans are rare and mainly associated with contact to infected cattle, C. ulcerans has become an emerging pathogen today. In Western Europe, cases of respiratory diphtheria caused by C. ulcerans have been reported more often than infections by Corynebacterium diphtheria, while systemic infections are also increasingly reported. Little is known about factors that contribute to host colonization and virulence of this zoonotic pathogen. Research in this field has received new impetus by the publication of several C. ulcerans genome sequences in the past years. This review gives a comprehensive overview of the basic knowledge of C. ulcerans, as well as the recent advances made in the analysis of putative virulence factors.

  16. Characterization of strains of Corynebacterium bovis.

    PubMed Central

    Brooks, B W; Barnum, D A

    1984-01-01

    The biochemical and morphological characteristics of 104 strains of Corynebacterium bovis isolated from bovine milk samples and the C. bovis reference strain were found to be uniform. Valuable criteria for identification were presence of catalase and oxidase, production of acid from glucose and fructose and a requirement for enriched basal media. Six strains of human and three strains of bovine origin were found to be inconsistent with the reference strain. PMID:6722650

  17. Cutaneous infections due to Corynebacterium diphtheriae

    PubMed Central

    Cockcroft, W. H.; Boyko, W. J.; Allen, D. E.

    1973-01-01

    Toxigenic Corynebacterium diphtheriae was grown from skin lesions of 44 indigent patients seen at the emergency or out-patient departments of this hospital, 43 of them within the last 16 months of the study period. In all cases staphylococci or hemolytic streptococci were also present in the wounds. An increase in the incidence of clinical diphtheria occurred in the few months preceding and overlapping the period of recognition of the cutaneous infections. The gravis strains, which accounted for the majority of the infections, were sensitive to erythromycin and to penicillin, but were relatively resistant to cloxacillin. PMID:4632361

  18. The killing of macrophages by Corynebacterium ulcerans.

    PubMed

    Hacker, Elena; Ott, Lisa; Schulze-Luehrmann, Jan; Lührmann, Anja; Wiesmann, Veit; Wittenberg, Thomas; Burkovski, Andreas

    2016-01-01

    Corynebacterium ulcerans is an emerging pathogen transmitted by a zoonotic pathway with a very broad host spectrum to humans. Despite rising numbers of infections and potentially fatal outcomes, data on the molecular basis of pathogenicity are scarce. In this study, the interaction of 2 C. ulcerans isolates - one from an asymptomatic dog, one from a fatal case of human infection - with human macrophages was investigated. C. ulcerans strains were able to survive in macrophages for at least 20 hours. Uptake led to delay of phagolysosome maturation and detrimental effects on the macrophages as deduced from cytotoxicity measurements and FACS analyses. The data presented here indicate a high infectious potential of this emerging pathogen.

  19. [Breast cancer treated by antibiotherapy? Granulomatous mastitis with Corynebacterium].

    PubMed

    Buhler, J; Grignon, Y; Gallon, F

    2015-09-01

    Granulomatous mastitis is a rare disease, often associated with Corynebacterium infection. It raises the problem of diagnosis of breast tumor with a fast evolution and inflammatory character. We report two cases of granulomatous mastitis with Corynebacterium. It concerns the clinical and radiological description, followed by the therapeutic alternatives and future of the patients. The clinical presentation is variable. The treatment consists in a surgical procedure of resection. The medical treatment based of corticosteroids also proves efficient. The association between Corynebacterium presence and this pathology seems frequent and needs a specific bacteriological search.

  20. Septic arthritis in a native knee due to Corynebacterium striatum.

    PubMed

    Molina Collada, Juan; Rico Nieto, Alicia; Díaz de Bustamante Ussia, Macarena; Balsa Criado, Alejandro

    2017-03-07

    We describe a case of septic arthritis in a native knee due to Corynebacterium striatum, gram-positive bacilli that are usually commensal organisms of skin and mucosal membranes, but are seldom implicated in native septic arthritis. An 84-year-old man with Corynebacterium striatum septic arthritis of his native left knee and no response to conventional antibiotic therapy. Thus, the patient was allowed to take dalbavancin for compassionate use, with an excellent clinical outcome. This case emphasizes de role of Corynebacterium striatum in native joint infections and highlights the importance of early detection and appropriate treatment in improving the clinical outcome.

  1. Non-diphtheriae Corynebacterium species: an emerging respiratory pathogen.

    PubMed

    Díez-Aguilar, M; Ruiz-Garbajosa, P; Fernández-Olmos, A; Guisado, P; Del Campo, R; Quereda, C; Cantón, R; Meseguer, M A

    2013-06-01

    The purpose of the study was to describe the microbiological and clinical features of ten cases of lower respiratory tract infection due to Corynebacterium striatum, Corynebacterium propinquum and Corynebacterium pseudodiphtheriticum. Respiratory samples were recovered from hospitalised patients who were diagnosed of pneumonia and exacerbations of chronic respiratory infections. The samples were Gram-stained and seeded on conventional bacterial growing media. Bacteria were identified by matrix-assisted linear desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility was tested by the disk diffusion method. All patients presented an acute respiratory onset, most of them in the context of an underlying disease and/or immunosuppression. In all patients, the microscopical examination of Gram-stained respiratory samples showed numerous polymorphonuclear cells and Gram-positive bacilli, suggestive of the Corynebacterium morphotype. A pure culture growth of Corynebacterium was obtained in the majority (72 %) of samples. The conclusions are that non-diphtheriae Corynebacterium species are an emerging cause of respiratory infection among patients with chronic respiratory disease and/or immunosuppression, and cannot always be considered as mere colonisers. The microorganism's predominance in Gram-stained purulent respiratory samples together with abundant growth in the culture is the key for the microbiological diagnosis.

  2. Complete genome sequence, lifestyle, and multi-drug resistance of the human pathogen Corynebacterium resistens DSM 45100 isolated from blood samples of a leukemia patient

    PubMed Central

    2012-01-01

    Background Corynebacterium resistens was initially recovered from human infections and recognized as a new coryneform species that is highly resistant to antimicrobial agents. Bacteremia associated with this organism in immunocompromised patients was rapidly fatal as standard minocycline therapies failed. C. resistens DSM 45100 was isolated from a blood culture of samples taken from a patient with acute myelocytic leukemia. The complete genome sequence of C. resistens DSM 45100 was determined by pyrosequencing to identify genes contributing to multi-drug resistance, virulence, and the lipophilic lifestyle of this newly described human pathogen. Results The genome of C. resistens DSM 45100 consists of a circular chromosome of 2,601,311 bp in size and the 28,312-bp plasmid pJA144188. Metabolic analysis showed that the genome of C. resistens DSM 45100 lacks genes for typical sugar uptake systems, anaplerotic functions, and a fatty acid synthase, explaining the strict lipophilic lifestyle of this species. The genome encodes a broad spectrum of enzymes ensuring the availability of exogenous fatty acids for growth, including predicted virulence factors that probably contribute to fatty acid metabolism by damaging host tissue. C. resistens DSM 45100 is able to use external L-histidine as a combined carbon and nitrogen source, presumably as a result of adaptation to the hitherto unknown habitat on the human skin. Plasmid pJA144188 harbors several genes contributing to antibiotic resistance of C. resistens DSM 45100, including a tetracycline resistance region of the Tet W type known from Lactobacillus reuteri and Streptococcus suis. The tet(W) gene of pJA144188 was cloned in Corynebacterium glutamicum and was shown to confer high levels of resistance to tetracycline, doxycycline, and minocycline in vitro. Conclusions The detected gene repertoire of C. resistens DSM 45100 provides insights into the lipophilic lifestyle and virulence functions of this newly recognized

  3. The killing of macrophages by Corynebacterium ulcerans

    PubMed Central

    Hacker, Elena; Ott, Lisa; Schulze-Luehrmann, Jan; Lührmann, Anja; Wiesmann, Veit; Wittenberg, Thomas; Burkovski, Andreas

    2016-01-01

    Corynebacterium ulcerans is an emerging pathogen transmitted by a zoonotic pathway with a very broad host spectrum to humans. Despite rising numbers of infections and potentially fatal outcomes, data on the molecular basis of pathogenicity are scarce. In this study, the interaction of 2 C. ulcerans isolates - one from an asymptomatic dog, one from a fatal case of human infection - with human macrophages was investigated. C. ulcerans strains were able to survive in macrophages for at least 20 hours. Uptake led to delay of phagolysosome maturation and detrimental effects on the macrophages as deduced from cytotoxicity measurements and FACS analyses. The data presented here indicate a high infectious potential of this emerging pathogen. PMID:26632348

  4. [Pneumonia caused byCorynebacterium pseudodiphtheriticum].

    PubMed

    Furiasse, Daniela; Gasparotto, Ana M; Monterisi, Aída; Castellano, Gabriela; Rocchi, Marta

    Microorganisms of the genera Corynebacterium, specie pseudodiphtheriticum are a part of the indigenous microbiota of human skin and oropharinx. Nevertheless in recent decades these bacilli are emerging as opportunistic pathogens causing clinically significant infections in patients with previous compromise. We report the case of a 76 years old female patient, with a history of hypertension, hypothyroidism, type 2 diabetes and chronic renal failure, who presented pneumonia during their stay at the intensive care unit. The induced sputum revealed a representative sample with monomicrobial gram positive pleomorphic coryneform rods (Gram stain) and cultures demonstrated the presence of C. pseudodiphtheriticum as the only bacteria recovered. The pacient received an empirical third generation cephalosporin medication with a succesfull recovery.

  5. Corynebacterium jeikeium bacteremia in a hemodialyzed patient.

    PubMed

    Ifantidou, Athina M; Diamantidis, Michael D; Tseliki, Georgia; Angelou, Argiri S; Christidou, Photini; Papa, Anna; Pentilas, Demetrius

    2010-09-01

    Corynebacterium jeikeium, frequently encountered in clinical specimens, is part of the normal skin flora. Nevertheless, a few cases of C. jeikeium bacteremia followed by severe clinical manifestations have been reported. C. jeikeium has been reported to cause endocarditis, septicemia, meningitis, pneumonia and osteomyelitis, along with soft tissue and trauma infections. Herein we describe a case of C. jeikeium bacteremia in Greece. The isolation of a coryneform bacterium from a clinical specimen should not immediately be considered a superinfection by the skin flora. Clinical and laboratory investigations are essential in order to evaluate such cases before applying appropriate treatment. On the other hand, the association of coryneform bacteria and disease should be critically investigated, with a thorough identification of the strain, ideally beyond the classical methods, at a specialized center.

  6. Staphylococcus aureus Shifts toward Commensalism in Response to Corynebacterium Species

    PubMed Central

    Ramsey, Matthew M.; Freire, Marcelo O.; Gabrilska, Rebecca A.; Rumbaugh, Kendra P.; Lemon, Katherine P.

    2016-01-01

    Staphylococcus aureus–human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe–microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr) system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence toward a commensal state when exposed to commensal Corynebacterium species. PMID:27582729

  7. Staphylococcus aureus Shifts toward Commensalism in Response to Corynebacterium Species.

    PubMed

    Ramsey, Matthew M; Freire, Marcelo O; Gabrilska, Rebecca A; Rumbaugh, Kendra P; Lemon, Katherine P

    2016-01-01

    Staphylococcus aureus-human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe-microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr) system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence toward a commensal state when exposed to commensal Corynebacterium species.

  8. Corynebacterium pseudotuberculosis infection in Israeli dairy cattle.

    PubMed Central

    Yeruham, I.; Elad, D.; Friedman, S.; Perl, S.

    2003-01-01

    Two forms of Corynebacterium pseudotuberculosis infection in Israeli dairy cattle herds during a survey period of 13 years (1989-2001) are described. The more common form, which was diagnosed in 45 herds, was characterized by ulcerative granulomatous lesions which occurred either sporadically--in 26 herds (with a morbidity rate of up to 5%)--or in an epidemic course in 19 herds. Most (80.6%) of the affected animals were cows; the rest were first-calving cows (16.2%) and heifers (3.2%). The morbidity occurred mostly during the summer months. The ulcerative granulomatous lesions appeared in three clinical forms: cutaneous, mastitic and visceral. Mixed forms were also observed. The morbidity rate was 6.4% and the culling rate reached 16.3% of the affected animals. Most of the strains of C. pseudotuberculosis which were isolated from the abscesses in the cutaneous form of the disease and from milk samples failed to reduce nitrate. A decrease in milk production (6%) and an increase in bulk-milk somatic cell count were noted. Necrotic and ulcerative dermatitis on the heel of the foot occurred in an epidemic course in heifers in only two herds during the winter months, with morbidity rates of 7.5 and 76.2%, respectively. C. pseudotuberculosis isolates from skin lesions and from the soil did reduce nitrate. Clinical, epizootiological and microbiological aspects of the infection are described. PMID:14596537

  9. Inherited resistance to Corynebacterium kutscheri in mice.

    PubMed Central

    Hirst, R G; Wallace, M E

    1976-01-01

    An analysis of the factors responsible for inherited resistance to Corynebacterium kutscheri was undertaken. Various inbred mouse strains were examined; these included the Swiss Lynch and C57Bl/l mice, their F1 and F2 progeny, and the progeny of the F1 backcrossed to each parent strain. Two modes of inherited resistance are described. An examination suggested that resistance as measured by the mean lethal dose of C. kutscheri was under polygenic control and was inherited continuously. However, the efficiency with which C. kutscheri was eliminated by the mononuclear phagocyte cells of the liver over 3 days differed markedly among strains. A genetic analysis of this mononuclear phagocyte microbicidal efficiency (MPME) in Swiss Lynch and C57Bl/6 mice was undertaken. The trait, MPME, was present, but did not segregate, in the F1 progeny or in the progeny of the backcross to the resistant C57Bl/6 parent; this was clear evidence of dominance. Moreover, MPME segregated in a ratio of 1:1 in the progeny of the backcross to the sensitive Swiss Lynch parent and in a ratio of 3:1 in the F2 progeny. It was concluded that MPME was inherited discontinuously and was controlled by a single dominant autosomal gene (or closely linked group); the recessive allele was assigned the gene symbol ack. Linkage experiments showed there to be no association between the ack locus and any of the immune-response genes. PMID:971958

  10. Enhanced poly(3-hydroxypropionate) production via β-alanine pathway in recombinant Escherichia coli

    PubMed Central

    Lacmata, Stephen Tamekou; Kuiate, Jules-Roger; Ding, Yamei; Xian, Mo; Liu, Huizhou; Boudjeko, Thaddée; Feng, Xinjun; Zhao, Guang

    2017-01-01

    Poly(3-hydroxypropionate) (P3HP) is a thermoplastic with great compostability and biocompatibility, and can be produced through several biosynthetic pathways, in which the glycerol pathway achieved the highest P3HP production. However, exogenous supply of vitamin B12 was required to maintain the activity of glycerol dehydratase, resulting in high production cost. To avoid the addition of VB12, we have previously constructed a P3HP biosynthetic route with β-alanine as intermediate, and the present study aimed to improve the P3HP production of this pathway. L-aspartate decarboxylase PanD was found to be the rate-limiting enzyme in the β-alanine pathway firstly. To improve the pathway efficiency, PanD was screened from four different sources (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Corynebacterium glutamicum). And PanD from C. glutamicum was found to have the highest activity, the P3HP production was improved in flask cultivation with this enzyme. To further improve the production, the host strain was screened and the culture condition was optimized. Under optimal conditions, production and content of P3HP reached to 10.2 g/L and 39.1% (wt/wt [cell dry weight]) in an aerobic fed-batch fermentation. To date, this is the highest P3HP production without VB12. PMID:28253372

  11. Application of granular activated carbon/MnFe₂O₄ composite immobilized on C. glutamicum MTCC 2745 to remove As(III) and As(V): Kinetic, mechanistic and thermodynamic studies.

    PubMed

    Podder, M S; Majumder, C B

    2016-01-15

    The main objective of the present study was to investigate the efficiency of Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 (GAC/MnFe2O4) composite to treat high concentration of arsenic bearing wastewater. Non-linear regression analysis was done for determining the best-fit kinetic model on the basis of three correlation coefficients and three error functions and also for predicting the parameters involved in kinetic models. The results showed that Fractal-like mixed 1,2 order model for As(III) and Brouser-Weron-Sototlongo as well as Fractal-like pseudo second order models for As(V) were proficient to provide realistic description of biosorption/bioaccumulation kinetic. Applicability of mechanistic models in the current study exhibited that the rate governing step in biosorption/bioaccumulation of both As(III) and As(V) was film diffusion rather than intraparticle diffusion. The evaluated thermodynamic parameters ΔG(0), ΔH(0) and ΔS(0) revealed that biosorption/bioaccumulation of both As(III) and As(V) was feasible, spontaneous and exothermic under studied conditions.

  12. Application of granular activated carbon/MnFe2O4 composite immobilized on C. glutamicum MTCC 2745 to remove As(III) and As(V): Kinetic, mechanistic and thermodynamic studies

    NASA Astrophysics Data System (ADS)

    Podder, M. S.; Majumder, C. B.

    2016-01-01

    The main objective of the present study was to investigate the efficiency of Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 (GAC/MnFe2O4) composite to treat high concentration of arsenic bearing wastewater. Non-linear regression analysis was done for determining the best-fit kinetic model on the basis of three correlation coefficients and three error functions and also for predicting the parameters involved in kinetic models. The results showed that Fractal-like mixed 1,2 order model for As(III) and Brouser-Weron-Sototlongo as well as Fractal-like pseudo second order models for As(V) were proficient to provide realistic description of biosorption/bioaccumulation kinetic. Applicability of mechanistic models in the current study exhibited that the rate governing step in biosorption/bioaccumulation of both As(III) and As(V) was film diffusion rather than intraparticle diffusion. The evaluated thermodynamic parameters ΔG0, ΔH0 and ΔS0 revealed that biosorption/bioaccumulation of both As(III) and As(V) was feasible, spontaneous and exothermic under studied conditions.

  13. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  14. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  15. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  16. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  17. 21 CFR 866.3140 - Corynebacterium spp. serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Corynebacterium spp. serological reagents. 866.3140 Section 866.3140 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  18. Early prosthetic valve endocarditis caused by Corynebacterium kroppenstedtii.

    PubMed

    Hagemann, Jürgen Benjamin; Essig, Andreas; Herrmann, Manuel; Liebold, Andreas; Quader, Mohamed Abo

    2015-12-01

    Corynebacterium (C.) kroppenstedtii is a rarely detected agent of bacterial infections in humans. Here, we describe the first case of prosthetic valve endocarditis caused by C. kroppenstedtii. Application of molecular methods using surgically excised valve tissue was a cornerstone for the establishment of the microbiological diagnosis, which is crucial for targeted antimicrobial treatment.

  19. Native valve endocarditis due to Corynebacterium group JK.

    PubMed

    Moffie, B G; Veenendaal, R A; Thompson, J

    1990-12-01

    We report a case of a 32-yr-old woman on chronic intermittent haemodialysis, who developed endocarditis due to a Corynebacterium group JK, involving both the native aortic and mitral valves. Despite a four-week treatment with vancomycin, an aortic root abscess developed. The diagnosis was confirmed on autopsy.

  20. Experimental transmission of Corynebacterium pseudotuberculosis in horses by house flies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The route of infection of pigeon fever remains undetermined. The purpose of this study was to investigate house flies (Musca domestica L.) as vectors of Corynebacterium pseudotuberculosis in horses. Eight ponies were used in a randomized, controlled, blinded experimental study. Ten wounds were creat...

  1. Corynebacterium pseudotuberculosis liver abscess in a mature alpaca (Lama pacos)

    PubMed Central

    Sprake, Philippa; Gold, Jenifer R.

    2012-01-01

    A mature female alpaca was evaluated for weight loss and a 10-day history of anorexia, diarrhea, abdominal distension, and ventral edema. Ultrasonography revealed a hepatic mass, culture of which identified Corynebacterium pseudotuberculosis. This is the first reported case of an internal caseous lymphadenitis lesion resulting in clinical disease in a camelid. PMID:23024384

  2. Caseous lymphadenitis caused by Corynebacterium ulcerans in the dromedary camel.

    PubMed Central

    Tejedor, M T; Martin, J L; Lupiola, P; Gutierrez, C

    2000-01-01

    Caseous lymphadenitis that affected the dorsal and ventral superficial lymph nodes in the left cervicothoracic region of a young dromedary camel is described. The agent isolated was Corynebacterium ulcerans. To our knowledge, this is the first description of purulent lymphadenitis caused by C. ulcerans in a species belonging to the Camelidae. Images p127-a PMID:10723599

  3. Assessing the Genotypic Differences between Strains of Corynebacterium pseudotuberculosis biovar equi through Comparative Genomics

    PubMed Central

    Ramos, Rommel T. J.; Veras, Adonney A. O.; Pinheiro, Kenny C.; Benevides, Leandro J.; Edman, Judy M.; Spier, Sharon J.; Azevedo, Vasco; Silva, Artur

    2017-01-01

    Seven genomes of Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, generating high-quality scaffolds over 2.35 Mbp. This bacterium is the causative agent of disease known as “pigeon fever” which commonly affects horses worldwide. The pangenome of biovar equi was calculated and two phylogenomic approaches were used to identify clustering patterns within Corynebacterium genus. Furthermore, other comparative analyses were performed including the prediction of genomic islands and prophages, and SNP-based phylogeny. In the phylogenomic tree, C. pseudotuberculosis was divided into two distinct clades, one formed by nitrate non-reducing species (biovar ovis) and another formed by nitrate-reducing species (biovar equi). In the latter group, the strains isolated from California were more related to each other, while the strains CIP 52.97 and 1/06-A formed the outermost clade of the biovar equi. A total of 1,355 core genes were identified, corresponding to 42.5% of the pangenome. This pangenome has one of the smallest core genomes described in the literature, suggesting a high genetic variability of biovar equi of C. pseudotuberculosis. The analysis of the similarity between the resistance islands identified a higher proximity between the strains that caused more severe infectious conditions (infection in the internal organs). Pathogenicity islands were largely conserved between strains. Several genes that modulate the pathogenicity of C. pseudotuberculosis were described including peptidases, recombination enzymes, micoside synthesis enzymes, bacteriocins with antimicrobial activity and several others. Finally, no genotypic differences were observed between the strains that caused the three different types of infection (external abscess formation, infection with abscess formation in the internal organs, and ulcerative lymphangitis). Instead, it was noted that there is a higher phenetic correlation between strains isolated at

  4. Human clinical isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans collected in Canada from 1999 to 2003 but not fitting reporting criteria for cases of diphtheria.

    PubMed

    Dewinter, Leanne M; Bernard, Kathryn A; Romney, Marc G

    2005-07-01

    A 5-year collection of Corynebacterium diphtheriae and Corynebacterium ulcerans human clinical isolates yielded nine isolates from blood cultures of patients with invasive infections, stressing the importance of C. diphtheriae as a serious blood-borne pathogen. Seven percent of C. diphtheriae and 100% of C. ulcerans isolates produced diphtheria toxin, demonstrating that toxigenic corynebacteria continue to circulate.

  5. Evolution, epidemiology and diversity of Corynebacterium diphtheriae: New perspectives on an old foe.

    PubMed

    Sangal, Vartul; Hoskisson, Paul A

    2016-09-01

    Diphtheria is a debilitating disease caused by toxigenic Corynebacterium diphtheriae strains and has been effectively controlled by the toxoid vaccine, yet several recent outbreaks have been reported across the globe. Moreover, non-toxigenic C. diphtheriae strains are emerging as a major global health concern by causing severe pharyngitis and tonsillitis, endocarditis, septic arthritis and osteomyelitis. Molecular epidemiological investigations suggest the existence of outbreak-associated clones with multiple genotypes circulating around the world. Evolution and pathogenesis appears to be driven by recombination as major virulence factors, including the tox gene and pilus gene clusters, are found within genomic islands that appear to be mobile between strains. The number of pilus gene clusters and variation introduced by gain or loss of gene function correlate with the variable adhesive and invasive properties of C. diphtheriae strains. Genomic variation does not support the separation of C. diphtheriae strains into biovars which correlates well with findings of studies based on multilocus sequence typing. Genomic analyses of a relatively small number of strains also revealed a recombination driven diversification of strains within a sequence type and indicate a wider diversity among C. diphtheriae strains than previously appreciated. This suggests that there is a need for increased effort from the scientific community to study C. diphtheriae to help understand the genomic diversity and pathogenicity within the population of this important human pathogen.

  6. Identification and functional characterization of the NanH extracellular sialidase from Corynebacterium diphtheriae.

    PubMed

    Kim, Seonghun; Oh, Doo-Byoung; Kwon, Ohsuk; Kang, Hyun Ah

    2010-04-01

    Corynebacterium diphtheriae, a pathogenic Gram-positive bacterium, contains sialic acids on its cell surface, but no genes related to sialic acid decoration or metabolism have been reported in C. diphtheriae. In the present study, we have identified a putative sialidase gene, nanH, from C. diphtheriae KCTC3075 and characterized its product for enzyme activity. Interestingly, the recombinant NanH protein was secreted as a catalytically active sialidase into the periplasmic space in Escherichia coli, while the short region at its C-terminus was truncated by proteolysis. We reconstructed a truncated NanH protein (His(6)-NanH(DeltaN)) devoid of its signal sequence as a mature enzyme fused with the 6xHis tag at the N-terminal region. The purified His(6)-NanH(DeltaN) can cleave alpha-2,3- and alpha-2,6-linked sialic acid from sialic acid-containing substrates. In addition, even though the efficiency was low, the recombinant His(6)-NanH(DeltaN) was able to catalyse the transfer of sialic acid using several sialoconjugates as donor, suggesting that the reversible nature of C. diphtheriae NanH can be used for the synthesis of sialyl oligosaccharides via transglycosylation reaction.

  7. Corynebacterium tapiri sp. nov. and Corynebacterium nasicanis sp. nov., isolated from a tapir and a dog, respectively.

    PubMed

    Baumgardt, Sandra; Loncaric, Igor; Kämpfer, Peter; Busse, Hans-Jürgen

    2015-11-01

    Two Gram-stain-positive bacterial isolates, strain 2385/12T and strain 2673/12T were isolated from a tapir and a dog's nose, respectively. The two strains were rod to coccoid-shaped, catalase-positive and oxidase-negative. The highest 16S rRNA gene sequence similarity identified Corynebacterium singulare CCUG 37330T (96.3% similarity) as the nearest relative of strain 2385/12T and suggested the isolate represented a novel species. Corynebacterium humireducens DSM 45392T (98.7% 16S rRNA gene sequence similarity) was identified as the nearest relative of strain 2673/12T. Results from DNA-DNA hybridization with the type strain of C. humireducens demonstrated that strain 2673/12T also represented a novel species. Strain 2385/12T showed a quinone system consisting predominantly of menaquinones MK-8(H2) and MK-9(H2) whereas strain 2673/12T contained only MK-8(H2) as predominant quinone. The polar lipid profiles of the two strains showed the major compounds phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid. Phosphatidylinositol was identified as another major lipid in 2673/12T whereas it was only found in moderate amounts in strain 2385/12T. Furthermore, moderate to minor amounts of phosphatidylinositol-mannoside, β-gentiobiosyl diacylglycerol and variable counts of several unidentified lipids were detected in the two strains. Both strains contained corynemycolic acids. The polyamine patterns were characterized by the major compound putrescine in strain 2385/12T and spermidine in strain 2673/12T. In the fatty acid profiles, predominantly C18:1ω9c and C16:0 were detected. The two strains are distinguishable from each other and the nearest related established species of the genus Corynebacterium phylogenetically and phenotypically. In conclusion, two novel species of the genus Corynebacterium are proposed, namely Corynebacterium tapiri sp. nov. (type strain, 2385/12T = CCUG 65456T = LMG 28165T) and Corynebacterium nasicanis sp. nov. (type

  8. Draft genome sequence of Corynebacterium diphtheriae biovar intermedius NCTC 5011.

    PubMed

    Sangal, Vartul; Tucker, Nicholas P; Burkovski, Andreas; Hoskisson, Paul A

    2012-09-01

    We report an annotated draft genome of the human pathogen Corynebacterium diphtheriae bv. intermedius NCTC 5011. This strain is the first C. diphtheriae bv. intermedius strain to be sequenced, and our results provide a useful comparison to the other primary disease-causing biovars, C. diphtheriae bv. gravis and C. diphtheriae bv. mitis. The sequence has been deposited at DDBJ/EMBL/GenBank with the accession number AJVH01000000.

  9. Gangrenous dermatitis caused by Corynebacterium ulcerans in Richardson ground squirrels.

    PubMed

    Olson, M E; Goemans, I; Bolingbroke, D; Lundberg, S

    1988-08-01

    Gangrenous dermatitis caused by Corynebacterium ulcerans developed in 63 of 350 wild Richardson ground squirrels (Spermophilus richardsonii). Six squirrels died of toxemia and/or septicemia, but 57 responded to topical and parenteral administration of antibiotics. The epizo-otic was believed to be associated with fighting; infected and carrier ground squirrels most likely transmitted the C ulcerans through bite wounds. Individuals handling ground squirrels should be cautioned that C ulcerans may produce a diphtheria-like disease in human beings.

  10. Corynebacterium pseudodiphtheriticum keratitis and conjunctivitis: a case report.

    PubMed

    Li, A; Lal, S

    2000-02-01

    A case of keratitis and conjunctivitis in an 86-year-old man caused by Corynebacterium pseudodiphtheriticum is reported. Corynebacteria are uncommon causes of ocular surface infections. However, the presence of corneal and conjunctival epithelial defects in an immunocompromised patient can result in severe infection by a commensal organism such as C. pseudodiphtheriticum. The significance of a positive culture in these settings should not be overlooked.

  11. Corynebacterium urealyticum: a comprehensive review of an understated organism.

    PubMed

    Salem, Nagla; Salem, Lamyaa; Saber, Sally; Ismail, Ghada; Bluth, Martin H

    2015-01-01

    Corynebacterium urealyticum is a Gram positive, slow-growing, lipophilic, multi-drug resistant, urease positive micro-organism with diphtheroid morphology. It has been reported as an opportunistic nosocomial pathogen and as the cause of a variety of diseases including but not limited to cystitis, pyelonephritis, and bacteremia among others. This review serves to describe C. urealyticum with respect to its history, identification, laboratory investigation, relationship to disease and treatment in order to allow increased familiarity with this organism in clinical disease.

  12. Corynebacterium propinquum: A Rare Cause of Prosthetic Valve Endocarditis.

    PubMed

    Jangda, Umair; Upadhyay, Ankit; Bagheri, Farshad; Patel, Nilesh R; Mendelson, Robert I

    2016-01-01

    Nondiphtheria Corynebacterium species are often dismissed as culture contaminants, but they have recently become increasingly recognized as pathologic organisms. We present the case of a 48-year-old male patient on chronic prednisone therapy for rheumatoid arthritis with a history of mitral valve replacement with prosthetic valve. He presented with fever, dizziness, dyspnea on exertion, intermittent chest pain, and palpitations. Transesophageal echocardiography revealed two medium-sized densities along the inner aspect of the sewing ring and one larger density along the atrial surface of the sewing ring consistent with vegetation. Two separate blood cultures grew Corynebacterium propinquum, which were sensitive to ceftriaxone but highly resistant to vancomycin and daptomycin. The patient completed a course of ceftriaxone and repeat TEE study and after 6 weeks demonstrated near complete resolution of the vegetation. To our knowledge, this case represents the first in the literature of Corynebacterium propinquum causing prosthetic valve endocarditis. The ability of these organisms to cause deep-seated systemic infections should be recognized, especially in immune-compromised patients.

  13. Corynebacterium propinquum: A Rare Cause of Prosthetic Valve Endocarditis

    PubMed Central

    Bagheri, Farshad; Patel, Nilesh R.; Mendelson, Robert I.

    2016-01-01

    Nondiphtheria Corynebacterium species are often dismissed as culture contaminants, but they have recently become increasingly recognized as pathologic organisms. We present the case of a 48-year-old male patient on chronic prednisone therapy for rheumatoid arthritis with a history of mitral valve replacement with prosthetic valve. He presented with fever, dizziness, dyspnea on exertion, intermittent chest pain, and palpitations. Transesophageal echocardiography revealed two medium-sized densities along the inner aspect of the sewing ring and one larger density along the atrial surface of the sewing ring consistent with vegetation. Two separate blood cultures grew Corynebacterium propinquum, which were sensitive to ceftriaxone but highly resistant to vancomycin and daptomycin. The patient completed a course of ceftriaxone and repeat TEE study and after 6 weeks demonstrated near complete resolution of the vegetation. To our knowledge, this case represents the first in the literature of Corynebacterium propinquum causing prosthetic valve endocarditis. The ability of these organisms to cause deep-seated systemic infections should be recognized, especially in immune-compromised patients. PMID:27891149

  14. Rapid detection and molecular differentiation of toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans strains by LightCycler PCR.

    PubMed

    Sing, Andreas; Berger, Anja; Schneider-Brachert, Wulf; Holzmann, Thomas; Reischl, Udo

    2011-07-01

    The systemic symptoms of diphtheria are caused by the tox-encoded diphtheria toxin (DT) which is produced by toxigenic Corynebacterium spp. Besides the classical agent C. diphtheriae, the zoonotic pathogen C. ulcerans has increasingly been reported as an emerging pathogen for diphtheria. The reliable detection of toxigenic Corynebacterium spp. is of substantial importance for both diphtheria surveillance in the public health sector and the clinical workup of a patient with diphtherialike symptoms. Since the respective tox genes of C. diphtheriae and C. ulcerans differ from each other in both DNA and amino acid sequence, both tox genes should be covered by novel real-time PCR methods. We describe the development and validation of a LightCycler PCR assay which reliably recognizes tox genes from both C. diphtheriae and C. ulcerans and differentiates the respective target genes by fluorescence resonance energy transfer (FRET) hybridization probe melting curve analysis.

  15. Bloodstream infection caused by nontoxigenic Corynebacterium diphtheriae in an immunocompromised host in the United States.

    PubMed

    Wojewoda, Christina M; Koval, Christine E; Wilson, Deborah A; Chakos, Mary H; Harrington, Susan M

    2012-06-01

    Corynebacterium species are well-known causes of catheter-related bloodstream infections. Toxigenic strains of Corynebacterium diphtheriae cause respiratory diphtheria. We report a bloodstream infection caused by a nontoxigenic strain of C. diphtheriae and discuss the epidemiology, possible sources of the infection, and the implications of rapid species identification of corynebacteria.

  16. Expression of a functional single-chain antibody via Corynebacterium pseudodiphtheriticum.

    PubMed

    Sundaram, R K; Hurwitz, I; Matthews, S; Hoy, E; Kurapati, S; Crawford, C; Sundaram, P; Durvasula, R V

    2008-07-01

    Antibody-based therapeutics are effective against conditions ranging from acute infections to malignancy. They may prove crucial in combating bioterrorism and responding to drug-resistant and emerging pathogens. At present the cost of producing therapeutic monoclonal antibodies is between $1,000 to $6,000 per gram. The need to administer antibodies parenterally at frequent intervals further drives the cost of this treatment. Here we present an antibody delivery system, termed paratransgenesis, with the potential to overcome these limitations. The paratransgenic approach involves genetically transforming a commensal or symbiont bacterium to express foreign molecules that target pathogens. We describe transformation of Corynebacterium pseudodiptheriticum, a commensal bacterium found in the human respiratory tract, to express a murine single-chain antibody binding progesterone. The antibody was functional and bound specifically to progesterone in a concentration-dependent manner. This marker antibody system is the precursor to development of expression systems producing recombinant humanized single-chain antibodies. Studies are in progress evaluating fitness, transgene stablility, and pathogenecity of the genetically engineered C. pseudodiptheriticum. We anticipate developing a repertoire of expressed molecules targeting infectious agents and surface epitopes of pulmonary mass lesions. If expression systems for anti-pathogen molecules in C. pseudodiptheriticum and other respiratory commensal bacteria can be optimized, these bacteria have the potential for a range of therapeutic and prophylactic applications.

  17. Corynebacterium nuruki sp. nov., isolated from an alcohol fermentation starter.

    PubMed

    Shin, Na-Ri; Jung, Mi-Ja; Kim, Min-Soo; Roh, Seong Woon; Nam, Young-Do; Bae, Jin-Woo

    2011-10-01

    A novel Gram-positive, strictly aerobic and non-motile bacterial strain, S6-4(T), was isolated from a Korean alcohol fermentation starter. Optimal growth occurred at 37 °C, at pH 8 and in 1 % (w/v) NaCl. The isolate was positive for oxidase and catalase. It assimilated various sugars and acids were produced from several carbohydrates. The major cell-wall sugars were galactose and arabinose. The major fatty acids of strain S6-4(T) were C(16 : 0), C(17 : 1)ω9c, C(18 : 1)ω9c and 10-methyl C(18 : 0) (tuberculostearic acid). The predominant isoprenoid quinone was menaquinone MK-9(H(2)) and peptidoglycan amino acids were meso-diaminopimelic acid, alanine, glycine and glutamic acid. The strain contained mycolic acids. According to phylogenetic analysis based on 16S rRNA gene sequences, strain S6-4(T) was most closely related to Corynebacterium variabile DSM 20132(T) (98.1 % similarity). The genomic DNA G+C content of strain S6-4(T) was 73.6 mol% and DNA-DNA hybridization values with related strains were below 33±4 %. On the basis of phenotypic, genotypic and phylogenetic data, strain S6-4(T) represents a novel species in the genus Corynebacterium, for which the name Corynebacterium nuruki sp. nov. is proposed; the type strain is S6-4(T) ( = KACC 15032(T)  = JCM 17162(T)).

  18. A Case of Necrotizing Epiglottitis Due to Nontoxigenic Corynebacterium diphtheriae.

    PubMed

    Lake, Jessica A; Ehrhardt, Matthew J; Suchi, Mariko; Chun, Robert H; Willoughby, Rodney E

    2015-07-01

    Diphtheria is a rare cause of infection in highly vaccinated populations and may not be recognized by modern clinicians. Infections by nontoxigenic Corynebacterium diphtheriae are emerging. We report the first case of necrotizing epiglottitis secondary to nontoxigenic C diphtheriae. A fully vaccinated child developed fever, poor oral intake, and sore throat and was found to have necrotizing epiglottitis. Necrotizing epiglottitis predominantly occurs in the immunocompromised host. Laboratory evaluation revealed pancytopenia, and bone marrow biopsy was diagnostic for acute lymphoblastic leukemia. Clinicians should be aware of aggressive infections that identify immunocompromised patients. This case highlights the features of a reemerging pathogen, C diphtheriae.

  19. Structure modeling of a metalloendopeptidase from Corynebacterium pseudotuberculosis.

    PubMed

    Guimarães, Luis C; Silva, Natália F; Miyoshi, Anderson; Schneider, Maria P C; Silva, Artur; Azevedo, Vasco; Brasil, Davi S B; Lameira, Jerônimo; Alves, Cláudio N

    2012-05-01

    Metalloendopeptidases are zinc-dependent hydrolases enzymes with many different roles in biological systems, ranging from remodeling conjunctive tissue to removing signaling sequences from nascent proteins. Here, we describe the three-dimensional structure of the metalloendopeptidase from Corynebacterium pseudotuberculosis generated by homology modeling and molecular dynamics. Analysis of key distances shows that His-132, Asp-136, His-211, Leu-212 and one molecule of water play an important role in the protein-Zn(2+) ion interaction. The model obtained may provide structural insights into this enzyme and can be useful for the design of new caseous lymphadenitis vaccines based on genetic attenuation from key point mutation.

  20. Corynebacterium urealyticum: a comprehensive review of an understated organism

    PubMed Central

    Salem, Nagla; Salem, Lamyaa; Saber, Sally; Ismail, Ghada; Bluth, Martin H

    2015-01-01

    Corynebacterium urealyticum is a Gram positive, slow-growing, lipophilic, multi-drug resistant, urease positive micro-organism with diphtheroid morphology. It has been reported as an opportunistic nosocomial pathogen and as the cause of a variety of diseases including but not limited to cystitis, pyelonephritis, and bacteremia among others. This review serves to describe C. urealyticum with respect to its history, identification, laboratory investigation, relationship to disease and treatment in order to allow increased familiarity with this organism in clinical disease. PMID:26056481

  1. Corynebacterium CDC Group G Native and Prosthetic Valve Endocarditis.

    PubMed

    Sattar, Adil; Yu, Siegfried; Koirala, Janak

    2015-08-11

    We report the first case of native and recurrent prosthetic valve endocarditis with Corynebacterium CDC group G, a rarely reported cause of infective endocarditis (IE). Previously, there have been only two cases reported for prosthetic valve IE caused by these organisms. A 69-year-old female with a known history of mitral valve regurgitation presented with a 3-day history of high-grade fever, pleuritic chest pain and cough. Echocardiography confirmed findings of mitral valve thickening consistent with endocarditis, which subsequently progressed to become large and mobile vegetations. Both sets of blood cultures taken on admission were positive for Corynebacterium CDC group G. Despite removal of a long-term venous access port, the patient's presumed source of line associated bacteremia, mitral valve replacement, and aggressive antibiotic therapy, the patient had recurrence of vegetations on the prosthetic valve. She underwent replacement of her prosthetic mitral valve in the subsequent 2 weeks, before she progressed to disseminated intravascular coagulation and expired. Although they are typically considered contaminants, corynebacteria, in the appropriate clinical setting, should be recognized, identified, and treated as potentially life-threatening infections, particularly in the case of line-associated bacteremias, and native and prosthetic valve endocarditis.

  2. Corynebacterium equi: in vitro susceptibility to twenty-six antimicrobial agents.

    PubMed Central

    Woolcock, J B; Mutimer, M D

    1980-01-01

    The minimal concentrations of 26 antimicrobial agents required to inhibit growth of 100 isolates of Corynebacterium equi in vitro have been determined. The most active agents were penicillin G, doxycycline, erythromycin, lincomycin, and the aminoglycosides. PMID:7235683

  3. [Etiologic role of Corynebacterium non diphtheriae in patients with different pathology].

    PubMed

    Kraeva, L A; Manina, Zh N; Tseneva, G Ia; Radchenko, A G

    2007-01-01

    Bacteriologic examination of 1589 patients showed that, aside from C. diphtheriae, 11% of acute upper respiratory tract infections were caused by other Corynebacterium species. Such bacteria can cause infections of various localizations (bronchitis, pyelonephritis, urethritis, colpitis, dermatitis, arthritis, etc.). C. pseudodiphtheriticum and C. xerosis were isolated from clinical specimens most frequently. Corynebacterium spp. have adhesive, hemolytic, hemagglutinating, and neuraminidase activity; some of them are highly pathogenic. The most virulent, were following species: C. diphtheriae, C. pseudotuberculosis, C. urealyticum, and C. ulcerans. Corynebacterium non diphtheriae were frequently isolated from clinical specimens in association with staphylococci and streptococci. In such cases, factors of pathogenicity and resistance to antibiotics were more pronounced. Strains isolated with association with other bacteria have lost susceptibility to tetracycline, oleandomycin, penicillin, and erythromycin. It is important to be vigilant about bacteria from Corynebacterium genus in clinical settings, and thoroughly study their biologic characteristics, especially in immunocompromised patients.

  4. First Draft Genome Sequences of Malaysian Clinical Isolates of Corynebacterium diphtheriae

    PubMed Central

    Ahmad, Norazah; Mohd Khalid, Mohd Khairul Nizam; Abd Wahab, Muhammad Adib; Hashim, Rohaidah; Tang, Soo Nee; Liow, Yii Ling; Hamzah, Hazwani; Dahalan, Nurul Ain; Seradja, Valentinus

    2017-01-01

    ABSTRACT Corynebacterium diphtheriae has caused multiple isolated diphtheria cases in Malaysia over the years. Here, we report the first draft genome sequences of 15 Malaysia C. diphtheriae clinical isolates collected from the years 1981 to 2016. PMID:28254972

  5. First Draft Genome Sequences of Malaysian Clinical Isolates of Corynebacterium diphtheriae.

    PubMed

    Ahmad, Norazah; Hii, Shirley Yi Fen; Mohd Khalid, Mohd Khairul Nizam; Abd Wahab, Muhammad Adib; Hashim, Rohaidah; Tang, Soo Nee; Liow, Yii Ling; Hamzah, Hazwani; Dahalan, Nurul Ain; Seradja, Valentinus

    2017-03-02

    Corynebacterium diphtheriae has caused multiple isolated diphtheria cases in Malaysia over the years. Here, we report the first draft genome sequences of 15 Malaysia C. diphtheriae clinical isolates collected from the years 1981 to 2016.

  6. Experimental inoculation of house flies, Musca domestica L., with Corynebacterium pseudotuberculosis serovar equi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Corynebacterium pseudotuberculosis (Actinomycetales: Corynebacteriaceae) infection in horses causes three different disease syndromes: external abscesses, infection of internal organs and ulcerative lymphangitis. The route of infection in horses remains undetermined, but transmission by insect vecto...

  7. Experimental transmission of Corynebacterium pseudotuberculosis biovar equi in horses by house flies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The route of Corynebacterium pseudotuberculosis infection in horses remains undetermined, but transmission by insects is suspected. Scientists from CMAVE and Auburn University investigated house flies (Musca domestica L.) as possible vectors. Three ponies were directly inoculated with C. pseudotuber...

  8. Experimental inoculation of house flies Musca domestica with Corynebacterium pseudotuberculosis serovar equi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Corynebacterium pseudotuberculosis (Actinomycetales: Corynebacteriaceae) infection in horses causes external abscesses, infection of internal organs and ulcerative lymphangitis. The exact mechanism of infection remains unknown, but fly transmission is suspected. Scientists at Auburn University and U...

  9. Genome sequence of Corynebacterium nuruki S6-4 T, isolated from alcohol fermentation starter.

    PubMed

    Shin, Na-Ri; Whon, Tae Woong; Roh, Seong Woon; Kim, Min-Soo; Jung, Mi-Ja; Lee, Jina; Bae, Jin-Woo

    2011-08-01

    Corynebacterium nuruki S6-4(T), isolated from Korean alcohol fermentation starter, is a strictly aerobic, nonmotile, Gram-positive, and rod-shaped bacterium belonging to the genus Corynebacterium and the actinomycete group. We report here the draft genome sequence of C. nuruki strain S6-4(T) (3,106,595 bp, with a G+C content of 69.5%).

  10. Genome Sequence of Corynebacterium nuruki S6-4T, Isolated from Alcohol Fermentation Starter▿

    PubMed Central

    Shin, Na-Ri; Whon, Tae Woong; Roh, Seong Woon; Kim, Min-Soo; Jung, Mi-Ja; Lee, Jina; Bae, Jin-Woo

    2011-01-01

    Corynebacterium nuruki S6-4T, isolated from Korean alcohol fermentation starter, is a strictly aerobic, nonmotile, Gram-positive, and rod-shaped bacterium belonging to the genus Corynebacterium and the actinomycete group. We report here the draft genome sequence of C. nuruki strain S6-4T (3,106,595 bp, with a G+C content of 69.5%). PMID:21685278

  11. [THE COMPARATIVE ANALYSIS OF TECHNIQUES OF IDENTIFICATION OF CORYNEBACTERIUM NON DIPHTHERIAE].

    PubMed

    Kharseeva, G G; Voronina, N A; Mironov, A Yu; Alutina, E L

    2015-12-01

    The comparative analysis was carried out concerning effectiveness of three techniques of identification of Corynebacterium non diphtheriae: bacteriological, molecular genetic (sequenation on 16SpRNA) andmass-spectrometric (MALDI-ToFMS). The analysis covered 49 strains of Corynebacterium non diphtheriae (C.pseudodiphheriticum, C.amycolatum, C.propinquum, C.falsenii) and 2 strains of Corynebacterium diphtheriae isolated under various pathology form urogenital tract and upper respiratory ways. The corinbacteria were identified using bacteriologic technique, sequenation on 16SpRNA and mass-spectrometric technique (MALDIToF MS). The full concordance of results of species' identification was marked in 26 (51%) of strains of Corynebacterium non diphtheriae at using three analysis techniques; in 43 (84.3%) strains--at comparison of bacteriologic technique with sequenation on 16S pRNA and in 29 (57%)--at mass-spectrometric analysis and sequenation on 16S pRNA. The bacteriologic technique is effective for identification of Corynebacterium diphtheriae. The precise establishment of species belonging of corynebacteria with variable biochemical characteristics the molecular genetic technique of analysis is to be applied. The mass-spectrometric technique (MALDI-ToF MS) requires further renewal of data bases for identifying larger spectrum of representatives of genus Corynebacterium.

  12. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  13. Outbreak of Corynebacterium pseudodiphtheriticum Infection in Cystic Fibrosis Patients, France

    PubMed Central

    Bittar, Fadi; Cassagne, Carole; Bosdure, Emmanuelle; Stremler, Nathalie; Dubus, Jean-Christophe; Sarles, Jacques; Reynaud-Gaubert, Martine; Raoult, Didier

    2010-01-01

    An increasing body of evidence indicates that nondiphtheria corynebacteria may be responsible for respiratory tract infections. We report an outbreak of Corynebacterium pseudodiphtheriticum infection in children with cystic fibrosis (CF). To identify 18 C. pseudodiphtheriticum strains isolated from 13 French children with CF, we used molecular methods (partial rpoB gene sequencing) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Clinical symptoms were exhibited by 10 children (76.9%), including cough, rhinitis, and lung exacerbations. The results of MALDI-TOF identification matched perfectly with those obtained from molecular identification. Retrospective analysis of sputum specimens by using specific real-time PCR showed that ≈20% of children with CF were colonized with these bacteria, whereas children who did not have CF had negative test results. Our study reemphasizes the conclusion that correctly identifying bacteria at the species level facilitates detection of an outbreak of new or emerging infections in humans. PMID:20678316

  14. Outbreak of Corynebacterium pseudodiphtheriticum infection in cystic fibrosis patients, France.

    PubMed

    Bittar, Fadi; Cassagne, Carole; Bosdure, Emmanuelle; Stremler, Nathalie; Dubus, Jean Christophe; Sarles, Jacques; Reynaud-Gaubert, Martine; Raoult, Didier; Rolain, Jean-Marc

    2010-08-01

    An increasing body of evidence indicates that nondiphtheria corynebacteria may be responsible for respiratory tract infections. We report an outbreak of Corynebacterium pseudodiphtheriticum infection in children with cystic fibrosis (CF). To identify 18 C. pseudodiphtheriticum strains isolated from 13 French children with CF, we used molecular methods (partial rpoB gene sequencing) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Clinical symptoms were exhibited by 10 children (76.9%), including cough, rhinitis, and lung exacerbations. The results of MALDI-TOF identification matched perfectly with those obtained from molecular identification. Retrospective analysis of sputum specimens by using specific real-time PCR showed that approximately 20% of children with CF were colonized with these bacteria, whereas children who did not have CF had negative test results. Our study reemphasizes the conclusion that correctly identifying bacteria at the species level facilitates detection of an outbreak of new or emerging infections in humans.

  15. Tetracycline Inhibition of a Lipase from Corynebacterium acnes

    PubMed Central

    Weaber, K.; Freedman, R.; Eudy, W. W.

    1971-01-01

    A lipase which hydrolyzes triglycerides (tricaprylin and trilaurin) and naphthyl laurate was obtained from the broth of Corynebacterium acnes cultures by ammonium sulfate fractionation. Ca2+ and sodium taurocholate stimulated activity of the enzyme. Ethylenediaminetetraacetic acid (EDTA) did not inhibit activity of the Ca2+-activated enzyme, but lipolytic activity was inhibited by EDTA in the absence of Ca2+. Tetracycline (10−4m) produced a slight inhibition of the lipase activity with 5 × 10−5m or less showing no effect on the lipase activity. However, complete inhibition by tetracycline at 10−4m was observed for Ca2+-activated enzyme. Tetracycline inhibition of the C. acnes lipase could be demonstrated at concentrations as low as 10−6m. PMID:4252558

  16. Corynebacterium diphtheriae infections currently and in the past.

    PubMed

    Zasada, Aleksandra Anna

    2015-01-01

    Along with the introduction of common obligatory vaccinations against diphtheria, the disease has been limited in developed countries. However, diphtheria is still endemic in developing countries. Due to a growing popularity of visiting these countries, there is a risk of importation of the disease to Europe. Studies revealed that over 60% of persons aged >40 years in the Polish population do not have a protective level of antibodies against diphtheria. Furthermore, an access to diphtheria antitoxin, which is essential in diphtheria treatment, is now hardly accessible in Europe. On the other hand, in many countries, including Poland, new infections caused by non-toxigenic Corynebacterium diphtheriae have been emerged. Such infections are frequently manifested by bacteraemia and endocarditis with a high fatality rate, amounting even to 41%.

  17. Persistence of Corynebacterium diphtheriae in Delhi & National Capital Region (NCR).

    PubMed

    Bhagat, S; Grover, S S; Gupta, N; Roy, R D; Khare, S

    2015-10-01

    Despite the introduction of mass immunization, diphtheria continues to play a major role as a potentially lethal infectious disease in many countries. Delay in the specific therapy of diphtheria may result in death and, therefore, accurate diagnosis of diphtheria is imperative. This study was carried out at National Centre for Disease Control (NCDC), Delhi, India, on samples of suspected diphtheria cases referred from various government hospitals of Delhi and neighbouring areas during 2012-2014. Primary identification of Corynebacterium diphtheriae was done by standard culture, staining and biochemical tests followed by toxigenicity testing by Elek's test on samples positive for C. diphtheriae. The results showed persistence of toxigenic C. diphtheriae in our community indicating the possibility of inadequate immunization coverage.

  18. Exploration of Nitrate Reductase Metabolic Pathway in Corynebacterium pseudotuberculosis

    PubMed Central

    Abreu, Vinícius; Diniz, Carlos; Dorneles, Elaine M. S.; Barh, Debmalya

    2017-01-01

    Based on the ability of nitrate reductase synthesis, Corynebacterium pseudotuberculosis is classified into two biovars: Ovis and Equi. Due to the presence of nitrate reductase, the Equi biovar can survive in absence of oxygen. On the other hand, Ovis biovar that does not have nitrate reductase is able to adapt to various ecological niches and can grow on certain carbon sources. Apart from these two biovars, some other strains are also able to carry out the reduction of nitrate. The enzymes that are involved in electron transport chain are also identified by in silico methods. Findings about pathogen metabolism can contribute to the identification of relationship between nitrate reductase and the C. pseudotuberculosis pathogenicity, virulence factors, and discovery of drug targets. PMID:28316974

  19. Production of D-Alanine by Corynebacterium fascians

    PubMed Central

    Yamada, Shigeki; Maeshima, Haruko; Wada, Mitsuru; Chibata, Ichiro

    1973-01-01

    A strain identified as Corynebacterium fascians was found to accumulate extracellular D-alanine from glycerol. Cultural conditions for the accumulation of D-alanine were investigated and, as a result, a yield of 7 g of D-alanine per liter was obtained after a 96-h incubation in a medium containing 5% glycerol, 4% (NH4)2HPO4, and 0.3% corn steep liquor. Optical purity of D-alanine was dependent upon the concentration of corn steep liquor. At the optimal condition, almost optically pure D-alanine was formed and readily isolated (5 g/liter) from the fermentation broth. The product was not contaminated with any detectable amount of other amino acids, except for glycine which was present at a concentration of less than 1 percent. PMID:4699220

  20. Isolation of Corynebacterium ureicelerivorans from normally sterile sites in humans.

    PubMed

    Fernández-Natal, M I; Sáez-Nieto, J A; Valdezate, S; Rodríguez-Pollán, R H; Lapeña, S; Cachón, F; Soriano, F

    2009-06-01

    Fifteen Corynebacterium ureicelerivorans isolates were recovered in pure culture from six patients during a five-year period. Five patients had bacteremia and the other was an infection of ascitic fluid. The API Coryne numerical profile obtained corresponds to the profile for C. bovis, while Biolog GP2 identified four out of the six isolates as C. jeikeium. The organisms were molecular identified by 16S rDNA and rpoB. The present report also includes information on new phenotypic tests and, for the first time, antimicrobial susceptibility data of C. ureicelerivorans and their rpoB sequences. All macrolide-resistant isolates presented a constitutive MLS phenotype. This organism must be differentiated from other slow-growing, lipophilic, and urea-splitting corynebacteria.

  1. High cell density cultivation of recombinant yeasts and bacteria under non-pressurized and pressurized conditions in stirred tank bioreactors.

    PubMed

    Knoll, Arnd; Bartsch, Stefan; Husemann, Bernward; Engel, Philip; Schroer, Kirsten; Ribeiro, Betina; Stöckmann, Christoph; Seletzky, Juri; Büchs, Jochen

    2007-10-31

    This study demonstrates the applicability of pressurized stirred tank bioreactors for oxygen transfer enhancement in aerobic cultivation processes. The specific power input and the reactor pressure was employed as process variable. As model organism Escherichia coli, Arxula adeninivorans, Saccharomyces cerevisiae and Corynebacterium glutamicum were cultivated to high cell densities. By applying specific power inputs of approx. 48kWm(-3) the oxygen transfer rate of a E. coli culture in the non-pressurized stirred tank bioreactor was lifted up to values of 0.51moll(-1)h(-1). When a reactor pressure up to 10bar was applied, the oxygen transfer rate of a pressurized stirred tank bioreactor was lifted up to values of 0.89moll(-1)h(-1). The non-pressurized stirred tank bioreactor was able to support non-oxygen limited growth of cell densities of more than 40gl(-1) cell dry weight (CDW) of E. coli, whereas the pressurized stirred tank bioreactor was able to support non-oxygen limited growth of cell densities up to 225gl(-1) CDW of A. adeninivorans, 89gl(-1) CDW of S. cerevisiae, 226gl(-1) CDW of C. glutamicum and 110gl(-1) CDW of E. coli. Compared to literature data, some of these cell densities are the highest values ever achieved in high cell density cultivation of microorganisms in stirred tank bioreactors. By comparing the specific power inputs as well as the k(L)a values of both systems, it is demonstrated that only the pressure is a scaleable tool for oxygen transfer enhancement in industrial stirred tank bioreactors. Furthermore, it was shown that increased carbon dioxide partial pressures did not remarkably inhibit the growth of the investigated model organisms.

  2. Complete Genome Sequence of Corynebacterium pseudotuberculosis Strain 1/06-A, Isolated from a Horse in North America

    PubMed Central

    Pethick, Florence E.; Lainson, Alex F.; Yaga, Raja; Flockhart, Allen; Smith, David G. E.; Donachie, Willie; Cerdeira, Louise T.; Silva, Artur; Bol, Erik; Lopes, Thiago S.; Barbosa, Maria S.; Pinto, Anne C.; dos Santos, Anderson R.; Soares, Siomar C.; Almeida, Sintia S.; Guimaraes, Luis C.; Aburjaile, Flavia F.; Abreu, Vinicius A. C.; Ribeiro, Dayana; Fiaux, Karina K.; Diniz, Carlos A. A.; Barbosa, Eudes G. V.; Pereira, Ulisses P.; Hassan, Syed S.; Ali, Amjad; Bakhtiar, Syeda M.; Dorella, Fernanda A.; Carneiro, Adriana R.; Ramos, Rommel T. J.; Rocha, Flavia S.; Schneider, Maria P. C.; Miyoshi, Anderson; Azevedo, Vasco

    2012-01-01

    Corynebacterium pseudotuberculosis causes disease in several animal species, although distinct biovars exist that appear to be restricted to specific hosts. In order to facilitate a better understanding of the differences between biovars, we report here the complete genome sequence of the equine pathogen Corynebacterium pseudotuberculosis strain 1/06-A. PMID:22843601

  3. Genome Sequence of the Corynebacterium pseudotuberculosis Cp316 Strain, Isolated from the Abscess of a Californian Horse

    PubMed Central

    Ramos, Rommel Thiago Jucá; Carneiro, Adriana Ribeiro; Pinto, Anne Cybelle; Soares, Siomar de Castro; Santos, Anderson Rodrigues; Almeida, Sintia Silva; Guimarães, Luis Carlos; Aburjaile, Flávia Figueira; Barbosa, Eudes Guilherme Vieira; Dorella, Fernanda Alves; Rocha, Flávia Souza; Cerdeira, Louise Teixeira; Barbosa, Maria Silvanira; Tauch, Andreas; Edman, Judy; Spier, Sharon; Miyoshi, Anderson; Schneider, Maria Paula Cruz; Azevedo, Vasco

    2012-01-01

    The bacterium Corynebacterium pseudotuberculosis is of major veterinary importance because it affects livestock, particularly sheep, goats, and horses, in several countries, including Australia, Brazil, the United States, and Canada, resulting in significant economic losses. In the present study, we describe the complete genome of the Corynebacterium pseudotuberculosis Cp316 strain, biovar equi, isolated from the abscess of a North American horse. PMID:23144380

  4. CoryneBase: Corynebacterium Genomic Resources and Analysis Tools at Your Fingertips

    PubMed Central

    Tan, Mui Fern; Jakubovics, Nick S.; Wee, Wei Yee; Mutha, Naresh V. R.; Wong, Guat Jah; Ang, Mia Yang; Yazdi, Amir Hessam; Choo, Siew Woh

    2014-01-01

    Corynebacteria are used for a wide variety of industrial purposes but some species are associated with human diseases. With increasing number of corynebacterial genomes having been sequenced, comparative analysis of these strains may provide better understanding of their biology, phylogeny, virulence and taxonomy that may lead to the discoveries of beneficial industrial strains or contribute to better management of diseases. To facilitate the ongoing research of corynebacteria, a specialized central repository and analysis platform for the corynebacterial research community is needed to host the fast-growing amount of genomic data and facilitate the analysis of these data. Here we present CoryneBase, a genomic database for Corynebacterium with diverse functionality for the analysis of genomes aimed to provide: (1) annotated genome sequences of Corynebacterium where 165,918 coding sequences and 4,180 RNAs can be found in 27 species; (2) access to comprehensive Corynebacterium data through the use of advanced web technologies for interactive web interfaces; and (3) advanced bioinformatic analysis tools consisting of standard BLAST for homology search, VFDB BLAST for sequence homology search against the Virulence Factor Database (VFDB), Pairwise Genome Comparison (PGC) tool for comparative genomic analysis, and a newly designed Pathogenomics Profiling Tool (PathoProT) for comparative pathogenomic analysis. CoryneBase offers the access of a range of Corynebacterium genomic resources as well as analysis tools for comparative genomics and pathogenomics. It is publicly available at http://corynebacterium.um.edu.my/. PMID:24466021

  5. Encrusted Cystitis Secondary to Corynebacterium glucuronolyticum in a 57-Year-Old Man Without Predisposing Factors.

    PubMed

    Curry, Chelsea R; Saluja, Karan; Das, Sanchita; Thakral, Beenu; Dangle, Pankaj; Keeler, Thomas C; Watkin, William G

    2015-01-01

    Encrusted cystitis is a rare condition characterized by encrustation of the bladder mucosa with associated chronic inflammation induced by urea-splitting bacterial infection--most commonly, Corynebacterium urealyticum. Moreover, it usually occurs in immunocompromised patients, especially recipients of renal transplants or patients with a history of previous urological procedures. Due to the rarity of the entity and the slow growth of Corynebacterium species, appropriate treatment is often delayed due to difficulties in diagnosis and resistance to numerous antibiotics. We report a case of encrusted cystitis caused by Corynebacterium glucuronolyticum, another urea-splitting microbe, in a 57-year-old previously healthy Caucasian man with no known predisposing factors. The timely diagnosis and management in this otherwise healthy patient was facilitated by characteristic imaging, cystoscopy, and histologic findings confirmed by results of prolonged urine cultures and 16S ribosomal RNA (rRNA) gene sequencing of the microbe.

  6. Cultural characteristics and fatty acid composition of Corynebacterium acnes.

    PubMed

    Moss, C W; Dowell, V R; Lewis, V J; Schekter, M A

    1967-11-01

    A detailed study of the cultural characteristics and cellular fatty acid composition of 27 isolates of Corynebacterium acnes was performed to establish the properties by which this organism may be identified and characterized. The fatty acids were extracted directly from whole cells and examined as methyl esters by gas-liquid chromatography. Each strain possessed a similar fatty acid profile which was characterized by a large percentage of C15 branched-chain acid. Uniformity in certain biochemical reactions and cultural characteristics was also observed. All strains were catalase-positive, nonmotile, and urease-negative, reduced nitrate, liquefied gelatin, failed to hydrolyze esculin and starch, and gave a positive methyl red test. Glucose, fructose, and glycerol were fermented, but not lactose, salicin, sucrose, maltose, xylose, or arabinose. Production of hydrogen sulfide and indole, fermentation of mannitol, and hemolytic activity were variable characteristics. Two species of the genus Propionibacterium were also tested and found to be similar to C. acnes both in cultural characteristics and fatty acid composition. The results strengthen previous suggestions that C. acnes should be classified in the genus Propionibacterium.

  7. Plasticity of Corynebacterium diphtheriae pathogenicity islands revealed by PCR.

    PubMed

    Soares, S C; Dorella, F A; Pacheco, L G C; Hirata, R; Mattos-Guaraldi, A L; Azevedo, V; Miyoshi, A

    2011-06-28

    Despite the existence of a vaccine against diphtheria, this disease remains endemic and is reemerging in several regions due to many factors, including variations in genes coding for virulence factors. One common feature of virulence factors is their high concentration in pathogenicity islands (PAIs), very unstable regions acquired via horizontal gene transfer, which has lead to the emergence of various bacterial pathogens. The 13 putative PAIs in Corynebacterium diphtheriae NCTC 13129 and the reemergence of this disease point to the great variability in the PAIs of this species, which may reflect on bacterial life style and physiological versatility. We investigated the relationships between the large number of PAIs in C. diphtheriae and the possible implications of their plasticity in virulence. The GenoFrag software was used to design primers to analyze the genome plasticity of two pathogenicity islands of the reference strain (PiCds 3 and 8) in 11 different strains. We found that PiCd 3 was absent in only two strains, showing genes playing putative important roles in virulence and that only one strain harbored PiCd 8, due to its location in a putative "hotspot" for horizontal gene transfer events.

  8. Structure of a DsbF homologue from Corynebacterium diphtheriae.

    PubMed

    Um, Si-Hyeon; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2014-09-01

    Disulfide-bond formation, mediated by the Dsb family of proteins, is important in the correct folding of secreted or extracellular proteins in bacteria. In Gram-negative bacteria, disulfide bonds are introduced into the folding proteins in the periplasm by DsbA. DsbE from Escherichia coli has been implicated in the reduction of disulfide bonds in the maturation of cytochrome c. The Gram-positive bacterium Mycobacterium tuberculosis encodes DsbE and its homologue DsbF, the structures of which have been determined. However, the two mycobacterial proteins are able to oxidatively fold a protein in vitro, unlike DsbE from E. coli. In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site. Structural comparison with M. tuberculosis DsbF would help in understanding the function of the C. diphtheriae protein.

  9. Fibrinogen binds to nontoxigenic and toxigenic Corynebacterium diphtheriae strains.

    PubMed

    Sabbadini, Priscila Soares; Genovez, Marcia Rocha Novais; Silva, Cecília Ferreira da; Adelino, Thelma Lúcia Novaes; Santos, Cintia Silva dos; Pereira, Gabriela Andrade; Nagao, Prescilla Emy; Dias, Alexandre Alves de Souza de Oliveira; Mattos-Guaraldi, Ana Luiza; Hirata Júnior, Raphael

    2010-08-01

    The production of fibrinous exudates may play an important role in determining the outcome of bacterial infection. Although pseudomembrane formation is a characteristic feature of diphtheria, little is known about the fibrinogen (Fbn)-binding properties of Corynebacterium diphtheriae strains and the influence of the gene that codes for diphtheria toxin (tox gene) in this process. In this study we demonstrated the ability of C. diphtheriae strains to bind to Fbn and to convert Fbn to fibrin. Bacterial interaction with rabbit plasma was evaluated by both slide and tube tests. Interaction of microorganisms with human Fbn was evaluated by both enzyme linked immunosorbent assay (ELISA) and fluorescein isothiocyanate-conjugated (FITC) Fbn binding assays. Nontoxigenic and toxigenic strains formed bacterial aggregates in the presence of plasma in the slide tests. The ability to convert Fbn to a loose web of fibrin in the plasma solution in the tube tests appeared to be a common characteristic of the species, including strains that do not carry the tox gene. Fbn binding to C. diphtheriae strains occurred at varying intensities, as demonstrated by the FITC-Fbn and ELISA binding assays. Our data suggest that the capacity to bind to Fbn and to convert Fbn to fibrin may play a role in pseudomembrane formation and act as virulence determinants of both nontoxigenic and toxigenic strains.

  10. Tellurite resistance: a putative pitfall in Corynebacterium diphtheriae diagnosis?

    PubMed

    dos Santos, Louisy Sanches; Antunes, Camila Azevedo; de Oliveira, Daniel Martins; Sant'Anna, Lincoln de Oliveira; Pereira, José Augusto Adler; Hirata Júnior, Raphael; Burkovski, Andreas; Mattos-Guaraldi, Ana Luíza

    2015-11-01

    Corynebacterium diphtheriae strains continue to circulate worldwide causing diphtheria and invasive diseases, such as endocarditis, osteomyelitis, pneumonia and catheter-related infections. Presumptive C. diphtheriae infections diagnosis in a clinical microbiology laboratory requires a primary isolation consisting of a bacterial culture on blood agar and agar containing tellurite (TeO3(2-)). In this study, nine genome sequenced and four unsequenced strains of C. diphtheriae from different sources, including three samples from a recent outbreak in Brazil, were characterized with respect to their growth properties on tellurite-containing agar. Levels of tellurite-resistance (Te(R)) were evaluated by determining the minimum inhibitory concentrations of potassium tellurite (K2TeO3) and by a viability reduction test in solid culture medium with K2TeO3. Significant differences in Te(R) levels of C. diphtheriae strains were observed independent of origin, biovar or presence of the tox gene. Data indicated that the standard initial screening with TeO3(2-)-selective medium for diphtheria bacilli identification may lead to false-negative results in C. diphtheriae diagnosis laboratories.

  11. Structure of a DsbF homologue from Corynebacterium diphtheriae

    PubMed Central

    Um, Si-Hyeon; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2014-01-01

    Disulfide-bond formation, mediated by the Dsb family of proteins, is important in the correct folding of secreted or extracellular proteins in bacteria. In Gram-negative bacteria, disulfide bonds are introduced into the folding proteins in the periplasm by DsbA. DsbE from Escherichia coli has been implicated in the reduction of disulfide bonds in the maturation of cytochrome c. The Gram-positive bacterium Mycobacterium tuberculosis encodes DsbE and its homologue DsbF, the structures of which have been determined. However, the two mycobacterial proteins are able to oxidatively fold a protein in vitro, unlike DsbE from E. coli. In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site. Structural comparison with M. tuberculosis DsbF would help in understanding the function of the C. diphtheriae protein. PMID:25195886

  12. Production of a bacteriocin, ulceracin 378, by Corynebacterium ulcerans.

    PubMed Central

    Abrehem, K; Zamiri, I

    1983-01-01

    The production of the bacteriocin ulceracin 378 by Corynebacterium ulcerans 378 was demonstrated during the growth of the organism on solid medium. Ulceracin 378 was not found in broth cultures and could not be extracted from the organisms by various solvents and salt solutions. Ulceracin 378 was not inducible by UV irradiation or mitomycin C treatments. Ulceracin 378 was active against all of the C. ulcerans strains tested and some related species, but it was not autoinhibitory. The active material was not phage related and was extracted from cultures grown on semisolid media composed of proteose peptone, Tween 80, Casamino Acids, glycerol, and sodium chloride. The yield was significantly reduced by either increasing the agar concentration or omitting Tween 80. Ulceracin 378 was resistant to DNase, RNase, phospholipases C and D, and alkaline phosphatase but was susceptible to proteolytic enzymes. This suggests that the active principle of ulceracin is protein in nature. Ulceracin 378 was partially purified by ammonium sulfate fractionation, dialysis, and chromatography on DEAE-cellulose. PMID:6688939

  13. Biofilm production by multiresistant Corynebacterium striatum associated with nosocomial outbreak

    PubMed Central

    de Souza, Cassius; Faria, Yuri Vieira; Sant’Anna, Lincoln de Oliveira; Viana, Vanilda Gonçalves; Seabra, Sérgio Henrique; de Souza, Mônica Cristina; Vieira, Verônica Viana; Hirata, Raphael; Moreira, Lílian de Oliveira; de Mattos-Guaraldi, Ana Luíza

    2015-01-01

    Corynebacterium striatum is a potentially pathogenic microorganism that causes nosocomial outbreaks. However, little is known about its virulence factors that may contribute to healthcare-associated infections (HAIs). We investigated the biofilm production on abiotic surfaces of multidrug-resistant (MDR) and multidrug-susceptible (MDS) strains of C. striatum of pulsed-field gel electrophoresis types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak in Rio de Janeiro, Brazil. The results showed that C. striatum was able to adhere to hydrophilic and hydrophobic abiotic surfaces. The C. striatum 1987/I-MDR strain, predominantly isolated from patients undergoing endotracheal intubation procedures, showed the greatest ability to adhere to all surfaces. C. striatum bound fibrinogen to its surface, which contributed to biofilm formation. Scanning electron microscopy showed the production of mature biofilms on polyurethane catheters by all pulsotypes. In conclusion, biofilm production may contribute to the establishment of HAIs caused by C. striatum. PMID:25946249

  14. Sinus Microbiome Diversity Depletion and Corynebacterium tuberculostearicum Enrichment Mediates Rhinosinusitis

    PubMed Central

    Song, Yuanlin; Roediger, Frederick C.; Pletcher, Steven D.; Goldberg, Andrew N.; Lynch, Susan V.

    2016-01-01

    Persistent mucosal inflammation and microbial infection are characteristics of chronic rhinosinusitis (CRS). Mucosal microbiota dysbiosis is found in other chronic inflammatory diseases; however, the relationship between sinus microbiota composition and CRS is unknown. Using comparative microbiome profiling of a cohort of CRS patients and healthy subjects, we demonstrate that the sinus microbiota of CRS patients exhibits significantly reduced bacterial diversity compared with that of healthy controls. In our cohort of CRS patients, multiple, phylogenetically distinct lactic acid bacteria were depleted concomitant with an increase in the relative abundance of a single species, Corynebacterium tuberculostearicum. We recapitulated the conditions observed in our human cohort in a murine model and confirmed the pathogenic potential of C. tuberculostearicum and the critical necessity for a replete mucosal microbiota to protect against this species. Moreover, Lactobacillus sakei, which was identified from our comparative microbiome analyses as a potentially protective species, defended against C. tuberculostearicum sinus infection, even in the context of a depleted sinus bacterial community. These studies demonstrate that sinus mucosal health is highly dependent on the composition of the resident microbiota as well as identify both a new sino-pathogen and a strong bacterial candidate for therapeutic intervention. PMID:22972842

  15. Recombinant allergens

    PubMed Central

    Jutel, Marek; Solarewicz-Madejek, Katarzyna; Smolinska, Sylwia

    2012-01-01

    Allergen specific immunotherapy (SIT) is the only known causative treatment of allergic diseases. Recombinant allergen-based vaccination strategies arose from a strong need to both to improve safety and enhance efficacy of SIT. In addition, new vaccines can be effective in allergies including food allergy or atopic dermatitis, which poorly respond to the current treatment with allergen extracts. A number of successful clinical studies with both wild-type and hypoallergenic derivatives of recombinant allergens vaccines have been reported for the last decade. They showed high efficacy and safety profile as well as very strong modulation of T and B cell responses to specific allergens. PMID:23095874

  16. Corynebacterium pelargi sp. nov., isolated from the trachea of white stork nestlings.

    PubMed

    Kämpfer, Peter; Jerzak, Leszek; Bochenski, Marcin; Kasprzak, Mariusz; Wilharm, Gottfried; Golke, Jan; Busse, Hans-Jürgen; Glaeser, Stefanie P

    2015-05-01

    A Gram-stain-positive, pleomorphic, oxidase-negative, non-motile isolate from the trachea of a white stork from Poland, designated strain 136/3(T), was subjected to a comprehensive taxonomic investigation. A comparative analysis of the 16S rRNA gene sequence showed highest similarities to Corynebacterium mustelae , Corynebacterium pseudotuberculosis , Corynebacterium vitaeruminis and Corynebacterium ulcerans (96.0-96.3%). The quinone system consisted of major amounts of MK-8(H2), minor amounts of MK-9(H2) and traces of MK-8 and MK-9. The polar lipid profile of strain 136/3(T) contained phosphatidylinositol and phosphatidylinositol-mannoside as major lipids and phosphatidylglycerol and an acidic glycolipid in moderate amounts. In addition small amounts of diphosphatidylglycerol, a phospholipid, an aminolipid and two lipids of unknown group affiliation were found. The polyamine pattern was composed of the major components spermidine and spermine. Putrescine, 1,3-diaminopropane, cadaverine, sym-homospermidine and tyramine were found in minor or trace amounts. The diamino acid of the peptidoglycan was meso-diaminopimelic acid. In the fatty acid profile straight-chain, saturated and mono-unsaturated fatty acids predominated (C(18 : 1)ω9c, C(16 : 1)ω7c, C16 : 0, C(18  : 0)). Corynemycolic acids were detected. Physiological traits as well as unique traits of the polar lipid profile and the fatty acid pattern distinguished strain 136/3(T) from the most closely related species. All these results indicate that strain 136/3(T) represents a novel species of the genus Corynebacterium for which we propose the name Corynebacterium pelargi sp. nov. The type strain is 136/3(T) ( =CIP 110778(T) =CCM 8517(T) =LMG 28174(T)).

  17. Recombinant gonadotropins.

    PubMed

    Lathi, R B; Milki, A A

    2001-10-01

    Recombinant DNA technology makes it possible to produce large amounts of human gene products for pharmacologic applications, supplanting the need for human tissues. The genes for the alpha and beta subunits of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) have been characterized and cloned. Recombinant FSH (rFSH) has been shown to be safe and effective in the treatment of fertility disorders. In comparison with the urinary gonadotropin products, human menopausal gonadotropins (HMG), and urinary follitropins (uFSH), rFSH is more potent and better tolerated by patients. Recombinant HCG appears to be as efficacious as urinary HCG with the benefit of improved local tolerance. Recombinant LH (rLH) is likely to be recommended as a supplement to rFSH for ovulation induction in hypogonadotropic women. It may also benefit in vitro fertilization patients undergoing controlled ovarian hyperstimulation with rFSH combined with pituitary suppression, with a gonadotropin-releasing hormone agonist or antagonist.

  18. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  19. Molecular characterization of Corynebacterium pseudotuberculosis isolates using ERIC-PCR.

    PubMed

    Guimarães, Alessandro de Sá; Dorneles, Elaine Maria Seles; Andrade, Giovanna Ivo; Lage, Andrey Pereira; Miyoshi, Anderson; Azevedo, Vasco; Gouveia, Aurora Maria Guimarães; Heinemann, Marcos Bryan

    2011-12-15

    Caseous lymphadenitis is an infectious sheep and goats disease caused by Corynebacterium pseudotuberculosis and characterized by abscesses in superficial and visceral lymph nodes. C. pseudotuberculosis strains isolated from these hosts have been shown to be very difficult to type by the existing methods. The aim of this study is evaluating the potential of the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis strains isolated in sheep. One hundred and twenty seven isolates of C. pseudotuberculosis were isolated from lesions suspected to have had caseous lymphadenitis collected from sheep at the slaughterhouse. Animals were from 24 flocks in 13 municipalities of the Minas Gerais State, Brazil. Species identification of the isolates was performed by routine biochemical tests and mPCR. Fingerprint was performed by RAPD using ERIC-1R, ERIC-2 and ERIC-1R+ERIC-2 primers. Seventeen different genotypes were generated by ERIC 1-PCR, 21 genotypes by ERIC 2-PCR and 21 genotypes by ERIC 1+2-PCR. Hunter-Gaston Discrimination Index (HGDI) found for ERIC 1, ERIC 2, ERIC 1+2 PCR were 0.69, 0.87, and 0.84, respectively. For most herds evaluated observed at most three different genotypes among isolates from animals of these property, in all ERIC-PCR assays. However a few flocks observed between four and nine genotypes per flock. The W Kendall value found for correlation among the three techniques of ERIC-PCR was 0.91 (P<5.0 x 10(-6)). The results show that ERIC-PCR has good discriminatory power and advantages over other DNA-based typing methods, making it a useful tool to discriminate C. pseudotuberculosis isolates.

  20. Rapid detection of Corynebacterium pseudotuberculosis in clinical samples from sheep.

    PubMed

    Kumar, Jyoti; Tripathi, Bhupendra Nath; Kumar, Rajiv; Sonawane, Ganesh Gangaram; Dixit, Shivendra Kumar

    2013-08-01

    Corynebacterium pseudotuberculosis, a Gram-positive bacterium is the causative agent of caseous lymphadenitis (CLA), a chronic disease of sheep, goats and other warm blooded animals. In the present study, a total of 1,080 sheep reared under semi-intensive system on organized farms situated in the semi arid tropical region of Rajasthan, India, was clinically examined. Pus samples from superficial lymph nodes of 25 (2.31%) adult sheep showing clinical lesions similar to CLA were collected for laboratory analyses. On the basis of morphological, cultural and biochemical characteristics 12 (48%) bacterial isolates from pus identified it as C. pseudotuberculosis. A polymerase chain reaction (PCR) assay targeting Putative oligopeptide/dipeptide ABC transporter, nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase coenzyme F420-dependent and proline iminopeptidase (PIP) genes of C. pseudotuberculosis was developed that showed 14 pus samples as positive. All C. pseudotuberculosis isolates were also found positive for these genes in the PCR. The specificity of the PCR products was confirmed by sequencing of the amplified products that showed 98-100% homology with published sequences available in the NCBI database. The present study shows the incidence of CLA as 2.31%, 1.1% and 1.29% based on clinical, bacterial culture and direct pus PCR assay, respectively. The PCR assay was rapid, specific and as significant as bacterial culture in detecting bacteria directly in the clinical pus samples. The PCR assay developed in the study can be applied for the diagnosis and control of CLA. Furthermore, the assay can also be applied to detect C. pseudotuberculosis in various clinical samples.

  1. Corynebacterium diphtheriae: genome diversity, population structure and genotyping perspectives.

    PubMed

    Mokrousov, Igor

    2009-01-01

    The epidemic re-emergence of diphtheria in Russia and the Newly Independent States (NIS) of the former Soviet Union in the 1990s demonstrated the continued threat of this thought to be rare disease. The bacteriophage encoded toxin is a main virulence factor of Corynebacterium diphtheriae, however, an analysis of the first complete genome sequence of C. diphtheriae revealed a recent acquisition of other pathogenicity factors including iron-uptake systems, adhesins and fimbrial proteins as indeed this extracellular pathogen has more possibilities for lateral gene transfer than, e.g., its close relative, mainly intracellular Mycobacterium tuberculosis. C. diphtheriae appears to have a phylogeographical structure mainly represented by area-specific variants whose circulation is under strong influence of human host factors, including health control measures, first of all, vaccination, and social economic conditions. This framework core population structure may be challenged by importation of the endemic and eventually toxigenic strains from new areas thus leading to localized or large epidemics caused directly by imported strains or by bacteriophage-lysogenized indigenous strains converted into toxin production. A feature of C. diphtheriae co-existence with humans is its periodicity: following large epidemic in the 1990s, the present period is marked by increasing heterogeneity of the circulating populations whereas re-emergence of new toxigenic variants along with persistent circulation of invasive non-toxigenic strains appear alarming. To identify and rapidly monitor subtle changes in the genome structure at an infraclonal level during and between epidemics, portable and discriminatory typing methods of C. diphtheriae are still needed. In this view, CRISPRs and minisatellites are promising genomic markers for development of high-resolution typing schemes and databasing of C. diphtheriae.

  2. Purification and structural characterization of siderophore (corynebactin) from Corynebacterium diphtheriae.

    PubMed

    Zajdowicz, Sheryl; Haller, Jon C; Krafft, Amy E; Hunsucker, Steve W; Mant, Colin T; Duncan, Mark W; Hodges, Robert S; Jones, David N M; Holmes, Randall K

    2012-01-01

    During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin.

  3. Structural analysis of FAD synthetase from Corynebacterium ammoniagenes

    PubMed Central

    Frago, Susana; Martínez-Júlvez, Marta; Serrano, Ana; Medina, Milagros

    2008-01-01

    Background The prokaryotic FAD synthetase family – a group of bifunctional enzymes that catalyse riboflavin phosphorylation and FMN adenylylation within a single polypeptide chain- was analysed in terms of sequence and structure. Results Sequences of nearly 800 prokaryotic species were aligned. Those related with bifunctional FAD synthetase activities showed conservation of several consensus regions and highly conserved residues. A 3D model for the FAD synthetase from Corynebacterium ammoniagenes (CaFADS) was generated. This model confirms that the N-terminal and C-terminal domains are related to nucleotydyltransferases and riboflavin kinases, respectively. Models for the interaction of CaFADS with its substrates were also produced, allowing location of all the protein substrates in their putative binding pockets. These include two independent flavin binding sites for each CaFADS activity. Conclusion For the first time, the putative presence of a flavin binding site for the adenylylation activity, independent from that related with the phosphorylation activity, is shown. Additionally, these models suggest the functional relevance of some residues putatively involved in the catalytic processes. Their relevant roles were analysed by site-directed mutagenesis. A role was confirmed for H28, H31, S164 and T165 in the stabilisation of the P groups and the adenine moiety of ATP and, the P of FMN for the adenylylation. Similarly, T208, N210 and E268 appear critical for accommodation of the P groups of ATP and the ribityl end of RF in the active site for the phosphorylation process. Finally, the C-terminal domain was shown to catalyse the phosphorylation process on its own, but no reaction at all was observed with the individually expressed N-terminal domain. PMID:18811972

  4. Protection in alpacas against Corynebacterium pseudotuberculosis using different bacterial components.

    PubMed

    Braga, Walter U

    2007-01-31

    Corynebacterium pseudotuberculosis is a Gram positive bacterium that produces caseous lymphadenitis in sheep and goats, and a granulomatous lymphadenitis in llamas and alpacas. To evaluate the immune potential of different doses of cell wall and toxin components of C. pseudotuberculosis from alpaca origin, 12 adult alpacas were allotted at random to four groups, and SC inoculated in the left flank with vaccines composed of low and high doses of bacterial crude antigens, cell wall: 250 and 500 microg/ml and toxin: 133 and 265 microg/ml, respectively. The vaccines were supplemented with 20 microg/ml of muramyl dipeptide as adjuvant. Three alpacas were sham inoculated with adjuvant as a control. After 3 weeks, immunized and naive alpacas were challenged intradermally in the right flank with 1 x 10(6) colony forming units (CFU) of C. pseudotuberculosis. The alpacas were sacrificed at days 28, 58 and 112 after inoculation, and the degree of protection induced by vaccines was demonstrated by the absence of abscesses and/or bacteria. The alpacas vaccinated with high dose of toxin, did not show abscesses. In contrast, the alpacas vaccinated with a low dose of toxin showed abscesses at the inoculation site, regional, and renal lymph nodes. The cell wall vaccinated alpacas showed a lesser degree of protection than the other groups with superficial and internal abscesses. The control alpacas had persistent fever and abscesses at the inoculation site, regional, and internal lymph nodes. In addition, a robust and early humoral response was observed in all vaccinated alpacas after challenge, lasting at least 3 months. The results suggest that the toxin of C. pseudotuberculosis is a very important antigen, inducing a dose dependant protective immunity against this bacterium in alpacas.

  5. Purification and Structural Characterization of Siderophore (Corynebactin) from Corynebacterium diphtheriae

    PubMed Central

    Zajdowicz, Sheryl; Haller, Jon C.; Krafft, Amy E.; Hunsucker, Steve W.; Mant, Colin T.; Duncan, Mark W.; Hodges, Robert S.; Jones, David N. M.; Holmes, Randall K.

    2012-01-01

    During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin. PMID:22514641

  6. Molecular epidemiology of Corynebacterium pseudotuberculosis isolated from horses in California.

    PubMed

    Haas, Dionei J; Dorneles, Elaine M S; Spier, Sharon J; Carroll, Scott P; Edman, Judy; Azevedo, Vasco A; Heinemann, Marcos B; Lage, Andrey P

    2017-04-01

    Corynebacterium pseudotuberculosis biovar Equi is an important pathogen of horses. It is increasing in frequency in the United States, and is responsible for various clinical forms of infection, including external abscesses, internal abscesses of the abdominal or thoracic cavities, and ulcerative lymphangitis. The host/pathogen factors dictating the form or severity of infection are currently unknown. Our recent investigations have shown that genotyping C. pseudotuberculosis isolates using enterobacterial repetitive intergenic consensus (ERIC)-PCR is useful for understanding the evolutionary genetics of the species as well for molecular epidemiology studies. The aims of the present study were to assess (i) the genetic diversity of C. pseudotuberculosis strains isolated from horses in California, United States and (ii) the epidemiologic relationships among isolates. One hundred and seven C. pseudotuberculosis biovar Equi isolates from ninety-five horses, and two C. pseudotuberculosis biovar Ovis strains, C. pseudotuberculosis ATCC 19410(T) type strain and C. pseudotuberculosis 1002 vaccine strain, were fingerprinted using the ERIC 1+2-PCR. C. pseudotuberculosis isolated from horses showed a high genetic diversity, clustering in twenty-seven genotypes with a diversity index of 0.91. Minimal spanning tree showed four major clonal complexes with a pattern of temporal clustering. Strains isolated from the same horse showed identical ERIC 1+2-PCR genotype, with the exception of two strains isolated from the same animal that showed distinct genotypes, suggesting a co-infection. We found no strong genetic signals related to clinical form (including internal versus external infections). However, temporal clustering of genotypes was observed.

  7. Genome shuffling improves thermotolerance and glutamic acid production of Corynebacteria glutamicum.

    PubMed

    Zheng, Pu; Liu, Miao; Liu, Xiao-de; Du, Qiao-Yan; Ni, Ye; Sun, Zhi-Hao

    2012-03-01

    Genome shuffling was used to improve the thermotolerance of L: -glutamic acid-producing strain Corynebacteria glutamicum. Five strains with subtle improvements in high temperature tolerance and productivity were selected by ultraviolet irradiation and diethyl sulfate mutagenesis. An improved strain (F343) was obtained by three rounds of genome shuffling of the five strains as mentioned above. The cell density of F343 was four times higher than that of ancestor strains after 24 h of cultivation at 44°C, and importantly, the yield of L: -glutamic acid was increased by 1.8-times comparing with that of the ancestor strain at 38°C in a 5-L fermentor. With glucose supplement and two-stage pH control, the L: -glutamate acid concentration of F343 reached 119 g/L after fermentation for 30 h. The genetic diversity between F343 and its ancestors was also evaluated by amplified fragment length polymorphism analysis. Results suggest that the phenotypes for both thermotolerance and L: -glutamic acid production in F343 were evolved.

  8. Genome Sequence of Corynebacterium pseudotuberculosis Strain PA02 Isolated from an Ovine Host in the Amazon

    PubMed Central

    Muge, Gabriel R. S.; Veras, Adonney A. O.; de Sá, Pablo H. C. G.; Cavalcante, Ana Lídia Queiroz; Alves, Jorianne Thyeska Castro; Morais, Ezequiel; Silva, André G. M.; Azevedo, Vasco; Folador, Adriana Ribeiro Carneiro; Silva, Artur

    2016-01-01

    In this work, we report the complete genome sequence of Corynebacterium pseudotuberculosis strain PA02 isolated from an ovine host. The genome contains 2,328,435 bp, a 52.2% G+C content, 2,035 coding sequences, 12 rRNA operons, 45 tRNAs, and 14 predicted pseudogenes. PMID:27516524

  9. An unusual etiological agent of implantable cardioverter device endocarditis: Corynebacterium mucifaciens

    PubMed Central

    Kaya, Adnan; Tekkesin, Ahmet Ilker; Kalenderoglu, Koray; Alper, Ahmet Taha

    2016-01-01

    Cardiac pacing devices and implantable cardioverter defibrillator (ICD) are becoming the mainstay of therapy in cardiology and infective endocarditis (IE) and pocket infection; however, these devices require careful monitoring. Here, we describe a case of a 68-year-old female with an ICD presenting with a previously unknown etiological agent of IE, Corynebacterium mucifaciens. PMID:27133333

  10. Nontoxigenic highly pathogenic clone of Corynebacterium diphtheriae, Poland, 2004-2012.

    PubMed

    Zasada, Aleksandra A

    2013-11-01

    Twenty-five cases of nontoxigenic Corynebacterium diphtheriae infection were recorded in Poland during 2004-2012, of which 18 were invasive. Alcoholism, homelessness, hepatic cirrhosis, and dental caries were predisposing factors for infection. However, for 17% of cases, no concomitant diseases or predisposing factors were found.

  11. Sequence Analysis of Toxin Gene-Bearing Corynebacterium diphtheriae Strains, Australia.

    PubMed

    Doyle, Christine J; Mazins, Adam; Graham, Rikki M A; Fang, Ning-Xia; Smith, Helen V; Jennison, Amy V

    2017-01-01

    By conducting a molecular characterization of Corynebacterium diphtheriae strains in Australia, we identified novel sequences, nonfunctional toxin genes, and 5 recent cases of toxigenic cutaneous diphtheria. These findings highlight the importance of extrapharyngeal infections for toxin gene-bearing (functional or not) and non-toxin gene-bearing C. diphtheriae strains. Continued surveillance is recommended.

  12. Characterization of Corynebacterium diphtheriae isolates from infected skin lesions in the Northern Territory of Australia.

    PubMed

    Gordon, Claire L; Fagan, Peter; Hennessy, Jann; Baird, Robert

    2011-11-01

    Corynebacterium diphtheriae is commonly isolated from cutaneous skin lesions in the Northern Territory of Australia. We prospectively assessed 32 recent isolates from infected skin lesions, in addition to reviewing 192 isolates collected over 5 years for toxin status. No isolates carried the toxin gene. Toxigenic C. diphtheriae is now a rare occurrence in the Northern Territory.

  13. Draft Genome Sequence of Toxigenic Corynebacterium ulcerans Strain 03-8664 Isolated from a Human Throat

    PubMed Central

    Viana, Marcus V. C.; Benevides, Leandro J.; Mariano, Diego C. B.; Veras, Adonney A. O.; Sá, Pablo H. C.; Rocha, Flávia S.; Vilas Boas, Priscilla C. B.; Soares, Siomar C.; Barbosa, Maria S.; Guiso, Nicole; Badell, Edgar; Azevedo, Vasco; Ramos, Rommel T. J.

    2016-01-01

    Corynebacterium ulcerans is an emergent pathogen infecting wild and domesticated animals worldwide that may serve as reservoirs for zoonotic infections. In this study, we present the draft genome of C. ulcerans strain 03-8664. The draft genome has 2,428,683 bp, 2,262 coding sequences, and 12 rRNA genes. PMID:27469956

  14. Characterization and comparison of invasive Corynebacterium diphtheriae isolates from France and Poland.

    PubMed

    Farfour, E; Badell, E; Zasada, A; Hotzel, H; Tomaso, H; Guillot, S; Guiso, N

    2012-01-01

    Corynebacterium diphtheriae, the agent of diphtheria, is rarely responsible for bacteremia. However, high numbers of bacteremia have been reported in countries with extensive immunization coverage. Here, we used molecular and phenotypic tools to characterize and compare 42 invasive isolates collected in France (including New Caledonia) and Poland over a 23-year period.

  15. First Report on the Draft Genome Sequences of Corynebacterium diphtheriae Isolates from India

    PubMed Central

    Anandan, Shalini; Rajamani Sekar, Suresh Kumar; Gopi, Radha; Devanga Ragupathi, Naveen Kumar; Ramesh, Srilekha; Verghese, Valsan Philip; Korulla, Sophy; Mathai, Sarah; Sangal, Lucky; Joshi, Sudhir

    2016-01-01

    We report here the draft genome sequences of five Corynebacterium diphtheriae isolates of Indian origin. The C. diphtheriae isolates TH1141, TH510, TH1526, TH1337, and TH2031 belong to sequence type ST-50, ST-295, ST-377, ST-405, and ST-405, with an average genome size of 2.5 Mbp. PMID:27881543

  16. Sequence Analysis of Toxin Gene–Bearing Corynebacterium diphtheriae Strains, Australia

    PubMed Central

    Mazins, Adam; Graham, Rikki M.A.; Fang, Ning-Xia; Smith, Helen V.; Jennison, Amy V.

    2017-01-01

    By conducting a molecular characterization of Corynebacterium diphtheriae strains in Australia, we identified novel sequences, nonfunctional toxin genes, and 5 recent cases of toxigenic cutaneous diphtheria. These findings highlight the importance of extrapharyngeal infections for toxin gene–bearing (functional or not) and non–toxin gene–bearing C. diphtheriae strains. Continued surveillance is recommended. PMID:27983494

  17. Genomic analysis of a nontoxigenic, invasive Corynebacterium diphtheriae strain from Brazil.

    PubMed

    Encinas, Fernando; Marin, Michel A; Ramos, Juliana N; Vieira, Verônica V; Mattos-Guaraldi, Ana Luiza; Vicente, Ana Carolina P

    2015-09-01

    We report the complete genome sequence and analysis of an invasive Corynebacterium diphtheriae strain that caused endocarditis in Rio de Janeiro, Brazil. It was selected for sequencing on the basis of the current relevance of nontoxigenic strains for public health. The genomic information was explored in the context of diversity, plasticity and genetic relatedness with other contemporary strains.

  18. Whole-Genome Sequences of Four Corynebacterium CDC Group F-1 Strains Isolated from Urine

    PubMed Central

    Bernier, Anne-Marie; Peters, Geoffrey A.

    2017-01-01

    ABSTRACT Three draft and one complete genome sequence from strains isolated from urine and consistent with Corynebacterium CDC group F-1 were assembled and studied. Genome sizes ranged between 2.3 and 2.44 Mb, with G+C content between 60.4% and 60.7%. PMID:28153894

  19. Draft Genome Sequence of Corynebacterium pseudodiphtheriticum Strain 090104 “Sokolov”

    PubMed Central

    Melnikov, Vyacheslav G.

    2013-01-01

    This report describes the first draft genome sequence of a Corynebacterium pseudodiphtheriticum strain. The information on the genome organization and putative gene products will assist in better understanding of the molecular mechanisms involved in the beneficial probiotic effects of this bacterium. PMID:24201200

  20. Nontoxigenic tox-bearing Corynebacterium ulcerans Infection among Game Animals, Germany

    PubMed Central

    Kutzer, Peter; Peters, Martin; Sing, Andreas; Contzen, Matthias; Rau, Jörg

    2014-01-01

    Corynebacterium ulcerans may cause diphtheria in humans and caseous lymphadenitis in animals. We isolated nontoxigenic tox-bearing C. ulcerans from 13 game animals in Germany. Our results indicate a role for game animals as reservoirs for zoonotic C. ulcerans. PMID:24572455

  1. Complete Genome Sequence of Corynebacterium minutissimum, an Opportunistic Pathogen and the Causative Agent of Erythrasma.

    PubMed

    Penton, Patricia K; Tyagi, Eishita; Humrighouse, Ben W; McQuiston, John R

    2015-03-19

    Corynebacterium minutissimum was first isolated in 1961 from infection sites of patients presenting with erythrasma, a common cutaneous infection characterized by a rash. Since its discovery, C. minutissimum has been identified as an opportunistic pathogen in immunosuppressed cancer and HIV patients. Here, we report the whole-genome sequence of C. minutissimum.

  2. Genome Sequence of Corynebacterium pseudotuberculosis Strain XH02 Isolated from a Boer Goat in Xuanhan, China

    PubMed Central

    Li, Hexian; Zhang, Mengsi; Wang, Zhiying; Zhou, Rongqiong; Hu, Shijun; Li, Xiaoxia; Song, Xinyue; Zhu, Zheng

    2016-01-01

    We report here the genome sequence of Corynebacterium pseudotuberculosis strain XH02, isolated from a Boer goat in China. The genome consists of 2,357,671 bp, with a 52.18% G+C content, 2,263 coding sequences, 21 rRNAs, 49 tRNAs, and 44 predicted pseudogenes. PMID:27881549

  3. Successful treatment of Corynebacterium urealyticum encrusting cystitis with systemic and intravesical antimicrobial therapy

    PubMed Central

    Raab, Oriana; Béraud, Romain; Tefft, Karen M.; Muckle, C. Anne

    2015-01-01

    A 6-year-old Saint Bernard dog was diagnosed with encrusting cystitis caused by Corynebacterium urealyticum. The infection persisted despite the prolonged use of antimicrobials and surgical debridement of the urinary bladder. Resolution occurred following intravenous vancomycin, urine acidification, and intravesical gentamicin. The challenges involved in the treatment of encrusting cystitis are described. PMID:25969578

  4. Draft Genome Sequence of Corynebacterium ulcerans Strain 04-3911, Isolated from Humans

    PubMed Central

    Viana, Marcus V. C.; Benevides, Leandro J.; Mariano, Diego C. B.; Veras, Adooney A. O.; Sá, Pablo H. C.; Rocha, Flávia S.; Vilas Boas, Priscilla C. B.; Soares, Siomar C.; Barbosa, Maria S.; Guiso, Nicole; Badell, Edgar; Carneiro, Adriana R.; Azevedo, Vasco; Ramos, Rommel T. J.

    2016-01-01

    Corynebacterium ulcerans is a pathogenic bacterium infecting wild and domesticated animals; some infection cases in humans have increased throughout the world. The current study describes the draft genome of strain 04-3911, isolated from humans. The draft genome has 2,492,680 bp, 2,143 coding sequences, 12 rRNA genes, and 50 tRNA genes. PMID:27034486

  5. Is Corynebacterium pseudotuberculosis infection (pigeon fever) in horses an emerging disease in western Canada?

    PubMed

    Corbeil, Louise E; Morrissey, Jennifer F; Léguillette, Renaud

    2016-10-01

    This report describes 5 horses in the southern Alberta region with typical and atypical external abscessation due to Corynebacterium pseudotuberculosis (pigeon fever). "Pigeon fever" has recently been diagnosed in new geographic regions in North America and should be kept as a differential diagnosis by practitioners when an external or internal abscess is identified in a horse.

  6. Is Corynebacterium pseudotuberculosis infection (pigeon fever) in horses an emerging disease in western Canada?

    PubMed Central

    Corbeil, Louise E.; Morrissey, Jennifer K.; Léguillette, Renaud

    2016-01-01

    This report describes 5 horses in the southern Alberta region with typical and atypical external abscessation due to Corynebacterium pseudotuberculosis (pigeon fever). “Pigeon fever” has recently been diagnosed in new geographic regions in North America and should be kept as a differential diagnosis by practitioners when an external or internal abscess is identified in a horse. PMID:27708444

  7. Indirect Fluorescent-Antibody Method for the Identification of Corynebacterium vaginale

    PubMed Central

    Vice, John L.; Smaron, Mary F.

    1973-01-01

    The indirect fluorescent-antibody technique was employed in an attempt to develop a rapid method of identification of Corynebacterium vaginale. Six reference strains and ten clinical isolates selected on the basis of morphology and conventional biochemical tests were compared. Antisera were prepared in rabbits against the six reference strains. The most satisfactory antiserum was that prepared using strain 14018 grown diphasically (14018 Di) as the antigen. Certain of the antisera did exhibit a cross-reacting titer when reacted against Corynebacterium diptheriae, Corynebacterium xerosis, or Lactobacillus acidophilus. However, antisera adsorbed with these bacteria did not exhibit a significant decrease in titer when reacted against the homologous strain. Various other species of Corynebacterium as well as species of Nocardia, Actinomyces, Hemophilus, and Streptococcus did not fluoresce with the antisera. A specific antiserum was prepared by adsorbing anti-14018 Di with L. acidophilus. The adsorption removed the cross-reacting antibody but did not affect the staining reaction with C. vaginale strains. All reference strains and clinical isolates characterized as C. vaginale gave a definite positive reaction with the adsorbed anti-14018 Di. The specificity of the reactions was assessed by adsorbing the antiserum with the homologous strain. The data suggest that the indirect staining method will be of value in the rapid presumptive identification of C. vaginale. Images PMID:4197767

  8. Complete Genome Sequence of Corynebacterium pseudotuberculosis Strain E19, Isolated from a Horse in Chile.

    PubMed

    Cavalcante, Ana Lídia Q; Dias, Larissa M; Alves, Jorianne T C; Veras, Adonney A O; Guimarães, Luis C; Rocha, Flávia S; Gala-García, Alfonso; Retamal, Patricio; Ramos, Rommel T J; Azevedo, Vasco; Silva, Artur; Carneiro, Adriana R

    2015-11-25

    Corynebacterium pseudotuberculosis is related to several diseases infecting horses and small ruminants, causing economic losses to agribusiness. Here, we present the genome sequence of C. pseudotuberculosis strain E19. The genome includes one circular chromosome 2,367,956 bp (52.1% G+C content), with 2,112 genes predicted, 12 rRNAs, and 48 tRNAs.

  9. Corynebacterium pseudotuberculosis Pneumonia in a Veterinary Student Infected During Laboratory Work

    PubMed Central

    Heggelund, Lars; Gaustad, Peter; Håvelsrud, Othilde Elise; Blom, Jochen; Borgen, Lars; Sundset, Arve; Sørum, Henning; Frøland, Stig Sophus

    2015-01-01

    We present a case of Corynebacterium pseudotuberculosis pneumonia in a veterinary student, with molecular genetic evidence of acquisition during laboratory work, an observation relevant for laboratory personnel working with C pseudotuberculosis isolates. The patient was clinically cured with 14 months trimethoprim/sulfamethoxazole and rifampicin combination treatment. PMID:26380345

  10. Urosepsis caused by Globicatella sanguinis and Corynebacterium riegelii in an adult: case report and literature review.

    PubMed

    Matsunami, Masatoshi; Matusnami, Masatoshi; Otsuka, Yoshihito; Ohkusu, Kiyofumi; Sogi, Misa; Kitazono, Hidetaka; Hosokawa, Naoto

    2012-08-01

    We report an extremely rare case of urosepsis caused by Globicatella sanguinis and Corynebacterium riegelii coinfection in a 94-year-old Japanese man with nephrolithiasis. Prompt identification of this coinfection is important so that effective antimicrobial coverage can be initiated.

  11. Guanosine Diphosphate-l-Fucose Glycopeptide Fucosyltransferase Activity in Corynebacterium insidiosum1

    PubMed Central

    Sadowski, Peter L.; Strobel, Gary A.

    1973-01-01

    The biosynthesis of a phytotoxic glycopeptide of Corynebacterium insidiosum involves guanosine diphosphate-l-fucosyltransferase activity. This enzyme activity is most consistently associated with the cellular membranes fraction. The optimal pH for the transfer reaction is 7.5. The partially hydrolyzed toxin serves as an acceptor (primer) of l-fucose. PMID:4199136

  12. Complete Genome Sequence of Corynebacterium pseudotuberculosis Strain PA01, Isolated from Sheep in Pará, Brazil

    PubMed Central

    Alves, Jorianne T. C.; Veras, Adonney A. O.; Cavalcante, Ana Lídia Q.; de Sá, Pablo H. C. G.; Dias, Larissa M.; Guimarães, Luis C.; Morais, Ezequiel; Silva, André G. M.; Azevedo, Vasco; Ramos, Rommel T. J.; Silva, Artur

    2016-01-01

    Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis disease. In this work, we present the first complete genome sequence of Corynebacterium pseudotuberculosis strain PA01, isolated in northern Brazil from an infected sheep. The genome length is 2,337,920 bp, and 2,003 coding sequences (CDS), 12 rRNAs, and 49 tRNAs were predicted. PMID:26823595

  13. Identification of non-diphtheriae corynebacterium by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

    PubMed

    Alatoom, Adnan A; Cazanave, Charles J; Cunningham, Scott A; Ihde, Sherry M; Patel, Robin

    2012-01-01

    We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for identification of 92 clinical isolates of Corynebacterium species in comparison to identification using rpoB or 16S rRNA gene sequencing. Eighty isolates (87%) yielded a score of ≥1.700, and all of these were correctly identified to the species level with the exception of Corynebacterium aurimucosum being misidentified as the closely related Corynebacterium minutissimum.

  14. Screening for Corynebacterium diphtheriae and Corynebacterium ulcerans in patients with upper respiratory tract infections 2007-2008: a multicentre European study.

    PubMed

    Wagner, K S; White, J M; Neal, S; Crowcroft, N S; Kuprevičiene, N; Paberza, R; Lucenko, I; Jõks, U; Akbaş, E; Alexandrou-Athanassoulis, H; Detcheva, A; Vuopio, J; von Hunolstein, C; Murphy, P G; Andrews, N; Efstratiou, A

    2011-04-01

    Diphtheria is now rare in most European countries but, when cases do arise, the case fatality rate is high (5-10%). Because few countries continue to routinely screen for the causative organisms of diphtheria, the extent to which they are circulating amongst different European populations is largely unknown. During 2007-2008, ten European countries each screened between 968 and 8551 throat swabs from patients with upper respiratory tract infections. Six toxigenic strains of Corynebacterium diphtheriae were identified: two from symptomatic patients in Latvia (the country with the highest reported incidence of diphtheria in the European Union) and four from Lithuania (two cases, two carriers); the last reported case of diphtheria in Lithuania was in 2002. Carriage rates of non-toxigenic organisms ranged from 0 (Bulgaria, Finland, Greece, Ireland, Italy) to 4.0 per 1000 (95% CI 2.0-7.1) in Turkey. A total of 28 non-toxigenic strains were identified during the study (26 C. diphtheriae, one Corynebacterium ulcerans, one Corynebacterium pseudotuberculosis). The non-toxigenic C. ulcerans strain was isolated from the UK, the country with the highest reported incidence of cases due to C. ulcerans. Of the eleven ribotypes detected, Cluj was seen most frequently in the non-toxigenic isolates and, amongst toxigenic isolates, the major epidemic clone, Sankt-Petersburg, is still in circulation. Isolation of toxigenic C. diphtheriae and non-toxigenic C. diphtheriae and C. ulcerans in highly-vaccinated populations highlights the need to maintain microbiological surveillance, laboratory expertise and an awareness of these organisms amongst public health specialists, microbiologists and clinicians.

  15. Exudative pharyngitis possibly due to Corynebacterium pseudodiphtheriticum, a new challenge in the differential diagnosis of diphtheria.

    PubMed Central

    Izurieta, H. S.; Strebel, P. M.; Youngblood, T.; Hollis, D. G.; Popovic, T.

    1997-01-01

    Corynebacterium pseudodiphtheriticum has rarely been reported to cause disease in humans, despite its common presence in the flora of the upper respiratory tract. We report here a case of exudative pharyngitis with pseudomembrane possibly caused by C. pseudodiphtheriticum in a 4-year-old girl. The case initially triggered clinical and laboratory suspicion of diphtheria. Because C. pseudodiphtheriticum can be easily confused with Corynebacterium diphtheriae in Gram stain, clarification of its role in the pathogenesis of exudative pharyngitis in otherwise healthy persons is of public health importance. Simple and rapid screening tests to differentiate C. pseudodiphtheriticum from C. diphtheriae should be performed to prevent unnecessary concern in the community and unnecessary outbreak control measures. PMID:9126447

  16. Colonisation with toxigenic Corynebacterium diphtheriae in a Scottish burns patient, June 2015.

    PubMed

    Deshpande, Ashutosh; Inkster, Teresa; Hamilton, Kate; Litt, David; Fry, Norman; Kennedy, Iain T R; Shookhye-Dickson, Jacqueline; Hill, Robert L R

    2015-01-01

    On 12 June 2015, Corynebacterium diphtheriae was identified in a skin swab from a burns patient in Scotland. The isolate was confirmed to be genotypically and phenotypically toxigenic. Multilocus sequence typing of three patient isolates yielded sequence type ST 125. The patient was clinically well. We summarise findings of this case, and results of close contact identification and screening: 12 family and close contacts and 32 hospital staff have been found negative for C. diphtheriae.

  17. Late-onset prosthetic valve endocarditis caused by nontoxigenic Corynebacterium diphtheriae.

    PubMed

    El-Hazmi, Malak M

    2015-08-29

    In developed countries, Corynebacterium diphtheriae infection is rare due to efficient immunization programs. However, cases of nontoxigenic strains of C. diphtheriae infections, including endocarditis, have been reported recently. Although the incidence remains low, these infections are associated with high morbidity and mortality. This report describes the first and atypical case of bacteremia and endocarditis caused by nontoxigenic C. diphtheriae var. gravis after introduction of immunization in the Kingdom of Saudi Arabia (KSA).

  18. Description of Corynebacterium trachiae sp. nov., isolated from a white stork (Ciconia ciconia).

    PubMed

    Kämpfer, Peter; Jerzak, Leszek; Wilharm, Gottfried; Golke, Jan; Busse, Hans-Jürgen; Glaeser, Stefanie P

    2015-03-01

    A Gram-stain-positive bacterial isolate, strain 280/10(T) was isolated from the trachea of a white stork. The isolate was morphologically rod- to coccoid-shaped, non-motile and showed no oxidase activity. The highest 16S rRNA gene sequence similarity was found to the type strain of Corynebacterium ciconiae (97.3 % similarity) as the nearest relative of strain 280/10(T), all other 16S rRNA gene sequence similarities to type strains of species of the genus Corynebacterium were below 94.2 %. Strain 280/10(T) had a quinone system consisting predominantly of menaquinone MK-8(H2), minor quantities of MK-9(H2) and small amounts of MK-8. The diamino acid of the peptidoglycan was meso-diaminopimelic acid. The major compounds in the polar lipid profiles were diphosphatidylglycerol, phoshatidylglycerol, phosphatidylinositol and an acidic glycolipid. Two phosphatidylinositol-mannosides and several unidentified lipids were also present. The strain contained corynemycolic acids, while only small amounts of cellular polyamines were detected. Spermidine and spermine were predominant in the polyamine pattern of 280/10(T) and putrescine was present in moderate amounts. In the fatty acid profile C18 : 1ω9c, and C16 : 0 were predominant. The strain was distinguishable from C. ciconiae, which is the most closely related species. In conclusion, strain 280/10(T) is proposed to represent a novel species of the genus Corynebacterium with the name Corynebacterium trachiae sp. nov. and the type strain 280/10(T) ( = CIP 110796(T) = LMG 28214(T)).

  19. Complete Genome Sequence of the Attenuated Corynebacterium pseudotuberculosis Strain T1

    PubMed Central

    Almeida, Sintia; Loureiro, Dan; Mariano, Diego C. B.; Sousa, Thiago J.; Pereira, Felipe L.; Dorella, Fernanda A.; Carvalho, Alex F.; Moura-Costa, Lilia F.; Leal, Carlos A. G.; Figueiredo, Henrique C.; Meyer, Roberto; Azevedo, Vasco

    2016-01-01

    We present here the genome sequence of the attenuated Corynebacterium pseudotuberculosis strain T1. The sequencing was performed with an Ion Torrent Personal Genome Machine platform. The genome is a circular chromosome of 2,337,201 bp, with a G+C content of 52.85% and a total of 2,125 coding sequences (CDSs), 12 rRNAs, 49 tRNAs, and 24 pseudogenes. PMID:27609922

  20. Complete Genome Sequence of Corynebacterium pseudotuberculosis Cp31, Isolated from an Egyptian Buffalo

    PubMed Central

    Ramos, Rommel Thiago Jucá; Ribeiro Carneiro, Adriana; Cybelle Pinto, Anne; de Castro Soares, Siomar; Rodrigues Santos, Anderson; Silva Almeida, Sintia; Guimarães, Luis Carlos; Figueira Aburjaile, Flávia; Vieira Barbosa, Eudes Guilherme; Alves Dorella, Fernanda; Souza Rocha, Flávia; Souza Lopes, Thiago; Kawasaki, Regiane; Gomes Sá, Pablo; da Rocha Coimbra, Nilson Antônio; Teixeira Cerdeira, Louise; Silvanira Barbosa, Maria; Cruz Schneider, Maria Paula; Miyoshi, Anderson; Selim, Salah Abdel Karim; Moawad, Mohamed Salah; Azevedo, Vasco

    2012-01-01

    Corynebacterium pseudotuberculosis is of major veterinary importance because it affects many animal species, causing economically significant livestock diseases and losses. Therefore, the genomic sequencing of various lines of this organism, isolated from different hosts, will aid in the development of diagnostic methods and new prevention and treatment strategies and improve our knowledge of the biology of this microorganism. In this study, we present the genome of C. pseudotuberculosis Cp31, isolated from a buffalo in Egypt. PMID:23144408

  1. Complete Genome Sequence of Corynebacterium pseudotuberculosis Strain E19, Isolated from a Horse in Chile

    PubMed Central

    Cavalcante, Ana Lídia Q.; Dias, Larissa M.; Alves, Jorianne T. C.; Veras, Adonney A. O.; Guimarães, Luis C.; Rocha, Flávia S.; Gala-García, Alfonso; Ramos, Rommel T. J.; Azevedo, Vasco; Silva, Artur

    2015-01-01

    Corynebacterium pseudotuberculosis is related to several diseases infecting horses and small ruminants, causing economic losses to agribusiness. Here, we present the genome sequence of C. pseudotuberculosis strain E19. The genome includes one circular chromosome 2,367,956 bp (52.1% G+C content), with 2,112 genes predicted, 12 rRNAs, and 48 tRNAs. PMID:26607893

  2. Whole-Genome Sequence of Corynebacterium pseudotuberculosis 262 Biovar equi Isolated from Cow Milk

    PubMed Central

    Araújo, Carlos Leonardo de A.; Dias, Larissa M.; Veras, Adonney A. O.; Alves, Jorianne T. C.; Cavalcante, Ana Lídia Q.; Dowson, Christopher G.; Azevedo, Vasco; Ramos, Rommel T. J.; Silva, Artur

    2016-01-01

    We report the complete genome sequence of Corynebacterium pseudotuberculosis 262, isolated from a bovine host. C. pseudotuberculosis is an etiological agent of diseases with medical and veterinary relevance. The genome contains 2,325,749 bp, 52.8% G+C content, 2,022 coding sequences (CDS), 50 pseudogenes, 48 tRNAs, and 12 rRNAs. PMID:27013052

  3. Draft Genome Sequence of Corynebacterium amycolatum Strain ICIS 53 Isolated from a Female Urogenital Tract.

    PubMed

    Gladysheva, Irina V; Cherkasov, Sergey V; Khlopko, Yuriy A; Plotnikov, Andrey O; Gogoleva, Natalya E

    2016-11-10

    This report describes the draft genome sequence of Corynebacterium amycolatum strain ICIS 53, isolated from the reproductive tract of a healthy woman. The size of the genome was 2,460,257 bp (58.98% G+C content). Annotation revealed 2,173 coding sequences, including 2,076 proteins, 7 rRNA genes, and 53 tRNA genes.

  4. Draft Genome Sequence of Corynebacterium amycolatum Strain ICIS 53 Isolated from a Female Urogenital Tract

    PubMed Central

    Cherkasov, Sergey V.; Khlopko, Yuriy A.; Plotnikov, Andrey O.; Gogoleva, Natalya E.

    2016-01-01

    This report describes the draft genome sequence of Corynebacterium amycolatum strain ICIS 53, isolated from the reproductive tract of a healthy woman. The size of the genome was 2,460,257 bp (58.98% G+C content). Annotation revealed 2,173 coding sequences, including 2,076 proteins, 7 rRNA genes, and 53 tRNA genes. PMID:27834713

  5. Corynebacterium glucuronolyticum causing genitourinary tract infection: Case report and review of the literature

    PubMed Central

    Gherardi, G.; Di Bonaventura, G.; Pompilio, A.; Savini, V.

    2015-01-01

    Corynebacterium species are increasingly recognized as opportunistic pathogens. A growing number of taxonomic studies has yielded a description of numerous new Corynebacterium species, such as those related to the urogenital tract, with Corynebacterium glucuronolyticum found to be rarely involved in genitourinary tract infections, particularly in male individuals. In this report, we describe a urethritis case caused by C. glucuronolyticum in a 37-year-old, apparently healthy male, who complained mild pain in the lower abdomen, with several urinary symptoms. While urethral and semen specimens did not yield positive results for microbiological evaluation, cultures of urine samples revealed the monomicrobial growth on blood-containing media of tiny colonies after 24 h of incubation, clearly evident only after 48 h of incubation under CO2-enriched atmosphere. Colonies were identified as C. glucuronolyticum both by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and 16S rRNA gene sequencing. Oral ciprofloxacin gradually led to clinical improvement and, finally, to a complete recovery, in accordance with microbiological findings. In spite of its infrequent detection, C. glucuronolyticum might be a potential urogenital pathogen in males more commonly that what believed, perhaps due to slow growth leading to underrecognition; we suggest therefore to consider the organism in the differential diagnostics of bacterial diseases of the urinary tract. PMID:26793456

  6. Improving baculovirus recombination

    PubMed Central

    Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infection unless rescued by recombination with a baculovirus transfer vector. Modified viral DNA allows 100% recombinant virus formation, obviates the need for further virus purification and offers an efficient means of mass parallel recombinant formation. PMID:12527795

  7. Microbial Production of Amino Acid-Related Compounds.

    PubMed

    Wendisch, Volker F

    2016-11-22

    Corynebacterium glutamicum is the workhorse of the production of proteinogenic amino acids used in food and feed biotechnology. After more than 50 years of safe amino acid production, C. glutamicum has recently also been engineered for the production of amino acid-derived compounds, which find various applications, e.g., as synthons for the chemical industry in several markets including the polymer market. The amino acid-derived compounds such as non-proteinogenic ω-amino acids, α,ω-diamines, and cyclic or hydroxylated amino acids have similar carbon backbones and functional groups as their amino acid precursors. Decarboxylation of amino acids may yield ω-amino acids such as β-alanine, γ-aminobutyrate, and δ-aminovalerate as well as α,ω-diamines such as putrescine and cadaverine. Since transamination is the final step in several amino acid biosynthesis pathways, 2-keto acids as immediate amino acid precursors are also amenable to production using recombinant C. glutamicum strains. Approaches for metabolic engineering of C. glutamicum for production of amino acid-derived compounds will be described, and where applicable, production from alternative carbon sources or use of genome streamline will be referred to. The excellent large-scale fermentation experience with C. glutamicum offers the possibility that these amino acid-derived speciality products may enter large-volume markets.

  8. Reengineering of the feedback-inhibition enzyme N-acetyl-L-glutamate kinase to enhance L-arginine production in Corynebacterium crenatum.

    PubMed

    Zhang, Jingjing; Xu, Meijuan; Ge, Xiaoxun; Zhang, Xian; Yang, Taowei; Xu, Zhenghong; Rao, Zhiming

    2017-02-01

    N-acetyl-L-glutamate kinase (NAGK) catalyzes the second step of L-arginine biosynthesis and is inhibited by L-arginine in Corynebacterium crenatum. To ascertain the basis for the arginine sensitivity of CcNAGK, residue E19 which located at the entrance of the Arginine-ring was subjected to site-saturated mutagenesis and we successfully illustrated the inhibition-resistant mechanism. Typically, the E19Y mutant displayed the greatest deregulation of L-arginine feedback inhibition. An equally important strategy is to improve the catalytic activity and thermostability of CcNAGK. For further strain improvement, we used site-directed mutagenesis to identify mutations that improve CcNAGK. Results identified variants I74V, F91H and K234T display higher specific activity and thermostability. The L-arginine yield and productivity of the recombinant strain C. crenatum SYPA-EH3 (which possesses a combination of all four mutant sites, E19Y/I74V/F91H/K234T) reached 61.2 and 0.638 g/L/h, respectively, after 96 h in 5 L bioreactor fermentation, an increase of approximately 41.8% compared with the initial strain.

  9. Corynebacterium seminale sp. nov., a new species associated with genital infections in male patients.

    PubMed

    Riegel, P; Ruimy, R; de Briel, D; Prévost, G; Jehl, F; Bimet, F; Christen, R; Monteil, H

    1995-09-01

    We studied 12 coryneform isolates having similar biochemical profiles which did not permit their assignment to any recognized taxa. Human semen was the source for seven of these strains, whereas the other strains were isolated from urethra, urine, and blood specimens of adult male patients. These bacteria were found in significant quantities (10(4) to 10(5) CFU/ml) in semen specimens from infertile male patients with the diagnosis of prostatitis. These strains had characteristics of the genus Corynebacterium, such as 60 mol% G + C in the DNA and corynemycolic acids, meso-diaminopimelic acid, arabinose, and galactose in the cell wall. Quantitative DNA-DNA hybridizations (S1 nuclease procedure) and phylogenies based on comparisons of almost-complete small-subunit ribosomal DNA sequences confirmed that these strains constitute a single new species within the genus Corynebacterium. All 12 strains showed similar phenotypic features, i.e., good growth on sheep blood agar in contrast with poor growth on the same medium supplemented with 1% Tween 80, a positive CAMP test in the presence of Staphylococcus aureus, glucose and sucrose fermentation, and the presence of beta-glucuronidase. Some strains reduced nitrate and hydrolyzed urea or esculin. These features allowed us to distinguish these strains from members of any other coryneform taxon, and the proposed name is Corynebacterium seminale with strain IBS B12915 (CIP 104297) as the type strain. The description and delineation of these strains as a new species should be useful for further studies, including evaluations of their prevalence among the normal flora and their clinical implications.

  10. Toxigenic Corynebacterium ulcerans isolated from a free-roaming red fox (Vulpes vulpes).

    PubMed

    Sting, Reinhard; Ketterer-Pintur, Sandra; Contzen, Matthias; Mauder, Norman; Süss-Dombrowski, Christine

    2015-01-01

    Corynebacterium (C.) ulcerans could be isolated from the spleen of a red fox (Vulpes vulpes) that had been found dead in the state of Baden-Württemberg, Germany. Pathohistological examination suggested that the fox had died of distemper, as was confirmed by PCR. The isolate was identified biochemically, by MALDI-TOF MS, FT-IR and by partial 16S rRNA, rpoB and tox gene sequencing. Using the Elek test the C. ulcerans isolate demonstrated diphtheria toxin production. FT-IR and sequencing data obtained from the C. ulcerans isolate from the red fox showed higher similarity to isolates from humans than to those from wild game.

  11. Crystallization and preliminary X-ray studies of the diphtheria Tox repressor from Corynebacterium diphtheriae.

    PubMed

    Schiering, N; Tao, X; Murphy, J R; Petsko, G A; Ringe, D

    1994-12-16

    Crystals of the diphtheria tox repressor (DtxR) from Corynebacterium diphtheriae suitable for structure determination have been obtained. DtxR activated with transition metal ions represses the expression of the structural gene for the diphtheria toxin, tox, which is encoded on the genome of a family of closely related corynebacteriophages. The space group of the obtained crystals is trigonal P3(1)21 or its enantiomorph P3(2)21 with a = b = 64.2 A, c = 220.5 A, alpha = beta = 90 degrees, gamma = 120 degrees. Two monomers comprise the asymmetric unit. The crystals diffract to a resolution of better than 3 A.

  12. Detection of Corynebacterium bovis infection in athymic nude mice from a research animal facility in Korea.

    PubMed

    Kim, Tae-Hyoun; Kim, Dong-Su; Han, Ju-Hee; Chang, Seo-Na; Kim, Kyung-Sul; Seok, Seung-Hyeok; Kim, Dong-Jae; Park, Jong-Hwan; Park, Jae-Hak

    2014-12-01

    Corynebacterium (C.) bovis infection in nude mice causes hyperkeratosis and weight loss and has been reported worldwide but not in Korea. In 2011, nude mice from an animal facility in Korea were found to have white flakes on their dorsal skin. Histopathological testing revealed that the mice had hyperkeratosis and Gram-positive bacteria were found in the skin. We identified isolated bacteria from the skin lesions as C. bovis using PCR and 16S rRNA sequencing. To the best of our knowledge, this is the first report of C. bovis infection in nude mice from Korea.

  13. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  14. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  15. In vitro susceptibility of equine-obtained isolates of Corynebacterium pseudotuberculosis to gallium maltolate and 20 other antimicrobial agents.

    PubMed

    Norman, T E; Batista, M; Lawhon, S D; Zhang, S; Kuskie, K R; Swinford, A K; Bernstein, L R; Cohen, N D

    2014-07-01

    This study's objective was to determine the in vitro antimicrobial activities of gallium maltolate (GaM) and 20 other antimicrobial agents against clinical equine isolates of Corynebacterium pseudotuberculosis. The growth of cultured isolates was not inhibited by any concentration of GaM. MIC data revealed susceptibility to commonly used antimicrobials.

  16. In Vitro Susceptibility of Equine-Obtained Isolates of Corynebacterium pseudotuberculosis to Gallium Maltolate and 20 Other Antimicrobial Agents

    PubMed Central

    Batista, M.; Lawhon, S. D.; Zhang, S.; Kuskie, K. R.; Swinford, A. K.; Bernstein, L. R.; Cohen, N. D.

    2014-01-01

    This study's objective was to determine the in vitro antimicrobial activities of gallium maltolate (GaM) and 20 other antimicrobial agents against clinical equine isolates of Corynebacterium pseudotuberculosis. The growth of cultured isolates was not inhibited by any concentration of GaM. MIC data revealed susceptibility to commonly used antimicrobials. PMID:24829243

  17. Differential Chemical Protection of Mammalian Cells from the Exotoxins of ’Corynebacterium diphtheriae’ and ’Pseudomonas aeruginosa’,

    DTIC Science & Technology

    Many drugs or chemicals had markedly different effects on the cytotoxicity induced by Pseudomonas aeruginosa exotoxin A (PE) or Corynebacterium ... diphtheriae exotoxin (DE). The glycolytic inhibitor NaF protected cells from DE but potentiated the cytotoxicity of PE. Another energy inhibitor, salicylic

  18. Draft Genome Sequence of Corynebacterium ulcerans FRC58, Isolated from the Bronchitic Aspiration of a Patient in France

    PubMed Central

    Silva, Andréia do Socorro de Sousa; Baraúna, Rafael Azevedo; de Sá, Pablo Caracciolo Gomes; das Graças, Diego Assis; Carneiro, Adriana Ribeiro; Thouvenin, Maxime; Azevedo, Vasco; Badell, Edgar; Guiso, Nicole; da Silva, Artur Luiz da Costa

    2014-01-01

    Corynebacterium ulcerans is a bacterial species with high importance because it causes infections in animals and, rarely, in humans. Its virulence mechanisms remain unclear. The current study describes the draft genome of C. ulcerans FRC58, which was isolated from the bronchitic aspiration of a patient in France. PMID:24407640

  19. Draft Genome Sequence of Toxigenic Corynebacterium ulcerans Strain 04-7514, Isolated from a Dog in France

    PubMed Central

    Viana, Marcus V. C.; Benevides, Leandro J.; Mariano, Diego C. B.; Veras, Adooney A. O.; Sá, Pablo H. C.; Rocha, Flávia S.; Vilas Boas, Priscilla C. B.; Soares, Siomar C.; Barbosa, Maria S.; Guiso, Nicole; Badell, Edgar; Carneiro, Adriana R.; Azevedo, Vasco; Ramos, Rommel T. J.

    2016-01-01

    Here, we present the draft genome of toxigenic Corynebacterium ulcerans strain 04-7514. The draft genome has 2,497,845 bp, 2,059 coding sequences, 12 rRNA genes, 46 tRNA genes, 150 pseudogenes, 1 clustered regularly interspaced short palindromic repeat (CRISPR) array, and a G+C content of 53.50%. PMID:27034487

  20. Corynebacterium equi Infections in Horses, 1958-1984: A Review of 131 Cases.

    PubMed

    Zink, M C; Yager, J A; Smart, N L

    1986-05-01

    Of 131 cases of Corynebacterium equi infection in horses submitted for necropsy to the Ontario Veterinary College or Veterinary Laboratory Services, OMAF, Guelph, Ontario from 1958 to 1984, 115 were diagnosed as suppurative pneumonia, and of these 55 had associated ulcerative enterocolitis. Only five animals had intestinal involvement without pulmonary lesions. The remaining 11 cases included arthritis/cellulitis, skin abscesses and submandibular lymphadenitis. While the lung, intestine and associated lymph nodes yielded C. equi most frequently, in 21% of cases C. equi was also cultured from parenchymatous organs (spleen, liver or kidney) or blood. Corynebacterium equi infection accounted for 10% of all foals submitted for postmortem examination and 45% of all foals with pneumonia. Affected foals were one to four months of age. Submissions occurred between the months of May and August with a peak during July. There was a significantly greater prevalence of C. equi infection in Standardbreds when compared with other breeds. Of foals in this study, 36% were from farms which had had other horses succumb to this disease. Of the foals with pulmonary involvement, 21% did not have fever or clinical signs referable to the respiratory or gastrointestinal systems, findings which indicated that a large percentage of cases were subclinical.

  1. Desulfurization of dibenzothiophene by a newly isolated Corynebacterium sp. ZD-1 in aqueous phase.

    PubMed

    Wang, Miao-Dong; Li, Wei; Wang, Da-Hui; Shi, Yao

    2004-01-01

    Sulfur emission through fuel combustion is a global problem because it is a major cause of acid rain. Crud oil contains many heterocyclic organic sulfur compounds, among which dibenzothiophene (DBT) and DBTs bearing alkyl substitutions usually are representative compounds. A strain was isolated from refinery sludge and identified as Corynebacterium ZD-1. The behavior of DBT degradation by ZD-1 in aqueous phase was investigated. Corynebacterium ZD-1 could metabolize DBT to 2-hydroxybiphenyl(2-HBP) as the dead-end metabolite through a sulfur-specific pathway. In shake flask culture, ZD-1 had its maximal desulfurization activity in the late exponential growth phase and the specific production rate of 2-HBP was about 0.14 (mmol x kg dry cell(-1) x min(-1), mmol x KDC(-1) x min(-1)). Active resting cells for desulfurization should be prepared only in this period. 2-HBP inhibited the growth of strain ZD-1, the production of DBT degradation enzymes, and the activity of enzymes. Sulfate inhibited the production of dibenzothiophene (DBT) degradation enzymes but had no effect on the enzymes' activity. The production rates of 2-HBP at lower cell densities were higher and the maximum amount conversion of DBT to 2-HBP (0.067 mmol/L) after 8 h was gained at 9.2 g dry cell/L rather higher cell density. The results indicated that this newly isolated strain could be a promising biocatalyst for DBT desulfurization.

  2. AAC(3)-XI, a New Aminoglycoside 3-N-Acetyltransferase from Corynebacterium striatum

    PubMed Central

    Galimand, Marc; Fishovitz, Jennifer; Lambert, Thierry; Barbe, Valérie; Zajicek, Jaroslav

    2015-01-01

    Corynebacterium striatum BM4687 was resistant to gentamicin and tobramycin but susceptible to kanamycin A and amikacin, a phenotype distinct among Gram-positive bacteria. Analysis of the entire genome of this strain did not detect any genes for known aminoglycoside resistance enzymes. Yet, annotation of the coding sequences identified 12 putative acetyltransferases or GCN5-related N-acetyltransferases. A total of 11 of these coding sequences were also present in the genomes of other Corynebacterium spp. The 12th coding sequence had 55 to 60% amino acid identity with acetyltransferases in Actinomycetales. The gene was cloned in Escherichia coli, where it conferred resistance to aminoglycosides by acetylation. The protein was purified to homogeneity, and its steady-state kinetic parameters were determined for dibekacin and kanamycin B. The product of the turnover of dibekacin was purified, and its structure was elucidated by high-field nuclear magnetic resonance (NMR), indicating transfer of the acetyl group to the amine at the C-3 position. Due to the unique profile of the reaction, it was designated aminoglycoside 3-N-acetyltransferase type XI. PMID:26149994

  3. Tips and tricks for the assembly of a Corynebacterium pseudotuberculosis genome using a semiconductor sequencer

    PubMed Central

    Ramos, Rommel Thiago Jucá; Carneiro, Adriana Ribeiro; Soares, Siomar de Castro; dos Santos, Anderson Rodrigues; Almeida, Sintia; Guimarães, Luis; Figueira, Flávia; Barbosa, Eudes; Tauch, Andreas; Azevedo, Vasco; Silva, Artur

    2013-01-01

    New sequencing platforms have enabled rapid decoding of complete prokaryotic genomes at relatively low cost. The Ion Torrent platform is an example of these technologies, characterized by lower coverage, generating challenges for the genome assembly. One particular problem is the lack of genomes that enable reference-based assembly, such as the one used in the present study, Corynebacterium pseudotuberculosis biovar equi, which causes high economic losses in the US equine industry. The quality treatment strategy incorporated into the assembly pipeline enabled a 16-fold greater use of the sequencing data obtained compared with traditional quality filter approaches. Data preprocessing prior to the de novo assembly enabled the use of known methodologies in the next-generation sequencing data assembly. Moreover, manual curation was proved to be essential for ensuring a quality assembly, which was validated by comparative genomics with other species of the genus Corynebacterium. The present study presents a modus operandi that enables a greater and better use of data obtained from semiconductor sequencing for obtaining the complete genome from a prokaryotic microorganism, C. pseudotuberculosis, which is not a traditional biological model such as Escherichia coli. PMID:23199210

  4. Microbiological and clinical features of Corynebacterium urealyticum: urinary tract stones and genomics as the Rosetta Stone.

    PubMed

    Soriano, F; Tauch, A

    2008-07-01

    Corynebacterium urealyticum, formerly known as coryneform CDC group D2, was first recognized to be involved in human infections 30 years ago. It is a slow-growing, lipophilic, asaccharolytic and usually multidrug-resistant organism with potent urease activity. Its cell wall peptidoglycan, menaquinone, mycolic and cellular fatty acid composition is consistent with that of the genus Corynebacterium. DNA-DNA hybridization studies and 16S rDNA sequencing analysis have been used to determine the degree of relatedness of C. urealyticum to other corynebacterial species. The genome of the type strain consists of a circular chromosome with a size of 2 369 219 bp and a mean G + C content of 64.2%, and analysis of its genome explains the bacterium's lifestyle. C. urealyticum is a common skin colonizer of hospitalized elderly individuals who are receiving broad-spectrum antibiotics. It is an opportunistic pathogen causing mainly acute cystitis, pyelonephritis, encrusted cystitis, and encrusted pyelitis. More infrequently, it causes other infections, but mainly in patients with urological diseases. Infections are more common in males than in females, and treatment requires administration of antibiotics active against the organism in vitro, mainly glycopeptides, as well as surgical intervention, the latter mostly in cases of chronic infection. Mortality directly associated with infection by this organism is not frequent, but encrusted pyelitis in kidney-recipient patients may cause graft loss. The outcome of infection by this organism is reasonably good if the microbiological diagnosis is made and patients are treated appropriately.

  5. Characterization of Staphylococcus and Corynebacterium Clusters in the Human Axillary Region

    PubMed Central

    Callewaert, Chris; Kerckhof, Frederiek-Maarten; Granitsiotis, Michael S.; Van Gele, Mireille; Van de Wiele, Tom; Boon, Nico

    2013-01-01

    The skin microbial community is regarded as essential for human health and well-being, but likewise plays an important role in the formation of body odor in, for instance, the axillae. Few molecular-based research was done on the axillary microbiome. This study typified the axillary microbiome of a group of 53 healthy subjects. A profound view was obtained of the interpersonal, intrapersonal and temporal diversity of the human axillary microbiota. Denaturing gradient gel electrophoresis (DGGE) and next generation sequencing on 16S rRNA gene region were combined and used as extent to each other. Two important clusters were characterized, where Staphylococcus and Corynebacterium species were the abundant species. Females predominantly clustered within the Staphylococcus cluster (87%, n = 17), whereas males clustered more in the Corynebacterium cluster (39%, n = 36). The axillary microbiota was unique to each individual. Left-right asymmetry occurred in about half of the human population. For the first time, an elaborate study was performed on the dynamics of the axillary microbiome. A relatively stable axillary microbiome was noticed, although a few subjects evolved towards another stable community. The deodorant usage had a proportional linear influence on the species diversity of the axillary microbiome. PMID:23950955

  6. Corynebacterium equi Infections in Horses, 1958-1984: A Review of 131 Cases

    PubMed Central

    Zink, M. Christine; Yager, Julie A.; Smart, Nonie L.

    1986-01-01

    Of 131 cases of Corynebacterium equi infection in horses submitted for necropsy to the Ontario Veterinary College or Veterinary Laboratory Services, OMAF, Guelph, Ontario from 1958 to 1984, 115 were diagnosed as suppurative pneumonia, and of these 55 had associated ulcerative enterocolitis. Only five animals had intestinal involvement without pulmonary lesions. The remaining 11 cases included arthritis/cellulitis, skin abscesses and submandibular lymphadenitis. While the lung, intestine and associated lymph nodes yielded C. equi most frequently, in 21% of cases C. equi was also cultured from parenchymatous organs (spleen, liver or kidney) or blood. Corynebacterium equi infection accounted for 10% of all foals submitted for postmortem examination and 45% of all foals with pneumonia. Affected foals were one to four months of age. Submissions occurred between the months of May and August with a peak during July. There was a significantly greater prevalence of C. equi infection in Standardbreds when compared with other breeds. Of foals in this study, 36% were from farms which had had other horses succumb to this disease. Of the foals with pulmonary involvement, 21% did not have fever or clinical signs referable to the respiratory or gastrointestinal systems, findings which indicated that a large percentage of cases were subclinical. PMID:17422658

  7. Corynebacterium accolens Releases Antipneumococcal Free Fatty Acids from Human Nostril and Skin Surface Triacylglycerols

    PubMed Central

    Bomar, Lindsey; Brugger, Silvio D.; Yost, Brian H.; Davies, Sean S.

    2016-01-01

    ABSTRACT Bacterial interspecies interactions play clinically important roles in shaping microbial community composition. We observed that Corynebacterium spp. are overrepresented in children free of Streptococcus pneumoniae (pneumococcus), a common pediatric nasal colonizer and an important infectious agent. Corynebacterium accolens, a benign lipid-requiring species, inhibits pneumococcal growth during in vitro cocultivation on medium supplemented with human skin surface triacylglycerols (TAGs) that are likely present in the nostrils. This inhibition depends on LipS1, a TAG lipase necessary for C. accolens growth on TAGs such as triolein. We determined that C. accolens hydrolysis of triolein releases oleic acid, which inhibits pneumococcus, as do other free fatty acids (FFAs) that might be released by LipS1 from human skin surface TAGs. Our results support a model in which C. accolens hydrolyzes skin surface TAGS in vivo releasing antipneumococcal FFAs. These data indicate that C. accolens may play a beneficial role in sculpting the human microbiome. PMID:26733066

  8. Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

    PubMed

    Katsukawa, Chihiro; Komiya, Takako; Umeda, Kaoru; Goto, Minami; Yanai, Tokuma; Takahashi, Motohide; Yamamoto, Akihiko; Iwaki, Masaaki

    2016-03-01

    Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.

  9. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  10. The draft genome sequence of Corynebacterium diphtheriae bv. mitis NCTC 3529 reveals significant diversity between the primary disease-causing biovars.

    PubMed

    Sangal, Vartul; Tucker, Nicholas P; Burkovski, Andreas; Hoskisson, Paul A

    2012-06-01

    We report the draft genome of the human pathogen Corynebacterium diphtheriae bv. mitis NCTC 3529. This is the first C. diphtheriae bv. mitis strain to be sequenced and reveals significant differences from the other primary biovar, C. diphtheriae bv. gravis.

  11. Recombination of cluster ions

    NASA Technical Reports Server (NTRS)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  12. A slow-forming isopeptide bond in the structure of the major pilin SpaD from Corynebacterium diphtheriae has implications for pilus assembly

    SciTech Connect

    Kang, Hae Joo; Paterson, Neil G.; Kim, Chae Un; Middleditch, Martin; Chang, Chungyu; Ton-That, Hung; Baker, Edward N.

    2014-05-01

    Two crystal structures of the major pilin SpaD from C. diphtheriae have been determined at 1.87 and 2.5 Å resolution. The N-terminal domain is found to contain an isopeptide bond that forms slowly over time in the recombinant protein. Given its structural context, this provides insight into the relationship between internal isopeptide-bond formation and pilus assembly. The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys–Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys–Asn isopeptide-bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its isopeptide bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.

  13. Strategies used for genetically modifying bacterial genome: ite-directed mutagenesis, gene inactivation, and gene over-expression*

    PubMed Central

    Xu, Jian-zhong; Zhang, Wei-guo

    2016-01-01

    With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators. PMID:26834010

  14. Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes

    PubMed Central

    Herguedas, Beatriz; Martínez-Júlvez, Marta; Frago, Susana; Medina, Milagros; Hermoso, Juan A.

    2009-01-01

    FAD synthetase from Corynebacterium ammoniagenes (CaFADS), a prokary­otic bifunctional enzyme that catalyses the phosphorylation of riboflavin as well as the adenylylation of FMN, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. Diffraction-quality cubic crystals of native and selenomethionine-labelled (SeMet-CaFADS) protein belonged to the cubic space group P213, with unit-cell parameters a = b = c = 133.47 Å and a = b = c = 133.40 Å, respectively. Data sets for native and SeMet-containing crystals were collected to 1.95 and 2.42 Å resolution, respectively. PMID:20054130

  15. Corynebacterium striatum Bacteremia Associated with a Catheter-Related Blood Stream Infection

    PubMed Central

    Oishi, Tomohiro; Yamane, Kunikazu; Terada, Kihei

    2017-01-01

    A 49-year-old woman visited our emergency department because of exertional dyspnea due to severe left ventricular functional failure. It progressed to disseminated intravascular coagulation and disturbance of consciousness on day 67 of admission. Gram-positive bacilli were detected from two different blood culture samples on day 67 of admission. An API-Coryne test and sequencing (1~615 bp) of the 16S rRNA gene were performed, and the strain was identified as Corynebacterium striatum. The bacterium was detected from the removed central venous catheter tip too, and the patient was diagnosed with catheter-related bloodstream infection by C. striatum. However, treatment was not effective, and the patient died on day 73 of admission. PMID:28197349

  16. Multilocus sequence types of invasive Corynebacterium diphtheriae isolated in the Rio de Janeiro urban area, Brazil.

    PubMed

    Viguetti, S Z; Pacheco, L G C; Santos, L S; Soares, S C; Bolt, F; Baldwin, A; Dowson, C G; Rosso, M L; Guiso, N; Miyoshi, A; Hirata, R; Mattos-Guaraldi, A L; Azevedo, V

    2012-04-01

    Invasive infections caused by Corynebacterium diphtheriae in vaccinated and non-vaccinated individuals have been reported increasingly. In this study we used multilocus sequence typing (MLST) to study genetic relationships between six invasive strains of this bacterium isolated solely in the urban area of Rio de Janeiro, Brazil, during a 10-year period. Of note, all the strains rendered negative results in PCR reactions for the tox gene, and four strains presented an atypical sucrose-fermenting ability. Five strains represented new sequence types. MLST results did not support the hypothesis that invasive (sucrose-positive) strains of C. diphtheriae are part of a single clonal complex. Instead, one of the main findings of the study was that such strains can be normally found in clonal complexes with strains related to non-invasive disease. Comparative analyses with C. diphtheriae isolated in different countries provided further information on the geographical circulation of some sequence types.

  17. [Adhesion of corynebacterium diphtheriae: the role of surface structures and formation mechanism].

    PubMed

    Kharseeva, G G; Alieva, A A

    2014-01-01

    The paper is devoted to the study of surface structures including pili (fimbriae) 67-72p surface protein, DIP 1281 surface protein, lipoarabinomannan CdiLAM and their role in the adhesion and colonization of the mucous membrane of the throat by Corynebacterium diphtheriae. A description is offered for the main stages in the adhesion process of diphtheria causative agent and the ability of its adhesins to stimulate the effect of innate and acquired immunity factors. The paper stresses prospectiveness of the development of vaccines forming immunoprotection of the organism against adhesive activity of C. diphtheriae and also preventing their colonization and reproduction. That would facilitate a solution for the problem of diphtheria carrier state, which cannot be solved using the existing means of preventive vaccination.

  18. Extended Characterization of Corynebacterium pyruviciproducens Based on Clinical Strains from Canada and Switzerland

    PubMed Central

    Hinić, V.; Turan, S.; Schultheiss, E.; Pacheco, A. L.; Frei, R.; Bernard, K.

    2014-01-01

    The species Corynebacterium pyruviciproducens was described in 2010 based on the features of a single strain. In this report, we describe the chemotaxonomic and phenotypic characteristics of 11 C. pyruviciproducens clinical strains isolated in Switzerland and Canada in comparison to the strain 06-17730T. Heterogeneities within the type strain were found in the 16S rRNA gene and in biochemical markers. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification of this species could not be achieved since currently this bacterial species is not included in the corresponding database. Reliable identification is obtained only with sequence-based identification tools. Results of antimicrobial susceptibility testing of this species with an extended panel of antimicrobials are presented here for the first time. PMID:24951802

  19. Expression, purification and characterization of cold shock protein A of Corynebacterium pseudotuberculosis.

    PubMed

    Lindae, Antje; Eberle, Raphael J; Caruso, Icaro P; Coronado, Monika A; de Moraes, Fabio R; Azevedo, Vasco; Arni, Raghuvir K

    2015-08-01

    The gram-positive bacterium Corynebacterium pseudotuberculosis is the causative agent of different diseases that cause dramatically reduced yields of wool and milk, and results in weight loss, carcass condemnation and also death mainly in sheep, equids, cattle and goats and therefore globally results in considerable economical loss. Cold shock proteins are conserved in many bacteria and eukaryotic cells and they help to restore normal cell functions after cold shock in which some appear to have specific functions at normal growth temperature as well. Cold shock protein A from C. pseudotuberculosis was expressed in Escherichia coli and purified. The thermal unfolding/refolding process characterized by circular dichroism, differential scanning calorimetry and NMR spectroscopy techniques indicated that the refolding process was almost completely reversible.

  20. Clonal multidrug-resistant Corynebacterium striatum within a nosocomial environment, Rio de Janeiro, Brazil

    PubMed Central

    Baio, Paulo Victor Pereira; Mota, Higor Franceschi; Freitas, Andréa D'avila; Gomes, Débora Leandro Rama; Ramos, Juliana Nunes; Sant'Anna, Lincoln Oliveira; Souza, Mônica Cristina; Camello, Thereza Cristina Ferreira; Hirata, Raphael; Vieira, Verônica Viana; Mattos-Guaraldi, Ana Luíza

    2013-01-01

    Corynebacterium striatum is a potentially pathogenic microorganism with the ability to produce outbreaks of nosocomial infections. Here, we document a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro, Brazil. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. Fifteen C. striatum strains were isolated from adults (half of whom were 50 years of age and older). C. striatum was mostly isolated in pure culture from tracheal aspirates of patients undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE type I, comprising 11 MDR isolates that were mostly isolated from intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE types I and II) infection was observed in five cases. PMID:23440110

  1. Diphthericin types, bacteriophage types and serotypes of Corynebacterium diphtheriae strains isolated in Australia

    PubMed Central

    Gibson, L. F.; Colman, G.

    1973-01-01

    A dipthericin typing scheme has been constructed using 441 strains of Corynebacterium diptheriae isolated in eastern Australia from 1962 to 1971. Ten types have been distinguished using seven strains of C. diphtheriae and two strains of C. belfanti as indicators of the diphthericins produced by the newly isolated strains. Strains grouped into types L2, L3 and L3a were found only in Melbourne and types L1 and L4 were predominant in Sydney. Type L5 strains were isolated intermittently throughout the period of study and were found in all eastern states. Numerical analysis of the characteristics of the strains suggests that associations exist between, on the one hand, diphthericin type and, on the other hand, bacteriophage type, serotype and biochemical activity. PMID:4203597

  2. An observational study of Corynebacterium bovis in selected Ontario dairy herds.

    PubMed Central

    Brooks, B W; Barnum, D A; Meek, A H

    1983-01-01

    An observational study of Corynebacterium bovis was conducted in 74 Ontario dairy herds. The levels of infection with C. bovis were 19.9, 36.2 and 85.6% at the quarter, cow and herd level, respectively. Teat disinfection was found to be the variable best able to distinguish between herds with a high or low C. bovis quarter infection rate. Mean total milk somatic cell counts for 1103 quarters and 107 cows infected with only C. bovis ranged between 150,000 and 200,000/mL and were significantly higher than for uninfected quarters or cows. The rate of infection with mastitis pathogens was not significantly different in quarters previously colonized with only C. bovis compared to previously uninfected quarters. PMID:6831308

  3. Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains

    PubMed Central

    Silva, Artur; Ali, Amjad; Pinto, Anne C.; Santos, Anderson R.; Rocha, Aryanne A. M. C.; Lopes, Débora O.; Dorella, Fernanda A.; Pacheco, Luis G. C.; Costa, Marcília P.; Turk, Meritxell Z.; Seyffert, Núbia; Moraes, Pablo M. R. O.; Soares, Siomar C.; Almeida, Sintia S.; Castro, Thiago L. P.; Abreu, Vinicius A. C.; Trost, Eva; Baumbach, Jan; Tauch, Andreas; Schneider, Maria Paula C.; McCulloch, John; Cerdeira, Louise T.; Ramos, Rommel T. J.; Zerlotini, Adhemar; Dominitini, Anderson; Resende, Daniela M.; Coser, Elisângela M.; Oliveira, Luciana M.; Pedrosa, André L.; Vieira, Carlos U.; Guimarães, Cláudia T.; Bartholomeu, Daniela C.; Oliveira, Diana M.; Santos, Fabrício R.; Rabelo, Élida Mara; Lobo, Francisco P.; Franco, Glória R.; Costa, Ana Flávia; Castro, Ieso M.; Dias, Sílvia Regina Costa; Ferro, Jesus A.; Ortega, José Miguel; Paiva, Luciano V.; Goulart, Luiz R.; Almeida, Juliana Franco; Ferro, Maria Inês T.; Carneiro, Newton P.; Falcão, Paula R. K.; Grynberg, Priscila; Teixeira, Santuza M. R.; Brommonschenkel, Sérgio; Oliveira, Sérgio C.; Meyer, Roberto; Moore, Robert J.; Miyoshi, Anderson; Oliveira, Guilherme C.

    2011-01-01

    Background Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. Methodology and Findings We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. Conclusions These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829. PMID:21533164

  4. The core stimulon of Corynebacterium pseudotuberculosis strain 1002 identified using ab initio methodologies.

    PubMed

    Pinto, Anne Cybelle; Ramos, Rommel T J; Silva, Wanderson Marques; Rocha, Flávia Souza; Barbosa, Silvanira; Miyoshi, Anderson; Schneider, Maria P C; Silva, Artur; Azevedo, Vasco

    2012-07-01

    Corynebacterium pseudotuberculosis is a bacterium which causes diseases such as caseous lymphadenitis in small ruminants, resulting in large-scale economic losses for agribusiness worldwide. Consequently, this bacterium including its transcriptional profile analysis has been the focus of various studies. Identification of the transcripts that appear under conditions that simulate the environment encountered by this bacterial species in the host is of great importance in discovering new targets for the production of more efficient vaccines. We sequenced the cDNA of Corynebacterium pseudotuberculosis strain 1002, using the SOLiD V3 system, under the following conditions: osmotic stress (2 M), acidity (pH), heat shock (50 °C) and control condition (N). To identify the transcripts shared among the stimulons and integrate this information with the results from BLAST and BLAST2GO, we developed the software CoreStImulon (CSI) which allows the user to individually distinguish the genes in terms of their participation in biological processes, their function and cellular location. In the biosynthetic processes, eleven genes represented in the core stimulon and twenty genes in the control were observed. This validates the hypothesis that the organisms strategy for surviving in a hostile environment is through growth reduction. The oxidation reduction process, response to stress process, and cell adhesion are controlled by genes that contribute to bacterial cell maintenance under stress conditions; these could be involved in their pathogenicity. The methodology for identification of transcripts obtained by ab initio assembly and shared among the stimulons permitted candidates selection for vaccine studies. CSI is available at https://sourceforge.net/projects/corestimulon/.

  5. Recombinant Baculovirus Isolation.

    PubMed

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2016-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3 ), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac(®) or BaculoDirect™, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Previously, many methods required a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. Fortunately, this step is no longer required for most systems currently available. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay or real-time polymerase chain reaction (PCR). Methods unique to the Bac-to-Bac(®) system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

  6. Similarity of rpoB gene sequences of sucrose-fermenting and non-fermenting Corynebacterium diphtheriae strains.

    PubMed

    Hirata, R; Pacheco, L G; Soares, S C; Santos, L S; Moreira, L O; Sabbadini, P S; Santos, C S; Miyoshi, A; Azevedo, V A; Mattos-Guaraldi, A L

    2011-03-01

    During the last decades, the majority of Brazilian Corynebacterium diphtheriae isolates were shown to be capable to metabolize sucrose, sometimes leading to erroneous identification as a non-diphtheric Corynebacterium species. The sequencing of the polymorphic region of the RNA polymerase beta subunit-encoding gene (rpoB) is an important taxonomic tool for identification of corynebacteria. The present study aimed to investigate the rpoB gene polymorphic features of sucrose-fermenting and non sucrose-fermenting strains. The results showed that sucrose-fermenting strains presented rpoB gene polymorphic regions with more than 98% similarity with the sequences deposited in the gene bank corresponding to non sucrose-fermenting strains. Data indicate that sucrose-fermenting isolates may act as a variant of C. diphtheriae biotype mitis. In addition we alert that sucrose-fermenting strains should not be discarded as contaminants mainly in countries where the possibility of isolation of this variant is higher.

  7. High quality draft genome sequence of Corynebacterium ulceribovis type strain IMMIB-L1395(T) (DSM 45146(T)).

    PubMed

    Yassin, Atteyet F; Lapidus, Alla; Han, James; Reddy, T B K; Huntemann, Marcel; Pati, Amrita; Ivanova, Natalia; Markowitz, Victor; Woyke, Tanja; Klenk, Hans-Peter; Kyrpides, Nikos C

    2015-01-01

    Corynebacterium ulceribovis strain IMMIB L-1395(T) (= DSM 45146(T)) is an aerobic to facultative anaerobic, Gram-positive, non-spore-forming, non-motile rod-shaped bacterium that was isolated from the skin of the udder of a cow, in Schleswig Holstein, Germany. The cell wall of C. ulceribovis contains corynemycolic acids. The cellular fatty acids are those described for the genus Corynebacterium, but tuberculostearic acid is not present. Here we describe the features of C. ulceribovis strain IMMIB L-1395(T), together with genome sequence information and its annotation. The 2,300,451 bp long genome containing 2,104 protein-coding genes and 54 RNA-encoding genes and is part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG) project.

  8. High quality draft genome sequence of Corynebacterium ulceribovis type strain IMMIB-L1395T (DSM 45146T)

    SciTech Connect

    Yassin, Atteyet F.; Lapidus, Alla; Han, James; Reddy, T. B. K.; Huntemann, Marcel; Pati, Amrita; Ivanova, Natalia; Markowitz, Victor; Woyke, Tanja; Klenk, Hans-Peter; Kyrpides, Nikos C.

    2015-08-05

    We report that the Corynebacterium ulceribovis strain IMMIB L-1395T (= DSM 45146T) is an aerobic to facultative anaerobic, Gram-positive, non-spore-forming, non-motile rod-shaped bacterium that was isolated from the skin of the udder of a cow, in Schleswig Holstein, Germany. The cell wall of C. ulceribovis contains corynemycolic acids. The cellular fatty acids are those described for the genus Corynebacterium, but tuberculostearic acid is not present. Here we describe the features of C. ulceribovis strain IMMIB L-1395T, together with genome sequence information and its annotation. The 2,300,451 bp long genome containing 2,104 protein-coding genes and 54 RNA-encoding genes and is part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG) project.

  9. High quality draft genome sequence of Corynebacterium ulceribovis type strain IMMIB-L1395T (DSM 45146T)

    DOE PAGES

    Yassin, Atteyet F.; Lapidus, Alla; Han, James; ...

    2015-08-05

    We report that the Corynebacterium ulceribovis strain IMMIB L-1395T (= DSM 45146T) is an aerobic to facultative anaerobic, Gram-positive, non-spore-forming, non-motile rod-shaped bacterium that was isolated from the skin of the udder of a cow, in Schleswig Holstein, Germany. The cell wall of C. ulceribovis contains corynemycolic acids. The cellular fatty acids are those described for the genus Corynebacterium, but tuberculostearic acid is not present. Here we describe the features of C. ulceribovis strain IMMIB L-1395T, together with genome sequence information and its annotation. The 2,300,451 bp long genome containing 2,104 protein-coding genes and 54 RNA-encoding genes and is partmore » of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG) project.« less

  10. Draft Genome Sequence of Corynebacterium pseudotuberculosis Strain PA06 Isolated from a Subauricular Abscess in an Ovine Host

    PubMed Central

    de Moura, Vitória Almeida Gonçalves; Lima, Alyne Cristina Sodré; Paixão, Carla Thais Moreira; Lobato, Amália Raiana Fonseca; Alves, Jorianne Thyeska Castro; Guaraldi, Ana Luiza de Mattos; Folador, Adriana Ribeiro Carneiro; Ramos, Rommel T. J.; Silva, Artur

    2017-01-01

    ABSTRACT We report here the draft genome sequence of Corynebacterium pseudotuberculosis PA06, isolated from a subauricular abscess in an ovine host. C. pseudotuberculosis is a worldwide pathogen of small and large ruminants. The genome comprises 2,320,074 bp, with a G+C content of 52.2%, 2,195 coding sequences, 48 tRNAs, and three rRNAs. PMID:28360159

  11. Usefulness of 16S rDNA sequencing for the diagnosis of infective endocarditis caused by Corynebacterium diphtheriae.

    PubMed

    Pathipati, Padmaja; Menon, Thangam; Kumar, Naveen; Francis, Thara; Sekar, Prem; Cherian, Kotturathu Mammen

    2012-08-01

    We report a rare case of infective endocarditis caused by Corynebacterium diphtheriae in an 8-year-old boy, 2 years after a right ventricular outflow tract reconstruction with a bovine Contegra valved conduit. The patient recovered well after an RV-PA conduit enblock explantation and replacement with an aortic homograft with antibiotic treatment. All bacteriological cultures of excised tissue and blood were negative. The aetiological agent was identified as C. diphtheriae subsp. gravis by 16s rDNA sequencing.

  12. A lack of genetic basis for biovar differentiation in clinically important Corynebacterium diphtheriae from whole genome sequencing.

    PubMed

    Sangal, Vartul; Burkovski, Andreas; Hunt, Alison C; Edwards, Becky; Blom, Jochen; Hoskisson, Paul A

    2014-01-01

    The differentiation of clinically important Corynebacterium diphtheriae into specific biovars is complex and phylogenetically unclear. Comparative genomic analyses of 17 strains indicate that the division of C. diphtheriae into different biovars does not correlate with the variation in the gene content in the relevant metabolic categories that are potentially involved in the biovar discrimination. The biochemical separation is also not supported by phylogenetic analyses, suggesting molecular methods of typing C. diphtheriae strains should be adopted much more widely.

  13. Draft Genome Sequence of Corynebacterium pseudotuberculosis Strain PA05 Isolated from an Ovine Host in Pará State, Brazil

    PubMed Central

    Lima, Alyne Cristina Sodré; de Moura, Vitória Almeida Gonçalves; Pinheiro, Kenny da Costa; Paixão, Carla Thais Moreira; da Costa, Wana Lailan Oliveira; Folador, Adriana Ribeiro Carneiro; Guaraldi, Ana Luiza de Mattos; Ramos, Rommel T. J.; Silva, Artur

    2017-01-01

    ABSTRACT We report here the draft genome sequence of Corynebacterium pseudotuberculosis PA05, isolated from an ovine host in Pará State, Brazil. C. pseudotuberculosis is an etiological agent of diseases with veterinary and medical importance. The genome contains 2,435,137 bp, a G+C content of 52.2%, 2,295 coding sequences, five pseudogenes, 53 tRNAs, and six rRNAs. PMID:28360158

  14. Draft Genome Sequence of Corynebacterium pseudotuberculosis Strain PA07 Biovar ovis, Isolated from a Sheep Udder in Amazonia

    PubMed Central

    Araújo, Fabrício Almeida; Marques, Joana Montezano; de Moura, Vitória Almeida Gonçalves; Schneider, Maria Paula Cruz; Andrade, Soraya Silva; Lima, Alyne Cristina Sodré; Folador, Adriana Ribeiro Carneiro; Silva, Artur

    2017-01-01

    ABSTRACT In this work, we present the draft genome sequence of Corynebacterium pseudotuberculosis strain PA07 biovar ovis, isolated from a caseous secretion from a sheep udder in Pará, Brazil. The genome contains 2,320,235 bp, 52.2% G+C content, 2,191 coding sequences (CDSs), five pseudogenes, 48 tRNAs, and three rRNAs. PMID:28336591

  15. Complete Genome Sequence of Corynebacterium pseudotuberculosis Strain CIP 52.97, Isolated from a Horse in Kenya

    PubMed Central

    Cerdeira, Louise Teixeira; Schneider, Maria Paula Cruz; Pinto, Anne Cybelle; de Almeida, Sintia Silva; dos Santos, Anderson Rodrigues; Barbosa, Eudes Guilherme Vieira; Ali, Amjad; Aburjaile, Flávia Figueira; de Abreu, Vinicius Augusto Carvalho; Guimarães, Luis Carlos; Soares, Siomar de Castro; Dorella, Fernanda Alves; Rocha, Flávia Souza; Bol, Erick; Gomes de Sá, Pablo Henrique Caracciolo; Lopes, Thiago Souza; Barbosa, Maria Silvanira; Carneiro, Adriana Ribeiro; Jucá Ramos, Rommel Thiago; Coimbra, Nilson Antônio da Rocha; Lima, Alex Ranieri Jerônimo; Barh, Debmalya; Jain, Neha; Tiwari, Sandeep; Raja, Rathiram; Zambare, Vasudeo; Ghosh, Preetam; Trost, Eva; Tauch, Andreas; Miyoshi, Anderson; Azevedo, Vasco; Silva, Artur

    2011-01-01

    In this work, we report the whole-genome sequence of Corynebacterium pseudotuberculosis bv. equi strain CIP 52.97 (Collection Institut Pasteur), isolated in 1952 from a case of ulcerative lymphangitis in a Kenyan horse, which has evidently caused significant losses to agribusiness. Therefore, obtaining this genome will allow the detection of important targets for postgenomic studies, with the aim of minimizing problems caused by this microorganism. PMID:22123771

  16. Complete genome sequence of Corynebacterium pseudotuberculosis strain CIP 52.97, isolated from a horse in Kenya.

    PubMed

    Cerdeira, Louise Teixeira; Schneider, Maria Paula Cruz; Pinto, Anne Cybelle; de Almeida, Sintia Silva; dos Santos, Anderson Rodrigues; Barbosa, Eudes Guilherme Vieira; Ali, Amjad; Aburjaile, Flávia Figueira; de Abreu, Vinicius Augusto Carvalho; Guimarães, Luis Carlos; Soares, Siomar de Castro; Dorella, Fernanda Alves; Rocha, Flávia Souza; Bol, Erick; Gomes de Sá, Pablo Henrique Caracciolo; Lopes, Thiago Souza; Barbosa, Maria Silvanira; Carneiro, Adriana Ribeiro; Jucá Ramos, Rommel Thiago; Coimbra, Nilson Antônio da Rocha; Lima, Alex Ranieri Jerônimo; Barh, Debmalya; Jain, Neha; Tiwari, Sandeep; Raja, Rathiram; Zambare, Vasudeo; Ghosh, Preetam; Trost, Eva; Tauch, Andreas; Miyoshi, Anderson; Azevedo, Vasco; Silva, Artur

    2011-12-01

    In this work, we report the whole-genome sequence of Corynebacterium pseudotuberculosis bv. equi strain CIP 52.97 (Collection Institut Pasteur), isolated in 1952 from a case of ulcerative lymphangitis in a Kenyan horse, which has evidently caused significant losses to agribusiness. Therefore, obtaining this genome will allow the detection of important targets for postgenomic studies, with the aim of minimizing problems caused by this microorganism.

  17. Native Valve Endocarditis due to Corynebacterium striatum confirmed by 16S Ribosomal RNA Sequencing: A Case Report and Literature Review

    PubMed Central

    2016-01-01

    Corynebacterium species are non-fermentous Gram-positive bacilli that are normal flora of human skin and mucous membranes and are commonly isolated in clinical specimens. Non-diphtheriae Corynebacterium are regarded as contaminants when found in blood culture. Currently, Corynebacterium striatum is considered one of the emerging nosocomial agents implicated in endocarditis and serious infections. We report a case of native-valve infective endocarditis caused by C. striatum, which was misidentified by automated identification system but identified accurately by 16S ribosomal RNA sequencing, in a 55-year-old male patient. The patient had two mobile vegetations on his mitral valve, both of which had high embolic risk. Through surgical valve replacement and an antibiotic regimen, the patient recovered completely. In unusual clinical scenarios, C. striatum should not be simply dismissed as a contaminant when isolated from clinical specimens. The possibility of C. striatum infection should be considered even in an immunocompetent patient, and we suggest a genotypic assay, such as 16S rRNA sequencing, to confirm species identity. PMID:27659439

  18. Dissociative recombination in aeronomy

    NASA Technical Reports Server (NTRS)

    Fox, J. L.

    1989-01-01

    The importance of dissociative recombination in planetary aeronomy is summarized, and two examples are discussed. The first is the role of dissociative recombination of N2(+) in the escape of nitrogen from Mars. A previous model is updated to reflect new experimental data on the electronic states of N produced in this process. Second, the intensity of the atomic oxygen green line on the nightside of Venus is modeled. Use is made of theoretical rate coefficients for production of O (1S) in dissociative recombination from different vibrational levels of O2(+).

  19. Draft Genome Sequences of Corynebacterium kroppenstedtii CNM633/14 and CNM632/14, Multidrug-Resistant and Antibiotic-Sensitive Isolates from Nodules of Granulomatous Mastitis Patients

    PubMed Central

    Soriano, Francisco; Ariza-Miguel, Jaime; Marrodan-Ciordia, Teresa; Acedo, Alberto; Hernandez, Marta; Tauch, Andreas

    2015-01-01

    Corynebacterium kroppenstedtii has been associated with infections of the female breast. Genome sequencing of two strains revealed a specific genomic island in the multidrug-resistant isolate CNM633/14 with similarity to the R plasmid pJA144188 of Corynebacterium resistens DSM 45100, being indicative of the horizontal transfer of antibiotic resistance genes to C. kroppenstedtii. PMID:25999560

  20. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  1. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  2. Multiphoton Assisted Recombination

    NASA Astrophysics Data System (ADS)

    Shuman, E. S.; Jones, R. R.; Gallagher, T. F.

    2008-12-01

    We have observed multiphoton assisted recombination in the presence of a 38.8 GHz microwave field. Stimulated emission of up to ten microwave photons results in energy transfer from continuum electrons, enabling recombination. The maximum electron energy loss is far greater than the 2Up predicted by the standard “simpleman’s” model. The data are well reproduced by both an approximate analytic expression and numerical simulations in which the combined Coulomb and radiation fields are taken into account.

  3. Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris

    PubMed Central

    Gurkan, Cemal; Ellar, David J

    2005-01-01

    The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins. The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promoter that is particularly suitable for heterologous expression of foreign genes. While recombinant production of bacterial toxins and their derivatives is highly desirable, attempts at their heterologous expression using the traditional E. coli expression system can be problematic due to the formation of inclusion bodies that often severely limit the final yields of biologically active products. However, recent literature now suggests that P. pastoris may be an attractive alternative host for the heterologous production of bacterial toxins, such as those from the genera Bacillus, Clostridium, and Corynebacterium, as well as their more complex derivatives. Here, we review the recombinant production of bacterial toxins and their derivatives in P. pastoris with special emphasis on their potential clinical applications. Considering that de novo design and construction of synthetic toxin genes have often been necessary to achieve optimal heterologous expression in P. pastoris, we also present general guidelines to this end based on our experience with the P. pastoris expression of the Bacillus thuringiensis Cyt2Aa1 toxin. PMID:16336647

  4. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  5. Atom Recombination on Surface

    NASA Astrophysics Data System (ADS)

    Kim, Young Chai

    Upon high speed re-entry of the Space Shuttle Orbiter (SSO) through the earth's atmosphere, oxygen and nitrogen atoms produced in the shock wave in front of the SSO recombine on the surface of the SSO, releasing heat. To minimize the rise of surface temperature due to the reaction, surface material of the SSO should have a low recombination probability, gamma, of atoms impinging on it. To design such material, it is necessary to understand the mechanism of atom recombination. With this in mind, gamma values were measured for recombination of O, N, and H atoms in a diffusion tube reactor between 700 and 1250 K (HT), 300 and 700 K (MT), and at 194 K (LT) on silica. The rate of recombination was first order with respect to the atom concentration from LT to HT. The Arrhenius plots, gamma vs. 1/T, were very complex. All observations are explained by assuming a surface with a small fraction of active sites that irreversibly bind chemisorbed atoms. Everything happens as if the active sites were surrounded by collection zones within which all atoms striking the surface are adsorbed reversibly with an assumed sticking probability of unity. These atoms then diffuse on the surface. Some of them reach the active sites where they can recombine with the chemisorbed atoms. At LT, all atoms striking the surface reach the active sites. As a result of desorption at MT, the collection zones shrink with increasing temperature. At HT, only atoms striking active sites directly from the gas phase lead to recombination. An analytical solution of the diffusion-reaction problem obtained for a model where the active sites are distributed uniformly fits with the experimental data from LT to HT. The two novel features of this work are the identification of the active sites on silica for recombination of H on silica at HT as surface OH groups and the suggestion that another kind of active site is responsible for recombination of O and N atoms at HT as well as for H atoms at LT and MT. Although

  6. Improvement of the ammonia assimilation for enhancing L-arginine production of Corynebacterium crenatum.

    PubMed

    Guo, Jing; Man, Zaiwei; Rao, Zhiming; Xu, Meijuan; Yang, Taowei; Zhang, Xian; Xu, Zhenghong

    2017-03-01

    There are four nitrogen atoms in L-arginine molecule and the nitrogen content is 32.1%. By now, metabolic engineering for L-arginine production strain improvement was focused on carbon flux optimization. In previous work, we obtained an L-arginine-producing Corynebacterium crenatum SDNN403 (ARG) through screening and mutation breeding. In this paper, a strain engineering strategy focusing on nitrogen supply and ammonium assimilation for L-arginine production was performed. Firstly, the effects of nitrogen atom donor (L-glutamate, L-glutamine and L-aspartate) addition on L-arginine production of ARG were studied, and the addition of L-glutamine and L-aspartate was beneficial for L-arginine production. Then, the glutamine synthetase gene glnA and aspartase gene aspA from E. coli were overexpressed in ARG for increasing the L-glutamine and L-aspartate synthesis, and the L-arginine production was effectively increased. In addition, the L-glutamate supply re-emerged as a limiting factor for L-arginine biosynthesis. Finally, the glutamate dehydrogenase gene gdh was co-overexpressed for further enhancement of L-arginine production. The final strain could produce 53.2 g l(-1) of L-arginine, which was increased by 41.5% compared to ARG in fed-batch fermentation.

  7. Corynebacterium bovis: epizootiologic features and environmental contamination in an enzootically infected rodent room.

    PubMed

    Burr, Holly N; Wolf, Felix R; Lipman, Neil S

    2012-03-01

    Corynebacterium bovis is a common pathogen in athymic nude mouse colonies. Control and eradication of the organism are challenging because depopulation and restricted colony access are often not options within vivaria. We evaluated potential sources and dissemination routes of C. bovis in an enzootically infected colony. Immunocompetent mice and personnel were evaluated for their potential to carry C. bovis, and husbandry and sanitation methods were evaluated for their efficacy in preventing cross-contamination. C. bovis was detected in furred immunocompetent mice previously exposed to infected athymic nude mice and in the nasopharynx of humans. Microisolation cages were not effective in maintaining athymic nude mice C. bovis-free when they were housed in a room known to contain immunodeficient mice with C. bovis infections. A tunnel washer that provided a ≥180 °F final rinse provided effective elimination of C. bovis from cage components. Passive and active air sampling techniques showed airborne dispersal of C. bovis despite the use of individually ventilated caging systems and stringent operational standards. Bacterial growth was not observed in settle plates placed inside autoclaved individually ventilated microisolation cages on various ventilated racks for 24-h periods. C. bovis aerosolization was shown to be a means of spread of the bacterium during cage-change procedures inside a class II type A2 biosafety cabinet. Our findings indicate that C. bovis can be a pervasive environmental contaminant in infected rodent holding rooms and successful eradication strategies must include environmental decontamination and attention to air quality.

  8. Induction of the NFκ-B signal transduction pathway in response to Corynebacterium diphtheriae infection.

    PubMed

    Ott, Lisa; Scholz, Brigitte; Höller, Martina; Hasselt, Kristin; Ensser, Armin; Burkovski, Andreas

    2013-01-01

    Corynebacterium diphtheriae, the causative agent of diphtheria, has been thoroughly studied with respect to toxin production and pili formation, while knowledge on host responses to C. diphtheriae infection is limited. In this study, we studied adhesion to and invasion of epithelial cells by different C. diphtheriae isolates. When NFκ-B reporter cell lines were used to monitor the effect of C. diphtheriae infection on human cells, strain-specific differences were observed. While adhesion to host cells had no effect, a correlation of invasion rate with NFκ-B induction was found, which indicates that internalization of bacteria is crucial for NFκ-B induction. Immunofluorescence microscopy experiments used to support the reporter assays showed that translocation of p65, as a hallmark of NFκ-B induction, was only observed in association with cell invasion by C. diphtheriae. Our data indicate that the response of epithelial cells to C. diphtheriae infection is determined by internalization of bacteria and that invasion of these cells is an active process; tetracycline-treated C. diphtheriae was still able to attach to host cells, but lost its ability to invade the cytoplasm. Recognition of pathogen-associated molecular patterns such as pili subunits by membrane-bound receptors facing the outside of the cell is not sufficient for NFκ-B induction.

  9. Genome organization and pathogenicity of Corynebacterium diphtheriae C7(-) and PW8 strains.

    PubMed

    Iwaki, Masaaki; Komiya, Takako; Yamamoto, Akihiko; Ishiwa, Akiko; Nagata, Noriyo; Arakawa, Yoshichika; Takahashi, Motohide

    2010-09-01

    Corynebacterium diphtheriae is the causative agent of diphtheria. In 2003, the complete genomic nucleotide sequence of an isolate (NCTC13129) from a large outbreak in the former Soviet Union was published, in which the presence of 13 putative pathogenicity islands (PAIs) was demonstrated. In contrast, earlier work on diphtheria mainly employed the C7(-) strain for genetic analysis; therefore, current knowledge of the molecular genetics of the bacterium is limited to that strain. However, genomic information on the NCTC13129 strain has scarcely been compared to strain C7(-). Another important C. diphtheriae strain is Park-Williams no. 8 (PW8), which has been the only major strain used in toxoid vaccine production and for which genomic information also is not available. Here, we show by comparative genomic hybridization that at least 37 regions from the reference genome, including 11 of the 13 PAIs, are considered to be absent in the C7(-) genome. Despite this, the C7(-) strain still retained signs of pathogenicity, showing a degree of adhesion to Detroit 562 cells, as well as the formation of and persistence in abscesses in animal skin comparable to that of the NCTC13129 strain. In contrast, the PW8 strain, suggested to lack 14 genomic regions, including 3 PAIs, exhibited more reduced signs of pathogenicity. These results, together with great diversity in the presence of the 37 genomic regions among various C. diphtheriae strains shown by PCR analyses, suggest great heterogeneity of this pathogen, not only in genome organization, but also in pathogenicity.

  10. Microbiological changes and diversity in autochthonous non-toxigenic Corynebacterium diphtheriae isolated in France.

    PubMed

    Farfour, E; Badell, E; Dinu, S; Guillot, S; Guiso, N

    2013-10-01

    Autochtonous toxigenic Corynebacterium diphtheriae have disappeared in mainland France, but non-toxigenic C. diphtheriae are still circulating. Using phenotypic and molecular tools, we retrospectively characterized 103 non-toxigenic C. diphtheriae collected in mainland France and highlight several changes. The proportion of C. diphtheriae belfanti increased between 1977 and 2011 and it is the most frequent biotype recovered in recent years. Resistance to ciprofloxacin has increased and most isolates with decreased sensitivity belong to the belfanti biotype. Using multilocus sequence typing, we demonstrate that French isolates are distributed in a large number of sequence types and identify three distinct lineages. C. diphtheriae mitis and gravis form lineage I while C. diphtheriae belfanti forms lineages II and III. Almost all isolates of lineage II are part of a unique clonal complex or are very close to it. Most French isolates have a dtxR sequence homologous to that of toxigenic isolates, suggesting that if lyzogenised by a corynephage, they can express diphtheria toxin.

  11. Opposite nucleotide usage biases in different parts of the Corynebacterium diphtheriae spaC gene.

    PubMed

    Khrustalev, Vladislav Victorovich; Barkovsky, Eugene Victorovich; Kolodkina, Valentina Leonidovna; Khrustaleva, Tatyana Aleksandrovna

    2015-01-01

    In this work we described a bacterial open reading frame with two different directions of nucleotide usage biases in its two parts. The level of GC-content in third codon positions (3GC) is equal to 40.17 ± 0.22% during the most of the length of Corynebacterium diphtheriae spaC gene. However, in the 3'-end of the same gene (from codon #1600 to codon #1873) 3GC level is equal to 64.61 ± 0.91%. Using original methodology ('VVTAK Sliding window' and 'VVTAK VarInvar') we approved that there is an ongoing mutational AT-pressure during the most of the length of spaC gene (up to codon #1599), and there is an ongoing mutational G-pressure in the 3′-end of spaC. Intragenic promoters predicted by three different methods may be the cause of the differences in preferable types of nucleotide mutations in spaC parts because of their autonomous transcription.

  12. Identification of Corynebacterium diphtheriae gene involved in adherence to epithelial cells.

    PubMed

    Kolodkina, Valentina; Denisevich, Tatyana; Titov, Leonid

    2011-03-01

    Corynebacterium diphtheriae the causative pathogen of human diphtheria infects the nasopharynx or skin. Although diphtheria has been extensively studied, little is known about the two key aspects of C. diphtheriae invasiveness: colonization and invasion. The role of adhesive properties in establishing the infection of C. diphtheriae strains, independent of toxin production, still needs to be clarified. In this study, we describe a novel gene involved in adherence to epithelial cells. Transformation of C. diphtheriae 225, biotype gravis, ribotype St-Petersburg by EZ:TN(KAN-2)Tnp Transposome was undertaken. A C. diphtheriae 225 Tn5 insertion library of 2800 mutants was created. Five hundred and eighty five transformants were qualitatively screened for reduced adherence to HEp-2 cells by an adherence assay. One mutant strain consistently exhibiting 15.2% of the wild-type adherence was isolated. The DNA flanking the transposon was identified by inverse PCR and subsequent sequencing. The disrupted gene was 94% identical to the C. diphtheriae DIP1621 gene that belongs to unclassified genes. In conclusion, the disruption of the C. diphtheriae DIP1621 gene led to decreased adherence to epithelial cells; its exact function remains to be established.

  13. Regulation and activity of a zinc uptake regulator, Zur, in Corynebacterium diphtheriae.

    PubMed

    Smith, Kelsy F; Bibb, Lori A; Schmitt, Michael P; Oram, Diana M

    2009-03-01

    Regulation of metal ion homeostasis is essential to bacterial cell survival, and in most species it is controlled by metal-dependent transcriptional regulators. In this study, we describe a Corynebacterium diphtheriae ferric uptake regulator-family protein, Zur, that controls expression of genes involved in zinc uptake. By measuring promoter activities and mRNA levels, we demonstrate that Zur represses transcription of three genes (zrg, cmrA, and troA) in zinc-replete conditions. All three of these genes have similarity to genes involved in zinc uptake. Transcription of zrg and cmrA was also shown to be regulated in response to iron and manganese, respectively, by mechanisms that are independent of Zur. We demonstrate that the activity of the zur promoter is slightly decreased under low zinc conditions in a process that is dependent on Zur itself. This regulation of zur transcription is distinctive and has not yet been described for any other zur. An adjacent gene, predicted to encode a metal-dependent transcriptional regulator in the ArsR/SmtB family, is transcribed from a separate promoter whose activity is unaffected by Zur. A C. diphtheriae zur mutant was more sensitive to peroxide stress, which suggests that zur has a role in protecting the bacterium from oxidative damage. Our studies provide the first evidence of a zinc specific transcriptional regulator in C. diphtheriae and give new insights into the intricate regulatory network responsible for regulating metal ion concentrations in this toxigenic human pathogen.

  14. Pathogenic properties of a Corynebacterium diphtheriae strain isolated from a case of osteomyelitis.

    PubMed

    Peixoto, Renata Stavracakis; Hacker, Elena; Antunes, Camila Azevedo; Weerasekera, Dulanthi; Dias, Alexandre Alves de Souza de Oliveira; Martins, Carlos Alberto; Hirata, Raphael; Santos, Kátia Regina Netto Dos; Burkovski, Andreas; Mattos-Guaraldi, Ana Luíza

    2016-11-01

    Corynebacterium diphtheriae is typically recognized as a colonizer of the upper respiratory tract (respiratory diphtheria) and the skin (cutaneous diphtheria). However, different strains of Corynebacteriumdiphtheriae can also cause invasive infections. In this study, the characterization of a non-toxigenic Corynebacteriumdiphtheriae strain (designated BR-INCA5015) isolated from osteomyelitis in the frontal bone of a patient with adenoid cystic carcinoma was performed. Pathogenic properties of the strain BR-INCA5015 were tested in a Caenorhabditis elegans survival assay showing strong colonization and killing by this strain. Survival rates of 3.8±2.7 %, 33.6±7.3 % and 0 % were observed for strains ATCC 27010T, ATCC 27012 and BR-INCA5015, respectively, at day 7. BR-INCA5015 was able to colonize epithelial cells, showing elevated capacity to adhere to and survive within HeLa cells compared to other Corynebacteriumdiphtheriae isolates. Intracellular survival in macrophages (THP-1 and RAW 264.7) was significantly higher compared to control strains ATCC 27010T (non-toxigenic) and ATCC 27012 (toxigenic). Furthermore, the ability of BR-INCA5015 to induce osteomyelitis was confirmed by in vivo assay using Swiss Webster mice.

  15. Characterization of DIP0733, a multi-functional virulence factor of Corynebacterium diphtheriae.

    PubMed

    Antunes, Camila Azevedo; Sanches dos Santos, Louisy; Hacker, Elena; Köhler, Stefanie; Bösl, Korbinian; Ott, Lisa; de Luna, Maria das Graças; Hirata, Raphael; Azevedo, Vasco Ariston de Carvalho; Mattos-Guaraldi, Ana-Luíza; Burkovski, Andreas

    2015-03-01

    Corynebacterium diphtheriae is typically recognized as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen-host interaction. The molecular mechanisms controlling and facilitating internalization of Cor. diphtheriae are poorly understood. In this study, we investigated the role of DIP0733 as virulence factor to elucidate how it contributes to the process of pathogen-host cell interaction. Based on in vitro experiments, it was suggested recently that the DIP0733 protein might be involved in adhesion, invasion of epithelial cells and induction of apoptosis. A corresponding Cor. diphtheriae mutant strain generated in this study was attenuated in its ability to colonize and kill the host in a Caenorhabditis elegans infection model system. Furthermore, the mutant showed an altered adhesion pattern and a drastically reduced ability to adhere and invade epithelial cells. Subsequent experiments showed an influence of DIP0733 on binding of Cor. diphtheriae to extracellular matrix proteins such as collagen and fibronectin. Furthermore, based on its fibrinogen-binding activity, DIP0733 may play a role in avoiding recognition of Cor. diphtheriae by the immune system. In summary, our findings support the idea that DIP0733 is a multi-functional virulence factor of Cor. diphtheriae.

  16. [The sensitivity to antibiotics of biofilm cultures of toxigenic strains Corynebacterium diphtheriae].

    PubMed

    Frolova, Ya N; Kharseyeva, G G; Mironov, A Yu

    2014-06-01

    The article presents analysis of sensitivity to antibacterial preparations of typical and biofilm culture of museum strain of Corynebacterium diphtheriae gravis tox+ SV-665. The strain was obtained from the L.A. Tarasevitch state research institute of standardization and control of medical biological preparations. The second strain C. diphtheriaecirculates gravis tox+ circulates in population of the Rostov oblast and it was recovered from patient with diagnosis of "localized form of diphtheria" by bacteriologic laboratory "1002 CGSEN SKVO" of Rostov-on-Don. The week and month biofilm cultures of both strains of C. diphtheriae gravis tox+ were used. The sensitivity to antibacterial preparations of typical and biofilm cultures of museum and circulating in population strains of agent of diphtheria were detected using minimal suppressing concentration by technique of serial dilutions in fluid growth medium. It is demonstrated that the most effective in respect of C. diphtheriae are such preparations as cefotaxinum, gentamycinum, lincomycin, canamycin and cefasolin. The sensitivity of pathogen in composition of biofilm to these preparations has no changes.

  17. Process optimization for an industrial-scale production of Diphtheria toxin by Corynebacterium diphtheriae PW8.

    PubMed

    Suwanpatcharakul, Maethichai; Pakdeecharoen, Chompunut; Visuttitewin, Supitcha; Pesirikan, Norapath; Chauvatcharin, Somchai; Pongtharangkul, Thunyarat

    2016-11-01

    In this study, several parameters affecting the toxin production of Corynebacterium diphtheriae Parke Williams 8 (PW8) were investigated in detail. The comparison studies of amino acid profile in NZ Amine A-based medium (NZ medium) and beef digest-based medium (BD medium) suggested that an insufficient supply of amino acids was not responsible for low toxin yield observed in NZ medium. Supplementation of additional amino acids and growth promoting nutrient (in a form of yeast extract) into NZ medium enhanced only cell growth but not toxin production. Thus, BD medium was selected as the most suitable base medium for toxin production as it gave a significantly higher limit of flocculation (93 ± 0 Lf/ml) than NZ medium (46 ± 0 Lf/ml). Interestingly, a supplementation of 0.2% YE into BD medium resulted in a significant increase in growth as well as toxin production (235 ± 5 Lf/ml). In conclusion, consistently high toxin titer (174-239 Lf/ml) could be obtained from BD medium at a 5 L-scale production as long as 1) the protein content of BD medium was at least 24 g/L, 2) the iron content was below 0.15 ppm and 3) 0.2% YE was supplemented into the medium.

  18. New diphtheria toxin repressor types depicted in a Romanian collection of Corynebacterium diphtheriae isolates.

    PubMed

    Dinu, Sorin; Damian, Maria; Badell, Edgar; Dragomirescu, Cristiana Cerasella; Guiso, Nicole

    2014-10-01

    Corynebacterium diphtheriae is the etiological agent of diphtheria, a potential fatal disease caused by a corynephage toxin. The expression of this diphtheria toxin is controlled via an iron-dependent repressor with various functions (DtxR). Some mutations in the dtxR gene are associated with diminished activity or even with total loss of DtxR function. We conducted a molecular study to characterize the dtxR alleles harbored by 34 isolates of C. diphtheriae recovered from Romanian patients between 1961 and 2007. Three of the seven alleles identified in this study have not previously been described. Two new DtxR types were identified, one of which has an unusual polypeptide length. All the new DtxR types were found in toxigenic isolates, suggesting that they effectively regulate the expression of diphtheria toxin. Furthermore, one of the new DtxR identified was also found in a non-toxigenic isolate, making it a potential source of toxigenic isolates after lysogenic conversion.

  19. Serology and clinical relevance of Corynebacterium pseudotuberculosis in native Korean goats (Capra hircus coreanae).

    PubMed

    Jung, Byeong Yeal; Lee, Seung-Hun; Kim, Ha-Young; Byun, Jae-Won; Shin, Dong-Ho; Kim, Daekeun; Kwak, Dongmi

    2015-04-01

    This study was conducted to assess the seroprevalence and clinical relevance of Corynebacterium pseudotuberculosis, which is the causative agent of caseous lymphadenitis (CLA), in native Korean goats (Capra hircus coreanae). A total of 466 native Korean goats from 40 herds (11 to 12 samples per herd) were randomly selected throughout the nation and evaluated by direct palpation, bacterial isolation, ELISA, and PCR. In serological examinations, 267 (57.3 %) of the goats tested were positive against C. pseudotuberculosis. When seroprevalence was analyzed according to age, region, and season, statistically significant differences were observed in relation to all three parameters (P < 0.05). For clinical examination, the superficial lymph nodes of all goats were palpated to diagnose CLA. Pus samples taken from superficial abscesses were used for bacterial isolation. Among the 466 goats tested, 34 (7.3 %) were presumptively diagnosed with CLA, and C. pseudotuberculosis was isolated from 24 goats (70.6 % of goats with CLA lesions) whose infections were confirmed by PCR. Considering the high seroprevalence and bacterial isolation rate from most of the superficial CLA lesions, it is suspected that many internal CLA lesions exist in this goat population. These results suggest that C. pseudotuberculosis infection is widespread in native Korean goats, and appropriate control programs need to be established.

  20. Detection and Elimination of Corynebacterium bovis from Barrier Rooms by Using an Environmental Sampling Surveillance Program.

    PubMed

    Manuel, Christopher; Pugazhenthi, Umarani; Spiegel, Shannon; Leszczynski, Jori

    2017-02-16

    Rodent health-monitoring programs based on sampling an IVC system's exhaust air dust (EAD) has enhanced and evenreplaced traditional sentinels for some rodent pathogens. EAD testing by qPCR assay is an optimal surveillance methodfor the rapid detection of Corynebacterium bovis-infected immunodeficient mice. Here we demonstrate that an active EADsurveillance program for C. bovis can be used to maintain nude mice C. bovis-free after the transition from historically enzootically infected colonies. During 3 events over 3 y, rapid detection of infection, elimination of infected mice, aggressivequarantine measures, and local decontamination prevented the spread of C. bovis within 2 barrier rooms. In total, 4 cages ofinfected nude mice were identified and removed, preventing the spread of infection to 469 other cages of immunodeficientmice. In addition, we present data regarding a refinement to EAD testing which enables row-specific surveillance of an IVCrack. This technique systemically decreases the amount of testing required to locate an individually infected cage. Due to ourability to rapidly detect and localize an infected cage, we were able to investigate the route of C. bovis introduction into ourbarrier rooms. Our epidemiologic investigation suggested that the transmission of C. bovis occurred through contaminated,cryopreserved, patient-derived xenograft tumor tissue. This previously unknown source of C. bovis can infect mice used topropagate these tumors. Together, these data demonstrate that a remediation program that combines rapid detection, testand-cull, and local decontamination under quarantine conditions can eliminate C. bovis from a mouse colony.

  1. Procedure for Horizontal Transfer of Patient-Derived Xenograft Tumors to Eliminate Corynebacterium bovis.

    PubMed

    Manuel, Christopher; Bagby, Stacey; Reisinger, Julie; Pugazhenthi, Umarani; Pitts, Todd; Keysar, Stephen; Arcaroli, John; Leszczynski, Jori

    2017-02-16

    Human patient-derived xenograft (PDX) tumors, propagated in immunodeficient mice, are rapidly growing in use as amodelfor cancer research. Horizontal transfer between mice, without in vitro cell culture, allows these tumors to retainmany of their unique characteristics from their individual patient of origin. However, the immunodeficient mouse strainsused to grow these tumors are susceptible to numerous opportunistic pathogens, including Corynebacterium bovis. At ourinstitution, 2 in vivo tumor banks of PDX tumors had been maintained within nude mouse colonies enzootically infectedwith C. bovis. Elimination of C. bovis from these colonies required the aseptic harvest and horizontal transfer of tumor tissue between infected and naïve recipient mice without cross-contamination. Out of necessity, we developed a standard operating procedure using enhancements to traditional aseptic surgical technique with concurrent application of both procedural and physical barriers to prevent C. bovis transmission. By using these methods, all 61 unique PDX tumor models were successfullyharvested from C. bovis-infected mice and transferred into recipient mice without transmission of infection. Our datademonstrate that, in situations where C. bovis-free colonies can be established and maintained, this procedure can successfullybe used to eliminate C. bovis from an in vivo tumor bank of valuable PDX tumors.

  2. Procedure for Horizontal Transfer of Patient-Derived Xenograft Tumors to Eliminate Corynebacterium bovis.

    PubMed

    Manuel, Christopher A; Bagby, Stacey M; Reisinger, Julie A; Pugazhenthi, Umarani; Pitts, Todd M; Keysar, Stephen B; Arcaroli, John J; Leszczynski, Jori K

    2017-03-01

    Human patient-derived xenograft (PDX) tumors, propagated in immunodeficient mice, are rapidly growing in use as a model for cancer research. Horizontal transfer between mice, without in vitro cell culture, allows these tumors to retain many of their unique characteristics from their individual patient of origin. However, the immunodeficient mouse strains used to grow these tumors are susceptible to numerous opportunistic pathogens, including Corynebacterium bovis. At our institution, 2 in vivo tumor banks of PDX tumors had been maintained within nude mouse colonies enzootically infected with C. bovis. Elimination of C. bovis from these colonies required the aseptic harvest and horizontal transfer of tumor tissue between infected and naïve recipient mice without cross-contamination. Out of necessity, we developed a standard operating procedure using enhancements to traditional aseptic surgical technique with concurrent application of both procedural and physical barriers to prevent C. bovis transmission. By using these methods, all 61 unique PDX tumor models were successfully harvested from C. bovis-infected mice and transferred into recipient mice without transmission of infection. Our data demonstrate that, in situations where C. bovis-free colonies can be established and maintained, this procedure can successfully be used to eliminate C. bovis from an in vivo tumor bank of valuable PDX tumors.

  3. Rapid emergence of daptomycin resistance in clinical isolates of Corynebacterium striatum… a cautionary tale.

    PubMed

    McElvania TeKippe, E; Thomas, B S; Ewald, G A; Lawrence, S J; Burnham, C-A D

    2014-12-01

    The objective of this study was to investigate the observation of daptomycin resistance in Corynebacterium striatum, both in vivo and in vitro. We describe a case of C. striatum bacteremia in a patient with a left ventricular assist device (LVAD); the initial isolate recovered was daptomycin susceptible with a minimum inhibitory concentration (MIC) of 0.125 μg/ml. Two months later, and after daptomycin therapy, the individual became bacteremic with an isolate of C. striatum with a daptomycin MIC of >256 μg/ml. To study the prevalence of daptomycin resistance in C. striatum, clinical isolates of C. striatum were grown in broth culture containing daptomycin to investigate the emergence of resistance to this antimicrobial. Molecular typing was used to evaluate serial isolates from the index patient and the clinical isolates of C. striatum we assayed. In vitro analysis of isolates from the index patient and 7 of 11 additional C. striatum isolates exhibited the emergence of high-level daptomycin resistance, despite initially demonstrating low MICs to this antimicrobial agent. This phenotype was persistent even after serial subculture in the absence of daptomycin. Together, these data demonstrate that caution should be taken when using daptomycin to treat high-inoculum infections and/or infections of indwelling medical devices with C. striatum. To our knowledge, this is the first report characterizing the emergence of daptomycin resistance in C. striatum.

  4. [Survival capacity of Corynebacterium pseudotuberculosis biovar ovis in different soil types from Chubut, Argentine Patagonia].

    PubMed

    Alvarez, Laura; William, Aillin; Castro, Isabel; Valenzuela, Fernanda; Estevao Belchior, Silvia

    Corynebacterium pseudotuberculosis is transmitted among sheep in Argentine Patagonia causing pseudotuberculosis. The bacterium penetrates the skin or mucous membrane wounds, infecting the superficial lymph nodes and viscera. When surface abscesses are cut during shearing, they drain their purulent contents and contaminate tools and the soil. The objective of this work was to evaluate the survival capacity of C. pseudotuberculosis over time, in soils from the extra-Andean Patagonia region. Five types of superficial soils were collected from different areas in Chubut province (extra-Andean Patagonia), having distinctive physicochemical properties including organic matter content (very high to nonexistent), pH (neutral to strongly alkaline), electrical conductivity (saline to non-saline) and texture (sandy, clayey, silty loam). Different aliquots of each type of soil were inoculated with C. pseudotuberculosis PAT10 strain isolated from a Patagonian sheep, and were stored at room temperature. The number of surviving bacteria was determined at various times. Sixty percent (60%) of the inoculated C. pseudotuberculosis population survived for 80 to 210 days in soils with moderate to high organic matter content respectively. Silty soils favored bacterial survival, whereas the variables pH and salinity had no effect on survival.

  5. Reproductive Pathological Changes Associated with Experimental Subchronic Corynebacterium pseudotuberculosis Infection in Nonpregnant Boer Does

    PubMed Central

    Othman, A. M.; Abba, Y.; Jesse, F. F. A.; Ilyasu, Y. M.; Saharee, A. A.; Haron, A. W.; Zamri-Saad, M.; Lila, M. A. M.

    2016-01-01

    Corynebacterium pseudotuberculosis causes caseous lymphadenitis (CLA), which is a contagious and chronic disease in sheep and goats. In order to assess the histopathological changes observed in the reproductive organs of nonpregnant does infected with the bacteria, 20 apparently healthy adult Boer does were divided into four inoculation groups, intradermal, intranasal, oral, and control, consisting of five goats each. Excluding the control group, which was unexposed, other does were inoculated with 107 CFU/1 mL of live C. pseudotuberculosis through the various routes stated above. Thirty days after infection, the ovaries, uterus, and iliac lymph nodes were collected for bacterial recovery and molecular detection, as well as histopathological examination. The mean changes in necrosis, congestion, inflammatory cell infiltration, and oedema varied in severity among the ovaries, uterus, and iliac lymph nodes following different inoculation routes. Overall, the intranasal route of inoculation showed more severe (p < 0.05) lesions in all the organs examined. The findings of this study have shown that C. pseudotuberculosis could predispose to infertility resulting from pathological lesions in the uterus and ovaries of does. PMID:27006831

  6. Evaluation of three methods for DNA fingerprinting of Corynebacterium pseudotuberculosis strains isolated from goats in Poland.

    PubMed

    Stefańska, Ilona; Rzewuska, Magdalena; Binek, Marian

    2008-01-01

    Phenotypic approaches based on metabolic and biological characteristics of Corynebacterium pseudotuberculosis have been limited due to insufficient discrimination between closely related isolates. In this paper we present performance and convenience of three molecular typing methods: BOX-PCR, random amplification of polymorphic DNA (RAPD) and amplification of DNA fragments surrounding rare restriction site (ADSRRS-fingerprinting) in genome analysis of these bacteria. Among examined 61 strains there were distinguished four, eight and 10 different genotypes by BOX-PCR, RAPD and ADSRRS-fingerprinting, respectively. The value of discrimination index was the lowest for BOX-PCR (D = 0.265), much bigger for RAPD (D = 0.539) and the highest for ADSRRS-fingerprinting (D = 0.604). The good discriminatory ability and reproducibility of RAPD and ADSRRS-fingerprinting indicates that those techniques may be particularly applied for epidemiological studies of C. pseudotuberculosis isolates. We found that ADSRRS-fingerprinting is a rapid method offering good discrimination power, excellent reproducibility and may be applied for epidemiological studies of intraspecific genetic relatedness of C. pseudotuberculosis strains.

  7. The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes.

    PubMed

    Serrano, Ana; Sebastián, María; Arilla-Luna, Sonia; Baquedano, Silvia; Herguedas, Beatriz; Velázquez-Campoy, Adrián; Martínez-Júlvez, Marta; Medina, Milagros

    2017-03-24

    Bifunctional FAD synthetases (FADSs) fold in two independent modules; The C-terminal riboflavin kinase (RFK) catalyzes the RFK activity, while the N-terminal FMN-adenylyltransferase (FMNAT) exhibits the FMNAT activity. The search for macromolecular interfaces in the Corynebacterium ammoniagenes FADS (CaFADS) crystal structure predicts a dimer of trimers organization. Within each trimer, a head-to-tail arrangement causes the RFK and FMNAT catalytic sites of the two neighboring protomers to approach, in agreement with active site residues of one module influencing the activity at the other. We analyze the relevance of the CaFADS head-to-tail macromolecular interfaces to stabilization of assemblies, catalysis and ligand binding. With this aim, we evaluate the effect of point mutations in loop L1c-FlapI, loop L6c, and helix α1c of the RFK module (positions K202, E203, F206, D298, V300, E301 and L304), regions at the macromolecular interface between two protomers within the trimer. Although none of the studied residues is critical in the formation and dissociation of assemblies, residues at L1c-FlapI and helix α1c particularly modulate quaternary architecture, as well as ligand binding and kinetic parameters involved with RFK and FMNAT activities. These data support the influence of transient oligomeric structures on substrate accommodation and catalysis at both CaFADS active sites.

  8. Corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique mycothiol redox relay mechanism.

    PubMed

    Tossounian, Maria-Armineh; Pedre, Brandán; Wahni, Khadija; Erdogan, Huriye; Vertommen, Didier; Van Molle, Inge; Messens, Joris

    2015-05-01

    Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.

  9. Effect of 1,1-dimethylhydrazine (UDMH) on Corynebacterium parvum-associated immunosuppression in mice.

    PubMed

    Frazier, D E; Bauer, R M; Tarr, M J; Olsen, R G

    1992-01-01

    These studies further investigate the immunoenhancement properties of UDMH by utilizing Corynebacterium parvum-induced immunosuppressed mice as well as evaluating activated macrophage production of reactive oxygen intermediates or their effects. Forty-eight hour Con A-induced lymphoblastogenic responses from splenocytes isolated from C. parvum and UDMH-treated Balb/C mice were significantly increased compared with C. parvum alone, although less than normal control mice (no treatment). In vitro bioassay of IL-2 production in cell culture supernatant isolated from these same treatment groups exhibited a pattern of stimulation similar to that of lymphocyte blastogenesis. In addition, UDMH did not interfere with H2O2-mediated suppression of either Con A- or LPS-induced lymphocyte blastogenesis and actually enhanced suppression of Con A-induced lymphocyte cultures at 25 micrograms/ml. We also report that production of superoxide anion from TPA-activated peritoneal macrophages exposed to various concentrations of UDMH in vitro was not affected. Although in vivo exposure to UDMH partially reversed C. parvum-induced immunosuppression in mice, the exact mechanism by which UDMH acts to reverse this immune suppression is not clear. UDMH does not appear to interfere with either activated peritoneal macrophage production of superoxide anion or H2O2-induced suppression of lymphocyte blastogenesis to elicit immune enhancement.

  10. Culture-negative prosthetic valve endocarditis with concomitant septicemia due to a nontoxigenic Corynebacterium diphtheriae biotype gravis isolate in a patient with multiple risk factors.

    PubMed

    Clinton, Lani Kai; Bankowski, Matthew J; Shimasaki, Teppei; Sae-Ow, Wichit; Whelen, A Christian; O'Connor, Norman; Kim, Wesley; Young, Royden

    2013-11-01

    A 54-year-old female with a prosthetic mitral valve presented with a 3-day history of dizziness, subjective fever, and chills. Blood cultures were positive for a pleomorphic Gram-positive rod. Initial phenotypic testing could only support the identification of a Corynebacterium species. Nucleic acid sequencing (16S rRNA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) were conclusive for Corynebacterium diphtheriae. Definitive phenotypic testing classified the strain as nontoxigenic C. diphtheriae biotype Gravis.

  11. Susceptibility to Aminoglycosides and Distribution of aph and aac(3)-XI Genes among Corynebacterium striatum Clinical Isolates

    PubMed Central

    Navas, Jesús; Fernández-Martínez, Marta; Salas, Carlos; Cano, María Eliecer; Martínez-Martínez, Luis

    2016-01-01

    Corynebacterium striatum is an opportunistic pathogen, often multidrug-resistant, which has been associated with serious infections in humans. Aminoglycosides are second-line or complementary antibiotics used for the treatment of Corynebacterium infections. We investigated the susceptibility to six aminoglycosides and the molecular mechanisms involved in aminoglycoside resistance in a collection of 64 Corynebacterium striatum isolated in our laboratory during the period 2005–2009. Antimicrobial susceptibility was determined using E-test. The mechanisms of aminoglycoside resistance were investigated by PCR and sequencing. The 64 C. striatum were assessed for the possibility of clonal spreading by Pulsed-field Gel Electrophoresis (PFGE). Netilmicin and amikacin were active against the 64 C. striatum isolates (MICs90 = 0.38 and 0.5 mg/L, respectively). Twenty-seven of the 64 C. striatum strains showed a MIC90 for kanamycin > 256 mg/L, and 26 out the 27 were positive for the aph(3’)-Ic gene. Thirty-six out of our 64 C. striatum were streptomycin resistant, and 23 out of the 36 carried both the aph(3”)-Ib and aph(6)-Id genes. The gene aac(3)-XI encoding a new aminoglycoside 3-N acetyl transferase from C. striatum was present in 44 out of the 64 isolates, all of them showing MICs of gentamicin and tobramycin > 1 mg/L. CS4933, a C. striatum showing very low susceptibility to kanamycin and streptomycin, contains an aminoglycoside resistance region that includes the aph(3’)-Ic gene, and the tandem of genes aph(3”)-Ib and aph(6)-Id. Forty-six major PFGE types were identified among the 64 C. striatum isolates, indicating that they were mainly not clonal. Our results showed that the 64 clinical C. striatum were highly resistant to aminoglycosides and mostly unrelated. PMID:27936101

  12. Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere.

    PubMed Central

    Sebastian, J; Chandra, A K; Kolattukudy, P E

    1987-01-01

    A phyllospheric bacterial culture, previously reported to partially replace nitrogen fertilizer (B. R. Patti and A. K. Chandra, Plant Soil 61:419-427, 1981) was found to contain a fluorescent pseudomonas which was identified as Pseudomonas putida and a Corynebacterium sp. The P. putida isolate was found to produce an extracellular cutinase when grown in a medium containing cutin, the polyester structural component of plant cuticle. The Corynebacterium sp. grew on nitrogen-free medium but could not produce cutinase under any induction conditions tested, whereas P. putida could not grow on nitrogen-free medium. When cocultured with the nitrogen-fixing Corynebacterium sp., the P. putida isolate grew in a nitrogen-free medium, suggesting that the former provided fixed N2 for the latter. These results suggest that the two species coexist on the plant surface, with one providing carbon and the other providing reduced nitrogen for their growth. The presence of cutin in the medium induced cutinase production by P. putida. However, unlike the previously studied fungal systems, cutin hydrolysate did not induce cutinase. Thin-layer chromatographic analysis of the products released from labeled apple fruit cutin showed that the extracellular enzyme released all classes of cutin monomers. This enzyme also catalyzed hydrolysis of the model ester substrates, p-nitrophenyl esters of fatty acids, and optimal conditions were determined for a spectrophotometric assay with p-nitrophenyl butyrate as the substrate. It did not hydrolyze triacyl glycerols, indicating that the cutinase activity was not due to a nonspecific lipase. It showed a broad pH optimum between 8.0 and 10.5 with 3H-labeled apple cutin as the substrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3793714

  13. Laboratory guidelines for the diagnosis of infections caused by Corynebacterium diphtheriae and C. ulcerans. World Health Organization.

    PubMed

    Efstratiou, A; George, R C

    1999-12-01

    These guidelines represent an application of the World Health Organization European Region's manual for the laboratory diagnosis of diphtheria for laboratories in the United Kingdom (UK), but they could be applied to laboratories overseas. The manual was rewritten in response to the re-emergence of diphtheria in eastern Europe and the emergence of other infections caused by Corynebacterium diphtheriae and C. ulcerans in the UK and overseas. The guidelines summarise our current recommendations and procedures for the microbiological diagnosis of infections caused by toxigenic and non-toxigenic isolates of corynebacteria, with particular reference to C. diphtheriae and C. ulcerans.

  14. Direct polymerase chain reaction for detection of toxigenic Corynebacterium diphtheriae strains from the Republic of Georgia after prolonged storage.

    PubMed

    Kobaidze, K; Popovic, T; Nakao, H; Quick, L

    2000-02-01

    A total of 226 paired nose and throat swab specimens from 113 clinical diphtheria cases from the republic of Georgia were analyzed by direct polymerase chain reaction targeting both A and B subunits of the diphtheria toxin gene, tox. Even after prolonged transport and extensive storage (7-14 months) of the clinical specimens in silica gel packages, direct polymerase chain reaction detected the diphtheria tox gene in 54% of the specimens. Specimens obtained by throat swab were three times more likely than those obtained by nose swab to be positive for Corynebacterium diphtheriae.

  15. Recombinant renewable polyclonal antibodies.

    PubMed

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  16. The dissociative recombination of ?

    NASA Astrophysics Data System (ADS)

    Laubé, S.; Lehfaoui, L.; Rowe, B. R.; Mitchell, J. B. A.

    1998-09-01

    The dissociative recombination rate coefficient for 0953-4075/31/18/016/img2 has been measured at 300 K using a flowing afterglow Langmuir probe-mass spectrometer apparatus. A value of 0953-4075/31/18/016/img3 has been found.

  17. Introduction to dissociative recombination

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.; Mitchell, J. Brian A.

    1989-01-01

    Dissociative recombination (DR) of molecular ions with electrons has important consequences in many areas of physical science. Ab-initio calculations coupled with resonant scattering theory and multichannel quantum defect studies have produced detailed results illuminating the role of ion vibrational excitation, the quantum yields of the DR products, and the role of Rydberg states. The theoretical and experimental results are discussed.

  18. Recombineering linear BACs.

    PubMed

    Chen, Qingwen; Narayanan, Kumaran

    2015-01-01

    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

  19. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  20. Identification and diversity of multiresistant Corynebacterium striatum clinical isolates by MALDI-TOF mass spectrometry and by a multigene sequencing approach

    PubMed Central

    2012-01-01

    Background The genus Corynebacterium is composed of Gram-positive bacteria that are widely distributed throughout the environment; these bacteria are also part of the normal microbiota of human skin and mucous membranes. Multiple studies have shown that species of this genus, including C. striatum, become pathogenic to humans under special conditions. Our aim was to determine the characteristics of clinical multiresistant strains of C. striatum that were isolated in our geographical region, to determine their diversity, and to compare them with the type strain and with related species. We studied fifty-two strains of C. striatum isolated from different hospitals from Mallorca, Spain, mainly from the Hospital Joan March in Bunyola, Mallorca. Most of the strains were isolated from sputum cultures of respiratory samples from patients with chronic obstructive pulmonary disease. To gain further insight into the genetic diversity of the strains, we analysed several housekeeping genes and other genes associated with antibiotic resistance. Strains were also characterised phenotypically by their antibiotic resistance profiles and by MALDI-TOF mass spectrometry analysis. Results The ITS1 region, gyrA and rpoB were chosen as the appropriate genes in the C. striatum genome to study the genetic diversity of C. striatum species and to discriminate between strains. After analysing these three genes, four sequence types (ST2, ST4, ST1 and ST11) were found to be the most abundant. Splits tree analysis of the strains demonstrated that these clinical isolates did not share any alleles with the type strain of the species. Recombination was detected within all of the C. striatum isolates, and different clonal populations were detected within the samples. Conclusions Our results demonstrate that the isolates were best identified using gene-based molecular methods; using these methods, the isolated strains were determined to be different from the type strain of C. striatum. The ITS1

  1. Strategies to Prevent, Treat, and Provoke Corynebacterium-Associated Hyperkeratosis in Athymic Nude Mice

    PubMed Central

    Burr, Holly N; Lipman, Neil S; White, Julie R; Zheng, Junting; Wolf, Felix R

    2011-01-01

    Athymic nude mice infected with Corynebacterium bovis typically exhibit transient hyperkeratotic dermatitis. Our vivarium experienced an increased incidence of disease characterized by persistent skin lesions and increased mortality, leading to this study. For detection of infection, skin and buccal swab methods showed comparable sensitivities in nude mice. Various prevention, treatment, and eradication strategies were evaluated through clinical assessment, microbiology, and histopathology. In experimentally naïve athymic nude mice, a 2-wk course of prophylactic amoxicillin-containing diet (1200 ppm amoxicillin; effective dose, 200 mg/kg) was ineffective at preventing infection or disease. There was also no significant difference in disease duration or severity in athymic nude mice that received amoxicillin diet or penicillin–streptomycin topical spray (penicillin, 2500 U/mL; streptomycin, 2500 µg/mL). Prolonged treatment with 4 or 8 wk of amoxicillin diet cleared only a small number of athymic nude mice that had subclinical C. bovis infections. Antibiotic sensitivity of C. bovis isolates demonstrated a small colony isolate with less susceptibility to all antibiotics compared with a large colony isolate. Resistance did not appear to develop after prolonged treatment with amoxicillin. Provocation testing by administration of cyclophosphamide (50 mg/kg IP every 48 to 72 h for 90 d) to subclinically infected athymic nude mice resulted in prolonged clinical disease that waxed and waned without progression to severe disease. Our findings suggest that antibiotic prophylaxis and treatment of clinical disease in experimentally naïve mice is unrewarding, eradication of bacterial infection is difficult, and severe disease associated with C. bovis is likely multifactorial. PMID:21640035

  2. Microbiological and molecular characterization of Corynebacterium diphtheriae isolated in Algeria between 1992 and 2015.

    PubMed

    Benamrouche, N; Hasnaoui, S; Badell, E; Guettou, B; Lazri, M; Guiso, N; Rahal, K

    2016-12-01

    The objectives of this study were to undertake the microbiological and molecular characterization of Corynebacterium diphtheriae isolates collected in Algeria during epidemic and post-epidemic periods between 1992 and 2015. Microbiological characterization includes the determination of biotype and toxigenicity status using phenotypic and genotypic methods. Antimicrobial susceptibility was determined by the E-test method. Molecular characterization was performed by multi-locus sequence typing. In total, there were 157 cases of C. diphtheriae isolates, 127 in patients with respiratory diphtheria and 30 with ozena. Isolates with a mitis biotype were predominant (122 out of 157; 77.7%) followed by belfanti (28 out of 157; 17.8%) and gravis biotype (seven out of 157; 4.5%). Toxigenic isolates were predominant in the period 1992-2006 (74 out of 134) whereas in the period 2007-2015, only non-toxigenic isolates circulated (23 out of 23). All 157 isolates were susceptible to erythromycin, gentamicin, vancomycin and cotrimoxazole. Reduced susceptibility to penicillin G, cefotaxime, tetracycline and chloramphenicol was detected in 90 (57.3%), 88 (56.1%), 112 (71.3%) and 90 (57.3%) isolates, respectively. Multi-locus sequence typing analysis indicates that sequence type 116 (ST-116) was the most frequent, with 65 out of 100 isolates analysed, in particular during the epidemic period 1992-1999 (57 out of 65 isolates). In the post-epidemic period, 2000-2015, 13 different sequence types were isolated. All belfanti isolates (ten out of 100 isolates) belonged to closely related sequence types grouped in a phylogenetically distinct eBurst group and were collected exclusively in ozena cases. In conclusion, the epidemic period was associated with ST-116 while the post-epidemic period was characterized by more diversity. Belfanti isolates are grouped in a phylogenetically distinct clonal complex.

  3. Invasion of endothelial cells and arthritogenic potential of endocarditis-associated Corynebacterium diphtheriae.

    PubMed

    Peixoto, Renata Stavracakis; Pereira, Gabriela Andrade; Sanches dos Santos, Louisy; Rocha-de-Souza, Cláudio Marcos; Gomes, Débora Leandro Rama; Silva Dos Santos, Cintia; Werneck, Lucia Maria Correa; Dias, Alexandre Alves de Souza de Oliveira; Hirata, Raphael; Nagao, Prescilla Emy; Mattos-Guaraldi, Ana Luíza

    2014-03-01

    Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, different clones have been also associated with invasive infections such as sepsis, endocarditis, septic arthritis and osteomyelitis. The mechanisms that promote C. diphtheriae infection and haematogenic dissemination need further investigation. In this study we evaluated the association and invasion mechanisms with human umbilical vein endothelial cells (HUVECs) and experimental arthritis in mice of endocarditis-associated strains and control non-invasive strains. C. diphtheriae strains were able to adhere to and invade HUVECs at different levels. The endocarditis-associated strains displayed an aggregative adherence pattern and a higher number of internalized viable cells in HUVECs. Transmission electron microscopy (TEM) analysis revealed intracellular bacteria free in the cytoplasm and/or contained in a host-membrane-confined compartment as single micro-organisms. Data showed bacterial internalization dependent on microfilament and microtubule stability and involvement of protein phosphorylation in the HUVEC signalling pathway. A high number of affected joints and high arthritis index in addition to the histopathological features indicated a strain-dependent ability of C. diphtheriae to cause severe polyarthritis. A correlation between the arthritis index and increased systemic levels of IL-6 and TNF-α was observed for endocarditis-associated strains. In conclusion, higher incidence of potential mechanisms by which C. diphtheriae may access the bloodstream through the endothelial barrier and stimulate the production of pro-inflammatory cytokines such as IL-6 and TNF-α, in addition to the ability to affect the joints and induce arthritis through haematogenic spread are thought to be related to the pathogenesis of endocarditis-associated strains.

  4. SubMICs of penicillin and erythromycin enhance biofilm formation and hydrophobicity of Corynebacterium diphtheriae strains.

    PubMed

    Gomes, D L R; Peixoto, R S; Barbosa, E A B; Napoleão, F; Sabbadini, P S; dos Santos, K R N; Mattos-Guaraldi, A L; Hirata, R

    2013-05-01

    Subinhibitory concentrations (subMICs) of antibiotics may alter bacterial surface properties and change microbial physiology. This study aimed to investigate the effect of a subMIC (⅛ MIC) of penicillin (PEN) and erythromycin (ERY) on bacterial morphology, haemagglutinating activity, cell-surface hydrophobicity (CSH) and biofilm formation on glass and polystyrene surfaces, as well as the distribution of cell-surface acidic anionic residues of Corynebacterium diphtheriae strains (HC01 tox(-) strain; CDC-E8392 and 241 tox(+) strains). All micro-organisms tested were susceptible to PEN and ERY. Growth in the presence of PEN induced bacterial filamentation, whereas subMIC of ERY caused cell-size reduction of strains 241 and CDC-E8392. Adherence to human erythrocytes was reduced after growth in the presence of ERY, while CSH was increased by a subMIC of both antibiotics in bacterial adherence to n-hexadecane assays. Conversely, antibiotic inhibition of biofilm formation was not observed. All strains enhanced biofilm formation on glass after treatment with ERY, while only strain 241 increased glass adherence after cultivation in the presence of PEN. Biofilm production on polystyrene surfaces was improved by ⅛ MIC of ERY. After growth in the presence of both antimicrobial agents, strains 241 and CDC-E8392 exhibited anionic surface charges with focal distribution. In conclusion, subMICs of PEN and ERY modified bacterial surface properties and enhanced not only biofilm formation but also cell-surface hydrophobicity. Antibiotic-induced biofilm formation may contribute to the inconsistent success of antimicrobial therapy for C. diphtheriae infections.

  5. Potential pathogenic role of aggregative-adhering Corynebacterium diphtheriae of different clonal groups in endocarditis.

    PubMed

    Hirata Jr, R; Pereira, G A; Filardy, A A; Gomes, D L R; Damasco, P V; Rosa, A C P; Nagao, P E; Pimenta, F P; Mattos-Guaraldi, A L

    2008-11-01

    Invasive diseases caused by Corynebacterium diphtheriae have been described increasingly. Several reports indicate the destructive feature of endocarditis attributable to nontoxigenic strains. However, few reports have dealt with the pathogenicity of invasive strains. The present investigation demonstrates a phenotypic trait that may be used to identify potentially invasive strains. The study also draws attention to clinical and microbiological aspects observed in 5 cases of endocarditis due to C. diphtheriae that occurred outside Europe. Four cases occurred in female school-age children (7-14 years) treated at different hospitals in Rio de Janeiro, Brazil. All patients developed other complications including septicemia, renal failure and/or arthritis. Surgical treatment was performed on 2 patients for valve replacement. Lethality was observed in 40% of the cases. Microorganisms isolated from 5 blood samples and identified as C. diphtheriae subsp mitis (N = 4) and C. diphtheriae subsp gravis (N = 1) displayed an aggregative adherence pattern to HEp-2 cells and identical one-dimensional SDS-PAGE protein profiles. Aggregative-adhering invasive strains of C. diphtheriae showed 5 distinct RAPD profiles. Despite the clonal diversity, all 5 C. diphtheriae invasive isolates seemed to display special bacterial adhesive properties that may favor blood-barrier disruption and systemic dissemination of bacteria. In conclusion, blood isolates from patients with endocarditis exhibited a unique adhering pattern, suggesting a pathogenic role of aggregative-adhering C. diphtheriae of different clones in endocarditis. Accordingly, the aggregative-adherence pattern may be used as an indication of some invasive potential of C. diphtheriae strains.

  6. Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment.

    PubMed

    Draganova, Elizabeth B; Akbas, Neval; Adrian, Seth A; Lukat-Rodgers, Gudrun S; Collins, Daniel P; Dawson, John H; Allen, Courtni E; Schmitt, Michael P; Rodgers, Kenton R; Dixon, Dabney W

    2015-11-03

    The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type (WT) HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicates that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A is reduced readily in the presence of dithionite. The FeCO Raman shifts in WT, H136A, and Y235A HmuT-CO complexes provide further evidence of the axial ligand assignments. Additionally, these frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A-CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.

  7. Detection and Elimination of Corynebacterium bovis from Barrier Rooms by Using an Environmental Sampling Surveillance Program.

    PubMed

    Manuel, Christopher A; Pugazhenthi, Umarani; Spiegel, Shannon P; Leszczynski, Jori K

    2017-03-01

    Rodent health-monitoring programs based on sampling an IVC system's exhaust air dust (EAD) has enhanced and even replaced traditional sentinels for some rodent pathogens. EAD testing by qPCR assay is an optimal surveillance method for the rapid detection of Corynebacterium bovis-infected immunodeficient mice. Here we demonstrate that an active EAD surveillance program for C. bovis can be used to maintain nude mice C. bovis-free after the transition from historically enzootically infected colonies. During 3 events over 3 y, rapid detection of infection, elimination of infected mice, aggressive quarantine measures, and local decontamination prevented the spread of C. bovis within 2 barrier rooms. In total, 4 cages of infected nude mice were identified and removed, preventing the spread of infection to 469 other cages of immunodeficient mice. In addition, we present data regarding a refinement to EAD testing which enables row-specific surveillance of an IVC rack. This technique systemically decreases the amount of testing required to locate an individually infected cage. Due to our ability to rapidly detect and localize an infected cage, we were able to investigate the route of C. bovis introduction into our barrier rooms. Our epidemiologic investigation suggested that the transmission of C. bovis occurred through contaminated, cryopreserved, patient-derived xenograft tumor tissue. This previously unknown source of C. bovis can infect mice used to propagate these tumors. Together, these data demonstrate that a remediation program that combines rapid detection, test-and-cull, and local decontamination under quarantine conditions can eliminate C. bovis from a mouse colony.

  8. Surveillance of a Ventilated Rack System for Corynebacterium bovis by Sampling Exhaust-Air Manifolds.

    PubMed

    Manuel, Christopher A; Pugazhenthi, Umarani; Leszczynski, Jori K

    2016-01-01

    Corynebacterium bovis causes an opportunistic infection of nude (Foxn1, nu/nu) mice, leading to nude mouse hyperkeratotic dermatitis (scaly skin disease). Enzootic in many nude mouse colonies, C. bovis spreads rapidly to naive nude mice, despite modern husbandry practices, and is very difficult to eradicate. To facilitate rapid detection in support of eradication efforts, we investigated a surveillance method based on quantitative real-time PCR (qPCR) evaluation of swabs collected from the horizontal exhaust manifold (HEM) of an IVC rack system. We first evaluated the efficacy of rack sanitation methods for removing C. bovis DNA from the HEM of racks housing endemic colonies of infected nude mice. Pressurized water used to flush the racks' air exhaust system followed by a standard rack-washer cycle was ineffective in eliminating C. bovis DNA. Only after autoclaving did all sanitized racks test negative for C. bovis DNA. We then measured the effects of stage of infection (early or established), cage density, and cage location on the rack on time-to-detection at the HEM. Stage of infection significantly affected time-to-detection, independent of cage location. Early infections required 7.3 ± 1.2 d whereas established infections required 1 ± 0 d for detection of C. bovis at the HEM. Cage density influenced the quantity of C. bovis DNA detected but not time-to-detection. The location of the cage on the rack affected the time-to-detection only during early C. bovis infections. We suggest that qPCR swabs of HEM are useful during the routine surveillance of nude mouse colonies for C. bovis infection.

  9. Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase

    PubMed Central

    Sanli, Gulsah; Banta, Scott; Anderson, Stephen; Blaber, Michael

    2004-01-01

    Corynebacterium 2,5-Diketo-D-gluconic acid reductase (2,5-DKGR) catalyzes the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-Keto-L-gulonic acid (2-KLG). 2-KLG is an immediate precursor to L-ascorbic acid (vitamin C), and 2,5-DKGR is, therefore, an important enzyme in a novel industrial method for the production of vitamin C. 2,5-DKGR, as with most other members of the aldo-keto reductase (AKR) superfamily, exhibits a preference for NADPH compared to NADH as a cofactor in the stereo-specific reduction of substrate. The application of 2,5-DKGR in the industrial production of vitamin C would be greatly enhanced if NADH could be efficiently utilized as a cofactor. A mutant form of 2,5-DKGR has previously been identified that exhibits two orders of magnitude higher activity with NADH in comparison to the wild-type enzyme, while retaining a high level of activity with NADPH. We report here an X-ray crystal structure of the holo form of this mutant in complex with NADH cofactor, as well as thermodynamic stability data. By comparing the results to our previously reported X-ray structure of the holo form of wild-type 2,5-DKGR in complex with NADPH, the structural basis of the differential NAD(P)H selectivity of wild-type and mutant 2,5-DKGR enzymes has been identified. PMID:14718658

  10. The role of houseflies (Musca domestica) in harbouring Corynebacterium pseudotuberculosis in dairy herds in Israel.

    PubMed

    Braverman, Y; Chizov-Ginzburg, A; Saran, A; Winkler, M

    1999-12-01

    A study was conducted to assess the role of houseflies, Musca domestica L. in harbouring Corynebacterium pseudotuberculosis in dairy farms in Israel. The bacterium was isolated in June 1993 from 40 wild houseflies which had fed on a lesion on a cow, and from 28 laboratory flies fed on contaminated milk from a cow infected with mastitis. The bacterium was recovered from the body surface of 10 flies (of a total of 160) 10 min after being dipped entirely in a bacterial broth. The bacterium was recovered from the body surface of 10 flies (of a total of 40) 5 min after being fed on contaminated milk. When 110 flies were fed on contaminated sugar cubes, the bacterium was recovered externally from 70 flies 5 min later, and from an additional 20 flies 10 min after feeding. Of 110 flies, 80 excreted bacteria in saliva from 5 min to 3 h after feeding on contaminated milk. Bacteria were isolated from the intestine of 40 of 60 flies between 1 h and 4 h after feeding on contaminated milk. Bacteria were found in the faeces of 30 of 60 flies, between 1 h and 4 h after feeding on contaminated milk. In the light of these findings, and given the fact that this species of fly has a predilection to feed on milk residues of cow teats, the authors concluded that the housefly plays an important role in harbouring and disseminating C. pseudotuberculosis in dairy herds in Israel. In contrast, stable flies (Stomoxys calcitrans L.) are not important in the habouring and dissemination of the bacteria, since bacteria were not recovered 5, 10, 15, 30 min, 2 h or 24 h after membrane feeding on a mixture of bacterial broth and blood.

  11. Bayesian Geostatistical Analysis and Ecoclimatic Determinants of Corynebacterium pseudotuberculosis Infection among Horses

    PubMed Central

    Boysen, Courtney; Davis, Elizabeth G.; Beard, Laurie A.; Lubbers, Brian V.; Raghavan, Ram K.

    2015-01-01

    Kansas witnessed an unprecedented outbreak in Corynebacterium pseudotuberculosis infection among horses, a disease commonly referred to as pigeon fever during fall 2012. Bayesian geostatistical models were developed to identify key environmental and climatic risk factors associated with C. pseudotuberculosis infection in horses. Positive infection status among horses (cases) was determined by positive test results for characteristic abscess formation, positive bacterial culture on purulent material obtained from a lanced abscess (n = 82), or positive serologic evidence of exposure to organism (≥1:512)(n = 11). Horses negative for these tests (n = 172)(controls) were considered free of infection. Information pertaining to horse demographics and stabled location were obtained through review of medical records and/or contact with horse owners via telephone. Covariate information for environmental and climatic determinants were obtained from USDA (soil attributes), USGS (land use/land cover), and NASA MODIS and NASA Prediction of Worldwide Renewable Resources (climate). Candidate covariates were screened using univariate regression models followed by Bayesian geostatistical models with and without covariates. The best performing model indicated a protective effect for higher soil moisture content (OR = 0.53, 95% CrI = 0.25, 0.71), and detrimental effects for higher land surface temperature (≥35°C) (OR = 2.81, 95% CrI = 2.21, 3.85) and habitat fragmentation (OR = 1.31, 95% CrI = 1.27, 2.22) for C. pseudotuberculosis infection status in horses, while age, gender and breed had no effect. Preventative and ecoclimatic significance of these findings are discussed. PMID:26473728

  12. Surveillance of a Ventilated Rack System for Corynebacterium bovis by Sampling Exhaust-Air Manifolds

    PubMed Central

    Manuel, Christopher A; Pugazhenthi, Umarani; Leszczynski, Jori K

    2016-01-01

    Corynebacterium bovis causes an opportunistic infection of nude (Foxn1, nu/nu) mice, leading to nude mouse hyperkeratotic dermatitis (scaly skin disease). Enzootic in many nude mouse colonies, C. bovis spreads rapidly to naive nude mice, despite modern husbandry practices, and is very difficult to eradicate. To facilitate rapid detection in support of eradication efforts, we investigated a surveillance method based on quantitative real-time PCR (qPCR) evaluation of swabs collected from the horizontal exhaust manifold (HEM) of an IVC rack system. We first evaluated the efficacy of rack sanitation methods for removing C. bovis DNA from the HEM of racks housing endemic colonies of infected nude mice. Pressurized water used to flush the racks’ air exhaust system followed by a standard rack-washer cycle was ineffective in eliminating C. bovis DNA. Only after autoclaving did all sanitized racks test negative for C. bovis DNA. We then measured the effects of stage of infection (early or established), cage density, and cage location on the rack on time-to-detection at the HEM. Stage of infection significantly affected time-to-detection, independent of cage location. Early infections required 7.3 ± 1.2 d whereas established infections required 1 ± 0 d for detection of C. bovis at the HEM. Cage density influenced the quantity of C. bovis DNA detected but not time-to-detection. The location of the cage on the rack affected the time-to-detection only during early C. bovis infections. We suggest that qPCR swabs of HEM are useful during the routine surveillance of nude mouse colonies for C. bovis infection. PMID:26817981

  13. Experimental Studies on the Pathogenesis of Corynebacterium equi Infection in Foals

    PubMed Central

    Prescott, J. F.; Johnson, J. A.; Markham, R. J. F.

    1980-01-01

    Four month-old foals were infected orally with 75 mL of a suspension of 5.0 × 108 Corynebacterium equi per mL. Two foals were killed after ten days and had scanty number of C. equi in the caeco-colic lymph nodes. No C. equi were recovered from the other two foals, killed 20 days after infection. No gross pathological change was detected in these four foals, although mild microscopic lesions were seen in the ileum of one foal. Results of lymphocyte blastogenesis using peripheral blood lymphocytes and C. equi antigens showed, however, that lymphocytes became sensitized to C. equi following this challenge. In a second experiment four month-old foals were given orally the same dose of organisms but on five consecutive days. Two foals were killed ten days after infection and showed mild histological changes in the large bowel mucosa and C. equi could be recovered from all intestinal lymph nodes cultured. In one of these foals moderate numbers of C. equi were present in the bronchial lymph node. Of the other two foals, one died after 22 days with severe ulcerative enterocolitis and intestinal lymphadenitis. Only one small pulmonary abscess was detected despite large numbers of C. equi in the lungs. The other foal developed similar intestinal changes and was euthanized 25 days after infection. No C. equi were detected in the lungs or bronchial lymph node. Lymphocyte blastogenesis in these animals showed a rapid rise in response to C. equi antigens. These studies suggest that C. equi pneumonia in foals does not always arise from an intestinal infection, that minor intestinal infection causes a cellular immune response and that massive exposure of the bowel over a sustained period is necessary to induce intestinal lesions. ImagesFig. 2.Fig. 3. PMID:7427776

  14. Strategies to prevent, treat, and provoke Corynebacterium-associated hyperkeratosis in athymic nude mice.

    PubMed

    Burr, Holly N; Lipman, Neil S; White, Julie R; Zheng, Junting; Wolf, Felix R

    2011-05-01

    Athymic nude mice infected with Corynebacterium bovis typically exhibit transient hyperkeratotic dermatitis. Our vivarium experienced an increased incidence of disease characterized by persistent skin lesions and increased mortality, leading to this study. For detection of infection, skin and buccal swab methods showed comparable sensitivities in nude mice. Various prevention, treatment, and eradication strategies were evaluated through clinical assessment, microbiology, and histopathology. In experimentally naïve athymic nude mice, a 2-wk course of prophylactic amoxicillin-containing diet (1200 ppm amoxicillin; effective dose, 200 mg/kg) was ineffective at preventing infection or disease. There was also no significant difference in disease duration or severity in athymic nude mice that received amoxicillin diet or penicillin-streptomycin topical spray (penicillin, 2500 U/mL; streptomycin, 2500 μg/mL). Prolonged treatment with 4 or 8 wk of amoxicillin diet cleared only a small number of athymic nude mice that had subclinical C. bovis infections. Antibiotic sensitivity of C. bovis isolates demonstrated a small colony isolate with less susceptibility to all antibiotics compared with a large colony isolate. Resistance did not appear to develop after prolonged treatment with amoxicillin. Provocation testing by administration of cyclophosphamide (50 mg/kg i.p. every 48 to 72 h for 90 d) to subclinically infected athymic nude mice resulted in prolonged clinical disease that waxed and waned without progression to severe disease. Our findings suggest that antibiotic prophylaxis and treatment of clinical disease in experimentally naïve mice is unrewarding, eradication of bacterial infection is difficult, and severe disease associated with C. bovis is likely multifactorial.

  15. Proteome scale comparative modeling for conserved drug and vaccine targets identification in Corynebacterium pseudotuberculosis.

    PubMed

    Hassan, Syed Shah; Tiwari, Sandeep; Guimarães, Luís Carlos; Jamal, Syed Babar; Folador, Edson; Sharma, Neha Barve; de Castro Soares, Siomar; Almeida, Síntia; Ali, Amjad; Islam, Arshad; Póvoa, Fabiana Dias; de Abreu, Vinicius Augusto Carvalho; Jain, Neha; Bhattacharya, Antaripa; Juneja, Lucky; Miyoshi, Anderson; Silva, Artur; Barh, Debmalya; Turjanski, Adrian Gustavo; Azevedo, Vasco; Ferreira, Rafaela Salgado

    2014-01-01

    Corynebacterium pseudotuberculosis (Cp) is a pathogenic bacterium that causes caseous lymphadenitis (CLA), ulcerative lymphangitis, mastitis, and edematous to a broad spectrum of hosts, including ruminants, thereby threatening economic and dairy industries worldwide. Currently there is no effective drug or vaccine available against Cp. To identify new targets, we adopted a novel integrative strategy, which began with the prediction of the modelome (tridimensional protein structures for the proteome of an organism, generated through comparative modeling) for 15 previously sequenced C. pseudotuberculosis strains. This pan-modelomics approach identified a set of 331 conserved proteins having 95-100% intra-species sequence similarity. Next, we combined subtractive proteomics and modelomics to reveal a set of 10 Cp proteins, which may be essential for the bacteria. Of these, 4 proteins (tcsR, mtrA, nrdI, and ispH) were essential and non-host homologs (considering man, horse, cow and sheep as hosts) and satisfied all criteria of being putative targets. Additionally, we subjected these 4 proteins to virtual screening of a drug-like compound library. In all cases, molecules predicted to form favorable interactions and which showed high complementarity to the target were found among the top ranking compounds. The remaining 6 essential proteins (adk, gapA, glyA, fumC, gnd, and aspA) have homologs in the host proteomes. Their active site cavities were compared to the respective cavities in host proteins. We propose that some of these proteins can be selectively targeted using structure-based drug design approaches (SBDD). Our results facilitate the selection of C. pseudotuberculosis putative proteins for developing broad-spectrum novel drugs and vaccines. A few of the targets identified here have been validated in other microorganisms, suggesting that our modelome strategy is effective and can also be applicable to other pathogens.

  16. Implantation of Corynebacterium pseudodiphtheriticum for elimination of Staphylococcus aureus from the nasal cavity in volunteers

    NASA Astrophysics Data System (ADS)

    Viacheslav, Ilyin; Kiryukhina, Nataliya

    Nasal carriage of Staphylococcus aureus is a well-documented risk factor of infection and inflammation of the skin, soft tissues and bacteremia. It is also known that most often etiology of these disorders is associated with autoinfection. The present-day methods of opportunistic pathogens eradication from the nasal cavity are based principally on the use of antiseptic and antibacterial agents. For instance, a local antibiotic mupirocin in the form of nasal ointment is considered to be the gold standard for the treatment of S. aureus carriage. The literature describes investigations showing how mupirocin can strengthen antibiotic resistance in S. aureus strains, including those with methicillin resistance (MRSA). It is also common knowledge that recolonization of the nasal mucous membrane takes place within several months after mupirocin treatment. This circumstance dictates the necessity to look for alternative ways of preventing the S. aureus carriage and methods of elimination. One of the methods of nasal S. aureus elimination is implantation of nonpathogenic microorganisms which will extrude opportunistic pathogens without impinging the symbiotic microbiota. Effectiveness of saline suspension of Corynebacterium pseudodiphtheriticum containing spray was assessed in a several chamber experiments with simulation of some spaceflight factors (dry immersion, isolation). Various schemes of application of preparations were applied. In all cases of corynebacteria application the strong inhibiting effect against S. aureus was detected. This fact opens a prospect of using nonpathogenic corynebacteria as a nasal probiotic. Administration of the nasal corynebacteria spray possibly prevented cross-infection by MRSA and appearance of staphylococcal infection. Further pre-clinical and clinical study of this bacterial therapy method is under development.

  17. Bayesian Geostatistical Analysis and Ecoclimatic Determinants of Corynebacterium pseudotuberculosis Infection among Horses.

    PubMed

    Boysen, Courtney; Davis, Elizabeth G; Beard, Laurie A; Lubbers, Brian V; Raghavan, Ram K

    2015-01-01

    Kansas witnessed an unprecedented outbreak in Corynebacterium pseudotuberculosis infection among horses, a disease commonly referred to as pigeon fever during fall 2012. Bayesian geostatistical models were developed to identify key environmental and climatic risk factors associated with C. pseudotuberculosis infection in horses. Positive infection status among horses (cases) was determined by positive test results for characteristic abscess formation, positive bacterial culture on purulent material obtained from a lanced abscess (n = 82), or positive serologic evidence of exposure to organism (≥ 1:512)(n = 11). Horses negative for these tests (n = 172)(controls) were considered free of infection. Information pertaining to horse demographics and stabled location were obtained through review of medical records and/or contact with horse owners via telephone. Covariate information for environmental and climatic determinants were obtained from USDA (soil attributes), USGS (land use/land cover), and NASA MODIS and NASA Prediction of Worldwide Renewable Resources (climate). Candidate covariates were screened using univariate regression models followed by Bayesian geostatistical models with and without covariates. The best performing model indicated a protective effect for higher soil moisture content (OR = 0.53, 95% CrI = 0.25, 0.71), and detrimental effects for higher land surface temperature (≥ 35°C) (OR = 2.81, 95% CrI = 2.21, 3.85) and habitat fragmentation (OR = 1.31, 95% CrI = 1.27, 2.22) for C. pseudotuberculosis infection status in horses, while age, gender and breed had no effect. Preventative and ecoclimatic significance of these findings are discussed.

  18. Complete Genome Sequences of Corynebacterium pseudotuberculosis Strains 3/99-5 and 42/02-A, Isolated from Sheep in Scotland and Australia, Respectively

    PubMed Central

    Pethick, Florence E.; Lainson, Alex F.; Yaga, Raja; Flockhart, Allen; Smith, David G. E.; Donachie, Willie; Cerdeira, Louise T.; Silva, Artur; Bol, Erik; Lopes, Thiago S.; Barbosa, Maria S.; Pinto, Anne C.; dos Santos, Anderson R.; Soares, Siomar C.; Almeida, Sintia S.; Guimaraes, Luis C.; Aburjaile, Flavia F.; Abreu, Vinicius A. C.; Ribeiro, Dayana; Fiaux, Karina K.; Diniz, Carlos A. A.; Barbosa, Eudes G. V.; Pereira, Ulisses P.; Hassan, Syed S.; Ali, Amjad; Bakhtiar, Syeda M.; Dorella, Fernanda A.; Carneiro, Adriana R.; Ramos, Rommel T. J.; Rocha, Flavia S.; Schneider, Maria P. C.; Miyoshi, Anderson; Azevedo, Vasco

    2012-01-01

    Here, we report the whole-genome sequences of two ovine-pathogenic Corynebacterium pseudotuberculosis isolates: strain 3/99-5, which represents the first C. pseudotuberculosis genome originating from the United Kingdom, and 42/02-A, the second from Australia. These genome sequences will contribute to the objective of determining the global pan-genome of this bacterium. PMID:22887652

  19. Identification of clinically relevant Corynebacterium strains by Api Coryne, MALDI-TOF-mass spectrometry and molecular approaches.

    PubMed

    Alibi, S; Ferjani, A; Gaillot, O; Marzouk, M; Courcol, R; Boukadida, J

    2015-09-01

    We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 97 Corynebacterium clinical in comparison to identification strains by Api Coryne and MALDI-TOF-MS using 16S rRNA gene and hypervariable region of rpoB genes sequencing as a reference method. C. striatum was the predominant species isolated followed by C. amycolatum. There was an agreement between Api Coryne strips and MALDI-TOF-MS identification in 88.65% of cases. MALDI-TOF-MS was unable to differentiate C. aurimucosum from C. minutissimum and C. minutissimum from C. singulare but reliably identify 92 of 97 (94.84%) strains. Two strains remained incompletely identified to the species level by MALDI-TOF-MS and molecular approaches. They belonged to Cellulomonas and Pseudoclavibacter genus. In conclusion, MALDI-TOF-MS is a rapid and reliable method for the identification of Corynebacterium species. However, some limits have been noted and have to be resolved by the application of molecular methods.

  20. A PCR for dtxR gene: application to diagnosis of non-toxigenic and toxigenic Corynebacterium diphtheriae.

    PubMed

    Pimenta, Fabricia P; Matias, Gisele A M; Pereira, Gabriela A; Camello, Thereza C F; Alves, Gabriela B; Rosa, Ana C P; Hirata, Raphael; Mattos-Guaraldi, Ana L

    2008-06-01

    The significant rise in the percentage of adults susceptible to diphtheria and the emergence of non-toxigenic Corynebacterium diphtheriae strains as the causative agent of endocarditis and other systemic infections emphasize the need for alternative laboratory diagnostic procedures. In this study, for the first time, the value of a species-specific PCR assay that targets the dtxR gene is documented as a procedure for differentiating C. diphtheriae from Corynebacterium-like colonies. The results of the PCR-dtxR were all positive for 91 C. diphtheriae (54 non-toxigenic and 37 toxigenic) strains. PCR-dtxR completely correlated with the standard biochemical and commercial identification for all C. diphtheriae strains tested. Conversely, the PCR-dtxR results were negative in 100% of the 111 non-diphtherial Gram-positive rod strains obtained during identification procedures in a hospital laboratory. Thus, the PCR-dtxR assay emerged as viable, cost-effective screening method for C. diphtheriae laboratory identification.