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

PubMed Central

Maeda, Tomoya; Tanaka, Yuya; Wachi, Masaaki

2016-01-01

ABSTRACT 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

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 Δ rneG Δ pnp 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 Δ pnp , Δ rneG , and Δ ybeY 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

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.

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

Patchoulol Production with Metabolically Engineered Corynebacterium glutamicum

PubMed Central

Wichmann, Julian; Baier, Thomas; Frohwitter, Jonas; Risse, Joe M.; Peters-Wendisch, Petra; Kruse, Olaf

2018-01-01

Patchoulol is a sesquiterpene alcohol and an important natural product for the perfume industry. Corynebacterium glutamicum is the prominent host for the fermentative production of amino acids with an average annual production volume of ~6 million tons. Due to its robustness and well established large-scale fermentation, C. glutamicum has been engineered for the production of a number of value-added compounds including terpenoids. Both C40 and C50 carotenoids, including the industrially relevant astaxanthin, and short-chain terpenes such as the sesquiterpene valencene can be produced with this organism. In this study, systematic metabolic engineering enabled construction of a patchoulol producing C. glutamicum strain by applying the following strategies: (i) construction of a farnesyl pyrophosphate-producing platform strain by combining genomic deletions with heterologous expression of ispA from Escherichia coli; (ii) prevention of carotenoid-like byproduct formation; (iii) overproduction of limiting enzymes from the 2-c-methyl-d-erythritol 4-phosphate (MEP)-pathway to increase precursor supply; and (iv) heterologous expression of the plant patchoulol synthase gene PcPS from Pogostemon cablin. Additionally, a proof of principle liter-scale fermentation with a two-phase organic overlay-culture medium system for terpenoid capture was performed. To the best of our knowledge, the patchoulol titers demonstrated here are the highest reported to date with up to 60 mg L−1 and volumetric productivities of up to 18 mg L−1 d−1. PMID:29673223

Succinic acid production from corn cob hydrolysates by genetically engineered Corynebacterium glutamicum.

PubMed

Wang, Chen; Zhang, Hengli; Cai, Heng; Zhou, Zhihui; Chen, Yilu; Chen, Yali; Ouyang, Pingkai

2014-01-01

Corynebacterium glutamicum wild type lacks the ability to utilize the xylose fractions of lignocellulosic hydrolysates. In the present work, we constructed a xylose metabolic pathway in C. glutamicum by heterologous expression of the xylA and xylB genes coming from Escherichia coli. Dilute-acid hydrolysates of corn cobs containing xylose and glucose were used as a substrate for succinic acid production by recombinant C. glutamicum NC-2. The results indicated that the available activated charcoal pretreatment in dilute-acid hydrolysates of corn cobs could be able to overcome the inhibitory effect in succinic acid production. Succinic acid was shown to be efficiently produced from corn cob hydrolysates (55 g l(-1) xylose and 4 g l(-1) glucose) under oxygen deprivation with addition of sodium carbonate. Succinic acid concentration reached 40.8 g l(-1) with a yield of 0.69 g g(-1) total sugars within 48 h. It was the first report of succinic acid production from corn cob hydrolysates by metabolically engineered C. glutamicum. This study suggested that dilute-acid hydrolysates of corn cobs may be an alternative substrate for the efficient production of succinic acid by C. glutamicum.

CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum.

PubMed

Cho, Jae Sung; Choi, Kyeong Rok; Prabowo, Cindy Pricilia Surya; Shin, Jae Ho; Yang, Dongsoo; Jang, Jaedong; Lee, Sang Yup

2017-07-01

Genome engineering of Corynebacterium glutamicum, an important industrial microorganism for amino acids production, currently relies on random mutagenesis and inefficient double crossover events. Here we report a rapid genome engineering strategy to scarlessly knock out one or more genes in C. glutamicum in sequential and iterative manner. Recombinase RecT is used to incorporate synthetic single-stranded oligodeoxyribonucleotides into the genome and CRISPR/Cas9 to counter-select negative mutants. We completed the system by engineering the respective plasmids harboring CRISPR/Cas9 and RecT for efficient curing such that multiple gene targets can be done iteratively and final strains will be free of plasmids. To demonstrate the system, seven different mutants were constructed within two weeks to study the combinatorial deletion effects of three different genes on the production of γ-aminobutyric acid, an industrially relevant chemical of much interest. This genome engineering strategy will expedite metabolic engineering of C. glutamicum. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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

Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets.

PubMed

Bott, Michael; Brocker, Melanie

2012-06-01

In bacteria, adaptation to changing environmental conditions is often mediated by two-component signal transduction systems. In the prototypical case, a specific stimulus is sensed by a membrane-bound histidine kinase and triggers autophosphorylation of a histidine residue. Subsequently, the phosphoryl group is transferred to an aspartate residue of the cognate response regulator, which then becomes active and mediates a specific response, usually by activating and/or repressing a set of target genes. In this review, we summarize the current knowledge on two-component signal transduction in Corynebacterium glutamicum. This Gram-positive soil bacterium is used for the large-scale biotechnological production of amino acids and can also be applied for the synthesis of a wide variety of other products, such as organic acids, biofuels, or proteins. Therefore, C. glutamicum has become an important model organism in industrial biotechnology and in systems biology. The type strain ATCC 13032 possesses 13 two-component systems and the role of five has been elucidated in recent years. They are involved in citrate utilization (CitAB), osmoregulation and cell wall homeostasis (MtrAB), adaptation to phosphate starvation (PhoSR), adaptation to copper stress (CopSR), and heme homeostasis (HrrSA). As C. glutamicum does not only face changing conditions in its natural environment, but also during cultivation in industrial bioreactors of up to 500 m(3) volume, adaptability can also be crucial for good performance in biotechnological production processes. Detailed knowledge on two-component signal transduction and regulatory networks therefore will contribute to both the application and the systemic understanding of C. glutamicum and related species.

Comparative analysis of the Corynebacterium glutamicum transcriptome in response to changes in dissolved oxygen levels.

PubMed

Liu, Xiuxia; Yang, Sun; Wang, Fen; Dai, Xiaofeng; Yang, Yankun; Bai, Zhonghu

2017-02-01

The dissolved oxygen (DO) level of a culture of Corynebacterium glutamicum (C. glutamicum) in a bioreactor has a significant impact on the cellular redox potential and the distribution of energy and metabolites. In this study, to gain a deeper understanding of the effects of DO on the metabolism of C. glutamicum, we sought to systematically explore the influence of different DO concentrations on genetic regulation and metabolism through transcriptomic analysis. The results revealed that after 20 h of fermentation, oxygen limitation enhanced the glucose metabolism, pyruvate metabolism and carbon overflow, and restricted NAD + availability. A high oxygen supply enhanced the TCA cycle and reduced glyoxylate metabolism. Several key genes involved in response of C. glutamicum to different oxygen concentrations were examined, which provided suggestions for target site modifications in developing optimized oxygen supply strategies. These data provided new insights into the relationship between oxygen supply and metabolism of C. glutamicum.

The Zur regulon of Corynebacterium glutamicum ATCC 13032

PubMed Central

2010-01-01

Background Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators. Results The cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays. Conclusion Whole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc

Metabolic evolution of Corynebacterium glutamicum for increased production of L-ornithine

PubMed Central

2013-01-01

Background L-ornithine is effective in the treatment of liver diseases and helps strengthen the heart. The commercial applications mean that efficient biotechnological production of L-ornithine has become increasingly necessary. Adaptive evolution strategies have been proven a feasible and efficient technique to achieve improved cellular properties without requiring metabolic or regulatory details of the strain. The evolved strains can be further optimised by metabolic engineering. Thus, metabolic evolution strategy was used for engineering Corynebacterium glutamicum to enhance L-ornithine production. Results A C. glutamicum strain was engineered by using a combination of gene deletions and adaptive evolution with 70 passages of growth-based selection. The metabolically evolved C. glutamicum strain, named ΔAPE6937R42, produced 24.1 g/L of L-ornithine in a 5-L bioreactor. The mechanism used by C. glutamicum ΔAPE6937R42 to produce L-ornithine was investigated by analysing transcriptional levels of select genes and NADPH contents. The upregulation of the transcription levels of genes involved in the upstream pathway of glutamate biosynthesis and the elevated NADPH concentration caused by the upregulation of the transcriptional level of the ppnK gene promoted L-ornithine production in C. glutamicum ΔAPE6937R42. Conclusions The availability of NADPH plays an important role in L-ornithine production in C. glutamicum. Our results demonstrated that the combination of growth-coupled evolution with analysis of transcript abundances provides a strategy to engineer microbial strains for improving production of target compounds. PMID:23725060

Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum.

PubMed

Liu, Jiao; Wang, Yu; Lu, Yujiao; Zheng, Ping; Sun, Jibin; Ma, Yanhe

2017-11-16

Corynebacterium glutamicum is an important industrial workhorse and advanced genetic engineering tools are urgently demanded. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) have revolutionized the field of genome engineering. The CRISPR/Cas9 system that utilizes NGG as protospacer adjacent motif (PAM) and has good targeting specificity can be developed into a powerful tool for efficient and precise genome editing of C. glutamicum. Herein, we developed a versatile CRISPR/Cas9 genome editing toolbox for C. glutamicum. Cas9 and gRNA expression cassettes were reconstituted to combat Cas9 toxicity and facilitate effective termination of gRNA transcription. Co-transformation of Cas9 and gRNA expression plasmids was exploited to overcome high-frequency mutation of cas9, allowing not only highly efficient gene deletion and insertion with plasmid-borne editing templates (efficiencies up to 60.0 and 62.5%, respectively) but also simple and time-saving operation. Furthermore, CRISPR/Cas9-mediated ssDNA recombineering was developed to precisely introduce small modifications and single-nucleotide changes into the genome of C. glutamicum with efficiencies over 80.0%. Notably, double-locus editing was also achieved in C. glutamicum. This toolbox works well in several C. glutamicum strains including the widely-used strains ATCC 13032 and ATCC 13869. In this study, we developed a CRISPR/Cas9 toolbox that could facilitate markerless gene deletion, gene insertion, precise base editing, and double-locus editing in C. glutamicum. The CRISPR/Cas9 toolbox holds promise for accelerating the engineering of C. glutamicum and advancing its application in the production of biochemicals and biofuels.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kyung-Jin, E-mail: kkj@postech.ac.kr; Kim, Sujin; Lee, Sujin

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 crystalmore » 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.« less

Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system.

PubMed

Peng, Feng; Wang, Xinyue; Sun, Yang; Dong, Guibin; Yang, Yankun; Liu, Xiuxia; Bai, Zhonghu

2017-11-14

Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.

Use of In Vitro Transcription System for Analysis of Corynebacterium glutamicum Promoters Recognized by Two Sigma Factors.

PubMed

Šilar, Radoslav; Holátko, Jiří; Rucká, Lenka; Rapoport, Andrey; Dostálová, Hana; Kadeřábková, Pavla; Nešvera, Jan; Pátek, Miroslav

2016-09-01

Promoter activities in Corynebacterium glutamicum strains with deletions of genes encoding sigma factors of RNA polymerase suggested that transcription from some promoters is controlled by two sigma factors. To prove that different sigma factors are involved in the recognition of selected Corynebacterium glutamicum promoters, in vitro transcription system was applied. It was found that a typical housekeeping promoter Pper interacts with the alternative sigma factor σ(B) in addition to the primary sigma factor σ(A). On the other way round, the σ(B)-dependent promoter of the pqo gene that is expressed mainly in the stationary growth phase was active also with σ(A). Some promoters of genes involved in stress responses (P1clgR, P2dnaK, and P2dnaJ2) were found to be recognized by two stress-responding sigma factors, σ(H) and σ(E). In vitro transcription system thus proved to be a useful direct technique for demonstrating the overlap of different sigma factors in recognition of individual promoters in C. glutamicum.

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.

Engineering Corynebacterium glutamicum for the production of 2,3-butanediol.

PubMed

Radoš, Dušica; Carvalho, Ana Lúcia; Wieschalka, Stefan; Neves, Ana Rute; Blombach, Bastian; Eikmanns, Bernhard J; Santos, Helena

2015-10-29

2,3-Butanediol is an important bulk chemical with a wide range of applications. In bacteria, this metabolite is synthesised from pyruvate via a three-step pathway involving α-acetolactate synthase, α-acetolactate decarboxylase and 2,3-butanediol dehydrogenase. Thus far, the best producers of 2,3-butanediol are pathogenic strains, hence, the development of more suitable organisms for industrial scale fermentation is needed. Herein, 2,3-butanediol production was engineered in the Generally Regarded As Safe (GRAS) organism Corynebacterium glutamicum. A two-stage fermentation process was implemented: first, cells were grown aerobically on acetate; in the subsequent production stage cells were used to convert glucose into 2,3-butanediol under non-growing and oxygen-limiting conditions. A gene cluster, encoding the 2,3-butanediol biosynthetic pathway of Lactococcus lactis, was assembled and expressed in background strains, C. glutamicum ΔldhA, C. glutamicum ΔaceEΔpqoΔldhA and C. glutamicum ΔaceEΔpqoΔldhAΔmdh, tailored to minimize pyruvate-consuming reactions, i.e., to prevent carbon loss in lactic, acetic and succinic acids. Producer strains were characterized in terms of activity of the relevant enzymes in the 2,3-butanediol forming pathway, growth, and production of 2,3-butanediol under oxygen-limited conditions. Productivity was maximized by manipulating the aeration rate in the production phase. The final strain, C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,Ptuf butA), under optimized conditions produced 2,3-butanediol with a 0.66 mol mol(-1) yield on glucose, an overall productivity of 0.2 g L(-1) h(-1) and a titer of 6.3 g L(-1). We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production. The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.

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.

Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beskrovnaya, O.Yu.; Fonshtein, M.Yu.; Kolibaba, L.G.

1989-01-01

Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector /lambda/pSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB/sup +/ clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA/sup +/ transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmidsmore » consistently transduced the markers thrB/sup +/ and lysA/sup +/. The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes.« less

A RecET-assisted CRISPR-Cas9 genome editing in Corynebacterium glutamicum.

PubMed

Wang, Bo; Hu, Qitiao; Zhang, Yu; Shi, Ruilin; Chai, Xin; Liu, Zhe; Shang, Xiuling; Zhang, Yun; Wen, Tingyi

2018-04-23

Extensive modification of genome is an efficient manner to regulate the metabolic network for producing target metabolites or non-native products using Corynebacterium glutamicum as a cell factory. Genome editing approaches by means of homologous recombination and counter-selection markers are laborious and time consuming due to multiple round manipulations and low editing efficiencies. The current two-plasmid-based CRISPR-Cas9 editing methods generate false positives due to the potential instability of Cas9 on the plasmid, and require a high transformation efficiency for co-occurrence of two plasmids transformation. Here, we developed a RecET-assisted CRISPR-Cas9 genome editing method using a chromosome-borne Cas9-RecET and a single plasmid harboring sgRNA and repair templates. The inducible expression of chromosomal RecET promoted the frequencies of homologous recombination, and increased the efficiency for gene deletion. Due to the high transformation efficiency of a single plasmid, this method enabled 10- and 20-kb region deletion, 2.5-, 5.7- and 7.5-kb expression cassette insertion and precise site-specific mutation, suggesting a versatility of this method. Deletion of argR and farR regulators as well as site-directed mutation of argB and pgi genes generated the mutant capable of accumulating L-arginine, indicating the stability of chromosome-borne Cas9 for iterative genome editing. Using this method, the model-predicted target genes were modified to redirect metabolic flux towards 1,2-propanediol biosynthetic pathway. The final engineered strain produced 6.75 ± 0.46 g/L of 1,2-propanediol that is the highest titer reported in C. glutamicum. Furthermore, this method is available for Corynebacterium pekinense 1.563, suggesting its universal applicability in other Corynebacterium species. The RecET-assisted CRISPR-Cas9 genome editing method will facilitate engineering of metabolic networks for the synthesis of interested bio-based products from renewable

Functional Analysis of the Twin-Arginine Translocation Pathway in Corynebacterium glutamicum ATCC 13869▿

PubMed Central

Kikuchi, Yoshimi; Date, Masayo; Itaya, Hiroshi; Matsui, Kazuhiko; Wu, Long-Fei

2006-01-01

Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins. PMID:16997984

Arabitol Metabolism of Corynebacterium glutamicum and Its Regulation by AtlR

PubMed Central

Laslo, Tanja; von Zaluskowski, Philipp; Gabris, Christina; Lodd, Elisabeth; Rückert, Christian; Dangel, Petra; Kalinowski, Jörn; Auchter, Marc; Seibold, Gerd

2012-01-01

Expression profiling of Corynebacterium glutamicum in comparison to a derivative deficient in the transcriptional regulator AtlR (previously known as SucR or MtlR) revealed eight genes showing more than 4-fold higher mRNA levels in the mutant. Four of these genes are located in the direct vicinity of the atlR gene, i.e., xylB, rbtT, mtlD, and sixA, annotated as encoding xylulokinase, the ribitol transporter, mannitol 2-dehydrogenase, and phosphohistidine phosphatase, respectively. Transcriptional analysis indicated that atlR and the four genes are organized as atlR-xylB and rbtT-mtlD-sixA operons. Growth experiments with C. glutamicum and C. glutamicum ΔatlR, ΔxylB, ΔrbtT, ΔmtlD, and ΔsixA derivatives with sugar alcohols revealed that (i) wild-type C. glutamicum grows on d-arabitol but not on other sugar alcohols, (ii) growth in the presence of d-arabitol allows subsequent growth on d-mannitol, (iii) d-arabitol is cometabolized with glucose and preferentially utilized over d-mannitol, (iv) RbtT and XylB are involved in d-arabitol but not in d-mannitol metabolism, (v) MtlD is required for d-arabitol and d-mannitol metabolism, and (vi) SixA is not required for growth on any of the substrates tested. Furthermore, we show that MtlD confers d-arabitol and d-mannitol dehydrogenase activities, that the levels of these and also xylulokinase activities are generally high in the C. glutamicum ΔatlR mutant, whereas in the parental strain, they were high when cells were grown in the presence of d-arabitol and very low when cells were grown in its absence. Our results show that the XylB, RbtT, and MtlD proteins allow the growth of C. glutamicum on d-arabitol and that d-arabitol metabolism is subject to arabitol-dependent derepression by AtlR. PMID:22178972

Biotransformation of ferulic acid to protocatechuic acid by Corynebacterium glutamicum ATCC 21420 engineered to express vanillate O-demethylase.

PubMed

Okai, Naoko; Masuda, Takaya; Takeshima, Yasunobu; Tanaka, Kosei; Yoshida, Ken-Ichi; Miyamoto, Masanori; Ogino, Chiaki; Kondo, Akihiko

2017-12-01

Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a lignin-derived phenolic compound abundant in plant biomass. The utilization of FA and its conversion to valuable compounds is desired. Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a precursor of polymers and plastics and a constituent of food. A microbial conversion system to produce PCA from FA was developed in this study using a PCA-producing strain of Corynebacterium glutamicum F (ATCC 21420). C. glutamicum strain F grown at 30 °C for 48 h utilized 2 mM each of FA and vanillic acid (4-hydroxy-3-methoxybenzoic acid, VA) to produce PCA, which was secreted into the medium. FA may be catabolized by C. glutamicum through proposed (I) non-β-oxidative, CoA-dependent or (II) β-oxidative, CoA-dependent phenylpropanoid pathways. The conversion of VA to PCA is the last step in each pathway. Therefore, the vanillate O-demethylase gene (vanAB) from Corynebacterium efficiens NBRC 100395 was expressed in C. glutamicum F (designated strain FVan) cultured at 30 °C in AF medium containing FA. Strain C. glutamicum FVan converted 4.57 ± 0.07 mM of FA into 2.87 ± 0.01 mM PCA after 48 h with yields of 62.8% (mol/mol), and 6.91 mM (1064 mg/L) of PCA was produced from 16.0 mM of FA after 12 h of fed-batch biotransformation. Genomic analysis of C. glutamicum ATCC 21420 revealed that the PCA-utilization genes (pca cluster) were conserved in strain ATCC 21420 and that mutations were present in the PCA importer gene pcaK.

Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions.

PubMed

Tsuge, Yota; Hori, Yoshimi; Kudou, Motonori; Ishii, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

2014-10-01

The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD(+) and NADPH/NADP(+) ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.

Metabolic engineering of Corynebacterium glutamicum ATCC13869 for L-valine production.

PubMed

Chen, Cheng; Li, Yanyan; Hu, Jinyu; Dong, Xunyan; Wang, Xiaoyuan

2015-05-01

In this study, an L-valine-producing strain was developed from Corynebacterium glutamicum ATCC13869 through deletion of the three genes aceE, alaT and ilvA combined with the overexpression of six genes ilvB, ilvN, ilvC, lrp1, brnF and brnE. Overexpression of lrp1 alone increased L-valine production by 16-fold. Deletion of the aceE, alaT and ilvA increased L-valine production by 44-fold. Overexpression of the six genes ilvB, ilvN, ilvC, lrp1, brnE and brnF in the triple deletion mutant WCC003 further increased L-valine production. The strain WCC003/pJYW-4-ilvBNC1-lrp1-brnFE produced 243mM L-valine in flask cultivation and 437mM (51g/L) L-valine in fed-batch fermentation and lacked detectable amino-acid byproduct such as l-alanine and l-isoleucine that are usually found in the fermentation of L-valine-producing C. glutamicum. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Cloning, expression, purification and preliminary crystallographic characterization of a shikimate dehydrogenase from Corynebacterium glutamicum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schoepe, Jan, E-mail: jschoepe@smail.uni-koeln.de; Niefind, Karsten; Chatterjee, Shivani

2006-07-01

The crystallization and preliminary X-ray characterization of a shikimate dehydrogenase from C. glutamicum is presented. The shikimate dehydrogenase from Corynebacterium glutamicum has been cloned into an Escherichia coli expression vector, overexpressed and purified. Native crystals were obtained by the vapour-diffusion technique using 2-methyl-2,4-pentanediol as a precipitant. The crystals belong to the centred monoclinic space group C2, with unit-cell parameters a = 118.77, b = 63.17, c = 35.67 Å, β = 92.26° (at 100 K), and diffract to 1.64 Å on a synchrotron X-ray source. The asymmetric unit is likely to contain one molecule, corresponding to a packing density ofmore » 2.08 Å{sup 3} Da{sup −1} and a solvent content of about 41%.« less

Isoprenoid Pyrophosphate-Dependent Transcriptional Regulation of Carotenogenesis in Corynebacterium glutamicum

PubMed Central

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

2017-01-01

Corynebacterium glutamicum is a natural producer of the C50 carotenoid decaprenoxanthin. The crtEcg0722crtBIYEb operon comprises most of its genes for terpenoid biosynthesis. The MarR-type regulator encoded upstream and in divergent orientation of the carotenoid biosynthesis operon has not yet been characterized. This regulator, named CrtR in this study, is encoded in many actinobacterial genomes co-occurring with terpenoid biosynthesis genes. CrtR was shown to repress the crt operon of C. glutamicum since DNA microarray experiments revealed that transcript levels of crt operon genes were increased 10 to 70-fold in its absence. Transcriptional fusions of a promoter-less gfp gene with the crt operon and crtR promoters confirmed that CrtR represses its own gene and the crt operon. Gel mobility shift assays with purified His-tagged CrtR showed that CrtR binds to a region overlapping with the −10 and −35 promoter sequences of the crt operon. Isoprenoid pyrophosphates interfered with binding of CrtR to its target DNA, a so far unknown mechanism for regulation of carotenogenesis. The molecular details of protein-ligand interactions remain to be studied. Decaprenoxanthin synthesis by C. glutamicum wild type was enhanced 10 to 30-fold upon deletion of crtR and was decreased 5 to 6-fold as result of crtR overexpression. Moreover, deletion of crtR was shown as metabolic engineering strategy to improve production of native and non-native carotenoids including lycopene, β-carotene, C.p. 450 and sarcinaxanthin. PMID:28484430

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

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

PubMed Central

Miyamoto, Aya; Mutoh, Sumire; Kitano, Yuko; Tajima, Mei; Shirakura, Daisuke; Takasaki, Manami; Mitsuhashi, Satoshi; Takeno, Seiki

2013-01-01

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally

Development of biotin-prototrophic and -hyperauxotrophic Corynebacterium glutamicum strains.

PubMed

Ikeda, Masato; Miyamoto, Aya; Mutoh, Sumire; Kitano, Yuko; Tajima, Mei; Shirakura, Daisuke; Takasaki, Manami; Mitsuhashi, Satoshi; Takeno, Seiki

2013-08-01

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally

Expression, purification, crystallization and initial crystallographic characterization of the p-hydroxybenzoate hydroxylase from Corynebacterium glutamicum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Soo-Young; Kang, Beom Sik; Kim, Ghyung-Hwa

2007-11-01

PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collectedmore » to a maximum resolution of 2.5 Å on a synchrotron beamline. The crystal belongs to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = b = 94.72, c = 359.68 Å, γ = 120°. The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 Å{sup 3} Da{sup −1}. The structure was solved by molecular replacement. Structure refinement is in progress.« less

A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression.

PubMed

Lausberg, Frank; Chattopadhyay, Ava Rebecca; Heyer, Antonia; Eggeling, Lothar; Freudl, Roland

2012-09-01

Here we report on the construction of a tetracycline inducible expression vector that allows a tightly regulable gene expression in Corynebacterium glutamicum which is used in industry for production of small molecules such as amino acids. Using the green fluorescent protein (GFP) as a reporter protein we show that this vector, named pCLTON1, is characterized by tight repression under non-induced conditions as compared to a conventional IPTG inducible expression vector, and that it allows gradual GFP synthesis upon gradual increase of anhydrotetracycline addition. Copyright © 2012 Elsevier Inc. All rights reserved.

Distinct roles of two anaplerotic pathways in glutamate production induced by biotin limitation in Corynebacterium glutamicum.

PubMed

Sato, Hiroki; Orishimo, Keita; Shirai, Tomokazu; Hirasawa, Takashi; Nagahisa, Keisuke; Shimizu, Hiroshi; Wachi, Masaaki

2008-07-01

Corynebacterium glutamicum is a biotin auxotrophic bacterium in which glutamate production is induced under biotin-limited conditions. During glutamate production, anaplerotic reactions catalyzed by phosphoenolpyruvate carboxylase (PEPC) and a biotin-containing enzyme pyruvate carboxylase (PC) are believed to play an important role in supplying oxaloacetate in the tricarboxylic acid cycle. To understand the distinct roles of PEPC and PC on glutamate production by C. glutamicum, we observed glutamate production induced under biotin-limited conditions in the disruptants of the genes encoding PEPC (ppc) and PC (pyc), respectively. The pyc disruptant retained the ability to produce high amounts of glutamate, and lactate was simultaneously produced probably due to the increased intracellular pyruvate levels. On the other hand, the ppc knockout mutant could not produce glutamate. Additionally, glutamate production in the pyc disruptant was enhanced by overexpression of ppc rather than disruption of the lactate dehydrogenase gene (ldh), which is involved in lactate production. Metabolic flux analysis based on the 13C-labeling experiment and measurement of 13C-enrichment in glutamate using nuclear magnetic resonance spectroscopy revealed that the flux for anaplerotic reactions in the pyc disruptant was lower than that in the wild type, concomitantly increasing the flux for lactate formation. Moreover, overexpression of ppc increased this flux in both the pyc disruptant and the wild type. Our results suggest that the PEPC-catalyzed anaplerotic reaction is necessary for glutamate production induced under biotin-limited conditions, because PC is not active during glutamate production, and overexpression of ppc effectively enhances glutamate production under biotin-limited conditions.

Metabolic engineering of Corynebacterium glutamicum for fermentative production of chemicals in biorefinery.

PubMed

Baritugo, Kei-Anne; Kim, Hee Taek; David, Yokimiko; Choi, Jong-Il; Hong, Soon Ho; Jeong, Ki Jun; Choi, Jong Hyun; Joo, Jeong Chan; Park, Si Jae

2018-05-01

Bio-based production of industrially important chemicals provides an eco-friendly alternative to current petrochemical-based processes. Because of the limited supply of fossil fuel reserves, various technologies utilizing microbial host strains for the sustainable production of platform chemicals from renewable biomass have been developed. Corynebacterium glutamicum is a non-pathogenic industrial microbial species traditionally used for L-glutamate and L-lysine production. It is a promising species for industrial production of bio-based chemicals because of its flexible metabolism that allows the utilization of a broad spectrum of carbon sources and the production of various amino acids. Classical breeding, systems, synthetic biology, and metabolic engineering approaches have been used to improve its applications, ranging from traditional amino-acid production to modern biorefinery systems for production of value-added platform chemicals. This review describes recent advances in the development of genetic engineering tools and techniques for the establishment and optimization of metabolic pathways for bio-based production of major C2-C6 platform chemicals using recombinant C. glutamicum.

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

PubMed

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.

Mu-driven transposition of recombinant mini-Mu unit DNA in the Corynebacterium glutamicum chromosome.

PubMed

Gorshkova, Natalya V; Lobanova, Juliya S; Tokmakova, Irina L; Smirnov, Sergey V; Akhverdyan, Valerii Z; Krylov, Alexander A; Mashko, Sergey V

2018-03-01

A dual-component Mu-transposition system was modified for the integration/amplification of genes in Corynebacterium. The system consists of two types of plasmids: (i) a non-replicative integrative plasmid that contains the transposing mini-Mu(LR) unit bracketed by the L/R Mu ends or the mini-Mu(LER) unit, which additionally contains the enhancer element, E, and (ii) an integration helper plasmid that expresses the transposition factor genes for MuA and MuB. Efficient transposition in the C. glutamicum chromosome (≈ 2 × 10 -4 per cell) occurred mainly through the replicative pathway via cointegrate formation followed by possible resolution. Optimizing the E location in the mini-Mu unit significantly increased the efficiency of Mu-driven intramolecular transposition-amplification in C. glutamicum as well as in gram-negative bacteria. The new C. glutamicum genome modification strategy that was developed allows the consequent independent integration/amplification/fixation of target genes at high copy numbers. After integration/amplification of the first mini-Mu(LER) unit in the C. glutamicum chromosome, the E-element, which is bracketed by lox-like sites, is excised by Cre-mediated fashion, thereby fixing the truncated mini-Mu(LR) unit in its position for the subsequent integration/amplification of new mini-Mu(LER) units. This strategy was demonstrated using the genes for the citrine and green fluorescent proteins, yECitrine and yEGFP, respectively.

Rational engineering of multiple module pathways for the production of L-phenylalanine in Corynebacterium glutamicum.

PubMed

Zhang, Chuanzhi; Zhang, Junli; Kang, Zhen; Du, Guocheng; Chen, Jian

2015-05-01

Microbial production of L-phenylalanine (L-Phe) from renewable sources has attracted much attention recently. In the present study, Corynebacterium glutamicum 13032 was rationally engineered to produce L-Phe from inexpensive glucose. First, all the L-Phe biosynthesis pathway genes were investigated and the results demonstrated that in addition to AroF and PheA, the native PpsA, TktA, AroE and AroA, and the heterologous AroL and TyrB were also the key enzymes for L-Phe biosynthesis. Through combinational expression of these key enzymes, the L-Phe production was increased to 6.33 ± 0.13 g l(-1) which was about 1.48-fold of that of the parent strain C. glutamicum (pXM-pheA (fbr)-aroF (fbr)) (fbr, feedback-inhibition resistance). Furthermore, the production of L-Phe was improved to 9.14 ± 0.21 g l(-1) by modifying the glucose and L-Phe transport systems and blocking the acetate and lactate biosynthesis pathways. Eventually, the titer of L-Phe was enhanced to 15.76 ± 0.23 g l(-1) with a fed-batch fermentation strategy. To the best of our knowledge, this was the highest value reported in rationally engineered C. glutamicum 13032 strains. The results obtained will also contribute to rational engineering of C. glutamicum for production of other valuable aromatic compounds.

Engineering of Corynebacterium glutamicum for growth and succinate production from levoglucosan, a pyrolytic sugar substrate.

PubMed

Kim, Eun-Mi; Um, Youngsoon; Bott, Michael; Woo, Han Min

2015-10-01

Thermochemical processing provides continuous production of bio-oils from lignocellulosic biomass. Levoglucosan, a pyrolytic sugar substrate C6H10O5 in a bio-oil, has been used for ethanol production using engineered Escherichia coli. Here we provide the first example for succinate production from levoglucosan with Corynebacterium glutamicum, a well-known industrial amino acid producer. Heterologous expression of a gene encoding a sugar kinase from Lipomyces starkeyi, Gibberella zeae or Pseudomonas aeruginosa was employed for levoglucosan conversion in C. glutamicum because the wild type was unable to utilize levoglucosan as sole carbon source. As result, expression of a levoglucosan kinase (LGK) of L. starkeyi only enabled growth with levoglucosan as sole carbon source in CgXII minimal medium by catalyzing conversion of levoglucosan to glucose-6-phosphate. Subsequently, the lgk gene was expressed in an aerobic succinate producer of C. glutamicum, strain BL-1. The recombinant strain showed a higher succinate yield (0.25 g g(-1)) from 2% (w/v) levoglucosan than the reference strain BL-1 from 2% (w/v) glucose (0.19 g g(-1)), confirming that levoglucosan is an attractive carbon substrate for C. glutamicum producer strains. In summary, we demonstrated that a pyrolytic sugar could be a potential carbon source for microbial cell factories. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A Deficiency in Arabinogalactan Biosynthesis Affects Corynebacterium glutamicum Mycolate Outer Membrane Stability▿

PubMed Central

Bou Raad, Roland; Méniche, Xavier; de Sousa-d'Auria, Celia; Chami, Mohamed; Salmeron, Christophe; Tropis, Marielle; Labarre, Cecile; Daffé, Mamadou; Houssin, Christine; Bayan, Nicolas

2010-01-01

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The ultrastructure of the cell envelope is very atypical. It is composed of a heteropolymer of peptidoglycan and arabinogalactan (AG) covalently associated to an outer membrane. Five arabinosyltransferases are involved in the biosynthesis of AG in C. glutamicum. AftB catalyzes the transfer of Araf (arabinofuranosyl) onto the arabinan domain of the arabinogalactan to form terminal β(1 → 2)-linked Araf residues. Here we show that ΔaftB cells lack half of the arabinogalactan mycoloylation sites but are still able to assemble an outer membrane. In addition, we show that a ΔaftB mutant grown on a rich medium has a perturbed cell envelope and sheds a significant amount of membrane fragments in the external culture medium. These fragments contain mono- and dimycolate of trehalose and PorA/H, the major porin of C. glutamicum, but lack conventional phospholipids that typify the plasma membrane, suggesting that they are derived from the atypical mycolate outer membrane of the cell envelope. This is the first report of outer membrane destabilization in the Corynebacterineae, and it suggests that a strong interaction between the mycolate outer membrane and the underlying polymer is essential for cell envelope integrity. The presence of outer membrane-derived fragments (OMFs) in the external medium of the ΔaftB mutant is also a very promising tool for outer membrane characterization. Indeed, fingerprint analysis of major OMF-associated proteins has already led to the identification of 3 associated mycoloyltransferases and an unknown protein with a C-terminal hydrophobic anchoring domain reminiscent of that found for the S-layer protein PS2 of C. glutamicum. PMID:20363942

Modular pathway engineering of Corynebacterium glutamicum to improve xylose utilization and succinate production.

PubMed

Jo, Suah; Yoon, Jinkyung; Lee, Sun-Mi; Um, Youngsoon; Han, Sung Ok; Woo, Han Min

2017-09-20

Xylose-negative Corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (XI) pathway or the Weimberg pathway. Heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. In this study, we show that two functionally-redundant transcriptional regulators (GntR1 and GntR2) present on xylose repress the pentose phosphate pathway genes. For efficient xylose utilization, pentose phosphate pathway genes and a phosphoketolase gene were overexpressed with the XI pathway in C. glutamicum. Overexpression of the genes encoding for transaldolase (Tal), 6-phosphogluconate dehydrogenase (Gnd), or phosphoketolase (XpkA) enhanced the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. However, co-expression of these genes did not have a synergetic effect on xylose utilization. For the succinate production from xylose, overexpression of the tal gene with the XI pathway in a succinate-producing strain improved xylose utilization and increased the specific succinate production rate by 2.5-fold compared to wild-type with the XI pathway alone. Thus, overexpression of the tal, gnd, or xpkA gene could be helpful for engineering C. glutamicum toward production of value-added chemicals with efficient xylose utilization. Copyright © 2017 Elsevier B.V. All rights reserved.

Heat-killed cell preparation of Corynebacterium glutamicum stimulates the immune activity and improves survival of mice against enterohemorrhagic Escherichia coli.

PubMed

Ebisawa, Makoto; Tsukahara, Takamitsu; Fudou, Ryosuke; Ohta, Yasuhiro; Tokura, Mitsunori; Onishi, Norimasa; Fujieda, Takeshi

2017-05-01

Fermentation by Corynebacterium glutamicum is used by various industries to produce L-Glutamate, and the heat-killed cell preparation of this bacterium (HCCG) is a by-product of the fermentation process. In present study, we evaluated the immunostimulating and survival effects against enterohemorrhagic Escherichia coli (STEC) infection of HCCG. HCCG significantly stimulated in vitro IgA and interleukin-12 p70 production in murine Peyer's patch cells and peritoneal macrophages, respectively. Oral administration of 10 mg/kg body weight (BW) of HCCG for seven consecutive days stimulated IgA concentration in murine cecal digesta. Mice were orally administered HCCG for 17 consecutive days (d0-d17), and challenged with STEC on d4 to d6. Survival of mice tended to improve by 100 mg/kg BW of HCCG administration compared with those in control group. In conclusion, HCCG supplementation was found to prevent STEC infection in mice, and thus it may have the potential to stimulate the immune status of mammals.

Enhanced production of recombinant proteins with Corynebacterium glutamicum by deletion of insertion sequences (IS elements).

PubMed

Choi, Jae Woong; Yim, Sung Sun; Kim, Min Jeong; Jeong, Ki Jun

2015-12-29

In most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain. From the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA). Our findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C

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

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.

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. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

PubMed Central

Elišáková, Veronika; Pátek, Miroslav; Holátko, Jiří; Nešvera, Jan; Leyval, Damien; Goergen, Jean-Louis; Delaunay, Stéphane

2005-01-01

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Kmr). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ΔilvA ΔpanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions. PMID:15640189

Analysis of SOS-Induced Spontaneous Prophage Induction in Corynebacterium glutamicum at the Single-Cell Level

PubMed Central

Nanda, Arun M.; Heyer, Antonia; Krämer, Christina; Grünberger, Alexander; Kohlheyer, Dietrich

2014-01-01

The genome of the Gram-positive soil bacterium Corynebacterium glutamicum ATCC 13032 contains three integrated prophage elements (CGP1 to -3). Recently, it was shown that the large lysogenic prophage CGP3 (∼187 kbp) is excised spontaneously in a small number of cells. In this study, we provide evidence that a spontaneously induced SOS response is partly responsible for the observed spontaneous CGP3 induction. Whereas previous studies focused mainly on the induction of prophages at the population level, we analyzed the spontaneous CGP3 induction at the single-cell level using promoters of phage genes (Pint2 and Plysin) fused to reporter genes encoding fluorescent proteins. Flow-cytometric analysis revealed a spontaneous CGP3 activity in about 0.01 to 0.08% of the cells grown in standard minimal medium, which displayed a significantly reduced viability. A PrecA-eyfp promoter fusion revealed that a small fraction of C. glutamicum cells (∼0.2%) exhibited a spontaneous induction of the SOS response. Correlation of PrecA to the activity of downstream SOS genes (PdivS and PrecN) confirmed a bona fide induction of this stress response rather than stochastic gene expression. Interestingly, the reporter output of PrecA and CGP3 promoter fusions displayed a positive correlation at the single-cell level (ρ = 0.44 to 0.77). Furthermore, analysis of the PrecA-eyfp/Pint2-e2-crimson strain during growth revealed the highest percentage of spontaneous PrecA and Pint2 activity in the early exponential phase, when fast replication occurs. Based on these studies, we postulate that spontaneously occurring DNA damage induces the SOS response, which in turn triggers the induction of lysogenic prophages. PMID:24163339

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.

Functional architecture and global properties of the Corynebacterium glutamicum regulatory network: Novel insights from a dataset with a high genomic coverage.

PubMed

Freyre-González, Julio A; Tauch, Andreas

2017-09-10

Corynebacterium glutamicum is a Gram-positive, anaerobic, rod-shaped soil bacterium able to grow on a diversity of carbon sources like sugars and organic acids. It is a biotechnological relevant organism because of its highly efficient ability to biosynthesize amino acids, such as l-glutamic acid and l-lysine. Here, we reconstructed the most complete C. glutamicum regulatory network to date and comprehensively analyzed its global organizational properties, systems-level features and functional architecture. Our analyses show the tremendous power of Abasy Atlas to study the functional organization of regulatory networks. We created two models of the C. glutamicum regulatory network: all-evidences (containing both weak and strong supported interactions, genomic coverage=73%) and strongly-supported (only accounting for strongly supported evidences, genomic coverage=71%). Using state-of-the-art methodologies, we prove that power-law behaviors truly govern the connectivity and clustering coefficient distributions. We found a non-previously reported circuit motif that we named complex feed-forward motif. We highlighted the importance of feedback loops for the functional architecture, beyond whether they are statistically over-represented or not in the network. We show that the previously reported top-down approach is inadequate to infer the hierarchy governing a regulatory network because feedback bridges different hierarchical layers, and the top-down approach disregards the presence of intermodular genes shaping the integration layer. Our findings all together further support a diamond-shaped, three-layered hierarchy exhibiting some feedback between processing and coordination layers, which is shaped by four classes of systems-level elements: global regulators, locally autonomous modules, basal machinery and intermodular genes. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Characterization and chromosomal organization of the murD-murC-ftsQ region of Corynebacterium glutamicum ATCC 13869.

PubMed

Ramos, Angelina; Honrubia, Maria P; Vega, Daniel; Ayala, Juan A; Bouhss, Ahmed; Mengin-Lecreulx, Dominique; Gil, José A

2004-04-01

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

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

PubMed

Becker, Judith; Schäfer, Rudolf; Kohlstedt, Michael; Harder, Björn J; Borchert, Nicole S; Stöveken, Nadine; Bremer, Erhard; Wittmann, Christoph

2013-11-15

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. 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. 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 industrial workhorse C

Osmosensor and osmoregulator properties of the betaine carrier BetP from Corynebacterium glutamicum in proteoliposomes.

PubMed

Rübenhagen, R; Rönsch, H; Jung, H; Krämer, R; Morbach, S

2000-01-14

The secondary glycine betaine uptake system BetP of Corynebacterium glutamicum was purified from Escherichia coli membranes in strep-tagged form after heterologous expression of the betP gene and was reconstituted in E. coli lipids. BetP retained its kinetic properties (V(max) and K(m) for betaine and Na(+)) as compared with intact cells. The influence of driving forces (Na(+) gradient and/or electrical potential) on betaine uptake was quantified in proteoliposomes. BetP was effectively regulated by the external osmolality and was stimulated by the local anesthetic tetracaine. A shift of the optimum of osmotic stimulation to higher osmolalities was linearly correlated with an increasing share of phosphatidyl glycerol, the major lipid of the C. glutamicum plasma membrane in the E. coli lipid proteoliposomes. This finding correlates with results demonstrating an identical shift when betP was expressed in E. coli instead of C. glutamicum. These data indicate that (i) BetP comprises all elements of osmosensing and osmoregulatory mechanisms of betaine uptake, (ii) osmoregulation of BetP is directly related to protein/membrane interactions, (iii) the turgor pressure presumably plays no major role in osmoregulation of BetP, and (iv) the regulatory properties of BetP may be related to the physical state of the surrounding membrane.

Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield in Corynebacterium glutamicum.

PubMed

Zhang, Xin; Zhang, Xiaomei; Xu, Guoqiang; Zhang, Xiaojuan; Shi, Jinsong; Xu, Zhenghong

2018-05-03

L-Serine is widely used in the pharmaceutical, food, and cosmetics industries. Although direct fermentative production of L-serine from sugar in Corynebacterium glutamicum has been achieved, the L-serine yield remains relatively low. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the L-serine yield based on engineered C. glutamicum ΔSSAAI strain. Subsequently, we developed a novel high-throughput screening method using a biosensor constructed based on NCgl0581, a transcriptional factor specifically responsive to L-serine, so that L-serine concentration within single cell of C. glutamicum can be monitored via fluorescence-activated cell sorting (FACS). Novel L-serine-producing mutants were isolated from a large library of mutagenized cells. The mutant strain A36-pDser was screened from 1.2 × 10 5 cells, and the magnesium ion concentration in the medium was optimized specifically for this mutant. C. glutamicum A36-pDser accumulated 34.78 g/L L-serine with a yield of 0.35 g/g sucrose, which were 35.9 and 66.7% higher than those of the parent C. glutamicum ΔSSAAI-pDser strain, respectively. The L-serine yield achieved in this mutant was the highest of all reported L-serine-producing strains of C. glutamicum. Moreover, the whole-genome sequencing identified 11 non-synonymous mutations of genes associated with metabolic and transport pathways, which might be responsible for the higher L-serine production and better cell growth in C. glutamicum A36-pDser. This study explored an effective mutagenesis strategy and reported a novel high-throughput screening method for the development of L-serine-producing strains.

Deregulation of Feedback Inhibition of Phosphoenolpyruvate Carboxylase for Improved Lysine Production in Corynebacterium glutamicum

PubMed Central

Chen, Zhen; Bommareddy, Rajesh Reddy; Frank, Doinita; Rappert, Sugima

2014-01-01

Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and 13C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products. PMID:24334667

Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum.

PubMed

Chen, Zhen; Bommareddy, Rajesh Reddy; Frank, Doinita; Rappert, Sugima; Zeng, An-Ping

2014-02-01

Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and (13)C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products.

(L)-Valine production with minimization of by-products' synthesis in Corynebacterium glutamicum and Brevibacterium flavum.

PubMed

Hou, Xiaohu; Chen, Xinde; Zhang, Yue; Qian, He; Zhang, Weiguo

2012-12-01

Corynebacterium glutamicum ATCC13032 and Brevibacterium flavum JV16 were engineered for L-valine production by over-expressing ilvEBN ( r ) C genes at 31 °C in 72 h fermentation. Different strategies were carried out to reduce the by-products' accumulation in L-valine fermentation and also to increase the availability of precursor for L-valine biosynthesis. The native promoter of ilvA of C. glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of L-isoleucine. Effect of different relative dissolved oxygen on L-valine production and by-products' formation was recorded, indicating that 15 % saturation may be the most appropriate relative dissolved oxygen for L-valine fermentation with almost no L-lactic acid and L-glutamate formed. To minimize L-alanine accumulation, alaT and/or avtA was inactivated in C. glutamicum and B. flavum, respectively. Compared to high concentration of L-alanine accumulated by alaT inactivated strains harboring ilvEBN ( r ) C genes, L-alanine concentration was reduced to 0.18 g/L by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ( r ) C, and 0.22 g/L by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ( r ) C. Meanwhile, L-valine production and conversion efficiency were enhanced to 31.15 g/L and 0.173 g/g by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ( r ) C, 38.82 g/L and 0.252 g/g by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ( r ) C. This study provides combined strategies to improve L-valine yield by minimization of by-products' production.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Dual production of poly(3-hydroxybutyrate) and glutamate using variable biotin concentrations in Corynebacterium glutamicum.

PubMed

Jo, Sung-Jin; Leong, Chean Ring; Matsumoto, Ken'ichiro; Taguchi, Seiichi

2009-04-01

We previously synthesized poly(3-hydroxybutyrate) [P(3HB)] in recombinant Corynebacterium glutamicum, a prominent producer of amino acids. In this study, a two-step cultivation was established for the dual production of glutamate and P(3HB) due to the differences in the optimal concentration of biotin. Glutamate was extracellularly produced first under the biotin-limited condition of 0.3 microg/L. Production was then shifted to P(3HB) by addition of biotin to a total concentration of 9 microg/L. The final products obtained were 18 g/L glutamate and 36 wt% of P(3HB).

Disruption of pknG enhances production of gamma-aminobutyric acid by Corynebacterium glutamicum expressing glutamate decarboxylase.

PubMed

Okai, Naoko; Takahashi, Chihiro; Hatada, Kazuki; Ogino, Chiaki; Kondo, Akihiko

2014-01-01

Gamma-aminobutyric acid (GABA), a building block of the biodegradable plastic polyamide 4, is synthesized from glucose by Corynebacterium glutamicum that expresses Escherichia coli glutamate decarboxylase (GAD) B encoded by gadB. This strain was engineered to produce GABA more efficiently from biomass-derived sugars. To enhance GABA production further by increasing the intracellular concentration of its precursor glutamate, we focused on engineering pknG (encoding serine/threonine protein kinase G), which controls the activity of 2-oxoglutarate dehydrogenase (Odh) in the tricarboxylic acid cycle branch point leading to glutamate synthesis. We succeeded in expressing GadB in a C. glutamicum strain harboring a deletion of pknG. C. glutamicum strains GAD and GAD ∆pknG were cultured in GP2 medium containing 100 g L(-1) glucose and 0.1 mM pyridoxal 5'-phosphate. Strain GAD∆pknG produced 31.1 ± 0.41 g L(-1) (0.259 g L(-1) h(-1)) of GABA in 120 hours, representing a 2.29-fold higher level compared with GAD. The production yield of GABA from glucose by GAD∆pknG reached 0.893 mol mol(-1).

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.

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

Promoter library-based module combination (PLMC) technology for optimization of threonine biosynthesis in Corynebacterium glutamicum.

PubMed

Wei, Liang; Xu, Ning; Wang, Yiran; Zhou, Wei; Han, Guoqiang; Ma, Yanhe; Liu, Jun

2018-05-01

Due to the lack of efficient control elements and tools, the fine-tuning of gene expression in the multi-gene metabolic pathways is still a great challenge for engineering microbial cell factories, especially for the important industrial microorganism Corynebacterium glutamicum. In this study, the promoter library-based module combination (PLMC) technology was developed to efficiently optimize the expression of genes in C. glutamicum. A random promoter library was designed to contain the putative - 10 (NNTANANT) and - 35 (NNGNCN) consensus motifs, and refined through a three-step screening procedure to achieve numerous genetic control elements with different strength levels, including fluorescence-activated cell sorting (FACS) screening, agar plate screening, and 96-well plate screening. Multiple conventional strategies were employed for further precise characterizations of the promoter library, such as real-time quantitative PCR, sodium dodecyl sulfate polyacrylamide gel electrophoresis, FACS analysis, and the lacZ reporter system. These results suggested that the established promoter elements effectively regulated gene expression and showed varying strengths over a wide range. Subsequently, a multi-module combination technology was created based on the efficient promoter elements for combination and optimization of modules in the multi-gene pathways. Using this technology, the threonine biosynthesis pathway was reconstructed and optimized by predictable tuning expression of five modules in C. glutamicum. The threonine titer of the optimized strain was significantly improved to 12.8 g/L, an approximate 6.1-fold higher than that of the control strain. Overall, the PLMC technology presented in this study provides a rapid and effective method for combination and optimization of multi-gene pathways in C. glutamicum.

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

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

PubMed

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

2014-07-01

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. 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. 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. 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.

A role of the transcriptional regulator LldR (NCgl2814) in glutamate metabolism under biotin-limited conditions in Corynebacterium glutamicum.

PubMed

Supkulsutra, Tanyanut; Maeda, Tomoya; Kumagai, Kosuke; Wachi, Masaaki

2013-01-01

Corynebacterium glutamicum is a Gram-positive, rod-shaped, aerobic bacterium used for the fermentative production of L-glutamate. LldR (NCgl2814) is known as a repressor for ldhA and lldD encoding lactate dehydrogenases. LdhA is responsible for production of L-lactate, while LldD is for its assimilation. Since L-lactate production was observed as a by-product of glutamate production under biotin-limited conditions, LldR might play a regulatory role in the glutamate metabolism. Here for the first time, we investigated effects of overproduction or deletion of LldR on the glutamate metabolism under biotin-limited conditions in C. glutamicum. It was found that glutamate production under biotin-limited conditions was decreased by overproduction of LldR. In the wild-type cells, L-lactate was produced in the first 24 h and it was re-consumed thereafter. On the other hand, in the overproduced cells, L-lactate was produced like the wild type, but it was not re-consumed. This means that L-lactate assimilation, which is catalyzed by LldD, was suppressed by the overproduction of LldR, but L-lactate production, which is catalyzed by LdhA, was not affected, indicating that LldR mainly controls the expression of lldD but not of ldhA under biotin-limited conditions. This was confirmed by quantitative real-time RT-PCR. From these results, it is suggested that L-lactate metabolism, which is controlled by LldR, has a buffering function of the pyruvate pool for glutamate production.

Characterization and use of catabolite-repressed promoters from gluconate genes in Corynebacterium glutamicum.

PubMed

Letek, Michal; Valbuena, Noelia; Ramos, Angelina; Ordóñez, Efrén; Gil, José A; Mateos, Luís M

2006-01-01

The genes involved in gluconate catabolism (gntP and gntK) in Corynebacterium glutamicum are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the gnt operon in Escherichia coli and Bacillus subtilis. In C. glutamicum, gntP and gntK are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of gntP and gntK is not induced by gluconate, and the gnt genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (PgntK and PgntP) was performed using bifunctional promoter probe vectors containing mel (involved in melanin production) or egfp2 (encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the divIVA gene in C. glutamicum is shown, corroborating the possible use of the gnt promoters to control gene expression. glxR (which encodes GlxR, the hypothetical CRP protein) was subcloned from the C. glutamicum chromosomal DNA and overexpressed in corynebacteria; we found that the level of gnt expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on PgntP and PgntK fragments was detected only in the presence of cAMP.

Analysis of optimal phenotypic space using elementary modes as applied to Corynebacterium glutamicum

PubMed Central

Gayen, Kalyan; Venkatesh, KV

2006-01-01

Background Quantification of the metabolic network of an organism offers insights into possible ways of developing mutant strain for better productivity of an extracellular metabolite. The first step in this quantification is the enumeration of stoichiometries of all reactions occurring in a metabolic network. The structural details of the network in combination with experimentally observed accumulation rates of external metabolites can yield flux distribution at steady state. One such methodology for quantification is the use of elementary modes, which are minimal set of enzymes connecting external metabolites. Here, we have used a linear objective function subject to elementary modes as constraint to determine the fluxes in the metabolic network of Corynebacterium glutamicum. The feasible phenotypic space was evaluated at various combinations of oxygen and ammonia uptake rates. Results Quantification of the fluxes of the elementary modes in the metabolism of C. glutamicum was formulated as linear programming. The analysis demonstrated that the solution was dependent on the criteria of objective function when less than four accumulation rates of the external metabolites were considered. The analysis yielded feasible ranges of fluxes of elementary modes that satisfy the experimental accumulation rates. In C. glutamicum, the elementary modes relating to biomass synthesis through glycolysis and TCA cycle were predominantly operational in the initial growth phase. At a later time, the elementary modes contributing to lysine synthesis became active. The oxygen and ammonia uptake rates were shown to be bounded in the phenotypic space due to the stoichiometric constraint of the elementary modes. Conclusion We have demonstrated the use of elementary modes and the linear programming to quantify a metabolic network. We have used the methodology to quantify the network of C. glutamicum, which evaluates the set of operational elementary modes at different phases of

Integrated Analysis of the Transcriptome and Metabolome of Corynebacterium glutamicum during Penicillin-Induced Glutamic Acid Production.

PubMed

Hirasawa, Takashi; Saito, Masaki; Yoshikawa, Katsunori; Furusawa, Chikara; Shmizu, Hiroshi

2018-05-01

Corynebacterium glutamicum is known for its ability to produce glutamic acid and has been utilized for the fermentative production of various amino acids. Glutamic acid production in C. glutamicum is induced by penicillin. In this study, the transcriptome and metabolome of C. glutamicum is analyzed to understand the mechanism of penicillin-induced glutamic acid production. Transcriptomic analysis with DNA microarray revealed that expression of some glycolysis- and TCA cycle-related genes, which include those encoding the enzymes involved in conversion of glucose to 2-oxoglutaric acid, is upregulated after penicillin addition. Meanwhile, expression of some TCA cycle-related genes, encoding the enzymes for conversion of 2-oxoglutaric acid to oxaloacetic acid, and the anaplerotic reactions decreased. In addition, expression of NCgl1221 and odhI, encoding proteins involved in glutamic acid excretion and inhibition of the 2-oxoglutarate dehydrogenase, respectively, is upregulated. Functional category enrichment analysis of genes upregulated and downregulated after penicillin addition revealed that genes for signal transduction systems are enriched among upregulated genes, whereas those for energy production and carbohydrate and amino acid metabolisms are enriched among the downregulated genes. As for the metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry, the intracellular content of most metabolites of the glycolysis and the TCA cycle decreased dramatically after penicillin addition. Overall, these results indicate that the cellular metabolism and glutamic acid excretion are mainly optimized at the transcription level during penicillin-induced glutamic acid production by C. glutamicum. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Linking Central Metabolism with Increased Pathway Flux: l-Valine Accumulation by Corynebacterium glutamicum

PubMed Central

Radmacher, Eva; Vaitsikova, Adela; Burger, Udo; Krumbach, Karin; Sahm, Hermann; Eggeling, Lothar

2002-01-01

Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE, which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete l-valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since l-valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate. The same genes, ilvBNCD, overexpressed in an ilvA deletion mutant which is unable to synthesize l-isoleucine increased the concentration of this amino acid to 58 mM. A further dramatic increase was obtained when panBC was deleted, making the resulting mutant auxotrophic for d-pantothenate. When the resulting strain, C. glutamicum 13032ΔilvAΔpanBC with ilvBNCD overexpressed, was grown under limiting conditions it accumulated 91 mM l-valine. This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the l-valine biosynthesis pathway. PMID:11976094

Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis

PubMed Central

Liu, Dong-Xin; Fan, Chang-Sheng; Tao, Ju-Hong; Liang, Guo-Xin; Gao, Shan-E; Wang, Hai-Jiao; Li, Xin; Song, Da-Xin

2004-01-01

AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine. METHODS: By nitrosoguanidine mutagenesis, five p-fluorophenylalanine (FP)-resistant mutants of C.glutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the PBF-aroG-pheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pTK and pTGAK, respectively. Then, they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays. RESULTS: Engineering strains of C.glutamicum (Tyr-) were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold, and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, respectively. CONCLUSION: Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production. PMID:15534933

Influence of membrane composition on osmosensing by the betaine carrier BetP from Corynebacterium glutamicum.

PubMed

Schiller, Dirk; Ott, Vera; Krämer, Reinhard; Morbach, Susanne

2006-03-24

The glycine betaine carrier BetP from Corynebacterium glutamicum was recently shown to function as both an osmosensor and osmoregulator in proteoliposomes made from Escherichia coli phospholipids by sensing changes in the internal K+ concentration as a measure of hyperosmotic stress (Rübenhagen, R., Morbach, S., and Krämer, R. (2001) EMBO J. 20, 5412-5420). Furthermore, evidence was provided that a stretch of 25 amino acids of the C-terminal domain of BetP is critically involved in K+ sensing. This K+-sensitive region has been further characterized. Glu572 turned out to be important for osmosensing in E. coli cells and in proteoliposomes made from E. coli phospholipids. BetP mutants E572K, E572P, and E572A/H573A/R574A were unable to detect an increase in the internal K+ concentration in this membrane environment. However, these BetP variants regained their ability to detect osmotic stress in membranes with increased phosphatidylglycerol content, i.e. in intact C. glutamicum cells or in proteoliposomes mimicking the composition of the C. glutamicum membrane. Mutants E572P and Y550P were still insensitive to osmotic stress also in this membrane background. These results led to the following conclusions. (i) The K+ sensor in mutants E572Q, E572D, and E572K is only partially impaired. (ii) Restoration of activity regulation is not possible if the correct conformation or orientation of the C-terminal domain is compromised by a proline residue at position 572 or 550. (iii) Phosphatidylglycerol in the membrane of C. glutamicum seems to stabilize the inactive conformation of BetP C252T and other mutants.

Maltose Uptake by the Novel ABC Transport System MusEFGK2I Causes Increased Expression of ptsG in Corynebacterium glutamicum

PubMed Central

Henrich, Alexander; Kuhlmann, Nora; Eck, Alexander W.; Krämer, Reinhard

2013-01-01

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 [14C]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. PMID:23543710

New insights into the biogenesis of the cell envelope of corynebacteria: identification and functional characterization of five new mycoloyltransferase genes in Corynebacterium glutamicum.

PubMed

De Sousa-D'Auria, Célia; Kacem, Raoudha; Puech, Virginie; Tropis, Marielle; Leblon, Gérard; Houssin, Christine; Daffé, Mamadou

2003-07-15

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

[Metabolic flux analysis of L-serine synthesis by Corynebacterium glutamicum SYPS-062].

PubMed

Zhang, Xiaomei; Dou, Wenfang; Xu, Hongyu; Xu, Zhenghong

2010-10-01

Corynebacterium glutamicum SYPS-062 was an L-serine producing strain stored at our lab and could produce L-serine directly from sugar. We studied the effects of cofactors in one carbon unit metabolism-folate and VB12 on the cell growth, sucrose consumption and L-serine production by SYPS-062. In the same time, the metabolic flux distribution was determined in different conditions. The supplementation of folate or VB12 enhanced the cell growth, energy synthesis, and finally increased the flux of pentose phosphate pathway (HMP), whereas the carbon flux to L-serine was decreased. The addition of VB12 not only increased the ratio of L-serine synthesis pathway on G3P joint, but also caused the insufficiency of tricarboxylic acid cycle (TCA) flux, which needed more anaplerotic reaction flux to replenish TCA cycle, that was an important limiting factor for the further increasing of the L-serine productivity.

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.

Metabolic Design of Corynebacterium glutamicum for Production of l-Cysteine with Consideration of Sulfur-Supplemented Animal Feed.

PubMed

Joo, Young-Chul; Hyeon, Jeong Eun; Han, Sung Ok

2017-06-14

l-Cysteine is a valuable sulfur-containing amino acid widely used as a nutrition supplement in industrial food production, agriculture, and animal feed. However, this amino acid is mostly produced by acid hydrolysis and extraction from human or animal hairs. In this study, we constructed recombinant Corynebacterium glutamicum strains that overexpress combinatorial genes for l-cysteine production. The aims of this work were to investigate the effect of the combined overexpression of serine acetyltransferase (CysE), O-acetylserine sulfhydrylase (CysK), and the transcriptional regulator CysR on l-cysteine production. The CysR-overexpressing strain accumulated approximately 2.7-fold more intracellular sulfide than the control strain (empty pMT-tac vector). Moreover, in the resulting CysEKR recombinant strain, combinatorial overexpression of genes involved in l-cysteine production successfully enhanced its production by approximately 3.0-fold relative to that in the control strain. This study demonstrates a biotechnological model for the production of animal feed supplements such as l-cysteine using metabolically engineered C. glutamicum.

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vetting,M.; Frantom, P.; Blanchard, J.

2008-01-01

The glycosyltransferase termed MshA catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to 1-l-myo-inositol-1-phosphate in the first committed step of mycothiol biosynthesis. The structure of MshA from Corynebacterium glutamicum was determined both in the absence of substrates and in a complex with UDP and 1-l-myo-inositol-1-phosphate. MshA belongs to the GT-B structural family whose members have a two-domain structure with both domains exhibiting a Rossman-type fold. Binding of the donor sugar to the C-terminal domain produces a 97 rotational reorientation of the N-terminal domain relative to the C-terminal domain, clamping down on UDP and generating the binding site for 1-l-myo-inositol-1-phosphate. The structuremore » highlights the residues important in binding of UDP-N-acetylglucosamine and 1-l-myo-inositol-1-phosphate. Molecular models of the ternary complex suggest a mechanism in which the {beta}-phosphate of the substrate, UDP-N-acetylglucosamine, promotes the nucleophilic attack of the 3-hydroxyl group of 1-l-myo-inositol-1-phosphate while at the same time promoting the cleavage of the sugar nucleotide bond.« less

NrdH Redoxin Enhances Resistance to Multiple Oxidative Stresses by Acting as a Peroxidase Cofactor in Corynebacterium glutamicum

PubMed Central

Si, Mei-Ru; Zhang, Lei; Yang, Zhi-Fang; Xu, Yi-Xiang; Liu, Ying-Bao; Jiang, Cheng-Ying; Wang, Yao; Liu, Shuang-Jiang

2014-01-01

NrdH redoxins are small protein disulfide oxidoreductases behaving like thioredoxins but sharing a high amino acid sequence similarity to glutaredoxins. Although NrdH redoxins are supposed to be another candidate in the antioxidant system, their physiological roles in oxidative stress remain unclear. In this study, we confirmed that the Corynebacterium glutamicum NrdH redoxin catalytically reduces the disulfides in the class Ib ribonucleotide reductases (RNR), insulin and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), by exclusively receiving electrons from thioredoxin reductase. Overexpression of NrdH increased the resistance of C. glutamicum to multiple oxidative stresses by reducing ROS accumulation. Accordingly, elevated expression of the nrdH gene was observed when the C. glutamicum wild-type strain was exposed to oxidative stress conditions. It was discovered that the NrdH-mediated resistance to oxidative stresses was largely dependent on the presence of the thiol peroxidase Prx, as the increased resistance to oxidative stresses mediated by overexpression of NrdH was largely abrogated in the prx mutant. Furthermore, we showed that NrdH facilitated the hydroperoxide reduction activity of Prx by directly targeting and serving as its electron donor. Thus, we present evidence that the NrdH redoxin can protect against the damaging effects of reactive oxygen species (ROS) induced by various exogenous oxidative stresses by acting as a peroxidase cofactor. PMID:24375145

l-Valine Production during Growth of Pyruvate Dehydrogenase Complex- Deficient Corynebacterium glutamicum in the Presence of Ethanol or by Inactivation of the Transcriptional Regulator SugR▿

PubMed Central

Blombach, Bastian; Arndt, Annette; Auchter, Marc; Eikmanns, Bernhard J.

2009-01-01

Pyruvate dehydrogenase complex-deficient strains of Corynebacterium glutamicum produce l-valine from glucose only after depletion of the acetate required for growth. Here we show that inactivation of the DeoR-type transcriptional regulator SugR or replacement of acetate by ethanol already in course of the growth phase results in efficient l-valine production. PMID:19088318

Overexpression of ppc or deletion of mdh for improving production of γ-aminobutyric acid in recombinant Corynebacterium glutamicum.

PubMed

Shi, Feng; Zhang, Ming; Li, Yongfu

2017-06-01

L-Glutamate decarboxylase (GAD) transforms L-glutamate into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene(s) can synthesize GABA from its own produced L-glutamate. To enhance GABA production in recombinant C. glutamicum strain SH, metabolic engineering strategies were used to improve the supply of the GABA precursor, L-glutamate. Five new strains were constructed here. First, the ppc gene was coexpressed with two GAD genes (gadB1 and gadB2). Then, the mdh gene was deleted in C. glutamicum SH. Next, gadB1-gadB2 and gadB1-gadB2-ppc co-expression plasmids were transformed into C. glutamicum strains SH and Δmdh, resulting in four recombinant GAD strains SE1, SE2, SDE1, and SDE2, respectively. Finally, the mdh gene was overexpressed in mdh-deleted SDE1, generating the mdh-complemented GAD strain SDE3. After fermenting for 72 h, GABA production increased to 26.3 ± 3.4, 24.8 ± 0.7, and 25.5 ± 3.3 g/L in ppc-overexpressed SE2, mdh-deleted SDE1, and mdh-deleted ppc-overexpressed SDE2, respectively, which was higher than that in the control GAD strain SE1 (22.7 ± 0.5 g/L). While in the mdh-complemented SDE3, GABA production decreased to 20.0 ± 0.6 g/L. This study demonstrates that the recombinant strains SE2, SDE1, and SDE2 can be used as candidates for GABA production.

Trehalose acts as a uridine 5'-diphosphoglucose-competitive inhibitor of trehalose 6-phosphate synthase in Corynebacterium glutamicum.

PubMed

Oide, Shinichi; Inui, Masayuki

2017-12-01

Trehalose is a compatible solute widely distributed in nature. The most prevalent pathway for its synthesis starts from condensation of glucose 6-phosphate (Glc6P) and uridine 5'-diphosphoglucose (UDP-Glc) catalyzed by trehalose 6-phosphate synthase (TPS). A previous laboratory evolution experiment with the bacterium Corynebacterium glutamicum generated strains adapted to supraoptimal temperatures, and the R328H substitution of the TPS encoded by otsA was shown to be associated with thermotolerance acquired by the evolved strains. In this study, we found that the OtsA:R328H substitution promotes both intra- and extracellular trehalose accumulation and demonstrated that build-up of intracellular trehalose accounts for the OtsA R 328H -dependent thermotolerance, using the mycobacterial trehalose-specific transporter. Counterintuitively, characterization of the recombinant OtsA proteins revealed that the mutation downshifts the temperature optimum of OtsA. A search for the molecular basis of OtsA R 328H -dependent enhancement of trehalose synthesis led to the unexpected findings that trehalose is an effective inhibitor of OtsA and that OtsA R 328H is highly tolerant to the trehalose-mediated inhibition. The only available report on such feedback regulation of TPS is for the silk moth from over 50 years ago [Murphy TA and Wyatt GR (1965) J Biol Chem 240, 1500-1508]. While trehalose acts as a Glc6P-competitive inhibitor in the silk moth, the disaccharide was found to inhibit OtsA in a UDP-Glc-competitive manner in C. glutamicum, suggesting independent origins of the negative feedback regulations found for the two species. We showed that overexpression of the wild-type OtsA counteracts the trehalose-dependent regulation and restores the evolved strain-like phenotype to the isogenic wild-type otsA revertant, demonstrating that thermotolerance conferred by OtsA R 328H is attributable to its feedback-resistant property. © 2017 Federation of European Biochemical

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.

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

Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate.

PubMed

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

2013-09-01

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.

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

Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rice, E.A.; Bannon, G.A.; Glenn, K.C.

2008-11-21

The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysinemore » revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.« less

Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producing γ-aminobutyrate in Corynebacterium glutamicum.

PubMed

Shi, Feng; Luan, Mingyue; Li, Yongfu

2018-04-18

Glutamate decarboxylase (GAD) converts L-glutamate (Glu) into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene, gadB2 or gadB1, can synthesize GABA from its own produced Glu. To enhance GABA production in C. glutamicum, ribosomal binding site (RBS) sequence and promoter were searched and optimized for increasing the expression efficiency of gadB2. R4 exhibited the highest strength among RBS sequences tested, with 6 nt the optimal aligned spacing (AS) between RBS and start codon. This combination of RBS sequence and AS contributed to gadB2 expression, increased GAD activity by 156% and GABA production by 82% compared to normal strong RBS and AS combination. Then, a series of native promoters were selected for transcribing gadB2 under optimal RBS and AS combination. P dnaK , P dtsR , P odhI and P clgR expressed gadB2 and produced GABA as effectively as widely applied P tuf and P cspB promoters and more effectively than P sod promoter. However, each native promoter did not work as well as the synthetic strong promoter P tacM , which produced 20.2 ± 0.3 g/L GABA. Even with prolonged length and bicistronic architecture, the strength of P dnaK did not enhance. Finally, gadB2 and mutant gadB1 were co-expressed under the optimal promoter and RBS combination, thus converted Glu into GABA completely and improved GABA production to more than 25 g/L. This study provides useful promoters and RBS sequences for gene expression in C. glutamicum.

Reprogramming One-Carbon Metabolic Pathways To Decouple l-Serine Catabolism from Cell Growth in Corynebacterium glutamicum.

PubMed

Zhang, Yun; Shang, Xiuling; Lai, Shujuan; Zhang, Yu; Hu, Qitiao; Chai, Xin; Wang, Bo; Liu, Shuwen; Wen, Tingyi

2018-02-16

l-Serine, the principal one-carbon source for DNA biosynthesis, is difficult for microorganisms to accumulate due to the coupling of l-serine catabolism and microbial growth. Here, we reprogrammed the one-carbon unit metabolic pathways in Corynebacterium glutamicum to decouple l-serine catabolism from cell growth. In silico model-based simulation showed a negative influence on glyA-encoding serine hydroxymethyltransferase flux with l-serine productivity. Attenuation of glyA transcription resulted in increased l-serine accumulation, and a decrease in purine pools, poor growth and longer cell shapes. The gcvTHP-encoded glycine cleavage (Gcv) system from Escherichia coli was introduced into C. glutamicum, allowing glycine-derived 13 CH 2 to be assimilated into intracellular purine synthesis, which resulted in an increased amount of one-carbon units. Gcv introduction not only restored cell viability and morphology but also increased l-serine accumulation. Moreover, comparative proteomic analysis indicated that abundance changes of the enzymes involved in one-carbon unit cycles might be responsible for maintaining one-carbon unit homeostasis. Reprogramming of the one-carbon metabolic pathways allowed cells to reach a comparable growth rate to accumulate 13.21 g/L l-serine by fed-batch fermentation in minimal medium. This novel strategy provides new insights into the regulation of cellular properties and essential metabolite accumulation by introducing an extrinsic pathway.

Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacterium glutamicum for lysine production.

PubMed

Chen, Zhen; Meyer, Weiqian; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

2011-07-01

Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter L-lysine within 30 h of fed-batch fermentation in a bioreactor.

Rich biotin content in lignocellulose biomass plays the key role in determining cellulosic glutamic acid accumulation by Corynebacterium glutamicum.

PubMed

Wen, Jingbai; Xiao, Yanqiu; Liu, Ting; Gao, Qiuqiang; Bao, Jie

2018-01-01

Lignocellulose is one of the most promising alternative feedstocks for glutamic acid production as commodity building block chemical, but the efforts by the dominant industrial fermentation strain Corynebacterium glutamicum failed for accumulating glutamic acid using lignocellulose feedstock. We identified the existence of surprisingly high biotin concentration in corn stover hydrolysate as the determining factor for the failure of glutamic acid accumulation by Corynebacterium glutamicum . Under excessive biotin content, induction by penicillin resulted in 41.7 ± 0.1 g/L of glutamic acid with the yield of 0.50 g glutamic acid/g glucose. Our further investigation revealed that corn stover contained 353 ± 16 μg of biotin per kg dry solids, approximately one order of magnitude greater than the biotin in corn grain. Most of the biotin remained stable during the biorefining chain and the rich biotin content in corn stover hydrolysate almost completely blocked the glutamic acid accumulation. This rich biotin existence was found to be a common phenomenon in the wide range of lignocellulose biomass and this may be the key reason why the previous studies failed in cellulosic glutamic acid fermentation from lignocellulose biomass. The extended recording of the complete members of all eight vitamin B compounds in lignocellulose biomass further reveals that the major vitamin B members were also under the high concentration levels even after harsh pretreatment. The high content of biotin in wide range of lignocellulose biomass feedstocks and the corresponding hydrolysates was discovered and it was found to be the key factor in determining the cellulosic glutamic acid accumulation. The highly reserved biotin and the high content of their other vitamin B compounds in biorefining process might act as the potential nutrients to biorefining fermentations. This study creates a new insight that lignocellulose biorefining not only generates inhibitors, but also keeps nutrients

Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains.

PubMed

Mustafi, Nurije; Grünberger, Alexander; Mahr, Regina; Helfrich, Stefan; Nöh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia

2014-01-01

The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains.

Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains

PubMed Central

Mahr, Regina; Helfrich, Stefan; Nöh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia

2014-01-01

The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains. PMID:24465669

Biotypes analysis of Corynebacterium glutamicum growing in dicarboxylic acids demonstrates the existence of industrially-relevant intra-species variations.

PubMed

Pérez-García, Fernando; Vasco-Cárdenas, María F; Barreiro, Carlos

2016-09-02

Production enhancement of industrial microbial products or strains has been traditionally tackled by mutagenesis with chemical methods, irradiation or genetic manipulation. However, the final yield increase must go hand in hand with the resistance increasing against the usual inherent toxicity of the final products. Few studies have been carried out on resistance improvement and even fewer on the initial selection of naturally-generated biotypes, which could decrease the artificial mutagenesis. This fact is vital in the case of GRAS microorganisms as Corynebacterium glutamicum involved in food, feed and cosmetics production. The characteristic wide diversity and plasticity in terms of their genetic material of Actinobacteria eases the biotypes generation. Thus, differences in morphology, glutamate and lysine production and growth in media supplemented with dicarboxylic acids were analysed in four biotypes of C. glutamicum ATCC 13032. A 2D-DIGE analysis of these biotypes growing with itaconic acid allowed us to define their differences. Thus, an optimized central metabolism and better protection against the generated stress conditions present the CgL biotype as a suitable platform for production of itaconic acid, which is used as a building block (e.g.: acrylic plastic). This analysis highlights the preliminary biotypes screening as a way to reach optimal industrial productions.

Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum

PubMed Central

2012-01-01

Background The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 might be involved in biotin uptake by C. glutamicum. Results By comparison of global gene expression patterns of cells grown with limiting or excess supply of biotin or with dethiobiotin as supplement replacing biotin revealed that expression of genes coding for enzymes of biotin ring assembly and for the putative uptake system was regulated according to biotin availability. RT-PCR and 5'-RACE experiments demonstrated that the genes bioY, bioM, and bioN are transcribed from one promoter as a single transcript. Biochemical analyses revealed that BioYMN catalyzes the effective uptake of biotin with a concentration of 60 nM biotin supporting a half-maximal transport rate. Maximal biotin uptake rates were at least five fold higher in biotin-limited cells as compared to cells grown with excess biotin. Overexpression of bioYMN led to an at least 50 fold higher biotin uptake rate as compared to the empty vector control. Overproduction of BioYMN alleviated biotin limitation and interfered with triggering L-glutamate production by biotin limitation. Conclusions The operon bioYMN from C. glutamicum was shown to be induced by biotin limitation. Transport assays with radio-labeled biotin revealed that BioYMN functions as a biotin uptake system. Overexpression of bioYMN affected L-glutamate production triggered by biotin limitation. PMID:22243621

Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum.

PubMed

Schneider, Jens; Peters-Wendisch, Petra; Stansen, K Corinna; Götker, Susanne; Maximow, Stanislav; Krämer, Reinhard; Wendisch, Volker F

2012-01-13

The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 might be involved in biotin uptake by C. glutamicum. By comparison of global gene expression patterns of cells grown with limiting or excess supply of biotin or with dethiobiotin as supplement replacing biotin revealed that expression of genes coding for enzymes of biotin ring assembly and for the putative uptake system was regulated according to biotin availability. RT-PCR and 5'-RACE experiments demonstrated that the genes bioY, bioM, and bioN are transcribed from one promoter as a single transcript. Biochemical analyses revealed that BioYMN catalyzes the effective uptake of biotin with a concentration of 60 nM biotin supporting a half-maximal transport rate. Maximal biotin uptake rates were at least five fold higher in biotin-limited cells as compared to cells grown with excess biotin. Overexpression of bioYMN led to an at least 50 fold higher biotin uptake rate as compared to the empty vector control. Overproduction of BioYMN alleviated biotin limitation and interfered with triggering L-glutamate production by biotin limitation. The operon bioYMN from C. glutamicum was shown to be induced by biotin limitation. Transport assays with radio-labeled biotin revealed that BioYMN functions as a biotin uptake system. Overexpression of bioYMN affected L-glutamate production triggered by biotin limitation.

Glucose consumption rate critically depends on redox state in Corynebacterium glutamicum under oxygen deprivation.

PubMed

Tsuge, Yota; Uematsu, Kimio; Yamamoto, Shogo; Suda, Masako; Yukawa, Hideaki; Inui, Masayuki

2015-07-01

Rapid sugar consumption is important for the microbial production of chemicals and fuels. Here, we show that overexpression of the NADH dehydrogenase gene (ndh) increased glucose consumption rate in Corynebacterium glutamicum under oxygen-deprived conditions through investigating the relationship between the glucose consumption rate and intracellular NADH/NAD(+) ratio in various mutant strains. The NADH/NAD(+) ratio was strongly repressed under oxygen deprivation when glucose consumption was accelerated by the addition of pyruvate or sodium hydrogen carbonate. Overexpression of the ndh gene in the wild-type strain under oxygen deprivation decreased the NADH/NAD(+) ratio from 0.32 to 0.13, whereas the glucose consumption rate increased by 27%. Similarly, in phosphoenolpyruvate carboxylase gene (ppc)- or malate dehydrogenase gene (mdh)-deficient strains, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.66 to 0.37 and 2.20 to 0.57, respectively, whereas the glucose consumption rate increased by 57 and 330%, respectively. However, in a lactate dehydrogenase gene (L-ldhA)-deficient strain, although the NADH/NAD(+) ratio decreased from 5.62 to 1.13, the glucose consumption rate was not markedly altered. In a tailored D-lactate-producing strain, which lacked ppc and L-ldhA genes, but expressed D-ldhA from Lactobacillus delbrueckii, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.77 to 0.56, and increased the glucose consumption rate by 50%. Overall, the glucose consumption rate was found to be inversely proportional to the NADH/NAD(+) ratio in C. glutamicum cultured under oxygen deprivation. These findings could provide an option to increase the productivity of chemicals and fuels under oxygen deprivation.

Extreme furfural tolerance of a soil bacterium Enterobacter cloacae GGT036.

PubMed

Choi, Sun Young; Gong, Gyeongtaek; Park, Hong-Sil; Um, Youngsoon; Sim, Sang Jun; Woo, Han Min

2015-01-10

Detoxification process of cellular inhibitors including furfural is essential for production of bio-based chemicals from lignocellulosic biomass. Here we isolated an extreme furfural-tolerant bacterium Enterobacter cloacae GGT036 from soil sample collected in Mt. Gwanak, Republic of Korea. Among isolated bacteria, only E. cloacae GGT036 showed cell growth with 35 mM furfural under aerobic culture. Compared to the maximal half inhibitory concentration (IC50) of well-known industrial strains Escherichia coli (24.9 mM furfural) and Corynebacterium glutamicum (10 mM furfural) based on the cell density, IC50 of E. cloacae GGT036 (47.7 mM) was significantly higher after 24 h, compared to E. coli and C. glutamicum. Since bacterial cell growth was exponentially inhibited depending on linearly increased furfural concentrations in the medium, we concluded that E. cloacae GGT036 is an extreme furfural-tolerant bacterium. Recently, the complete genome sequence of E. cloacae GGT036 was announced and this could provide an insight for engineering of E. cloacae GGT036 itself or other industrially relevant bacteria. Copyright © 2014 Elsevier B.V. All rights reserved.

Coevolutionary Analysis Enabled Rational Deregulation of Allosteric Enzyme Inhibition in Corynebacterium glutamicum for Lysine Production ▿

PubMed Central

Chen, Zhen; Meyer, Weiqian; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

2011-01-01

Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter l-lysine within 30 h of fed-batch fermentation in a bioreactor. PMID:21531824

Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

PubMed Central

2012-01-01

Background The expression of genes in Corynebacterium glutamicum, a Gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors of RNA polymerase, including the stress-responsive ECF-sigma factor SigH. The sigH gene is located in a gene cluster together with the rshA gene, putatively encoding an anti-sigma factor. The aim of this study was to analyze the transcriptional regulation of the sigH and rshA gene cluster and the effects of RshA on the SigH regulon, in order to refine the model describing the role of SigH and RshA during stress response. Results Transcription analyses revealed that the sigH gene and rshA gene are cotranscribed from four sigH housekeeping promoters in C. glutamicum. In addition, a SigH-controlled rshA promoter was found to only drive the transcription of the rshA gene. To test the role of the putative anti-sigma factor gene rshA under normal growth conditions, a C. glutamicum rshA deletion strain was constructed and used for genome-wide transcription profiling with DNA microarrays. In total, 83 genes organized in 61 putative transcriptional units, including those previously detected using sigH mutant strains, exhibited increased transcript levels in the rshA deletion mutant compared to its parental strain. The genes encoding proteins related to disulphide stress response, heat stress proteins, components of the SOS-response to DNA damage and proteasome components were the most markedly upregulated gene groups. Altogether six SigH-dependent promoters upstream of the identified genes were determined by primer extension and a refined consensus promoter consisting of 45 original promoter sequences was constructed. Conclusions The rshA gene codes for an anti-sigma factor controlling the function of the stress-responsive sigma factor SigH in C. glutamicum. Transcription of rshA from a SigH-dependent promoter may serve to quickly shutdown the SigH-dependent stress

Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum.

PubMed

Busche, Tobias; Silar, Radoslav; Pičmanová, Martina; Pátek, Miroslav; Kalinowski, Jörn

2012-09-03

The expression of genes in Corynebacterium glutamicum, a Gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors of RNA polymerase, including the stress-responsive ECF-sigma factor SigH. The sigH gene is located in a gene cluster together with the rshA gene, putatively encoding an anti-sigma factor. The aim of this study was to analyze the transcriptional regulation of the sigH and rshA gene cluster and the effects of RshA on the SigH regulon, in order to refine the model describing the role of SigH and RshA during stress response. Transcription analyses revealed that the sigH gene and rshA gene are cotranscribed from four sigH housekeeping promoters in C. glutamicum. In addition, a SigH-controlled rshA promoter was found to only drive the transcription of the rshA gene. To test the role of the putative anti-sigma factor gene rshA under normal growth conditions, a C. glutamicum rshA deletion strain was constructed and used for genome-wide transcription profiling with DNA microarrays. In total, 83 genes organized in 61 putative transcriptional units, including those previously detected using sigH mutant strains, exhibited increased transcript levels in the rshA deletion mutant compared to its parental strain. The genes encoding proteins related to disulphide stress response, heat stress proteins, components of the SOS-response to DNA damage and proteasome components were the most markedly upregulated gene groups. Altogether six SigH-dependent promoters upstream of the identified genes were determined by primer extension and a refined consensus promoter consisting of 45 original promoter sequences was constructed. The rshA gene codes for an anti-sigma factor controlling the function of the stress-responsive sigma factor SigH in C. glutamicum. Transcription of rshA from a SigH-dependent promoter may serve to quickly shutdown the SigH-dependent stress response after the cells have

Valorization of pyrolysis water: a biorefinery side stream, for 1,2-propanediol production with engineered Corynebacterium glutamicum.

PubMed

Lange, Julian; Müller, Felix; Bernecker, Kerstin; Dahmen, Nicolaus; Takors, Ralf; Blombach, Bastian

2017-01-01

A future bioeconomy relies on the efficient use of renewable resources for energy and material product supply. In this context, biorefineries have been developed and play a key role in converting lignocellulosic residues. Although a holistic use of the biomass feed is desired, side streams evoke in current biorefinery approaches. To ensure profitability, efficiency, and sustainability of the overall conversion process, a meaningful valorization of these materials is needed. Here, a so far unexploited side stream derived from fast pyrolysis of wheat straw-pyrolysis water-was used for production of 1,2-propanediol in microbial fermentation with engineered Corynebacterium glutamicum . A protocol for pretreatment of pyrolysis water was established and enabled growth on its major constituents, acetate and acetol, with rates up to 0.36 ± 0.04 h -1 . To convert acetol to 1,2-propanediol, the plasmid pJUL gldA expressing the glycerol dehydrogenase from Escherichia coli was introduced into C. glutamicum . 1,2-propanediol was formed in a growth-coupled biotransformation and production was further increased by construction of C. glutamicum Δ pqo Δ aceE Δ ldhA Δ mdh pJUL gldA . In a two-phase aerobic/microaerobic fed-batch process with pyrolysis water as substrate, this strain produced 18.3 ± 1.2 mM 1,2-propanediol with a yield of 0.96 ± 0.05 mol 1,2-propanediol per mol acetol and showed an overall volumetric productivity of 1.4 ± 0.1 mmol 1,2-propanediol L -1  h -1 . This study implements microbial fermentation into a biorefinery based on pyrolytic liquefaction of lignocellulosic biomass and accesses a novel value chain by valorizing the side stream pyrolysis water for 1,2-PDO production with engineered C. glutamicum . The established bioprocess operated at maximal product yield and accomplished the so far highest overall volumetric productivity for microbial 1,2-PDO production with an engineered producer strain. Besides, the results highlight the

Metabolic engineering of an ATP-neutral Embden-Meyerhof-Parnas pathway in Corynebacterium glutamicum: growth restoration by an adaptive point mutation in NADH dehydrogenase.

PubMed

Komati Reddy, Gajendar; Lindner, Steffen N; Wendisch, Volker F

2015-03-01

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

Enhancing the supply of oxaloacetate for L-glutamate production by pyc overexpression in different Corynebacterium glutamicum.

PubMed

Guo, Xuan; Wang, Jing; Xie, Xixian; Xu, Qingyang; Zhang, Chenglin; Chen, Ning

2013-06-01

During L-glutamate production, phosphoenolpyruvate carboxylase and pyruvate carboxylase (PCx) play important roles in supplying oxaloacetate to the tricarboxylic acid cycle. To explore the significance of PCx for L-glutamate overproduction, the pyc gene encoding PCx was amplified in Corynebacterium glutamicum GDK-9 triggered by biotin limitation and CN1021 triggered by a temperature shock, respectively. In the fed-batch cultures, GDK-9pXMJ19pyc exhibited 7.4 % lower L-alanine excretion and no improved L-glutamate production. In contrast, CN1021pXMJ19pyc finally exhibited 13 % lower L-alanine excretion and identical L-glutamate production, however, 8.5 % higher L-glutamate production was detected during a short period of the fermentation. It was indicated that pyc overexpression in L-glutamate producer strains, especially CN1021, increased the supply of oxaloacetate for L-glutamate synthesis and decreased byproduct excretion at the pyruvate node.

Heterologous expression and localization of gentisate transporter Ncg12922 from Corynebacterium glutamicum ATCC 13032

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Ying; Graduate School of Chinese Academy of Sciences, Beijing 100049; Yan Dazhong

2006-07-28

Ralstonia sp. strain U2 metabolizes naphthalene via gentisate (2,5-dihydroxybenzoate) to central metabolites, but it was found unable to utilize gentisate as growth substrate. A putative gentisate transporter encoded by ncg12922 from Corynebacterium glutamicum ATCC 13032 was functionally expressed in Ralstonia sp. strain U2, converting strain U2 to a gentisate utilizer. After ncg12922 was inserted into plasmid pGFPe with green fluorescence protein gene gfp, the expressed fusion protein Ncg12922-GFP could be visualized in the periphery of Escherichia coli cells under confocal microscope, consistent with a cytoplasmic membrane location. In contrast, GFP was ubiquitous in the cytoplasm of E. coli cells carryingmore » pGFPe only. Gentisate 1,2-dioxygenase activity was present in the cell extract from strain U2 induced with gentisate but at a much lower level (one-fifth) than that obtained with salicylate. However, it exhibited a similar level in strain U2 containing Ncg12922 induced either by salicylate or gentisate.« less

Reengineering of a Corynebacterium glutamicum L-arginine and L-citrulline producer.

PubMed

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

2009-03-01

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, argG92(up), 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 (Delta argR), the production was further increased. The best mutation set, Delta argR 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

Increased Production of Food-Grade d-Tagatose from d-Galactose by Permeabilized and Immobilized Cells of Corynebacterium glutamicum, a GRAS Host, Expressing d-Galactose Isomerase from Geobacillus thermodenitrificans.

PubMed

Shin, Kyung-Chul; Sim, Dong-Hyun; Seo, Min-Ju; Oh, Deok-Kun

2016-11-02

The generally recognized as safe microorganism Corynebacterium glutamicum expressing Geobacillus thermodenitrificans d-galactose isomerase (d-GaI) was an efficient host for the production of d-tagatose, a functional sweetener. The d-tagatose production at 500 g/L d-galactose by the host was 1.4-fold higher than that by Escherichia coli expressing d-GaI. The d-tagatose-producing activity of permeabilized C. glutamicum (PCG) cells treated with 1% (w/v) Triton X-100 was 2.1-fold higher than that of untreated cells. Permeabilized and immobilized C. glutamicum (PICG) cells in 3% (w/v) alginate showed a 3.1-fold longer half-life at 50 °C and 3.1-fold higher total d-tagatose concentration in repeated batch reactions than PCG cells. PICG cells, which produced 165 g/L d-tagatose after 3 h, with a conversion of 55% (w/w) and a productivity of 55 g/L/h, showed significantly higher d-tagatose productivity than that reported for other cells. Thus, d-tagatose production by PICG cells may be an economical process to produce food-grade d-tagatose.

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. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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). Copyright © 2016 Elsevier B.V. All rights reserved.

Protein S-Mycothiolation Functions as Redox-Switch and Thiol Protection Mechanism in Corynebacterium glutamicum Under Hypochlorite Stress

PubMed Central

Chi, Bui Khanh; Busche, Tobias; Van Laer, Koen; Bäsell, Katrin; Becher, Dörte; Clermont, Lina; Seibold, Gerd M.; Persicke, Marcus; Kalinowski, Jörn; Messens, Joris

2014-01-01

Abstract Aims: Protein S-bacillithiolation was recently discovered as important thiol protection and redox-switch mechanism in response to hypochlorite stress in Firmicutes bacteria. Here we used transcriptomics to analyze the NaOCl stress response in the mycothiol (MSH)-producing Corynebacterium glutamicum. We further applied thiol-redox proteomics and mass spectrometry (MS) to identify protein S-mycothiolation. Results: Transcriptomics revealed the strong upregulation of the disulfide stress σH regulon by NaOCl stress in C. glutamicum, including genes for the anti sigma factor (rshA), the thioredoxin and MSH pathways (trxB1, trxC, cg1375, trxB, mshC, mca, mtr) that maintain the redox balance. We identified 25 S-mycothiolated proteins in NaOCl-treated cells by liquid chromatography–tandem mass spectrometry (LC-MS/MS), including 16 proteins that are reversibly oxidized by NaOCl in the thiol-redox proteome. The S-mycothiolome includes the methionine synthase (MetE), the maltodextrin phosphorylase (MalP), the myoinositol-1-phosphate synthase (Ino1), enzymes for the biosynthesis of nucleotides (GuaB1, GuaB2, PurL, NadC), and thiamine (ThiD), translation proteins (TufA, PheT, RpsF, RplM, RpsM, RpsC), and antioxidant enzymes (Tpx, Gpx, MsrA). We further show that S-mycothiolation of the thiol peroxidase (Tpx) affects its peroxiredoxin activity in vitro that can be restored by mycoredoxin1. LC-MS/MS analysis further identified 8 proteins with S-cysteinylations in the mshC mutant suggesting that cysteine can be used for S-thiolations in the absence of MSH. Innovation and Conclusion: We identified widespread protein S-mycothiolations in the MSH-producing C. glutamicum and demonstrate that S-mycothiolation reversibly affects the peroxidase activity of Tpx. Interestingly, many targets are conserved S-thiolated across bacillithiol- and MSH-producing bacteria, which could become future drug targets in related pathogenic Gram-positives. Antioxid. Redox Signal. 20, 589–605

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.

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. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Export of l-Isoleucine from Corynebacterium glutamicum: a Two-Gene-Encoded Member of a New Translocator Family

PubMed Central

Kennerknecht, Nicole; Sahm, Hermann; Yen, Ming-Ren; Pátek, Miroslav; Saier, Jr., Milton H.; Eggeling, Lothar

2002-01-01

Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes l-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for l-isoleucine export, we have used transposon mutagenesis to isolate mutants of C. glutamicum sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices. brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports l-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of l-leucine and l-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in α-proteobacteria but not in other prokaryotes analyzed. PMID:12081967

Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes.

PubMed

Park, Jong-Uk; Jo, Jae-Hyung; Kim, Young-Ji; Chung, So-Sun; Lee, Jin-Ho; Lee, Hyune Hwan

2008-04-01

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the lambdaOL1 and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes.. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the OL1 from the lambdaPL promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one lambdaOL1, and CJ1OX2, which has two successive lambdaOL1, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

Boosting Anaplerotic Reactions by Pyruvate Kinase Gene Deletion and Phosphoenolpyruvate Carboxylase Desensitization for Glutamic Acid and Lysine Production in Corynebacterium glutamicum.

PubMed

Yokota, Atsushi; Sawada, Kazunori; Wada, Masaru

In the 1980s, Shiio and coworkers demonstrated using random mutagenesis that the following three phenotypes were effective for boosting lysine production by Corynebacterium glutamicum: (1) low-activity-level citrate synthase (CS L ), (2) phosphoenolpyruvate carboxylase (PEPC) resistant to feedback inhibition by aspartic acid (PEPC R ), and (3) pyruvate kinase (PYK) deficiency. Here, we reevaluated these phenotypes and their interrelationship in lysine production using recombinant DNA techniques.The pyk deletion and PEPC R (D299N in ppc) independently showed marginal effects on lysine production, but both phenotypes synergistically increased lysine yield, demonstrating the importance of PEPC as an anaplerotic enzyme in lysine production. Similar effects were also found for glutamic acid production. CS L (S252C in gltA) further increased lysine yield. Thus, using molecular techniques, the combination of these three phenotypes was reconfirmed to be effective for lysine production. However, a simple CS L mutant showed instabilities in growth and lysine yield.Surprisingly, the pyk deletion was found to increase biomass production in wild-type C. glutamicum ATCC13032 under biotin-sufficient conditions. The mutant showed a 37% increase in growth (based on OD 660 ) compared with the ATCC13032 strain in a complex medium containing 100 g/L glucose. Metabolome analysis revealed the intracellular accumulation of excess precursor metabolites. Thus, their conversion into biomass was considered to relieve the metabolic distortion in the pyk-deleted mutant. Detailed physiological studies of various pyk-deleted mutants also suggested that malate:quinone oxidoreductase (MQO) is important to control both the intracellular oxaloacetic acid (OAA) level and respiration rate. These findings may facilitate the rational use of C. glutamicum in fermentation industries.

Heme Derived from Corynebacterium glutamicum: A Potential Iron Additive for Swine and an Electron Carrier Additive for Lactic Acid Bacterial Culture.

PubMed

Choi, Su-In; Park, Jihoon; Kim, Pil

2017-03-28

To investigate the potential applications of bacterial heme, aminolevulinic acid synthase (HemA) was expressed in a Corynebacterium glutamicum HA strain that had been adaptively evolved against oxidative stress. The red pigment from the constructed strain was extracted and it exhibited the typical heme absorbance at 408 nm from the spectrum. To investigate the potential of this strain as an iron additive for swine, a prototype feed additive was manufactured in pilot scale by culturing the strain in a 5 ton fermenter followed by spray-drying the biomass with flour as an excipient (biomass: flour = 1:10 (w/w)). The 10% prototype additive along with regular feed was supplied to a pig, resulting in a 1.1 kg greater increase in weight gain with no diarrhea in 3 weeks as compared with that in a control pig that was fed an additive containing only flour. To verify if C. glutamicum -synthesized heme is a potential electron carrier, lactic acid bacteria were cultured under aerobic conditions with the extracted heme. The biomasses of the aerobically grown Lactococcus lactis , Lactobacillus rhamosus , and Lactobacillus casei were 97%, 15%, and 4% greater, respectively, than those under fermentative growth conditions. As a potential preservative, cultures of the four strains of lactic acid bacteria were stored at 4°C with the extracted heme and living lactic acid bacterial cells were counted. There were more L. lactis and L. plantarum live cells when stored with heme, whereas L. rhamosus and L. casei showed no significant differences in live-cell numbers. The potential uses of the heme from C. glutamicum are further discussed.

Enhancing poly-γ-glutamic acid production in Bacillus amyloliquefaciens by introducing the glutamate synthesis features from Corynebacterium glutamicum.

PubMed

Feng, Jun; Quan, Yufen; Gu, Yanyan; Liu, Fenghong; Huang, Xiaozhong; Shen, Haosheng; Dang, Yulei; Cao, Mingfeng; Gao, Weixia; Lu, Xiaoyun; Wang, Yi; Song, Cunjiang; Wang, Shufang

2017-05-22

Poly-γ-glutamic acid (γ-PGA) is a valuable polymer with glutamate as its sole precursor. Enhancement of the intracellular glutamate synthesis is a very important strategy for the improvement of γ-PGA production, especially for those glutamate-independent γ-PGA producing strains. Corynebacterium glutamicum has long been used for industrial glutamate production and it exhibits some unique features for glutamate synthesis; therefore introduction of these metabolic characters into the γ-PGA producing strain might lead to increased intracellular glutamate availability, and thus ultimate γ-PGA production. In this study, the unique glutamate synthesis features from C. glutamicum was introduced into the glutamate-independent γ-PGA producing Bacillus amyloliquefaciens NK-1 strain. After introducing the energy-saving NADPH-dependent glutamate dehydrogenase (NADPH-GDH) pathway, the NK-1 (pHT315-gdh) strain showed slightly increase (by 9.1%) in γ-PGA production. Moreover, an optimized metabolic toggle switch for controlling the expression of ɑ-oxoglutarate dehydrogenase complex (ODHC) was introduced into the NK-1 strain, because it was previously shown that the ODHC in C. glutamicum was completely inhibited when glutamate was actively produced. The obtained NK-PO1 (pHT01-xylR) strain showed 66.2% higher γ-PGA production than the NK-1 strain. However, the further combination of these two strategies (introducing both NADPH-GDH pathway and the metabolic toggle switch) did not lead to further increase of γ-PGA production but rather the resultant γ-PGA production was even lower than that in the NK-1 strain. We proposed new metabolic engineering strategies to improve the γ-PGA production in B. amyloliquefaciens. The NK-1 (pHT315-gdh) strain with the introduction of NADPH-GDH pathway showed 9.1% improvement in γ-PGA production. The NK-PO1 (pHT01-xylR) strain with the introduction of a metabolic toggle switch for controlling the expression of ODHC showed 66.2% higher �

In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum

PubMed Central

Nagashima, Takashi; Nakamura, Eri; Kato, Ryosuke; Ohshita, Masakazu; Hayashi, Mikiro; Takeno, Seiki

2017-01-01

ABSTRACT For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI. Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA. These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicum. IMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis, which use an individual nonaggregating type II fatty

Sequence-based identification of inositol monophosphatase-like histidinol-phosphate phosphatases (HisN) in Corynebacterium glutamicum, Actinobacteria, and beyond.

PubMed

Kulis-Horn, Robert Kasimir; Rückert, Christian; Kalinowski, Jörn; Persicke, Marcus

2017-07-18

The eighth step of L-histidine biosynthesis is carried out by an enzyme called histidinol-phosphate phosphatase (HolPase). Three unrelated HolPase families are known so far. Two of them are well studied: HAD-type HolPases known from Gammaproteobacteria like Escherichia coli or Salmonella enterica and PHP-type HolPases known from yeast and Firmicutes like Bacillus subtilis. However, the third family of HolPases, the inositol monophosphatase (IMPase)-like HolPases, present in Actinobacteria like Corynebacterium glutamicum (HisN) and plants, are poorly characterized. Moreover, there exist several IMPase-like proteins in bacteria (e.g. CysQ, ImpA, and SuhB) which are very similar to HisN but most likely do not participate in L-histidine biosynthesis. Deletion of hisN, the gene encoding the IMPase-like HolPase in C. glutamicum, does not result in complete L-histidine auxotrophy. Out of four hisN homologs present in the genome of C. glutamicum (impA, suhB, cysQ, and cg0911), only cg0911 encodes an enzyme with HolPase activity. The enzymatic properties of HisN and Cg0911 were determined, delivering the first available kinetic data for IMPase-like HolPases. Additionally, we analyzed the amino acid sequences of potential HisN, ImpA, SuhB, CysQ and Cg0911 orthologs from bacteria and identified six conserved sequence motifs for each group of orthologs. Mutational studies confirmed the importance of a highly conserved aspartate residue accompanied by several aromatic amino acid residues present in motif 5 for HolPase activity. Several bacterial proteins containing all identified HolPase motifs, but showing only moderate sequence similarity to HisN from C. glutamicum, were experimentally confirmed as IMPase-like HolPases, demonstrating the value of the identified motifs. Based on the confirmed IMPase-like HolPases two profile Hidden Markov Models (HMMs) were build using an iterative approach. These HMMs allow the fast, reliable detection and differentiation of the two

The α-Glucan Phosphorylase MalP of Corynebacterium glutamicum Is Subject to Transcriptional Regulation and Competitive Inhibition by ADP-Glucose

PubMed Central

Clermont, Lina; Macha, Arthur; Müller, Laura M.; Derya, Sami M.; von Zaluskowski, Philipp; Eck, Alexander; Eikmanns, Bernhard J.

2015-01-01

ABSTRACT α-Glucan phosphorylases contribute to degradation of glycogen and maltodextrins formed in the course of maltose metabolism in bacteria. Accordingly, bacterial α-glucan phosphorylases are classified as either glycogen or maltodextrin phosphorylase, GlgP or MalP, respectively. GlgP and MalP enzymes follow the same catalytic mechanism, and thus their substrate spectra overlap; however, they differ in their regulation: GlgP genes are constitutively expressed and the enzymes are controlled on the activity level, whereas expression of MalP genes are transcriptionally controlled in response to the carbon source used for cultivation. We characterize here the modes of control of the α-glucan phosphorylase MalP of the Gram-positive Corynebacterium glutamicum. In accordance to the proposed function of the malP gene product as MalP, we found transcription of malP to be regulated in response to the carbon source. Moreover, malP transcription is shown to depend on the growth phase and to occur independently of the cell glycogen content. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. Since the latter is considered a typical feature of GlgPs, we propose that C. glutamicum MalP acts as both maltodextrin and glycogen phosphorylase and, based on these findings, we question the current system for classification of bacterial α-glucan phosphorylases. IMPORTANCE Bacterial α-glucan phosphorylases have been classified conferring to their purpose as either glycogen or maltodextrin phosphorylases. We found transcription of malP in C. glutamicum to be regulated in response to the carbon source, which is recognized as typical for maltodextrin phosphorylases. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. The latter is considered a typical feature of GlgPs. These findings

The Crystal Structures of Apo and cAMP-Bound GlxR from Corynebacterium glutamicum Reveal Structural and Dynamic Changes upon cAMP Binding in CRP/FNR Family Transcription Factors

PubMed Central

Townsend, Philip D.; Jungwirth, Britta; Pojer, Florence; Bußmann, Michael; Money, Victoria A.; Cole, Stewart T.; Pühler, Alfred; Tauch, Andreas; Bott, Michael; Cann, Martin J.; Pohl, Ehmke

2014-01-01

The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator) transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition. PMID:25469635

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.

Corynebacterium pilbarense sp. nov., a non-lipophilic corynebacterium isolated from a human ankle aspirate.

PubMed

Aravena-Roman, M; Spröer, C; Sträubler, B; Inglis, T; Yassin, A F

2010-07-01

A non-lipophilic coryneform bacterium isolated from an anaerobic Bactec bottle inoculated with an ankle aspirate from a male patient was characterized by phenotypic and molecular taxonomic methods. Chemotaxonomic investigations revealed the presence of short-chain mycolic acids in the cell wall of the bacterium, a feature consistent with members of the genus Corynebacterium. Comparative 16S rRNA gene sequence analysis demonstrated that the isolate displayed 92.0-99.0 % gene sequence similarity with members of the genus Corynebacterium, with Corynebacterium ureicelerivorans as the most closely related phylogenetic species (99.0 % gene sequence similarity). However, the isolate could be genomically separated from C. ureicelerivorans on the basis of DNA-DNA hybridization studies (39.5 % relatedness). Furthermore, the isolate could also be differentiated from C. ureicelerivorans and other species of the genus Corynebacterium on the basis of biochemical properties. Based on both phenotypic and phylogenetic evidence, it is proposed that this isolate be classified as representing a novel species, Corynebacterium pilbarense sp. nov. (type strain IMMIB WACC 658(T)=DSM 45350(T)=CCUG 57942(T)).

Improvement of the intracellular environment for enhancing l-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply.

PubMed

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

2016-11-01

l-arginine, a semi essential amino acid, is an important amino acid in food flavoring and pharmaceutical industries. Its production by microbial fermentation is gaining more and more attention. In previous work, we obtained a new l-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through mutation breeding. In this work, we enhanced l-arginine production through improvement of the intracellular environment. First, two NAD(P)H-dependent H 2 O 2 -forming flavin reductases Frd181 (encoded by frd1 gene) and Frd188 (encoded by frd2) in C. glutamicum were identified for the first time. Next, the roles of Frd181 and Frd188 in C. glutamicum were studied by overexpression and deletion of the encoding genes, and the results showed that the inactivation of Frd181 and Frd188 was beneficial for cell growth and l-arginine production, owing to the decreased H 2 O 2 synthesis and intracellular reactive oxygen species (ROS) level, and increased intracellular NADH and ATP levels. Then, the ATP level was further increased by deletion of noxA (encoding NADH oxidase) and amn (encoding AMP nucleosidase), and overexpression of pgk (encoding 3-phosphoglycerate kinase) and pyk (encoding pyruvate kinase), and the l-arginine production and yield from glucose were significantly increased. In fed-batch fermentation, the l-arginine production and yield from glucose of the final strain reached 57.3g/L and 0.326g/g, respectively, which were 49.2% and 34.2% higher than those of the parent strain, respectively. ROS and ATP are important elements of the intracellular environment, and l-arginine biosynthesis requires a large amount of ATP. For the first time, we enhanced l-arginine production and yield from glucose through reducing the H 2 O 2 synthesis and increasing the ATP supply. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum.

PubMed

Ikeda, Masato; Nagashima, Takashi; Nakamura, Eri; Kato, Ryosuke; Ohshita, Masakazu; Hayashi, Mikiro; Takeno, Seiki

2017-10-01

For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicum IMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis , which use an individual nonaggregating type II fatty acid synthase

Improvement of succinate production by release of end-product inhibition in Corynebacterium glutamicum.

PubMed

Chung, Soon-Chun; Park, Joon-Song; Yun, Jiae; Park, Jin Hwan

2017-03-01

Succinate is a renewable-based platform chemical that may be used to produce a wide range of chemicals including 1,4-butanediol, tetrahydrofurane, and γ-butyrolactone. However, industrial fermentation of organic acids is often subject to end-product inhibition, which significantly retards cell growth and limits metabolic activities and final productivity. In this study, we report the development of metabolically engineered Corynebacterium glutamicum for high production of succinate by release of end-product inhibition coupled with an increase of key metabolic flux. It was found that the rates of glucose consumption and succinate production were significantly reduced by extracellular succinate in an engineered strain, S003. To understand the mechanism underlying the inhibition by succinate, comparative transcriptome analysis was performed. Among the downregulated genes, overexpression of the NCgl0275 gene was found to suppress the inhibition of glucose consumption and succinate production, resulting in a 37.7% increase in succinate production up to 55.4g/L in fed-batch fermentation. Further improvement was achieved by increasing the metabolic flux from PEP to OAA. The final engineered strain was able to produce 152.2g/L succinate, the highest production reported to date, with a yield of 1.1g/g glucose under anaerobic condition. These results suggest that the release of end-product inhibition coupled with an increase in key metabolic flux is a promising strategy for enhancing production of succinate. Copyright © 2017. Published by Elsevier Inc.

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

PubMed Central

2014-01-01

Background Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. Results The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. Conclusion There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system. PMID:24731213

The linkage between nutrient supply, intracellular enzyme abundances and bacterial growth: New evidences from the central carbon metabolism of Corynebacterium glutamicum.

PubMed

Noack, Stephan; Voges, Raphael; Gätgens, Jochem; Wiechert, Wolfgang

2017-09-20

Corynebacterium glutamicum serves as important production host for small molecular compounds that are derived from precursor molecules of the central carbon metabolism. It is therefore a well-studied model organism of industrial biotechnology. However, a deeper understanding of the regulatory principles underlying the synthesis of central metabolic enzymes under different environmental conditions as well as its impact on cell growth is still missing. We studied enzyme abundances in C. glutamicum in response to growth on: (i) one limiting carbon source by sampling chemostat and fed-batch cultivations and (ii) changing carbon sources provided in excess by sampling batch cultivations. The targeted quantification of 20 central metabolic enzymes by isotope dilution mass spectrometry revealed that cells maintain stable enzyme concentrations when grown on d-glucose as single carbon and energy source and, most importantly, independent of its availability. By contrast, switching from d-glucose to d-fructose, d-mannose, d-arabitol, acetate, l-lactate or l-glutamate results in highly specific enzyme regulation patterns that can partly be explained by the activity of known transcriptional regulators. Based on these experimental results we propose a simple framework for modeling cell population growth as a nested function of nutrient supply and intracellular enzyme abundances. In summary, our study extends the basis for the formulation of predictive mechanistic models of bacterial growth, applicable in industrial bioprocess development. Copyright © 2017 Elsevier B.V. All rights reserved.

Glutamine-rich toxic proteins GrtA, GrtB and GrtC together with the antisense RNA AsgR constitute a toxin-antitoxin-like system in Corynebacterium glutamicum.

PubMed

Maeda, Tomoya; Tanaka, Yuya; Inui, Masayuki

2018-06-01

The Corynebacterium glutamicum R grtA (cgR_2936), grtB (cgR_2934) and grtC (cgR_2933) genes were identified as paralogs encoding glutamine-rich toxic proteins. We also identified a new antisense small RNA AsgR (antisense sRNA for grtA) that overlaps the 3' end of the grtA gene. Single over-expressions of grtA, grtB and grtC resulted in complete inhibition of Escherichia coli cell growth. This growth was rescued by co-expression of AsgR. Similar effects were observed in C. glutamicum, although the toxicities of these proteins were moderate. Inhibition of AsgR transcription resulted in increased levels and prolonged half-lives of grtA, grtB and grtC mRNAs. We also found that the expression levels of grtA, grtB and grtC were increased in an RNase III deletion mutant. Primer extension analysis revealed the RNase III cleavage site to be in the 3' untranslated region (3'-UTR) of the grtA mRNA. The expression levels of grtA, grtB and grtC were increased after exposure to several stresses, including heat shock, treatment with penicillin G, lysozyme or H 2 O 2 . The deletions of grtABC and asgR genes resulted in decreased survival rate under several stresses. These results indicate that GrtABC and AsgR constitute a type I toxin-antitoxin-like system in C. glutamicum. © 2018 John Wiley & Sons Ltd.

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.

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

Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions.

PubMed

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

2013-02-01

We previously demonstrated efficient L-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of l-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the L-valine yield. Eliminating these by-products therefore was deemed key to improving theL-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and L-valine production dropped considerably due to the severely elevated intracellular NADH/NAD(+) ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher L-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM L-valine at a yield of 88% mol mol of glucose(-1) after 24 h under oxygen deprivation, a vastly improved yield over our previous best.

Engineering of Corynebacterium glutamicum for High-Yield l-Valine Production under Oxygen Deprivation Conditions

PubMed Central

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

2013-01-01

We previously demonstrated efficient l-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of l-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the l-valine yield. Eliminating these by-products therefore was deemed key to improving the l-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and l-valine production dropped considerably due to the severely elevated intracellular NADH/NAD+ ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher l-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM l-valine at a yield of 88% mol mol of glucose−1 after 24 h under oxygen deprivation, a vastly improved yield over our previous best. PMID:23241971

Pyruvate kinase deletion as an effective phenotype to enhance lysine production in Corynebacterium glutamicum ATCC13032: Redirecting the carbon flow to a precursor metabolite.

PubMed

Yanase, Masaki; Aikoh, Tohru; Sawada, Kazunori; Ogura, Kotaro; Hagiwara, Takuya; Imai, Keita; Wada, Masaru; Yokota, Atsushi

2016-08-01

Various attempts have been made to enhance lysine production in Corynebacterium glutamicum. Pyruvate kinase (PYK) defect is one of the strategies used to enhance the supply of oxaloacetic acid (OAA), a precursor metabolite for lysine biosynthesis. However, inconsistent effects of this mutation have been reported: positive effects of PYK defect in mutants having phosphoenolpyruvate carboxylase (PEPC) desensitized to feedback inhibition by aspartic acid, while negative effects in simple PYK gene (pyk) knockout mutants. To address these discrepancies, the effects of pyk deletion on lysine yield were investigated with or without the D299N mutation in ppc rendering PEPC desensitization. C. glutamicum ATCC13032 mutant strain P with a feedback inhibition-desensitized aspartokinase was used as the parent strain, producing 9.36 g/L lysine from 100 g/L glucose in a jar fermentor culture. Under these conditions, while the simple mutant D2 with pyk deletion or R2 with the PEPC-desensitization mutation showed marginally increased lysine yield (∼1.1-fold, not significant), the mutant DR2 strain having both mutations showed synergistically increased lysine productivity (1.38-fold, 12.9 g/L). Therefore, the pyk deletion is effective under a PEPC-desensitized background, which ensures enhanced supply of OAA, thus clarifying the discrepancies. A citrate synthase defective mutation (S252C in gltA) further increased the lysine yield in strain DR2 (1.68-fold, 15.7 g/L). Thus, these three mutations coordinately enhanced the lysine yield. Both the malate:quinone oxidoreductase activity and respiration rate were significantly reduced in strains D2 and DR2. Overall, these results provide valuable knowledge for engineering the anaplerotic reaction to increase lysine yield in C. glutamicum. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

L-Serine overproduction with minimization of by-product synthesis by engineered Corynebacterium glutamicum.

PubMed

Zhu, Qinjian; Zhang, Xiaomei; Luo, Yuchang; Guo, Wen; Xu, Guoqiang; Shi, Jinsong; Xu, Zhenghong

2015-02-01

The direct fermentative production of L-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low L-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing L-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both L-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products L-alanine and L-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards L-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as L-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the L-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of L-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of L-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve L-serine productivity.

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. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Corynebacterium riegelii sp. nov., an Unusual Species Isolated from Female Patients with Urinary Tract Infections

PubMed Central

Funke, Guido; Lawson, Paul A.; Collins, Matthew D.

1998-01-01

Four strains of an unknown coryneform bacterium were isolated in pure culture from females with urinary tract infections. Strong urease activity and the ability to slowly ferment maltose but not glucose were the most significant phenotypic features of this catalase-positive, nonmotile, nonlipophilic, rod-shaped bacterium which served to distinguish it from all other presently defined coryneform bacteria. Chemotaxonomic investigations demonstrated that the unknown bacterium belonged to the genus Corynebacterium. Comparative 16S rRNA gene sequence analysis revealed that the isolates were genealogically identical and represented a new subline within the genus Corynebacterium, for which the designation Corynebacterium riegelii sp. nov. is proposed. The type strain of Corynebacterium riegelii is CCUG 38180 (DSM 44326, CIP 105310). PMID:9508284

Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of CO2 on anaerobic succinate production by Corynebacterium glutamicum.

PubMed

Radoš, Dušica; Turner, David L; Fonseca, Luís L; Carvalho, Ana Lúcia; Blombach, Bastian; Eikmanns, Bernhard J; Neves, Ana Rute; Santos, Helena

2014-05-01

Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2 was estimated by using (13)C-labeled glucose and (13)C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (~5%) regardless of the presence or absence of CO2. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H(+):organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation.

Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae*

PubMed Central

Wesener, Darryl A.; Levengood, Matthew R.

2017-01-01

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. PMID:28039359

Biosynthesis of rare ketoses through constructing a recombination pathway in an engineered Corynebacterium glutamicum.

PubMed

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

2015-01-01

Rare sugars have various known biological functions and potential for applications in pharmaceutical, cosmetics, and food industries. Here we designed and constructed a recombination pathway in Corynebacterium glutamicum, in which dihydroxyacetone phosphate (DHAP), an intermediate of the glycolytic pathway, and a variety of aldehydes were condensed to synthesize rare ketoses sequentially by rhamnulose-1-phosphate aldolase (RhaD) and fructose-1-phosphatase (YqaB) obtained from Escherichia coli. A wild-type strain harboring this artificial pathway had the ability to produce D-sorbose and D-psicose using D-glyceraldehyde and glucose as the substrates. The tpi gene, encoding triose phosphate isomerase was further deleted, and the concentration of DHAP increased to nearly 20-fold relative to that of the wild-type. After additional optimization of expression levels from rhaD and yqaB genes and of the fermentation conditions, the engineered strain SY6(pVRTY) exhibited preferable performance for rare ketoses production. Its yield increased to 0.59 mol/mol D-glyceraldehyde from 0.33 mol/mol D-glyceraldehyde and productivity to 2.35 g/L h from 0.58 g/L h. Moreover, this strain accumulated 19.5 g/L of D-sorbose and 13.4 g/L of D-psicose using a fed-batch culture mode under the optimal conditions. In addition, it was verified that the strain SY6(pVRTY) meanwhile had the ability to synthesize C4, C5, C6, and C7 rare ketoses when a range of representative achiral and homochiral aldehydes were applied as the substrates. Therefore, the platform strain exhibited the potential for microbial production of rare ketoses and deoxysugars. © 2014 Wiley Periodicals, Inc.

l-Valine Production with Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum▿

PubMed Central

Blombach, Bastian; Schreiner, Mark E.; Holátko, Jiří; Bartek, Tobias; Oldiges, Marco; Eikmanns, Bernhard J.

2007-01-01

Corynebacterium glutamicum was engineered for the production of l-valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum ΔaceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amounts of pyruvate, l-alanine, and l-valine from glucose as the sole carbon source. Lactate or acetate was not formed. Plasmid-bound overexpression of ilvBNCE in C. glutamicum ΔaceE resulted in an approximately 10-fold-lower intracellular pyruvate concentration (2.3 mM) and a shift of the extracellular product pattern from pyruvate and l-alanine towards l-valine. In fed-batch fermentations at high cell densities and an excess of glucose, C. glutamicum ΔaceE(pJC4ilvBNCE) produced up to 210 mM l-valine with a volumetric productivity of 10.0 mM h−1 (1.17 g l−1 h−1) and a maximum yield of about 0.6 mol per mol (0.4 g per g) of glucose. PMID:17293513

Characterization of OxyR as a Negative Transcriptional Regulator That Represses Catalase Production in Corynebacterium diphtheriae

PubMed Central

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 H2O2. 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 H2O2. In contrast, exposure of C. diphtheriae C7(β) to H2O2 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 H2O2 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 H2O2. 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 H2O2 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 H2O2. PMID:22438866

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

Improvement of the redox balance increases L-valine production by Corynebacterium glutamicum under oxygen deprivation conditions.

PubMed

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

2012-02-01

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.

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

Global gene expression during stringent response in Corynebacterium glutamicum in presence and absence of the rel gene encoding (p)ppGpp synthase

PubMed Central

Brockmann-Gretza, Olaf; Kalinowski, Jörn

2006-01-01

Background The stringent response is the initial reaction of microorganisms to nutritional stress. During stringent response the small nucleotides (p)ppGpp act as global regulators and reprogram bacterial transcription. In this work, the genetic network controlled by the stringent response was characterized in the amino acid-producing Corynebacterium glutamicum. Results The transcriptome of a C. glutamicum rel gene deletion mutant, unable to synthesize (p)ppGpp and to induce the stringent response, was compared with that of its rel-proficient parent strain by microarray analysis. A total of 357 genes were found to be transcribed differentially in the rel-deficient mutant strain. In a second experiment, the stringent response was induced by addition of DL-serine hydroxamate (SHX) in early exponential growth phase. The time point of the maximal effect on transcription was determined by real-time RT-PCR using the histidine and serine biosynthetic genes. Transcription of all of these genes reached a maximum at 10 minutes after SHX addition. Microarray experiments were performed comparing the transcriptomes of SHX-induced cultures of the rel-proficient strain and the rel mutant. The differentially expressed genes were grouped into three classes. Class A comprises genes which are differentially regulated only in the presence of an intact rel gene. This class includes the non-essential sigma factor gene sigB which was upregulated and a large number of genes involved in nitrogen metabolism which were downregulated. Class B comprises genes which were differentially regulated in response to SHX in both strains, independent of the rel gene. A large number of genes encoding ribosomal proteins fall into this class, all being downregulated. Class C comprises genes which were differentially regulated in response to SHX only in the rel mutant. This class includes genes encoding putative stress proteins and global transcriptional regulators that might be responsible for the complex

Chassis organism from Corynebacterium glutamicum--a top-down approach to identify and delete irrelevant gene clusters.

PubMed

Unthan, Simon; Baumgart, Meike; Radek, Andreas; Herbst, Marius; Siebert, Daniel; Brühl, Natalie; Bartsch, Anna; Bott, Michael; Wiechert, Wolfgang; Marin, Kay; Hans, Stephan; Krämer, Reinhard; Seibold, Gerd; Frunzke, Julia; Kalinowski, Jörn; Rückert, Christian; Wendisch, Volker F; Noack, Stephan

2015-02-01

For synthetic biology applications, a robust structural basis is required, which can be constructed either from scratch or in a top-down approach starting from any existing organism. In this study, we initiated the top-down construction of a chassis organism from Corynebacterium glutamicum ATCC 13032, aiming for the relevant gene set to maintain its fast growth on defined medium. We evaluated each native gene for its essentiality considering expression levels, phylogenetic conservation, and knockout data. Based on this classification, we determined 41 gene clusters ranging from 3.7 to 49.7 kbp as target sites for deletion. 36 deletions were successful and 10 genome-reduced strains showed impaired growth rates, indicating that genes were hit, which are relevant to maintain biological fitness at wild-type level. In contrast, 26 deleted clusters were found to include exclusively irrelevant genes for growth on defined medium. A combinatory deletion of all irrelevant gene clusters would, in a prophage-free strain, decrease the size of the native genome by about 722 kbp (22%) to 2561 kbp. Finally, five combinatory deletions of irrelevant gene clusters were investigated. The study introduces the novel concept of relevant genes and demonstrates general strategies to construct a chassis suitable for biotechnological application. © 2014 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-Non-Commercial-NoDerivs Licence, which permits use and distribution in any medium, provided the original work is properly cited, the use is non- commercial and no modifications or adaptations are made.

Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing.

PubMed

Marx, A; de Graaf, A A; Wiechert, W; Eggeling, L; Sahm, H

1996-01-20

To determine the in vivo fluxes of the central metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of (1)H-detected (13)C nuclear magnetic resonance spectroscopy to follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [1-(13)C]glucose and determined the fractional enrichments in 27 carbon atoms of 11 amino acids isolated from the cell. Since precursor metabolites of the central metabolism are incorporated in an exactly defined manner in the carbon skeleton of amino acids, the fractional enrichments in carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology to C. glutamicum, all major fluxes within the central metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 13.7%. Due to the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase

Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of l-Valine

PubMed Central

Lange, C.; Rittmann, D.; Wendisch, V. F.; Bott, M.; Sahm, H.

2003-01-01

Addition of l-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 ΔilvA ΔpanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of l-isoleucine could relieve the valine effect on VAL1 whereas l-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain. PMID:12732517

Complete nucleotide sequences and annotations of φ673 and φ674, two newly characterised lytic phages of Corynebacterium glutamicum ATCC 13032.

PubMed

Yomantas, Yurgis A V; Abalakina, Elena G; Lobanova, Juliya S; Mamontov, Victor A; Stoynova, Nataliya V; Mashko, Sergey V

2018-05-15

The genomes of two new lytic phages of Corynebacterium glutamicum ATCC 13032, φ673 and φ674, were sequenced and annotated (GenBank: MG324353, MG324354). Electron microscopy studies of both virions revealed that taxonomically they belong to the Siphoviridae family and have a polyhedral head with a width of 50 nm and a non-contractile tail with a length of 250 nm. The genomes of φ673 and φ674 consist of linear double-stranded DNA molecules with lengths of 44,530 bp (G+C = 51.1%) and 43,193 bp (G+C = 50.7%) and identical, protruding, cohesive 3' ends 13 nt in length. The level of identity between the φ673 and φ674 genomes is 85.2%. Two major structural proteins of each virion were separated via SDS-PAGE and identified using peptide mass fingerprinting. Based on bioinformatic analysis, 56 and 54 ORFs were predicted for φ673 and φ674, respectively. Only 20 of the putative gene products of φ673 and 20 of φ674 could be assigned to known functions. Both genomes were divided into functional modules. Nine putative promoters in the φ673 genome and eight in the φ674 genome were predicted. One bidirectional Rho-independent transcription terminator was identified and experimentally confirmed in each phage genome.

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.

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 Central

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

2012-01-01

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. PMID:22505259

Toward Homosuccinate Fermentation: Metabolic Engineering of Corynebacterium glutamicum for Anaerobic Production of Succinate from Glucose and Formate

PubMed Central

Litsanov, Boris; Brocker, Melanie

2012-01-01

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 pycP458S 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). PMID:22389371

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.

Metabolic engineering to guide evolution - Creating a novel mode for L-valine production with Corynebacterium glutamicum.

PubMed

Schwentner, Andreas; Feith, André; Münch, Eugenia; Busche, Tobias; Rückert, Christian; Kalinowski, Jörn; Takors, Ralf; Blombach, Bastian

2018-03-06

Evolutionary approaches are often undirected and mutagen-based yielding numerous mutations, which need elaborate screenings to identify relevant targets. We here apply Metabolic engineering to Guide Evolution (MGE), an evolutionary approach evolving and identifying new targets to improve microbial producer strains. MGE is based on the idea to impair the cell's metabolism by metabolic engineering, thereby generating guided evolutionary pressure. It consists of three distinct phases: (i) metabolic engineering to create the evolutionary pressure on the applied strain followed by (ii) a cultivation phase with growth as straightforward screening indicator for the evolutionary event, and (iii) comparative whole genome sequencing (WGS), to identify mutations in the evolved strains, which are eventually re-engineered for verification. Applying MGE, we evolved the PEP and pyruvate carboxylase-deficient strain C. glutamicum Δppc Δpyc to grow on glucose as substrate with rates up to 0.31 ± 0.02 h -1 which corresponds to 80% of the growth rate of the wildtype strain. The intersection of the mutations identified by WGS revealed isocitrate dehydrogenase (ICD) as consistent target in three independently evolved mutants. Upon re-engineering in C. glutamicum Δppc Δpyc, the identified mutations led to diminished ICD activities and activated the glyoxylate shunt replenishing oxaloacetate required for growth. Intracellular relative quantitative metabolome analysis showed that the pools of citrate, isocitrate, cis-aconitate, and L-valine were significantly higher compared to the WT control. As an alternative to existing L-valine producer strains based on inactivated or attenuated pyruvate dehydrogenase complex, we finally engineered the PEP and pyruvate carboxylase-deficient C. glutamicum strains with identified ICD mutations for L-valine production by overexpression of the L-valine biosynthesis genes. Among them, C. glutamicum Δppc Δpyc ICD G407S (pJC4ilvBNCE) produced up

Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in l-lactate and sugar utilization

PubMed Central

Gao, Yong-Gui; Suzuki, Hiroaki; Itou, Hiroshi; Zhou, Yong; Tanaka, Yoshikazu; Wachi, Masaaki; Watanabe, Nobuhisa; Tanaka, Isao; Yao, Min

2008-01-01

LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-Å resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn2+ binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed. PMID:18988622

The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum.

PubMed

Fiuza, Maria; Canova, Marc J; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A

2008-12-26

The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (L-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.

Rational modification of Corynebacterium glutamicum dihydrodipicolinate reductase to switch the nucleotide-cofactor specificity for increasing l-lysine production.

PubMed

Xu, Jian-Zhong; Yang, Han-Kun; Liu, Li-Ming; Wang, Ying-Yu; Zhang, Wei-Guo

2018-03-25

l-lysine is an important amino acid in animals and humans and NADPH is a vital cofactor for maximizing the efficiency of l-lysine fermentation. Dihydrodipicolinate reductase (DHDPR), an NAD(P)H-dependent enzyme, shows a variance in nucleotide-cofactor affinity in bacteria. In this study, we rationally engineered Corynebacterium glutamicum DHDPR (CgDHDPR) to switch its nucleotide-cofactor specificity resulting in an increase in final titer (from 82.6 to 117.3 g L -1 ), carbon yield (from 0.35 to 0.44 g [g glucose] -1 ) and productivity (from 2.07 to 2.93 g L -1  hr -1 ) of l-lysine in JL-6 ΔdapB::Ec-dapB C115G,G116C in fed-batch fermentation. To do this, we comparatively analyzed the characteristics of CgDHDPR and Escherichia coli DHDPR (EcDHDPR), indicating that hetero-expression of NADH-dependent EcDHDPR increased l-lysine production. Subsequently, we rationally modified the conserved structure of cofactor-binding motif, and results indicated that introducing the mutation K11A or R13A in CgDHDPR and introducing the mutation R16A or R39A in EcDHDPR modifies the nucleotide-cofactor affinity of DHDPR. Lastly, the effects of these mutated DHDPRs on l-lysine production were investigated. The highest increase (26.2%) in l-lysine production was observed for JL-6 ΔdapB::Ec-dapB C115G,G116C , followed by JL-6 Cg-dapB C37G,G38C (21.4%) and JL-6 ΔdapB::Ec-dapB C46G,G47C (15.2%). This is the first report of a rational modification of DHDPR that enhances the l-lysine production and yield through the modulation of nucleotide-cofactor specificity. © 2018 Wiley Periodicals, Inc.

Identification and characterization of smallest pore-forming protein in the cell wall of pathogenic Corynebacterium urealyticum DSM 7109.

PubMed

Abdali, Narges; Younas, Farhan; Mafakheri, Samaneh; Pothula, Karunakar R; Kleinekathöfer, Ulrich; Tauch, Andreas; Benz, Roland

2018-05-09

Corynebacterium urealyticum, a pathogenic, multidrug resistant member of the mycolata, is known as causative agent of urinary tract infections although it is a bacterium of the skin flora. This pathogenic bacterium shares with the mycolata the property of having an unusual cell envelope composition and architecture, typical for the genus Corynebacterium. The cell wall of members of the mycolata contains channel-forming proteins for the uptake of solutes. In this study, we provide novel information on the identification and characterization of a pore-forming protein in the cell wall of C. urealyticum DSM 7109. Detergent extracts of whole C. urealyticum cultures formed in lipid bilayer membranes slightly cation-selective pores with a single-channel conductance of 1.75 nS in 1 M KCl. Experiments with different salts and non-electrolytes suggested that the cell wall pore of C. urealyticum is wide and water-filled and has a diameter of about 1.8 nm. Molecular modelling and dynamics has been performed to obtain a model of the pore. For the search of the gene coding for the cell wall pore of C. urealyticum we looked in the known genome of C. urealyticum for a similar chromosomal localization of the porin gene to known porH and porA genes of other Corynebacterium strains. Three genes are located between the genes coding for GroEL2 and polyphosphate kinase (PKK2). Two of the genes (cur_1714 and cur_1715) were expressed in different constructs in C. glutamicum ΔporAΔporH and in porin-deficient BL21 DE3 Omp8 E. coli strains. The results suggested that the gene cur_1714 codes alone for the cell wall channel. The cell wall porin of C. urealyticum termed PorACur was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 4 kDa on tricine-containing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Biophysical characterization of the purified protein (PorACur) suggested indeed that cur_1714 is the gene

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.

The MurC Ligase Essential for Peptidoglycan Biosynthesis Is Regulated by the Serine/Threonine Protein Kinase PknA in Corynebacterium glutamicum*

PubMed Central

Fiuza, Maria; Canova, Marc J.; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M.; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A.

2008-01-01

The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (l-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis. PMID:18974047

Proteomics of corynebacteria: From biotechnology workhorses to pathogens.

PubMed

Poetsch, Ansgar; Haussmann, Ute; Burkovski, Andreas

2011-08-01

Corynebacteria belong to the high G+C Gram-positive bacteria (Actinobacteria) and are closely related to Mycobacterium and Nocardia species. The best investigated member of this group of almost seventy species is Corynebacterium glutamicum, a soil bacterium isolated in 1957, which is used for the industrial production of more than two million tons of amino acids per year. This review focuses on the technical advances made in proteomics approaches during the last years and summarizes applications of these techniques with respect to C. glutamicum metabolic pathways and stress response. Additionally, selected proteome applications for other biotechnologically important or pathogenic corynebacteria are described. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Systems metabolic engineering of Corynebacterium glutamicum for the production of the carbon-5 platform chemicals 5-aminovalerate and glutarate.

PubMed

Rohles, Christina Maria; Gießelmann, Gideon; Kohlstedt, Michael; Wittmann, Christoph; Becker, Judith

2016-09-13

The steadily growing world population and our ever luxurious life style, along with the simultaneously decreasing fossil resources has confronted modern society with the issue and need of finding renewable routes to accommodate for our demands. Shifting the production pipeline from raw oil to biomass requires efficient processes for numerous platform chemicals being produced with high yield, high titer and high productivity. In the present work, we established a de novo bio-based production process for the two carbon-5 platform chemicals 5-aminovalerate and glutarate on basis of the lysine-hyperproducing strain Corynebacterium glutamicum LYS-12. Upon heterologous implementation of the Pseudomonas putida genes davA, encoding 5-aminovaleramidase and davB, encoding lysine monooxygenase, 5-aminovalerate production was established. Related to the presence of endogenous genes coding for 5-aminovalerate transaminase (gabT) and glutarate semialdehyde dehydrogenase, 5-aminovalerate was partially converted to glutarate. Moreover, residual L-lysine was secreted as by-product. The issue of by-product formation was then addressed by deletion of the lysE gene, encoding the L-lysine exporter. Additionally, a putative gabT gene was deleted to enhance 5-aminovalerate production. To fully exploit the performance of the optimized strain, fed-batch fermentation was carried out producing 28 g L(-1) 5-aminovalerate with a maximal space-time yield of 0.9 g L(-1) h(-1). The present study describes the construction of a recombinant microbial cell factory for the production of carbon-5 platform chemicals. Beyond a basic proof-of-concept, we were able to specifically increase the production flux of 5-aminovalerate thereby generating a strain with excellent production performance. Additional improvement can be expected by removal of remaining by-product formation and bottlenecks, associated to the terminal pathway, to generate a strain being applicable as centerpiece for a bio

[Corynebacterium ulcerans pulmonary infection].

PubMed

Thouvenin, Maxime; Beilouny, Bassam; Badell, Edgar; Guiso, Nicole

2016-01-01

Corynebacterium ulcerans is a bacterium able to infect humans by inducing a disease close to diphtheria. We describe the case of a 83-year-old patient hospitalized as a matter of urgency in intensive care for which C. ulcerans was isolated in pure culture in its bronchial samples. Even if the isolate was not secreting toxin in vitro, it possesses the tox gene which motivated the use of specific antitoxin serum. After two months of intensive care the patient went out of the service. It is about a remarkable case of clinicobiologic collaboration.

A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.

PubMed

Bommareddy, Rajesh Reddy; Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

2014-09-01

Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Metabolome analysis-based design and engineering of a metabolic pathway in Corynebacterium glutamicum to match rates of simultaneous utilization of D-glucose and L-arabinose.

PubMed

Kawaguchi, Hideo; Yoshihara, Kumiko; Hara, Kiyotaka Y; Hasunuma, Tomohisa; Ogino, Chiaki; Kondo, Akihiko

2018-05-17

L-Arabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to D-xylose. However, few microorganisms are capable of utilizing pentoses, and catabolic genes and operons enabling bacterial utilization of pentoses are typically subject to carbon catabolite repression by more-preferred carbon sources, such as D-glucose, leading to a preferential utilization of D-glucose over pentoses. In order to simultaneously utilize both D-glucose and L-arabinose at the same rate, a modified metabolic pathway was rationally designed based on metabolome analysis. Corynebacterium glutamicum ATCC 31831 utilized D-glucose and L-arabinose simultaneously at a low concentration (3.6 g/L each) but preferentially utilized D-glucose over L-arabinose at a high concentration (15 g/L each), although L-arabinose and D-glucose were consumed at comparable rates in the absence of the second carbon source. Metabolome analysis revealed that phosphofructokinase and pyruvate kinase were major bottlenecks for D-glucose and L-arabinose metabolism, respectively. Based on the results of metabolome analysis, a metabolic pathway was engineered by overexpressing pyruvate kinase in combination with deletion of araR, which encodes a repressor of L-arabinose uptake and catabolism. The recombinant strain utilized high concentrations of D-glucose and L-arabinose (15 g/L each) at the same consumption rate. During simultaneous utilization of both carbon sources at high concentrations, intracellular levels of phosphoenolpyruvate declined and acetyl-CoA levels increased significantly as compared with the wild-type strain that preferentially utilized D-glucose. These results suggest that overexpression of pyruvate kinase in the araR deletion strain increased the specific consumption rate of L-arabinose and that citrate synthase activity becomes a new bottleneck in the engineered pathway during the simultaneous utilization of D-glucose and L-arabinose. Metabolome analysis

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Nitrogen metabolism and nitrogen control in corynebacteria: variations of a common theme.

PubMed

Walter, Britta; Hänssler, Eva; Kalinowski, Jörn; Burkovski, Andreas

2007-01-01

The published genome sequences of Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum and Corynebacterium jeikeium were screened for genes encoding central components of nitrogen source uptake, nitrogen assimilation and nitrogen control systems. Interestingly, the soil-living species C. efficiens and C. glutamicum exhibit a broader spectrum of genes for nitrogen transport and metabolism than the pathogenic species C. diphtheriae and C. jeikeium. The latter are characterized by gene decay and loss of functions like urea metabolism and nitrogen-dependent transcription control. The global regulator of nitrogen regulation AmtR and its DNA-binding motif are conserved in C. diphtheriae, C. efficiens and C. glutamicum, while in C. jeikeium, an AmtR-encoding gene as well as putative AmtR-binding motifs are missing. Copyright (c) 2007 S. Karger AG, Basel.

Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum.

PubMed

Wang, Zhihao; Chan, Siu Hung Joshua; Sudarsan, Suresh; Blank, Lars M; Jensen, Peter Ruhdal; Solem, Christian

2016-11-01

The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

[Serological affinity of some species of nonpathogenic corynebacteria].

PubMed

Furtat, I M; Nohina, T M; Mikhal's'kyĭ, L O; Vedenieieva, O A

2002-01-01

Serological peculiarities of the species strains Corynebacterium glutamicum, C. ammoniagenes, C. vitaeruminis, C. variabilis and strain of Corynebacterium sp. (Brevibacterium stationis) UCM Ac-719 have been investigated with the help of immunoenzyme analysis ELISA with the use of mice immune serum, specific to C. ammoniagenes UCM Ac-732T, C. vitaeruminis UCM Ac-718T, C. variabilis UCM Ac-717T, C. glutamicum UCM Ac-733 and Corynebacterium sp. UCM Ac-719. It has been established that the species of nonpathogenic corynebacteria differ between themselves as to the degree of serological affinity. C. variabilis, C. ammoniagenes and C. glutamicum are the least similar as to this indication. Weak antigenic relations have been revealed in C. vitaeruminis and C. ammoniagenes. The latter displayed the higher, as compared with other strains, affinity for Corynebacterium sp. UCM Ac-719. The highest degree of serological affinity within the species was registered in strains C. glutamicum and C. variabilis. Data obtained evidence that the ELISA method permits conducting the high-reliability species diagnosis of nonpathogenic corynebacteria on the basis of their antigenic characteristics.

Isolation of Corynebacterium tuscaniae sp. nov. from Blood Cultures of a Patient with Endocarditis

PubMed Central

Riegel, Philippe; Creti, Roberta; Mattei, Romano; Nieri, Alfredo; von Hunolstein, Christina

2006-01-01

A strain of an unknown coryneform bacterium was repeatedly isolated in pure culture from the blood of a patient affected by endocarditis. Comparative 16S rRNA gene sequence analysis revealed that this isolate represented a new subline within the genus Corynebacterium. This new taxon can be identified by the presence of corynomycolic acids and its enzymatic activities and fermentation of sugars. Acid production from glucose and maltose, pyrazinamidase and alkaline phoshatase activities, and hippurate hydrolysis were the most characteristic phenotypic features of the bacterium. On the basis of both phenotypic and phylogenetic evidence, it is proposed that this isolate be classified as a novel species, Corynebacterium tuscaniae sp. nov. The type strain, ISS-5309, has been deposited in the American Type Culture Collection (ATCC BAA-1141) and in the Culture Collection of the University of Göteborg (CCUG 51321). PMID:16455875

The C-terminal domain of the betaine carrier BetP of Corynebacterium glutamicum is directly involved in sensing K+ as an osmotic stimulus.

PubMed

Schiller, Dirk; Rübenhagen, René; Krämer, Reinhard; Morbach, Susanne

2004-05-18

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

Characterization and Use of Catabolite-Repressed Promoters from Gluconate Genes in Corynebacterium glutamicum†

PubMed Central

Letek, Michal; Valbuena, Noelia; Ramos, Angelina; Ordóñez, Efrén; Gil, José A.; Mateos, Luís M.

2006-01-01

The genes involved in gluconate catabolism (gntP and gntK) in Corynebacterium glutamicum are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the gnt operon in Escherichia coli and Bacillus subtilis. In C. glutamicum, gntP and gntK are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of gntP and gntK is not induced by gluconate, and the gnt genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (PgntK and PgntP) was performed using bifunctional promoter probe vectors containing mel (involved in melanin production) or egfp2 (encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the divIVA gene in C. glutamicum is shown, corroborating the possible use of the gnt promoters to control gene expression. glxR (which encodes GlxR, the hypothetical CRP protein) was subcloned from the C. glutamicum chromosomal DNA and overexpressed in corynebacteria; we found that the level of gnt expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on PgntP and PgntK fragments was detected only in the presence of cAMP. PMID:16385030

Corynebacterium godavarianum sp. nov., isolated from the Godavari river, India.

PubMed

Jani, Kunal; Khare, Kaustubh; Senik, Svetlana; Karodi, Prachi; Vemuluri, Venkata Ramana; Bandal, Jayashree; Shouche, Yogesh; Rale, Vinay; Sharma, Avinash

2018-01-01

A Gram-stain-positive, rod-shaped, non-motile bacterium, strain PRD07 T , was isolated from Godavari river, India during the world's largest spiritual and religious mass bathing event 'Kumbh Mela'. Molecular analysis using 16S rRNA gene sequencing and phylogenetic analysis reveals the distinct phylogenetic positioning of strain PRD07 T within the genus Corynebacterium. The strain demonstrated highest sequence similarity to Corynebacterium imitans DSM 44264 T (97.9 %), Corynebacterium appendicis DSM 44531 T (97.1 %) and <96.7 % with all other members of the genus Corynebacterium. The G+C content of PRD07 T was 68.5 mol% (Tm) and the DNA-DNA hybridization depicts 61.09 % genomic relatedness with C. imitans DSM 44264 T . Chemotaxonomic assessment of strain PRD07 T suggested presence of C16 : 0 (31.6 %), C18 : 0 (3.5 %) and C18 : 1ω9c (58.6 %) as the major cellular fatty acids. The major polar lipids of strain PRD07 T were phosphatidylglycerol, diphosphatidylglycerol and glycophospholipid. Differentiating molecular, phylogenetic and chemotaxonomic characteristics of strain PRD07 T with its closest relatives necessitated the description of strain PRD07 T as a novel species of genus Corynebacterium for which the name Corynebacteriumgodavarianum sp. nov., has been proposed. The type strain is PRD07 T (=MCC 3388 T =KCTC 39803 T =LMG 29598 T ).

Engineered coryneform bacteria as a bio-tool for arsenic remediation.

PubMed

Villadangos, Almudena F; Ordóñez, Efrén; Pedre, Brandán; Messens, Joris; Gil, Jose A; Mateos, Luis M

2014-12-01

Despite current remediation efforts, arsenic contamination in water sources is still a major health problem, highlighting the need for new approaches. In this work, strains of the nonpathogenic and highly arsenic-resistant bacterium Corynebacterium glutamicum were used as inexpensive tools to accumulate inorganic arsenic, either as arsenate (As(V)) or arsenite (As(III)) species. The assays made use of "resting cells" from these strains, which were assessed under well-established conditions and compared with C. glutamicum background controls. The two mutant As(V)-accumulating strains were those used in a previously published study: (i) ArsC1/C2, in which the gene/s encoding the mycothiol-dependent arsenate reductases is/are disrupted, and (ii) MshA/C mutants unable to produce mycothiol, the low molecular weight thiol essential for arsenate reduction. The As(III)-accumulating strains were either those lacking the arsenite permease activities (Acr3-1 and Acr3-2) needed in As(III) release or recombinant strains overexpressing the aquaglyceroporin genes (glpF) from Corynebacterium diphtheriae or Streptomyces coelicolor, to improve As(III) uptake. Both genetically modified strains accumulated 30-fold more As(V) and 15-fold more As(III) than the controls. The arsenic resistance of the modified strains was inversely proportional to their metal accumulation ability. Our results provide the basis for investigations into the use of these modified C. glutamicum strains as a new bio-tool in arsenic remediation efforts.

Inactivation of Cg10062, a cis-3-chloroacrylic acid dehalogenase homologue in Corynebacterium glutamicum, by (R)- and (S)-oxirane-2-carboxylate: analysis and implications.

PubMed

Robertson, Brooklyn A; Johnson, William H; Lo, Herng-Hsiang; Whitman, Christian P

2008-08-19

( R)- and ( S)-oxirane-2-carboxylate were determined to be active site-directed irreversible inhibitors of the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) homologue Cg10062 found in Corynebacterium glutamicum. Kinetic analysis indicates that the ( R) enantiomer binds more tightly and is the more potent inhibitor, likely reflecting more favorable interactions with active site residues. Pro-1 is the sole site of covalent modification by the ( R) and ( S) enantiomers. Pro-1, Arg-70, Arg-73, and Glu-114, previously identified as catalytic residues in Cg10062, have also been implicated in the inactivation mechanism. Pro-1, Arg-70, and Arg-73 are essential residues for the process as indicated by the observation that the enzymes with the corresponding alanine mutations are not covalently modified by either enantiomer. The E114Q mutant slows covalent modification of Cg10062 but does not prevent it. The results are comparable to those found for the irreversible inactivation of cis-CaaD by ( R)-oxirane-2-carboxylate with two important distinctions: the alkylation of cis-CaaD is stereospecific, and Glu-114 does not take part in the cis-CaaD inactivation mechanism. Cg10062 exhibits low-level cis-CaaD and trans-3-chloroacrylic acid dehalogenase (CaaD) activities, with the cis-CaaD activity predominating. Hence, the preference of Cg10062 for the cis isomer correlates with the observation that the ( R) enantiomer is the more potent inactivator. Moreover, the factors responsible for the relaxed substrate specificity of Cg10062 may account for the stereoselective inactivation by the enantiomeric epoxides. Delineation of these factors would provide a more complete picture of the substrate specificity determinants for cis-CaaD. This study represents an important step toward this goal by setting the stage for a crystallographic analysis of inactivated Cg10062.

Corynebacterium Prosthetic Joint Infection

PubMed Central

Cazanave, Charles; Greenwood-Quaintance, Kerryl E.; Hanssen, Arlen D.

2012-01-01

Identification of Corynebacterium species may be challenging. Corynebacterium species are occasional causes of prosthetic joint infection (PJI), but few data are available on the subject. Based on the literature, C. amycolatum, C. aurimucosum, C. jeikeium, and C. striatum are the most common Corynebacterium species that cause PJI. We designed a rapid PCR assay to detect the most common human Corynebacterium species, with a specific focus on PJI. A polyphosphate kinase gene identified using whole-genome sequence was targeted. The assay differentiates the antibiotic-resistant species C. jeikeium and C. urealyticum from other species in a single assay. The assay was applied to a collection of human Corynebacterium isolates from multiple clinical sources, and clinically relevant species were detected. The assay was then tested on Corynebacterium isolates specifically associated with PJI; all were detected. We also describe the first case of C. simulans PJI. PMID:22337986

Ribosomal RNA and ribosomal proteins in corynebacteria.

PubMed

Martín, Juan F; Barreiro, Carlos; González-Lavado, Eva; Barriuso, Mónica

2003-09-04

Ribosomal RNAs (rRNAs) (16S, 23S, 5S) encoded by the rrn operons and ribosomal proteins play a very important role in the formation of ribosomes and in the control of translation. Five copies of the rrn operon were reported by hybridization studies in Brevibacterium (Corynebacterium) lactofermentum but the genome sequence of Corynebacterium glutamicum provided evidence for six rrn copies. All six copies of the C. glutamicum 16S rRNA have a size of 1523 bp and each of the six copies of the 5S contain 120 bp whereas size differences are found between the six copies of the 23S rRNA. The anti-Shine-Dalgarno sequence at the 3'-end of the 16S rRNA was 5'-CCUCCUUUC-3'. Each rrn operon is transcribed as a large precursor rRNA (pre-rRNA) that is processed by RNaseIII and other RNases at specific cleavage boxes that have been identified in the C. glutamicum pre-rRNA. A secondary structure of the C. glutamicum 16S rRNA is proposed. The 16S rRNA sequence has been used as a molecular evolution clock allowing the deduction of a phylogenetic tree of all Corynebacterium species. In C. glutamicum, there are 11 ribosomal protein gene clusters encoding 42 ribosomal proteins. The organization of some of the ribosomal protein gene cluster is identical to that of Escherichia coli whereas in other clusters the organization of the genes is rather different. Some specific ribosomal protein genes are located in a different cluster in C. glutamicum when compared with E. coli, indicating that the control of expression of these genes is different in E. coli and C. glutamicum.

Corynebacterium timonense sp. nov. and Corynebacterium massiliense sp. nov., isolated from human blood and human articular hip fluid.

PubMed

Merhej, Vicky; Falsen, Enevold; Raoult, Didier; Roux, Véronique

2009-08-01

Gram-positive, facultatively anaerobic, rod-shaped bacteria were isolated from the blood of a patient with endocarditis (strain 5401744T) and from the hip joint fluid of a patient with an infected orthopaedic prosthesis (strain 5402485T). These strains were characterized by using a polyphasic taxonomic approach. Based on cellular morphology and biochemical criteria the two isolates were tentatively assigned to the genus Corynebacterium, although they did not correspond to any recognized species. The predominant fatty acids were a mix of C18:2omega6,9c and anteiso-C18:0 (32.1% of the total), C16:0 (26.3%) and C18:1omega9c (22.5%) for strain 5402485T and C18:1omega9c (36.4%), C17:1omega9c (27.1%) and C16:0 (10.9%) for strain 5401744T. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain 5401744T was closely related to the type strains of Corynebacterium auris, Corynebacterium capitovis, Corynebacterium lipophiloflavum and Corynebacterium mycetoides (97.0, 96.6, 96.5 and 96.3% similarity, respectively) and strain 5402485T was closely related to the type strains of Corynebacterium macginleyi, Corynebacterium accolens, Corynebacterium tuberculostearicum, Corynebacterium confusum, Corynebacterium mastitidis and Corynebacterium renale (95.6, 95.3, 95.3, 94.5, 94.0 and 93.5%, respectively). On the basis of phenotypic data and phylogenetic inference, these isolates are considered to represent two novel species of the genus Corynebacterium, for which the names Corynebacterium timonense sp. nov. (type strain, 5401744T=CSUR P20T=CIP 109424T=CCUG 53856T) and Corynebacterium massiliense sp. nov. (type strain, 5402485T=CSUR P19T=CIP 109423T=CCUG 53857T) are proposed.

Searching whole genome sequences for biochemical identification features of emerging and reemerging pathogenic Corynebacterium species.

PubMed

Santos, André S; Ramos, Rommel T; Silva, Artur; Hirata, Raphael; Mattos-Guaraldi, Ana L; Meyer, Roberto; Azevedo, Vasco; Felicori, Liza; Pacheco, Luis G C

2018-05-11

Biochemical tests are traditionally used for bacterial identification at the species level in clinical microbiology laboratories. While biochemical profiles are generally efficient for the identification of the most important corynebacterial pathogen Corynebacterium diphtheriae, their ability to differentiate between biovars of this bacterium is still controversial. Besides, the unambiguous identification of emerging human pathogenic species of the genus Corynebacterium may be hampered by highly variable biochemical profiles commonly reported for these species, including Corynebacterium striatum, Corynebacterium amycolatum, Corynebacterium minutissimum, and Corynebacterium xerosis. In order to identify the genomic basis contributing for the biochemical variabilities observed in phenotypic identification methods of these bacteria, we combined a comprehensive literature review with a bioinformatics approach based on reconstruction of six specific biochemical reactions/pathways in 33 recently released whole genome sequences. We used data retrieved from curated databases (MetaCyc, PathoSystems Resource Integration Center (PATRIC), The SEED, TransportDB, UniProtKB) associated with homology searches by BLAST and profile Hidden Markov Models (HMMs) to detect enzymes participating in the various pathways and performed ab initio protein structure modeling and molecular docking to confirm specific results. We found a differential distribution among the various strains of genes that code for some important enzymes, such as beta-phosphoglucomutase and fructokinase, and also for individual components of carbohydrate transport systems, including the fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase (PTS) and the ribose-specific ATP-binging cassette (ABC) transporter. Horizontal gene transfer plays a role in the biochemical variability of the isolates, as some genes needed for sucrose fermentation were seen to be present in genomic islands. Noteworthy

[The serological properties of saprophytic corynebacteria studied by immunoenzyme analysis].

PubMed

Mikhal'skiĭ, L A; Nogina, T M; Furtat, I M

1997-01-01

The degree of serological similarity of saprophytic corynebacteria have been studied using immunoassay ELISA analysis, that is seven collection strains, belonging to Corynebacterium glutamicum (3 strains), C. ammoniagenes (1 strain), C. vitarumen (1 strain), C. variabilis (2 strains) and three industrial strains-lysine producers. Intact and heated bacteria cells have been used as antigens. It has been shown that industrial strain C. glutamicum 22L and collection strains C. glutamicum IMV AC-715, IMV AC-714, IMV AC-733 have the highest degree of serological relationship. C. vitarumen IMV AC-718, C variabilis IMV AC-716 as well as Corynebacterium sp. E531 and VNIIgenetics 90 are close to them according to their serological properties. C. ammoniagenes IMV AC-732 and C. variabilis IMV AC-717 strains have the lowest degree of similarity with other saprophytic corynebacteria which have been studied.

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

High quality draft genome sequence of Corynebacterium ulceribovis type strain IMMIB-L1395T (DSM 45146T)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yassin, Atteyet F.; Lapidus, Alla; Han, James

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

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Corynebacterium accolens Releases Antipneumococcal Free Fatty Acids from Human Nostril and Skin Surface Triacylglycerols.

PubMed

Bomar, Lindsey; Brugger, Silvio D; Yost, Brian H; Davies, Sean S; Lemon, Katherine P

2016-01-05

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. Little is known about how harmless Corynebacterium species that colonize the human nose and skin might impact pathogen colonization and proliferation at these sites. We show that Corynebacterium accolens, a common benign nasal bacterium, modifies its local habitat in vitro as it inhibits growth of Streptococcus pneumoniae by releasing antibacterial free fatty acids from host skin surface triacylglycerols. We further identify the primary C. accolens lipase required for this activity. We postulate a model in which higher numbers of C. accolens cells deter/limit S. pneumoniae nostril colonization, which might partly explain why children without S. pneumoniae colonization have higher levels of nasal Corynebacterium. This work narrows the gap between descriptive studies and the needed in-depth understanding of the molecular mechanisms of microbe-microbe interactions that help shape the human microbiome. It also lays the

A novel bioremediation strategy for petroleum hydrocarbon pollutants using salt tolerant Corynebacterium variabile HRJ4 and biochar.

PubMed

Zhang, Hairong; Tang, Jingchun; Wang, Lin; Liu, Juncheng; Gurav, Ranjit Gajanan; Sun, Kejing

2016-09-01

The present work aimed to develop a novel strategy to bioremediate the petroleum hydrocarbon contaminants in the environment. Salt tolerant bacterium was isolated from Dagang oilfield, China and identified as Corynebacterium variabile HRJ4 based on 16S rRNA gene sequence analysis. The bacterium had a high salt tolerant capability and biochar was developed as carrier for the bacterium. The bacteria with biochar were most effective in degradation of n-alkanes (C16, C18, C19, C26, C28) and polycyclic aromatic hydrocarbons (NAP, PYR) mixture. The result demonstrated that immobilization of C. variabile HRJ4 with biochar showed higher degradation of total petroleum hydrocarbons (THPs) up to 78.9% after 7-day of incubation as compared to the free leaving bacteria. The approach of this study will be helpful in clean-up of petroleum-contamination in the environments through bioremediation process using eco-friendly and cost effective materials like biochar. Copyright © 2016. Published by Elsevier B.V.

Novel Chromosome Organization Pattern in Actinomycetales-Overlapping Replication Cycles Combined with Diploidy.

PubMed

Böhm, Kati; Meyer, Fabian; Rhomberg, Agata; Kalinowski, Jörn; Donovan, Catriona; Bramkamp, Marc

2017-06-06

Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we found that C. glutamicum cells are at least diploid under all of the conditions tested and that these organisms have overlapping C periods during replication, with both origins initiating replication simultaneously. On the basis of experimental data, we propose growth rate-dependent cell cycle models for C. glutamicum IMPORTANCE Bacterial cell cycles are known for few model organisms and can vary significantly between species. Here, we studied the cell cycle of Corynebacterium glutamicum , an emerging cell biological model organism for mycolic acid-containing bacteria, including mycobacteria. Our data suggest that C. glutamicum carries two pole-attached chromosomes that replicate with overlapping C periods, thus initiating a new round of DNA replication before the previous one is terminated. The newly replicated origins segregate to midcell positions, where cell division occurs between the two new origins. Even after long starvation or under extremely slow-growth conditions, C. glutamicum cells are at least diploid, likely as an adaptation to environmental stress that may cause DNA damage. The cell cycle of C. glutamicum combines features of slow-growing organisms, such as polar origin localization, and fast-growing organisms, such as overlapping C periods. Copyright © 2017 Böhm et al.

Peptidoglycan from Fermentation By-Product Triggers Defense Responses in Grapevine

PubMed Central

Chen, Yang; Takeda, Taito; Aoki, Yoshinao; Fujita, Keiko; Suzuki, Shunji; Igarashi, Daisuke

2014-01-01

Plants are constantly under attack from a variety of microorganisms, and rely on a series of complex detection and response systems to protect themselves from infection. Here, we found that a by-product of glutamate fermentation triggered defense responses in grapevine, increasing the expression of defense response genes in cultured cells, foliar chitinase activity, and resistance to infection by downy mildew in leaf explants. To identify the molecule that triggered this innate immunity, we fractionated and purified candidates extracted from Corynebacterium glutamicum, a bacterium used in the production of amino acids by fermentation. Using hydrolysis by lysozyme, a silkworm larva plasma detection system, and gel filtration analysis, we identified peptidoglycan as inducing the defense responses. Peptidoglycans of Escherichia coli, Bacillus subtilis, and Staphylococcus aureus also generated similar defensive responses. PMID:25427192

Corynebacterium species isolated from patients with mastitis.

PubMed

Paviour, Sue; Musaad, Sahar; Roberts, Sally; Taylor, Graeme; Taylor, Susan; Shore, Keith; Lang, Selwyn; Holland, David

2002-12-01

Corynebacteria were isolated from breast tissue, pus, or deep wound swabs of 24 women; the most common species isolated was the newly described Corynebacterium kroppenstedtii, followed by Corynebacterium amycolatum and Corynebacterium tuberculostearicum. Gram-positive bacilli were seen in samples sent for culture or in histological specimens for 12 women, and 9 of the 12 women from whom adequate histological specimens were obtained had conditions that met the criteria for granulomatous lobular mastitis, a chronic inflammatory disease of unknown etiology.

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. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

Antimicrobial Treatment Options for Granulomatous Mastitis Caused by Corynebacterium Species

PubMed Central

Dobinson, Hazel C.; Anderson, Trevor P.; Chambers, Stephen T.; Doogue, Matthew P.; Seaward, Lois

2015-01-01

Corynebacterium species are increasingly recognized as important pathogens in granulomatous mastitis. Currently, there are no published treatment protocols for Corynebacterium breast infections. This study describes antimicrobial treatment options in the context of other management strategies used for granulomatous mastitis. Corynebacterium spp. isolated from breast tissue and aspirate samples stored from 2002 to 2013 were identified and determined to the species level using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), 16S RNA sequencing, and rpoB gene targets. The MICs for 12 antimicrobials were performed using Etest for each isolate. Correlations of these with antimicrobial characteristics, choice of antimicrobial, and disease outcome were evaluated. Corynebacterium spp. from breast tissue and aspirate samples were confirmed in 17 isolates from 16 patients. Based on EUCAST breakpoints, Corynebacterium kroppenstedtii isolates (n = 11) were susceptible to seven antibiotic classes but resistant to β-lactam antibiotics. Corynebacterium tuberculostearicum isolates (n = 4) were multidrug resistant. Two nonlipophilic species were isolated, Corynebacterium glucuronolyticum and Corynebacterium freneyi, both of which have various susceptibilities to antimicrobial agents. Short-course antimicrobial therapy was common (median, 6 courses per subject; range, 1 to 9 courses). Patients with C. kroppenstedtii presented with a hot painful breast mass and underwent multiple surgical procedures (median, 4 procedures; range, 2 to 6 procedures). The management of Corynebacterium breast infections requires a multidisciplinary approach and includes culture and appropriate sensitivity testing to guide antimicrobial therapy. Established infections have a poor outcome, possibly because adequate concentrations of some drugs will be difficult to achieve in lipophilic granulomata. Lipophilic antimicrobial therapy may offer a therapeutic

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

Novel Chromosome Organization Pattern in Actinomycetales—Overlapping Replication Cycles Combined with Diploidy

PubMed Central

Böhm, Kati; Meyer, Fabian; Rhomberg, Agata; Kalinowski, Jörn; Donovan, Catriona

2017-01-01

ABSTRACT Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we found that C. glutamicum cells are at least diploid under all of the conditions tested and that these organisms have overlapping C periods during replication, with both origins initiating replication simultaneously. On the basis of experimental data, we propose growth rate-dependent cell cycle models for C. glutamicum. PMID:28588128

Caenorhabditis elegans star formation and negative chemotaxis induced by infection with corynebacteria.

PubMed

Antunes, Camila Azevedo; Clark, Laura; Wanuske, Marie-Therès; Hacker, Elena; Ott, Lisa; Simpson-Louredo, Liliane; de Luna, Maria das Gracas; Hirata, Raphael; Mattos-Guaraldi, Ana Luíza; Hodgkin, Jonathan; Burkovski, Andreas

2016-01-01

Caenorhabditis elegans is one of the major model systems in biology based on advantageous properties such as short life span, transparency, genetic tractability and ease of culture using an Escherichia coli diet. In its natural habitat, compost and rotting plant material, this nematode lives on bacteria. However, C. elegans is a predator of bacteria, but can also be infected by nematopathogenic coryneform bacteria such Microbacterium and Leucobacter species, which display intriguing and diverse modes of pathogenicity. Depending on the nematode pathogen, aggregates of worms, termed worm-stars, can be formed, or severe rectal swelling, so-called Dar formation, can be induced. Using the human and animal pathogens Corynebacterium diphtheriae and Corynebacterium ulcerans as well as the non-pathogenic species Corynebacterium glutamicum, we show that these coryneform bacteria can also induce star formation slowly in worms, as well as a severe tail-swelling phenotype. While C. glutamicum had a significant, but minor influence on survival of C. elegans, nematodes were killed after infection with C. diphtheriae and C. ulcerans. The two pathogenic species were avoided by the nematodes and induced aversive learning in C. elegans.

Corynebacterium endocarditis species-specific risk factors and outcomes

PubMed Central

Belmares, Jaime; Detterline, Stephanie; Pak, Janet B; Parada, Jorge P

2007-01-01

Background Corynebacterium species are recognized as uncommon agents of endocarditis, but little is known regarding species-specific risk factors and outcomes in Corynebacterium endocarditis. Methods Case report and Medline search of English language journals for cases of Corynebacterium endocarditis. Inclusion criteria required that cases be identified as endocarditis, having persistent Corynebacterium bacteremia, murmurs described by the authors as identifying the affected valve, or vegetations found by echocardiography or in surgical or autopsy specimens. Cases also required patient-specific information on risk factors and outcomes (age, gender, prior prosthetic valve, other prior nosocomial risk factors (infected valve, involvement of native versus prosthetic valve, need for valve replacement, and death) to be included in the analysis. Publications of Corynebacterium endocarditis which reported aggregate data were excluded. Univariate analysis was conducted with chi-square and t-tests, as appropriate, with p = 0.05 considered significant. Results 129 cases of Corynebacterium endocarditis involving nine species met inclusion criteria. Corynebacterium endocarditis typically infects the left heart of adult males and nearly one third of patients have underlying valvular disease. One quarter of patients required valve replacement and one half of patients died. Toxigenic C. diphtheriae is associated with pediatric infections (p < 0.001). Only C. amycolatum has a predilection for women (p = 0.024), while C. pseudodiphtheriticum infections are most frequent in men (p = 0.023). C. striatum, C. jeikeium and C. hemolyticum are associated with nosocomial risk factors (p < 0.001, 0.028, and 0.024, respectively). No species was found to have a predilection for any particular heart valve. C. pseudodiphtheriticum is associated with a previous prosthetic valve replacement (p = 0.004). C. jeikeium infections are more likely to require valve replacement (p = 0.026). Infections

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

Cystic neutrophilic granulomatous mastitis associated with Corynebacterium including Corynebacterium kroppenstedtii.

PubMed

Johnstone, Kate J; Robson, Jennifer; Cherian, Sarah G; Wan Sai Cheong, Jenny; Kerr, Kris; Bligh, Judith F

2017-06-01

Granulomatous (lobular) mastitis is a rare inflammatory breast disease affecting parous reproductive-aged women. Once considered idiopathic, there is growing evidence of an association with corynebacteria infection, especially in the setting of a distinct histological pattern termed cystic neutrophilic granulomatous mastitis (CNGM). We describe 15 cases with histological features either confirming (n = 12) or suggesting (n = 3) CNGM, and concurrent microbiological evidence of Corynebacterium species. The organism was detected by culture or 16S rRNA gene sequencing of specimens obtained at surgery or fine needle aspiration. In seven cases, Gram-positive organisms were seen within vacuolated spaces. Speciation was performed in nine cases, with Corynebacterium kroppenstedtii subsequently identified. These cases provide further evidence in support of this association and in doing so highlight the importance of recognising these histological clues as well as the limitations of Gram stain and microbiological culture in detecting this previously under-recognised disease process. Copyright © 2017 Royal College of Pathologists of Australasia. All rights reserved.

Draft Genome Sequence of Corynebacterium kefirresidentii SB, Isolated from Kefir.

PubMed

Blasche, Sonja; Kim, Yongkyu; Patil, Kiran R

2017-09-14

The genus Corynebacterium includes Gram-positive species with a high G+C content. We report here a novel species, Corynebacterium kefirresidentii SB, isolated from kefir grains collected in Germany. Its draft genome sequence was remarkably dissimilar (average nucleotide identity, 76.54%) to those of other Corynebacterium spp., confirming that this is a unique novel species. Copyright © 2017 Blasche et al.

[Effect of Corynebacterium non diphtheriae on functional activity and apoptosis of macrophages].

PubMed

Kharseeva, G G; Voronina, N A; Tiukavkina, S Iu

2014-01-01

Determine the ability of Corynebacterium non diphtheriae to induce phagocytosis and apoptosis of macrophages and evaluate regulatory effect of nuetrophilokines (NPK) induced by Corynebacterium non diphtheriae on these processes. The ability of Corynebacterium non diphtheriae, isolated from upper respiratory tract, skin and urogenital tract (UGT) were studied for the ability to induce phagocytosis and apoptosis of mice macrophages (MP; in vitro during staining by May-Grunwald with additional staining by Romanowsky-Giemsa) before and after the addition of NPK induced by Corynebacterium non diphtheriae. Phagocytic index (PI) was the same for all the Corynebacterium non diphtheriae species, phagocytic number (PN) and index of phagocytosis completion (IPC)--were minimal relative to corynebacteria isolated from UGT. All the studied corynebacteria species induced MP apoptosis; the most pronounced apoptogenic effect was detected in Corynebacterium pseudotuberculosis isolated from UGT. NPK increased PN against corynebacteria isolated from the studied biotopes, IPC--only during studies of corynebacteria isolated from skin. The effect of NPK resulted in a reduction of apoptogenic effect for almost all the Corynebacterium non diphtheriae, regardless of the isolation location. A pronounced apoptogenic effect and insufficiency of phagocytosis processes induced by corynebacteria are the means of realization of Corynebacterium non diphtheriae pathogenic effect. NPK use is possible for immune correction of immune deficiency conditions developing against the background of diseases determined by Corynebacterium non diphtheriae.

Corynebacterium minutissimum vascular graft infection: case report and review of 281 cases of prosthetic device-related Corynebacterium infection.

PubMed

Reece, Rebecca M; Cunha, Cheston B; Rich, Josiah D

2014-09-01

Corynebacterium spp. have proven their pathogenic potential in causing infections, particularly in the setting of immunosuppression and prosthetic devices. We conducted a PubMed literature review of all cases of Corynebacterium prosthetic device infections published in the English language through December 2013. The majority of cases involved peritoneal dialysis and central venous catheters, but prosthetic joints and central nervous system shunts/drains were also involved. The management of these cases in terms of retention or removal of the device was not uniform; however, the overall mortality remained the same among both groups. All of these prosthetic device infections pose potential problems in management when the device cannot be removed safely for the patient, especially with the lack of data on the pathogenicity of Corynebacterium species. However with better identification of species and sensitivities, successful treatment is possible even with retention of the device.

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

In silico identification of essential proteins in Corynebacterium pseudotuberculosis based on protein-protein interaction networks.

PubMed

Folador, Edson Luiz; de Carvalho, Paulo Vinícius Sanches Daltro; Silva, Wanderson Marques; Ferreira, Rafaela Salgado; Silva, Artur; Gromiha, Michael; Ghosh, Preetam; Barh, Debmalya; Azevedo, Vasco; Röttger, Richard

2016-11-04

Corynebacterium pseudotuberculosis (Cp) is a gram-positive bacterium that is classified into equi and ovis serovars. The serovar ovis is the etiological agent of caseous lymphadenitis, a chronic infection affecting sheep and goats, causing economic losses due to carcass condemnation and decreased production of meat, wool, and milk. Current diagnosis or treatment protocols are not fully effective and, thus, require further research of Cp pathogenesis. Here, we mapped known protein-protein interactions (PPI) from various species to nine Cp strains to reconstruct parts of the potential Cp interactome and to identify potentially essential proteins serving as putative drug targets. On average, we predict 16,669 interactions for each of the nine strains (with 15,495 interactions shared among all strains). An in silico sanity check suggests that the potential networks were not formed by spurious interactions but have a strong biological bias. With the inferred Cp networks we identify 181 essential proteins, among which 41 are non-host homologous. The list of candidate interactions of the Cp strains lay the basis for developing novel hypotheses and designing according wet-lab studies. The non-host homologous essential proteins are attractive targets for therapeutic and diagnostic proposes. They allow for searching of small molecule inhibitors of binding interactions enabling modern drug discovery. Overall, the predicted Cp PPI networks form a valuable and versatile tool for researchers interested in Corynebacterium pseudotuberculosis.

Selective oxidation of aliphatic C-H bonds in alkylphenols by a chemomimetic biocatalytic system.

PubMed

Du, Lei; Dong, Sheng; Zhang, Xingwang; Jiang, Chengying; Chen, Jingfei; Yao, Lishan; Wang, Xiao; Wan, Xiaobo; Liu, Xi; Wang, Xinquan; Huang, Shaohua; Cui, Qiu; Feng, Yingang; Liu, Shuang-Jiang; Li, Shengying

2017-06-27

Selective oxidation of aliphatic C-H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. Chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. Here, we report the development of a unique chemomimetic biocatalytic system originated from the Gram-positive bacterium Corynebacterium glutamicum The system consisting of CreHI (for installation of a phosphate directing/anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols), and CreD (for directing/anchoring group offloading) is able to selectively oxidize the aliphatic C-H bonds of p - and m -alkylated phenols in a controllable manner. Moreover, the crystal structures of the central P450 biocatalyst CreJ in complex with two representative substrates provide significant structural insights into its substrate flexibility and reaction selectivity.

Selective oxidation of aliphatic C–H bonds in alkylphenols by a chemomimetic biocatalytic system

PubMed Central

Du, Lei; Dong, Sheng; Zhang, Xingwang; Jiang, Chengying; Chen, Jingfei; Yao, Lishan; Wang, Xiao; Wan, Xiaobo; Liu, Xi; Wang, Xinquan; Huang, Shaohua; Cui, Qiu; Liu, Shuang-Jiang; Li, Shengying

2017-01-01

Selective oxidation of aliphatic C–H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. Chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. Here, we report the development of a unique chemomimetic biocatalytic system originated from the Gram-positive bacterium Corynebacterium glutamicum. The system consisting of CreHI (for installation of a phosphate directing/anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols), and CreD (for directing/anchoring group offloading) is able to selectively oxidize the aliphatic C–H bonds of p- and m-alkylated phenols in a controllable manner. Moreover, the crystal structures of the central P450 biocatalyst CreJ in complex with two representative substrates provide significant structural insights into its substrate flexibility and reaction selectivity. PMID:28607077

Idiopathic Granulomatous Mastitis Associated with Corynebacterium Sp. Infection

PubMed Central

Lee, Yun Sun; Balfour, John

2011-01-01

Idiopathic granulomatous mastitis (IGM) is a rare inflammatory condition of the breast. The etiology and treatments options of IGM remain controversial. Previous case reports have suggested that Corynebacterium sp., a gram-positive bacillus endogenous to the skin, may be associated with IGM. In the present report, we describe the first case of IGM with a positive culture for Corynebacterium sp. reported in the United States. PMID:21857740

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.

Under the influence of the active deodorant ingredient 4-hydroxy-3-methoxybenzyl alcohol, the skin bacterium Corynebacterium jeikeium moderately responds with differential gene expression.

PubMed

Brune, Iris; Becker, Anke; Paarmann, Daniel; Albersmeier, Andreas; Kalinowski, Jörn; Pühler, Alfred; Tauch, Andreas

2006-12-15

A 70mer oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Corynebacterium jeikeium, a skin bacterium that is predominantly present in the human axilla and involved in axillary odor formation. Oligonucleotides representing 100% of the predicted coding regions of the C. jeikeium K411 genome were designed and spotted in quadruplicate onto epoxy-coated glass slides. The quality of the printed microarray was demonstrated by co-hybridization with fluorescently labeled cDNA probes obtained from exponentially growing C. jeikeium cultures. Accordingly, genes detected with different intensities resulting in log(2) transformed ratios greater than 0.8 or smaller than -0.8 can be regarded as differentially expressed with a confidence level greater than 99%. In an application example, we measured global changes of gene expression during growth of C. jeikeium in the presence of different concentrations of the deodorant component 4-hydroxy-3-methoxybenzyl alcohol that is active in preventing body odor formation. Global expression profiling revealed that low concentrations of 4-hydroxy-3-methoxybenzyl alcohol (0.5 and 2.5mg/ml) had almost no detectable effect on the transcriptome of C. jeikeium. A slightly higher concentration of 4-hydroxy-3-methoxybenzyl alcohol (5mg/ml) resulted in differential expression of 95 genes, 86 of which showed an enhanced expression when compared to a control culture. Besides many genes encoding proteins that apparently participate in transcription and translation, the drug resistance determinant cmx and the predicted virulence factors sapA and sapD showed significantly enhanced expression levels. Differential expression of relevant genes was validated by real-time reverse transcription PCR assays.

[Skin and Soft Tissue Infections Due to Corynebacterium ulcerans - Case Reports].

PubMed

Jenssen, Christian; Schwede, Ilona; Neumann, Volker; Pietsch, Cristine; Handrick, Werner

2017-10-01

History and clinical findings  We report on three patients suffering from skin and soft tissue infections of the legs due to toxigenic Corynebacterium ulcerans strains. In all three patients, there was a predisposition due to chronic diseases. Three patients had domestic animals (cat, dog) in their households. Investigations and diagnosis  A mixed bacterial flora including Corynebacterium ulcerans was found in wound swab samples. Diphtheric toxin was produced by the Corynebacterium ulcerans strains in all three cases. Treatment and course  In all three patients, successful handling of the skin and soft tissue infections was possible by combining local treatment with antibiotics. Diphtheria antitoxin was not administered in any case. Conclusion  Based on a review of the recent literature pathogenesis, clinical symptoms and signs, diagnostics and therapy of skin and soft tissue infections due to Corynebacterium ulcerans are discussed. Corynebacterium ulcerans should be considered as a potential cause of severe skin and soft tissue infections. Occupational or domestic animal contacts should be evaluated. © Georg Thieme Verlag KG Stuttgart · New York.

Seminal Corynebacterium strains in infertile men with and without leucocytospermia.

PubMed

Mashaly, M; Masallat, D T; Elkholy, A A; Abdel-Hamid, I A; Mostafa, T

2016-04-01

This study aimed to identify seminal Corynebacterium strains in infertile men with and without leucocytospermia. Semen samples from 60 infertile men were allocated into two equal groups: semen samples with leucocytospermia and semen samples without leucocytospermia. Semen culture for Corynebacterium species was carried out on Columbia agar medium confirmed by Gram-stained film and biochemical tests followed by analytical profile index biotyping and antibiotic susceptibility. Bacterial isolates were detected in 20/60 semen cultures (33.3%) as Corynebacteria, Staphylococci, Alpha haemolytic streptococci and E. coli. In all, 12/60 (20%) had Corynebacterium positive semen culture, whereas C. seminal was the major isolated species followed by C. amycolatum, C. jekium and C. urealyticum. There was nonsignificant difference between patients with/without Corynebacterium positive culture regarding sperm concentration and normal sperm morphology; however, in positive cultures sperm motility was significantly lower compared with negative cultures. Antimicrobial sensitivity among Corynebacteria strains was highest for vancomycin, rifampicin then imipenem, ampicillin + sulbactam, ciprofloxacin. It is concluded that positive semen cultures for different Corynebacteria species were demonstrated in infertile men, whereas Corynebacterium seminale was the most common isolated species. Vancomycin, rifampicin then imipenem and ampicillin + sulbactam are recommended as sensitive antibiotics. © 2015 Blackwell Verlag GmbH.

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.

Postgenomic approaches to using corynebacteria as biocatalysts.

PubMed

Vertès, Alain A; Inui, Masayuki; Yukawa, Hideaki

2012-01-01

Corynebacterium glutamicum exhibits numerous ideal intrinsic attributes as a factory of primary and secondary metabolites. The versatile capabilities of this organism have long been implemented at the industrial scale to produce an array of amino acids at high yields and conversion rates, thereby enabling the development of an entire industry. The postgenomic era provides a new technological platform not only to further optimize the intrinsic attributes of C. glutamicum whole cells as biocatalysts, but also to dramatically expand the product portfolio that can be manufactured by this organism, from amino acids to commodity chemicals. This review addresses the methods and strain optimization strategies enabled by genomic information and associated techniques. Their implementation has provided important additional incremental improvements to the economics of industry-scale manufacturing in which C. glutamicum and its episomal elements are used as a performing host-vector system.

Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host–pathogen interaction

PubMed Central

Mishra, Arun K; Driessen, Nicole N; Appelmelk, Ben J; Besra, Gurdyal S

2011-01-01

Approximately one third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. This bacterium has an unusual lipid-rich cell wall containing a vast repertoire of antigens, providing a hydrophobic impermeable barrier against chemical drugs, thus representing an attractive target for vaccine and drug development. Apart from the mycolyl–arabinogalactan–peptidoglycan complex, mycobacteria possess several immunomodulatory constituents, notably lipomannan and lipoarabinomannan. The availability of whole-genome sequences of M. tuberculosis and related bacilli over the past decade has led to the identification and functional characterization of various enzymes and the potential drug targets involved in the biosynthesis of these glycoconjugates. Both lipomannan and lipoarabinomannan possess highly variable chemical structures, which interact with different receptors of the immune system during host–pathogen interactions, such as Toll-like receptors-2 and C-type lectins. Recently, the availability of mutants defective in the synthesis of these glycoconjugates in mycobacteria and the closely related bacterium, Corynebacterium glutamicum, has paved the way for host–pathogen interaction studies, as well as, providing attenuated strains of mycobacteria for the development of new vaccine candidates. This review provides a comprehensive account of the structure, biosynthesis and immunomodulatory properties of these important glycoconjugates. PMID:21521247

Corynebacterium accolens isolated from breast abscess: possible association with granulomatous mastitis.

PubMed

Ang, Lei M N; Brown, Hamish

2007-05-01

Corynebacterium accolens is rarely isolated as a human pathogen. We describe here a case of C. accolens isolated from a breast abscess in a patient previously diagnosed with granulomatous mastitis. The possible association of Corynebacterium accolens and granulomatous mastitis in this patient is discussed.

Genome sequence and description of Corynebacterium ihumii sp. nov.

PubMed Central

Padmanabhan, Roshan; Dubourg, Grégory; Lagier, Jean-Christophe; Couderc, Carine; Michelle, Caroline; Raoult, Didier; Fournier, Pierre-Edouard

2014-01-01

Corynebacterium ihumii strain GD7T sp. nov. is proposed as the type strain of a new species, which belongs to the family Corynebacteriaceae of the class Actinobacteria. This strain was isolated from the fecal flora of a 62 year-old male patient, as a part of the culturomics study. Corynebacterium ihumii is a Gram positive, facultativly anaerobic, nonsporulating bacillus. Here, we describe the features of this organism, together with the high quality draft genome sequence, annotation and the comparison with other member of the genus Corynebacteria. C. ihumii genome is 2,232,265 bp long (one chromosome but no plasmid) containing 2,125 protein-coding and 53 RNA genes, including 4 rRNA genes. The whole-genome shotgun sequence of Corynebacterium ihumii strain GD7T sp. nov has been deposited in EMBL under accession number GCA_000403725. PMID:25197488

Corynebacterium accolens Isolated from Breast Abscess: Possible Association with Granulomatous Mastitis▿

PubMed Central

Ang, Lei M. N.; Brown, Hamish

2007-01-01

Corynebacterium accolens is rarely isolated as a human pathogen. We describe here a case of C. accolens isolated from a breast abscess in a patient previously diagnosed with granulomatous mastitis. The possible association of Corynebacterium accolens and granulomatous mastitis in this patient is discussed. PMID:17344355

Microbiological investigation and clinical significance of Corynebacterium spp. in respiratory specimens.

PubMed

Nhan, Tu-Xuan; Parienti, Jean-Jacques; Badiou, Guillaume; Leclercq, Roland; Cattoir, Vincent

2012-11-01

The purpose of this retrospective study was to evaluate the pathogenic role of Corynebacterium species in lower respiratory tract infections as well as their routine laboratory investigation. From April 2007 to August 2009, 27 clinical isolates were significantly recovered from respiratory specimens of 27 different patients clinically suspected of having lower respiratory tract infections. The average age of patients was 65 years, while 22 (81%) patients presented at least 1 predisposing condition. Of the 27 patients, 15 (56%) were classified as infected according to Centers for Disease Control and Prevention/National Healthcare Safety Network criteria, with 93% of infections being hospital acquired. All isolates were accurately identified to the species level using molecular methods (i.e., 17 Corynebacterium pseudodiphtheriticum, 7 Corynebacterium striatum, and 3 Corynebacterium accolens), whereas phenotypic methods remained frequently unreliable for identifying C. striatum and C. accolens strains. All tested isolates were susceptible to amoxicillin, imipenem, vancomycin, linezolid, and tigecycline, whereas most of them were resistant to erythromycin. Copyright © 2012 Elsevier Inc. All rights reserved.

Corynebacterium macginleyi Conjunctivitis in Canada▿

PubMed Central

Alsuwaidi, Ahmed R.; Wiebe, Deborah; Burdz, Tamara; Ng, Betty; Reimer, Aleisha; Singh, Cathleen; Bernard, Kathryn

2010-01-01

This report describes for the first time Corynebacterium macginleyi as a cause of conjunctivitis in Canada, where menaquinone analysis was done as part of the strain characterization. This species is typically isolated from ocular surfaces of patients from Europe and Japan. The isolate was resistant to erythromycin and clindamycin. PMID:20702661

Contextual control of skin immunity and inflammation by Corynebacterium.

PubMed

Ridaura, Vanessa K; Bouladoux, Nicolas; Claesen, Jan; Chen, Y Erin; Byrd, Allyson L; Constantinides, Michael G; Merrill, Eric D; Tamoutounour, Samira; Fischbach, Michael A; Belkaid, Yasmine

2018-03-05

How defined microbes influence the skin immune system remains poorly understood. Here we demonstrate that Corynebacteria , dominant members of the skin microbiota, promote a dramatic increase in the number and activation of a defined subset of γδ T cells. This effect is long-lasting, occurs independently of other microbes, and is, in part, mediated by interleukin (IL)-23. Under steady-state conditions, the impact of Corynebacterium is discrete and noninflammatory. However, when applied to the skin of a host fed a high-fat diet, Corynebacterium accolens alone promotes inflammation in an IL-23-dependent manner. Such effect is highly conserved among species of Corynebacterium and dependent on the expression of a dominant component of the cell envelope, mycolic acid. Our data uncover a mode of communication between the immune system and a dominant genus of the skin microbiota and reveal that the functional impact of canonical skin microbial determinants is contextually controlled by the inflammatory and metabolic state of the host. © 2018 Ridaura et al.

Gene expression systems in corynebacteria.

PubMed

Srivastava, Preeti; Deb, J K

2005-04-01

Corynebacterium belongs to a group of gram-positive bacteria having moderate to high G+C content, the other members being Mycobacterium, Nocardia, and Rhodococcus. Considerable information is now available on the plasmids, gene regulatory elements, and gene expression in corynebacteria, especially in soil corynebacteria such as Corynebacterium glutamicum. These bacteria are non-pathogenic and, unlike Bacillus and Streptomyces, are low in proteolytic activity and thus have the potential of becoming attractive systems for expression of heterologous proteins. This review discusses recent advances in our understanding of the organization of various regulatory elements, such as promoters, transcription terminators, and development of vectors for cloning and gene expression.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aich, Sanjukta; Prasad, Lata; Delbaere, Louis T.J.

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2{sub 1} with four molecules per asymmetric unit. The 2.3 {angstrom} resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guaninemore » binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.« less

Insight of Genus Corynebacterium: Ascertaining the Role of Pathogenic and Non-pathogenic Species

PubMed Central

Oliveira, Alberto; Oliveira, Leticia C.; Aburjaile, Flavia; Benevides, Leandro; Tiwari, Sandeep; Jamal, Syed B.; Silva, Arthur; Figueiredo, Henrique C. P.; Ghosh, Preetam; Portela, Ricardo W.; De Carvalho Azevedo, Vasco A.; Wattam, Alice R.

2017-01-01

This review gathers recent information about genomic and transcriptomic studies in the Corynebacterium genus, exploring, for example, prediction of pathogenicity islands and stress response in different pathogenic and non-pathogenic species. In addition, is described several phylogeny studies to Corynebacterium, exploring since the identification of species until biological speciation in one species belonging to the genus Corynebacterium. Important concepts associated with virulence highlighting the role of Pld protein and Tox gene. The adhesion, characteristic of virulence factor, was described using the sortase mechanism that is associated to anchorage to the cell wall. In addition, survival inside the host cell and some diseases, were too addressed for pathogenic corynebacteria, while important biochemical pathways and biotechnological applications retain the focus of this review for non-pathogenic corynebacteria. Concluding, this review broadly explores characteristics in genus Corynebacterium showing to have strong relevance inside the medical, veterinary, and biotechnology field. PMID:29075239

Insight of Genus Corynebacterium: Ascertaining the Role of Pathogenic and Non-pathogenic Species.

PubMed

Oliveira, Alberto; Oliveira, Leticia C; Aburjaile, Flavia; Benevides, Leandro; Tiwari, Sandeep; Jamal, Syed B; Silva, Arthur; Figueiredo, Henrique C P; Ghosh, Preetam; Portela, Ricardo W; De Carvalho Azevedo, Vasco A; Wattam, Alice R

2017-01-01

This review gathers recent information about genomic and transcriptomic studies in the Corynebacterium genus, exploring, for example, prediction of pathogenicity islands and stress response in different pathogenic and non-pathogenic species. In addition, is described several phylogeny studies to Corynebacterium , exploring since the identification of species until biological speciation in one species belonging to the genus Corynebacterium . Important concepts associated with virulence highlighting the role of Pld protein and Tox gene. The adhesion, characteristic of virulence factor, was described using the sortase mechanism that is associated to anchorage to the cell wall. In addition, survival inside the host cell and some diseases, were too addressed for pathogenic corynebacteria, while important biochemical pathways and biotechnological applications retain the focus of this review for non-pathogenic corynebacteria. Concluding, this review broadly explores characteristics in genus Corynebacterium showing to have strong relevance inside the medical, veterinary, and biotechnology field.

SIALIDASE (NEURAMINIDASE) OF CORYNEBACTERIUM DIPHTHERIAE.

PubMed

WARREN, L; SPEARING, C W

1963-11-01

Warren, Leonard (National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.) and C. W. Spearing. Sialidase (neuraminidase) of Corynebacterium diphtheriae. J. Bacteriol. 86:950-955. 1963.-The characteristics of a sialidase produced by Corynebacterium diphtheriae were studied. The enzyme was partially purified from preparations of diphtheria toxin on a column of Sephadex G-75. By this means the lethal factor of diphtheria toxin was separated, in part, from the sialidase activity. There appeared to be a close immunological relationship between the sialidases of C. diphtheriae and clostridia, since a preparation of diphtheria antitoxin was as effective an inhibitor of diphtheria sialidase as of the sialidase of three species of clostridia. Conversely, antitoxin to clostridia inhibited diphtheria sialidase. Diphtheria antitoxin was essentially inactive toward influenza virus sialidase, and was completely inactive against purified sialidase of Vibrio cholerae. Removal of sialic acid from the proteins in a preparation of diphtheria antitoxin did not alter the inhibitory activity of the antitoxin against diphtheria sialidase. The enzyme operated optimally at pH 5.5 and did not require calcium ions for activity. The substrate specificity of diphtheria sialidase appears to be the same as that of other previously described sialidases.

Rapid Detection and Molecular Differentiation of Toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans Strains by LightCycler PCR ▿

PubMed Central

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

2011-01-01

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. PMID:21593261

Comparative evolutionary genomics of Corynebacterium with special reference to codon and amino acid usage diversities.

PubMed

Pal, Shilpee; Sarkar, Indrani; Roy, Ayan; Mohapatra, Pradeep K Das; Mondal, Keshab C; Sen, Arnab

2018-02-01

The present study has been aimed to the comparative analysis of high GC composition containing Corynebacterium genomes and their evolutionary study by exploring codon and amino acid usage patterns. Phylogenetic study by MLSA approach, indel analysis and BLAST matrix differentiated Corynebacterium species in pathogenic and non-pathogenic clusters. Correspondence analysis on synonymous codon usage reveals that, gene length, optimal codon frequencies and tRNA abundance affect the gene expression of Corynebacterium. Most of the optimal codons as well as translationally optimal codons are C ending i.e. RNY (R-purine, N-any nucleotide base, and Y-pyrimidine) and reveal translational selection pressure on codon bias of Corynebacterium. Amino acid usage is affected by hydrophobicity, aromaticity, protein energy cost, etc. Highly expressed genes followed the cost minimization hypothesis and are less diverged at their synonymous positions of codons. Functional analysis of core genes shows significant difference in pathogenic and non-pathogenic Corynebacterium. The study reveals close relationship between non-pathogenic and opportunistic pathogenic Corynebaterium as well as between molecular evolution and survival niches of the organism.

Corynebacterium tuberculostearicum: a Potentially Misidentified and Multiresistant Corynebacterium Species Isolated from Clinical Specimens

PubMed Central

Hinić, V.; Lang, C.; Weisser, M.; Straub, C.; Frei, R.

2012-01-01

Corynebacterium tuberculostearicum is a lipophilic corynebacterium validly characterized in 2004. We provide clinical information on 18 patients from whom this organism was isolated. The majority of the patients were hospitalized and had a history of prolonged treatment with broad-spectrum antimicrobials. In 7 (38.9%) of the 18 cases, the isolates were found to be clinically relevant. The present report also includes detailed data on the biochemical and molecular identification of C. tuberculostearicum, as well as its identification by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Our data demonstrate that routine biochemical tests do not provide reliable identification of C. tuberculostearicum. MALDI-TOF MS represents a helpful tool for the identification of this species, since all of the strains matched C. tuberculostearicum as the first choice and 58.3% (7/12) of the strains processed with the full extraction protocol generated scores of >2.000. Nevertheless, partial 16S rRNA gene sequencing still represents the gold standard for the identification of this species. Due to the challenging identification of C. tuberculostearicum, we presume that this organism is often misidentified and its clinical relevance is underestimated. The antimicrobial susceptibility profile of C. tuberculostearicum presented here reveals that 14 (87.5%) of the 16 strains analyzed exhibited multidrug resistance. PMID:22593594

Identification of Corynebacterium amycolatum and other nonlipophilic fermentative corynebacteria of human origin.

PubMed

Wauters, G; Van Bosterhaut, B; Janssens, M; Verhaegen, J

1998-05-01

Four identification tests, proposed in addition to conventional methods, were evaluated with 320 fermentative nonlipophilic Corynebacterium strains: growth at 20 degrees C, glucose fermentation at 42 degrees C, alkalinization of sodium formate, and acid production from ethylene glycol. These tests were highly discriminant. Corynebacterium amycolatum displayed a unique profile, allowing it to be distinguished from similar species, such as C. xerosis, C. striatum, and C. minutissimum.

Identification of Corynebacterium amycolatum and Other Nonlipophilic Fermentative Corynebacteria of Human Origin

PubMed Central

Wauters, Georges; Van Bosterhaut, Bernard; Janssens, Michèle; Verhaegen, Jan

1998-01-01

Four identification tests, proposed in addition to conventional methods, were evaluated with 320 fermentative nonlipophilic Corynebacterium strains: growth at 20°C, glucose fermentation at 42°C, alkalinization of sodium formate, and acid production from ethylene glycol. These tests were highly discriminant. Corynebacterium amycolatum displayed a unique profile, allowing it to be distinguished from similar species, such as C. xerosis, C. striatum, and C. minutissimum. PMID:9574722

SIALIDASE (NEURAMINIDASE) OF CORYNEBACTERIUM DIPHTHERIAE

PubMed Central

Warren, Leonard; Spearing, C. W.

1963-01-01

Warren, Leonard (National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.) and C. W. Spearing. Sialidase (neuraminidase) of Corynebacterium diphtheriae. J. Bacteriol. 86:950–955. 1963.—The characteristics of a sialidase produced by Corynebacterium diphtheriae were studied. The enzyme was partially purified from preparations of diphtheria toxin on a column of Sephadex G-75. By this means the lethal factor of diphtheria toxin was separated, in part, from the sialidase activity. There appeared to be a close immunological relationship between the sialidases of C. diphtheriae and clostridia, since a preparation of diphtheria antitoxin was as effective an inhibitor of diphtheria sialidase as of the sialidase of three species of clostridia. Conversely, antitoxin to clostridia inhibited diphtheria sialidase. Diphtheria antitoxin was essentially inactive toward influenza virus sialidase, and was completely inactive against purified sialidase of Vibrio cholerae. Removal of sialic acid from the proteins in a preparation of diphtheria antitoxin did not alter the inhibitory activity of the antitoxin against diphtheria sialidase. The enzyme operated optimally at pH 5.5 and did not require calcium ions for activity. The substrate specificity of diphtheria sialidase appears to be the same as that of other previously described sialidases. PMID:14080806

Rapid microbiochemical identification of Corynebacterium diphtheriae and other medically important corynebacteria.

PubMed Central

Thompson, J S; Gates-Davis, D R; Yong, D C

1983-01-01

A rapid biochemical method based on the fermentation of carbohydrates, the hydrolysis of urea, and the reduction of nitrate was used to identify Corynebacterium diphtheriae, C. ulcerans, C. pseudodiphtheriticum, C. haemolyticum, C. pseudotuberculosis, C. pyogenes, C. ovis, the Centers for Disease Control JK group, and Rhodococcus (Corynebacterium) equi. With this procedure identification was confirmed for 133 stock cultures and clinical isolates of corynebacteria. Most were identified within 1 h and all were identified within 4 h after inoculation into the test substrates. PMID:6355166

rpoB Gene Sequencing for Identification of Corynebacterium Species

PubMed Central

Khamis, Atieh; Raoult, Didier; La Scola, Bernard

2004-01-01

The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species. PMID:15364970

Genomic diversity and phylogenetic relationships among lipid-requiring diphtheroids from humans and characterization of Corynebacterium macginleyi sp. nov.

PubMed

Riegel, P; Ruimy, R; de Briel, D; Prévost, G; Jehl, F; Christen, R; Monteil, H

1995-01-01

DNA relatedness experiments were performed with 38 clinical isolates and 13 reference strains of coryneform taxa exhibiting a lipid requirement for optimal growth. Forty-five of these strains split into five genomic groups at the species level, whereas six other strains remained unclustered. Genomospecies II fits Corynebacterium accolens, but the other genomospecies were not genetically related to any of the defined Corynebacterium species. Phylogenetic analyses of genes coding for small-subunit rRNA sequences revealed that two genomospecies (I and III) and C. accolens form a tight cluster within the robust branch that groups all Corynebacterium species presently sequenced. Reference strains of biotypes C-1, C-2, and C-3 of "Corynebacterium pseudogenitalium" were found to fall into genomospecies I, as well as "Corynebacterium tuberculostearicum," Centers for Disease Control and Prevention (CDC) coryneform group G-1, and CDC coryneform group G-2 reference strains. Biochemical tests allowed differentiation between genomospecies except between genomospecies IV and V and between six unclustered strains and genomospecies I. We propose a new classification for these lipid-requiring diphtheroids within the genus Corynebacterium with the delineation of some CDC coryneform group G-1 strains (genomospecies III) as a new species for which the name Corynebacterium macginleyi is proposed. The type strain is strain JCL-2 (CIP 104099), isolated from a human corneal ulcer.

The Arsenic Detoxification System in Corynebacteria: Basis and Application for Bioremediation and Redox Control.

PubMed

Mateos, Luis M; Villadangos, Almudena F; de la Rubia, Alfonso G; Mourenza, Alvaro; Marcos-Pascual, Laura; Letek, Michal; Pedre, Brandán; Messens, Joris; Gil, Jose A

2017-01-01

Arsenic (As) is widespread in the environment and highly toxic. It has been released by volcanic and anthropogenic activities and causes serious health problems worldwide. To survive arsenic-rich environments, soil and saprophytic microorganisms have developed molecular detoxification mechanisms to survive arsenic-rich environments, mainly by the enzymatic conversion of inorganic arsenate (As V ) to arsenite (As III ) by arsenate reductases, which is then extruded by arsenite permeases. One of these Gram-positive bacteria, Corynebacterium glutamicum, the workhorse of biotechnological research, is also resistant to arsenic. To sanitize contaminated soils and waters, C. glutamicum strains were modified to work as arsenic "biocontainers." Two chromosomally encoded ars operons (ars1 and ars2) are responsible for As resistance. The genes within these operons encode for metalloregulatory proteins (ArsR1/R2), arsenite permeases (Acr3-1/-2), and arsenate reductases (ArsC1/C2/C1'). ArsC1/C2 arsenate reductases are coupled to the low molecular weight thiol mycothiol (MSH) and to the recently discovered mycoredoxin-1 (Mrx-1) present in most Actinobacteria. This MSH/Mrx-1 redox system protects cells against different forms of stress, including reactive oxygen species (ROS), metals, and antibiotics. ROS can modify functional sulfur cysteines by oxidizing the thiol (-SH) to a sulfenic acid (-SOH). These oxidation-sensitive protein cysteine thiols are redox regulated by the MSH/Mrx-1 couple in Corynebacterium and Mycobacterium. In summary, the molecular mechanisms involved in arsenic resistance system in C. glutamicum have paved the way for understanding the cellular response against oxidative stress in Actinobacteria. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of antimicrobial susceptibilities of Corynebacterium species by broth microdilution and disk diffusion methods.

PubMed Central

Weiss, K; Laverdière, M; Rivest, R

1996-01-01

Corynebacterium species are increasingly being implicated in foreign-body infections and in immunocompromised-host infections. However, there are no specific recommendations on the method or the criteria to use in order to determine the in vitro activities of the antibiotics commonly used to treat Corynebacterium infections. The first aim of our study was to compare the susceptibilities of various species of Corynebacterium to vancomycin, erythromycin, and penicillin by using a broth microdilution method and a disk diffusion method. Second, the activity of penicillin against our isolates was assessed by using the interpretative criteria recommended by the National Committee for Clinical Laboratory Standards for the determination of the susceptibility of streptococci and Listeria monocytogenes to penicillin. Overall, 100% of the isolates were susceptible to vancomycin, while considerable variations in the activities of erythromycin and penicillin were noted for the different species tested, including the non-Corynebacterium jeikeium species. A good correlation in the susceptibilities of vancomycin and erythromycin between the disk diffusion and the microdilution methods was observed. However, a 5% rate of major or very major errors was detected with the Listeria criteria, while a high rate of minor errors (18%) was noted when the streptococcus criteria were used. Our findings indicate considerable variations in the activities of erythromycin and penicillin against the various species of Corynebacterium. Because of the absence of definite recommendations, important discrepancies were observed between the methods and the interpretations of the penicillin activity. PMID:8849254

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.

Comparative 13C Metabolic Flux Analysis of Pyruvate Dehydrogenase Complex-Deficient, l-Valine-Producing Corynebacterium glutamicum▿†

PubMed Central

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

2011-01-01

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 13C 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. PMID:21784914

Bloodstream Infection Caused by Nontoxigenic Corynebacterium diphtheriae in an Immunocompromised Host in the United States

PubMed Central

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

2012-01-01

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. PMID:22493337

Otitis in a cat associated with Corynebacterium provencense.

PubMed

Kittl, Sonja; Brodard, Isabelle; Rychener, Lorenz; Jores, Jörg; Roosje, Petra; Gobeli Brawand, Stefanie

2018-06-25

The role of corynebacteria in canine and feline otitis has not been investigated in detail; however, members of this genus are increasingly recognized as pathogens of otitis in both human and veterinary medicine. Here we report the first case of feline otitis associated with the recently described species Corynebacterium provencense. A seven-month old cat presented with a head tilt and ataxia was diagnosed with peripheral vestibular syndrome associated with an otitis media/interna. This took place 6 weeks after resection of a polyp, having initially shown a full recovery with topical ofloxacin and glucocorticoid treatment. Bacteriology of an ear swab yielded a pure culture of corynebacteria, which could not be identified at the species level using routine methods. However, the 16S rRNA gene sequence was 100% identical to the recently published novel corynebacterium species, Corynebacterium provencense. Whole genome sequencing of the cat isolate and calculation of average nucleotide identity (99.1%) confirmed this finding. The cat isolate was found to contain additional presumptive iron acquisition genes that are likely to encode virulence factors. Furthermore, the strain tested resistant to clindamycin, penicillin and ciprofloxacin. The cat was subsequently treated with chloramphenicol, which lead to clinical improvement. Corynebacteria from otitis cases are not routinely identified at the species level and not tested for antimicrobial susceptibility in veterinary laboratories, as they are not considered major pathogens. This may lead to underreporting of this genus or animals being treated with inappropriate antimicrobials since corynebacteria are often resistant to multiple drugs.

Clonal Multidrug-Resistant Corynebacterium striatum Strains, Italy

PubMed Central

Campanile, Floriana; Carretto, Edoardo; Barbarini, Daniela; Grigis, Annalisa; Falcone, Marco; Goglio, Antonio; Venditti, Mario

2009-01-01

We assessed the clinical relevance and performed molecular characterization of 36 multidrug-resistant strains of Corynebacterium striatum. Pulsed-field gel electrophoresis confirmed a single clone, possessing erm(X), tetA/B, cmxA/B, and aphA1 genes, but few related subclones. This strain is emerging as a pathogen in Italy. PMID:19116057

Suture-Related Keratitis Caused by Corynebacterium macginleyi▿

PubMed Central

Suzuki, Takashi; Iihara, Hirotoshi; Uno, Toshihiko; Hara, Yuko; Ohkusu, Kiyofumi; Hata, Hiroyuki; Shudo, Masachika; Ohashi, Yuichi

2007-01-01

We report two cases of suture-related keratitis following penetrating keratoplasty. In both cases, Corynebacterium macginleyi was isolated from corneal specimens. Scanning electron microscopy revealed that corynebacteria could aggregate and form a biofilm. The MICs of sulbenicillin and fluoroquinolones were high for both isolates. Our findings show that C. macginleyi can cause keratitis with biofilm formation. PMID:17913935

Identification of Non-diphtheriae Corynebacterium by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Alatoom, Adnan A.; Cazanave, Charles J.; Cunningham, Scott A.; Ihde, Sherry M.

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. PMID:22075579

Systems metabolic engineering strategies for the production of amino acids.

PubMed

Ma, Qian; Zhang, Quanwei; Xu, Qingyang; Zhang, Chenglin; Li, Yanjun; Fan, Xiaoguang; Xie, Xixian; Chen, Ning

2017-06-01

Systems metabolic engineering is a multidisciplinary area that integrates systems biology, synthetic biology and evolutionary engineering. It is an efficient approach for strain improvement and process optimization, and has been successfully applied in the microbial production of various chemicals including amino acids. In this review, systems metabolic engineering strategies including pathway-focused approaches, systems biology-based approaches, evolutionary approaches and their applications in two major amino acid producing microorganisms: Corynebacterium glutamicum and Escherichia coli, are summarized.

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 § 866.3140...

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 § 866.3140...

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 § 866.3140...

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 § 866.3140...

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 § 866.3140...

Corynebacterium species causing breast abscesses among patients attending a tertiary care hospital in Chennai, South India.

PubMed

Poojary, Indira; Kurian, Ann; V A, Jayalekshmi; Devapriya J, Debora; M A, Thirunarayan

2017-07-01

Corynebacterium species other than Corynebacterium diphtheriae were mostly considered contaminants in the past, but there are reports of their association with wide variety of human infections lately. In this study, we look into Corynebacterium species isolated from breast abscess patients and assess their antimicrobial susceptibility pattern and treatment outcomes. Pus samples from suspected breast abscess cases were examined from October 2014 to September 2015. Growth of Gram-positive bacilli morphologically resembling Corynebacterium species were identified by matrix-assisted laser desorption/ionization- time of flight mass spectrometry identifications generated by the Vitek MS system (bioMérieux, France) (MALDI-TOF Vitek MS system) and antimicrobial susceptibility was done. Corynebacterium species were isolated from 10 female breast abscess patients with median age of 36 years (range 25-59 years). Out of the 10 isolates four isolates were identified as C. kroppenstedtii; one isolate as C. striatum and five isolates were identified as C. amycolatum/C.xerosis. Out of four isolates of C .kroppenstedtii, two isolates were resistant to cotrimoxazole and one C. striatum isolate was resistant to penicillin, ampicillin, cotrimoxazole and clindamycin. Of the five isolates identified as C amycolatum/C xerosis, all were sensitive to vancomycin and linezolid but resistant to clindamycin. All the patients were treated with incision, drainage and antibiotics based on the sensitivity pattern; eight were cured and two patients did not come for follow-up. Corynebacterium species should be considered one of the causative agents of breast abscess and a varied susceptibility profile amongst the different species makes susceptibility testing important. Identification by MALDI-TOF Vitek MS system may not differentiate between C. amycolatum and C. xerosis.

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.

Fatal case of bacteremia caused by an atypical strain of Corynebacterium mucifaciens.

PubMed

Cantarelli, Vlademir Vicente; Brodt, Teresa Cristina Z; Secchi, Carina; Inamine, Everton; Pereira, Fabiana de Souza; Pilger, Diogo Andre

2006-12-01

Corynebacterium species have often been considered normal skin flora or contaminants; however, in recent years they have been increasingly implicated in serious infections. Moreover, many new species have been discovered and old species renamed, especially after molecular biology techniques were introduced. Corynebacterium mucifaciens is mainly isolated from blood and from other normally-sterile body fluids; it forms slightly yellow, mucoid colonies on blood agar. We report a fatal case of bacteremia due to an atypical strain of C. mucifaciens. This strain had atypical colony morphology; analysis of the 16S rRNA gene was used to define the species.

Genomic diversity among Corynebacterium jeikeium strains and comparison with biochemical characteristics and antimicrobial susceptibilities.

PubMed Central

Riegel, P; de Briel, D; Prévost, G; Jehl, F; Monteil, H

1994-01-01

Levels of DNA relatedness were determined by performing DNA-DNA hybridization experiments (S1 nuclease procedure) with 13 human isolates exhibiting various antimicrobial susceptibility patterns which had been identified as Corynebacterium jeikeium by classical tests and the API Coryne system and with reference strains of C. jeikeium and related taxa. Twelve of 13 isolates which formed three genomic groups showed between 22 and 75% relatedness with the type strain of C. jeikeium. One of these genomic groups included all the strains resistant to penicillin and gentamicin and is genomically related to the C. jeikeium type strain at the species level. In addition, the reference strain of "Corynebacterium genitalium" biotype II was found to belong to this genospecies and therefore can be considered as a synonym of C. jeikeium. In contrast, one isolate and the reference strains of "Corynebacterium pseudogenitalium" biotypes C-3 and C-4 which were assigned to C. jeikeium by the API Coryne system were less than 10% related to the C. jeikeium type strain. These nongenomically related strains can be differentiated from the jeikeium-related strains on the basis of positive acidification from fructose and growth under anaerobic conditions. Furthermore, these strains exhibited full susceptibility to penicillin whereas the strains related to the C. jeikeium type strain are resistant to or only moderately susceptible to penicillin. No genomic relationship was found between C. jeikeium-related strains and other lipophilic coryneforms, identified as Corynebacterium accolens or Corynebacterium group G or F. Our study demonstrates the necessity to perform the fructose fermentation test or respiratory-type test for the correct identification of lipophilic coryneforms as C. jeikeium. Although these strains show genomic diversity at the species level, in a practical aspect, biochemical properties as well as antimicrobial susceptibility may allow the classification of such isolates in

Genomic diversity among Corynebacterium jeikeium strains and comparison with biochemical characteristics and antimicrobial susceptibilities.

PubMed

Riegel, P; de Briel, D; Prévost, G; Jehl, F; Monteil, H

1994-08-01

Levels of DNA relatedness were determined by performing DNA-DNA hybridization experiments (S1 nuclease procedure) with 13 human isolates exhibiting various antimicrobial susceptibility patterns which had been identified as Corynebacterium jeikeium by classical tests and the API Coryne system and with reference strains of C. jeikeium and related taxa. Twelve of 13 isolates which formed three genomic groups showed between 22 and 75% relatedness with the type strain of C. jeikeium. One of these genomic groups included all the strains resistant to penicillin and gentamicin and is genomically related to the C. jeikeium type strain at the species level. In addition, the reference strain of "Corynebacterium genitalium" biotype II was found to belong to this genospecies and therefore can be considered as a synonym of C. jeikeium. In contrast, one isolate and the reference strains of "Corynebacterium pseudogenitalium" biotypes C-3 and C-4 which were assigned to C. jeikeium by the API Coryne system were less than 10% related to the C. jeikeium type strain. These nongenomically related strains can be differentiated from the jeikeium-related strains on the basis of positive acidification from fructose and growth under anaerobic conditions. Furthermore, these strains exhibited full susceptibility to penicillin whereas the strains related to the C. jeikeium type strain are resistant to or only moderately susceptible to penicillin. No genomic relationship was found between C. jeikeium-related strains and other lipophilic coryneforms, identified as Corynebacterium accolens or Corynebacterium group G or F. Our study demonstrates the necessity to perform the fructose fermentation test or respiratory-type test for the correct identification of lipophilic coryneforms as C. jeikeium. Although these strains show genomic diversity at the species level, in a practical aspect, biochemical properties as well as antimicrobial susceptibility may allow the classification of such isolates in

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.

Characteristics of Rare or Recently Described Corynebacterium Species Recovered from Human Clinical Material in Canada

PubMed Central

Bernard, K. A.; Munro, C.; Wiebe, D.; Ongsansoy, E.

2002-01-01

Nineteen new Corynebacterium species or taxa described since 1995 have been associated with human disease. We report the characteristics of 72 strains identified as or most closely resembling 14 of these newer, medically relevant Corynebacterium species or taxa, as well as describe in brief an isolate of Corynebacterium bovis, a rare pathogen for humans. The bacteria studied in this report were nearly all derived from human clinical specimens and were identified by a polyphasic approach. Most were characterized by nearly full 16S rRNA gene sequence analysis. Some isolates were recovered from previously unreported sources and exhibited unusual phenotypes or represented the first isolates found outside Europe. Products of fermentation, with emphasis on the presence or absence of propionic acid, were also studied in order to provide an additional characteristic with which to differentiate among phenotypically similar species. PMID:12409436

l-lysine production by Bacillus methanolicus: Genome-based mutational analysis and l-lysine secretion engineering.

PubMed

Nærdal, Ingemar; Netzer, Roman; Irla, Marta; Krog, Anne; Heggeset, Tonje Marita Bjerkan; Wendisch, Volker F; Brautaset, Trygve

2017-02-20

Bacillus methanolicus is a methylotrophic bacterium with an increasing interest in academic research and for biotechnological applications. This bacterium was previously applied for methanol-based production of l-glutamate, l-lysine and the five-carbon diamine cadaverine by wild type, classical mutant and recombinant strains. The genomes of two different l-lysine secreting B. methanolicus classical mutant strains, NOA2#13A52-8A66 and M168-20, were sequenced. We focused on mutational mapping in genes present in l-lysine and other relevant amino acid biosynthetic pathways, as well as in the primary cell metabolism important for precursor supply. In addition to mutations in the aspartate pathway genes dapG, lysA and hom-1, new mutational target genes like alr, proA, proB1, leuC, odhA and pdhD were identified. Surprisingly, no mutations were found in the putative l-lysine transporter gene lysE MGA3 . Inspection of the wild type B. methanolicus strain PB1 genome sequence identified two homologous putative l-lysine transporter genes, lysE PB1 and lysE2 PB1 . The biological role of these putative l-lysine transporter genes, together with the heterologous l-lysine exporter gene lysE Cg from Corynebacterium glutamicum, were therefore investigated. Our results demonstrated that the titer of secreted l-lysine in B. methanolicus was significantly increased by overexpression of lysE Cg while overexpression of lysE MGA3 , lysE PB1 and lysE2 PB1 had no measurable effect. Copyright © 2017 Elsevier B.V. All rights reserved.

Turicella otitidis and Corynebacterium auris: 20 years on.

PubMed

von Graevenitz, A; Funke, G

2014-02-01

Turicella otitidis and Corynebacterium auris, described as new species 20 years ago, have been isolated mainly from the external ear canal and middle ear fluid. While their taxonomic position has been clearly established, their diagnosis in the routine laboratory is difficult. The question of their pathogenic potential in otitis is still open but might be elucidated better if corynebacteria are speciated more often.

Secrets of soil survival revealed by the genome sequence of Arthrobacter aurescens TC1.

PubMed

Mongodin, Emmanuel F; Shapir, Nir; Daugherty, Sean C; DeBoy, Robert T; Emerson, Joanne B; Shvartzbeyn, Alla; Radune, Diana; Vamathevan, Jessica; Riggs, Florenta; Grinberg, Viktoria; Khouri, Hoda; Wackett, Lawrence P; Nelson, Karen E; Sadowsky, Michael J

2006-12-01

Arthrobacter sp. strains are among the most frequently isolated, indigenous, aerobic bacterial genera found in soils. Member of the genus are metabolically and ecologically diverse and have the ability to survive in environmentally harsh conditions for extended periods of time. The genome of Arthrobacter aurescens strain TC1, which was originally isolated from soil at an atrazine spill site, is composed of a single 4,597,686 basepair (bp) circular chromosome and two circular plasmids, pTC1 and pTC2, which are 408,237 bp and 300,725 bp, respectively. Over 66% of the 4,702 open reading frames (ORFs) present in the TC1 genome could be assigned a putative function, and 13.2% (623 genes) appear to be unique to this bacterium, suggesting niche specialization. The genome of TC1 is most similar to that of Tropheryma, Leifsonia, Streptomyces, and Corynebacterium glutamicum, and analyses suggest that A. aurescens TC1 has expanded its metabolic abilities by relying on the duplication of catabolic genes and by funneling metabolic intermediates generated by plasmid-borne genes to chromosomally encoded pathways. The data presented here suggest that Arthrobacter's environmental prevalence may be due to its ability to survive under stressful conditions induced by starvation, ionizing radiation, oxygen radicals, and toxic chemicals.

Single Upconversion Nanoparticle-Bacterium Cotrapping for Single-Bacterium Labeling and Analysis.

PubMed

Xin, Hongbao; Li, Yuchao; Xu, Dekang; Zhang, Yueli; Chen, Chia-Hung; Li, Baojun

2017-04-01

Detecting and analyzing pathogenic bacteria in an effective and reliable manner is crucial for the diagnosis of acute bacterial infection and initial antibiotic therapy. However, the precise labeling and analysis of bacteria at the single-bacterium level are a technical challenge but very important to reveal important details about the heterogeneity of cells and responds to environment. This study demonstrates an optical strategy for single-bacterium labeling and analysis by the cotrapping of single upconversion nanoparticles (UCNPs) and bacteria together. A single UCNP with an average size of ≈120 nm is first optically trapped. Both ends of a single bacterium are then trapped and labeled with single UCNPs emitting green light. The labeled bacterium can be flexibly moved to designated locations for further analysis. Signals from bacteria of different sizes are detected in real time for single-bacterium analysis. This cotrapping method provides a new approach for single-pathogenic-bacterium labeling, detection, and real-time analysis at the single-particle and single-bacterium level. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Experimental transmission of Corynebacterium pseudotuberculosis in horses by house flies

USDA-ARS?s Scientific Manuscript database

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...

Identification of low-molecular-weight compounds inhibiting growth of corynebacteria: potential lead compounds for antibiotics.

PubMed

Stark, Jaime L; Copeland, Jennifer C; Eletsky, Alexander; Somerville, Greg A; Szyperski, Thomas; Powers, Robert

2014-02-01

The bacterial genus Corynebacteria contains several pathogenic species that cause diseases such as diphtheria in humans and "cheesy gland" in goats and sheep. Thus, identifying new therapeutic targets to treat Corynebacteria infections is both medically and economically important. CG2496, a functionally uncharacterized protein from Corynebacterium glutamicum, was evaluated using an NMR ligand-affinity screen. A total of 11 compounds from a library of 460 biologically active compounds were shown to selectively bind CG2496 in a highly conserved region of the protein. The best binder was identified to be methiothepin (KD =54 ± 19 µM), an FDA-approved serotonin receptor antagonist. Methiothepin was also shown to inhibit the growth of C. glutamicum, but not bacteria that lack CG2496 homologs. Our results suggest that CG2496 is a novel therapeutic target and methiothepin is a potential lead compound or structural scaffold for developing new antibiotics specifically targeting Corynebacteria. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis, structural elucidation, and biochemical analysis of immunoactive glucuronosyl diacylglycerides of mycobacteria and corynebacteria.

PubMed

Cao, Benjamin; Chen, Xingqiang; Yamaryo-Botte, Yoshiki; Richardson, Mark B; Martin, Kirstee L; Khairallah, George N; Rupasinghe, Thusita W T; O'Flaherty, Roisin M; O'Hair, Richard A J; Ralton, Julie E; Crellin, Paul K; Coppel, Ross L; McConville, Malcolm J; Williams, Spencer J

2013-03-15

Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop "rules" allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg(2+)/Mn(2+)-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.

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

Metabolic engineering of industrial platform microorganisms for biorefinery applications--optimization of substrate spectrum and process robustness by rational and evolutive strategies.

PubMed

Buschke, Nele; Schäfer, Rudolf; Becker, Judith; Wittmann, Christoph

2013-05-01

Bio-based production promises a sustainable route to myriads of chemicals, materials and fuels. With regard to eco-efficiency, its future success strongly depends on a next level of bio-processes using raw materials beyond glucose. Such renewables, i.e., polymers, complex substrate mixtures and diluted waste streams, often cannot be metabolized naturally by the producing organisms. This particularly holds for well-known microorganisms from the traditional sugar-based biotechnology, including Escherichia coli, Corynebacterium glutamicum and Saccharomyces cerevisiae which have been engineered successfully to produce a broad range of products from glucose. In order to make full use of their production potential within the bio-refinery value chain, they have to be adapted to various feed-stocks of interest. This review focuses on the strategies to be applied for this purpose which combine rational and evolutive approaches. Hereby, the three industrial platform microorganisms, E. coli, C. glutamicum and S. cerevisiae are highlighted due to their particular importance. Copyright © 2012 Elsevier Ltd. All rights reserved.

Application of metabolic engineering for the biotechnological production of L-valine.

PubMed

Oldiges, Marco; Eikmanns, Bernhard J; Blombach, Bastian

2014-07-01

The branched chain amino acid L-valine is an essential nutrient for higher organisms, such as animals and humans. Besides the pharmaceutical application in parenteral nutrition and as synthon for the chemical synthesis of e.g. herbicides or anti-viral drugs, L-valine is now emerging into the feed market, and significant increase of sales and world production is expected. In accordance, well-known microbial production bacteria, such as Escherichia coli and Corynebacterium glutamicum strains, have recently been metabolically engineered for efficient L-valine production under aerobic or anaerobic conditions, and the respective cultivation and production conditions have been optimized. This review summarizes the state of the art in L-valine biosynthesis and its regulation in E. coli and C. glutamicum with respect to optimal metabolic network for microbial L-valine production, genetic strain engineering and bioprocess development for L-valine production, and finally, it will shed light on emerging technologies that have the potential to accelerate strain and bioprocess engineering in the near future.

Advanced biotechnology: metabolically engineered cells for the bio-based production of chemicals and fuels, materials, and health-care products.

PubMed

Becker, Judith; Wittmann, Christoph

2015-03-09

Corynebacterium glutamicum, Escherichia coli, and Saccharomyces cerevisiae in particular, have become established as important industrial workhorses in biotechnology. Recent years have seen tremendous progress in their advance into tailor-made producers, driven by the upcoming demand for sustainable processes and renewable raw materials. Here, the diversity and complexity of nature is simultaneously a challenge and a benefit. Harnessing biodiversity in the right manner through synergistic progress in systems metabolic engineering and chemical synthesis promises a future innovative bio-economy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nosocomial endocarditis caused by Corynebacterium amycolatum and other nondiphtheriae corynebacteria.

PubMed

Knox, Karen L; Holmes, Alison H

2002-01-01

The nondiphtheriae corynebacteria are uncommon but increasingly recognized as agents of endocarditis in patients with underlying structural heart disease or prosthetic-valves. We describe three cases of nosocomial endocarditis caused by nondiphtheriae corynebacteria, including the first reported case of Corynebacterium amycolatum, endocarditis. These all occurred in association with indwelling intravascular devices.

Experimental inoculation of house flies Musca domestica with Corynebacterium pseudotuberculosis serovar equi

USDA-ARS?s Scientific Manuscript database

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...

[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. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

From Corynebacterium glutamicum to Mycobacterium tuberculosis—towards transfers of gene regulatory networks and integrated data analyses with MycoRegNet

PubMed Central

Krawczyk, Justina; Kohl, Thomas A.; Goesmann, Alexander; Kalinowski, Jörn; Baumbach, Jan

2009-01-01

Year by year, approximately two million people die from tuberculosis, a disease caused by the bacterium Mycobacterium tuberculosis. There is a tremendous need for new anti-tuberculosis therapies (antituberculotica) and drugs to cope with the spread of tuberculosis. Despite many efforts to obtain a better understanding of M. tuberculosis' pathogenicity and its survival strategy in humans, many questions are still unresolved. Among other cellular processes in bacteria, pathogenicity is controlled by transcriptional regulation. Thus, various studies on M. tuberculosis concentrate on the analysis of transcriptional regulation in order to gain new insights on pathogenicity and other essential processes ensuring mycobacterial survival. We designed a bioinformatics pipeline for the reliable transfer of gene regulations between taxonomically closely related organisms that incorporates (i) a prediction of orthologous genes and (ii) the prediction of transcription factor binding sites. In total, 460 regulatory interactions were identified for M. tuberculosis using our comparative approach. Based on that, we designed a publicly available platform that aims to data integration, analysis, visualization and finally the reconstruction of mycobacterial transcriptional gene regulatory networks: MycoRegNet. It is a comprehensive database system and analysis platform that offers several methods for data exploration and the generation of novel hypotheses. MycoRegNet is publicly available at http://mycoregnet.cebitec.uni-bielefeld.de. PMID:19494184

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

Experimental transmission of Corynebacterium pseudotuberculosis biovar equi in horses by house flies

USDA-ARS?s Scientific Manuscript database

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...

Experimental inoculation of house flies, Musca domestica L., with Corynebacterium pseudotuberculosis serovar equi

USDA-ARS?s Scientific Manuscript database

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...

Oligonucleotide recombination in corynebacteria without the expression of exogenous recombinases.

PubMed

Krylov, Alexander A; Kolontaevsky, Egor E; Mashko, Sergey V

2014-10-01

Brevibacterium lactofermentum and Corynebacterium glutamicum are important biotechnology species of the genus Corynebacterium. The single-strand DNA annealing protein (SSAP)-independent oligonucleotide-mediated recombination procedure was successfully applied to the commonly used wild-type strains B. lactofermentum AJ1511 and C. glutamicum ATCC13032. When the rpsL gene was used as a target, the optimized protocol yielded up to (1.4±0.3)×10(3) and (6.7±1.3)×10(3) streptomycin-resistant colonies per 10(8) viable cells for the corresponding strains. We tested the influence of several parameters that are known to enhance the efficiency of oligonucleotide-mediated recombination in other bacterial species. Among them, increasing the concentration of oligonucleotides and targeting the lagging strand of the chromosome have proven to have positive effects on both of the tested species. No difference in the efficiency of recombination was observed between the oligonucleotides phosphorothiorated at the 5' ends and the unmodified oligonucleotides or between the oligonucleotides with four mutated nucleotides and those with one mutated nucleotide. The described approach demonstrates that during the adaptation of the recombineering technique, testing SSAP-independent oligonucleotide-mediated recombination could be a good starting point. Such testing could decrease the probability of an incorrect interpretation of the effect of exogenous protein factors (such as SSAP and/or corresponding exonucleases) due to non-optimal experimental conditions. In addition, SSAP-independent recombination itself could be useful in combination with suitable selection/enrichment methods. Copyright © 2014 Elsevier B.V. All rights reserved.

IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria.

PubMed

Baumgart, Meike; Luder, Kerstin; Grover, Shipra; Gätgens, Cornelia; Besra, Gurdyal S; Frunzke, Julia

2013-12-30

The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown. Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An ipsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Further studies revealed the absence of inositol-derived lipids in the cell wall and a complete loss of mycothiol biosynthesis. The phenotype of the C. glutamicum ipsA deletion mutant was complemented to different extend by homologs from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. Myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development.

Corynebacterium species nasopharyngeal carriage in asymptomatic individuals aged ≥ 65 years in Germany.

PubMed

Teutsch, Barbara; Berger, Anja; Marosevic, Durdica; Schönberger, Katharina; Lâm, Thiên-Trí; Hubert, Kerstin; Beer, Steffi; Wienert, Peter; Ackermann, Nikolaus; Claus, Heike; Drayß, Maria; Thiel, Kathrin; van der Linden, Mark; Vogel, Ulrich; Sing, Andreas

2017-10-01

The prevalence of protective anti-diphtheria toxin antibodies decreases with age. Therefore, the elderly might serve as reservoir for potentially toxigenic Corynebacterium (C.) species (C. diphtheriae, C. ulcerans, and C. pseudotuberculosis). This study aimed to examine the colonization rate of the nasopharynx with corynebacteria of individuals aged 65 years and older. In the period from October 2012 to June 2013, nasal and throat swabs were taken from 714 asymptomatic subjects aged 65-106 years (average age 77.2) at three regions in Germany and investigated for Corynebacterium species. A total of 402 strains of Corynebacterium species were isolated from 388 out of 714 asymptomatic subjects (carriage rate 54.3%). The carriage rate was significantly higher in study participants living in retirement homes (68.4%) compared to those living autonomously at home (51.1%). Strains were isolated mostly from the nose (99%). Corynebacterium accolens was the most often isolated species (39.8%), followed by C. propinquum (24.1%), C. pseudodiphtheriticum (19.4%), and C. tuberculostearicum (10.2%). No C. diphtheriae, C. ulcerans, and C. pseudotuberculosis strains were isolated. A subsample of 74 subjects was tested serologically for anti-diphtheria antibodies. Protective anti-diphtheria toxin antibodies were found in 29.7% of the subjects; 70.3% showed no protective immunity. These results suggest that carriage of potentially toxigenic corynebacteria is very rare among people aged 65 and older in Germany. However, the low prevalence of protective anti-diphtheria toxin antibodies might pose a risk for acquiring diphtheria especially for the elderly.

Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS.

PubMed

Peifer, Susanne; Schneider, Konstantin; Nürenberg, Gudrun; Volmer, Dietrich A; Heinzle, Elmar

2012-11-01

Intermediates of the purine biosynthesis pathway play key roles in cellular metabolism including nucleic acid synthesis and signal mediation. In addition, they are also of major interest to the biotechnological industry as several intermediates either possess flavor-enhancing characteristics or are applied in medical therapy. In this study, we have developed an analytical method for quantitation of 12 intermediates from the purine biosynthesis pathway including important nucleotides and their corresponding nucleosides and nucleobases. The approach comprised a single-step acidic extraction/quenching procedure, followed by quantitative electrospray LC-MS/MS analysis. The assay was validated in terms of accuracy, precision, reproducibility, and applicability for complex biological matrices. The method was subsequently applied for determination of free intracellular pool sizes of purine biosynthetic pathway intermediates in the two Gram-positive bacteria Corynebacterium glutamicum and Corynebacterium ammoniagenes. Importantly, no ion pair reagents were applied in this approach as usually required for liquid chromatography analysis of large classes of diverse metabolites.

Nosocomial Endocarditis Caused by Corynebacterium amycolatum and Other Nondiphtheriae Corynebacteria

PubMed Central

Holmes, Alison H.

2002-01-01

The nondiphtheriae corynebacteria are uncommon but increasingly recognized as important agents of community-acquired endocarditis in patients with underlying structural heart disease, as well as of prosthetic-valve endocarditis. We describe three cases of nondiphtheriae corynebacterial endocarditis, including the first reported case of endocarditis caused by Corynebacterium amycolatum, occurring over an 18-month period, all in association with indwelling intravascular devices. PMID:11749760

Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane.

PubMed

Carel, Clément; Marcoux, Julien; Réat, Valérie; Parra, Julien; Latgé, Guillaume; Laval, Françoise; Demange, Pascal; Burlet-Schiltz, Odile; Milon, Alain; Daffé, Mamadou; Tropis, Maryelle G; Renault, Marie A M

2017-04-18

The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum , a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum , we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O -mycoloylation, pyroglutamylation, and N -formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O -acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.

Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane

PubMed Central

Carel, Clément; Réat, Valérie; Parra, Julien; Latgé, Guillaume; Laval, Françoise; Burlet-Schiltz, Odile; Milon, Alain; Daffé, Mamadou; Tropis, Maryelle G.; Renault, Marie A. M.

2017-01-01

The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment. PMID:28373551

Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains

PubMed Central

Nærdal, Ingemar; Pfeifenschneider, Johannes; Brautaset, Trygve; Wendisch, Volker F

2015-01-01

Methanol is regarded as an attractive substrate for biotechnological production of value-added bulk products, such as amino acids and polyamines. In the present study, the methylotrophic and thermophilic bacterium Bacillus methanolicus was engineered into a microbial cell factory for the production of the platform chemical 1,5-diaminopentane (cadaverine) from methanol. This was achieved by the heterologous expression of the Escherichia coli genes cadA and ldcC encoding two different lysine decarboxylase enzymes, and by increasing the overall L-lysine production levels in this host. Both CadA and LdcC were functional in B. methanolicus cultivated at 50°C and expression of cadA resulted in cadaverine production levels up to 500 mg l−1 during shake flask conditions. A volume-corrected concentration of 11.3 g l−1 of cadaverine was obtained by high-cell density fed-batch methanol fermentation. Our results demonstrated that efficient conversion of L-lysine into cadaverine presumably has severe effects on feedback regulation of the L-lysine biosynthetic pathway in B. methanolicus. By also investigating the cadaverine tolerance level, B. methanolicus proved to be an exciting alternative host and comparable to the well-known bacterial hosts E. coli and Corynebacterium glutamicum. This study represents the first demonstration of microbial production of cadaverine from methanol. PMID:25644214

[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. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Complete nucleotide sequence and annotation of the temperate corynephage ϕ16 genome.

PubMed

Lobanova, Juliya S; Gak, Evgueni R; Andreeva, Irina G; Rybak, Konstantin V; Krylov, Alexander A; Mashko, Sergey V

2017-08-01

The complete genome of ϕ16, a temperate corynephage from Corynebacterium glutamicum ATCC 21792, was sequenced and annotated (GenBank: KY250482). The electron microscopy study of ϕ16 virion confirmed that it belongs to the family Siphoviridae. The ϕ16 genome consists of a linear double-stranded DNA molecule of 58,200 bp (G+C = 52.2%) with protruding cohesive 3'-ends of 14 nt. Four major structural proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting technique. Using bioinformatics analysis, 101 putative ORFs and 5 tRNA genes were predicted. Only 27 putative gene products could be assigned to known biological functions. The ϕ16 genome was divided into functional modules. Seven putative promoters and eight putative unidirectional intrinsic terminators were predicted. One site of putative «-1» programmed ribosomal frameshifting was proposed in the phage tail assembly genome region. C. glutamicum genetic tools could be broadened by exploiting the known integrase gene (gp33) and the newly identified excisionase gene (gp47), participating in site-specific recombination between ϕ16-attP/attB.

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

Use of Amplified Fragment Length Polymorphisms for Typing Corynebacterium diphtheriae

PubMed Central

De Zoysa, Aruni; Efstratiou, Androulla

2000-01-01

Amplified fragment length polymorphism (AFLP) was investigated for the differentiation of Corynebacterium diphtheriae isolates. Analysis using Taxotron revealed 10 distinct AFLP profiles among 57 isolates. Strains with ribotype patterns D1, D4, and D12 could not be distinguished; however, the technique discriminated isolates of ribotype patterns D3, D6, and D7 further. AFLP was rapid, fairly inexpensive, and reproducible and could be used as an alternative to ribotyping. PMID:11015416

IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria

PubMed Central

2013-01-01

Background The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown. Results Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An ipsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Further studies revealed the absence of inositol-derived lipids in the cell wall and a complete loss of mycothiol biosynthesis. The phenotype of the C. glutamicum ipsA deletion mutant was complemented to different extend by homologs from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. Myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. Conclusions This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development. PMID:24377418

Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii.

PubMed

Luong, Truc Thanh; Tirgar, Reyhaneh; Reardon-Robinson, Melissa E; Joachimiak, Andrzej; Osipiuk, Jerzy; Ton-That, Hung

2018-05-01

The actinobacterium Corynebacterium matruchotii has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the C. matruchotii genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA (MdbA Cd ). Crystallization studies uncovered that the 1.2-Å resolution structure of C. matruchotii MdbA (MdbA Cm ) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machine in vitro , we demonstrated that MdbA Cm catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that mdbA is essential in C. matruchotii Remarkably, heterologous expression of MdbA Cm in the C. diphtheriae Δ mdbA mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbA Cm is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in C. matruchotii by a mechanism that is conserved in Actinobacteria IMPORTANCE The actinobacterium Corynebacterium matruchotii has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a C. matruchotii oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that C. matruchotii MdbA catalyzes disulfide

A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level.

PubMed

Grünberger, Alexander; Paczia, Nicole; Probst, Christopher; Schendzielorz, Georg; Eggeling, Lothar; Noack, Stephan; Wiechert, Wolfgang; Kohlheyer, Dietrich

2012-05-08

In the continuously growing field of industrial biotechnology the scale-up from lab to industrial scale is still a major hurdle to develop competitive bioprocesses. During scale-up the productivity of single cells might be affected by bioreactor inhomogeneity and population heterogeneity. Currently, these complex interactions are difficult to investigate. In this report, design, fabrication and operation of a disposable picolitre cultivation system is described, in which environmental conditions can be well controlled on a short time scale and bacterial microcolony growth experiments can be observed by time-lapse microscopy. Three exemplary investigations will be discussed emphasizing the applicability and versatility of the device. Growth and analysis of industrially relevant bacteria with single cell resolution (in particular Escherichia coli and Corynebacterium glutamicum) starting from one single mother cell to densely packed cultures is demonstrated. Applying the picolitre bioreactor, 1.5-fold increased growth rates of C. glutamicum wild type cells were observed compared to typical 1 litre lab-scale batch cultivation. Moreover, the device was used to analyse and quantify the morphological changes of an industrially relevant l-lysine producer C. glutamicum after artificially inducing starvation conditions. Instead of a one week lab-scale experiment, only 1 h was sufficient to reveal the same information. Furthermore, time lapse microscopy during 24 h picolitre cultivation of an arginine producing strain containing a genetically encoded fluorescence sensor disclosed time dependent single cell productivity and growth, which was not possible with conventional methods.

Relationship between group JK corynebacteria and the biotypes of Corynebacterium genitalium and Corynebacterium pseudogenitalium.

PubMed

Evangelista, A T; Coppola, K M; Furness, G

1984-08-01

Twenty-six strains of group JK corynebacteria had the same colonial morphology and biological reactions as the biotypes of the biovars of Corynebacterium genitalium and C. pseudogenitalium. Therefore, group JK corynebacteria can be assigned to the biovars of C. genitalium or C. pseudogenitalium. Although the strains differed in sensitivity to 16 antibiotics tested by Sensi-Discs or by the Micro-Media technique, they are uniformly sensitive to 4-5 micrograms/mL of vancomycin. Medium containing 10 micrograms vancomycin/mL was bactericidal and the killing time was dependent on the concentration. The rate of mutation to resistance to 10 micrograms vancomycin was greater than 1 in 10(10) corynebacteria. Therefore, vancomycin sensitivity is a stable characteristic of these corynebacteria which also indicates that group JK corynebacteria are strains of either C. genitalium or C. pseudogenitalium. Since group JK corynebacteria are considered pathogens, this finding supports the belief that C. genitalium is a pathogen and suggests that some biotypes of the commensal C. pseudogenitalium may infect compromised hosts.

The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin.

PubMed

Küberl, Andreas; Polen, Tino; Bott, Michael

2016-04-26

The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation.

The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin

PubMed Central

Küberl, Andreas; Polen, Tino; Bott, Michael

2016-01-01

The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation. PMID:27078093

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

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

Endocarditis of native aortic and mitral valves due to Corynebacterium accolens: report of a case and application of phenotypic and genotypic techniques for identification.

PubMed

Claeys, G; Vanhouteghem, H; Riegel, P; Wauters, G; Hamerlynck, R; Dierick, J; de Witte, J; Verschraegen, G; Vaneechoutte, M

1996-05-01

Endocarditis of native aortic and mitral valves due to an organism identified as Corynebacterium accolens developed in a 73-year-old patient without predisposing factors. The organism was identified as C. accolens by biochemical identification, amplified rRNA gene restriction analysis, and DNA-DNA hybridization. This is the first case of C. accolens endocarditis reported, adding to the increasing number of Corynebacterium-related cases of endocarditis.

Detection of mycoloylglycerol by thin-layer chromatography as a tool for the rapid inclusion of corynebacteria of clinical origin in the genus Corynebacterium.

PubMed

Yagüe, G; Segovia, M; Valero-Guillén, P L

2000-01-28

A chemotaxonomic study of some corynebacteria isolated from clinical samples revealed characteristic thin-layer chromatographic patterns for meso-diaminopimelic acid containing species included in the genera Corynebacterium, Dermabacter and Brevibacterium. Notably, a specific compound was consistently detected in mycolic acid containing species of the genus Corynebacterium. This compound was composed by glycerol and mycolic acids and structural analyses carried out by fast atom bombardment mass spectrometry in C. minutissimum confirmed its identification as mycoloylglycerol. The chain length of mycoloyl groups in this molecule ranged from 28 to 34 carbon atoms, being mono-, di- or triunsaturated. Detection of mycoloylglycerol by thin-layer chromatography may be thus useful for the rapid inclusion of a great variety of corynebacteria of clinical origin in the genus Corynebacterium in laboratories employing chromatographic techniques as an adjunct for the identification of these microorganisms.

Identification of EayjjPB encoding a dicarboxylate transporter important for succinate production under aerobic and anaerobic conditions in Enterobacter aerogenes.

PubMed

Fukui, Keita; Nanatani, Kei; Hara, Yoshihiko; Tokura, Mitsunori; Abe, Keietsu

2018-05-01

Enterobacter aerogenes, a gram-negative, rod-shaped bacterium, is an effective producer of succinate from glucose via the reductive tricarboxylic acid cycle under anaerobic conditions. However, to date, succinate-exporter genes have not been identified in E. aerogenes, although succinate exporters have a large impact on fermentative succinate production. Recently, we genetically identified yjjP and yjjB, as genes encoding a succinate transporter in Escherichia coli. Evaluation of the yjjPB homologs in E. aerogenes (EayjjPB genes) showed that succinate accumulation increased from 4.1 g L -1 to 9.1 g L -1 when the EayjjPB genes were expressed under aerobic conditions. Under anaerobic conditions, succinate yield increased from 53% to 60% by EayjjPB expression and decreased to 48% by deletion of EayjjPB. Furthermore, the production levels of fumarate and malate, which are intermediates of the succinate-biosynthesis pathway, were also increased by EayjjPB expression. A complementation assay conducted in Corynebacterium glutamicum strain AJ110655ΔsucE1 demonstrated that both EaYjjP and EaYjjB are required for the restoration of succinate production. Taken together, these results suggest that EaYjjPB function as a dicarboxylate transporter in E. aerogenes and that the products of both genes are required for dicarboxylate transport. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains.

PubMed

Naerdal, Ingemar; Pfeifenschneider, Johannes; Brautaset, Trygve; Wendisch, Volker F

2015-03-01

Methanol is regarded as an attractive substrate for biotechnological production of value-added bulk products, such as amino acids and polyamines. In the present study, the methylotrophic and thermophilic bacterium Bacillus methanolicus was engineered into a microbial cell factory for the production of the platform chemical 1,5-diaminopentane (cadaverine) from methanol. This was achieved by the heterologous expression of the Escherichia coli genes cadA and ldcC encoding two different lysine decarboxylase enzymes, and by increasing the overall L-lysine production levels in this host. Both CadA and LdcC were functional in B. methanolicus cultivated at 50°C and expression of cadA resulted in cadaverine production levels up to 500 mg l(-1) during shake flask conditions. A volume-corrected concentration of 11.3 g l(-1) of cadaverine was obtained by high-cell density fed-batch methanol fermentation. Our results demonstrated that efficient conversion of L-lysine into cadaverine presumably has severe effects on feedback regulation of the L-lysine biosynthetic pathway in B. methanolicus. By also investigating the cadaverine tolerance level, B. methanolicus proved to be an exciting alternative host and comparable to the well-known bacterial hosts E. coli and Corynebacterium glutamicum. This study represents the first demonstration of microbial production of cadaverine from methanol. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

In Vitro Analysis of d-Lactyl-CoA-Polymerizing Polyhydroxyalkanoate Synthase in Polylactate and Poly(lactate- co-3-hydroxybutyrate) Syntheses.

PubMed

Matsumoto, Ken'ichiro; Iijima, Midori; Hori, Chiaki; Utsunomia, Camila; Ooi, Toshihiko; Taguchi, Seiichi

2018-05-15

Engineered d-lactyl-coenzyme A (LA-CoA)-polymerizing polyhydroxyalkanoate synthase (PhaC1 Ps STQK) efficiently produces poly(lactate- co-3-hydroxybutyrate) [P(LA- co-3HB]) copolymer in recombinant Escherichia coli, while synthesizing tiny amounts of poly(lactate) (PLA)-like polymers in recombinant Corynebacterium glutamicum. To elucidate the mechanisms underlying the interesting phenomena, in vitro analysis of PhaC1 Ps STQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaC1 Ps STQK polymerized LA-CoA as a sole substrate. However, the extension of PLA chains completely stalled at a molecular weight of ∼3000, presumably due to the low mobility of the generated polymer. The copolymerization of these substrates only proceeded with a low concentration of LA-CoA. In fact, the intracellular LA-CoA concentration in P(LA- co-3HB)-producing E. coli was below the detection limit, while that in C. glutamicum was as high as acetyl-CoA levels. Therefore, it was concluded that the mobility of polymerized products and LA-CoA concentration are dominant factors characterizing PLA and P(LA- co-3HB) biosynthetic systems.

Biotechnological production of polyamines by bacteria: recent achievements and future perspectives.

PubMed

Schneider, Jens; Wendisch, Volker F

2011-07-01

In Bacteria, the pathways of polyamine biosynthesis start with the amino acids L-lysine, L-ornithine, L-arginine, or L-aspartic acid. Some of these polyamines are of special interest due to their use in the production of engineering plastics (e.g., polyamides) or as curing agents in polymer applications. At present, the polyamines for industrial use are mainly synthesized on chemical routes. However, since a commercial market for polyamines as well as an industry for the fermentative production of amino acid exist, and since bacterial strains overproducing the polyamine precursors L-lysine, L-ornithine, and L-arginine are known, it was envisioned to engineer these amino acid-producing strains for polyamine production. Only recently, researchers have investigated the potential of amino acid-producing strains of Corynebacterium glutamicum and Escherichia coli for polyamine production. This mini-review illustrates the current knowledge of polyamine metabolism in Bacteria, including anabolism, catabolism, uptake, and excretion. The recent advances in engineering the industrial model bacteria C. glutamicum and E. coli for efficient production of the most promising polyamines, putrescine (1,4-diaminobutane), and cadaverine (1,5-diaminopentane), are discussed in more detail.

Endocarditis of native aortic and mitral valves due to Corynebacterium accolens: report of a case and application of phenotypic and genotypic techniques for identification.

PubMed Central

Claeys, G; Vanhouteghem, H; Riegel, P; Wauters, G; Hamerlynck, R; Dierick, J; de Witte, J; Verschraegen, G; Vaneechoutte, M

1996-01-01

Endocarditis of native aortic and mitral valves due to an organism identified as Corynebacterium accolens developed in a 73-year-old patient without predisposing factors. The organism was identified as C. accolens by biochemical identification, amplified rRNA gene restriction analysis, and DNA-DNA hybridization. This is the first case of C. accolens endocarditis reported, adding to the increasing number of Corynebacterium-related cases of endocarditis. PMID:8727922

Laccase catalysed grafting of phenolic onto xylan to improve its applicability in films

NASA Astrophysics Data System (ADS)

Pei, Jicheng; Wang, Bing; Zhang, Fangdong; Li, Zhongyang; Yin, Yunbei; Zhang, Dongxu

2015-07-01

Xylan can be tailored for various value-added applications. However, its use in aqueous systems is hampered by its complex structure, and small molecular weight. This research aimed at improving the xylan molecular weight and changing its structure. Laccase-catalysed oxidation of 4-coumaric acid (PCA), ferulic acid (FA), syringaldehyde (SD), and vanillin (VA) onto xylan was grafted to study the changes in its structure, tensile properties, and antibacterial activities. A Fourier transform infrared (FTIR) spectrum analyser was used to observe the changes in functional groups of xylan. The results showed a band at 1635 cm-1 corresponding to the stretching vibration of conjugated carbonyl carboxy hemoglobin and a benzene ring structure were strengthened; the appearance of a new band between 1200 cm-1 and 1270 cm-1 corresponding to alkyl ethers on the aryl C-O stretching vibration was due to the fact that during the grafting process, the number of benzene ring structures increased and covalent connections occurred between phenols and xylan. The reaction mechanism for the laccase-catalysed oxidation of phenol compounds onto xylan was preliminary explored by 13C-NMR. The results showed that PCA-xylan, FA-xylan graft poly onto xylan by Cγ ester bond, SD-xylan graft poly onto xylan by ether bond and an ester bond, and VD-xylan graft poly onto xylan by ether bond. The film strength of xylan derivatives has been significantly increased, especially for the PCA-xylan derivative. The increases in tensile stress at break, tensile strength, tensile yield stress, and Young's modulus were: 24.04%, 31.30%, 55.56%, and 28.21%, respectively. After laccase/phenolics were modified, xylan had a good antibacterial effect to E. coli, Corynebacterium glutamicum, and Bacillus subtilis. The SD-xylan, FA-xylan, and PCA-xylan showed a greater efficacy against E. coli, Corynebacterium glutamicum, and Bacillus subtilis, respectively.

Complete Closed Genome Sequence of Nontoxigenic Invasive Corynebacterium diphtheriae bv. mitis Strain ISS 3319.

PubMed

Azevedo Antunes, Camila; Richardson, Emily J; Quick, Joshua; Fuentes-Utrilla, Pablo; Isom, Georgia L; Goodall, Emily C; Möller, Jens; Hoskisson, Paul A; Mattos-Guaraldi, Ana Luiza; Cunningham, Adam F; Loman, Nicholas J; Sangal, Vartul; Burkovski, Andreas; Henderson, Ian R

2018-02-01

The genome sequence of the human pathogen Corynebacterium diphtheriae bv. mitis strain ISS 3319 was determined and closed in this study. The genome is estimated to have 2,404,936 bp encoding 2,257 proteins. This strain also possesses a plasmid of 1,960 bp. Copyright © 2018 Azevedo Antunes et al.

Improved L-ornithine production in Corynebacterium crenatum by introducing an artificial linear transacetylation pathway.

PubMed

Shu, Qunfeng; Xu, Meijuan; Li, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong; Rao, Zhiming

2018-05-04

L-Ornithine is a non-protein amino acid with extensive applications in the food and pharmaceutical industries. In this study, we performed metabolic pathway engineering of an L-arginine hyper-producing strain of Corynebacterium crenatum for L-ornithine production. First, we amplified the L-ornithine biosynthetic pathway flux by blocking the competing branch of the pathway. To enhance L-ornithine synthesis, we performed site-directed mutagenesis of the ornithine-binding sites to solve the problem of L-ornithine feedback inhibition for ornithine acetyltransferase. Alternatively, the genes argA from Escherichia coli and argE from Serratia marcescens, encoding the enzymes N-acetyl glutamate synthase and N-acetyl-L-ornithine deacetylase, respectively, were introduced into Corynebacterium crenatum to mimic the linear pathway of L-ornithine biosynthesis. Fermentation of the resulting strain in a 5-L bioreactor allowed a dramatically increased production of L-ornithine, 40.4 g/L, with an overall productivity of 0.673 g/L/h over 60 h. This demonstrates that an increased level of transacetylation is beneficial for L-ornithine biosynthesis.

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.

Biofilm Formation by a Metabolically Versatile Bacterium

DTIC Science & Technology

2009-03-19

ABSTRACT Rhodopseudomonas palustris is a photosynthetic bacterium that has good potential as a biocatalyst for the production ofhydrogen gas, a biofuel...Biofilm formation by a metabolically versatile bacterium: final report Report Title ABSTRACT Rhodopseudomonas palustris is a photosynthetic bacterium...agricultural waste. We characterized five new Rhodopseudomonas genome sequences and isolated and described R. palustris mutant strains that produce

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.

[Comparison of the antibiotic sensitivity of lipophilic Corynebacterium sp. isolated from patients on the day of admission and during hospitalization].

PubMed

Ciok-Pater, Emilia; Mikucka, Agnieszka; Gospodarek, Eugenia

2005-01-01

Lipophilic species of Corynebacterium are increasing problem in hospital infections. The aim of this study was to evaluate occurrence of these microorganisms in the materials taken from patients in the day of admission and during the hospitalization as well as comparison of their antibiotic sensitivity. The investigation included 65 strains isolated from hospitalized patients and 48 strains isolated from unchanged skin. Using Api Coryne test 5 species were identified. C. urealyticum dominated, the other were C. subsp. lipophilum and C. jeikeium. Among strains isolated from unchanged diseased skin the most C. jeikeium and C. accolens occurred. All strains were sensitive to glycopeptide, quinupristin/dalphopristin. The strains isolated from hospitalized patients were usually sensitive to fuside acid, doxycycline as well as tetracycline. Strains isolated from unchanged skin were sensitive to almost all tested antibiotics. In the group of 65 strains isolated from hospitalized patients 99.0% were multiresistant. In the group of strains isolated from unchanged skin only two strains were multiresistant. Differences in antibiotic sensitivity among analysed Corynebacterium sp. were confirmed. Majority of the "hospital strains" were characterized by multiresistance. Basing on these results it is possible to suppose, that multiresistance is main factor that favours lipophilic Corynebacterium species in the process of colonization of mucous membranes, skins as well as developing infections.

The Genus Corynebacterium and Other Medically Relevant Coryneform-Like Bacteria

PubMed Central

2012-01-01

Catalase-positive Gram-positive bacilli, commonly called “diphtheroids” or “coryneform” bacteria were historically nearly always dismissed as contaminants when recovered from patients, but increasingly have been implicated as the cause of significant infections. These taxa have been underreported, and the taxa were taxonomically confusing. The mechanisms of pathogenesis, especially for newly described taxa, were rarely studied. Antibiotic susceptibility data were relatively scant. In this minireview, clinical relevance, phenotypic and genetic identification methods, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) evaluations, and antimicrobial susceptibility testing involving species in the genus Corynebacterium and other medically relevant Gram-positive rods, collectively called coryneforms, are described. PMID:22837327

Metabolic flux estimation using particle swarm optimization with penalty function.

PubMed

Long, Hai-Xia; Xu, Wen-Bo; Sun, Jun

2009-01-01

Metabolic flux estimation through 13C trace experiment is crucial for quantifying the intracellular metabolic fluxes. In fact, it corresponds to a constrained optimization problem that minimizes a weighted distance between measured and simulated results. In this paper, we propose particle swarm optimization (PSO) with penalty function to solve 13C-based metabolic flux estimation problem. The stoichiometric constraints are transformed to an unconstrained one, by penalizing the constraints and building a single objective function, which in turn is minimized using PSO algorithm for flux quantification. The proposed algorithm is applied to estimate the central metabolic fluxes of Corynebacterium glutamicum. From simulation results, it is shown that the proposed algorithm has superior performance and fast convergence ability when compared to other existing algorithms.

Microbial Production of l-Serine from Renewable Feedstocks.

PubMed

Zhang, Xiaomei; Xu, Guoqiang; Shi, Jinsong; Koffas, Mattheos A G; Xu, Zhenghong

2018-07-01

l-Serine is a non-essential amino acid that has wide and expanding applications in industry with a fast-growing market demand. Currently, extraction and enzymatic catalysis are the main processes for l-serine production. However, such approaches limit the industrial-scale applications of this important amino acid. Therefore, shifting to the direct fermentative production of l-serine from renewable feedstocks has attracted increasing attention. This review details the current status of microbial production of l-serine from renewable feedstocks. We also summarize the current trends in metabolic engineering strategies and techniques for the typical industrial organisms Corynebacterium glutamicum and Escherichia coli that have been developed to address and overcome major challenges in the l-serine production process. Copyright © 2018 Elsevier Ltd. All rights reserved.

Rapid identification of Corynebacterium vaginale in non-purulent vaginitis.

PubMed Central

Wells, J I; Goei, S H

1981-01-01

A simple set of tests is proposed to give excellent probability for the identification of Corynebacterium vaginale from clinical material. Using these tests, 380 C vaginale were isolated from genital tract specimens from 1402 patients. Of these isolates 70 were from symptomatic patients. These 70 isolates were subjected to a further set of tests to confirm their identity. The advantage of these primary tests is that they can be completed on the day of isolation of the organism. Of these 70 isolates 66 were confirmed as C vaginale thus giving the primary set of tests a 94% rate of accurate identification. However this rate may be increased beyond 97% by the promotion of one of the key secondary tests to the primary set. PMID:7024317

Formic Acid-Based Direct, On-Plate Testing of Yeast and Corynebacterium Species by Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Theel, Elitza S.; Schmitt, Bryan H.; Hall, Leslie; Cunningham, Scott A.; Walchak, Robert C.; Patel, Robin

2012-01-01

An on-plate testing method using formic acid was evaluated on the Bruker Biotyper matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry system using 90 yeast and 78 Corynebacterium species isolates, and 95.6 and 81.1% of yeast and 96.1 and 92.3% of Corynebacterium isolates were correctly identified to the genus and species levels, respectively. The on-plate method using formic acid yielded identification percentages similar to those for the conventional but more laborious tube-based extraction. PMID:22760034

Formic acid-based direct, on-plate testing of yeast and Corynebacterium species by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Theel, Elitza S; Schmitt, Bryan H; Hall, Leslie; Cunningham, Scott A; Walchak, Robert C; Patel, Robin; Wengenack, Nancy L

2012-09-01

An on-plate testing method using formic acid was evaluated on the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry system using 90 yeast and 78 Corynebacterium species isolates, and 95.6 and 81.1% of yeast and 96.1 and 92.3% of Corynebacterium isolates were correctly identified to the genus and species levels, respectively. The on-plate method using formic acid yielded identification percentages similar to those for the conventional but more laborious tube-based extraction.

Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

PubMed Central

Yamamoto, Yoko; Fukui, Keita; Nishio, Yousuke; Hashiguchi, Kenichi; Usuda, Yoshihiro; Sode, Koji

2014-01-01

Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production. PMID:25416770

Label-free proteomic analysis to confirm the predicted proteome of Corynebacterium pseudotuberculosis under nitrosative stress mediated by nitric oxide.

PubMed

Silva, Wanderson M; Carvalho, Rodrigo D; Soares, Siomar C; Bastos, Isabela Fs; Folador, Edson L; Souza, Gustavo Hmf; Le Loir, Yves; Miyoshi, Anderson; Silva, Artur; Azevedo, Vasco

2014-12-04

Corynebacterium pseudotuberculosis biovar ovis is a facultative intracellular pathogen, and the etiological agent of caseous lymphadenitis in small ruminants. During the infection process, the bacterium is subjected to several stress conditions, including nitrosative stress, which is caused by nitric oxide (NO). In silico analysis of the genome of C. pseudotuberculosis ovis 1002 predicted several genes that could influence the resistance of this pathogen to nitrosative stress. Here, we applied high-throughput proteomics using high definition mass spectrometry to characterize the functional genome of C. pseudotuberculosis ovis 1002 in the presence of NO-donor Diethylenetriamine/nitric oxide adduct (DETA/NO), with the aim of identifying proteins involved in nitrosative stress resistance. We characterized 835 proteins, representing approximately 41% of the predicted proteome of C. pseudotuberculosis ovis 1002, following exposure to nitrosative stress. In total, 102 proteins were exclusive to the proteome of DETA/NO-induced cells, and a further 58 proteins were differentially regulated between the DETA/NO and control conditions. An interactomic analysis of the differential proteome of C. pseudotuberculosis in response to nitrosative stress was also performed. Our proteomic data set suggested the activation of both a general stress response and a specific nitrosative stress response, as well as changes in proteins involved in cellular metabolism, detoxification, transcriptional regulation, and DNA synthesis and repair. Our proteomic analysis validated previously-determined in silico data for C. pseudotuberculosis ovis 1002. In addition, proteomic screening performed in the presence of NO enabled the identification of a set of factors that can influence the resistance and survival of C. pseudotuberculosis during exposure to nitrosative stress.

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. Copyright © 2015. Published by Elsevier SAS.

NREL Researchers Discover How a Bacterium, Clostridium thermocellum,

Science.gov Websites

containing the bacterium actually promotes the growth of C. thermocellum, yet its mechanistic details remained a puzzle. This enhanced growth implied the bacterium had the ability to use CO2 and prompted NREL researchers to investigate the phenomena enhancing the bacterium's growth. "It took us by surprise that

Microbe Profile: Corynebacterium diphtheriae - an old foe always ready to seize opportunity.

PubMed

Hoskisson, Paul A

2018-02-21

Corynebacterium diphtheriae is a globally important Gram-positive aerobic Actinobacterium capable of causing the toxin-mediated disease, diphtheria. Diphtheria was a major cause of childhood mortality prior to the introduction of the toxoid vaccine, yet it is capable of rapid resurgence following the breakdown of healthcare provision, vaccination or displacement of people. The mechanism and treatment of toxin-mediated disease is well understood, however there are key gaps in our knowledge on the basic biology of C. diphtheriae particularly relating to host colonisation, the nature of asymptomatic carriage, population genomics and host adaptation.

Toxigenic Corynebacterium ulcerans in a fatal human case and her feline contacts, France, March 2014.

PubMed

Vandentorren, S; Guiso, N; Badell, E; Boisrenoult, P; Micaelo, M; Troché, G; Lecouls, P; Moquet, M J; Patey, O; Belchior, E

2014-09-25

In March 2014, a person in their eighties who was diagnosed with extensive cellulitis due to toxigenic Corynebacterium ulcerans died from multiple organ failure. Environmental investigation also isolated C. ulcerans in biological samples from two stray cats in contact with the case. This finding provides further evidence that pets can carry toxigenic C. ulcerans and may be a source of the infection in humans.

Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1.

PubMed Central

Omori, T; Monna, L; Saiki, Y; Kodama, T

1992-01-01

Strain SY1, identified as a Corynebacterium sp., was isolated on the basis of the ability to utilize dibenzothiophene (DBT) as a sole source of sulfur. Strain SY1 could utilize a wide range of organic and inorganic sulfur compounds, such as DBT sulfone, dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone, CS2, FeS2, and even elemental sulfur. Strain SY1 metabolized DBT to dibenzothiophene-5-oxide, DBT sulfone, and 2-hydroxybiphenyl, which was subsequently nitrated to produce at least two different hydroxynitrobiphenyls during cultivation. These metabolites were separated by silica gel column chromatography and identified by nuclear magnetic resonance, UV, and mass spectral techniques. Resting cells of SY1 desulfurized toluenesulfonic acid and released sulfite anion. On the basis of these results, a new DBT degradation pathway is proposed. PMID:1575493

Corynebacterium macginleyi Has to Date Been Isolated Exclusively from Conjunctival Swabs

PubMed Central

Funke, Guido; Pagano-Niederer, Maja; Bernauer, Wolfgang

1998-01-01

Fifteen strains of Corynebacterium macginleyi were exclusively isolated from conjunctival swabs of patients with either conjunctivitis or corneal ulcers. Up to now, only three C. macginleyi strains had been described in the literature. The characteristics of the 15 patients from whom C. macginleyi was isolated are outlined, characteristics useful for the identification of C. macginleyi are described, and the antimicrobial susceptibility pattern of the species is provided. C. macginleyi is uniformly susceptible to penicillins, quinolones, and aminoglycosides. Although considered to be of rather low pathogenicity C. macginleyi seems to have the potential to cause superinfections in selected patients with ocular surface problems. PMID:9817893

Cystic Neutrophilic Granulomatous Mastitis: Further Characterization of a Distinctive Histopathologic Entity Not Always Demonstrably Attributable to Corynebacterium Infection.

PubMed

D'Alfonso, Timothy M; Moo, Tracy-Ann; Arleo, Elizabeth K; Cheng, Esther; Antonio, Lilian B; Hoda, Syed A

2015-10-01

Granulomatous lobular mastitis (GLM) is an uncommon condition that typically occurs in parous, reproductive-aged women and can simulate malignancy on the basis of clinical and imaging features. A distinctive histologic pattern termed cystic neutrophilic granulomatous mastitis (CNGM) is seen in some cases of GLM and has been associated with Corynebacterium infection. We sought to further characterize the clinical, imaging, and histopathologic features of CNGM by studying 12 cases and attempted to establish the relationship of this disease with Corynebacterium infection. Patients were women ranging in age from 25 to 49 years (median: 34 y), and all presented with a palpable mass that was painful in half of the cases. In 2 of 9 cases, imaging was highly suspicious for malignancy (BI-RADS 5). CNGM was characterized by lobulocentric granulomas with mixed inflammation and clear vacuoles lined by neutrophils within granulomas. Gram-positive bacilli were identified in 5/12 cases. In 4 patients, the disease process worsened after the diagnostic core biopsy, with the development of a draining sinus in 2 cases. No growth of bacteria was seen in any microbial cultures. No bacterial DNA was identified by 16S rDNA polymerase chain reaction for 1 case that showed gram-positive bacilli on histology. Patients were treated with variable combinations of surgery, antibiotics, and steroids. The time to significant resolution of symptoms ranged from 2 weeks to 6 months. Similar to other forms of GLM, CNGM can mimic malignancy clinically and on imaging. When encountered in a needle core biopsy sample, recognition of the characteristic histologic pattern and its possible association with Corynebacterium infection can help guide treatment.

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

Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Omori, Toshio; Monna, L.; Saiki, Yuko

1992-03-01

Strain SY1, identified as a Corynebacterium sp., was isolated on the basis of the ability to utilize dibenzothiophene (DBT) as a sole source of sulfur. Strain SY1 could utilize a wide range of organic and inorganic sulfur compounds, such as DBT sulfone, dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone, CS{sub 2}, FeS{sub 2}, and even elemental sulfur. Strain SY1 metabolized DBT to dibenzothiophene-5-oxide, DBT sulfone, and 2-hydroxybiphenyl, which was subsequently nitrated to produce at least two different hydroxynitrobiphenyls during cultivation. These metabolites were separated by silica gel column chromatography and identified by nuclear magnetic resonance, UV, and mass spectral techniques. Restingmore » cells of SY1 desulfurized toluenesulfonic acid and released sulfite anion. On the basis of these results, a new DBT degradation pathway is proposed.« less

The transcriptional regulatory network of Corynebacterium jeikeium K411 and its interaction with metabolic routes contributing to human body odor formation.

PubMed

Barzantny, Helena; Schröder, Jasmin; Strotmeier, Jasmin; Fredrich, Eugenie; Brune, Iris; Tauch, Andreas

2012-06-15

Lipophilic corynebacteria are involved in the generation of volatile odorous products in the process of human body odor formation by degrading skin lipids and specific odor precursors. Therefore, these bacteria represent appropriate model systems for the cosmetic industry to examine axillary malodor formation on the molecular level. To understand the transcriptional control of metabolic pathways involved in this process, the transcriptional regulatory network of the lipophilic axilla isolate Corynebacterium jeikeium K411 was reconstructed from the complete genome sequence. This bioinformatic approach detected a gene-regulatory repertoire of 83 candidate proteins, including 56 DNA-binding transcriptional regulators, nine two-component systems, nine sigma factors, and nine regulators with diverse physiological functions. Furthermore, a cross-genome comparison among selected corynebacterial species of the taxonomic cluster 3 revealed a common gene-regulatory repertoire of 44 transcriptional regulators, including the MarR-like regulator Jk0257, which is exclusively encoded in the genomes of this taxonomical subline. The current network reconstruction comprises 48 transcriptional regulators and 674 gene-regulatory interactions that were assigned to five interconnected functional modules. Most genes involved in lipid degradation are under the combined control of the global cAMP-sensing transcriptional regulator GlxR and the LuxR-family regulator RamA, probably reflecting the essential role of lipid degradation in C. jeikeium. This study provides the first genome-scale in silico analysis of the transcriptional regulation of metabolism in a lipophilic bacterium involved in the formation of human body odor. Copyright © 2012 Elsevier B.V. All rights reserved.

Rapid hybrid de novo assembly of a microbial genome using only short reads: Corynebacterium pseudotuberculosis I19 as a case study.

PubMed

Cerdeira, Louise Teixeira; Carneiro, Adriana Ribeiro; Ramos, Rommel Thiago Jucá; de Almeida, Sintia Silva; D'Afonseca, Vivian; Schneider, Maria Paula Cruz; Baumbach, Jan; Tauch, Andreas; McCulloch, John Anthony; Azevedo, Vasco Ariston Carvalho; Silva, Artur

2011-08-01

Due to the advent of the so-called Next-Generation Sequencing (NGS) technologies the amount of monetary and temporal resources for whole-genome sequencing has been reduced by several orders of magnitude. Sequence reads can be assembled either by anchoring them directly onto an available reference genome (classical reference assembly), or can be concatenated by overlap (de novo assembly). The latter strategy is preferable because it tends to maintain the architecture of the genome sequence the however, depending on the NGS platform used, the shortness of read lengths cause tremendous problems the in the subsequent genome assembly phase, impeding closing of the entire genome sequence. To address the problem, we developed a multi-pronged hybrid de novo strategy combining De Bruijn graph and Overlap-Layout-Consensus methods, which was used to assemble from short reads the entire genome of Corynebacterium pseudotuberculosis strain I19, a bacterium with immense importance in veterinary medicine that causes Caseous Lymphadenitis in ruminants, principally ovines and caprines. Briefly, contigs were assembled de novo from the short reads and were only oriented using a reference genome by anchoring. Remaining gaps were closed using iterative anchoring of short reads by craning to gap flanks. Finally, we compare the genome sequence assembled using our hybrid strategy to a classical reference assembly using the same data as input and show that with the availability of a reference genome, it pays off to use the hybrid de novo strategy, rather than a classical reference assembly, because more genome sequences are preserved using the former. Copyright © 2011 Elsevier B.V. All rights reserved.

Characterization of the cellulose-degrading bacterium NCIMB 10462

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dees, C.; Scott, T.C.; Phelps, T.J.

The gram-negative cellulase-producing bacterium NCIMB 10462 has been previously named Pseudomonas fluorescens subsp. or var. cellulose. Because of renewed interest in cellulose-degrading bacteria for use in the bioconversion of cellulose to chemical feed stocks and fuels, we re-examined the characteristics of this microorganism to determine its true metabolic potential. Metabolic and physical characterization of NCIMB 10462 revealed that this is an alkalophilic, non-fermentative, gram-negative, oxidase-positive, motile, cellulose-degrading bacterium. The aerobic substrate utilization profile of this bacterium has few characteristics consistent with a classification of P. fluorescens and a very low probability match with the genus Sphingomonas. However, total lipid analysismore » did not reveal that any sphingolipid bases are produced by this bacterium. NCIMB 10462 grows best aerobically, but also grows well in complex media under reducing conditions. NCIMB 10462 grows slowly under anaerobic conditions on complex media, but growth on cellulosic media occurred only under aerobic conditions. Total fatty acid analysis (MIDI) of NCIMB 10462 failed to group this bacterium with a known pseudomonas species. However, fatty acid analysis of the bacteria when grown at temperatures below 37{degrees}C suggest that the organism is a pseudomonad. Since a predominant characteristic of this bacterium is its ability to degrade cellulose, we suggest that it be called Pseudomonas cellulosa.« less

Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria.

PubMed

Wittmann, Christoph; Heinzle, Elmar

2002-12-01

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

Genealogy Profiling through Strain Improvement by Using Metabolic Network Analysis: Metabolic Flux Genealogy of Several Generations of Lysine-Producing Corynebacteria

PubMed Central

Wittmann, Christoph; Heinzle, Elmar

2002-01-01

A comprehensive approach of metabolite balancing, 13C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C3 carboxylation and C4 decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C3 carboxylation and C4 decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that

The obligate respiratory supercomplex from Actinobacteria.

PubMed

Kao, Wei-Chun; Kleinschroth, Thomas; Nitschke, Wolfgang; Baymann, Frauke; Neehaul, Yashvin; Hellwig, Petra; Richers, Sebastian; Vonck, Janet; Bott, Michael; Hunte, Carola

2016-10-01

Actinobacteria are closely linked to human life as industrial producers of bioactive molecules and as human pathogens. Respiratory cytochrome bcc complex and cytochrome aa3 oxidase are key components of their aerobic energy metabolism. They form a supercomplex in the actinobacterial species Corynebacterium glutamicum. With comprehensive bioinformatics and phylogenetic analysis we show that genes for cyt bcc-aa3 supercomplex are characteristic for Actinobacteria (Actinobacteria and Acidimicrobiia, except the anaerobic orders Actinomycetales and Bifidobacteriales). An obligatory supercomplex is likely, due to the lack of genes encoding alternative electron transfer partners such as mono-heme cyt c. Instead, subunit QcrC of bcc complex, here classified as short di-heme cyt c, will provide the exclusive electron transfer link between the complexes as in C. glutamicum. Purified to high homogeneity, the C. glutamicum bcc-aa3 supercomplex contained all subunits and cofactors as analyzed by SDS-PAGE, BN-PAGE, absorption and EPR spectroscopy. Highly uniform supercomplex particles in electron microscopy analysis support a distinct structural composition. The supercomplex possesses a dimeric stoichiometry with a ratio of a-type, b-type and c-type hemes close to 1:1:1. Redox titrations revealed a low potential bcc complex (Em(ISP)=+160mV, Em(bL)=-291mV, Em(bH)=-163mV, Em(cc)=+100mV) fined-tuned for oxidation of menaquinol and a mixed potential aa3 oxidase (Em(CuA)=+150mV, Em(a/a3)=+143/+317mV) mediating between low and high redox potential to accomplish dioxygen reduction. The generated molecular model supports a stable assembled supercomplex with defined architecture which permits energetically efficient coupling of menaquinol oxidation and dioxygen reduction in one supramolecular entity. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Biotechnological production of aromatic compounds of the extended shikimate pathway from renewable biomass.

PubMed

Lee, Jin-Ho; Wendisch, Volker F

2017-09-10

Aromatic chemicals that contain an unsaturated ring with alternating double and single bonds find numerous applications in a wide range of industries, e.g. paper and dye manufacture, as fuel additives, electrical insulation, resins, pharmaceuticals, agrochemicals, in food, feed and cosmetics. Their chemical production is based on petroleum (BTX; benzene, toluene, and xylene), but they can also be obtained from plants by extraction. Due to petroleum depletion, health compliance, or environmental issues such as global warming, the biotechnological production of aromatics from renewable biomass came more and more into focus. Lignin, a complex polymeric aromatic molecule itself, is a natural source of aromatic compounds. Many microorganisms are able to catabolize a plethora of aromatic compounds and interception of these pathways may lead to the biotechnological production of value-added aromatic compounds which will be discussed for Corynebacterium glutamicum. Biosynthesis of aromatic amino acids not only gives rise to l-tryptophan, L-tyrosine and l-phenylalanine, but also to aromatic intermediates such as dehydroshikimate or chorismate from which value-added aromatic compounds can be derived. In this review, we will summarize recent strategies for the biotechnological production of aromatic and related compounds from renewable biomass by Escherichia coli, Pseudomonas putida, C. glutamicum and Saccharomyces cerevisiae. In particular, we will focus on metabolic engineering of the extended shikimate pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Single Bacterium Detection Using Sers

NASA Astrophysics Data System (ADS)

Gonchukov, S. A.; Baikova, T. V.; Alushin, M. V.; Svistunova, T. S.; Minaeva, S. A.; Ionin, A. A.; Kudryashov, S. I.; Saraeva, I. N.; Zayarny, D. A.

2016-02-01

This work is devoted to the study of a single Staphylococcus aureus bacterium detection using surface-enhanced Raman spectroscopy (SERS) and resonant Raman spectroscopy (RS). It was shown that SERS allows increasing sensitivity of predominantly low frequency lines connected with the vibrations of Amide, Proteins and DNA. At the same time the lines of carotenoids inherent to this kind of bacterium are well-detected due to the resonance Raman scattering mechanism. The reproducibility and stability of Raman spectra strongly depend on the characteristics of nanostructured substrate, and molecular structure and size of the tested biological object.

Pathogenicity and Genetic Variation of 3 Strains of Corynebacterium bovis in Immunodeficient Mice

PubMed Central

Dole, Vandana S; Henderson, Kenneth S; Fister, Richard D; Pietrowski, Michael T; Maldonado, Geomaris; Clifford, Charles B

2013-01-01

Corynebacterium bovis has been associated with hyperkeratotic dermatitis and acanthosis in mice. We studied 3 different strains of C. bovis: one previously described to cause hyperkeratotic dermatitis (HAC), one that infected athymic nude mice without leading to the classic clinical signs, and one of bovine origin (ATCC 7715). The 3 strains showed a few biochemical and genetic differences. Immunodeficient nude mice were housed in 3 independent isolators and inoculated with pure cultures of the 3 strains. We studied the transmission of these C. bovis studies to isolator-bedding and contact sentinels housed for 5 to 12 wk in filter-top or wire-top cages in the respective isolators. Using a 16S rRNA-based qPCR assay, we did not find consistent differences in growth and transmission among the 3 C. bovis strains, and neither the incidence nor severity of hyperkeratosis or acanthosis differed between strains. Housing in filter-top compared with wire-top cages did not alter the morbidity associated with any of the strains. Our findings confirmed the variability in the gross and histologic changes associated with C. bovis infection of mice. Although bacteriology was a sensitive method for the detection of Corynebacterium spp., standard algorithms occasionally misidentified C. bovis and several related species. Our study demonstrates that PCR of skin swabs or feces is a sensitive and specific method for the detection of C. bovis infection in mice. An rpoB-based screen of samples from North American vivaria revealed that HAC is the predominant C. bovis strain in laboratory mice. PMID:23849444

Pathogenicity and genetic variation of 3 strains of Corynebacterium bovis in immunodeficient mice.

PubMed

Dole, Vandana S; Henderson, Kenneth S; Fister, Richard D; Pietrowski, Michael T; Maldonado, Geomaris; Clifford, Charles B

2013-07-01

Corynebacterium bovis has been associated with hyperkeratotic dermatitis and acanthosis in mice. We studied 3 different strains of C. bovis: one previously described to cause hyperkeratotic dermatitis (HAC), one that infected athymic nude mice without leading to the classic clinical signs, and one of bovine origin (ATCC 7715). The 3 strains showed a few biochemical and genetic differences. Immunodeficient nude mice were housed in 3 independent isolators and inoculated with pure cultures of the 3 strains. We studied the transmission of these C. bovis studies to isolator-bedding and contact sentinels housed for 5 to 12 wk in filter-top or wire-top cages in the respective isolators. Using a 16S rRNA-based qPCR assay, we did not find consistent differences in growth and transmission among the 3 C. bovis strains, and neither the incidence nor severity of hyperkeratosis or acanthosis differed between strains. Housing in filter-top compared with wire-top cages did not alter the morbidity associated with any of the strains. Our findings confirmed the variability in the gross and histologic changes associated with C. bovis infection of mice. Although bacteriology was a sensitive method for the detection of Corynebacterium spp., standard algorithms occasionally misidentified C. bovis and several related species. Our study demonstrates that PCR of skin swabs or feces is a sensitive and specific method for the detection of C. bovis infection in mice. An rpoB-based screen of samples from North American vivaria revealed that HAC is the predominant C. bovis strain in laboratory mice.

Tips and tricks for the assembly of a Corynebacterium pseudotuberculosis genome using a semiconductor sequencer.

PubMed

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

2013-03-01

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. © 2012 The Authors. Published by Society for Applied Microbiology and Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Effects of eliminating pyruvate node pathways and of coexpression of heterogeneous carboxylation enzymes on succinate production by Enterobacter aerogenes.

PubMed

Tajima, Yoshinori; Yamamoto, Yoko; Fukui, Keita; Nishio, Yousuke; Hashiguchi, Kenichi; Usuda, Yoshihiro; Sode, Koji

2015-02-01

Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Crystal structure of heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ida, Koh; E-mail: idakoh@sci.kitasato-u.ac.jp; Moriguchi, Tomotaka

2005-07-29

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme. Here we report the crystal structures of the enzyme in complex with dimethylglycine and folinic acid. The {alpha} subunit is composed of two domains, contains NAD{sup +}, and binds folinic acid. The {beta} subunit contains dimethylglycine, FAD, and FMN, and these flavins are approximately 10 A apart. The {gamma} subunit is in contact with two domains of {alpha} subunit and has possibly a folate-binding structure. The {delta} subunit contains a single atom of zinc and has a Cys{sub 3}His zinc finger structure. Based on the structures determined and on themore » previous works, the structure-function relationship on the heterotetrameric sarcosine oxidase is discussed.« less

Production of succinic acid by metabolically engineered microorganisms.

PubMed

Ahn, Jung Ho; Jang, Yu-Sin; Lee, Sang Yup

2016-12-01

Succinic acid (SA) has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. For the economical bio-based production of SA, extensive research works have been performed on developing microbial strains by metabolic engineering as well as fermentation and downstream processes. Here we review metabolic engineering strategies applied for bio-based production of SA using representative microorganisms, including Saccharomyces cerevisiae, Pichia kudriavzevii, Escherichia coli, Mannheimia succiniciproducens, Basfia succiniciproducens, Actinobacillus succinogenes, and Corynebacterium glutamicum. In particular, strategies employed for developing engineered strains of these microorganisms leading to the best performance indices (titer, yield, and productivity) are showcased based on the published papers as well as patents. Those processes currently under commercialization are also analyzed and future perspectives are provided. Copyright © 2016 Elsevier Ltd. All rights reserved.

Escherichia coli yjjPB genes encode a succinate transporter important for succinate production.

PubMed

Fukui, Keita; Nanatani, Kei; Hara, Yoshihiko; Yamakami, Suguru; Yahagi, Daiki; Chinen, Akito; Tokura, Mitsunori; Abe, Keietsu

2017-09-01

Under anaerobic conditions, Escherichia coli produces succinate from glucose via the reductive tricarboxylic acid cycle. To date, however, no genes encoding succinate exporters have been established in E. coli. Therefore, we attempted to identify genes encoding succinate exporters by screening an E. coli MG1655 genome library. We identified the yjjPB genes as candidates encoding a succinate transporter, which enhanced succinate production in Pantoea ananatis under aerobic conditions. A complementation assay conducted in Corynebacterium glutamicum strain AJ110655ΔsucE1 demonstrated that both YjjP and YjjB are required for the restoration of succinate production. Furthermore, deletion of yjjPB decreased succinate production in E. coli by 70% under anaerobic conditions. Taken together, these results suggest that YjjPB constitutes a succinate transporter in E. coli and that the products of both genes are required for succinate export.

Detection of Salmonella bacterium in drinking water using microring resonator.

PubMed

Bahadoran, Mahdi; Noorden, Ahmad Fakhrurrazi Ahmad; Mohajer, Faeze Sadat; Abd Mubin, Mohamad Helmi; Chaudhary, Kashif; Jalil, Muhammad Arif; Ali, Jalil; Yupapin, Preecha

2016-01-01

A new microring resonator system is proposed for the detection of the Salmonella bacterium in drinking water, which is made up of SiO2-TiO2 waveguide embedded inside thin film layer of the flagellin. The change in refractive index due to the binding of the Salmonella bacterium with flagellin layer causes a shift in the output signal wavelength and the variation in through and drop port's intensities, which leads to the detection of Salmonella bacterium in drinking water. The sensitivity of proposed sensor for detecting of Salmonella bacterium in water solution is 149 nm/RIU and the limit of detection is 7 × 10(-4)RIU.

High-level fluoroquinolone resistance in ophthalmic clinical isolates belonging to the species Corynebacterium macginleyi.

PubMed

Eguchi, Hiroshi; Kuwahara, Tomomi; Miyamoto, Tatsuro; Nakayama-Imaohji, Haruyuki; Ichimura, Minoru; Hayashi, Tetsuya; Shiota, Hiroshi

2008-02-01

The clinical importance of nondiphtherial Corynebacterium, a ubiquitous member of the normal human microflora of the skin and mucous membrane, for ocular surface infections has been recognized recently. We performed an antimicrobial susceptibility test with Etest strips for three fluoroquinolones (ciprofloxacin, norfloxacin, and levofloxacin) and a taxonomic analysis on 21 isolates of Corynebacterium from ophthalmic samples. Of these, 16 isolates were identified as C. macginleyi at the species level on the basis of 16S rRNA gene sequence comparisons. The remaining five isolates were determined to be C. mastitidis (four) or C. accolens (one). Eleven of the C. macginleyi isolates showed high levels of resistance to all of the fluoroquinolones tested, and one isolate was resistant to norfloxacin alone. An analysis of the amplified quinolone-resistance-determining regions of the gyrA genes revealed that a single amino acid substitution in position 83 of the gyrA product was sufficient to generate the norfloxacin resistance phenotype, and double mutations leading to amino acid changes in positions 83 and 87 were necessary for high-level resistance against the other fluoroquinolones. We conducted the first example of multilocus sequence typing (MLST) analysis on C. macginleyi. The MLST analysis grouped the majority of C. macginleyi isolates into a single lineage, and another molecular strain typing by random amplified polymorphic DNA fragment patterns supported the finding, indicating that a particular lineage of C. macginleyi is dominant on the human ocular surface. This type of population might be particularly adaptable to the milieu on the human ocular surface.

High-Level Fluoroquinolone Resistance in Ophthalmic Clinical Isolates Belonging to the Species Corynebacterium macginleyi▿

PubMed Central

Eguchi, Hiroshi; Kuwahara, Tomomi; Miyamoto, Tatsuro; Nakayama-Imaohji, Haruyuki; Ichimura, Minoru; Hayashi, Tetsuya; Shiota, Hiroshi

2008-01-01

The clinical importance of nondiphtherial Corynebacterium, a ubiquitous member of the normal human microflora of the skin and mucous membrane, for ocular surface infections has been recognized recently. We performed an antimicrobial susceptibility test with Etest strips for three fluoroquinolones (ciprofloxacin, norfloxacin, and levofloxacin) and a taxonomic analysis on 21 isolates of Corynebacterium from ophthalmic samples. Of these, 16 isolates were identified as C. macginleyi at the species level on the basis of 16S rRNA gene sequence comparisons. The remaining five isolates were determined to be C. mastitidis (four) or C. accolens (one). Eleven of the C. macginleyi isolates showed high levels of resistance to all of the fluoroquinolones tested, and one isolate was resistant to norfloxacin alone. An analysis of the amplified quinolone-resistance-determining regions of the gyrA genes revealed that a single amino acid substitution in position 83 of the gyrA product was sufficient to generate the norfloxacin resistance phenotype, and double mutations leading to amino acid changes in positions 83 and 87 were necessary for high-level resistance against the other fluoroquinolones. We conducted the first example of multilocus sequence typing (MLST) analysis on C. macginleyi. The MLST analysis grouped the majority of C. macginleyi isolates into a single lineage, and another molecular strain typing by random amplified polymorphic DNA fragment patterns supported the finding, indicating that a particular lineage of C. macginleyi is dominant on the human ocular surface. This type of population might be particularly adaptable to the milieu on the human ocular surface. PMID:18077650

Taxonomic characterization of the cellulose-degrading bacterium NCIB 10462

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dees, C.; Ringleberg, D.; Scott, T.C.

The gram negative cellulase-producing bacterium NCIB 10462 has been previously named Pseudomonas fluorescens subsp. or var. cellulosa. Since there is renewed interest in cellulose-degrading bacteria for use in bioconversion of cellulose to chemical feed stocks and fuels, we re-examined the characteristics of this microorganism to determine its proper taxonomic characterization and to further define it`s true metabolic potential. Metabolic and physical characterization of NCIB 10462 revealed that this was an alkalophilic, non-fermentative, gram negative, oxidase positive, motile, cellulose-degrading bacterium. The aerobic substrate utilization profile of this bacterium was found to have few characteristics consistent with a classification of P. fluorescensmore » with a very low probability match with the genus Sphingomonas. Total lipid analysis did not reveal that any sphingolipid bases are produced by this bacterium. NCIB 10462 was found to grow best aerobically but also grows well in complex media under reducing conditions. NCIB 10462 grew slowly under full anaerobic conditions on complex media but growth on cellulosic media was found only under aerobic conditions. Total fatty acid analysis (MIDI) of NCIB 10462 failed to group this bacterium with a known pseudomonas species. However, fatty acid analysis of the bacteria when grown at temperatures below 37{degrees}C suggest that the organism is a pseudomonad. Since a predominant characteristic of this bacterium is it`s ability to degrade cellulose, we suggest it be called Pseudomonas cellulosa.« less

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

Pathway Construction in Corynebacterium glutamicum and Strain Engineering To Produce Rare Sugars from Glycerol.

PubMed

Yang, Jiangang; Zhu, Yueming; Men, Yan; Sun, Shangshang; Zeng, Yan; Zhang, Ying; Sun, Yuanxia; Ma, Yanhe

2016-12-21

Rare sugars are valuable natural products widely used in pharmaceutical and food industries. In this study, we expected to synthesize rare ketoses from abundant glycerol using dihydroxyacetone phosphate (DHAP)-dependent aldolases. First, a new glycerol assimilation pathway was constructed to synthesize DHAP. The enzymes which convert glycerol to 3-hydroxypropionaldehyde and l-glyceraldehyde were selected, and their corresponding aldehyde synthesis pathways were constructed in vivo. Four aldol pathways based on different aldolases and phosphorylase were gathered. Next, three pathways were assembled and the resulting strains synthesized 5-deoxypsicose, 5-deoxysorbose, and 5-deoxyfructose from glucose and glycerol and produce l-fructose, l-tagatose, l-sorbose, and l-psicose with glycerol as the only carbon source. To achieve higher product titer and yield, the recombinant strains were further engineered and fermentation conditions were optimized. Fed-batch culture of engineered strains obtained 38.1 g/L 5-deoxypsicose with a yield of 0.91 ± 0.04 mol product per mol of glycerol and synthesized 20.8 g/L l-fructose, 10.3 g/L l-tagatose, 1.2 g/L l-sorbose, and 0.95 g/L l-psicose.

Whole-Genome Sequence of Corynebacterium auriscanis Strain CIP 106629 Isolated from a Dog with Bilateral Otitis from the United Kingdom.

PubMed

Tiwari, Sandeep; Jamal, Syed Babar; Oliveira, Leticia Castro; Clermont, Dominique; Bizet, Chantal; Mariano, Diego; de Carvalho, Paulo Vinicius Sanches Daltro; Souza, Flavia; Pereira, Felipe Luiz; de Castro Soares, Siomar; Guimarães, Luis C; Dorella, Fernanda; Carvalho, Alex; Leal, Carlos; Barh, Debmalya; Figueiredo, Henrique; Hassan, Syed Shah; Azevedo, Vasco; Silva, Artur

2016-08-11

In this work, we describe a set of features of Corynebacterium auriscanis CIP 106629 and details of the draft genome sequence and annotation. The genome comprises a 2.5-Mbp-long single circular genome with 1,797 protein-coding genes, 5 rRNA, 50 tRNA, and 403 pseudogenes, with a G+C content of 58.50%. Copyright © 2016 Tiwari et al.

Metabolism of tetralin (1,2,3,4-tetrahydronaphthalene) in Corynebacterium sp. strain C125

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sikkema, J.; Bont, J.A.M. de

1993-02-01

Tetralin, widely used as a solvent in the petrochemical industry and in paints and waxes, degrades slowly in mixed cultures of microorganisms or in the presence of cosubstrates. This study reports on the metabolism of tetralin in the o-xylene-isolated Corynebacterium sp. strain C125. The researchers found that this organism attacks tetralin by an initial oxidation of the aromatic nucleus at positions C-5 and C-6 and they propose a four step inducible degradation pathway for tetralin starting at that point. The presence of the pathway makes this bacteria an excellent catalyst for the specific production of special cis-dihydro diols.

Notes from the field: respiratory diphtheria-like illness caused by toxigenic Corynebacterium ulcerans --- Idaho, 2010.

PubMed

2011-01-28

On September 12, 2010, the Idaho Department of Health and Welfare was notified of a case of respiratory diphtheria-like illness in an Idaho man aged 80 years whose pharyngeal specimens yielded Corynebacterium ulcerans. Although C. ulcerans is zoonotic, the patient reported no animal contact or consumption of an unpasteurized dairy product. His vaccination history was unknown. Respiratory diphtheria-like illness from C. ulcerans is uncommon but has been reported in industrialized countries where respiratory diphtheria is rare. The last case of diphtheria-like illness caused by C. ulcerans in the United States was reported in 2005.

Diagnosis in France of a Non-Toxigenic tox Gene-Bearing Strain of Corynebacterium diphtheriae in a Young Male Back From Senegal

PubMed Central

Rouyer, Cécile; Walewski, Violaine; Badell-Ocando, Edgar; Dumas, Marc; Zumelzu, Coralie; Jaureguy, Françoise; Brisse, Sylvain; Caux, Frédéric; Bouchaud, Olivier; Carbonnelle, Etienne

2017-01-01

Abstract Cutaneous diphtheria is uncommon in Europe. In this study, we report a case of imported cutaneous infection due to a non-toxigenic but tox gene-bearing (NTTB) strain of Corynebacterium diphtheriae. The NTTB strains are recognized as emerging pathogens across Europe, and physicians and bacteriologists should be aware of the circulation of these strains. PMID:28480263

Persistence of a Distinct Corynebacterium diphtheriae Clonal Group within Two Communities in the United States and Canada Where Diphtheria Is Endemic

PubMed Central

Marston, Chung K.; Jamieson, Frances; Cahoon, Fred; Lesiak, Gail; Golaz, Anne; Reeves, Mike; Popovic, Tanja

2001-01-01

Molecular characterization of 53 U.S. and Canadian Corynebacterium diphtheriae isolates by multilocus enzyme electrophoresis, ribotyping, and random amplified polymorphic DNA showed that strains with distinct molecular subtypes have persisted in the United States and Canada for at least 25 years. These strains are endemic rather than imported from countries with current endemic or epidemic diphtheria. PMID:11283092

Production of amino acids - Genetic and metabolic engineering approaches.

PubMed

Lee, Jin-Ho; Wendisch, Volker F

2017-12-01

The biotechnological production of amino acids occurs at the million-ton scale and annually about 6milliontons of l-glutamate and l-lysine are produced by Escherichia coli and Corynebacterium glutamicum strains. l-glutamate and l-lysine production from starch hydrolysates and molasses is very efficient and access to alternative carbon sources and new products has been enabled by metabolic engineering. This review focusses on genetic and metabolic engineering of amino acid producing strains. In particular, rational approaches involving modulation of transcriptional regulators, regulons, and attenuators will be discussed. To address current limitations of metabolic engineering, this article gives insights on recent systems metabolic engineering approaches based on functional tools and method such as genome reduction, amino acid sensors based on transcriptional regulators and riboswitches, CRISPR interference, small regulatory RNAs, DNA scaffolding, and optogenetic control, and discusses future prospects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biotin-independent strains of Escherichia coli for enhanced streptavidin production.

PubMed

Jeschek, Markus; Bahls, Maximilian O; Schneider, Veronika; Marlière, Philippe; Ward, Thomas R; Panke, Sven

2017-03-01

Biotin is an archetypal vitamin used as cofactor for carboxylation reactions found in all forms of life. However, biotin biosynthesis is an elaborate multi-enzymatic process and metabolically costly. Moreover, many industrially relevant organisms are incapable of biotin synthesis resulting in the requirement to supplement defined media. Here we describe the creation of biotin-independent strains of Escherichia coli and Corynebacterium glutamicum through installation of an optimized malonyl-CoA bypass, which re-routes natural fatty acid synthesis, rendering the previously essential vitamin completely obsolete. We utilize biotin-independent E. coli for the production of the high-value protein streptavidin which was hitherto restricted because of toxic effects due to biotin depletion. The engineered strain revealed significantly improved streptavidin production resulting in the highest titers and productivities reported for this protein to date. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Detoxification of acidic biorefinery waste liquor for production of high value amino acid.

PubMed

Christopher, Meera; Anusree, Murali; Mathew, Anil K; Nampoothiri, K Madhavan; Sukumaran, Rajeev Kumar; Pandey, Ashok

2016-08-01

The current study evaluates the detoxification of acid pretreatment liquor (APL) using adsorbent (ADS 400 & ADS 800) or ion-exchange (A-27MP & A-72MP) resins and its potential for amino acid production. The APL is generated as a by-product from the pretreatment of lignocellulosic biomass and is rich monomeric sugars as well as sugar degradation products (fermentation inhibitors) such as furfural and hydroxymethyl furfural (HMF). Of the four resins compared, ADS 800 removed approximately 85% and 60% of furfural and HMF, respectively. ADS 800 could be reused for up to six cycles after regeneration without losing its adsorption properties. The study was further extended by assessing the fermentability of detoxified APL for l-lysine production using wild and mutant strains of Corynebacterium glutamicum. The detoxified APL was superior to APL for l-lysine production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Oxidation of Ethylene Glycol by a Salt-Requiring Bacterium

PubMed Central

Caskey, William H.; Taber, Willard A.

1981-01-01

Bacterium T-52, cultured on ethylene glycol, readily oxidized glycolate and glyoxylate and exhibited elevated activities of ethylene glycol dehydrogenase and glycolate oxidase. Labeled glyoxylate was identified in reaction mixtures containing [14C]-ethylene glycol, but no glycolate was detected. The most likely pathway of ethylene glycol catabolism by bacterium T-52 is sequential oxidation to glycolate and glyoxylate. PMID:16345810

Microbial Transformations of Selenium

PubMed Central

Doran, J. W.; Alexander, M.

1977-01-01

Resting cell suspensions of a strain of Corynebacterium isolated from soil formed dimethyl selenide from selenate, selenite, elemental selenium, selenomethionine, selenocystine, and methaneseleninate. Extracts of the bacterium catalyzed the production of dimethyl selenide from selenite, elemental selenium, and methaneseleninate, and methylation of the inorganic Se compounds was enhanced by S-adenosylmethionine. Neither trimethylselenonium nor methaneselenonate was metabolized by the Corynebacterium. Resting cell suspensions of a methionine-utilizing pseudomonad converted selenomethionine to dimethyl diselenide. Six of 10 microorganisms able to grow on cystine used selenocystine as a sole source of carbon and formed elemental selenium, and one of the isolates, a pseudomonad, was found also to produce selenide. Soil enrichments converted trimethylselenonium to dimethyl selenide. Bacteria capable of utilizing trimethylselenonium, dimethyl selenide, and dimethyl diselenide as carbon sources were isolated from soil. PMID:16345188

Solution 1H NMR investigation of the active site molecular and electronic structures of substrate-bound, cyanide-inhibited HmuO, a bacterial heme oxygenase from Corynebacterium diphtheriae.

PubMed

Li, Yiming; Syvitski, Ray T; Chu, Grace C; Ikeda-Saito, Masao; Mar, Gerd N La

2003-02-28

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

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.

Responsiveness of senescent mice to the antitumor properties of Corynebacterium parvum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuhas, J.M.; Ullrich, R.L.

1976-01-01

The antitumor properties of Corynebacterium parvum have been studied in young (3- to 8-month-old) and aged (18 or more months old) BALB/c mice given s.c., i.m., i.p., or i.v. transplants of the highly malignant, weakly immunogenic line 1 lung carcinoma, and aged (25- to 33-month-old) BALB/c mice bearing primary mammary tumors. These aged BALB/c mice were shown to be less immunoresponsive than their younger counterparts, and this, in combination with nonimmunological factors, made them more sensitive to the lethal effects of the line 1 carcinoma. Correspondingly, C. parvum proved to have less antitumor activity in aged mice than it didmore » in young mice. In spite of this relatively weaker, antitumor activity for C. parvum in aged mice, repeated injections of this agent were able to induce temporary regressions of the primary mammary tumors studied and thereby prolong survival time.« less

Tumour growth, phagocytic activity and antibody response in corynebacterium parvum-treated mice

PubMed Central

Woodruff, M. F. A.; McBride, W. H.; Dunbar, Noreen

1974-01-01

Serum from both normal and T cell-deprived female adult CBA mice shows a background titre of antibody to Corynebacterium parvum of about 2–4 log2 units by a latex agglutination test. Intraperitoneal injection of C. parvum causes a marked rise in titre which reaches its peak after about a month, and a second injection at that time evokes a further response. Treatment with mercaptoethanol reduces the background titre, and also the titre 1–3 weeks after immunization by 1–2 log units. Subcutaneous injection of C. parvum on the other hand evokes little or no antibody response. Both the antitumour effect of C. parvum, and its effect on clearance of colloidal carbon from the blood stream, can occur in the presence of high levels of antibody directed against the organism. Theoretical and possible therapeutic implications of these findings are discussed. PMID:4549691

Dynamic Transmission of Protein Allostery without Structural Change: Spatial Pathways or Global Modes?

PubMed Central

McLeish, Tom C.B.; Cann, Martin J.; Rodgers, Thomas L.

2015-01-01

We examine the contrast between mechanisms for allosteric signaling that involve structural change, and those that do not, from the perspective of allosteric pathways. In particular we treat in detail the case of fluctuation-allostery by which amplitude modulation of the thermal fluctuations of the elastic normal modes conveys the allosteric signal, and address the question of what an allosteric pathway means in this case. We find that a perturbation theory of thermal elastic solids and nonperturbative approach (by super-coarse-graining elasticity into internal bending modes) have opposite signatures in their structure of correlated pathways. We illustrate the effect from analysis of previous results from GlxR of Corynebacterium glutamicum, an example of the CRP/FNR transcription family of allosteric homodimers. We find that the visibility of both correlated pathways and disconnected sites of correlated motion in this protein suggests that mechanisms of local elastic stretch and bend are recruited for the purpose of creating and controlling allosteric cooperativity. PMID:26338443

Recovery of high-purity metallic Pd from Pd(II)-sorbed biosorbents by incineration.

PubMed

Won, Sung Wook; Lim, Areum; Yun, Yeoung-Sang

2013-06-01

This work reports a direct way to recover metallic palladium with high purity from Pd(II)-sorbed polyethylenimine-modified Corynebacterium glutamicum biosorbent using a combined method of biosorption and incineration. This study is focused on the incineration part which affects the purity of recovered Pd. The incineration temperature and the amount of Pd loaded on the biosorbent were considered as major factors in the incineration process, and their effects were examined. The results showed that both factors significantly affected the enhancement of the recovery efficiency and purity of the recovered Pd. SEM-EDX and XRD analyses were used to confirm that Pd phase existed in the ash. As a result, the recovered Pd was changed from PdO to zero-valent Pd as the incineration temperature was increased from 600 to 900°C. Almost 100% pure metallic Pd was recovered with recovery efficiency above 99.0% under the conditions of 900°C and 136.9 mg/g. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enhanced production of 3-hydroxypropionic acid from glucose via malonyl-CoA pathway by engineered Escherichia coli.

PubMed

Cheng, Zhuan; Jiang, Jiaqi; Wu, Hui; Li, Zhimin; Ye, Qin

2016-01-01

In this study, production of 3-HP via malonyl-CoA was investigated by using metabolically engineered Escherichia coli carrying heterogeneous acetyl-CoA carboxylase (Acc) from Corynebacterium glutamicum and codon-optimized malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus. Three engineered E. coli strains with different host-vector systems were constructed and investigated. The results indicated that the combination of E. coli BL21(DE3) and pET28a was the most efficient host-vector system for 3-HP production, and the highest concentration of 3-HP attained in shake flask cultivation reached 1.80g/L by the strain BE-MDA with induction at 0.25mM IPTG and 25°C, and supplementation of NaHCO3 and biotin. In fed-batch fermentation performed in a 5-L reactor, the concentration of 3-HP achieved 10.08g/L in 36h. Copyright © 2015 Elsevier Ltd. All rights reserved.

Discovery and History of Amino Acid Fermentation.

PubMed

Hashimoto, Shin-Ichi

There has been a strong demand in Japan and East Asia for L-glutamic acid as a seasoning since monosodium glutamate was found to present umami taste in 1907. The discovery of glutamate fermentation by Corynebacterium glutamicum in 1956 enabled abundant and low-cost production of the amino acid, creating a large market. The discovery also prompted researchers to develop fermentative production processes for other L-amino acids, such as lysine. Currently, the amino acid fermentation industry is so huge that more than 5 million metric tons of amino acids are manufactured annually all over the world, and this number continues to grow. Research on amino acid fermentation fostered the notion and skills of metabolic engineering which has been applied for the production of other compounds from renewable resources. The discovery of glutamate fermentation has had revolutionary impacts on both the industry and science. In this chapter, the history and development of glutamate fermentation, including the very early stage of fermentation of other amino acids, are reviewed.

Identification and molecular characterization of Corynebacterium xerosis isolated from a sheep cutaneous abscess: first case report in Mexico.

PubMed

Hernández-León, Fernando; Acosta-Dibarrat, Jorge; Vázquez-Chagoyán, Juan Carlos; Rosas, Pomposo Fernandez; de Oca-Jiménez, Roberto Montes

2016-07-22

Corynebacterium xerosis is a commensal organism found in skin and mucous membranes of humans. It is considered an unusual pathogen, and it is rarely found in human and animal clinical samples. Here we describe the isolation of C. xerosis from a 4-months-old Pelifolk lamb located in Tesistán, central western Mexico. This microorganism should be considered for differential diagnosis in cutaneous abscessed lesions in sheep, as it represents a zoonotic risk factor for human infection in sheep farms. The animal exhibited a hard-consistency, 5 cm diameter abscess, without drainage, in the neck. The presumptive clinical diagnosis was caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis. Samples were obtained by puncture and cultured in 8 % sheep blood agar under microaerophilic conditions. Colonies were non-haemolytic, brown-yellowish and showed microscopic and biochemical features similar to C. pseudotuberculosis, except for the urea test. A multiplex-PCR for the amplification of partial sequences of the pld, rpoB and intergenic fragment from 16S to 23S genes suggested that isolate could be C. xerosis, which was later confirmed by sequencing analysis of the rpoB gene. This study shows for the first time isolation and molecular characterization of C. xerosis from a clinical sample of an ovine cutaneous abscess in Mexico. This finding highlights the need for differential diagnosis of this pathogen in ovine skin abscesses, as well as epidemiological and control studies of this pathogen in sheep farms.

Capsule-Transmitted Gut Symbiotic Bacterium of the Japanese Common Plataspid Stinkbug, Megacopta punctatissima

PubMed Central

Fukatsu, Takema; Hosokawa, Takahiro

2002-01-01

The Japanese common plataspid stinkbug, Megacopta punctatissima, deposits small brown particles, or symbiont capsules, on the underside of the egg mass for the purpose of transmission of symbiotic bacteria to the offspring. We investigated the microbiological aspects of the bacteria contained in the capsule, such as microbial diversity, phylogenetic placement, localization in vivo, and fitness effects on the host insect. Restriction fragment length polymorphism analysis of 16S ribosomal DNA clones revealed that a single bacterial species dominates the microbiota in the capsule. The bacterium was not detected in the eggs but in the capsules, which unequivocally demonstrated that the bacterium is transmitted to the offspring of the insect orally rather than transovarially, through probing of the capsule content. Molecular phylogenetic analysis showed that the bacterium belongs to the γ-subdivision of the Proteobacteria. In adult insects the bacterium was localized in the posterior section of the midgut. Deprivation of the bacterium from the nymphs resulted in retarded development, arrested growth, abnormal body coloration, and other symptoms, suggesting that the bacterium is essential for normal development and growth of the host insect. PMID:11772649

Lysine Fermentation: History and Genome Breeding.

PubMed

Ikeda, Masato

Lysine fermentation by Corynebacterium glutamicum was developed in 1958 by Kyowa Hakko Kogyo Co. Ltd. (current Kyowa Hakko Bio Co. Ltd.) and is the second oldest amino acid fermentation process after glutamate fermentation. The fundamental mechanism of lysine production, discovered in the early stages of the process's history, gave birth to the concept known as "metabolic regulatory fermentation," which is now widely applied to metabolite production. After the development of rational metabolic engineering, research on lysine production first highlighted the need for engineering of the central metabolism from the viewpoints of precursor supply and NADPH regeneration. Furthermore, the existence of active export systems for amino acids was first demonstrated for lysine in C. glutamicum, and this discovery has resulted in the current recognition of such exporters as an important consideration in metabolite production. Lysine fermentation is also notable as the first process to which genomics was successfully applied to improve amino acid production. The first global "genome breeding" strategy was developed using a lysine producer as a model; this has since led to new lysine producers that are more efficient than classical industrial producers. These advances in strain development technology, combined with recent systems-level approaches, have almost achieved the optimization of entire cellular systems as cell factories for lysine production. In parallel, the continuous improvement of the process has resulted not only in fermentation processes with reduced load on downstream processing but also in commercialization of various product forms according to their intended uses. Nowadays lysine fermentation underpins a giant lysine demand of more than 2 million metric tons per year.

Swimming efficiency of bacterium Escherichia coli

PubMed Central

Chattopadhyay, Suddhashil; Moldovan, Radu; Yeung, Chuck; Wu, X. L.

2006-01-01

We use measurements of swimming bacteria in an optical trap to determine fundamental properties of bacterial propulsion. In particular, we directly measure the force required to hold the bacterium in the optical trap and determine the propulsion matrix, which relates the translational and angular velocity of the flagellum to the torques and forces propelling the bacterium. From the propulsion matrix, dynamical properties such as torques, swimming speed, and power can be obtained by measuring the angular velocity of the motor. We find significant heterogeneities among different individuals even though all bacteria started from a single colony. The propulsive efficiency, defined as the ratio of the propulsive power output to the rotary power input provided by the motors, is found to be ≈2%, which is consistent with the efficiency predicted theoretically for a rigid helical coil. PMID:16954194

Application of PCR for detection of toxigenic Corynebacterium diphtheriae strains isolated during the Russian diphtheria epidemic, 1990 through 1994.

PubMed Central

Mikhailovich, V M; Melnikov, V G; Mazurova, I K; Wachsmuth, I K; Wenger, J D; Wharton, M; Nakao, H; Popovic, T

1995-01-01

A total of 250 Corynebacterium diphtheriae isolates from clinical cases and carriers in Russia were assayed by PCR directed at the A subunit of the diphtheria toxin gene to distinguish toxigenic from nontoxigenic strains; 170 strains were positive as indicated by the presence of the 248-bp amplicon. The results of this PCR assay were in complete concordance with those of the standard immunoprecipitation assay (Elek), and the PCR assay is a useful tool for rapid identification in clinical laboratories. PMID:8576378

Coiled to diffuse: Brownian motion of a helical bacterium.

PubMed

Butenko, Alexander V; Mogilko, Emma; Amitai, Lee; Pokroy, Boaz; Sloutskin, Eli

2012-09-11

We employ real-time three-dimensional confocal microscopy to follow the Brownian motion of a fixed helically shaped Leptospira interrogans (LI) bacterium. We extract from our measurements the translational and the rotational diffusion coefficients of this bacterium. A simple theoretical model is suggested, perfectly reproducing the experimental diffusion coefficients, with no tunable parameters. An older theoretical model, where edge effects are neglected, dramatically underestimates the observed rates of translation. Interestingly, the coiling of LI increases its rotational diffusion coefficient by a factor of 5, compared to a (hypothetical) rectified bacterium of the same contour length. Moreover, the translational diffusion coefficients would have decreased by a factor of ~1.5, if LI were rectified. This suggests that the spiral shape of the spirochaete bacteria, in addition to being employed for their active twisting motion, may also increase the ability of these bacteria to explore the surrounding fluid by passive Brownian diffusion.

Increased fertility of Corynebacterium glutamicum recipients in intergeneric matings with Escherichia coli after stress exposure.

PubMed Central

Schäfer, A; Kalinowski, J; Pühler, A

1994-01-01

Corynebacterial recipient cells exposed to heat, organic solvents, pH shifts, or detergents show an increased fertility in subsequent interspecific matings with Escherichia coli. This effect is independent of de novo protein biosynthesis and seems to be due to a direct inactivation of a restriction system active against foreign DNA that enters the cell by IncP-mediated conjugation. Images PMID:8135527

[Partial biological characteristics and algicidal activity of an algicidal bacterium].

PubMed

Li, San-Hua; Zhang, Qi-Ya

2013-02-01

An algicidal bacterium was isolated from freshwater (Lake Donghu in Wuhan) and coded as A01. The morphology of the algicidal bacterium was observed using optical microscope and electron microscopes, the results showed that A01 was rod-shaped, approximately 1.5 microm in length and 0.45 microm in width and with no flagella structure. A01 was Gram-negative and belongs to the family Acinetobacter sp. though identification by Gram's staining and 16S rDNA gene analysis. A01 exhibited strong algicidal activity on the bloom-forming cyanobacterium Anabaena eucompacta under laboratory conditions. The removal rate of chlorophyll a after 7-day incubation with the culture supernatant of A01 and thalli were 77% and 61%, respectively. Microscopic observation showed that almost all cyanobacterial cells were destroyed within 3 d of co-incubation with the supernatant of algicidal bacterium, but a mass of the cyanobacterial cell lysis was observed only after 5 d of co-incubation with the thalli of algicidal bacterium. These results indicated that the main algicidal component of A01 was in its culture supernatant. In other words, the strain A01 could secrete algicidal component against Anabaena eucompacta.

In Vitro Activity of TD-1792, a Multivalent Glycopeptide-Cephalosporin Antibiotic, against 377 Strains of Anaerobic Bacteria and 34 Strains of Corynebacterium Species

PubMed Central

Citron, Diane M.; Warren, Yumi A.; Goldstein, Ellie J. C.

2012-01-01

TD-1792 is a multivalent glycopeptide-cephalosporin heterodimer antibiotic with potent activity against Gram-positive bacteria. We tested TD-1792 against 377 anaerobes and 34 strains of Corynebacterium species. Against nearly all Gram-positive strains, TD-1792 had an MIC90 of 0.25 μg/ml and was typically 3 to 7 dilutions more active than vancomycin and daptomycin. PMID:22290981

Complete genome sequence of Corynebacterium variabile DSM 44702 isolated from the surface of smear-ripened cheeses and insights into cheese ripening and flavor generation

PubMed Central

2011-01-01

Background Corynebacterium variabile is part of the complex microflora on the surface of smear-ripened cheeses and contributes to the development of flavor and textural properties during cheese ripening. Still little is known about the metabolic processes and microbial interactions during the production of smear-ripened cheeses. Therefore, the gene repertoire contributing to the lifestyle of the cheese isolate C. variabile DSM 44702 was deduced from the complete genome sequence to get a better understanding of this industrial process. Results The chromosome of C. variabile DSM 44702 is composed of 3, 433, 007 bp and contains 3, 071 protein-coding regions. A comparative analysis of this gene repertoire with that of other corynebacteria detected 1, 534 predicted genes to be specific for the cheese isolate. These genes might contribute to distinct metabolic capabilities of C. variabile, as several of them are associated with metabolic functions in cheese habitats by playing roles in the utilization of alternative carbon and sulphur sources, in amino acid metabolism, and fatty acid degradation. Relevant C. variabile genes confer the capability to catabolize gluconate, lactate, propionate, taurine, and gamma-aminobutyric acid and to utilize external caseins. In addition, C. variabile is equipped with several siderophore biosynthesis gene clusters for iron acquisition and an exceptional repertoire of AraC-regulated iron uptake systems. Moreover, C. variabile can produce acetoin, butanediol, and methanethiol, which are important flavor compounds in smear-ripened cheeses. Conclusions The genome sequence of C. variabile provides detailed insights into the distinct metabolic features of this bacterium, implying a strong adaption to the iron-depleted cheese surface habitat. By combining in silico data obtained from the genome annotation with previous experimental knowledge, occasional observations on genes that are involved in the complex metabolic capacity of C. variabile

Molecular Characterization of Corynebacterium diphtheriae Outbreak Isolates, South Africa, March-June 2015.

PubMed

du Plessis, Mignon; Wolter, Nicole; Allam, Mushal; de Gouveia, Linda; Moosa, Fahima; Ntshoe, Genevie; Blumberg, Lucille; Cohen, Cheryl; Smith, Marshagne; Mutevedzi, Portia; Thomas, Juno; Horne, Valentino; Moodley, Prashini; Archary, Moherndran; Mahabeer, Yesholata; Mahomed, Saajida; Kuhn, Warren; Mlisana, Koleka; McCarthy, Kerrigan; von Gottberg, Anne

2017-08-01

In 2015, a cluster of respiratory diphtheria cases was reported from KwaZulu-Natal Province in South Africa. By using whole-genome analysis, we characterized 21 Corynebacterium diphtheriae isolates collected from 20 patients and contacts during the outbreak (1 patient was infected with 2 variants of C. diphtheriae). In addition, we included 1 cutaneous isolate, 2 endocarditis isolates, and 2 archived clinical isolates (ca. 1980) for comparison. Two novel lineages were identified, namely, toxigenic sequence type (ST) ST-378 (n = 17) and nontoxigenic ST-395 (n = 3). One archived isolate and the cutaneous isolate were ST-395, suggesting ongoing circulation of this lineage for >30 years. The absence of preexisting molecular sequence data limits drawing conclusions pertaining to the origin of these strains; however, these findings provide baseline genotypic data for future cases and outbreaks. Neither ST has been reported in any other country; this ST appears to be endemic only in South Africa.

Gut bacterium of Dendrobaena veneta (Annelida: Oligochaeta) possesses antimycobacterial activity.

PubMed

Fiołka, Marta J; Zagaja, Mirosław P; Piersiak, Tomasz D; Wróbel, Marek; Pawelec, Jarosław

2010-09-01

The new bacterial strain with antimycobacterial activity has been isolated from the midgut of Dendrobaena veneta (Annelida). Biochemical and molecular characterization of isolates from 18 individuals identified all as Raoultella ornithinolytica genus with 99% similarity. The bacterium is a possible symbiont of the earthworm D. veneta. The isolated microorganism has shown the activity against four strains of fast-growing mycobacteria: Mycobacterium butiricum, Mycobacterium jucho, Mycobacterium smegmatis and Mycobacterium phlei. The multiplication of the gut bacterium on plates with Sauton medium containing mycobacteria has caused a lytic effect. After the incubation of the cell free extract prepared from the gut bacterium with four strains of mycobacteria in liquid Sauton medium, the cells of all tested strains were deformed and divided to small oval forms and sometimes created long filaments. The effect was observed by the use of light, transmission and scanning microscopy. Viability of all examined species of mycobacteria was significantly decreased. The antimycobacterial effect was probably the result of the antibiotic action produced by the gut bacterium of the earthworm. The application of ultrafiltration procedure allowed to demonstrate that antimicrobial substance with strong antimycobacterial activity from bacterial culture supernatant, is a protein with the molecular mass above 100 kDa. Copyright 2010 Elsevier Inc. All rights reserved.

Evidence of increased carriage of Corynebacterium spp. in healthy individuals with low antibody titres against diphtheria toxoid.

PubMed Central

Bergamini, M.; Fabrizi, P.; Pagani, S.; Grilli, A.; Severini, R.; Contini, C.

2000-01-01

This study evaluated whether a correlation exists between carriage of corynebacteria and the lack of immunity to diphtheria toxoid. Samples of both nasal and pharyngeal secretions were taken from 500 apparently healthy subjects of both sexes and of all ages and inoculated onto Tinsdale's medium. A serum sample was also taken for ELISA test to determine the titre of diphtheria toxin antibodies. None of the subjects carried Corynebacterium diphtheriae. Ninety-three strains of Corynebacterium spp. were isolated from 93 subjects and 86 of these were classified to species or group level by biochemical tests. C. xerosis was the most common (25.8%) followed by C. pseudodiphthericum (16.1%), C. jeikeium and C. striatum (both 10.8%), and C. urealyticum (9.7%). Three other species accounted for approximately 20% of strains and seven were unclassified as biochemically atypical corynebacteria. Non-protective antibodies to diphtheria toxin were found in 80 of the 93 subjects and a strong statistical association was demonstrated between carriage of corynebacteria and non-protective levels of anti-toxin antibodies. The remaining 13 subjects had protective levels of antitoxin antibodies. In contrast, only 45 of the 407 non-colonized subjects had non-protective antitoxin titres. The prevalence of carriage increased with age among males as did the percentage of non-protected subjects. The prevalence of female carriers of corynebacteria was significantly lower. Serum samples from 12 subjects with different antibody titres to diphtheria toxoid reacted to varying degrees with whole-cell lysates of a number of species of corynebacteria. The results suggest that a causal relationship may exist between nasopharyngeal carriage of corynebacteria and a low anti-diphtheria toxin immune response. PMID:11057966

Biological effects of Corynebacterium parvum. IV. Adjuvant and inhibitory activities on B lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Howard, J.G.; Christie, G.H.; Scott, M.T.

1973-05-01

The PFC response to the thymus-independent antigen SIII (type 3 pneumococcal polysaccharide) was amplified in mice injected 4 days previously with killed Corynebacterium parvum. This adjuvant activity was demonstrable with high (2 to 50 mu g) but not low (0.1 to 0.5 mu g) doses of SIII. Induction of tolerance was unaffected. Depression of the response resulted from simultaneous injection of SIII with either C. parvum or Bordetella pertussis, while prior treatment with the latter was without effect. Responsiveness to SIII was transiently but potently suppressed in spleen cells transferred into lethally irradiated, C. parvum pretreated mice. Although C. parvummore » is an effective B cell adjuvant, other data imply that it acts indirectly on these lymphocytes. It is argued that both adjuvant and suppressive activities of C. parvum on the B cell response to SIII are most probably mediated by activated macrophages. (auth)« less

Biological effects of Corynebacterium parvum. IV. Adjuvant and inhibitory activities on B lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Howard, J.G.; Christie, G.H.; Scott, M.T.

1973-05-01

The PFC response to the thymus-independent antigen SIII(type 3 pneumococcal polysaccharide) was amplified in mice injected 4 days previously with killed Corynebacterium parvum. This adjuvant activity was demonstrable with high (2 to 50 mu g) but not low (0.1 to 0.5 mu g) doses of SIII. Induction of tolerance was unaffected. Depression of the response resulted from simultaneous injection of SIII with either C. parvum or Bordetella pertussis, while prior treatment with the latter was without effect. Responsiveness to SIII was transiently but potently suppressed in spleen cells transferred into lethally irradiated, C. parvum pretreated mice. Although C. parvum ismore » an effective B cell adjuvant, other data imply that it acts indirectly on these iymphocytes. It is argued that both adjuvant and suppressive activities of C. parvum on the B cell response to SIII are most probably mediated by activated macrophages. (auth)« less

Modeling and experimental design for metabolic flux analysis of lysine-producing Corynebacteria by mass spectrometry.

PubMed

Wittmann, C; Heinzle, E

2001-04-01

Experimental design of (13)C-tracer studies for metabolic flux analysis with mass spectrometric determination of labeling patterns was performed for the central metabolism of Corynebacterium glutamicum comprising various flux scenarios. Ratio measurement of mass isotopomer pools of Corynebacterium products lysine, alanine, and trehalose is sufficient to quantify the flux partitioning ratios (i) between glycolysis and pentose phosphate pathways (Phi(PPP)), (ii) between the split pathways in the lysine biosynthesis (Phi(DH)), (iii) at the pyruvate node (Phi(PC)), and reversibilities of (iv) glucose 6-phosphate isomerase (zeta(PGI)), (v) at the pyruvate node (zeta(PC/PEPCK)), and (vi) of transaldolase and transketolases in the PPP. Weighted sensitivities for flux parameters were derived from partial derivatives to quantitatively evaluate experimental approaches and predict precision for estimated flux parameters. Deviation of intensity ratios from ideal values of 1 was used as weighting function. Weighted flux sensitivities can be used to identify optimal type and degree of tracer labeling or potential intensity ratios to be measured. Experimental design for lysine-producing strain C. glutamicum MH 20-22B (Marx et al., Biotechnol. Bioeng. 49, 111-129, 1996) and various potential mutants with different alterations in the flux pattern showed that specific tracer labelings are optimal to quantify a certain flux parameter uninfluenced by the overall flux situation. Identified substrates of choice are [1-(13)C]glucose for the estimation of Phi(PPP) and zeta(PGI) and a 1 : 1 mixture of [U-(12)C/U-(13)C]glucose for the determination of zeta(PC/PEPCK). Phi(PC) can be quantified by feeding [4-(13)C]glucose or [U-(12)C/U-(13)C]glucose (1 : 1), whereas Phi(DH) is accessible via [4-(13)C]glucose. The sensitivity for the quantification of a certain flux parameter can be influenced by superposition through other flux parameters in the network, but substrate and measured mass

Characterization of the promising poly(3-hydroxybutyrate) producing halophilic bacterium Halomonas halophila.

PubMed

Kucera, Dan; Pernicová, Iva; Kovalcik, Adriana; Koller, Martin; Mullerova, Lucie; Sedlacek, Petr; Mravec, Filip; Nebesarova, Jana; Kalina, Michal; Marova, Ivana; Krzyzanek, Vladislav; Obruca, Stanislav

2018-05-01

This work explores molecular, morphological as well as biotechnological features of the highly promising polyhydroxyalkanoates (PHA) producer Halomonas halophila. Unlike many other halophiles, this bacterium does not require expensive complex media components and it is capable to accumulate high intracellular poly(3-hydroxybutyrate) (PHB) fractions up to 82% of cell dry mass. Most remarkably, regulating the concentration of NaCl apart from PHB yields influences also the polymer's molecular mass and polydispersity. The bacterium metabolizes various carbohydrates including sugars predominant in lignocelluloses and other inexpensive substrates. Therefore, the bacterium was employed for PHB production on hydrolysates of cheese whey, spent coffee grounds, sawdust and corn stover, which were hydrolyzed by HCl; required salinity of cultivation media was set up during neutralization by NaOH. The bacterium was capable to use all the tested hydrolysates as well as sugar beet molasses for PHB biosynthesis, indicating its potential for industrial PHB production. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Study on anti-bacterium activity of ginkgolic acids and their momomers].

PubMed

Yang, Xiaoming; Zhu, Wei; Chen, Jun; Qian, Zhiyu; Xie, Jimin

2004-09-01

Ginkgolic acids and their three monomers were separated from ginkgo sarcotestas. The anti-bacterium activity of ginkgolic acids were tested. The relation between the anti-bacterium activity and side chain of ginkgolic acid were studied. The MIC of ginkgolic acids and their three monomers and salicylic acid were tested. Ginkgolic acid has strong inhibitive effect on G+-bacterium. Salicylic acid has no side chain, so no anti-bacterial activity. When the length of gingkolic acid side chain is C13:0, it has the strongest anti-bacterial activity in three monomers. The side chain of ginkgolic acid is the key functional group that possessed anti-bacterial activity. The length of Ginkgolic acid was the main effective factor of anti-bacterial activity.

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

Bacteriophage-based vectors for site-specific insertion of DNA in the chromosome of Corynebacteria.

PubMed

Oram, Mark; Woolston, Joelle E; Jacobson, Andrew D; Holmes, Randall K; Oram, Diana M

2007-04-15

In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as beta. beta-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally encoded genes, is regulated by the DtxR protein in response to Fe(2+) levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the beta-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae DeltadtxR strain. Additionally, strains of beta-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for beta, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species.

Bacteriophage-based Vectors for Site-specific Insertion of DNA in the Chromosome of Corynebacteria

PubMed Central

Oram, Mark; Woolston, Joelle E.; Jacobson, Andrew D.; Holmes, Randall K.; Oram, Diana M.

2007-01-01

In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as β. β-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally-encoded genes, is regulated by the DtxR protein in response to Fe2+ levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the β-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae ΔdtxR strain. Additionally, strains of β-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for β, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species. PMID:17275217

Endohyphal Bacterium Enhances Production of Indole-3-Acetic Acid by a Foliar Fungal Endophyte

PubMed Central

Hoffman, Michele T.; Gunatilaka, Malkanthi K.; Wijeratne, Kithsiri; Gunatilaka, Leslie; Arnold, A. Elizabeth

2013-01-01

Numerous plant pathogens, rhizosphere symbionts, and endophytic bacteria and yeasts produce the important phytohormone indole-3-acetic acid (IAA), often with profound effects on host plants. However, to date IAA production has not been documented among foliar endophytes -- the diverse guild of primarily filamentous Ascomycota that live within healthy, above-ground tissues of all plant species studied thus far. Recently bacteria that live within hyphae of endophytes (endohyphal bacteria) have been detected, but their effects have not been studied previously. Here we show not only that IAA is produced in vitro by a foliar endophyte (here identified as Pestalotiopsis aff. neglecta, Xylariales), but that IAA production is enhanced significantly when the endophyte hosts an endohyphal bacterium (here identified as Luteibacter sp., Xanthomonadales). Both the endophyte and the endophyte/bacterium complex appear to rely on an L-tryptophan dependent pathway for IAA synthesis. The bacterium can be isolated from the fungus when the symbiotic complex is cultivated at 36°C. In pure culture the bacterium does not produce IAA. Culture filtrate from the endophyte-bacterium complex significantly enhances growth of tomato in vitro relative to controls and to filtrate from the endophyte alone. Together these results speak to a facultative symbiosis between an endophyte and endohyphal bacterium that strongly influences IAA production, providing a new framework in which to explore endophyte-plant interactions. PMID:24086270

Direct measurement of interaction forces between a single bacterium and a flat plate.

PubMed

Klein, Jonah D; Clapp, Aaron R; Dickinson, Richard B

2003-05-15

A technique for precisely measuring the equilibrium and viscous interaction forces between a single bacterium and a flat surface as functions of separation distance is described. A single-beam gradient optical trap was used to micromanipulate the bacterium against a flat surface while evanescent wave light scattering was used to measure separation distances. Calibrating the optical trap far from the surface allowed the trapped bacterium to be used as a force probe. Equilibrium force-distance profiles were determined by measuring the deflection of the cell from the center of the optical trap at various trap positions. Simultaneously, viscous forces were determined by measuring the relaxation time for the fluctuating bacterium. Absolute distances were determined using a best-fit approximation to the theoretical prediction for the hindered mobility of a diffusing sphere near a wall. Using this approach, forces in the range from 0.01 to 4 pN were measured at near-nanometer resolution between Staphylococcus aureus and glass that was bare or coated with adsorbed protein.

Pancreatic abscess caused by Corynebacterium coyleae mimicking malignant neoplasm.

PubMed

Taguchi, Masashi; Nishikawa, Shoichiro; Matsuoka, Hidehiko; Narita, Ryoichi; Abe, Shintaro; Fukuda, Kazumasa; Miyamoto, Hiroshi; Taniguchi, Hatsumi; Otsuki, Makoto

2006-11-01

A 50-year-old female was referred to our hospital because of postprandial epigastric pain and pancreatic head mass. On admission, an elastic hard mass with tenderness was palpable in the epigastric region. Laboratory findings showed no abnormalities, except for a slightly elevated C-reactive protein value and iron deficiency anemia. Serum levels of pancreatic enzymes and tumor markers were also within the reference range. Computed tomography (CT) demonstrated a 5-cm heterogenous mass at the head of the pancreas. Angiography showed that gastroduodenal artery was transformed and narrowed by the mass. Smooth stenosis of portal vein was also observed. Fusion CT-positron emission tomography with 2-deoxy-2-[F]fluoro-D-glucose demonstrated a focus of increased uptake in the pancreatic head mass. We suspected the mass of malignancy but, surprisingly, tumor size was gradually decreased without any therapies. Biopsy specimens from the mass of the pancreas showed marked inflammatory cell infiltration and marked interstitial fibrosis without malignant cells. Thereafter, we could isolate Corynebacterium coyleae from the biopsy specimen. We diagnosed the mass as a pancreatic abscess caused by C. coyleae and started with the intravenous antibiotics therapy. Subsequent follow-up CT and ultrasonography showed dramatic improvement in pancreatic mass. We present here a case of pancreatic abscess which was difficult to differentiate from malignant lesion by various imaging studies. Moreover, we could culture and identify C. coyleae which had never been reported to be the source of pancreatic abscess.

The construction of an engineered bacterium to remove cadmium from wastewater.

PubMed

Chang, S; Shu, H

2014-01-01

The removal of cadmium (Cd) from wastewater before it is released from factories is important for protecting human health. Although some researchers have developed engineered bacteria, the resistance of these engineered bacteria to Cd have not been improved. In this study, two key genes involved in glutathione synthesis (gshA and gshB), a serine acetyltransferase gene (cysE), a Thlaspi caerulescens phytochelatin synthase gene (TcPCS1), and a heavy metal ATPase gene (TcHMA3) were transformed into Escherichia coli BL21. The resistance of the engineered bacterium to Cd was significantly greater than that of the initial bacterium and the Cd accumulation in the engineered bacterium was much higher than in the initial bacterium. In addition, the Cd resistance of the bacteria harboring gshB, gshA, cysE, and TcPCS1 was higher than that of the bacteria harboring gshA, cysE, and TcPCS1. This finding demonstrated that gshB played an important role in glutathione synthesis and that the reaction catalyzed by glutathione synthase was the limiting step for producing phytochelatins. Furthermore, TcPCS1 had a greater specificity and a higher capacity for removing Cd than SpPCS1, and TcHMA3 not only played a role in T. caerulescens but also functioned in E. coli.

Conjugative plasmid in Corynebacterium flaccumfaciens subsp. oortii that confers resistance to arsenite, arsenate, and antimony(III)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hendrick, C.A.; Haskins, W.P.; Vidaver, A.K.

1984-07-01

Gene transfer systems for phytopathogenic corynebacteria have not been reported previously. In this paper a conjugative 46-megadalton plasmid (pDG101) found in Corynebacterium flaccumfaciens subsp. oorii CO101 is described that mediates resistance to arsenite, arsenate, and antimony(III). Transfer of the plasmid from CO101 to four other strains from the C. flaccumfaciens group occurred between cells immobilized on nitrocellulose filters or on agar surfaces. Transconjugant strains expressed the same levels of metal resistance as the donor strain and were able to act as donor strains in subsequent matings. The physical presence of the plasmid was detected by agarose gel electrophoresis. Arsenite-sensitive derivativesmore » of the donor and transconjugant strains were obtained after heat treatment; these were cured of pDG101.« less

Evaluation of the applicability of amplified rDNA-restriction analysis (ARDRA) to identification of species of the genus Corynebacterium.

PubMed

Vaneechoutte, M; Riegel, P; de Briel, D; Monteil, H; Verschraegen, G; De Rouck, A; Claeys, G

1995-10-01

The 16S rRNA genes (rDNA) of 50 strains belonging to 26 different coryneform bacterial species and genomospecies and of the type strain of Rhodococcus equi were enzymatically amplified. Amplified rDNA restriction analysis (ARDRA) with the enzymes AluI, CfoI and RsaI was carried out. The combination of the ARDRA patterns obtained after restriction with these three different enzymes enabled the differentiation between the following species: Corynebacterium accolens (number of strains = 2), C. afermentans subsp. afermentans (2), C. afermentans subsp. lipophilum (2), C. amycolatum (3), CDC coryneform group ANF-1-like (1), CDC coryneform group ANF-3-like (1), C. cystitidis (1), C. diphtheriae (4), C. jeikeium (3), C. macginleyi (2), C. minutissimum (1), C. pilosum (1), C. pseudotuberculosis (2), C. renale (2), C. striatum (2), C. urealyticum (3), C. xerosis (1), CDC coryneform groups B-1 (2), B-3 (2), F-1, genomospecies 1 and 2 (6), G, genomospecies 1 (1) and G, genomospecies 2 (2). The following strains or species could not be differentiated from each other: C. pseudodiphtheriticum (2) from C. propinquum (former CDC coryneform group ANF-3) (2), CDC coryneform group F-1, genomospecies 1 (4) from genomospecies 2 (2) and C. jeikeium genomospecies A (1) from genomospecies C (2). ARDRA may represent a possible alternative for identification of coryneforms, since this technique enabled the identification of most coryneforms tested and since DNA extraction (i.e. cell lysis by boiling), amplification, restriction and electrophoresis can be carried out within 8 hours. This might allow quick identification of C. diphtheriae and other possible pathogens of the genus Corynebacterium.

Metabolomics evaluation of the impact of smokeless tobacco exposure on the oral bacterium Capnocytophaga sputigena

PubMed Central

Sun, Jinchun; Jin, Jinshan; Beger, Richard D.; Cerniglia, Carl E.; Yang, Maocheng; Chen, Huizhong

2017-01-01

The association between exposure to smokeless tobacco products (STP) and oral diseases is partially due to the physiological and pathological changes in the composition of the oral microbiome and its metabolic profile. However, it is not clear how STPs affect the physiology and ecology of oral microbiota. A UPLC/QTof-MS-based metabolomics study was employed to analyze metabolic alterations in oral bacterium, Capnocytophaga sputigena as a result of smokeless tobacco exposure and to assess the capability of the bacterium to metabolize nicotine. Pathway analysis of the metabolome profiles indicated that smokeless tobacco extracts caused oxidative stress in the bacterium. The metabolomics data also showed that the argininenitric oxide pathway was perturbed by the smokeless tobacco treatment. Results also showed that LC/MS was useful in identifying STP constituents and additives, including caffeine and many flavoring compounds. No significant changes in levels of nicotine and its major metabolites were found when C. sputigena was cultured in a nutrient rich medium, although hydroxylnicotine and cotinine N-oxide were detected in the bacterial metabolites suggesting that nicotine metabolism might be present as a minor degradation pathway in the bacterium. Study results provide new insights regarding the physiological and toxicological effects of smokeless tobacco on oral bacterium C. sputigena and associated oral health as well as measuring the ability of the oral bacterium to metabolize nicotine. PMID:27480511

Metabolomics evaluation of the impact of smokeless tobacco exposure on the oral bacterium Capnocytophaga sputigena.

PubMed

Sun, Jinchun; Jin, Jinshan; Beger, Richard D; Cerniglia, Carl E; Yang, Maocheng; Chen, Huizhong

2016-10-01

The association between exposure to smokeless tobacco products (STP) and oral diseases is partially due to the physiological and pathological changes in the composition of the oral microbiome and its metabolic profile. However, it is not clear how STPs affect the physiology and ecology of oral microbiota. A UPLC/QTof-MS-based metabolomics study was employed to analyze metabolic alterations in oral bacterium, Capnocytophaga sputigena as a result of smokeless tobacco exposure and to assess the capability of the bacterium to metabolize nicotine. Pathway analysis of the metabolome profiles indicated that smokeless tobacco extracts caused oxidative stress in the bacterium. The metabolomics data also showed that the arginine-nitric oxide pathway was perturbed by the smokeless tobacco treatment. Results also showed that LC/MS was useful in identifying STP constituents and additives, including caffeine and many flavoring compounds. No significant changes in levels of nicotine and its major metabolites were found when C. sputigena was cultured in a nutrient rich medium, although hydroxylnicotine and cotinine N-oxide were detected in the bacterial metabolites suggesting that nicotine metabolism might be present as a minor degradation pathway in the bacterium. Study results provide new insights regarding the physiological and toxicological effects of smokeless tobacco on oral bacterium C. sputigena and associated oral health as well as measuring the ability of the oral bacterium to metabolize nicotine. Published by Elsevier Ltd.

Near-complete genome sequence of the cellulolytic Bacterium Bacteroides ( Pseudobacteroides) cellulosolvens ATCC 35603

DOE PAGES

Dassa, Bareket; Utturkar, Sagar M.; Hurt, Richard A.; ...

2015-09-24

We report the single-contig genome sequence of the anaerobic, mesophilic, cellulolytic bacterium, Bacteroides cellulosolvens. The bacterium produces a particularly elaborate cellulosome system, whereas the types of cohesin-dockerin interactions are opposite of other known cellulosome systems: cell-surface attachment is thus mediated via type-I interactions whereas enzymes are integrated via type-II interactions.

[A rarely isolated bacterium in microbiology laboratories: Streptococcus uberis].

PubMed

Eryıldız, Canan; Bukavaz, Şebnem; Gürcan, Şaban; Hatipoğlu, Osman

2017-04-01

Streptococcus uberis is a gram-positive bacterium that is mostly responsible for mastitis in cattle. The bacterium rarely has been associated with human infections. Conventional phenotyphic methods can be inadequate for the identification of S.uberis; and in microbiology laboratories S.uberis is confused with the other streptococci and enterococci isolates. Recently, molecular methods are recommended for the accurate identification of S.uberis isolates. The aim of this report is to present a lower respiratory tract infection case caused by S.uberis and the microbiological methods for identification of this bacterium. A 66-year-old male patient with squamous cell lung cancer who received radiotherapy was admitted in our hospital for the control. According to the chest X-Ray, patient was hospitalized with the prediagnosis of ''cavitary tumor, pulmonary abscess''. In the first day of the hospitalization, blood and sputum cultures were drawn. Blood culture was negative, however, Candida albicans was isolated in the sputum culture and it was estimated to be due to oral lesions. After two weeks from the hospitalization, sputum sample was taken from the patient since he had abnormal respiratory sounds and cough complaint. In the Gram stained smear of the sputum there were abundant leucocytes and gram-positive cocci, and S.uberis was isolated in both 5% sheep blood and chocolate agar media. Bacterial identification and antibiotic susceptibility tests were performed by VITEK 2 (Biomerieux, France) and also, the bacterium was identified by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) based VITEK MS system as S.uberis. The isolate was determined susceptible to ampicillin, erythromycin, clindamycin, levofloxacin, linezolid, penicillin, cefotaxime, ceftriaxone, tetracycline and vancomycin. 16S, 23S ribosomal RNA and 16S-23S intergenic spacer gene regions were amplified with specific primers and partial DNA sequence analysis of 16S

Trichloroethylene Biodegradation by a Methane-Oxidizing Bacterium †

PubMed Central

Little, C. Deane; Palumbo, Anthony V.; Herbes, Stephen E.; Lidstrom, Mary E.; Tyndall, Richard L.; Gilmer, Penny J.

1988-01-01

Trichloroethylene (TCE), a common groundwater contaminant, is a suspected carcinogen that is highly resistant to aerobic biodegradation. An aerobic, methane-oxidizing bacterium was isolated that degrades TCE in pure culture at concentrations commonly observed in contaminated groundwater. Strain 46-1, a type I methanotrophic bacterium, degraded TCE if grown on methane or methanol, producing CO2 and water-soluble products. Gas chromatography and 14C radiotracer techniques were used to determine the rate, methane dependence, and mechanism of TCE biodegradation. TCE biodegradation by strain 46-1 appears to be a cometabolic process that occurs when the organism is actively metabolizing a suitable growth substrate such as methane or methanol. It is proposed that TCE biodegradation by methanotrophs occurs by formation of TCE epoxide, which breaks down spontaneously in water to form dichloroacetic and glyoxylic acids and one-carbon products. Images PMID:16347616

Combined radiotherapy and Corynebacterium parvum treatment of rat tumors with different immunogenicity. [X rays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moroson, H.; Stowe, S.; Rotman, M.

1978-01-01

Evidence is presented that combined radiotherapy and Corynebacterium parvum treatment gives better results than radiotherapy alone in rats bearing a chemically-induced highly-immunogenic transplanted fibrosarcoma termed BP 179; however, similar behavior is not observed with either of two weakly-immunogenic mammary carcinomas, 13762 or ME/H. Relative immunogenicity is determined by the ability of immunized rats to reject tumor cell challenge. Both 13762 and ME/H carcinomata grow progressively and metastasize early to the retroperitoneal cavity and lungs if they are left untreated. Local radiotherapy of the primary tumor has no influence on growth of metastases whether it is combined with C. parvum ormore » not. Results of cell-mediated cytotoxicity studies with lymphocytes from BP 179 and ME/H tumor bearing rats treated with radiation or radiation plus C. parvum support the in vivo findings of combined radiotherapy. These data suggest that unlike strongly immunogenic tumors, weakly immunogenic tumors will not respond better to C. parvum combined with radiation therapy.« less

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476.

PubMed

Srivastava, Preeti; Deb, J K

2002-07-02

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

Draft Genome Sequence of the Cellulolytic Bacterium Clostridium papyrosolvens C7 (ATCC 700395).

PubMed

Zepeda, Veronica; Dassa, Bareket; Borovok, Ilya; Lamed, Raphael; Bayer, Edward A; Cate, Jamie H D

2013-09-12

We report the draft genome sequence of the cellulose-degrading bacterium Clostridium papyrosolvens C7, originally isolated from mud collected below a freshwater pond in Massachusetts. This Gram-positive bacterium grows in a mesophilic anaerobic environment with filter paper as the only carbon source, and it has a simple cellulosome system with multiple carbohydrate-degrading enzymes.

Draft Genome Sequence of the Cellulolytic Bacterium Clostridium papyrosolvens C7 (ATCC 700395)

PubMed Central

Zepeda, Veronica; Dassa, Bareket; Borovok, Ilya; Lamed, Raphael; Bayer, Edward A.

2013-01-01

We report the draft genome sequence of the cellulose-degrading bacterium Clostridium papyrosolvens C7, originally isolated from mud collected below a freshwater pond in Massachusetts. This Gram-positive bacterium grows in a mesophilic anaerobic environment with filter paper as the only carbon source, and it has a simple cellulosome system with multiple carbohydrate-degrading enzymes. PMID:24029755

Fast Mechanically Driven Daughter Cell Separation Is Widespread in Actinobacteria.

PubMed

Zhou, Xiaoxue; Halladin, David K; Theriot, Julie A

2016-08-30

Dividing cells of the coccoid Gram-positive bacterium Staphylococcus aureus undergo extremely rapid (millisecond) daughter cell separation (DCS) driven by mechanical crack propagation, a strategy that is very distinct from the gradual, enzymatically driven cell wall remodeling process that has been well described in several rod-shaped model bacteria. To determine if other bacteria, especially those in the same phylum (Firmicutes) or with similar coccoid shapes as S. aureus, might use a similar mechanically driven strategy for DCS, we used high-resolution video microscopy to examine cytokinesis in a phylogenetically wide range of species with various cell shapes and sizes. We found that fast mechanically driven DCS is rather rare in the Firmicutes (low G+C Gram positives), observed only in Staphylococcus and its closest coccoid relatives in the Macrococcus genus, and we did not observe this division strategy among the Gram-negative Proteobacteria In contrast, several members of the high-G+C Gram-positive phylum Actinobacteria (Micrococcus luteus, Brachybacterium faecium, Corynebacterium glutamicum, and Mycobacterium smegmatis) with diverse shapes ranging from coccoid to rod all undergo fast mechanical DCS during cell division. Most intriguingly, similar fast mechanical DCS was also observed during the sporulation of the actinobacterium Streptomyces venezuelae Much of our knowledge on bacterial cytokinesis comes from studying rod-shaped model organisms such as Escherichia coli and Bacillus subtilis Less is known about variations in this process among different bacterial species. While cell division in many bacteria has been characterized to some extent genetically or biochemically, few species have been examined using video microscopy to uncover the kinetics of cytokinesis and daughter cell separation (DCS). In this work, we found that fast (millisecond) DCS is exhibited by species in two independent clades of Gram-positive bacteria and is particularly prevalent

Septicaemia secondary to infection by Corynebacterium macginleyi in an Indian python (Python molurus).

PubMed

Martínez, Jorge; Segura, Pablo; García, David; Aduriz, Gorka; Ibabe, José C; Peris, Bernardo; Corpa, Juan M

2006-09-01

A seven-year-old female Indian python (Python molurus) weighing about 35kg was euthanased after several clinical episodes of stomatitis, pneumonia, ophthalmitis and dystocia over a period of four years. The animal had been maintained in a terrarium in a circus truck at an adequate temperature. During shows, however, the snake was considered to be exposed to stressful conditions for several hours at a time at low temperatures and with noise and bright lights. A post-mortem examination indicated ulcerative stomatitis, osteomyelitis, severe pneumonia and numerous granulomata and multifocal necrosis in stomach and spleen. Corynebacterium macginleyi was isolated in pure culture from the ulcerative stomatitis, and mixed with Stenotrophomonas maltophilia from the lungs and spleen. The findings indicated that the snake had died from a septicaemic process caused by C. macginleyi, probably originating from the stomatitis. The role of S. maltophilia as a secondary agent is discussed. The stress of the circus show and poor husbandry may have predisposed the animal to infection and septicaemia. This is the first report of C. macginleyi causing disease in a snake.

Characteristics of opportunistic species of the Corynebacterium and related coryneforms isolated from different clinical materials.

PubMed

Chudnicka, Alina; Kozioł-Montewka, Maria

2003-01-01

Taking into account the increasing contribution of species, which enter into the composition of purely physiological flora of the organism, of the Corynebacterium type and related coryneforms in opportunistic infections in people, the analysis of strains was made from different clinical materials from patients. Their identification was made on the basis of biochemical properties and their antibiotic sensitivity was characterized. It was found that strains with similar biochemical properties (C.striatum, C.amycolatum ) should be identified by means of genetic methods, all the more that they were isolated from clinically important materials. Out of the examined strains the biggest number of infections were caused by C.pseudodiphtheriticum, next C. striatum/C. amycolatum, Brevibacterium sp., C.propinquum, one: C.afermentans, C.jeikeium, C.group G, C.group F1, C.accolens, C.macqinleyi. The highest sensitivity of isolated strains was to Vancomycin, Teicoplanin and Imipenem.

Overproduction of Hydrogen From an Anaerobic Bacterium

DTIC Science & Technology

2008-12-01

fixation of nitrogen ( Haber - Bosch process), mostly to produce fertilizer. Nitrogenase provides a catalytic alternative to the commercial fixation of...the culture and suggests a uniquely simple hydrogen reactor design based on renewable feedstocks. 1. INTRODUCTION Hydrogen is an ideal... renewable feedstocks. Clostridium phytofermentans is a recently- discovered anaerobic bacterium, reported to possess cellulase enzymes that degrade

A 2-step cooking method of searing and hot water pasteurization to maximize the safety of refrigerated, vacuum packaged, chicken breast meat.

PubMed

Enns, D K; Crandall, P G; O'Bryan, C A; Griffis, C L; Martin, E M

2007-05-01

Americans consume almost 40 kg per capita of chicken each year. Increasing consumption of chicken surpassed pork in 1982 and beef in 1992. The objectives of this study were to examine the effectiveness of a novel, 2-step cooking method of grilling, slicing, vacuum packaging, and hot water pasteurization to inhibit the growth of Listeria monocytogenes in chicken breast meat. Because this study required the use of pilot plant scale pasteurization equipment, Listeria innocua M1, a nonpathogen with slightly greater heat resistance than L. monocytogenes, was used as a surrogate. We first examined the lethal effects of grilling on a boneless skinless chicken breast to mimic cross-contaminated, surface-inoculated Listeria. Searing produced a mean reduction of 2.5 log CFU/g of Listeria and a moisture loss of only 7% (w/w). A 2nd experiment studied the lethal effect of pasteurization of the sliced seared chicken breast. L. innocua M1 inoculated between the slices mimicked contamination in deep muscle. Pasteurization in a 71 degrees C bath (final internal temperature of 66 degrees C) gave an additional 2.3 log CFU/g reduction. L. innocua M1 did not show significant regrowth during a wk of refrigerated storage. The combined 2-step cooking method of searing and pasteurization gave a combined 4.8 log reduction in LI M1. In parallel tests a non-Listeria indicator, Corynebacterium glutamicum, inoculated between sliced, seared chicken, showed a 3 log reduction after pasteurization for 10 min in a 71 degrees C bath compared to 2.3 log reduction of Listeria. Corynebacterium regrowth occurred much faster than did L. innocua M1.

Comparative analysis of the expression level of recombinant ginsenoside-transforming β-glucosidase in GRAS hosts and mass production of the ginsenoside Rh2-Mix

PubMed Central

Siddiqi, Muhammad Zubair; Cui, Chang-Hao; Park, Seul-Ki; Han, Nam Soo; Kim, Sun-Chang

2017-01-01

The ginsenoside Rh2, a pharmaceutically active component of ginseng, is known to have anticancer and antitumor effects. However, white ginseng and red ginseng have extremely low concentrations of Rh2 or Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, and Rh3]. To enhance the production of food-grade ginsenoside Rh2, an edible enzymatic bioconversion technique was developed adopting GRAS host strains. A β-glucosidase (BglPm), which has ginsenoside conversion ability, was expressed in three GRAS host strains (Corynebacterium glutamicum, Saccharomyces cerevisiae and Lactococus lactis) by using a different vector system. Enzyme activity in these three GRAS hosts were 75.4%, 11.5%, and 9.3%, respectively, compared to that in the E. coli pGEX 4T-1 expression system. The highly expressed BglPm_C in C. glutamicum can effectively transform the ginsenoside Rg3-Mix [20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5] to Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, Rh3] using a scaled-up biotransformation reaction, which was performed in a 10-L jar fermenter at pH 6.5/7.0 and 37°C for 24 h. To our knowledge, this is the first report in which 50 g of PPD-Mix (Rb1, Rb2, Rb3, Rc, and Rd) as a starting substrate was converted to ginsenoside Rg3-Mix by acid heat treatment and then 24.5-g Rh2-Mix was obtained by enzymatic transformation of Rg3-Mix through by BglPm_C. Utilization of this enzymatic method adopting a GRAS host could be usefully exploited in the preparation of ginsenoside Rh2-Mix in cosmetics, functional food, and pharmaceutical industries, thereby replacing the E. coli expression system. PMID:28423055

Determination of phenanthrene bioavailability by using a self-dying reporter bacterium: test with model solids and soil.

PubMed

Shin, Doyun; Nam, Kyoungphile

2012-02-20

The present study was conducted to investigate the performance and feasibility of a self-dying reporter bacterium to visualize and quantify phenanthrene bioavailability in soil. The self-dying reporter bacterium was designed to die on the initiation of phenanthrene biodegradation. The viability of the reporter bacterium was determined by a fluorescence live/dead cell staining method and visualized by confocal laser scanning microscopic observation. Phenanthrene was spiked into four types of model solids and a sandy loam. The bioavailability of phenanthrene to the reporter bacterium was remarkably declined with the hydrophobicity of the model solids: essentially no phenanthrene was biodegraded in the presence of 9-nm pores and about 35.8% of initial phenanthrene was biodegraded without pores. Decrease in bioavailability was not evident in the nonporous hydrophilic bead, but a small decrease was observed in the porous hydrophilic bead at 1000 mg/kg of phenanthrene. The fluorescence intensity was commensurate with the extent of phenanthrene biodegradation by the reporter bacterium at the concentration range from 50 to 500 mg/kg. Such a quantitative relationship was also confirmed with a sandy loam spiked up to 1000 mg/kg of phenanthrene. This reporter bacterium may be a useful means to determine phenanthrene bioavailability in soil. Copyright © 2011 Elsevier B.V. All rights reserved.

Phosphate enhances levan production in the endophytic bacterium Gluconacetobacter diazotrophicus Pal5

PubMed Central

Idogawa, Nao; Amamoto, Ryuta; Murata, Kousaku; Kawai, Shigeyuki

2014-01-01

Gluconacetobacter diazotrophicus is a gram-negative and endophytic nitrogen-fixing bacterium that has several beneficial effects in host plants; thus, utilization of this bacterium as a biofertilizer in agriculture may be possible. G. diazotrophicus synthesizes levan, a D-fructofuranosyl polymer with β-(2→6) linkages, as an exopolysaccharide and the synthesized levan improves the stress tolerance of the bacterium. In this study, we found that phosphate enhances levan production by G. diazotrophicus Pal5, a wild type strain that showed a stronger mucous phenotype on solid medium containing 28 mM phosphate than on solid medium containing 7 mM phosphate. A G. diazotrophicus Pal5 levansucrase disruptant showed only a weak mucous phenotype regardless of the phosphate concentration, indicating that the mucous phenotype observed on 28 mM phosphate medium was caused by levan. To our knowledge, this is the first report of the effect of a high concentration of phosphate on exopolysaccharide production. PMID:24717418

[Diversity analysis of desulfuration bacterium from the oxidation ditch of city sewage treatment plant with SO2 gas].

PubMed

Huang, Bing; Zhang, Shi-Ling; Zhang, Jiang-Hong; Ao, Yong; Shi, Zhe

2011-07-01

A group of removing SO2 bacterium was obtained from the oxidation ditch of city sewage treatment plant by inductive domestication over 6 d with low concentration SO2 gas, and they have an ability with biodegradation rate of 888 mg x (L x h)(-1) and a degradation efficiency of 85% during 1.5 h for SO2 dissolved in water with their synergy. The clone library and two phylogenetic trees of the removing SO2 bacterium communities were obtained based on 16S rRNA DNA comparison by DNA extraction of the sample and in situ polymerase chain reaction (PCR). The phylogenetic analysis showed that 8 dominant desulfuration bacterium occupy about 69% of all removing SO2 bacterium, and some of them have a kindred with discovered desulfuration bacterium but not homogeneity, and there are four belong to alpha-Proteobacteria, another four belong to beta-Proteobacteria in them. The gene information about 16S rRNA sequence of the dominant desulfuration bacteria and domestication method provide a basic of looking for or domesticating removing SO2 bacterium for development microbial desulfurization technology of contained SO2 tail gas.

Characterization of a bacterium of the genus Azospirillum from cellulolytic nitrogen-fixing mixed cultures.

PubMed

Wong, P P; Stenberg, N E; Edgar, L

1980-03-01

A bacterium with the taxonomic characteristics of the genus Azospirillum was isolated from celluloytic N2-fixing mixed cultures. Its characteristics fit the descriptions of both Azopirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. It may be a variant strain of A. lipoferum. In mixed cultures with cellulolytic organisms, the bacterium grew and fixed N2 with cellelose as a sole source of energy and carbon. The mixed cultures used cellulose from leaves of wheat (Triticum aestivum L.), corn (Zea mays L.), and big bluestem grass (Andropogon gerardii Vitm). Microaerophilic N2-fixing bacteria of the genus Azospirillum, such as the bacterium we isolated, may be important contributors of fixed N2 in soil with partial anaerobiosis and cellulose decomposition.

Immune-Informatic Analysis and Design of Peptide Vaccine From Multi-epitopes Against Corynebacterium pseudotuberculosis

PubMed Central

Droppa-Almeida, Daniela; Franceschi, Elton; Padilha, Francine Ferreira

2018-01-01

Caseous lymphadenitis (CLA) is a disease caused by Corynebacterium pseudotuberculosis bacteria that affects sheep and goats. The absence of a serologic diagnose is a factor that contributes for the disease dissemination, and due to the formation of granuloma, the treatment is very expensive. Therefore, prophylaxis is the approach with best cost-benefit relation; however, it still lacks an effective vaccine. In this sense, this work seeks to apply bioinformatic tools to design an effective vaccine against CLA, using CP40 protein as standard for the design of immunodominant epitopes, from which a total of 6 sequences were obtained, varying from 10 to 16 amino acid residues. The evaluation of different properties of the vaccines showed that the vaccine is a potent and nonallergenic antigen remaining stable in a wide range of temperatures. The initial tertiary structure of the vaccine was then predicted and a model selected. Later, the process of CP40 protein and TLR2 receptor binding was performed, presenting interaction with this receptor, which plays an important role in the activation of the immune response. PMID:29780242

Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells

PubMed Central

Mandlik, Anjali; Swierczynski, Arlene; Das, Asis; Ton-That, Hung

2010-01-01

Summary Adherence to host tissues mediated by pili is pivotal in the establishment of infection by many bacterial pathogens. Corynebacterium diphtheriae assembles on its surface three distinct pilus structures. The function and the mechanism of how various pili mediate adherence, however, have remained poorly understood. Here we show that the SpaA-type pilus is sufficient for the specific adherence of corynebacteria to human pharyngeal epithelial cells. The deletion of the spaA gene, which encodes the major pilin forming the pilus shaft, abolishes pilus assembly but not adherence to pharyngeal cells. In contrast, adherence is greatly diminished when either minor pilin SpaB or SpaC is absent. Antibodies directed against either SpaB or SpaC block bacterial adherence. Consistent with a direct role of the minor pilins, latex beads coated with SpaB or SpaC protein bind specifically to pharyngeal cells. Therefore, tissue tropism of corynebacteria for pharyngeal cells is governed by specific minor pilins. Importantly, immunoelectron microscopy and immunofluorescence studies reveal clusters of minor pilins that are anchored to cell surface in the absence of a pilus shaft. Thus, the minor pilins may also be cell wall anchored in addition to their incorporation into pilus structures that could facilitate tight binding to host cells during bacterial infection. PMID:17376076

Biofilm Formation by a Metabolically Versatile Bacterium

DTIC Science & Technology

2005-10-02

Rhodopseudomonas palustris is a photosynthetic bacterium that has good potential to be developed as a biocatalyst for the production of hydrogen, a...A for none) Samanta, S. K and C. S. Harwood. 2005. Use of the Rhodopseudomonas palustris genome to identify a single amino acid that contributes to...operon from Rhodopseudomonas palustris mediates dicarboxylic acid degradation and participates in anaerobic benzoate degradation. Microbiology 151

Corynebacterium parvum-induced radiosensitivity and cycling changes of hematopoietic spleen colony-forming units

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maruyama, Y.; Magura, C.; Feola, J.

1977-07-01

Ten days after total-body irradiation with 550 rads of /sup 60/Co, spleen colonies were observed in adult C57BL mice. A change in radiosensitivity induced by Corynebacterium parvum, as measured by increased numbers of colony-forming units that survived the 550 rads, began shortly after C. parvum stimulation and extended for at least 7 days before irradiation. C. parvum given 4-24 hours before, followed by high specific activity (/sup 3/H)thymidine (HSATT) 1 hour before total-body irradiation greatly reduced survival of the stem cells that formed spleen colonies (CFU/sub s/) and CFU/sub s/ radiosensitivity to control levels. The HSATT sensitivity by ''suicide'' assaymore » in vivo and the time-response change in radiosensitivity corresponded with the decrease in radiosensitivity, which showed that CFU/sub s/ were stimulated by C. parvum administration and entered the S-phase shortly after stimulation. The data indicated a resting population close to the S-phase. After stimulation, this population entered S-phase. Syngeneic mouse lymphoma cells injected iv 24 hours earlier did not elicit any effect as a stimulus to CFU/sub s/ radiosensitivity change.« less

Boron nitride nanotube-based biosensing of various bacterium/viruses: continuum modelling-based simulation approach.

PubMed

Panchal, Mitesh B; Upadhyay, Sanjay H

2014-09-01

In this study, the feasibility of single walled boron nitride nanotube (SWBNNT)-based biosensors has been ensured considering the continuum modelling-based simulation approach, for mass-based detection of various bacterium/viruses. Various types of bacterium or viruses have been taken into consideration at the free-end of the cantilevered configuration of the SWBNNT, as a biosensor. Resonant frequency shift-based analysis has been performed with the adsorption of various bacterium/viruses considered as additional mass to the SWBNNT-based sensor system. The continuum mechanics-based analytical approach, considering effective wall thickness has been considered to validate the finite element method (FEM)-based simulation results, based on continuum volume-based modelling of the SWBNNT. As a systematic analysis approach, the FEM-based simulation results are found in excellent agreement with the analytical results, to analyse the SWBNNTs for their wide range of applications such as nanoresonators, biosensors, gas-sensors, transducers and so on. The obtained results suggest that by using the SWBNNT of smaller size the sensitivity of the sensor system can be enhanced and detection of the bacterium/virus having mass of 4.28 × 10⁻²⁴ kg can be effectively performed.

Effect of arsenite-oxidizing bacterium B. laterosporus on arsenite toxicity and arsenic translocation in rice seedlings.

PubMed

Yang, Gui-Di; Xie, Wan-Ying; Zhu, Xi; Huang, Yi; Yang, Xiao-Jun; Qiu, Zong-Qing; Lv, Zhen-Mao; Wang, Wen-Na; Lin, Wen-Xiong

2015-10-01

Arsenite [As (III)] oxidation can be accelerated by bacterial catalysis, but the effects of the accelerated oxidation on arsenic toxicity and translocation in rice plants are poorly understood. Herein we investigated how an arsenite-oxidizing bacterium, namely Brevibacillus laterosporus, influences As (III) toxicity and translocation in rice plants. Rice seedlings of four cultivars, namely Guangyou Ming 118 (GM), Teyou Hang II (TH), Shanyou 63 (SY) and Minghui 63 (MH), inoculated with or without the bacterium were grown hydroponically with As (III) to investigate its effects on arsenic toxicity and translocation in the plants. Percentages of As (III) oxidation in the solutions with the bacterium (100%) were all significantly higher than those without (30-72%). The addition of the bacterium significantly decreased As (III) concentrations in SY root, GM root and shoot, while increased the As (III) concentrations in the shoot of SY, MH and TH and in the root of MH. Furthermore, the As (III) concentrations in the root and shoot of SY were both the lowest among the treatments with the bacterium. On the other hand, its addition significantly alleviated the As (III) toxicity on four rice cultivars. Among the treatments amended with B. laterosporus, the bacterium showed the best remediation on SY seedlings, with respect to the subdued As (III) toxicity and decreased As (III) concentration in its roots. These results indicated that As (III) oxidation accelerated by B. laterosporus could be an effective method to alleviate As (III) toxicity on rice seedlings. Copyright © 2015 Elsevier Inc. All rights reserved.

A comparative study on phyllosphere nitrogen fixation by newly isolated Corynebacterium sp. & Flavobacterium sp. and their potentialities as biofertilizer.

PubMed

Giri, S; Pati, B R

2004-01-01

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

Characterization of a novel extremely alkalophilic bacterium

NASA Technical Reports Server (NTRS)

Souza, K. A.; Deal, P. H.

1977-01-01

A new alkalophilic bacterium, isolated from a natural spring of high pH is characterized. It is a Gram-positive, non-sporulating, motile rod requiring aerobic and alkaline conditions for growth. The characteristics of this organism resemble those of the coryneform group of bacteria; however, there are no accepted genera within this group with which this organism can be closely matched. Therefore, a new genus may be warranted.

Extracellular nucleic acids of the marine bacterium Rhodovulum sulfidophilum and recombinant RNA production technology using bacteria.

PubMed

Kikuchi, Yo; Umekage, So

2018-02-01

Extracellular nucleic acids of high molecular weight are detected ubiquitously in seawater. Recent studies have indicated that these nucleic acids are, at least in part, derived from active production by some bacteria. The marine bacterium Rhodovulum sulfidophilum is one of those bacteria. Rhodovulumsulfidophilum is a non-sulfur phototrophic marine bacterium that is known to form structured communities of cells called flocs, and to produce extracellular nucleic acids in culture media. Recently, it has been revealed that this bacterium produces gene transfer agent-like particles and that this particle production may be related to the extracellular nucleic acid production mechanism. This review provides a summary of recent physiological and genetic studies of these phenomena and also introduces a new method for extracellular production of artificial and biologically functional RNAs using this bacterium. In addition, artificial RNA production using Escherichia coli, which is related to this topic, will also be described. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Complete Genome Sequence of the p-Nitrophenol-Degrading Bacterium Pseudomonas putida DLL-E4

PubMed Central

Hu, Xiaojun; Wang, Jue; Wang, Fei; Chen, Qiongzhen; Huang, Yan

2014-01-01

The first complete genome sequence of a p-nitrophenol (PNP)-degrading bacterium is reported here. Pseudomonas putida DLL-E4, a Gram-negative bacterium isolated from methyl-parathion-polluted soil, can utilize PNP as the sole carbon and nitrogen source. P. putida DLL-E4 has a 6,484,062 bp circular chromosome that contains 5,894 genes, with a G+C content of 62.46%. PMID:24948765

Pathogenicity of Moraxella osloensis, a bacterium associated with the nematode Phasmarhabditis hermaphrodita, to the slug Deroceras reticulatum.

PubMed

Tan, L; Grewal, P S

2001-11-01

Moraxella osloensis, a gram-negative bacterium, is associated with Phasmarhabditis hermaphrodita, a nematode parasite of slugs. This bacterium-feeding nematode has potential for the biological control of slugs, especially the grey garden slug, Deroceras reticulatum. Infective juveniles of P. hermaphrodita invade the shell cavity of the slug, develop into self-fertilizing hermaphrodites, and produce progeny, resulting in host death. However, the role of the associated bacterium in the pathogenicity of the nematode to the slug is unknown. We discovered that M. osloensis alone is pathogenic to D. reticulatum after injection into the shell cavity or hemocoel of the slug. The bacteria from 60-h cultures were more pathogenic than the bacteria from 40-h cultures, as indicated by the higher and more rapid mortality of the slugs injected with the former. Coinjection of penicillin and streptomycin with the 60-h bacterial culture reduced its pathogenicity to the slug. Further work suggested that the reduction and loss of pathogenicity of the aged infective juveniles of P. hermaphrodita to D. reticulatum result from the loss of M. osloensis from the aged nematodes. Also, axenic J1/J2 nematodes were nonpathogenic after injection into the shell cavity. Therefore, we conclude that the bacterium is the sole killing agent of D. reticulatum in the nematode-bacterium complex and that P. hermaphrodita acts only as a vector to transport the bacterium into the shell cavity of the slug. The identification of the toxic metabolites produced by M. osloensis is being pursued.

Halomonas maura is a physiologically versatile bacterium of both ecological and biotechnological interest.

PubMed

Llamas, Inmaculada; del Moral, Ana; Martínez-Checa, Fernando; Arco, Yolanda; Arias, Soledad; Quesada, Emilia

2006-01-01

Halomonas maura is a bacterium of great metabolic versatility. We summarise in this work some of the properties that make it a very interesting microorganism both from an ecological and biotechnological point of view. It plays an active role in the nitrogen cycle, is capable of anaerobic respiration in the presence of nitrate and has recently been identified as a diazotrophic bacterium. Of equal interest is mauran, the exopolysaccharide produced by H. maura, which contributes to the formation of biofilms and thus affords the bacterium advantages in the colonisation of its saline niches. Mauran is highly viscous, shows thixotropic and pseudoplastic behaviour, has the capacity to capture heavy metals and exerts a certain immunomodulator effect in medicine. All these attributes have prompted us to make further investigations into its molecular characteristics. To date we have described 15 open reading frames (ORF's) related to exopolysaccharide production, nitrogen fixation and nitrate reductase activity among others.

Deinococcus mumbaiensis sp. nov., a radiation-resistant pleomorphic bacterium isolated from Mumbai, India.

PubMed

Shashidhar, Ravindranath; Bandekar, Jayant R

2006-01-01

A radiation-resistant, Gram-negative and pleomorphic bacterium (CON-1) was isolated from a contaminated tryptone glucose yeast extract agar plate in the laboratory. It was red pigmented, nonmotile, nonsporulating, and aerobic, and contained MK-8 as respiratory quinone. The cell wall of this bacterium contained ornithine. The major fatty acids were C16:0, C16:1, C17:0, C18:1 and iso C18:0. The DNA of CON-1 had a G+C content of 70 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that CON-1 exhibited a maximum similarity (94.72%) with Deinococcus grandis. Based on the genotypic, phenotypic and chemotaxonomic characteristics, the bacterium CON-1 was identified as a new species of the genus Deinococcus, for which the name Deinococcus mumbaiensis sp. nov. is proposed. The type strain of D. mumbaiensis is CON-1 (MTCC 7297(T)=DSM 17424(T)).

From Genome to Function: Systematic Analysis of the Soil Bacterium Bacillus Subtilis

PubMed Central

Crawshaw, Samuel G.; Wipat, Anil

2001-01-01

Bacillus subtilis is a sporulating Gram-positive bacterium that lives primarily in the soil and associated water sources. Whilst this bacterium has been studied extensively in the laboratory, relatively few studies have been undertaken to study its activity in natural environments. The publication of the B. subtilis genome sequence and subsequent systematic functional analysis programme have provided an opportunity to develop tools for analysing the role and expression of Bacillus genes in situ. In this paper we discuss analytical approaches that are being developed to relate genes to function in environments such as the rhizosphere. PMID:18628943

Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Gary; Dalin, Eileen; Tice, Hope

Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 C and pH 5.0 and fer-ments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this sporogenic lactic acid bacterium to grow at 50-55 C and pH 5.0 makes this organism an attractive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemi-cellulose. This bacterium is also considered as a potential probiotic. Complete genome squence of a representative strain, B. coagulans strain 36D1, is presented and discussed.

Genome Sequence of the Soil Bacterium Janthinobacterium sp. KBS0711

PubMed Central

Shoemaker, William R.; Muscarella, Mario E.

2015-01-01

We present a draft genome of Janthinobacterium sp. KBS0711 that was isolated from agricultural soil. The genome provides insight into the ecological strategies of this bacterium in free-living and host-associated environments. PMID:26089434

New lipopeptide produced by Corynebacterium aquaticum from a low-cost substrate.

PubMed

Martins, Paola Chaves; Bastos, Cibele Garcia; Granjeiro, Paulo Afonso; Martins, Vilásia Guimarães

2018-04-26

Conventional biosurfactants have high production costs. Therefore, the use of low-cost carbon sources for their production is attractive for industry. The ability to remain stable under various environmental conditions further extends industrial application. Here we aimed to evaluate the stability of a new lipopeptide produced by Corynebacterium aquaticum using fish residue as an unconventional energy source. The biosurfactant was produced using 3% fish residue, 2% of the microorganism, and mineral medium. Biosurfactant characterization was performed by thin layer chromatography (TLC), as well as by testing its infrared, surface tension, emulsifying activity, and ionic character. The stability of the biosurfactant was evaluated by testing its surface tension at a range of temperatures, pH, and saline concentrations, as well as after 6 months of storage. The biosurfactant was characterized as a lipopeptide due to its retention time, which was coincident with the amino acid and lipid chains obtained in the TLC analysis, being confirmed by some regions of absorption verified in the infrared analysis. The surface tension and emulsifying activity of the biosurfactant were 27.8 mN/m and 87.6%, respectively, and showed anionic character. The biosurfactant was stable at temperatures of 20 to 121 °C, in saline concentrations of 1 to 7%, and at pH close to neutrality. Based on our findings, it is possible to use unconventional sources of energy to produce a lipopeptide biosurfactant that can act under various environments.

Structure and morphology of magnetite anaerobically-produced by a marine magnetotactic bacterium and a dissimilatory iron-reducing bacterium

USGS Publications Warehouse

Sparks, N.H.C.; Mann, S.; Bazylinski, D.A.; Lovley, D.R.; Jannasch, H.W.; Frankel, R.B.

1990-01-01

Intracellular crystals of magnetite synthesized by cells of the magnetotactic vibroid organism, MV-1, and extracellular crystals of magnetite produced by the non-magnetotactic dissimilatory iron-reducing bacterium strain GS-15, were examined using high-resolution transmission electron microscopy, electron diffraction and 57Fe Mo??ssbauer spectroscopy. The magnetotactic bacterium contained a single chain of approximately 10 crystals aligned along the long axis of the cell. The crystals were essentially pure stoichiometric magnetite. When viewed along the crystal long axis the particles had a hexagonal cross-section whereas side-on they appeared as rectangules or truncated rectangles of average dimension, 53 ?? 35 nm. These findings are explained in terms of a three-dimensional morphology comprising a hexagonal prism of {110} faces which are capped and truncated by {111} end faces. Electron diffraction and lattice imaging studies indicated that the particles were structurally well-defined single crystals. In contrast, magnetite particles produced by the strain, GS-15 were irregular in shape and had smaller mean dimensions (14 nm). Single crystals were imaged but these were not of high structural perfection. These results highlight the influence of intracellular control on the crystallochemical specificity of bacterial magnetites. The characterization of these crystals is important in aiding the identification of biogenic magnetic materials in paleomagnetism and in studies of sediment magnetization. ?? 1990.

Transcriptome analysis of the rhizosphere bacterium Azospirillum brasilense reveals an extensive auxin response.

PubMed

Van Puyvelde, Sandra; Cloots, Lore; Engelen, Kristof; Das, Frederik; Marchal, Kathleen; Vanderleyden, Jos; Spaepen, Stijn

2011-05-01

The rhizosphere bacterium Azospirillum brasilense produces the auxin indole-3-acetic acid (IAA) through the indole-3-pyruvate pathway. As we previously demonstrated that transcription of the indole-3-pyruvate decarboxylase (ipdC) gene is positively regulated by IAA, produced by A. brasilense itself or added exogenously, we performed a microarray analysis to study the overall effects of IAA on the transcriptome of A. brasilense. The transcriptomes of A. brasilense wild-type and the ipdC knockout mutant, both cultured in the absence and presence of exogenously added IAA, were compared.Interfering with the IAA biosynthesis/homeostasis in A. brasilense through inactivation of the ipdC gene or IAA addition results in much broader transcriptional changes than anticipated. Based on the multitude of changes observed by comparing the different transcriptomes, we can conclude that IAA is a signaling molecule in A. brasilense. It appears that the bacterium, when exposed to IAA, adapts itself to the plant rhizosphere, by changing its arsenal of transport proteins and cell surface proteins. A striking example of adaptation to IAA exposure, as happens in the rhizosphere, is the upregulation of a type VI secretion system (T6SS) in the presence of IAA. The T6SS is described as specifically involved in bacterium-eukaryotic host interactions. Additionally, many transcription factors show an altered regulation as well, indicating that the regulatory machinery of the bacterium is changing.

Analysis of corynomycolic acids and other fatty acids produced by Corynebacterium lepus grown on kerosene.

PubMed Central

Cooper, D G; Zajic, J E; Gracey, D E

1979-01-01

The saponifiable carboxylic acids of the extracellular product of Corynebacterium lepus grown on kerosene have been isolated and characterized. About 25% of these acids were a mixture of simple, saturated fatty acids ranging from C13 to C24 and including both even and odd homologues. The distribution of these acids was bimodal, with maxima at C15 and C21. The other 75% of the acids was a mixture of corynomycolic acids [R1--CH(OH)--CH(R2)--COOH] ranging from C28 to C43. The R1 alkyl fragments varied from C16 to C25, and R2 fragments varied from C6 to C14. Both even and odd corynomycolic acid homologues were observed, and the distribution had a single pronounced maximum at C32 and C33. Bacterial utilization of the carboxylic oxidation products of the kerosene substrate is suggested to account for the wide distribution in chain length of these saturated fatty acids and for the observation of both even and odd homologues. PMID:422512

Pathogenicity of Moraxella osloensis, a Bacterium Associated with the Nematode Phasmarhabditis hermaphrodita, to the Slug Deroceras reticulatum

PubMed Central

Tan, Li; Grewal, Parwinder S.

2001-01-01

Moraxella osloensis, a gram-negative bacterium, is associated with Phasmarhabditis hermaphrodita, a nematode parasite of slugs. This bacterium-feeding nematode has potential for the biological control of slugs, especially the grey garden slug, Deroceras reticulatum. Infective juveniles of P. hermaphrodita invade the shell cavity of the slug, develop into self-fertilizing hermaphrodites, and produce progeny, resulting in host death. However, the role of the associated bacterium in the pathogenicity of the nematode to the slug is unknown. We discovered that M. osloensis alone is pathogenic to D. reticulatum after injection into the shell cavity or hemocoel of the slug. The bacteria from 60-h cultures were more pathogenic than the bacteria from 40-h cultures, as indicated by the higher and more rapid mortality of the slugs injected with the former. Coinjection of penicillin and streptomycin with the 60-h bacterial culture reduced its pathogenicity to the slug. Further work suggested that the reduction and loss of pathogenicity of the aged infective juveniles of P. hermaphrodita to D. reticulatum result from the loss of M. osloensis from the aged nematodes. Also, axenic J1/J2 nematodes were nonpathogenic after injection into the shell cavity. Therefore, we conclude that the bacterium is the sole killing agent of D. reticulatum in the nematode-bacterium complex and that P. hermaphrodita acts only as a vector to transport the bacterium into the shell cavity of the slug. The identification of the toxic metabolites produced by M. osloensis is being pursued. PMID:11679319

Understanding the interaction between an obligate hyperparasitic bacterium, Pasteuria penetrans and its obligate plant-parasitic nematode host, Meloidogyne spp.

PubMed

Davies, Keith G

2009-01-01

Pasteuria penetrans is an endospore-forming bacterium, which is a hyperparasite of root-knot nematodes Meloidogyne spp. that are economically important pests of a wide range of crops. The life cycle of the bacterium and nematode are described with emphasis on the bacterium's potential as a biocontrol agent. Two aspects that currently prohibit the commercial development of the bacterium as a biocontrol agent are the inability to culture it outside its host and its host specificity. Vegetative growth of the bacterium is possible in vitro; however, getting the vegetative stages of the bacterium to enter sporogenesis has been problematic. Insights from genomic survey sequences regarding the role of cation concentration and the phosphorylation of Spo0F have proved useful in inducing vegetative bacteria to sporulate. Similarly, genomic data have also proved useful in understanding the attachment of endospores to the cuticle of infective nematode juveniles, and a Velcro-like model of spore attachment is proposed that involves collagen-like fibres on the surface of the endospore interacting with mucins on the nematode cuticle. Ecological studies of the interactions between Daphnia and Pasteuria ramosa are examined and similarities are drawn between the co-evolution of virulence in the Daphnia system and that of plant-parasitic nematodes.

Description of a bacterium associated with redmouth disease of rainbow trout (Salmo gairdneri)

USGS Publications Warehouse

Ross, A.J.; Rucker, R.R.; Ewing, W.H.

1966-01-01

A description was given of a gram-negative, peritrichously flagellated, fermentative bacterium that was isolated on numerous occasions from kidney tissues of rainbow trout (Salmo gairdneri) afflicted with redmouth disease. Although the bacteria apparently were members of the family Enterobacteriaceae, it was impossible to determine their taxonomic position within the family with certainty. Hence it was recommended that their taxonomic position remain sub judice for the present. As a temporary designation RM bacterium was used. Redmouth disease was transmitted from infected to normal fish through the medium of water.

Cadherin Domains in the Polysaccharide-Degrading Marine Bacterium Saccharophagus degradans 2-40 Are Carbohydrate-Binding Modules▿

PubMed Central

Fraiberg, Milana; Borovok, Ilya; Bayer, Edward A.; Weiner, Ronald M.; Lamed, Raphael

2011-01-01

The complex polysaccharide-degrading marine bacterium Saccharophagus degradans strain 2-40 produces putative proteins that contain numerous cadherin and cadherin-like domains involved in intercellular contact interactions. The current study reveals that both domain types exhibit reversible calcium-dependent binding to different complex polysaccharides which serve as growth substrates for the bacterium. PMID:21036994

MmpL Genes Are Associated with Mycolic Acid Metabolism in Mycobacteria and Corynebacteria

PubMed Central

Varela, Cristian; Rittmann, Doris; Singh, Albel; Krumbach, Karin; Bhatt, Kiranmai; Eggeling, Lothar; Besra, Gurdyal S.; Bhatt, Apoorva

2012-01-01

Summary Mycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development. PMID:22520756

MmpL genes are associated with mycolic acid metabolism in mycobacteria and corynebacteria.

PubMed

Varela, Cristian; Rittmann, Doris; Singh, Albel; Krumbach, Karin; Bhatt, Kiranmai; Eggeling, Lothar; Besra, Gurdyal S; Bhatt, Apoorva

2012-04-20

Mycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development. Copyright © 2012 Elsevier Ltd. All rights reserved.

CoryneRegNet 3.0--an interactive systems biology platform for the analysis of gene regulatory networks in corynebacteria and Escherichia coli.

PubMed

Baumbach, Jan; Wittkop, Tobias; Rademacher, Katrin; Rahmann, Sven; Brinkrolf, Karina; Tauch, Andreas

2007-04-30

CoryneRegNet is an ontology-based data warehouse for the reconstruction and visualization of transcriptional regulatory interactions in prokaryotes. To extend the biological content of CoryneRegNet, we added comprehensive data on transcriptional regulations in the model organism Escherichia coli K-12, originally deposited in the international reference database RegulonDB. The enhanced web interface of CoryneRegNet offers several types of search options. The results of a search are displayed in a table-based style and include a visualization of the genetic organization of the respective gene region. Information on DNA binding sites of transcriptional regulators is depicted by sequence logos. The results can also be displayed by several layouters implemented in the graphical user interface GraphVis, allowing, for instance, the visualization of genome-wide network reconstructions and the homology-based inter-species comparison of reconstructed gene regulatory networks. In an application example, we compare the composition of the gene regulatory networks involved in the SOS response of E. coli and Corynebacterium glutamicum. CoryneRegNet is available at the following URL: http://www.cebitec.uni-bielefeld.de/groups/gi/software/coryneregnet/.

Experimental determination of group flux control coefficients in metabolic networks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simpson, T.W.; Shimizu, Hiroshi; Stephanopoulos, G.

1998-04-20

Grouping of reactions around key metabolite branch points can facilitate the study of metabolic control of complex metabolic networks. This top-down Metabolic Control Analysis is exemplified through the introduction of group control coefficients whose magnitudes provide a measure of the relative impact of each reaction group on the overall network flux, as well as on the overall network stability, following enzymatic amplification. In this article, the authors demonstrate the application of previously developed theory to the determination of group flux control coefficients. Experimental data for the changes in metabolic fluxes obtained in response to the introduction of six different environmentalmore » perturbations are used to determine the group flux control coefficients for three reaction groups formed around the phosphoenolpyruvate/pyruvate branch point. The consistency of the obtained group flux control coefficient estimates is systematically analyzed to ensure that all necessary conditions are satisfied. The magnitudes of the determined control coefficients suggest that the control of lysine production flux in Corynebacterium glutamicum cells at a growth base state resides within the lysine biosynthetic pathway that begins with the PEP/PYR carboxylation anaplorotic pathway.« less

Cloning and sequence analysis of the meso-diaminopimelate decarboxylase gene from Bacillus methanolicus MGA3 and comparison to other decarboxylase genes.

PubMed

Mills, D A; Flickinger, M C

1993-09-01

The lysA gene of Bacillus methanolicus MGA3 was cloned by complementation of an auxotrophic Escherichia coli lysA22 mutant with a genomic library of B. methanolicus MGA3 chromosomal DNA. Subcloning localized the B. methanolicus MGA3 lysA gene into a 2.3-kb SmaI-SstI fragment. Sequence analysis of the 2.3-kb fragment indicated an open reading frame encoding a protein of 48,223 Da, which was similar to the meso-diaminopimelate (DAP) decarboxylase amino acid sequences of Bacillus subtilis (62%) and Corynebacterium glutamicum (40%). Amino acid sequence analysis indicated several regions of conservation among bacterial DAP decarboxylases, eukaryotic ornithine decarboxylases, and arginine decarboxylases, suggesting a common structural arrangement for positioning of substrate and the cofactor pyridoxal 5'-phosphate. The B. methanolicus MGA3 DAP decarboxylase was shown to be a dimer (M(r) 86,000) with a subunit molecular mass of approximately 50,000 Da. This decarboxylase is inhibited by lysine (Ki = 0.93 mM) with a Km of 0.8 mM for DAP. The inhibition pattern suggests that the activity of this enzyme in lysine-overproducing strains of B. methanolicus MGA3 may limit lysine synthesis.

Metabolic engineering of a laboratory-evolved Thermobifida fusca muC strain for malic acid production on cellulose and minimal treated lignocellulosic biomass.

PubMed

Deng, Yu; Mao, Yin; Zhang, Xiaojuan

2016-01-01

Malic acid is mainly used as an acidulant and taste enhancer in the beverage and food industry. Previously, a mutant strain Thermobifida fusca muC, obtained by adaptive evolution was found to accumulate malic acid on cellulose with low yield. In this study, the malic acid synthesis pathway in T. fusca muC was confirmed to be from phosphoenolpyruvate to oxaloacetate, followed by reduction of oxaloacetate to malate. To increase the yield of malic acid by the muC strain significantly, the carbon flux from pyruvate was redirected to oxaloacetate by expressing an exogenous pyruvate carboxylase (PCx) gene from Corynebacterium glutamicum ATCC 13032 in the chromosome of T. fusca muC-16. The yield of malic acid in the engineered strain muC-16 was increased by 47.9% compared to the parent strain muC. The muC-16 strain was then grown on ∼100 g/L cellulose and the highest titer of malic acid was 62.76 g/L by batch fermentation. T. fusca muC-16 strain converted milled corn stover to malic acid with the highest titer of 21.47 g/L with minimal treatment. © 2016 American Institute of Chemical Engineers.

Amino acids production focusing on fermentation technologies - A review.

PubMed

D'Este, Martina; Alvarado-Morales, Merlin; Angelidaki, Irini

Amino acids are attractive and promising biochemicals with market capacity requirements constantly increasing. Their applicability ranges from animal feed additives, flavour enhancers and ingredients in cosmetic to specialty nutrients in pharmaceutical and medical fields. This review gives an overview of the processes applied for amino acids production and points out the main advantages and disadvantages of each. Due to the advances made in the genetic engineering techniques, the biotechnological processes, and in particular the fermentation with the aid of strains such as Corynebacterium glutamicum or Escherichia coli, play a significant role in the industrial production of amino acids. Despite the numerous advantages of the fermentative amino acids production, the process still needs significant improvements leading to increased productivity and reduction of the production costs. Although the production processes of amino acids have been extensively investigated in previous studies, a comprehensive overview of the developments in bioprocess technology has not been reported yet. This review states the importance of the fermentation process for industrial amino acids production, underlining the strengths and the weaknesses of the process. Moreover, the potential of innovative approaches utilizing macro and microalgae or bacteria are presented. Copyright © 2017 Elsevier Inc. All rights reserved.

Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rhee, Mun Su; Moritz, Brelan E.; Xie, Gary

Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 C and pH 5.0 and fer- ments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this spo- rogenic lactic acid bacterium to grow at 50-55 C and pH 5.0 makes this organism an attrac- tive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemi- cellulose. This bacterium is also considered as a potential probiotic. Complete genome se- quence of a representative strain, B. coagulans strainmore » 36D1, is presented and discussed.« less

Complete Genome Sequence of a thermotolerant sporogenic lactic acid bacterium, Bacillus coagulans strain 36D1

PubMed Central

Rhee, Mun Su; Moritz, Brélan E.; Xie, Gary; Glavina del Rio, T.; Dalin, E.; Tice, H.; Bruce, D.; Goodwin, L.; Chertkov, O.; Brettin, T.; Han, C.; Detter, C.; Pitluck, S.; Land, Miriam L.; Patel, Milind; Ou, Mark; Harbrucker, Roberta; Ingram, Lonnie O.; Shanmugam, K. T.

2011-01-01

Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 °C and pH 5.0 and ferments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this sporogenic lactic acid bacterium to grow at 50-55 °C and pH 5.0 makes this organism an attractive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemicellulose. This bacterium is also considered as a potential probiotic. Complete genome sequence of a representative strain, B. coagulans strain 36D1, is presented and discussed. PMID:22675583

Paradigms: examples from the bacterium Xylella fastidiosa.

PubMed

Purcell, Alexander

2013-01-01

The history of advances in research on Xylella fastidiosa provides excellent examples of how paradigms both advance and limit our scientific understanding of plant pathogens and the plant diseases they cause. I describe this from a personal perspective, having been directly involved with many persons who made paradigm-changing discoveries, beginning with the discovery that a bacterium, not a virus, causes Pierce's disease of grape and other plant diseases in numerous plant species, including important crop and forest species.

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.

[Expression optimization and characterization of Tenebrio molitor antimicrobiol peptides TmAMP1m in Escherichia coli].

PubMed

Alimu, Reyihanguli; Mao, Xinfang; Liu, Zhongyuan

2013-06-01

To improve the expression level of tmAMP1m gene from Tenebrio molitor in Escherichia coli, we studied the effects of expression level and activity of the fusion protein HIS-TmAMP1m by conditions, such as culture temperature, inducing time and the final concentration of inductor Isopropyl beta-D-thiogalactopyranoside (IPTG). We analyzed the optimum expression conditions by Tricine-SDS-PAGE electrophoresis, meanwhile, detected its antibacterial activity by using agarose cavity diffusion method. The results suggest that when inducing the recombinant plasmid with a final IPTG concentration of 0.1 mmol/L at 37 degrees C for 4 h, there was the highest expression level of fusion protein HIS-TmAMP1m in Escherichia coli. Under these conditions, the expression of fusion protein accounted for 40% of the total cell lysate with the best antibacterial activity. We purified the fusion protein HIS-TmAMPlm with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices. Western blotting analysis indicates that the His monoclonal antibody could be specifically bound to fusion protein HIS-TmAMPlm. After expression by inducing, the fusion protein could inhibit the growth of host cell transformed by pET30a-tmAMP1m. The fusion protein HIS-TmAMP1m had better stability and remained higher antibacterial activities when incubated at 100 degrees C for 10 h, repeated freeze thawing at -20 degrees C, dissolved in strong acid and alkali, or treated by organic solvents and protease. Moreover, the minimum inhibitory concentration results demonstrated that the fusion protein HIS-TmAMP1m has a good antibacterial activity against Staphylococcus aureus, Staphylococcus sp., Corynebacterium glutamicum, Bacillus thuringiensis, Corynebacterium sp. This study laid the foundation to promote the application of insect antimicrobial peptides and further research.

A novel strategy for acetonitrile wastewater treatment by using a recombinant bacterium with biofilm-forming and nitrile-degrading capability.

PubMed

Li, Chunyan; Yue, Zhenlei; Feng, Fengzhao; Xi, Chuanwu; Zang, Hailian; An, Xuejiao; Liu, Keran

2016-10-01

There is a great need for efficient acetonitrile removal technology in wastewater treatment to reduce the discharge of this pollutant in untreated wastewater. In this study, a nitrilase gene (nit) isolated from a nitrile-degrading bacterium (Rhodococcus rhodochrous BX2) was cloned and transformed into a biofilm-forming bacterium (Bacillus subtilis N4) that expressed the recombinant protein upon isopropylthio-β-galactoside (IPTG) induction. The recombinant bacterium (B. subtilis N4-pHT01-nit) formed strong biofilms and had nitrile-degrading capability. Further testing demonstrated that biofilms formed by B. subtilis N4-pHT01-nit were highly resistant to loading shock from acetonitrile and almost completely degraded the initial concentration of acetonitrile (800 mg L(-1)) within 24 h in a moving bed biofilm reactor (MBBR) after operation for 35 d. The bacterial composition of the biofilm, identified by high-throughput sequencing, in a reactor in which the B. subtilis N4-pHT01-nit bacterium was introduced indicated that the engineered bacterium was successfully immobilized in the reactor and became dominant genus. This work demonstrates that an engineered bacterium with nitrile-degrading and biofilm-forming capacity can improve the degradation of contaminants in wastewater. This approach offers a novel strategy for enhancing the biological oxidation of toxic pollutants in wastewater. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome Sequence of Lactobacillus delbrueckii subsp. lactis CNRZ327, a Dairy Bacterium with Anti-Inflammatory Properties.

PubMed

El Kafsi, Hela; Binesse, Johan; Loux, Valentin; Buratti, Julien; Boudebbouze, Samira; Dervyn, Rozenn; Hammani, Amal; Maguin, Emmanuelle; van de Guchte, Maarten

2014-07-17

Lactobacillus delbrueckii subsp. lactis CNRZ327 is a dairy bacterium with anti-inflammatory properties both in vitro and in vivo. Here, we report the genome sequence of this bacterium, which appears to contain no less than 215 insertion sequence (IS) elements, an exceptionally high number regarding the small genome size of the strain. Copyright © 2014 El Kafsi et al.

Chitin utilization by the insect-transmitted bacterium Xylella fastidiosa.

PubMed

Killiny, Nabil; Prado, Simone S; Almeida, Rodrigo P P

2010-09-01

Xylella fastidiosa is an insect-borne bacterium that colonizes xylem vessels of a large number of host plants, including several crops of economic importance. Chitin is a polysaccharide present in the cuticle of leafhopper vectors of X. fastidiosa and may serve as a carbon source for this bacterium. Biological assays showed that X. fastidiosa reached larger populations in the presence of chitin. Additionally, chitin induced phenotypic changes in this bacterium, notably increasing adhesiveness. Quantitative PCR assays indicated transcriptional changes in the presence of chitin, and an enzymatic assay demonstrated chitinolytic activity by X. fastidiosa. An ortholog of the chitinase A gene (chiA) was identified in the X. fastidiosa genome. The in silico analysis revealed that the open reading frame of chiA encodes a protein of 351 amino acids with an estimated molecular mass of 40 kDa. chiA is in a locus that consists of genes implicated in polysaccharide degradation. Moreover, this locus was also found in the genomes of closely related bacteria in the genus Xanthomonas, which are plant but not insect associated. X. fastidiosa degraded chitin when grown on a solid chitin-yeast extract-agar medium and grew in liquid medium with chitin as the sole carbon source; ChiA was also determined to be secreted. The gene encoding ChiA was cloned into Escherichia coli, and endochitinase activity was detected in the transformant, showing that the gene is functional and involved in chitin degradation. The results suggest that X. fastidiosa may use its vectors' foregut surface as a carbon source. In addition, chitin may trigger X. fastidiosa's gene regulation and biofilm formation within vectors. Further work is necessary to characterize the role of chitin and its utilization in X. fastidiosa.

Complete genome of Martelella sp. AD-3, a moderately halophilic polycyclic aromatic hydrocarbons-degrading bacterium.

PubMed

Cui, Changzheng; Li, Zhijie; Qian, Jiangchao; Shi, Jie; Huang, Ling; Tang, Hongzhi; Chen, Xin; Lin, Kuangfei; Xu, Ping; Liu, Yongdi

2016-05-10

Martelella sp. strain AD-3, a moderate halophilic bacterium, was isolated from a petroleum-contaminated soil with high salinity in China. Here, we report the complete genome of strain AD-3, which contains one circular chromosome and two circular plasmids. An array of genes related to metabolism of polycyclic aromatic hydrocarbons and halophilic mechanism in this bacterium was identified by the whole genome analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

The susceptibility of bovine udder quarters colonized with Corynebacterium bovis to experimental infection with Staphylococcus aureus or Streptococcus agalactiae.

PubMed Central

Brooks, B W; Barnum, D A

1984-01-01

Twenty bovine udder quarters colonized with Corynebacterium bovis SR6 and 20 uncolonized quarters were challenged by inoculation of Staphylococcus aureus Newbould 305 (ATCC 29740) into the teat cistern. The percentage of infection in quarters colonized with C. bovis (50%) was significantly lower than that in controls (100%). By similar challenge no significant difference was observed between the percentage of infection with Streptococcus agalactiae ATCC 27956 in 33 quarters colonized with C. bovis (70%) compared to 33 controls (87.9%). A total of 37 quarters colonized with C. bovis and 37 control quarters were challenged with Staph. aureus Newbould 305 (ATCC 29740) and Maxi (ATCC 27543) and Strep. agalactiae (ATCC 27956) by exposure of the teat orifice. The percentage of teat ducts colonized with C. bovis which became infected with either pathogen was not different from that for controls. PMID:6372969

Complete genome of the cellulolytic ruminal bacterium Ruminococcus albus 7

USDA-ARS?s Scientific Manuscript database

Ruminococcus albus 7 is a highly cellulolytic rumen bacterium that is a member of the phylum Firmicutes. Here, we describe the complete genome for this microbe. This genome will be useful for rumen microbiology, cellulosome biology, and in biofuel production, as one of its major fermentation product...

Draft Genome Sequence of an Anaerobic and Extremophilic Bacterium, Caldanaerobacter yonseiensis, Isolated from a Geothermal Hot Stream

PubMed Central

Lee, Sang-Jae; Lee, Yong-Jik; Park, Gun-Seok; Kim, Byoung-Chan; Lee, Sang Jun; Shin, Jae-Ho

2013-01-01

Caldanaerobacter yonseiensis is a strictly anaerobic, thermophilic, spore-forming bacterium, which was isolated from a geothermal hot stream in Indonesia. This bacterium utilizes xylose and produces a variety of proteases. Here, we report the draft genome sequence of C. yonseiensis, which reveals insights into the pentose phosphate pathway and protein degradation metabolism in thermophilic microorganisms. PMID:24201201

Fine Structure and Host-Virus Relationship of a Marine Bacterium and Its Bacteriophage

PubMed Central

Valentine, Artrice F.; Chapman, George B.

1966-01-01

Valentine, Artrice F. (Georgetown University, Washington, D.C.), and George B. Chapman. Fine structure and host-virus relationship of a marine bacterium and its bacteriophage. J. Bacteriol. 92:1535–1554. 1966.—The fine structure of a gram-negative marine bacterium, Cytophaga marinoflava sp. n., has been revealed by ultrathin sectioning and electron microscopy. Stages in the morphogenesis of the bacterial virus NCMB 385, which has been shown to be highly specific for this organism, were also demonstrated in bacterial cells fixed according to the Kellenberger technique. The bacterium possessed a cell wall, cytoplasmic membrane, and nuclear and cytoplasmic regions typical of bacterial cells. Both the cell wall and the cytoplasmic membrane showed a tripartite structure, i.e., each was composed of two dense layers separated by a low-density zone. Intracytoplasmic membrane systems were also observed, especially in dividing cells and in cells in which new viruses were being formed. As many as 18 hexagonally shaped, empty phage heads (membranes only) were observed in untreated, infected bacterial cells. Phage heads, intermediate in density to empty heads and fully condensed ones, possibly representing stages in the morphological development of the virus, were also seen. Images PMID:5924277

Complete genome sequence of the haloalkaliphilic, hydrogen-producing bacterium Halanaerobium hydrogeniformans.

PubMed

Brown, Steven D; Begemann, Matthew B; Mormile, Melanie R; Wall, Judy D; Han, Cliff S; Goodwin, Lynne A; Pitluck, Samuel; Land, Miriam L; Hauser, Loren J; Elias, Dwayne A

2011-07-01

Halanaerobium hydrogenoformans is an alkaliphilic bacterium capable of biohydrogen production at pH 11 and 7% (wt/vol) salt. We present the 2.6-Mb genome sequence to provide insights into its physiology and potential for bioenergy applications.

Isolation and biological characteristics of aerobic marine magnetotactic bacterium YSC-1

NASA Astrophysics Data System (ADS)

Gao, Jun; Pan, Hongmiao; Yue, Haidong; Song, Tao; Zhao, Yong; Chen, Guanjun; Wu, Longfei; Xiao, Tian

2006-12-01

Magnetotactic bacteria have become a hot spot of research in microbiology attracting intensive interest of researchers in multiple disciplinary fields. However, the studies were limited in few fastidious bacteria. The objective of this study aims at isolating new marine magnetic bacteria and better comprehension of magnetotactic bacteria. In this study, an aerobic magnetotactic bacterium YSC-1 was isolated from sediments in the Yellow Sea Cold Water Mass (YSCWM). In TEM, magnetic cells have one or several circular magnetosomes in diameter of 100nm, and consist of Fe and Co shown on energy dispersive X-ray spectrum. The biological and physiological characteristics of this bacterium were also described. The colour of YSC-1 colony is white in small rod. The gram stain is negative. Results showed that Strain YSC-1 differs from microaerophile magnetotactic bacteria MS-1 and WD-1 in biology.

Factors influencing the immune response. III. The blocking effect of Corynebacterium parvum upon the induction of acquired immunological unresponsiveness to bovine serum albumin in the adult rabbit

PubMed Central

Pinckard, R. N.; Weir, D. M.; McBride, W. H.

1968-01-01

The intravenous injection of 10 mg aggregate-free, centrifuged bovine albumin into adult rabbits has been shown to induce an unresponsive state to bovine serum albumin (BSA) in the majority of rabbits. An intravenous injection of 15 mg of a heat-killed suspension of Corynebacterium parvum either 6 days prior to or simultaneously with centrifuged bovine albumin has been shown to significantly block the induction of unresponsiveness to BSA. Some effects of C. parvum upon the lymphoreticular tissues in the rabbit are reported. ImagesFig. 1Fig. 2 PMID:5301938

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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Species identities and antimicrobial susceptibilities of corynebacteria isolated from various clinical sources.

PubMed

Riegel, P; Ruimy, R; Christen, R; Monteil, H

1996-08-01

Over a 14-month period, 415 clinical isolates of coryneform gram-positive rods were recovered from various sources and identified to the species level according to recent identification schemes. Corynebacterium urealyticum, Corynebacterium striatum, Corynebacterium amycolatum, and Corynebacterium jeikeium predominated, accounting for 63% of all isolates. Corynebacterium accolens, Corynebacterium striatum, Corynebacterium argentoratense, Corynebacterium propinquum and Corynebacterium pseudodiphtheriticum were mostly recovered from the respiratory tract, whereas Corynebacterium afermentans, CDC group G, and Corynebacterium jeikeium were mainly isolated from blood. None of the isolates was identified as Corynebacterium diphtheriae or Corynebacterium xerosis. Ampicillin resistance was detected in Corynebacterium jeikeium (96%) and Corynebacterium urealyticum (99%) and varied among Corynebacterium amycolatum (56%) and CDC group G (26%). These data emphasize the need for an accurate identification of coryneform organisms at the species level and for antimicrobial susceptibility testing of these organisms.

Branimycins B and C, Antibiotics Produced by the Abyssal Actinobacterium Pseudonocardia carboxydivorans M-227.

PubMed

Braña, Alfredo F; Sarmiento-Vizcaíno, Aida; Pérez-Victoria, Ignacio; Otero, Luis; Fernández, Jonathan; Palacios, Juan José; Martín, Jesús; de la Cruz, Mercedes; Díaz, Caridad; Vicente, Francisca; Reyes, Fernando; García, Luis A; Blanco, Gloria

2017-02-24

Two new antibiotics, branimycins B (2) and C (3), were produced by fermentation of the abyssal actinobacterium Pseudonocardia carboxydivorans M-227, isolated from deep seawater of the Avilés submarine Canyon. Their structures were elucidated by HRMS and NMR analyses. These compounds exhibit antibacterial activities against a panel of Gram-positive bacteria, including Corynebacterium urealyticum, Clostridium perfringens, and Micrococcus luteus, and against the Gram-negative bacterium Neisseria meningitidis. Additionally, branimycin B displayed moderate antibacterial activity against other Gram-negative bacteria such as Bacteroides fragilis, Haemophilus influenzae, and Escherichia coli, and branimycin C against the Gram-positive Enterococcus faecalis and methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

Factors limiting success of inoculation to enhance biodegradation of low concentrations of organic chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zaidi, B.R.; Murakami, Y.; Alexander, M.

1988-12-01

Corynebacterium sp. added to lake water rapidly mineralized 100 ..mu..g and 1.0 mg of p-nitrophenol (PNP)/L but acted very slowly on the substrate present at 26 ..mu..g/L. The rate and extent of mineralization of the lowest PNP concentration in Beebe Lake water varied according to the time the sample was taken and were directly related to rainfall, and presumably runoff, in the watershed. The addition of high concentrations of inorganic P or N to water samples collected after a drought period, during which mineralization by the bacterium was slow, enhanced PNP decomposition. Mineralization in Cayuga Lake water was increased slightlymore » by 10 mg of K/sub 2/HPO/sub 4//L, but the enhancement was marked by 100 mg/L. The stimulation was a response to P and K. Glucose stimulated PNP mineralization in samples from Beebe and Cayuga Lakes, and K/sub 2/HOP/sub 4/ further increased the rate and extent of the transformation. The addition of either of two eucaryotic inhibitors increased the rate of Corynebacterium sp. growth in lake water amended with 26 ..mu..g of PNP/L but decreased the rate of mineralization.« less

Polysaccharide degradation systems of the saprophytic bacterium Cellvibrio japonicus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, Jeffrey G.

Study of recalcitrant polysaccharide degradation by bacterial systems is critical for understanding biological processes such as global carbon cycling, nutritional contributions of the human gut microbiome, and the production of renewable fuels and chemicals. One bacterium that has a robust ability to degrade polysaccharides is the Gram-negative saprophyte Cellvibrio japonicus. A bacterium with a circuitous history, C. japonicus underwent several taxonomy changes from an initially described Pseudomonas sp. Most of the enzymes described in the pre-genomics era have also been renamed. Furthermore, this review aims to consolidate the biochemical, structural, and genetic data published on C. japonicus and its remarkablemore » ability to degrade cellulose, xylan, and pectin substrates. Initially, C. japonicus carbohydrate-active enzymes were studied biochemically and structurally for their novel polysaccharide binding and degradation characteristics, while more recent systems biology approaches have begun to unravel the complex regulation required for lignocellulose degradation in an environmental context. Also included is a discussion for the future of C. japonicus as a model system, with emphasis on current areas unexplored in terms of polysaccharide degradation and emerging directions for C. japonicus in both environmental and biotechnological applications.« less

Polysaccharide degradation systems of the saprophytic bacterium Cellvibrio japonicus

DOE PAGES

Gardner, Jeffrey G.

2016-06-04

Study of recalcitrant polysaccharide degradation by bacterial systems is critical for understanding biological processes such as global carbon cycling, nutritional contributions of the human gut microbiome, and the production of renewable fuels and chemicals. One bacterium that has a robust ability to degrade polysaccharides is the Gram-negative saprophyte Cellvibrio japonicus. A bacterium with a circuitous history, C. japonicus underwent several taxonomy changes from an initially described Pseudomonas sp. Most of the enzymes described in the pre-genomics era have also been renamed. Furthermore, this review aims to consolidate the biochemical, structural, and genetic data published on C. japonicus and its remarkablemore » ability to degrade cellulose, xylan, and pectin substrates. Initially, C. japonicus carbohydrate-active enzymes were studied biochemically and structurally for their novel polysaccharide binding and degradation characteristics, while more recent systems biology approaches have begun to unravel the complex regulation required for lignocellulose degradation in an environmental context. Also included is a discussion for the future of C. japonicus as a model system, with emphasis on current areas unexplored in terms of polysaccharide degradation and emerging directions for C. japonicus in both environmental and biotechnological applications.« less

Biodegradation of polyethylene by the thermophilic bacterium Brevibacillus borstelensis.

PubMed

Hadad, D; Geresh, S; Sivan, A

2005-01-01

To select a polyethylene-degrading micro-organism and to study the factors affecting its biodegrading activity. A thermophilic bacterium Brevibaccillus borstelensis strain 707 (isolated from soil) utilized branched low-density polyethylene as the sole carbon source and degraded it. Incubation of polyethylene with B. borstelensis (30 days, 50 degrees C) reduced its gravimetric and molecular weights by 11 and 30% respectively. Brevibaccillus borstelensis also degraded polyethylene in the presence of mannitol. Biodegradation of u.v. photo-oxidized polyethylene increased with increasing irradiation time. Fourier Transform Infra-Red (FTIR) analysis of photo-oxidized polyethylene revealed a reduction in carbonyl groups after incubation with the bacteria. This study demonstrates that polyethylene--considered to be inert--can be biodegraded if the right microbial strain is isolated. Enrichment culture methods were effective for isolating a thermophilic bacterium capable of utilizing polyethylene as the sole carbon and energy source. Maximal biodegradation was obtained in combination with photo-oxidation, which showed that carbonyl residues formed by photo-oxidation play a role in biodegradation. Brevibaccillus borstelensis also degraded the CH2 backbone of nonirradiated polyethylene. Biodegradation of polyethylene by a single bacterial strain contributes to our understanding of the process and the factors affecting polyethylene biodegradation.

Aerobic Reduction of Arsenate by a Bacterium Isolated From Activated Sludge

NASA Astrophysics Data System (ADS)

Kozai, N.; Ohnuki, T.; Hanada, S.; Nakamura, K.; Francis, A. J.

2006-12-01

Microlunatus phosphovorus strain NM-1 is a polyphosphate-accumulating bacterium isolated from activated sludge. This bacterium takes up a large amount of polyphosphate under aerobic conditions and release phosphate ions by hydrolysis of polyphosphate to orthophosphate under anaerobic conditions to derive energy for taking up substrates. To understand the nature of this strain, especially, influence of potential contaminants in sewage and wastewater on growth, we have been investigating behavior of this bacterium in media containing arsenic. The present paper mainly reports reduction of arsenate by this bacterium under aerobic conditions. The strain NM-1 (JCM 9379) was aerobically cultured at 30 °C in a nutrient medium containing 2.5 g/l peptone, 0.5 g/l glucose, 1.5 g/l yeast extract, and arsenic [Na2HAsO4 (As(V)) or Na3AsO3 (As(III))] at concentrations between 0 and 50 mM. The cells collected from arsenic-free media were dispersed in buffer solutions containing 2mM HEPES, 10mM NaCl, prescribed concentrations of As(V), and 0-0.2 percent glucose. Then, this cell suspension was kept at 20 °C under aerobic or anaerobic conditions. The speciation of arsenic was carried out by ion chromatography and ICP-MS. The growth of the strain under aerobic conditions was enhanced by the addition of As(V) at the concentration between 1 and 10 mM. The maximum optical density of the culture in the medium containing 5mM As(V) was 1.4 times greater than that of the control culture. Below the As(V) concentration of 10mM, most of the As(V) was reduced to As(III). The growth of the strain under anaerobic conditions has not been observed so far. The cells in the buffer solutions reduced As(V) under aerobic condition. The reduction was enhanced by the addition of glucose. However, the cell did not reduce As(V) under anaerobic conditions. The strain NM-1 showed high resistance to As(V) and As(III). The maximum optical density of the culture grown in a medium containing 50 mM As(V) was only

Thermostable purified endoglucanase from thermophilic bacterium acidothermus cellulolyticus

DOEpatents

Tucker, Melvin P.; Grohmann, Karel; Himmel, Michael E.; Mohagheghi, Ali

1992-01-01

A substantially purified high molecular weight cellulase enzyme having a molecular weight of between about 156,000 to about 203,400 daltons isolated from the bacterium Acidothermus cellulolyticus (ATCC 43068) and a method of producing it are disclosed. The enzyme is water soluble, possesses both C.sub.1 and C.sub.x types of enzymatic activity, has a high degree of stability toward heat and exhibits both a high optimum temperature activity and high inactivation characteristics.

Genome sequence of the algicidal bacterium Kordia algicida OT-1.

PubMed

Lee, Hyun Sook; Kang, Sung Gyun; Kwon, Kae Kyoung; Lee, Jung-Hyun; Kim, Sang-Jin

2011-08-01

Kordia algicida OT-1 is an algicidal bacterium against the bloom-forming microalgae. The genome sequence of K. algicida revealed a number of interesting features, including the degradation of macromolecules, the biosynthesis of carotenoid pigment and secondary metabolites, and the capacity for gliding motility, which might facilitate the understanding of algicidal mechanisms.

Effects of sulfur dioxide on expansion of lesions caused by Corynebacterium nebraskense in maize and by Xanthomonas phaseoli var. sojensis in soybean

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laurence, J.A.; Aluisio, A.L.

1981-01-01

In order to assess the effects of air pollution on plant disease development, the authors investigated the effects of SO/sub 2/ on lesion development by two bacterial pathogens. Maize or soybean plants were exposed to sulfur dioxide (SO/sub 2/) at 524 ..mu..g m/sup -3/ or 262 ..mu..g m/sup -3/ before, after or before and after inoculation with Corynebacterium nebraskense or Xanthomonas phaseoli var. sojensis, respectively. Lesion development was inhibited in both cases, regardless of when the exposures occurred. The time of exposure, however, altered the subsequent effect on lesion size. Dry weight and sulfur content of host tissue were notmore » altered by the joint effects of the pollutant and the pathogens.« less

Evidence of carbon fixation pathway in a bacterium from candidate phylum SBR1093 revealed with genomic analysis.

PubMed

Wang, Zhiping; Guo, Feng; Liu, Lili; Zhang, Tong

2014-01-01

Autotrophic CO2 fixation is the most important biotransformation process in the biosphere. Research focusing on the diversity and distribution of relevant autotrophs is significant to our comprehension of the biosphere. In this study, a draft genome of a bacterium from candidate phylum SBR1093 was reconstructed with the metagenome of an industrial activated sludge. Based on comparative genomics, this autotrophy may occur via a newly discovered carbon fixation path, the hydroxypropionate-hydroxybutyrate (HPHB) cycle, which was demonstrated in a previous work to be uniquely possessed by some genera from Archaea. This bacterium possesses all of the thirteen enzymes required for the HPHB cycle; these enzymes share 30∼50% identity with those in the autotrophic species of Archaea that undergo the HPHB cycle and 30∼80% identity with the corresponding enzymes of the mixotrophic species within Bradyrhizobiaceae. Thus, this bacterium might have an autotrophic growth mode in certain conditions. A phylogenetic analysis based on the 16S rRNA gene reveals that the phylotypes within candidate phylum SBR1093 are primarily clustered into 5 clades with a shallow branching pattern. This bacterium is clustered with phylotypes from organically contaminated environments, implying a demand for organics in heterotrophic metabolism. Considering the types of regulators, such as FnR, Fur, and ArsR, this bacterium might be a facultative aerobic mixotroph with potential multi-antibiotic and heavy metal resistances. This is the first report on Bacteria that may perform potential carbon fixation via the HPHB cycle, thus may expand our knowledge of the distribution and importance of the HPHB cycle in the biosphere.

Characterization of a potentially novel 'blown pack' spoilage bacterium isolated from bovine hide.

PubMed

Moschonas, G; Bolton, D J

2013-03-01

To characterize a psychrotrophic bacterium, designated TC1, previously isolated from a cattle hide in Ireland, and to investigate the ability of this strain to cause 'blown pack' spoilage (BPS) of vacuum-packaged beef primals. TC1 was characterized using a combination of phenotypic, chemotaxonomic and genotypic analyses and was assessed for its ability to spoil vacuum-packaged beef at refrigerated temperatures. TC1 was Gram-positive and formed elliptical subterminal endospores. The strain was able to grow between 0 and 33 °C, with optimal growth between 23 and 24 °C. TC1 could be differentiated from its phylogenetically closest neighbour (Clostridium lituseburense DSM 797(T)) by 16S rRNA gene sequencing, pulsed-field gel electrophoresis and cellular fatty acid composition. TC1 spoiled (BPS) beef within 42 days when inoculated in cold-stored (1 °C) vacuum-packed beef. The phenotypic, chemotaxonomic and genotypic characterization indicated that TC1 may represent a potentially novel, cold-tolerant, gas-producing bacterium of considerable economic significance to the beef industry. This study reports and characterizes an emerging BPS bacterium, which should be considered in future activities designed to minimize the psychrophilic and psychrotrophic spoilage of vacuum-packaged beef. © 2012 The Society for Applied Microbiology.

The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum.

PubMed

Nagao, Nobuyoshi; Yamamoto, Junya; Komatsu, Hiroyuki; Suzuki, Hiromichi; Hirose, Yuu; Umekage, So; Ohyama, Takashi; Kikuchi, Yo

2015-12-01

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum , we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum . However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.

Chitin Utilization by the Insect-Transmitted Bacterium Xylella fastidiosa▿ †

PubMed Central

Killiny, Nabil; Prado, Simone S.; Almeida, Rodrigo P. P.

2010-01-01

Xylella fastidiosa is an insect-borne bacterium that colonizes xylem vessels of a large number of host plants, including several crops of economic importance. Chitin is a polysaccharide present in the cuticle of leafhopper vectors of X. fastidiosa and may serve as a carbon source for this bacterium. Biological assays showed that X. fastidiosa reached larger populations in the presence of chitin. Additionally, chitin induced phenotypic changes in this bacterium, notably increasing adhesiveness. Quantitative PCR assays indicated transcriptional changes in the presence of chitin, and an enzymatic assay demonstrated chitinolytic activity by X. fastidiosa. An ortholog of the chitinase A gene (chiA) was identified in the X. fastidiosa genome. The in silico analysis revealed that the open reading frame of chiA encodes a protein of 351 amino acids with an estimated molecular mass of 40 kDa. chiA is in a locus that consists of genes implicated in polysaccharide degradation. Moreover, this locus was also found in the genomes of closely related bacteria in the genus Xanthomonas, which are plant but not insect associated. X. fastidiosa degraded chitin when grown on a solid chitin-yeast extract-agar medium and grew in liquid medium with chitin as the sole carbon source; ChiA was also determined to be secreted. The gene encoding ChiA was cloned into Escherichia coli, and endochitinase activity was detected in the transformant, showing that the gene is functional and involved in chitin degradation. The results suggest that X. fastidiosa may use its vectors' foregut surface as a carbon source. In addition, chitin may trigger X. fastidiosa's gene regulation and biofilm formation within vectors. Further work is necessary to characterize the role of chitin and its utilization in X. fastidiosa. PMID:20656858

Investigations of Iron Minerals Formed by Dissimilatory Alkaliphilic Bacterium with 57Fe Mössbauer Spectroscopy

NASA Astrophysics Data System (ADS)

Chistyakova, N. I.; Rusakov, V. S.; Shapkin, A. A.; Zhilina, T. N.; Zavarzina, D. G.; Lančok, A.; Kohout, J.

2010-07-01

Anaerobic alkaliphilic bacterium of Geoalkalibacter ferrihydriticus type (strain Z-0531), isolated from a bottom sediment sample from the weakly mineralized soda Lake Khadyn, have been analyzed. The strain uses the amorphous Fe(III)-hydroxide (AFH) as an electron acceptor and acetate CH3COO- as an electron donor. Mössbauer investigations of solid phase samples obtained during the process of the bacterium growth were carried out at room temperature, 77.8 K, 4.2 K without and with the presence of an external magnetic field (6 T) applied perpendicular to the γ-bebam.

Bioprocess automation on a Mini Pilot Plant enables fast quantitative microbial phenotyping.

PubMed

Unthan, Simon; Radek, Andreas; Wiechert, Wolfgang; Oldiges, Marco; Noack, Stephan

2015-03-11

The throughput of cultivation experiments in bioprocess development has drastically increased in recent years due to the availability of sophisticated microliter scale cultivation devices. However, as these devices still require time-consuming manual work, the bottleneck was merely shifted to media preparation, inoculation and finally the analyses of cultivation samples. A first step towards solving these issues was undertaken in our former study by embedding a BioLector in a robotic workstation. This workstation already allowed for the optimization of heterologous protein production processes, but remained limited when aiming for the characterization of small molecule producer strains. In this work, we extended our workstation to a versatile Mini Pilot Plant (MPP) by integrating further robotic workflows and microtiter plate assays that now enable a fast and accurate phenotyping of a broad range of microbial production hosts. A fully automated harvest procedure was established, which repeatedly samples up to 48 wells from BioLector cultivations in response to individually defined trigger conditions. The samples are automatically clarified by centrifugation and finally frozen for subsequent analyses. Sensitive metabolite assays in 384-well plate scale were integrated on the MPP for the direct determination of substrate uptake (specifically D-glucose and D-xylose) and product formation (specifically amino acids). In a first application, we characterized a set of Corynebacterium glutamicum L-lysine producer strains and could rapidly identify a unique strain showing increased L-lysine titers, which was subsequently confirmed in lab-scale bioreactor experiments. In a second study, we analyzed the substrate uptake kinetics of a previously constructed D-xylose-converting C. glutamicum strain during cultivation on mixed carbon sources in a fully automated experiment. The presented MPP is designed to face the challenges typically encountered during early-stage bioprocess

Corynebacterium diphtheriae in a free-roaming red fox: case report and historical review on diphtheria in animals.

PubMed

Sing, Andreas; Konrad, Regina; Meinel, Dominik M; Mauder, Norman; Schwabe, Ingo; Sting, Reinhard

2016-08-01

Corynebacterium diphtheriae, the classical causative agent of diphtheria, is considered to be nearly restricted to humans. Here we report the first finding of a non-toxigenic C. diphtheriae biovar belfanti strain in a free-roaming wild animal. The strain obtained from the subcutis and mammary gland of a dead red fox (Vulpes vulpes) was characterized by biochemical and molecular methods including MALDI-TOF and Multi Locus Sequence Typing. Since C. diphtheriae infections of animals, usually with close contact to humans, are reported only very rarely, an intense review comprising also scientific literature from the beginning of the 20th century was performed. Besides the present case, only 11 previously reported C. diphtheriae animal infections could be verified using current scientific criteria. Our report is the first on the isolation of C. diphtheriae from a wildlife animal without any previous human contact. In contrast, the very few unambiguous publications on C. diphtheriae in animals referred to livestock or pet animals with close human contact. C. diphtheriae carriage in animals has to be considered as an exceptionally rare event.