Elucidation of a protein-protein interaction network involved in Corynebacterium glutamicum cell wall biosynthesis as determined by bacterial two-hybrid analysis.
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ABSTRACT:
SUBMITTER: Jankute M
PROVIDER: S-EPMC4213368 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Proteins of the LCP (LytR, CpsA, Psr) family have been shown to inherit important roles in bacterial cell wall biosynthesis. However, their exact function in the formation of the complex cell wall structures of the Corynebacteriales, including the prominent pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae, remains unclear. Here, we analyzed the role of the LCP proteins LcpA and LcpB of Corynebacterium glutamicum, both of which localize at regions of nascent cell wall biosynthesis. A strain lacking lcpB did not show any growth-related or morphological phenotype under the tested conditions. In contrast, conditional silencing of the essential lcpA gene resulted in severe growth defects and drastic morphological changes. Compared to the wild-type cell wall, the cell wall of this mutant contained significantly less mycolic acids and a reduced amount of arabinogalactan. In particular, rhamnose, a specific sugar component of the linker that connects arabinogalactan and peptidoglycan, was decreased. Complementation studies of the lcpA-silencing strain with several mutated and truncated LcpA variants suggested that both periplasmic domains are essential for function whereas the cytoplasmic N-terminal part is dispensable. Successful complementation experiments with proteins of M. tuberculosis and C. diphtheriae revealed a conserved function of LCP proteins in these species. Finally, pyrophosphatase activity of LcpA was shown in an in vitro assay. Taken together, our results suggest that LCP proteins are responsible for the transfer of arabinogalactan onto peptidoglycan in actinobacterial species and support a crucial function of a so-far-uncharacterized C-terminal domain (LytR_C domain) which is frequently found at the C terminus of the LCP domain in this prokaryotic phylum.ImportanceAbout one-third of the world's population is infected with Mycobacterium tuberculosis, and multiple-antibiotic resistance provokes the demand for novel antibiotics. The special cell wall architecture of Corynebacteriales is critical for treatments because it is either a direct target or a barrier that the drug has to cross. Here, we present the analysis of LcpA and LcpB of the closely related Corynebacterium glutamicum, the first of which is an essential protein involved in cell wall biogenesis. Our work provides a comprehensive characterization of the impact of LCP proteins on cell wall biogenesis in this medically and biotechnologically important class of bacteria. Special focus is set on the two periplasmic LcpA domains and their contributions to physiological function.
| S-EPMC5075034 | biostudies-literature
Project description:BackgroundCorynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin.ResultsHere, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 ε-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum ΔcrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum ΔcrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 ± 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum ΔcrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 ± 0.3 mg/g CDW were obtained.ConclusionC. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production.
| S-EPMC3598387 | biostudies-literature
Project description:Diaminopimelate epimerase (DapF) is one of the crucial enzymes involved in l-lysine biosynthesis, where it converts l,l-diaminopimelate (l,l-DAP) into d,l-DAP. DapF is also considered as an attractive target for the development of antibacterial drugs. Here, we report the crystal structure of DapF from Corynebacterium glutamicum (CgDapF). Structures of CgDapF obtained under both oxidized and reduced conditions reveal that the function of CgDapF is regulated by redox-switch modulation via reversible disulfide bond formation between two catalytic cysteine residues. Under oxidized condition, two catalytic cysteine residues form a disulfide bond; these same cysteine residues exist in reduced form under reduced condition. Disulfide bond formation also induces a subsequent structural change in the dynamic catalytic loop at the active site, which results in open/closed conformational change at the active site. We also determined the crystal structure of CgDapF in complex with its product d,l-DAP, and elucidated how the enzyme recognizes its substrate l,l-DAP as a substrate. Moreover, the structure in complex with the d,l-DAP product reveals that CgDapF undergoes a large open/closed domain movement upon substrate binding, resulting in a completely buried active site with the substrate bound.
| S-EPMC5296763 | biostudies-literature
Project description:The phosphate (P(i)) starvation stimulon of Corynebacterium glutamicum was characterized by global gene expression analysis by using DNA microarrays. Hierarchical cluster analysis of the genes showing altered expression 10 to 180 min after a shift from P(i)-sufficient to P(i)-limiting conditions led to identification of five groups comprising 92 genes. Four of these groups included genes which are not directly involved in P metabolism and changed expression presumably due to the reduced growth rate observed after the shift or to the exchange of medium. One group, however, comprised 25 genes, most of which are obviously related to phosphorus (P) uptake and metabolism and exhibited 4- to >30-fold-greater expression after the shift to P(i) limitation. Among these genes, the RNA levels of the pstSCAB (ABC-type P(i) uptake system), glpQ (glycerophosphoryldiester phosphodiesterase), ugpAEBC (ABC-type sn-glycerol 3-phosphate uptake system), phoH (unknown function), nucH (extracellular nuclease), and Cgl0328 (5'-nucleotidase or related esterase) genes were increased, and pstSCAB exhibited a faster response than the other genes. Transcriptional fusion analyses revealed that elevated expression of pstSCAB and ugpAEBC was primarily due to transcriptional regulation. Several genes also involved in P uptake and metabolism were not affected by P(i) starvation; these included the genes encoding a PitA-like P(i) uptake system and a putative Na(+)-dependent P(i) transporter and the genes involved in the metabolism of pyrophosphate and polyphosphate. In summary, a global, time-resolved picture of the response of C. glutamicum to P(i) starvation was obtained.
| S-EPMC165763 | biostudies-literature
Project description:l-Histidine biosynthesis is an ancient metabolic pathway present in bacteria, archaea, lower eukaryotes, and plants. For decades l-histidine biosynthesis has been studied mainly in Escherichia coli and Salmonella typhimurium, revealing fundamental regulatory processes in bacteria. Furthermore, in the last 15 years this pathway has been also investigated intensively in the industrial amino acid-producing bacterium Corynebacterium glutamicum, revealing similarities to E. coli and S. typhimurium, as well as differences. This review summarizes the current knowledge of l-histidine biosynthesis in C. glutamicum. The genes involved and corresponding enzymes are described, in particular focusing on the imidazoleglycerol-phosphate synthase (HisFH) and the histidinol-phosphate phosphatase (HisN). The transcriptional organization of his genes in C. glutamicum is also reported, including the four histidine operons and their promoters. Knowledge of transcriptional regulation during stringent response and by histidine itself is summarized and a translational regulation mechanism is discussed, as well as clues about a histidine transport system. Finally, we discuss the potential of using this knowledge to create or improve C. glutamicum strains for the industrial l-histidine production.
| S-EPMC3896937 | biostudies-literature
Project description:Elongation growth in actinobacteria is localized at the cell poles. This is in contrast to many classical model organisms where insertion of new cell wall material is localized around the lateral site. We previously described a role of RodA from Corynebacterium glutamicum in apical cell growth and morphogenesis. Deletion of rodA had drastic effects on morphology and growth, likely a result from misregulation of penicillin-binding proteins and cell wall precursor delivery. We identified the interaction of RodA with the polar scaffold protein DivIVA, thus explaining subcellular localization of RodA to the cell poles. In this study, we describe this interaction in detail and map the interaction sites of DivIVA and RodA. A single amino acid residue in the N-terminal domain of DivIVA was found to be crucial for the interaction with RodA. The interaction site of RodA was mapped to its cytoplasmic, C-terminal domain, in a region encompassing the last 10 amino acids (AAs). Deletion of these 10 AAs significantly decreased the interaction efficiency with DivIVA. Our results corroborate the interaction of DivIVA and RodA, underscoring the important role of DivIVA as a spatial organizer of the elongation machinery in Corynebacterineae.
| S-EPMC4285798 | biostudies-literature
Project description:BACKGROUND: Arginine biosynthesis in Corynebacterium glutamicum consists of eight enzymatic steps, starting with acetylation of glutamate, catalysed by N-acetylglutamate synthase (NAGS). There are different kinds of known NAGSs, for example, "classical" ArgA, bifunctional ArgJ, ArgO, and S-NAGS. However, since C. glutamicum possesses a monofunctional ArgJ, which catalyses only the fifth step of the arginine biosynthesis pathway, glutamate must be acetylated by an as of yet unknown NAGS gene. RESULTS: Arginine biosynthesis was investigated by metabolome profiling using defined gene deletion mutants that were expected to accumulate corresponding intracellular metabolites. HPLC-ESI-qTOF analyses gave detailed insights into arginine metabolism by detecting six out of seven intermediates of arginine biosynthesis. Accumulation of N-acetylglutamate in all mutants was a further confirmation of the unknown NAGS activity. To elucidate the identity of this gene, a genomic library of C. glutamicum was created and used to complement an Escherichia coli ?argA mutant. The plasmid identified, which allowed functional complementation, contained part of gene cg3035, which contains an acetyltransferase domain in its amino acid sequence. Deletion of cg3035 in the C. glutamicum genome led to a partial auxotrophy for arginine. Heterologous overexpression of the entire cg3035 gene verified its ability to complement the E. coli ?argA mutant in vivo and homologous overexpression led to a significantly higher intracellular N-acetylglutamate pool. Enzyme assays confirmed the N-acetylglutamate synthase activity of Cg3035 in vitro. However, the amino acid sequence of Cg3035 revealed no similarities to members of known NAGS gene families. CONCLUSIONS: The N-acetylglutamate synthase Cg3035 is able to catalyse the first step of arginine biosynthesis in C. glutamicum. It represents a novel class of NAGS genes apparently present only in bacteria of the suborder Corynebacterineae, comprising amongst others the genera Corynebacterium, Mycobacterium, and Nocardia. Therefore, the name C-NAGS (Corynebacterineae-type NAGS) is proposed for this new family.
| S-EPMC3827942 | biostudies-literature
Project description:BACKGROUND:Acetoin, especially the optically pure (3S)- or (3R)-enantiomer, is a high-value-added bio-based platform chemical and important potential pharmaceutical intermediate. Over the past decades, intense efforts have been devoted to the production of acetoin through green biotechniques. However, efficient and economical methods for the production of optically pure acetoin enantiomers are rarely reported. Previously, we systematically engineered the GRAS microorganism Corynebacterium glutamicum to efficiently produce (3R)-acetoin from glucose. Nevertheless, its yield and average productivity were still unsatisfactory for industrial bioprocesses. RESULTS:In this study, cellular carbon fluxes in the acetoin producer CGR6 were further redirected toward acetoin synthesis using several metabolic engineering strategies, including blocking anaplerotic pathways, attenuating key genes of the TCA cycle and integrating additional copies of the alsSD operon into the genome. Among them, the combination of attenuation of citrate synthase and inactivation of phosphoenolpyruvate carboxylase showed a significant synergistic effect on acetoin production. Finally, the optimal engineered strain CGS11 produced a titer of 102.45 g/L acetoin with a yield of 0.419 g/g glucose at a rate of 1.86 g/L/h in a 5 L fermenter. The optical purity of the resulting (3R)-acetoin surpassed 95%. CONCLUSION:To the best of our knowledge, this is the highest titer of highly enantiomerically enriched (3R)-acetoin, together with a competitive product yield and productivity, achieved in a simple, green processes without expensive additives or substrates. This process therefore opens the possibility to achieve easy, efficient, economical and environmentally-friendly production of (3R)-acetoin via microbial fermentation in the near future.
| S-EPMC7216327 | biostudies-literature
Project description:A direct sulfhydrylation pathway for methionine biosynthesis in Corynebacterium glutamicum was found. The pathway was catalyzed by metY encoding O-acetylhomoserine sulfhydrylase. The gene metY, located immediately upstream of metA, was found to encode a protein of 437 amino acids with a deduced molecular mass of 46,751 Da. In accordance with DNA and protein sequence data, the introduction of metY into C. glutamicum resulted in the accumulation of a 47-kDa protein in the cells and a 30-fold increase in O-acetylhomoserine sulfhydrylase activity, showing the efficient expression of the cloned gene. Although disruption of the metB gene, which encodes cystathionine gamma-synthase catalyzing the transsulfuration pathway of methionine biosynthesis, or the metY gene was not enough to lead to methionine auxotrophy, an additional mutation in the metY or the metB gene resulted in methionine auxotrophy. The growth pattern of the metY mutant strain was identical to that of the metB mutant strain, suggesting that both methionine biosynthetic pathways function equally well. In addition, an Escherichia coli metB mutant could be complemented by transformation of the strain with a DNA fragment carrying corynebacterial metY and metA genes. These data clearly show that C. glutamicum utilizes both transsulfuration and direct sulfhydrylation pathways for methionine biosynthesis. Although metY and metA are in close proximity to one another, separated by 143 bp on the chromosome, deletion analysis suggests that they are expressed independently. As with metA, methionine could also repress the expression of metY. The repression was also observed with metB, but the degree of repression was more severe with metY, which shows almost complete repression at 0.5 mM methionine in minimal medium. The data suggest a physiologically distinctive role of the direct sulfhydrylation pathway in C. glutamicum.
| S-EPMC134843 | biostudies-literature
Project description:Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons (ars1 and ars2) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum. The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC); operon ars1 contains an additional arsenate reductase gene (arsC1') located immediately downstream from arsC1. Additional arsenite permease and arsenate reductase genes (arsB3 and arsC4) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1, arsB1, arsC1, arsC1', arsR2, arsB2, and arsC2 being inducible by arsenite.
| S-EPMC1266000 | biostudies-literature