Dataset Information

Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans.

ABSTRACT: In work previously reported (J. A. Gutierrez, P. J. Crowley, D. P. Brown, J. D. Hillman, P. Youngman, and A. S. Bleiweis, J. Bacteriol. 178:4166-4175, 1996), a Tn917 transposon-generated mutant of Streptococcus mutans JH1005 unable to synthesize glutamate anaerobically was isolated and the insertion point of the transposon was determined to be in the icd gene encoding isocitrate dehydrogenase (ICDH). The intact icd gene of S. mutans has now been isolated from an S. mutans genomic plasmid library by complementation of an icd mutation in Escherichia coli host strain EB106. Genetic analysis of the complementing plasmid pJG400 revealed an open reading frame (ORF) of 1,182 nucleotides which encoded an enzyme of 393 amino acids with a predicted molecular mass of 43 kDa. The nucleotide sequence contained regions of high (60 to 72%) homology with icd genes from three other bacterial species. Immediately 5' of the icd gene, we discovered an ORF of 1,119 nucleotides in length, designated citZ, encoding a homolog of known citrate synthase genes from other bacteria. This ORF encoded a predicted protein of 372 amino acids with a molecular mass of 43 kDa. Furthermore, plasmid pJG400 was also able to complement a citrate synthase (gltA) mutation of E. coli W620. The enzyme activities of both ICDH, found to be NAD+ dependent, and citrate synthase were measured in cell extracts of wild-type S. mutans and E. coli mutants harboring plasmid pJG400. The region 5' from the citZ gene also revealed a partial ORF encoding 264 carboxy-terminal amino acids of a putative aconitase gene. The genetic and biochemical evidence indicates that S. mutans possesses the enzymes required to convert acetyl coenzyme A and oxalacetate to alpha-ketoglutarate, which is necessary for the synthesis of glutamic acid. Indeed, S. mutans JH1005 was shown to assimilate ammonia as a sole source of nitrogen in minimal medium devoid of organic nitrogen sources.

SUBMITTER: Cvitkovitch DG

PROVIDER: S-EPMC178743 | biostudies-other | 1997 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans.

Cvitkovitch D G DG Gutierrez J A JA Bleiweis A S AS

Journal of bacteriology 19970201 3

In work previously reported (J. A. Gutierrez, P. J. Crowley, D. P. Brown, J. D. Hillman, P. Youngman, and A. S. Bleiweis, J. Bacteriol. 178:4166-4175, 1996), a Tn917 transposon-generated mutant of Streptococcus mutans JH1005 unable to synthesize glutamate anaerobically was isolated and the insertion point of the transposon was determined to be in the icd gene encoding isocitrate dehydrogenase (ICDH). The intact icd gene of S. mutans has now been isolated from an S. mutans genomic plasmid library ...[more]

PMID: 9006016

Similar Datasets

Project description:Streptococcus mutans is a primary cariogenic pathogen in humans. Arginine metabolism is required for bacterial growth. In S. mutans, however, the involvement of transcription factors in regulating arginine metabolism is unclear. The purpose of this study was to investigate the function and mechanism of ArgR family transcription factors in S. mutans. Here, we identified an ArgR (arginine repressor) family transcription factor named AhrC, which negatively regulates arginine biosynthesis and biofilm formation in S. mutans. The ahrC in-frame deletion strain exhibited slow growth and significantly increased intracellular arginine content. The strain overexpressing ahrC showed reduced intracellular arginine content, decreased biofilm biomass, reduced production of water-insoluble exopolysaccharides (EPS), and different biofilm structures. Furthermore, global gene expression profiles revealed differential expression levels of 233 genes in the ahrC-deficient strain, among which genes related to arginine biosynthesis (argJ, argB, argC, argD, argF, argG, argH) were significantly upregulated. In the ahrC overexpression strain, there are 89 differentially expressed genes, mostly related to arginine biosynthesis. The conserved DNA patterns bound by AhrC were identified by electrophoretic mobility shift assay (EMSA) and DNase I footprinting. In addition, the analysis of β-galactosidase activity showed that AhrC acted as a negative regulator. Taken together, our findings suggest that AhrC is an important transcription factor that regulates arginine biosynthesis gene expression and biofilm formation in S. mutans. These findings add new aspects to the complexity of regulating the expression of genes involved in arginine biosynthesis and biofilm formation in S. mutans. IMPORTANCE Arginine metabolism is essential for bacterial growth. The regulation of intracellular arginine metabolism in Streptococcus mutans, one of the major pathogens of dental caries, is unclear. In this study, we found that the transcription factor AhrC can directly and negatively regulate the expression of N-acetyl-gamma-glutamyl-phosphate reductase (argC), thus regulating arginine biosynthesis in S. mutans. In addition, the ahrC overexpression strain exhibited a significant decrease in biofilm and water-insoluble extracellular polysaccharides (EPS). This study adds new support to our understanding of the regulation of intracellular arginine metabolism in S. mutans.

Project description:UnlabelledLantibiotics are ribosomally synthesized peptide antibiotics composed of an N-terminal leader peptide that is cleaved to yield the active antibacterial peptide. Significant advancements in molecular tools that promote the study of lantibiotic biosynthesis can be used in Streptococcus mutans. Herein, we further our understanding of leader peptide sequence and core peptide structural requirements for the biosynthesis and transport of the lantibiotic mutacin 1140. Our study on mutacin 1140 biosynthesis shows a dedicated secondary cleavage site within the leader peptide and the dependency of transport on core peptide posttranslational modifications (PTMs). The secondary cleavage site on the leader peptide is found at the -9 position, and secondary cleavage occurs before the core peptide is transported out of the cell. The coordinated cleavage at the -9 position was absent in a lanT deletion strain, suggesting that the core peptide interaction with the LanT transporter enables uniform cleavage at the -9 position. Following transport, the LanP protease was found to be tolerant to a wide variety of amino acid substitutions at the primary leader peptide cleavage site, with the exception of arginine at the -1 position. Several leader and core peptide mutations produced core peptide variants that had intermediate stages of PTM enzyme modifications, supporting the concept that PTM enzyme modifications, secondary cleavage, and transport are occurring in a highly coordinated fashion.ImportanceMutacin 1140 belongs to the class I lantibiotic family of ribosomally synthesized and posttranslationally modified peptides (RiPPs). The biosynthesis of mutacin 1140 is a highly efficient process which does not lead to a discernible level of production of partially modified core peptide variants. The products isolated from an extensive mutagenesis study on the leader and core peptides of mutacin 1140 show that the posttranslational modifications (PTMs) on the core peptide occur under a highly coordinated dynamic process. PTMs are dictated by the distance of the core peptide modifiable residues from PTM enzyme active sites. The formation of lanthionine rings aids in the formation of successive PTMs, as was observed in a peptide variant lacking a C-terminal decarboxylation.

Project description:The rhamnose-glucose cell wall polysaccharide (RGP) of Streptococcus mutans plays a significant role in cell division, virulence, and stress protection. Prior studies examined function of the RGP using strains carrying deletions in the machinery involved in RGP assembly. In this study, we explored loss of the substrate for RGP, l-rhamnose, via deletion of rmlD (encoding the protein responsible for the terminal step in l-rhamnose biosynthesis). We demonstrate that loss of rhamnose biosynthesis causes a phenotype similar to strains with disrupted RGP assembly (ΔrgpG and ΔrgpF strains). Deletion of rmlD not only caused a severe growth defect under nonstress growth conditions but also elevated susceptibility of the strain to acid and oxidative stress, common conditions found in the oral cavity. A genetic complement of the ΔrmlD strain completely restored wild-type levels of growth, whereas addition of exogenous rhamnose did not. The loss of rhamnose production also significantly disrupted biofilm formation, an important aspect of S. mutans growth in the oral cavity. Further, we demonstrate that loss of either rmlD or rgpG results in ablation of rhamnose content in the S. mutans cell wall. Taken together, these results highlight the importance of rhamnose production in both the fitness and the ability of S. mutans to overcome environmental stresses.IMPORTANCEStreptococcus mutans is a pathogenic bacterium that is the primary etiologic agent of dental caries, a disease that affects billions yearly. Rhamnose biosynthesis is conserved not only in streptococcal species but in other Gram-positive, as well as Gram-negative, organisms. This study highlights the importance of rhamnose biosynthesis in RGP production for protection of the organism against acid and oxidative stresses, the two major stressors that the organism encounters in the oral cavity. Loss of RGP also severely impacts biofilm formation, the first step in the onset of dental caries. The high conservation of the rhamnose synthesis enzymes, as well as their importance in S. mutans and other organisms, makes them favorable antibiotic targets for the treatment of disease.

Dataset Information

Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans.

Publications

Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets