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Instability of ackA (acetate kinase) mutations and their effects on acetyl phosphate and ATP amounts in Streptococcus pneumoniae D39.

ABSTRACT: Acetyl phosphate (AcP) is a small-molecule metabolite that can act as a phosphoryl group donor for response regulators of two-component systems (TCSs). The serious human respiratory pathogen Streptococcus pneumoniae (pneumococcus) synthesizes AcP by the conventional pathway involving phosphotransacetylase and acetate kinase, encoded by pta and ackA, respectively. In addition, pneumococcus synthesizes copious amounts of AcP and hydrogen peroxide (H(2)O(2)) by pyruvate oxidase, which is encoded by spxB. To assess possible roles of AcP in pneumococcal TCS regulation and metabolism, we constructed strains with combinations of spxB, pta, and ackA mutations and determined their effects on ATP, AcP, and H(2)O(2) production. Unexpectedly, ?ackA mutants were unstable and readily accumulated primary suppressor mutations in spxB or its positive regulator, spxR, thereby reducing H(2)O(2) and AcP levels, and secondary capsule mutations in cps2E or cps2C. ?ackA ?spxB mutants contained half the cellular amount of ATP as a ?spxB or spxB(+) strain. Acetate addition and anaerobic growth experiments suggested decreased ATP, rather than increased AcP, as a reason that ?ackA mutants accumulated spxB or spxR suppressors, although experimental manipulation of the AcP amount was limited. This finding and other considerations suggest that coping with endogenously produced H(2)O(2) may require energy. Starting with a ?spxB mutant, we constructed ?pta, ?ackA, and ?pta ?ackA mutants. Epistasis and microarray experiment results were consistent with a role for the SpxB-Pta-AckA pathway in expression of the regulons controlled by the WalRK(Spn), CiaRH(Spn), and LiaSR(Spn) TCSs involved in sensing cell wall status. However, AcP likely does not play a physiological role in TCS sensing in S. pneumoniae.

SUBMITTER: Ramos-Montanez S

PROVIDER: S-EPMC3008541 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Instability of ackA (acetate kinase) mutations and their effects on acetyl phosphate and ATP amounts in Streptococcus pneumoniae D39.

Ramos-Montañez Smirla S Kazmierczak Krystyna M KM Hentchel Kristy L KL Winkler Malcolm E ME

Journal of bacteriology 20101015 24

Acetyl phosphate (AcP) is a small-molecule metabolite that can act as a phosphoryl group donor for response regulators of two-component systems (TCSs). The serious human respiratory pathogen Streptococcus pneumoniae (pneumococcus) synthesizes AcP by the conventional pathway involving phosphotransacetylase and acetate kinase, encoded by pta and ackA, respectively. In addition, pneumococcus synthesizes copious amounts of AcP and hydrogen peroxide (H(2)O(2)) by pyruvate oxidase, which is encoded by ...[more]

PMID: 20952579

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Project description:Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen and a leading cause of bacterial pneumonia worldwide. Small regulatory RNAs (sRNAs), which often act by posttranscriptionally regulating gene expression, have been shown to be crucial for the virulence of S. pneumoniae and other bacterial pathogens. Over 170 putative sRNAs have been identified in the S. pneumoniae TIGR4 strain (serotype 4) through transcriptomic studies, and a subset of these sRNAs has been further implicated in regulating pneumococcal pathogenesis. However, there is little overlap in the sRNAs identified among these studies, which indicates that the approaches used for sRNA identification were not sufficiently sensitive and robust and that there are likely many more undiscovered sRNAs encoded in the S. pneumoniae genome. Here, we sought to comprehensively identify sRNAs in Avery's virulent S. pneumoniae strain D39 using two independent RNA sequencing (RNA-seq)-based approaches. We developed an unbiased method for identifying novel sRNAs from bacterial RNA-seq data and have further tested the specificity of our analysis program toward identifying sRNAs encoded by both strains D39 and TIGR4. Interestingly, the genes for 15% of the putative sRNAs identified in strain TIGR4, including ones previously implicated in virulence, are not present in the strain D39 genome, suggesting that the differences in sRNA repertoires between these two serotypes may contribute to their strain-specific virulence properties. Finally, this study has identified 66 new sRNA candidates in strain D39, 30 of which have been further validated, raising the total number of sRNAs that have been identified in strain D39 to 112.IMPORTANCE Recent work has shown that sRNAs play crucial roles in S. pneumoniae pathogenesis, as inactivation of nearly one-third of the putative sRNA genes identified in one study led to reduced fitness or virulence in a murine model. Yet our understanding of sRNA-mediated gene regulation in S. pneumoniae has been hindered by limited knowledge about these regulatory RNAs, including which sRNAs are synthesized by different S. pneumoniae strains. We sought to address this problem by developing a sensitive sRNA detection technique to identify sRNAs in S. pneumoniae D39. A comparison of our data set reported here to those of other RNA-seq studies for S. pneumoniae strain D39 and TIGR4 has provided new insights into the S. pneumoniae sRNA transcriptome.

Dataset Information

Instability of ackA (acetate kinase) mutations and their effects on acetyl phosphate and ATP amounts in Streptococcus pneumoniae D39.

Publications

Instability of ackA (acetate kinase) mutations and their effects on acetyl phosphate and ATP amounts in Streptococcus pneumoniae D39.

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