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NADH-Mediated Gene Expression in Streptococcus pneumoniae and Role of Rex as a Transcriptional Repressor of the Rex-Regulon.

ABSTRACT: Nicotinamide adenine dinucleotides (NAD(H)) play a vital role in various biological processes, including keeping the cellular redox balance. In this study, we investigate the regulatory responses of Streptococcus pneumoniae D39 to NADH and characterize the role of the Rex protein as a transcriptional repressor of the gapN, fba, pncB, adhB2, gap, and adhE genes. Transcriptomic analysis was used to observe the response of S. pneumoniae D39 to NADH. Our microarray studies revealed elevated expression of various genes/operons involved in transport and biosynthesis of niacin (gapN, fba, pncB, adhB2, gap, and adhE). Promoter lacZ-fusion assays and microarray studies with the rex mutant revealed the role of Rex as a transcriptional repressor of gapN, fba, pncB, adhB2, gap, and adhE involved in niacin uptake and biosynthesis, in the presence of NADH. We predict the operator site (5'-TTGTKAWAAWWTTCACAA-3') of Rex in the regulatory regions of Rex-regulated genes that was subsequently validated by promoter mutational experiments.

SUBMITTER: Afzal M

PROVIDER: S-EPMC6018154 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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NADH-Mediated Gene Expression in Streptococcus pneumoniae and Role of Rex as a Transcriptional Repressor of the Rex-Regulon.

Afzal Muhammad M Shafeeq Sulman S Kuipers Oscar P OP

Frontiers in microbiology 20180619

Nicotinamide adenine dinucleotides (NAD(H)) play a vital role in various biological processes, including keeping the cellular redox balance. In this study, we investigate the regulatory responses of Streptococcus pneumoniae D39 to NADH and characterize the role of the Rex protein as a transcriptional repressor of the gapN, fba, pncB, adhB2, gap, and adhE genes. Transcriptomic analysis was used to observe the response of S. pneumoniae D39 to NADH. Our microarray studie ...[more]

PMID: 29971050

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Project description:Reversible or phenotypic tolerance to antibiotics within microbial populations has been implicated in treatment failure of chronic infections and development of persister cells. However, the molecular mechanisms regulating phenotypic drug tolerance are largely unknown. In this study, we identified a four-gene operon in Streptococcus pneumoniae that contributes to phenotypic tolerance to vancomycin (ptv). RNA sequencing, quantiative reverse transcriptase PCR, and transcriptional luciferase reporter experiments revealed that transcription of the ptv operon (consisting of ptvR, ptvA, ptvB, and ptvC) is induced by exposure to vancomycin. Further investigation showed that transcription of the ptv operon is repressed by PtvR, a PadR family repressor. Transcriptional induction of the ptv operon by vancomycin was achieved by transcriptional derepression of this locus, which was mediated by PtvR. Importantly, fully derepressing ptvABC by deleting ptvR or overexpressing the ptv operon with an exogenous promoter significantly enhanced vancomycin tolerance. Gene deletion analysis revealed that PtvA, PtvB, and PtvC are all required for the PtvR-regulated phenotypic tolerance to vancomycin. Finally, the results of an electrophoretic mobility shift assay with recombinant PtvR showed that PtvR represses the transcription of the ptv operon by binding to two palindromic sequences within the ptv promoter. Together, the ptv locus represents an inducible system in S. pneumoniae in response to stressful conditions, including those caused by antibiotics.IMPORTANCE Reversible or phenotypic tolerance to antibiotics within microbial populations is associated with treatment failure of bacterial diseases, but the underlying mechanisms regulating phenotypic drug tolerance remain obscure. This study reports our finding of a multigene locus that contributes to inducible tolerance to vancomycin in Streptococcus pneumoniae, an important opportunistic human pathogen. The vancomycin tolerance phenotype depends on the PtvR transcriptional repressor and three predicted membrane-associated proteins encoded by the ptv locus. This represents the first example of a gene locus in S. pneumoniae that is responsible for antibiotic tolerance and has important implications for further understanding bacterial responses and phenotypic tolerance to antibiotic treatment in this and other pathogens.

Project description:Previous studies of the oral pathogen Streptococcus mutans have determined that this Gram-positive facultative anaerobe mounts robust responses to both acid and oxidative stresses. The water-forming NADH oxidase (Nox; encoded by nox) is thought to be critical for the regeneration of NAD(+), for use in glycolysis, and for the reduction of oxygen, thereby preventing the formation of damaging reactive oxygen species. In this study, the free NAD(+)/NADH ratio in a nox deletion strain (?nox) was discovered to be remarkably higher than that in the parent strain, UA159, when the strains were grown in continuous culture. This unanticipated result was explained by significantly elevated lactate dehydrogenase (Ldh; encoded by ldh) activity and ldh transcription in the ?nox strain, which was mediated in part by the redox-sensing regulator Rex. cDNA microarray analysis of S. mutans cultures exposed to simultaneous acid stress (growth at a low pH) and oxidative stress (generated through the deletion of nox or the addition of exogenous oxygen) revealed a stress response synergistically heightened over that with either stress alone. In the ?nox strain, this elevated stress response included increased glucose phosphoenolpyruvate phosphotransferase system (PTS) activity, which appeared to be due to elevated manL transcription, mediated in part, like elevated ldh transcription, by Rex. While the ?nox strain does possess a membrane composition different from that of the parent strain, it did not appear to have defects in either membrane permeability or ATPase activity. However, the altered transcriptome and metabolome of the ?nox strain were sufficient to impair its ability to compete with commensal peroxigenic oral streptococci during growth under aerobic conditions.Streptococcus mutans is an oral pathogen whose ability to outcompete commensal oral streptococci is strongly linked to the formation of dental caries. Previous work has demonstrated that the S. mutans water-forming NADH oxidase is critical for both carbon metabolism and the prevention of oxidative stress. The results of this study show that upregulation of lactate dehydrogenase, mediated through the redox sensor Rex, overcompensates for the loss of nox. Additionally, nox deletion led to the upregulation of mannose and glucose transport, also mediated through Rex. Importantly, the loss of nox rendered S. mutans defective in its ability to compete directly with two species of commensal streptococci, suggesting a role for nox in the pathogenic potential of this organism.

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NADH-Mediated Gene Expression in Streptococcus pneumoniae and Role of Rex as a Transcriptional Repressor of the Rex-Regulon.

Publications

NADH-Mediated Gene Expression in <i>Streptococcus pneumoniae</i> and Role of Rex as a Transcriptional Repressor of the Rex-Regulon.

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