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Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein.

ABSTRACT: Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158.

SUBMITTER: Solano-Collado V

PROVIDER: S-EPMC8264293 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Streptococcus pyogenes (group A streptococcus [GAS]) is responsible for over 500,000 deaths worldwide each year. The highly virulent M1T1 GAS clone is one of the most frequently isolated serotypes from streptococcal pharyngitis and invasive disease. The oral epithelial tract is a niche highly abundant in glycosylated structures, particularly those of the ABO(H) blood group antigen family. Using a high-throughput approach, we determined that a strain representative of the globally disseminated M1T1 GAS clone 5448 interacts with numerous, structurally diverse glycans. Preeminent among GAS virulence factors is the surface-expressed M protein. M1 protein showed high affinity for several terminal galactose blood group antigen structures. Deletion mutagenesis shows that M1 protein mediates glycan binding via its B repeat domains. Association of M1T1 GAS with oral epithelial cells varied significantly as a result of phenotypic differences in blood group antigen expression, with significantly higher adherence to those cells expressing H antigen structures compared to cells expressing A, B, or AB antigen structures. These data suggest a novel mechanism for GAS attachment to host cells and propose a link between host blood group antigen expression and M1T1 GAS colonization. There has been a resurgence in group A streptococcal (GAS) invasive disease, which has been paralleled by the emergence of the highly virulent M1T1 GAS clone. Intensive research has focused on mechanisms that contribute to the invasive nature of this serotype, while the mechanisms that contribute to host susceptibility to disease and bacterial colonization and persistence are still poorly understood. The M1T1 GAS clone is frequently isolated from the throat, an environment highly abundant in blood group antigen structures. This work examined the interaction of the M1 protein, the preeminent GAS surface protein, against a wide range of host-expressed glycan structures. Our data suggest that susceptibility to infection by GAS in the oral tract may correlate with phenotypic differences in host blood group antigen expression. Thus, variations in host blood group antigen expression may serve as a selective pressure contributing to the dissemination and overrepresentation of M1T1 GAS.

Project description:BackgroundThe highly conserved macromolecular synthesis operon (MMSO) contains both dnaG (primase) and sigA (primary sigma factor). However, in previously evaluated gram-positive species, the MMSO is divergent upstream of dnaG. The MMSO of Bacillus subtilis contains three open reading frames (ORFs) that are differentially regulated by multiple promoters. In conjunction with studies to determine the expression profile of dnaG, the MMSO of Staphylococus epidermidis was characterized.ResultsThe ORFs of S. epidermidis were compared to the previously described MMSO of B. subtilis and two additional ORFs in S. epidermidis, serp1129 and serp1130, were identified. The largest transcript, 4.8 kb in length, was expressed only in exponential growth and encompassed all four ORFs (serp1130, serp1129, dnaG, and sigA). A separate transcript (1.5 kb) comprising serp1130 and serp1129 was expressed in early exponential growth. Two smaller transcripts 1.3 and 1.2 kb in size were detected with a sigA probe in both exponential and post-exponential phases of growth. Western blot analysis correlated with the transcriptional profile and demonstrated that Serp1129 was detected only in the exponential phase of growth. Computational analysis identified that Serp1130 contained a CBS motif whereas Serp1129 contained an ATP/GTP binding motif. Functional studies of Serp1129 demonstrated that it was capable of binding both ATP and GTP. Comparisons with a sigB:dhfr mutant revealed that the 1.3 kb sigA transcript was regulated by a sigma(B)-dependent promoter.ConclusionsThese studies demonstrated that the S. epidermidis 1457 MMSO contains two ORFs (serp1129 and serp1130) not described within the B. subtilis MMSO and at least three promoters, one of which is sigma(beta)-dependent. The transcriptional regulation of sigA by sigma(B) provides evidence that the staphylococcal sigma(B)-dependent response is controlled at both the transcriptional and post-transcriptional level. The conservation of serp1129 across multiple gram-positive organisms and its capability to bind ATP and GTP support the need for further investigation of its role in bacterial growth.

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Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein.

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