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The rec locus, a competence-induced operon in Streptococcus pneumoniae.

ABSTRACT: To study competence and the process of transformation (TFN) in pneumococci, we developed a method for isolating TFN- mutants using insertional inactivation coupled with fusions to the gene for alkaline phosphatase (phoA). One TFN- mutant transformed 2 log units less efficiently than the parent strain. Reconstitution of the mutated region revealed a locus, rec, that contains two polycistronic genes, exp10 and the previously identified recA (B. Martin, J. M. Ruellan, J. F. Angulo, R. Devoret, and J. P. Claverys, Nucleic Acids Res. 20:6412, 1992). Exp10 is likely to be a membrane-associated protein, as it has a prokaryotic signal sequence and an Exp10-PhoA fusion localized with cell membranes. On the basis of sequence similarity, pneumococcal RecA is a member of bacterial RecA proteins responsible for homologous recombination of DNA. DNA-RNA hybridization analysis showed that this locus is transcribed as a polycistronic message, with increased transcription occurring during competence. With an Exp10-PhoA chimera used as a reporter, there was a 10-fold increase in the expression of the rec locus during competence while there was only minimal expression under growth conditions that repressed competence. The TFN- mutant containing the exp10-phoA fusion produced activator, a small extracellular polypeptide that induces competence, and the expression of rec was induced in response to activator. Therefore, the rec locus is directly required for genetic transformation and is regulated by the cell signaling mechanism that induces competence.

SUBMITTER: Pearce BJ

PROVIDER: S-EPMC176560 | biostudies-other | 1995 Jan

REPOSITORIES: biostudies-other

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The rec locus, a competence-induced operon in Streptococcus pneumoniae.

Pearce B J BJ Naughton A M AM Campbell E A EA Masure H R HR

Journal of bacteriology 19950101 1

To study competence and the process of transformation (TFN) in pneumococci, we developed a method for isolating TFN- mutants using insertional inactivation coupled with fusions to the gene for alkaline phosphatase (phoA). One TFN- mutant transformed 2 log units less efficiently than the parent strain. Reconstitution of the mutated region revealed a locus, rec, that contains two polycistronic genes, exp10 and the previously identified recA (B. Martin, J. M. Ruellan, J. F. Angulo, R. Devoret, and ...[more]

PMID: 7798154

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Project description:Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra-species competition within the human host. However, the triggers of pneumocin expression are poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39. Furthermore, by analogy with pneumococcal competence, we show that several antibiotics activate the blp-genes. Using real-time gene expression measurements we show that while the promoter driving expression of the two-component regulatory system blpR/H is constitutive, the remaining blp-promoters that control pneumocin expression, immunity and the inducer peptide BlpC, are pH-dependent and induced in the late exponential phase. Intriguingly, competence for genetic transformation, mediated by the paralogous ComD/E two-component quorum system, is induced by the same environmental cues. To test for interplay between these regulatory systems, we quantified the regulatory response to the addition of synthetic BlpC and competence-stimulating peptide (CSP). Supporting the idea of such interplay, we found that immediately upon addition of CSP, the blp-promoters were activated in a comD/E-dependent manner. After a delay, blp-expression was highly induced and was strictly dependent on blpRH and blpC. This raised the question of the mechanism of BlpC export, since bioinformatic analysis showed that the genes encoding the putative exporter for BlpC, blpAB, are not intact in strain D39 and most other strains. By contrast, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. Consistent with the idea that comAB mediate BlpC export, we finally show that high-level expression of the blp-genes requires comAB. Together, our results demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might therefore have unpredictable consequences on pneumococcal colonization dynamics by activating genes that mediate intra-specific interference competition.

Project description:UnlabelledParB proteins bind centromere-like DNA sequences called parS sites and are involved in plasmid and chromosome segregation in bacteria. We previously showed that the opportunistic human pathogen Streptococcus pneumoniae contains four parS sequences located close to the origin of replication which are bound by ParB. Using chromatin immunoprecipitation (ChIP), we found here that ParB spreads out from one of these parS sites, parS(-1.6°), for more than 5 kb and occupies the nearby comCDE operon, which drives competence development. Competence allows S. pneumoniae to take up DNA from its environment, thereby mediating horizontal gene transfer, and is also employed as a general stress response. Mutating parS(-1.6°) or deleting parB resulted in transcriptional up-regulation of comCDE and ssbB (a gene belonging to the competence regulon), demonstrating that ParB acts as a repressor of competence. However, genome-wide transcription analysis showed that ParB is not a global transcriptional regulator. Different factors, such as the composition of the growth medium and antibiotic-induced stress, can trigger the sensitive switch driving competence. This work shows that the ParB-parS chromosome segregation machinery also influences this developmental process.ImportanceStreptococcus pneumoniae (pneumococcus) is an important human pathogen responsible for more than a million deaths each year. Like all other organisms, S. pneumoniae must be able to segregate its chromosomes properly. Not only is understanding the molecular mechanisms underlying chromosome segregation in S. pneumoniae therefore of fundamental importance, but also, this knowledge might offer new leads for ways to target this pathogen. Here, we identified a link between the pneumococcal chromosome segregation system and the competence-developmental system. Competence allows S. pneumoniae to take up and integrate exogenous DNA in its chromosome. This process plays a crucial role in successful adaptation to--and escape from--host defenses, antibiotic treatments, and vaccination strategies. We show that the chromosome segregation protein ParB acts as a repressor of competence. To the best of our knowledge, this is the first example of a ParB protein controlling bacterial competence.

Project description:Bacterial communities are established through a combination of cooperative and antagonistic interactions between the inhabitants. Competitive interactions often involve the production of antimicrobial substances, including bacteriocins, which are small antimicrobial peptides that target other community members. Despite the nearly ubiquitous presence of bacteriocin-encoding loci, inhibitory activity has been attributed to only a small fraction of gene clusters. In this study, we characterized a novel locus (the pld locus) in the pathogen Streptococcus pneumoniae that drives the production of a bacteriocin called pneumolancidin, which has broad antimicrobial activity. The locus encodes an unusual tandem array of four inhibitory peptides, three of which are absolutely required for antibacterial activity. The three peptide sequences are similar but appear to play distinct roles in regulation and inhibition. A modification enzyme typically found in loci encoding a class of highly modified bacteriocins called lantibiotics was required for inhibitory activity. The production of pneumolancidin is controlled by a two-component regulatory system that is activated by the accumulation of modified peptides. The locus is located on a mobile element that has been found in many pneumococcal lineages, although not all elements carry the pld genes. Intriguingly, a minimal region containing only the genes required for pneumolancidin immunity was found in several Streptococcus mitis strains. The pneumolancidin-producing strain can inhibit nearly all pneumococci tested to date and provided a competitive advantage in vivo. These peptides not only represent a unique strategy for bacterial competition but also are an important resource to guide the development of new antimicrobials.Successful colonization of a polymicrobial host surface is a prerequisite for the subsequent development of disease for many bacterial pathogens. Bacterial factors that directly inhibit the growth of neighbors may provide an advantage during colonization if the inhibition of competitors outweighs the energy for production. In this work, we found that production of a potent antimicrobial called pneumolancidin conferred a competitive advantage to the pathogen Streptococcus pneumoniae. S. pneumoniae secreting pneumolancidin inhibits a wide array of Gram-positive organisms, including all but one tested pneumococcal strain. The pneumolancidin genetic locus is of particular interest because it encodes three similar modified peptides (lantibiotics), each of which has a distinct role in the function of the locus. Lantibiotics represent a relatively untapped resource for the development of clinically useful antibiotics which are desperately needed. The broad inhibitory activity of pneumolancidin makes it an ideal candidate for further characterization and development.

Dataset Information

The rec locus, a competence-induced operon in Streptococcus pneumoniae.

Publications

The rec locus, a competence-induced operon in Streptococcus pneumoniae.

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