The Phr peptides of Bacillus species mediate quorum sensing, but their identification and function in other species of bacteria has not been determined. We have identified a Phr peptide quorum sensing system (TprA/PhrA) that controls the expression of a lantibiotic gene cluster in the Gram-positive human pathogen, Streptococcus pneumoniae. Lantibiotics are highly modified peptides that are part of the bacteriocin family of antimicrobial peptides. We have characterized the basic mechanism for a Phr peptide signaling system in S. pneumoniae and found that it induces expression of the lantibiotic genes when pneumococcal cells are at high density in the presence of galactose, a main sugar of the human nasopharynx, a highly competitive microbial environment. Activity of the Phr peptide system is not seen when pneumococcal cells are grown with glucose, the preferred carbon source and the most prevalent sugar encountered by S. pneumoniae during invasive disease. Thus, the lantibiotic genes are expressed under the control of both cell density signals via the Phr peptide system and nutritional signals from the carbon source present, suggesting that quorum sensing and the lantibiotic machinery may help pneumococcal cells compete for space and resources during colonization of the nasopharynx.
In all bacterial species examined thus far, small regulatory RNAs (sRNAs) contribute to intricate patterns of dynamic genetic regulation. Many of the actions of these nucleic acids are mediated by well-characterized chaperones such as the Hfq protein, but genetic screens have also recently identified the 3′-to-5′ exoribonuclease polynucleotide phosphorylase (PNPase) as an unexpected stabilizer and facilitator of sRNAs in vivo. To understand how a ribonuclease might mediate these effects, we tested the interactions of PNPase with sRNAs and found that the enzyme can readily degrade these nucleic acids in vitro but, nonetheless, copurifies from cell extracts with the same sRNAs without discernible degradation or modification to their 3′ ends, suggesting that the associated RNA is protected against the destructive activity of the ribonuclease. In vitro, PNPase, Hfq, and sRNA can form a ternary complex in which the ribonuclease plays a nondestructive, structural role. Such ternary complexes might be formed transiently in vivo, but could help to stabilize particular sRNAs and remodel their population on Hfq. Taken together, our results indicate that PNPase can be programmed to act on RNA in either destructive or stabilizing modes in vivo and may form complex, protective ribonucleoprotein assemblies that shape the landscape of sRNAs available for action.
Unlike most bacteria, Streptococcus pneumoniae (pneumococcus) has two evolutionarily distinct ABC transporters (Pst1 and Pst2) for inorganic phosphate (Pi) uptake. The genes encoding a two-component regulator (PnpRS) are located immediately upstream of the pst1 operon. Both the pst1 and pst2 operons encode putative PhoU-family regulators (PhoU1 and PhoU2) at their ends. This study addresses why S. pneumoniae contains dual Pi uptake systems and the regulation and contribution of the Pst1 and Pst2 systems in conditions of high (mM) Pi amount and low (μM) Pi amount. We show that in unencapsulated mutants, both pst1 and pst2 can be deleted, and Pi is taken up by a third Na+/Pi co-transporter, designated as NptA. In contrast, either pst1 or pst2 is unexpectedly required for the growth of capsule producing strains. We used a combination of mutational analysis, transcript level determinations by qRT-PCR and RNA-Seq, assays for cellular PnpR~P amounts by SDS-PAGE, and pulse-Pi uptake experiments to study the regulation of Pi uptake. In high Pi medium, PhoU2 serves as the master negative regulator of Pst2 transporter function and PnpR~P levels (post-transcriptionally). ΔphoU2 mutants have high PnpR~P levels and induction of the pst1 operon, poor growth, and sensitivity to antibiotics, possibly due to high Pi accumulation. In low Pi medium, Pst2 is still active, but PnpR~P amount and pst1 operon levels increase. Together, these results support a model in which pneumococcus maintains high Pi transport in high and low Pi conditions that is required for optimal capsule biosynthesis.
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.
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