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Volume 166, Issue 3

Peptides encoded in the RcrRPQ operon are essential for thermotolerance Free

Abstract

The MarR-like transcriptional regulator and two ABC transporters encoded by the operon in the dental caries pathogen have important regulatory roles related to oxidative stress tolerance, genetic competence and (p)ppGpp metabolism. A unique feature of the operon, when compared to other bacteria, is the presence of two peptides, designated Pep1 and Pep2, encoded in alternative reading frames at the 3′ end of . Here, we show that the operon, including Pep1 and 2, is essential for to survive and maintain viability at elevated temperatures. No major changes in the levels of the heat shock proteins DnaK or GroEL that could account for the thermosensitivity of mutants were observed. By introducing a single amino acid substitution into the gene that deletes an internally encoded peptide, XrpA, we found that XrpA is a contributing factor to the thermosensitive phenotype of a Δ strain. Overexpression of XrpA on a plasmid also caused a significant growth defect at 42 °C. Interestingly, loss of the gene for the RelA/SpoT homologue (RSH) enzyme, , restored growth of the Δ strain at 42 °C. During heat stress and when a stringent response was induced, levels of (p)ppGpp were elevated in the Δ strain. Deletion of in the Δ strain lowered the basal levels of (p)ppGpp to those observed in wild-type . Thus, (p)ppGpp pools are dysregulated in Δ, which likely leads to aberrant control of transcriptional/translational processes and the thermosensitive phenotype. In summary, the genes and peptides encoded in the operon are critical for thermotolerance, and in some strains these phenotypes are related to altered (p)ppGpp metabolism and increased production of the XrpA peptide.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): ABC transporters , dental caries , genetic competence , MarR regulator and stress tolerance
Funding
This study was supported by the:
  • National Institute of Dental and Craniofacial Research (Award DE13239)
    • Principle Award Recipient: Robert A. Burne
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2020-01-10
2024-04-25
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References

  1. Hobbs EC, Fontaine F, Yin X, Storz G. An expanding universe of small proteins. Curr Opin Microbiol 2011; 14:167–173 [View Article][PubMed]
     [Google Scholar]
  2. Warren AS, Archuleta J, Feng W-C, Setubal JC. Missing genes in the annotation of prokaryotic genomes. BMC Bioinformatics 2010; 11:131 [View Article][PubMed]
     [Google Scholar]
  3. Handler AA, Lim JE, Losick R. Peptide inhibitor of cytokinesis during sporulation in Bacillus subtilis . Mol Microbiol 2008; 68:588–599 [View Article][PubMed]
     [Google Scholar]
  4. Alix E, Blanc-Potard A-B. Peptide-assisted degradation of the Salmonella MgtC virulence factor. Embo J 2008; 27:546–557 [View Article][PubMed]
     [Google Scholar]
  5. Wadler CS, Vanderpool CK. A dual function for a bacterial small RNA: SgrS performs base pairing-dependent regulation and encodes a functional polypeptide. Proc Natl Acad Sci U S A 2007; 104:20454–20459 [View Article][PubMed]
     [Google Scholar]
  6. Hobbs EC, Yin X, Paul BJ, Astarita JL, Storz G. Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance. Proc Natl Acad Sci U S A 2012; 109:16696–16701 [View Article][PubMed]
     [Google Scholar]
  7. Ahn S-J, Kaspar J, Kim JN, Seaton K, Burne RA. Discovery of novel peptides regulating competence development in Streptococcus mutans . J Bacteriol 2014; 196:3735–3745 [View Article]
     [Google Scholar]
  8. Seaton K, Ahn S-J, Sagstetter AM, Burne RA. A transcriptional regulator and ABC transporters link stress tolerance, (p)ppGpp, and genetic competence in Streptococcus mutans . J Bacteriol 2011; 193:862–874 [View Article][PubMed]
     [Google Scholar]
  9. Seaton K, Ahn S-J, Burne RA. Regulation of competence and gene expression in Streptococcus mutans by the RcrR transcriptional regulator. Mol Oral Microbiol 2015; 30:147–159 [View Article]
     [Google Scholar]
  10. Shields RC, Burne RA. Conserved and divergent functions of RcrRPQ in Streptococcus gordonii and S. mutans . FEMS Microbiol Lett 2015; 362:fnv119 [View Article]
     [Google Scholar]
  11. Sambrook J, Russell D. Molecular Cloning: A Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001
     [Google Scholar]
  12. Lau PCY, Sung CK, Lee JH, Morrison DA, Cvitkovitch DG. Pcr ligation mutagenesis in transformable streptococci: application and efficiency. J Microbiol Methods 2002; 49:193–205 [View Article][PubMed]
     [Google Scholar]
  13. Jayaraman GC, Burne RA. DnaK expression in response to heat shock of Streptococcus mutans . FEMS Microbiol Lett 1995; 131:255–261 [View Article]
     [Google Scholar]
  14. Lemos JA, Burne RA, Castro AC. Molecular cloning, purification and immunological responses of recombinants GroEL and DnaK from Streptococcus pyogenes . FEMS Immunol Med Microbiol 2000; 28:121–128 [View Article][PubMed]
     [Google Scholar]
  15. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R et al. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 2007; 35:W71–74 [View Article][PubMed]
     [Google Scholar]
  16. Ahn S-J, Lemos JAC, Burne RA. Role of HtrA in growth and competence of Streptococcus mutans UA159. J Bacteriol 2005; 187:3028–3038 [View Article]
     [Google Scholar]
  17. Lemos JA, Lin VK, Nascimento MM, Abranches J, Burne RA. Three gene products govern (p)ppGpp production by Streptococcus mutans . Mol Microbiol 2007; 65:1568–1581 [View Article]
     [Google Scholar]
  18. Terleckyj B, Willett NP, Shockman GD. Growth of several cariogenic strains of oral streptococci in a chemically defined medium. Infect Immun 1975; 11:649–655 [View Article]
     [Google Scholar]
  19. Nascimento MM, Lemos JA, Abranches J, Lin VK, Burne RA. Role of RelA of Streptococcus mutans in global control of gene expression. J Bacteriol 2008; 190:28–36 [View Article][PubMed]
     [Google Scholar]
  20. Perry JA, Cvitkovitch DG, Lévesque CM. Cell death in Streptococcus mutans biofilms: a link between CSP and extracellular DNA. FEMS Microbiol Lett 2009; 299:261–266 [View Article][PubMed]
     [Google Scholar]
  21. Sumei L, Klein MI, Heim KP, Fan Y, Bitoun JP et al. Streptococcus mutans eDNA is up-regulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery. J Bacteriol 2014; 196:2355–2366
     [Google Scholar]
  22. Deochand DK, Grove A. Marr family transcription factors: dynamic variations on a common scaffold. Crit Rev Biochem Mol Biol 2017; 52:595–613 [View Article][PubMed]
     [Google Scholar]
  23. Ahn S-J, Gu T, Koh J, Rice KC. Remodeling of the Streptococcus mutans proteome in response to LrgAB and external stresses. Sci Rep 2017; 7:14063 [View Article][PubMed]
     [Google Scholar]
  24. Liu C, Niu Y, Zhou X, Zheng X, Wang S et al. Streptococcus mutans copes with heat stress by multiple transcriptional regulons modulating virulence and energy metabolism. Sci Rep 2015; 5:12929 [View Article][PubMed]
     [Google Scholar]
  25. Rice KC, Turner ME, Carney O’neshia V, Gu T, Ahn S-J. Modification of the Streptococcus mutans transcriptome by LrgAB and environmental stressors. Microb Genom 2017; 3:e000104 [View Article]
     [Google Scholar]
  26. Lemos JA, Luzardo Y, Burne RA. Physiologic effects of forced down-regulation of dnaK and groEL expression in Streptococcus mutans . J Bacteriol 2007; 189:1582–1588 [View Article]
     [Google Scholar]
  27. Jayaraman GC, Penders JE, Burne RA. Transcriptional analysis of the Streptococcus mutans hrcA, grpE and dnaK genes and regulation of expression in response to heat shock and environmental acidification. Mol Microbiol 1997; 25:329–341 [View Article][PubMed]
     [Google Scholar]
  28. Lemos JA, Chen YY, Burne RA. Genetic and physiologic analysis of the groE operon and role of the HrcA repressor in stress gene regulation and acid tolerance in Streptococcus mutans . J Bacteriol 2001; 183:6074–6084 [View Article][PubMed]
     [Google Scholar]
  29. Lemos JAC, Burne RA. Regulation and physiological significance of ClpC and ClpP in Streptococcus mutans . J Bacteriol 2002; 184:6357–6366 [View Article][PubMed]
     [Google Scholar]
  30. Shields RC, Zeng L, Culp DJ, Burne RA. Genomewide identification of essential genes and fitness determinants of Streptococcus mutans UA159. mSphere 2018; 3:e00031–18 [View Article]
     [Google Scholar]
  31. Biswas I, Jha JK, Fromm N. Shuttle expression plasmids for genetic studies in Streptococcus mutans . Microbiology 2008; 154:2275–2282 [View Article]
     [Google Scholar]
  32. Dong G, Tian X-L, Gomez ZA, Li Y-H. Regulated proteolysis of the alternative sigma factor SigX in Streptococcus mutans: implication in the escape from competence. BMC Microbiol 2014; 14:183 [View Article][PubMed]
     [Google Scholar]
  33. Kaspar J, Ahn S-J, Palmer SR, Choi SC, Stanhope MJ et al. A unique open reading frame within the comX gene of S treptococcus mutans regulates genetic competence and oxidative stress tolerance. Mol Microbiol 2015; 96:463–482 [View Article]
     [Google Scholar]
  34. Kaspar J, Shields RC, Burne RA. Competence inhibition by the XrpA peptide encoded within the comX gene of Streptococcus mutans . Mol Microbiol 2018; 109:345–364 [View Article][PubMed]
     [Google Scholar]
  35. Irving SE, Corrigan RM. Triggering the stringent response: signals responsible for activating (p)ppGpp synthesis in bacteria. Microbiology 2018; 164:268–276 [View Article]
     [Google Scholar]
  36. Gaca AO, Colomer-Winter C, Lemos JA. Many means to a common end: the intricacies of (p)ppGpp metabolism and its control of bacterial homeostasis. J Bacteriol 2015; 197:1146–1156 [View Article][PubMed]
     [Google Scholar]
  37. Kim JN, Ahn S-J, Seaton K, Garrett S, Burne RA. Transcriptional organization and physiological contributions of the relQ operon of Streptococcus mutans . J Bacteriol 2012; 194:1968–1978 [View Article][PubMed]
     [Google Scholar]
  38. Kaspar J, Kim JN, Ahn S-J, Burne RA. An Essential Role for (p)ppGpp in the integration of stress tolerance, peptide signaling, and competence development in Streptococcus mutans . Front Microbiol 2016; 7:1162 [View Article][PubMed]
     [Google Scholar]
  39. Abranches J, Martinez AR, Kajfasz JK, Chávez V, Garsin DA et al. The molecular alarmone (p)ppGpp mediates stress responses, vancomycin tolerance, and virulence in Enterococcus faecalis . J Bacteriol 2009; 191:2248–2256 [View Article][PubMed]
     [Google Scholar]
  40. VanBogelen RA, Kelley PM, Neidhardt FC. Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli . J Bacteriol 1987; 169:26–32 [View Article][PubMed]
     [Google Scholar]
  41. Gallant J, Palmer L, Pao CC. Anomalous synthesis of ppGpp in growing cells. Cell 1977; 11:181–185 [View Article][PubMed]
     [Google Scholar]
  42. Kriel A, Bittner AN, Kim SH, Liu K, Tehranchi AK et al. Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance. Mol Cell 2012; 48:231–241 [View Article][PubMed]
     [Google Scholar]
  43. Lemos JA, Nascimento MM, Lin VK, Abranches J, Burne RA. Global regulation by (p)ppGpp and CodY in Streptococcus mutans . J Bacteriol 2008; 190:5291–5299 [View Article][PubMed]
     [Google Scholar]
  44. Hendriksen WT, Bootsma HJ, Estevão S, Hoogenboezem T, de Jong A et al. Cody of Streptococcus pneumoniae: link between nutritional gene regulation and colonization. J Bacteriol 2008; 190:590–601 [View Article][PubMed]
     [Google Scholar]
  45. Corrigan RM, Bellows LE, Wood A, Gründling A. ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria. Proc Natl Acad Sci U S A 2016; 113:E1710–1719 [View Article][PubMed]
     [Google Scholar]
  46. Ahn S-J, Wen ZT, Burne RA. Multilevel control of competence development and stress tolerance in Streptococcus mutans UA159. Infect Immun 2006; 74:1631–1642 [View Article][PubMed]
     [Google Scholar]
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Peptides encoded in the Streptococcus mutans RcrRPQ operon are essential for thermotolerance
Microbiology 166, 306 (2020); https://doi.org/10.1099/mic.0.000887
/content/journal/micro/10.1099/mic.0.000887
/content/journal/micro/10.1099/mic.0.000887
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