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Volume 5, Issue 9

The resuscitation-promoting factor (Rpf) from and its putative reaction product 1,6-anhydro-MurNAc increase culturability of environmental bacteria Open Access

Abstract

Dormant bacterial cells do not divide and are not immediately culturable, but they persist in a state of low metabolic activity, a physiological state having clinical relevance, for instance in latent tuberculosis. Resuscitation-promoting factors (Rpfs) are proteins that act as signalling molecules mediating growth and replication. In this study we aimed to test the effect of Rpfs from on the number and diversity of cultured bacteria using insect and soil samples, and to examine if the increase in culturability could be reproduced with the putative reaction product of Rpf, 1,6-anhydro--acetylmuramic acid (1,6-anhydro-MurNAc). The gene from was amplified and cloned into a pET21b expression vector and the protein was expressed in BL21(DE3) cells and purified by affinity chromatography using a hexa-histidine tag. 1,6-Anhydro-MurNAc was prepared using reported chemical synthesis methods. Recombinant Rpf protein or 1,6-anhydro-MurNAc were added to R2A cultivation media, and their effect on the culturability of bacteria from eight environmental samples including four cockroach guts and four soils was examined. Colony-forming units, 16S rRNA gene copies and Illumina amplicon sequencing of the 16S rRNA gene were measured for all eight samples subjected to three different treatments: Rpf, 1,6-anhydro-MurNAc or blank control. Both Rpf and 1,6-anhydro-MurNAc increased the number of colony-forming units and of 16S rRNA gene copies across the samples although the protein was more effective. The Rpf and 1,6-anhydro-MurNAc promoted the cultivation of a diverse set of bacteria and in particular certain clades of the phyla and . This study opens the path for improved cultivation strategies aiming to isolate and study yet undescribed living bacterial organisms.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Actinobacteria , cultivation , Micrococcus luteus and resuscitation-promoting factors
Funding
This study was supported by the:
  • LOEWE-Zentrum für Translationale Medizin und Pharmakologie
    • Principle Award Recipient: AndreasVilcinskas
  • Alexander von Humboldt-Stiftung
    • Principle Award Recipient: JuanGuzman
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2023-09-22
2024-05-04
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References

  1. Kell DB, Young M. Bacterial dormancy and culturability: the role of autocrine growth factors. Curr Opin Microbiol 2000; 3:238–243 [View Article] [PubMed]
     [Google Scholar]
  2. Rittershaus ESC, Baek S-H, Sassetti CM. The normalcy of dormancy: common themes in microbial quiescence. Cell Host Microbe 2013; 13:643–651 [View Article]
     [Google Scholar]
  3. Puspita ID, Kamagata Y, Tanaka M, Asano K, Nakatsu CH. Are uncultivated bacteria really uncultivable?. Microbes Environ 2012; 27:356–366 [View Article] [PubMed]
     [Google Scholar]
  4. Ravagnani A, Finan CL, Young M. A novel firmicute protein family related to the actinobacterial resuscitation-promoting factors by non-orthologous domain displacement. BMC Genomics 2005; 6:1–14 [View Article] [PubMed]
     [Google Scholar]
  5. Mukamolova GV, Kaprelyants AS, Young DI, Young M, Kell DB. A bacterial cytokine. Proc Natl Acad Sci USA 1998; 95:8916–8921 [View Article]
     [Google Scholar]
  6. Shleeva MO, Bagramyan K, Telkov MV, Mukamolova GV, Young M et al. Formation and resuscitation of “non-culturable” cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase. Microbiology 2002; 148:1581–1591 [View Article] [PubMed]
     [Google Scholar]
  7. Mukamolova GV, Turapov OA, Young DI, Kaprelyants AS, Kell DB et al. A family of autocrine growth factors in Mycobacterium tuberculosis. Mol Microbiol 2002; 46:623–635 [View Article] [PubMed]
     [Google Scholar]
  8. Sexton DL, St-Onge RJ, Haiser HJ, Yousef MR, Brady L et al. Resuscitation-promoting factors are cell wall-lytic enzymes with important roles in the germination and growth of Streptomyces coelicolor. J Bacteriol 2015; 197:848–860 [View Article] [PubMed]
     [Google Scholar]
  9. Schroeckh V, Martin K. Resuscitation-promoting factors: distribution among actinobacteria, synthesis during life-cycle and biological activity. Antonie van Leeuwenhoek 2006; 89:359–365 [View Article] [PubMed]
     [Google Scholar]
  10. Cohen-Gonsaud M, Barthe P, Bagnéris C, Henderson B, Ward J et al. The structure of a resuscitation-promoting factor domain from Mycobacterium tuberculosis shows homology to lysozymes. Nat Struct Mol Biol 2005; 12:270–273 [View Article]
     [Google Scholar]
  11. Mukamolova GV, Murzin AG, Salina EG, Demina GR, Kell DB et al. Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation. Mol Microbiol 2006; 59:84–98 [View Article] [PubMed]
     [Google Scholar]
  12. Telkov MV, Demina GR, Voloshin SA, Salina EG, Dudik TV et al. Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases. Biochemistry 2006; 71:414–422 [View Article] [PubMed]
     [Google Scholar]
  13. Sexton DL, Herlihey FA, Brott AS, Crisante DA, Shepherdson E et al. Roles of LysM and LytM domains in resuscitation-promoting factor (Rpf) activity and Rpf-mediated peptidoglycan cleavage and dormant spore reactivation. J Biol Chem 2020; 295:9171–9182 [View Article] [PubMed]
     [Google Scholar]
  14. Nikitushkin VD, Demina GR, Shleeva MO, Kaprelyants AS. Peptidoglycan fragments stimulate resuscitation of “non-culturable” mycobacteria. Antonie van Leeuwenhoek 2013; 103:37–46 [View Article] [PubMed]
     [Google Scholar]
  15. Nikitushkin VD, Demina GR, Shleeva MO, Guryanova SV, Ruggiero A et al. A product of RpfB and RipA joint enzymatic action promotes the resuscitation of dormant mycobacteria. FEBS J 2015; 282:2500–2511 [View Article] [PubMed]
     [Google Scholar]
  16. Guzman J, Ortúzar M, Poehlein A, Daniel R, Trujillo ME et al. Agromyces archimandritae sp. nov., isolated from the cockroach Archimandrita tessellata. Int J Syst Evol Microbiol 2022; 72:005283 [View Article]
     [Google Scholar]
  17. Li S-H, Yu X-Y, Park D-J, Hozzein WN, Kim C-J et al. Rhodococcus soli sp. nov., an actinobacterium isolated from soil using a resuscitative technique. Antonie van Leeuwenhoek 2015; 107:357–366 [View Article] [PubMed]
     [Google Scholar]
  18. Lopez Marin MA, Suman J, Jani K, Ulbrich P, Cajthaml T et al. Solicola gregarius gen. nov., sp. nov., a soil actinobacterium isolated after enhanced cultivation with Micrococcus luteus culture supernatant. Int J Syst Evol Microbiol 2023; 73:005678 [View Article] [PubMed]
     [Google Scholar]
  19. Yu X-Y, Zhang L, Ren B, Yang N, Liu M et al. Arthrobacter liuii sp. nov., resuscitated from Xinjiang desert soil. Int J Syst Evol Microbiol 2015; 65:896–901 [View Article] [PubMed]
     [Google Scholar]
  20. Cai J, Pan A, Li Y, Xiao Y, Zhou Y et al. A novel strategy for enhancing anaerobic biodegradation of an anthraquinone dye reactive blue 19 with resuscitation-promoting factors. Chemosphere 2021; 263:127922 [View Article] [PubMed]
     [Google Scholar]
  21. Su X, Li S, Xie M, Tao L, Zhou Y et al. Enhancement of polychlorinated biphenyl biodegradation by resuscitation promoting factor (Rpf) and Rpf-responsive bacterial community. Chemosphere 2021; 263:128283 [View Article] [PubMed]
     [Google Scholar]
  22. Lopez Marin MA, Strejcek M, Junkova P, Suman J, Santrucek J et al. Exploring the potential of Micrococcus luteus culture supernatant with resuscitation-promoting factor for enhancing the culturability of soil bacteria. Front Microbiol 2021; 12:685263 [View Article] [PubMed]
     [Google Scholar]
  23. Calvert MB, Mayer C, Titz A. An efficient synthesis of 1, 6-anhydro-N-acetylmuramic acid from N-acetylglucosamine. Beilstein J Org Chem 2017; 13:2631–2636
     [Google Scholar]
  24. Guzman J, Won M, Poehlein A, Sombolestani AS, Mayorga-Ch D et al. Aristophania vespae gen. nov., sp. nov., isolated from wasps, is related to Bombella and Oecophyllibacter, isolated from bees and ants. Int J Syst Evol Microbiol 2023; 73:005699 [View Article]
     [Google Scholar]
  25. Salvà Serra F, Salvà-Serra F, Svensson-Stadler L, Busquets A, Jaén-Luchoro D et al. A protocol for extraction and purification of high-quality and quantity bacterial DNA applicable for genome sequencing: a modified version of the marmur procedure. Protocol Exchange 2018 [View Article]
     [Google Scholar]
  26. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 1991; 173:697–703 [View Article]
     [Google Scholar]
  27. Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 2013; 41:e1 [View Article] [PubMed]
     [Google Scholar]
  28. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA et al. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods 2016; 13:581–583 [View Article] [PubMed]
     [Google Scholar]
  29. McMurdie PJ, Holmes S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One 2013; 8:e61217 [View Article] [PubMed]
     [Google Scholar]
  30. Mukamolova GV, Turapov OA, Kazarian K, Telkov M, Kaprelyants AS et al. The rpf gene of Micrococcus luteus encodes an essential secreted growth factor. Mol Microbiol 2002; 46:611–621 [View Article] [PubMed]
     [Google Scholar]
  31. Puspita ID, Kitagawa W, Kamagata Y, Tanaka M, Nakatsu CH. Increase in bacterial colony formation from a permafrost ice wedge dosed with a Tomitella biformata recombinant resuscitation-promoting factor protein. Microbes Environ 2015; 30:151–156 [View Article] [PubMed]
     [Google Scholar]
  32. Wang Y, Shi J, Tang L, Zhang Y, Zhang Y et al. Evaluation of Rpf protein of Micrococcus luteus for cultivation of soil actinobacteria. Syst Appl Microbiol 2021; 44:126234 [View Article] [PubMed]
     [Google Scholar]
  33. Kuo V, Lehmkuhl BK, Lennon JT. Resuscitation of the microbial seed bank alters plant-soil interactions. Mol Ecol 2021; 30:2905–2914 [View Article] [PubMed]
     [Google Scholar]
  34. Ye Z, Li H, Jia Y, Fan J, Wan J et al. Supplementing resuscitation-promoting factor (Rpf) enhanced biodegradation of polychlorinated biphenyls (PCBs) by Rhodococcus biphenylivorans strain TG9T. Environ Pollut 2020; 263:114488 [View Article] [PubMed]
     [Google Scholar]
  35. Shirling EB, Gottlieb D. Cooperative description of type cultures of Streptomyces. IV. Species descriptions from the second, third and fourth studies. Int J Syst Bacteriol 1969; 19:391–512 [View Article]
     [Google Scholar]
  36. Chevrette MG, Carlson CM, Ortega HE, Thomas C, Ananiev GE et al. The antimicrobial potential of Streptomyces from insect microbiomes. Nat Commun 2019; 10:516 [View Article] [PubMed]
     [Google Scholar]
  37. Chouvenc T, Elliott ML, Šobotník J, Efstathion CA, Su N-Y. The termite fecal nest: a framework for the opportunistic acquisition of beneficial soil Streptomyces (Actinomycetales: Streptomycetaceae). Environ Entomol 2018; 47:1431–1439 [View Article] [PubMed]
     [Google Scholar]
  38. Kato M, Asamizu S, Onaka H. Intimate relationships among actinomycetes and mycolic acid-containing bacteria. Sci Rep 2022; 12:7222 [View Article] [PubMed]
     [Google Scholar]
  39. Meier-Kolthoff JP, Carbasse JS, Peinado-Olarte RL, Göker M. TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acids Res 2022; 50:D801–D807 [View Article] [PubMed]
     [Google Scholar]
  40. Shen F-T, Young L-S, Hsieh M-F, Lin S-Y, Young C-C. Molecular detection and phylogenetic analysis of the alkane 1-monooxygenase gene from Gordonia spp. Syst Appl Microbiol 2010; 33:53–59 [View Article] [PubMed]
     [Google Scholar]
  41. Wodzanowski KA, Hyland SN, Chinthamani S, Sandles L-MD, Honma K et al. Investigating peptidoglycan recycling pathways in Tannerella forsythia with N-acetylmuramic acid bioorthogonal probes. ACS Infect Dis 2022; 8:1831–1838 [View Article] [PubMed]
     [Google Scholar]
  42. Su X, Xue B, Wang Y, Hashmi MZ, Lin H et al. Bacterial community shifts evaluation in the sediments of Puyang River and its nitrogen removal capabilities exploration by resuscitation promoting factor. Ecotoxicol Environ Saf 2019; 179:188–197 [View Article] [PubMed]
     [Google Scholar]
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The resuscitation-promoting factor (Rpf) from Micrococcus luteus and its putative reaction product 1,6-anhydro-MurNAc increase culturability of environmental bacteria
Access Microbiology 5, 000647.v4 (2023); https://doi.org/10.1099/acmi.0.000647.v4
/content/journal/acmi/10.1099/acmi.0.000647.v4
/content/journal/acmi/10.1099/acmi.0.000647.v4
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