1887

Microbiology Society logo

Volume 167, Issue 8

Editor's Choice Characterization of an operon required for growth on cellobiose in Free

Abstract

Cellobiose metabolism is linked to the virulence properties in numerous bacterial pathogens. Here, we characterized a putative cellobiose PTS operon of to investigate the role of cellobiose metabolism in pathogenesis. Our gene knockout experiments demonstrated that the putative cellobiose operon enables uptake of cellobiose into and allows growth when cellobiose is provided as the sole carbon source in minimal medium. Additionally, using reporter gene fusion assays and DNA pulldown experiments, we show that its transcription is regulated by CelR, a novel transcriptional repressor protein, which directly binds to the upstream region of the cellobiose operon to control its expression. We have also identified cellobiose metabolism to play a significant role in physiology as observed by the reduction of sporulation efficiency when cellobiose uptake was compromised in the mutant strain. In corroboration to study findings, our hamster challenge experiment showed a significant reduction of pathogenicity by the cellobiose mutant strain in both the primary and the recurrent infection model – substantiating the role of cellobiose metabolism in pathogenesis.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): C. difficile , cellobiose , celR and Clostridioides difficile
Funding
This study was supported by the:
  • national institute of allergy and infectious diseases (Award 1R03AI135762-01A1)
    • Principle Award Recipient: RevathiGovind
  • national institute of allergy and infectious diseases (Award 1R15AI122173)
    • Principle Award Recipient: RevathiGovind
© 2021 The Authors
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001079
2021-08-19
2024-05-04
Loading full text...

Full text loading...

/deliver/fulltext/micro/167/8/mic001079.html?itemId=/content/journal/micro/10.1099/mic.0.001079&mimeType=html&fmt=ahah

References

  1. Cummings JH. Cellulose and the human gut. Gut 1984; 25:805–810 [View Article] [PubMed]
     [Google Scholar]
  2. Englyst HN, Anderson V, Cummings JH. Starch and non-starch polysaccharides in some cereal foods. J Sci Food Agric 1983; 34:1434–1440 [View Article] [PubMed]
     [Google Scholar]
  3. Flint HJ, Scott KP, Duncan SH, Louis P, Forano E. Microbial degradation of complex carbohydrates in the gut. Gut Microbes 2012; 3:289–306 [View Article] [PubMed]
     [Google Scholar]
  4. Chassard C, Delmas E, Robert C, Lawson PA, Bernalier-Donadille A. Ruminococcus champanellensis sp. nov., a cellulose-degrading bacterium from human gut microbiota. Int J Syst Evol Microbiol 2012; 62:138–143 [View Article] [PubMed]
     [Google Scholar]
  5. Gong J, Egbosimba EE, Forsberg CW. Cellulose-binding proteins of Fibrobacter succinogenes and the possible role of a 180-kDa cellulose-binding glycoprotein in adhesion to cellulose. Can J Microbiol 1996; 42:453–460 [View Article]
     [Google Scholar]
  6. Jun H-S, Qi M, Gong J, Egbosimba EE, Forsberg CW. Outer membrane proteins of Fibrobacter succinogenes with potential roles in adhesion to cellulose and in cellulose digestion. J Bacteriol 2007; 189:6806–6815 [View Article] [PubMed]
     [Google Scholar]
  7. Jenior ML, Leslie JL, Young VB, Schloss PD. Clostridium difficile alters the structure and metabolism of distinct cecal microbiomes during initial infection to promote sustained colonization. mSphere 2018; 3: [View Article] [PubMed]
     [Google Scholar]
  8. Collins J, Robinson C, Danhof H, Knetsch CW, van Leeuwen HC. Dietary trehalose enhances virulence of epidemic Clostridium difficile . Nature 2018; 553:291–294 [View Article] [PubMed]
     [Google Scholar]
  9. Antunes A, Camiade E, Monot M, Courtois E, Barbut F. Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile . Nucleic Acids Res 2012; 40:10701 [View Article] [PubMed]
     [Google Scholar]
  10. Karlsson S, Burman LG, Åkerlund T. Suppression of toxin production in Clostridium difficile VPI 10463 by amino acids. Microbiology (Reading) 1999; 145:1683–1693 [View Article] [PubMed]
     [Google Scholar]
  11. Francis IM, Jourdan S, Fanara S, Loria R, Rigali S. The cellobiose sensor CebR is the gatekeeper of Streptomyces scabies pathogenicity. mBio 2015; 6:e02018 [View Article] [PubMed]
     [Google Scholar]
  12. Wu M-C, Chen Y-C, Lin T-L, Hsieh P-F, Wang J-T. Cellobiose-specific phosphotransferase system of Klebsiella pneumoniae and its importance in biofilm formation and virulence. Infect Immun 2012; 80:2464–2472 [View Article] [PubMed]
     [Google Scholar]
  13. Park SF, Kroll RG. Expression of listeriolysin and phosphatidylinositol-specific phospholipase C is repressed by the plant-derived molecule cellobiose in Listeria monocytogenes . Mol Microbiol 1993; 8:653–661 [View Article] [PubMed]
     [Google Scholar]
  14. Cao TN, Joyet P, Aké FMD, Milohanic E, Deutscher J. Studies of the Listeria monocytogenes cellobiose transport components and their impact on virulence gene repression. J Mol Microbiol Biotechnol 2019; 29:10–26 [View Article] [PubMed]
     [Google Scholar]
  15. Maury MM, Tsai Y-H, Charlier C, Touchon M, Chenal-Francisque V et al. Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity. Nat Genet 2016; 48:308–313 [View Article] [PubMed]
     [Google Scholar]
  16. McAllister LJ, Ogunniyi AD, Stroeher UH, Paton JC. Contribution of a genomic accessory region encoding a putative cellobiose phosphotransferase system to virulence of Streptococcus pneumoniae . PLoS One 2012; 7:e32385 [View Article] [PubMed]
     [Google Scholar]
  17. Girinathan BP, Ou J, Dupuy B, Govind R. Pleiotropic roles of Clostridium difficile sin locus. PLoS Pathog 2018; 14:e1006940 [View Article] [PubMed]
     [Google Scholar]
  18. Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP. The ClosTron: A universal gene knock-out system for the genus Clostridium . J Microbiol Methods 2007; 70:452–464 [View Article] [PubMed]
     [Google Scholar]
  19. Karasawa T, Ikoma S, Yamakawa K, Nakamura S. A defined growth medium for Clostridium difficile . Microbiology (Reading) 1995; 141:371–375 [View Article] [PubMed]
     [Google Scholar]
  20. Fagan RP, Fairweather NF. Clostridium difficile has two parallel and essential Sec secretion systems. J Biol Chem 2011; 286:27483–27493 [View Article] [PubMed]
     [Google Scholar]
  21. Dupuy B, Sonenshein AL. Regulated transcription of Clostridium difficile toxin genes. Mol Microbiol 1998; 27:107–120 [View Article] [PubMed]
     [Google Scholar]
  22. Mani N, Lyras D, Barroso L, Howarth P, Wilkins T. Environmental response and autoregulation of Clostridium difficile TxeR, a sigma factor for toxin gene expression. J Bacteriol 2002; 184:5971–5978 [View Article] [PubMed]
     [Google Scholar]
  23. Jutras BL, Verma A, Stevenson B. Identification of novel DNA-binding proteins using DNA-affinity chromatography/pull down. Curr Protoc Microbiol 2012; Chapter 1:Unit1F.1:
     [Google Scholar]
  24. Sambol SP, Tang JK, Merrigan MM, Johnson S, Gerding DN. Infection of hamsters with epidemiologically important strains of Clostridium difficile. J Infect Dis 2001; 183:1760–1766 [View Article] [PubMed]
     [Google Scholar]
  25. Girinathan BP, Braun S, Sirigireddy AR, Espinola-Lopez J, Govind R. Correction: Importance of Glutamate Dehydrogenase (GDH) in Clostridium difficile colonization in vivo. PLoS One 2016; 11:e0165579 [View Article] [PubMed]
     [Google Scholar]
  26. Tobisch S, Stülke J, Hecker M. Regulation of the lic operon of Bacillus subtilis and characterization of potential phosphorylation sites of the Licr regulator protein by site-directed mutagenesis. J Bacteriol 1999; 181:4995–5003 [View Article] [PubMed]
     [Google Scholar]
  27. Saier MH, Reizer J. Proposed uniform nomenclature for the proteins and protein domains of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. J Bacteriol 1992; 174:1433–1438 [View Article] [PubMed]
     [Google Scholar]
  28. Thompson J, Ruvinov SB, Freedberg DI, Hall BG. Cellobiose-6-phosphate hydrolase (CELF) of Escherichia coli: Characterization and assignment to the unusual family 4 of glycosylhydrolases. J Bacteriol 1999; 181:7339–7345 [View Article] [PubMed]
     [Google Scholar]
  29. Kadokura K, Rokutani A, Yamamoto M, Ikegami T, Sugita H. Purification and characterization of Vibrio parahaemolyticus extracellular chitinase and chitin oligosaccharide deacetylase involved in the production of heterodisaccharide from chitin. Appl Microbiol Biotechnol 2007; 75:357–365 [View Article] [PubMed]
     [Google Scholar]
  30. Zheng M, Cooper DR, Grossoehme NE, Yu M, Hung LW. Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains. Acta Crystallogr D Biol Crystallogr 2009; 65:356–365 [View Article] [PubMed]
     [Google Scholar]
  31. Aravind L, Anantharaman V. HutC/FarR-like bacterial transcription factors of the GntR family contain a small molecule-binding domain of the chorismate lyase fold. FEMS Microbiol Lett 2003; 222:17–23 [View Article] [PubMed]
     [Google Scholar]
  32. Rigali S, Derouaux A, Giannotta F, Dusart J. Subdivision of the helix-turn-helix GntR family of bacterial regulators in the FadR, HutC, MocR, and YtrA subfamilies. J Biol Chem 2002; 277:12507–12515 [View Article] [PubMed]
     [Google Scholar]
  33. Haydon DJ, Guest JR. A new family of bacterial regulatory proteins. FEMS Microbiol Lett 1991; 63:291–295 [View Article] [PubMed]
     [Google Scholar]
  34. Lee MH, Scherer M, Rigali S, Golden JW. PlmA, a new member of the GntR family, has plasmid maintenance functions in Anabaena sp. strain PCC 7120. J Bacteriol 2003; 185:4315–4325 [View Article] [PubMed]
     [Google Scholar]
  35. Li Z, Xiang Z, Zeng J, Li Y, Li J. A GNTR family transcription factor in Streptococcus mutans regulates biofilm formation and expression of multiple sugar transporter genes. Front Microbiol 2018; 9:3224 [View Article] [PubMed]
     [Google Scholar]
  36. Tsypik O, Yushchuk O, Zaburannyi N, Flärdh K, Walker S. Transcriptional regulators of GntR family in Streptomyces coelicolor A3(2): analysis in silico and in vivo of YtrA subfamily. Folia Microbiol 2016; 61:209–220 [View Article]
     [Google Scholar]
  37. van Aalten DM, DiRusso CC, Knudsen J, Wierenga RK. Crystal structure of FadR, a fatty acid-responsive transcription factor with a novel acyl coenzyme A-binding fold. EMBO J 2000; 19:5167–5177 [View Article] [PubMed]
     [Google Scholar]
  38. Jain D. Allosteric control of transcription in GntR family of transcription regulators: A structural overview. IUBMB Life 2015; 67:556–563 [View Article] [PubMed]
     [Google Scholar]
  39. Afzal M, Shafeeq S, Ahmed H, Kuipers OP. N-acetylgalatosamine-mediated regulation of the aga operon by Agar in Streptococcus pneumoniae . Front Cell Infect Microbiol 2016; 6:101 [View Article] [PubMed]
     [Google Scholar]
  40. Shafeeq S, Kuipers OP, Kloosterman TG. Cellobiose-mediated gene expression in Streptococcus pneumoniae: a repressor function of the novel GntR-type regulator BguR. PLoS ONE 2013; 8:e57586 [View Article] [PubMed]
     [Google Scholar]
  41. Zeng L, Burne RA. Transcriptional regulation of the cellobiose operon of Streptococcus mutans . J Bacteriol 2009; 191:2153–2162 [View Article] [PubMed]
     [Google Scholar]
  42. Nie X, Yang B, Zhang L, Gu Y, Yang S et al. PTS regulation domain-containing transcriptional activator CelR and sigma factor σ(54) control cellobiose utilization in Clostridium acetobutylicum . Mol Microbiol 2016; 100:289–302 [View Article] [PubMed]
     [Google Scholar]
  43. Sadaie Y, Nakadate H, Fukui R, Yee LM, Asai K. Glucomannan utilization operon of Bacillus subtilis . FEMS Microbiol Lett 2008; 279:103–109 [View Article] [PubMed]
     [Google Scholar]
  44. Roy S, Patra T, Golder T, Chatterjee S, Koley H. Characterization of the gluconate utilization system of Vibrio cholerae with special reference to virulence modulation. Pathog Dis 74: [View Article] [PubMed]
     [Google Scholar]
  45. Shafeeq S, Kloosterman TG, Kuipers OP. CelR-mediated activation of the cellobiose-utilization gene cluster in Streptococcus pneumoniae . Microbiology (Reading) 2011; 157:2854–2861 [View Article] [PubMed]
     [Google Scholar]
  46. Néron S, Vadeboncoeur C. Two functionally different glucose phosphotransferase transport systems in Streptococcus mutans and Streptococcus sobrinus . Oral Microbiol Immunol 1987; 2:171–177 [View Article] [PubMed]
     [Google Scholar]
  47. Liberman ES, Bleiweis AS. Transport of glucose and mannose by a common phosphoenolpyruvate-dependent phosphotransferase system in Streptococcus mutans GS5. Infect Immun 1984; 43:1106–1109 [View Article] [PubMed]
     [Google Scholar]
  48. DiRusso CC, Heimert TL, Metzger AK. Characterization of FadR, a global transcriptional regulator of fatty acid metabolism in Escherichia coli. Interaction with the fadB promoter is prevented by long chain fatty acyl coenzyme A. J Biol Chem 1992; 267:8685–8691 [View Article] [PubMed]
     [Google Scholar]
  49. Suvorova IA, Korostelev YD, Gelfand MS. GNTR family of bacterial transcription factors and their DNA binding motifs: Structure, positioning and co-evolution. PLoS One 2015; 10:e0132618 [View Article] [PubMed]
     [Google Scholar]
  50. Soutourina O, Dubois T, Monot M, Shelyakin PV, Saujet L et al. Genome-wide transcription start site mapping and promoter assignments to a sigma factor in the human enteropathogen Clostridioides difficile . Front Microbiol 2020; 11:1939 [View Article] [PubMed]
     [Google Scholar]
  51. Neumann-Schaal M, Jahn D, Schmidt-Hohagen K. Metabolism the Difficile Way: The Key to the success of the pathogen Clostridioides difficile . Front Microbiol 2019; 10:219 [View Article] [PubMed]
     [Google Scholar]
  52. Daou N, Wang Y, Levdikov VM, Nandakumar M, Livny J. Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027. PLOS ONE 2019; 14:e0206896 [View Article] [PubMed]
     [Google Scholar]
  53. Grant CE, Bailey TL, Noble WS. FIMO: scanning for occurrences of a given motif. Bioinformatics 2011; 27:1017–1018 [View Article] [PubMed]
     [Google Scholar]
  54. Hall CL, Lee VT. Cyclic-di-GMP regulation of virulence in bacterial pathogens. Wiley Interdiscip Rev RNA 2018; 9: [View Article] [PubMed]
     [Google Scholar]
  55. Purcell EB, McKee RW, Courson DS, Garrett EM, McBride SM et al. A nutrient-regulated cyclic diguanylate phosphodiesterase controls Clostridium difficile biofilm and toxin production during stationary phase. Infect Immun 2017; 85: [View Article] [PubMed]
     [Google Scholar]
  56. Purcell EB, McKee RW, McBride SM, Waters CM, Tamayo R. Cyclic diguanylate inversely regulates motility and aggregation in Clostridium difficile . J Bacteriol 2012; 194:3307–3316 [View Article] [PubMed]
     [Google Scholar]
  57. McKee RW, Harvest CK, Tamayo R. Cyclic diguanylate regulates virulence factor genes via multiple riboswitches in Clostridium difficile . mSphere 2018; 3: [View Article] [PubMed]
     [Google Scholar]
  58. McKee RW, Mangalea MR, Purcell EB, Borchardt EK, Tamayo R. The second messenger cyclic Di-GMP regulates Clostridium difficile toxin production by controlling expression of sigD. J Bacteriol 2013; 195:5174–5185 [View Article] [PubMed]
     [Google Scholar]
  59. Bordeleau E, Burrus V. Cyclic-di-GMP signaling in the Gram-positive pathogen Clostridium difficile . Curr Genet 2015; 61:497–502 [View Article] [PubMed]
     [Google Scholar]
  60. Dhungel BA, Govind R. Phase variable expression of pdcB, a phosphodiesterase influences sporulation in Clostridioides difficile . bioRxiv 2021; 2021.05.24.445537:
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001079
Loading
Characterization of an operon required for growth on cellobiose in Clostridioides difficile
Microbiology 167, 001079 (2021); https://doi.org/10.1099/mic.0.001079
/content/journal/micro/10.1099/mic.0.001079
/content/journal/micro/10.1099/mic.0.001079
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error