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Clostridial Genetics: Genetic Manipulation of the Pathogenic Clostridia

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  • Authors: S. A. Kuehne1, J. I. Rood2, D. Lyras3
  • Editors: Vincent A. Fischetti4, Richard P. Novick5, Joseph J. Ferretti6, Daniel A. Portnoy7, Miriam Braunstein8, Julian I. Rood9
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: School of Dentistry and Institute for Microbiology and Infection, University of Birmingham, Birmingham, UK; 2: Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria, Australia 3800; 3: Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria, Australia 3800; 4: The Rockefeller University, New York, NY; 5: Skirball Institute for Molecular Medicine, NYU Medical Center, New York, NY; 6: Department of Microbiology & Immunology, University of Oklahoma Health Science Center, Oklahoma City, OK; 7: Department of Molecular and Cellular Microbiology, University of California, Berkeley, Berkeley, CA; 8: Department of Microbiology and Immunology, University of North Carolina-Chapel Hill, Chapel Hill, NC; 9: Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia
  • Source: microbiolspec June 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0040-2018
  • Received 15 August 2018 Accepted 10 December 2018 Published 07 June 2019
  • Sarah Kuehne, [email protected]
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  • Abstract:

    The past 10 years have been revolutionary for clostridial genetics. The rise of next-generation sequencing led to the availability of annotated whole-genome sequences of the important pathogenic clostridia: , () , and , but also () and . These sequences were a prerequisite for the development of functional, sophisticated genetic tools for the pathogenic clostridia. A breakthrough came in the early 2000s with the development of TargeTron-based technologies specific for the clostridia, such as ClosTron, an insertional gene inactivation tool. The following years saw a plethora of new technologies being developed, mostly for , but also for other members of the genus, including . A range of tools is now available, allowing researchers to precisely delete genes, change single nucleotides in the genome, complement deletions, integrate novel DNA into genomes, or overexpress genes. There are tools for forward genetics, including an inducible transposon mutagenesis system for . As the latest addition to the tool kit, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technologies have also been adopted for the construction of single and multiple gene deletions in . This article summarizes the key genetic technologies available to manipulate, study, and understand the pathogenic clostridia.

  • Citation: Kuehne S, Rood J, Lyras D. 2019. Clostridial Genetics: Genetic Manipulation of the Pathogenic Clostridia. Microbiol Spectrum 7(3):GPP3-0040-2018. doi:10.1128/microbiolspec.GPP3-0040-2018.

References

1. Brook I. 2012. Other Clostridium species, p 979–981. In Long SS (ed), Principles and Practice of Pediatric Infectious Diseases, 4th ed. Saunders, Philadelphia, PA. [PubMed]
2. Yutin N, Galperin MY. 2013. A genomic update on clostridial phylogeny: Gram-negative spore formers and other misplaced clostridia. Environ Microbiol 15:2631–2641.
3. Lawson PA, Citron DM, Tyrrell KL, Finegold SM. 2016. Reclassification of Clostridium difficile as Clostridioides difficile (Hall and O’Toole 1935) Prévot 1938. Anaerobe 40:95–99 http://dx.doi.org/10.1016/j.anaerobe.2016.06.008. [PubMed]
4. Guo H, Karberg M, Long M, Jones JP III, Sullenger B, Lambowitz AM. 2000. Group II introns designed to insert into therapeutically relevant DNA target sites in human cells. Science 289:452–457 http://dx.doi.org/10.1126/science.289.5478.452. [PubMed]
5. Sasi Jyothsna TS, Tushar L, Sasikala C, Ramana CV. 2016. Paraclostridium benzoelyticum gen. nov., sp. nov., isolated from marine sediment and reclassification of Clostridium bifermentans as Paraclostridium bifermentans comb. nov. Proposal of a new genus Paeniclostridium gen. nov. to accommodate Clostridium sordellii and Clostridium ghonii. Int J Syst Evol Microbiol 66:1268–1274 http://dx.doi.org/10.1099/ijsem.0.000874. [PubMed]
6. Chumbler NM, Farrow MA, Lapierre LA, Franklin JL, Lacy DB. 2016. Clostridium difficile toxins TcdA and TcdB cause colonic tissue damage by distinct mechanisms. Infect Immun 84:2871–2877 http://dx.doi.org/10.1128/IAI.00583-16. [PubMed]
7. Navarro MA, McClane BA, Uzal FA. 2018. Mechanisms of action and cell death associated with Clostridium perfringens toxins. Toxins (Basel) 10:E212 http://dx.doi.org/10.3390/toxins10050212. [PubMed]
8. Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, Shiba T, Ogasawara N, Hattori M, Kuhara S, Hayashi H. 2002. Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci U S A 99:996–1001 http://dx.doi.org/10.1073/pnas.022493799. [PubMed]
9. Bruggemann H, Baumer S, Fricke WF, Wiezer A, Liesegang H, Decker I, Herzberg C, Martinez-Arias R, Merkl R, Henne A, Gottschalk G. 2003. The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc Natl Acad Sci U S A 100:1316–1321 http://dx.doi.org/10.1073/pnas.0335853100. [PubMed]
10. Sebaihia M, Wren BW, Mullany P, Fairweather NF, Minton N, Stabler R, Thomson NR, Roberts AP, Cerdeño-Tárraga AM, Wang H, Holden MT, Wright A, Churcher C, Quail MA, Baker S, Bason N, Brooks K, Chillingworth T, Cronin A, Davis P, Dowd L, Fraser A, Feltwell T, Hance Z, Holroyd S, Jagels K, Moule S, Mungall K, Price C, Rabbinowitsch E, Sharp S, Simmonds M, Stevens K, Unwin L, Whithead S, Dupuy B, Dougan G, Barrell B, Parkhill J. 2006. The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet 38:779–786 http://dx.doi.org/10.1038/ng1830. [PubMed]
11. Sebaihia M, Peck MW, Minton NP, Thomson NR, Holden MTG, Mitchell WJ, Carter AT, Bentley SD, Mason DR, Crossman L, Paul CJ, Ivens A, Wells-Bennik MHJ, Davis IJ, Cerdeño-Tárraga AM, Churcher C, Quail MA, Chillingworth T, Feltwell T, Fraser A, Goodhead I, Hance Z, Jagels K, Larke N, Maddison M, Moule S, Mungall K, Norbertczak H, Rabbinowitsch E, Sanders M, Simmonds M, White B, Whithead S, Parkhill J. 2007. Genome sequence of a proteolytic (group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes. Genome Res 17:1082–1092 http://dx.doi.org/10.1101/gr.6282807. [PubMed]
12. Myers GSA, Rasko DA, Cheung JK, Ravel J, Seshadri R, DeBoy RT, Ren Q, Varga J, Awad MM, Brinkac LM, Daugherty SC, Haft DH, Dodson RJ, Madupu R, Nelson WC, Rosovitz MJ, Sullivan SA, Khouri H, Dimitrov GI, Watkins KL, Mulligan S, Benton J, Radune D, Fisher DJ, Atkins HS, Hiscox T, Jost BH, Billington SJ, Songer JG, McClane BA, Titball RW, Rood JI, Melville SB, Paulsen IT. 2006. Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens. Genome Res 16:1031–1040 http://dx.doi.org/10.1101/gr.5238106. [PubMed]
13. Hassan KA, Elbourne LD, Tetu SG, Melville SB, Rood JI, Paulsen IT. 2015. Genomic analyses of Clostridium perfringens isolates from five toxinotypes. Res Microbiol 166:255–263 http://dx.doi.org/10.1016/j.resmic.2014.10.003. [PubMed]
14. Lacey JA, Allnutt TR, Vezina B, Van TTH, Stent T, Han X, Rood JI, Wade B, Keyburn AL, Seemann T, Chen H, Haring V, Johanesen PA, Lyras D, Moore RJ. 2018. Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens. BMC Genomics 19:379 http://dx.doi.org/10.1186/s12864-018-4771-1. [PubMed]
15. Cohen JE, Wang R, Shen RF, Wu WW, Keller JE. 2017. Comparative pathogenomics of Clostridium tetani. PLoS One 12:e0182909 http://dx.doi.org/10.1371/journal.pone.0182909. [PubMed]
16. Brüggemann H, Brzuszkiewicz E, Chapeton-Montes D, Plourde L, Speck D, Popoff MR. 2015. Genomics of Clostridium tetani. Res Microbiol 166:326–331 http://dx.doi.org/10.1016/j.resmic.2015.01.002. [PubMed]
17. Williamson CH, Sahl JW, Smith TJ, Xie G, Foley BT, Smith LA, Fernández RA, Lindström M, Korkeala H, Keim P, Foster J, Hill K. 2016. Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing clostridia. BMC Genomics 17:180 http://dx.doi.org/10.1186/s12864-016-2502-z. [PubMed]
18. Eyre DW, Cule ML, Wilson DJ, Griffiths D, Vaughan A, O’Connor L, Ip CLC, Golubchik T, Batty EM, Finney JM, Wyllie DH, Didelot X, Piazza P, Bowden R, Dingle KE, Harding RM, Crook DW, Wilcox MH, Peto TEA, Walker AS. 2013. Diverse sources of C. difficile infection identified on whole-genome sequencing. N Engl J Med 369:1195–1205 http://dx.doi.org/10.1056/NEJMoa1216064. [PubMed]
19. He M, Miyajima F, Roberts P, Ellison L, Pickard DJ, Martin MJ, Connor TR, Harris SR, Fairley D, Bamford KB, D’Arc S, Brazier J, Brown D, Coia JE, Douce G, Gerding D, Kim HJ, Koh TH, Kato H, Senoh M, Louie T, Michell S, Butt E, Peacock SJ, Brown NM, Riley T, Songer G, Wilcox M, Pirmohamed M, Kuijper E, Hawkey P, Wren BW, Dougan G, Parkhill J, Lawley TD. 2013. Emergence and global spread of epidemic healthcare-associated Clostridium difficile. Nat Genet 45:109–113 http://dx.doi.org/10.1038/ng.2478. [PubMed]
20. Zhou Y, Sugiyama H, Johnson EA. 1993. Transfer of neurotoxigenicity from Clostridium butyricum to a nontoxigenic Clostridium botulinum type E-like strain. Appl Environ Microbiol 59:3825–3831.
21. Marvaud JC, Eisel U, Binz T, Niemann H, Popoff MR. 1998. TetR is a positive regulator of the tetanus toxin gene in Clostridium tetani and is homologous to BotR. Infect Immun 66:5698–5702.
22. Rood JI. 1997. Genetic analysis in Clostridium perfringens, p 65–71. In Rood JIMB, Songer JG, Titball RW (ed), The Clostridia: Molecular Biology and Pathogenesis. Academic Press Limited, London, United Kingdom. http://dx.doi.org/10.1016/B978-012595020-6/50007-3.
23. Scott PT, Rood JI. 1989. Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens. Gene 82:327–333 http://dx.doi.org/10.1016/0378-1119(89)90059-0.
24. Awad MM, Bryant AE, Stevens DL, Rood JI. 1995. Virulence studies on chromosomal alpha-toxin and theta-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of alpha-toxin in Clostridium perfringens-mediated gas gangrene. Mol Microbiol 15:191–202 http://dx.doi.org/10.1111/j.1365-2958.1995.tb02234.x. [PubMed]
25. Rood JI, Cole ST. 1991. Molecular genetics and pathogenesis of Clostridium perfringens. Microbiol Rev 55:621–648.
26. Lyras D, Rood JI. 1998. Conjugative transfer of RP4-oriT shuttle vectors from Escherichia coli to Clostridium perfringens. Plasmid 39:160–164 http://dx.doi.org/10.1006/plas.1997.1325. [PubMed]
27. Bannam TL, Rood JI. 1993. Clostridium perfringens- Escherichia coli shuttle vectors that carry single antibiotic resistance determinants. Plasmid 29:233–235 http://dx.doi.org/10.1006/plas.1993.1025. [PubMed]
28. Sloan J, Warner TA, Scott PT, Bannam TL, Berryman DI, Rood JI. 1992. Construction of a sequenced Clostridium perfringens- Escherichia coli shuttle plasmid. Plasmid 27:207–219 http://dx.doi.org/10.1016/0147-619X(92)90023-4.
29. Lyras D, Storie C, Huggins AS, Crellin PK, Bannam TL, Rood JI. 1998. Chloramphenicol resistance in Clostridium difficile is encoded on Tn 4453 transposons that are closely related to Tn 4451 from Clostridium perfringens. Antimicrob Agents Chemother 42:1563–1567 http://dx.doi.org/10.1128/AAC.42.7.1563. [PubMed]
30. Mani N, Lyras D, Barroso L, Howarth P, Wilkins T, Rood JI, Sonenshein AL, Dupuy B. 2002. Environmental response and autoregulation of Clostridium difficile TxeR, a sigma factor for toxin gene expression. J Bacteriol 184:5971–5978 http://dx.doi.org/10.1128/JB.184.21.5971-5978.2002. [PubMed]
31. O’Connor JR, Lyras D, Farrow KA, Adams V, Powell DR, Hinds J, Cheung JK, Rood JI. 2006. Construction and analysis of chromosomal Clostridium difficile mutants. Mol Microbiol 61:1335–1351 http://dx.doi.org/10.1111/j.1365-2958.2006.05315.x. [PubMed]
32. Lyras D, O’Connor JR, Howarth PM, Sambol SP, Carter GP, Phumoonna T, Poon R, Adams V, Vedantam G, Johnson S, Gerding DN, Rood JI. 2009. Toxin B is essential for virulence of Clostridium difficile. Nature 458:1176–1179 http://dx.doi.org/10.1038/nature07822. [PubMed]
33. Hussain HA, Roberts AP, Mullany P. 2005. Generation of an erythromycin-sensitive derivative of Clostridium difficile strain 630 (630Deltaerm) and demonstration that the conjugative transposon Tn 916DeltaE enters the genome of this strain at multiple sites. J Med Microbiol 54:137–141 http://dx.doi.org/10.1099/jmm.0.45790-0. [PubMed]
34. Collery MM, Kuehne SA, McBride SM, Kelly ML, Monot M, Cockayne A, Dupuy B, Minton NP. 2017. What’s a SNP between friends: the influence of single nucleotide polymorphisms on virulence and phenotypes of Clostridium difficile strain 630 and derivatives. Virulence 8:767–781 http://dx.doi.org/10.1080/21505594.2016.1237333. [PubMed]
35. Riedel T, Bunk B, Thürmer A, Spröer C, Brzuszkiewicz E, Abt B, Gronow S, Liesegang H, Daniel R, Overmann J. 2015. Genome resequencing of the virulent and multidrug-resistant reference strain Clostridium difficile 630. Genome Announc 3:e00276-15 http://dx.doi.org/10.1128/genomeA.00276-15. [PubMed]
36. van Eijk E, Anvar SY, Browne HP, Leung WY, Frank J, Schmitz AM, Roberts AP, Smits WK. 2015. Complete genome sequence of the Clostridium difficile laboratory strain 630Δerm reveals differences from strain 630, including translocation of the mobile element CTn5. BMC Genomics 16:31 http://dx.doi.org/10.1186/s12864-015-1252-7. [PubMed]
37. Minton NP, Ehsaan M, Humphreys CM, Little GT, Baker J, Henstra AM, Liew F, Kelly ML, Sheng L, Schwarz K, Zhang Y. 2016. A roadmap for gene system development in Clostridium. Anaerobe 41:104–112 http://dx.doi.org/10.1016/j.anaerobe.2016.05.011. [PubMed]
38. Johnston CD, Skeete CA, Fomenkov A, Roberts RJ, Rittling SR. 2017. Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia. PLoS One 12:e0185234 http://dx.doi.org/10.1371/journal.pone.0185234. [PubMed]
39. Mermelstein LD, Papoutsakis ET. 1993. In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 59:1077–1081.
40. Pyne ME, Moo-Young M, Chung DA, Chou CP. 2013. Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum. Biotechnol Biofuels 6:50 http://dx.doi.org/10.1186/1754-6834-6-50. [PubMed]
41. Lesiak JM, Liebl W, Ehrenreich A. 2014. Development of an in vivo methylation system for the solventogen Clostridium saccharobutylicum NCP 262 and analysis of two endonuclease mutants. J Biotechnol 188:97–99 http://dx.doi.org/10.1016/j.jbiotec.2014.07.005. [PubMed]
42. Yang X, Xu M, Yang ST. 2015. Metabolic and process engineering of Clostridium cellulovorans for biofuel production from cellulose. Metab Eng 32:39–48 http://dx.doi.org/10.1016/j.ymben.2015.09.001. [PubMed]
43. Yang X, Xu M, Yang ST. 2016. Restriction modification system analysis and development of in vivo methylation for the transformation of Clostridium cellulovorans. Appl Microbiol Biotechnol 100:2289–2299 http://dx.doi.org/10.1007/s00253-015-7141-9. [PubMed]
44. Uzal FA, Freedman JC, Shrestha A, Theoret JR, Garcia J, Awad MM, Adams V, Moore RJ, Rood JI, McClane BA. 2014. Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease. Future Microbiol 9:361–377 http://dx.doi.org/10.2217/fmb.13.168. [PubMed]
45. Lewis BB, Carter RA, Ling L, Leiner I, Taur Y, Kamboj M, Dubberke ER, Xavier J, Pamer EG. 2017. Pathogenicity locus, core genome, and accessory gene contributions to Clostridium difficile virulence. MBio 8:e00885-17 http://dx.doi.org/10.1128/mBio.00885-17. [PubMed]
46. Joseph RC, Kim NM, Sandoval NR. 2018. Recent developments of the synthetic biology toolkit for Clostridium. Front Microbiol 9:154 http://dx.doi.org/10.3389/fmicb.2018.00154. [PubMed]
47. Bradshaw M, Goodnough MC, Johnson EA. 1998. Conjugative transfer of the Escherichia coli- Clostridium perfringens shuttle vector pJIR1457 to Clostridium botulinum type A strains. Plasmid 40:233–237 http://dx.doi.org/10.1006/plas.1998.1366. [PubMed]
48. Carter GP, Awad MM, Hao Y, Thelen T, Bergin IL, Howarth PM, Seemann T, Rood JI, Aronoff DM, Lyras D. 2011. TcsL is an essential virulence factor in Clostridium sordellii ATCC 9714. Infect Immun 79:1025–1032 http://dx.doi.org/10.1128/IAI.00968-10. [PubMed]
49. Carter GP, Larcombe S, Li L, Jayawardena D, Awad MM, Songer JG, Lyras D. 2014. Expression of the large clostridial toxins is controlled by conserved regulatory mechanisms. Int J Med Microbiol 304:1147–1159 http://dx.doi.org/10.1016/j.ijmm.2014.08.008. [PubMed]
50. Awad MM, Cheung JK, Tan JE, McEwan AG, Lyras D, Rood JI. 2016. Functional analysis of an feoB mutant in Clostridium perfringens strain 13. Anaerobe 41:10–17 http://dx.doi.org/10.1016/j.anaerobe.2016.05.005. [PubMed]
51. Vidor CJ, Watts TD, Adams V, Bulach D, Couchman E, Rood JI, Fairweather NF, Awad M, Lyras D. 2018. Clostridium sordellii pathogenicity locus plasmid pCS1-1 encodes a novel clostridial conjugation locus. MBio 9:e01761-17 http://dx.doi.org/10.1128/mBio.01761-17. [PubMed]
52. Rabi R, Larcombe S, Mathias R, McGowan S, Awad M, Lyras D. 2018. Clostridium sordellii outer spore proteins maintain spore structural integrity and promote bacterial clearance from the gastrointestinal tract. PLoS Pathog 14:e1007004 http://dx.doi.org/10.1371/journal.ppat.1007004. [PubMed]
53. Carter GP, Douce GR, Govind R, Howarth PM, Mackin KE, Spencer J, Buckley AM, Antunes A, Kotsanas D, Jenkin GA, Dupuy B, Rood JI, Lyras D. 2011. The anti-sigma factor TcdC modulates hypervirulence in an epidemic BI/NAP1/027 clinical isolate of Clostridium difficile. PLoS Pathog 7:e1002317 http://dx.doi.org/10.1371/journal.ppat.1002317. [PubMed]
54. Mackin KE, Carter GP, Howarth P, Rood JI, Lyras D. 2013. Spo0A differentially regulates toxin production in evolutionarily diverse strains of Clostridium difficile. PLoS One 8:e79666 http://dx.doi.org/10.1371/journal.pone.0079666. [PubMed]
55. Kirk JA, Fagan RP. 2016. Heat shock increases conjugation efficiency in Clostridium difficile. Anaerobe 42:1–5 http://dx.doi.org/10.1016/j.anaerobe.2016.06.009. [PubMed]
56. Purdy D, O’Keeffe TA, Elmore M, Herbert M, McLeod A, Bokori-Brown M, Ostrowski A, Minton NP. 2002. Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier. Mol Microbiol 46:439–452 http://dx.doi.org/10.1046/j.1365-2958.2002.03134.x. [PubMed]
57. Fox ME, Lemmon MJ, Mauchline ML, Davis TO, Giaccia AJ, Minton NP, Brown JM. 1996. Anaerobic bacteria as a delivery system for cancer gene therapy: in vitro activation of 5-fluorocytosine by genetically engineered clostridia. Gene Ther 3:173–178.
58. Reynolds CB, Emerson JE, de la Riva L, Fagan RP, Fairweather NF. 2011. The Clostridium difficile cell wall protein CwpV is antigenically variable between strains, but exhibits conserved aggregation-promoting function. PLoS Pathog 7:e1002024 http://dx.doi.org/10.1371/journal.ppat.1002024. [PubMed]
59. Kovacs-Simon A, Leuzzi R, Kasendra M, Minton N, Titball RW, Michell SL. 2014. Lipoprotein CD0873 is a novel adhesin of Clostridium difficile. J Infect Dis 210:274–284 http://dx.doi.org/10.1093/infdis/jiu070. [PubMed]
60. de la Riva L, Willing SE, Tate EW, Fairweather NF. 2011. Roles of cysteine proteases Cwp84 and Cwp13 in biogenesis of the cell wall of Clostridium difficile. J Bacteriol 193:3276–3285 http://dx.doi.org/10.1128/JB.00248-11. [PubMed]
61. Heap JT, Pennington OJ, Cartman ST, Minton NP. 2009. A modular system for Clostridium shuttle plasmids. J Microbiol Methods 78:79–85 http://dx.doi.org/10.1016/j.mimet.2009.05.004. [PubMed]
62. Walter BM, Rupnik M, Hodnik V, Anderluh G, Dupuy B, Paulič N, Žgur-Bertok D, Butala M. 2014. The LexA regulated genes of the Clostridium difficile. BMC Microbiol 14:88 http://dx.doi.org/10.1186/1471-2180-14-88. [PubMed]
63. Martin MJ, Clare S, Goulding D, Faulds-Pain A, Barquist L, Browne HP, Pettit L, Dougan G, Lawley TD, Wren BW. 2013. The agr locus regulates virulence and colonization genes in Clostridium difficile 027. J Bacteriol 195:3672–3681 http://dx.doi.org/10.1128/JB.00473-13. [PubMed]
64. Dapa T, Unnikrishnan M. 2013. Biofilm formation by Clostridium difficile. Gut Microbes 4:397–402 http://dx.doi.org/10.4161/gmic.25862. [PubMed]
65. Zhang Z, Korkeala H, Dahlsten E, Sahala E, Heap JT, Minton NP, Lindström M. 2013. Two-Component Signal Transduction System CBO0787/CBO0786 Represses Transcription from Botulinum Neurotoxin Promoters in Clostridium botulinum ATCC 3502. PLoS Pathog 9(3): e1003252. http://dx.doi.org/10.1371/journal.ppat.1003252. [PubMed]
66. Little GT, Willson BJ, Heap JT, Winzer K, Minton NP. 2018. The butanol producing microbe Clostridium beijerinckii NCIMB 14988 manipulated using forward and reverse genetic tools. Biotechnol J 13:e1700711 http://dx.doi.org/10.1002/biot.201700711. [PubMed]
67. Liew F, Henstra AM, Köpke M, Winzer K, Simpson SD, Minton NP. 2017. Metabolic engineering of Clostridium autoethanogenum for selective alcohol production. Metab Eng 40:104–114 http://dx.doi.org/10.1016/j.ymben.2017.01.007. [PubMed]
68. Hartman AH, Liu H, Melville SB. 2011. Construction and characterization of a lactose-inducible promoter system for controlled gene expression in Clostridium perfringens. Appl Environ Microbiol 77:471–478 http://dx.doi.org/10.1128/AEM.01536-10. [PubMed]
69. Hendrick WA, Orr MW, Murray SR, Lee VT, Melville SB. 2017. Cyclic Di-GMP binding by an assembly ATPase (PilB2) and control of type IV pilin polymerization in the Gram-positive pathogen Clostridium perfringens. J Bacteriol 199:e00034-17 http://dx.doi.org/10.1128/JB.00034-17. [PubMed]
70. Obana N, Nakamura K, Nomura N. 2016. Role of RNase Y in Clostridium perfringens mRNA decay and processing. J Bacteriol 199:e00703-16. [PubMed]
71. Kumar RS, Hendrick W, Correll JB, Patterson AD, Melville SB, Ferry JG. 2013. Biochemistry and physiology of the β class carbonic anhydrase (Cpb) from Clostridium perfringens strain 13. J Bacteriol 195:2262–2269 http://dx.doi.org/10.1128/JB.02288-12. [PubMed]
72. Banerjee A, Leang C, Ueki T, Nevin KP, Lovley DR. 2014. Lactose-inducible system for metabolic engineering of Clostridium ljungdahlii. Appl Environ Microbiol 80:2410–2416 http://dx.doi.org/10.1128/AEM.03666-13. [PubMed]
73. Nariya H, Miyata S, Kuwahara T, Okabe A. 2011. Development and characterization of a xylose-inducible gene expression system for Clostridium perfringens. Appl Environ Microbiol 77:8439–8441 http://dx.doi.org/10.1128/AEM.05668-11. [PubMed]
74. Fagan RP, Fairweather NF. 2011. Clostridium difficile has two parallel and essential Sec secretion systems. J Biol Chem 286:27483–27493 http://dx.doi.org/10.1074/jbc.M111.263889. [PubMed]
75. Corrigan RM, Foster TJ. 2009. An improved tetracycline-inducible expression vector for Staphylococcus aureus. Plasmid 61:126–129 http://dx.doi.org/10.1016/j.plasmid.2008.10.001. [PubMed]
76. Govind R, Dupuy B. 2012. Secretion of Clostridium difficile toxins A and B requires the holin-like protein TcdE. PLoS Pathog 8:e1002727 http://dx.doi.org/10.1371/journal.ppat.1002727. [PubMed]
77. Pereira FC, Saujet L, Tomé AR, Serrano M, Monot M, Couture-Tosi E, Martin-Verstraete I, Dupuy B, Henriques AO. 2013. The spore differentiation pathway in the enteric pathogen Clostridium difficile. PLoS Genet 9:e1003782 http://dx.doi.org/10.1371/journal.pgen.1003782. [PubMed]
78. Dembek M, Willing SE, Hong HA, Hosseini S, Salgado PS, Cutting SM. 2017. Inducible expression of spo0A as a universal tool for studying sporulation in Clostridium difficile. Front Microbiol 8:1793 http://dx.doi.org/10.3389/fmicb.2017.01793. [PubMed]
79. Permpoonpattana P, Phetcharaburanin J, Mikelsone A, Dembek M, Tan S, Brisson MC, La Ragione R, Brisson AR, Fairweather N, Hong HA, Cutting SM. 2013. Functional characterization of Clostridium difficile spore coat proteins. J Bacteriol 195:1492–1503 http://dx.doi.org/10.1128/JB.02104-12. [PubMed]
80. Purcell EB, McKee RW, McBride SM, Waters CM, Tamayo R. 2012. Cyclic diguanylate inversely regulates motility and aggregation in Clostridium difficile. J Bacteriol 194:3307–3316 http://dx.doi.org/10.1128/JB.00100-12. [PubMed]
81. Adams V, Bantwal R, Stevenson L, Cheung JK, Awad MM, Nicholson J, Carter GP, Mackin KE, Rood JI, Lyras D. 2014. Utility of the clostridial site-specific recombinase TnpX to clone toxic-product-encoding genes and selectively remove genomic DNA fragments. Appl Environ Microbiol 80:3597–3603 http://dx.doi.org/10.1128/AEM.04285-13. [PubMed]
82. Bantwal R, Bannam TL, Porter CJ, Quinsey NS, Lyras D, Adams V, Rood JI. 2012. The peptidoglycan hydrolase TcpG is required for efficient conjugative transfer of pCW3 in Clostridium perfringens. Plasmid 67:139–147 http://dx.doi.org/10.1016/j.plasmid.2011.12.016. [PubMed]
83. Lyras D, Rood JI. 2000. Transposition of Tn 4451 and Tn 4453 involves a circular intermediate that forms a promoter for the large resolvase, TnpX. Mol Microbiol 38:588–601 http://dx.doi.org/10.1046/j.1365-2958.2000.02154.x.
84. Ransom EM, Ellermeier CD, Weiss DS. 2015. Use of mCherry Red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile. Appl Environ Microbiol 81:1652–1660 http://dx.doi.org/10.1128/AEM.03446-14. [PubMed]
85. Buckley AM, Jukes C, Candlish D, Irvine JJ, Spencer J, Fagan RP, Roe AJ, Christie JM, Fairweather NF, Douce GR. 2016. Lighting up Clostridium difficile: reporting gene expression using fluorescent LOV domains. Sci Rep 6:23463 http://dx.doi.org/10.1038/srep23463. [PubMed]
86. Ransom EM, Williams KB, Weiss DS, Ellermeier CD. 2014. Identification and characterization of a gene cluster required for proper rod shape, cell division, and pathogenesis in Clostridium difficile. J Bacteriol 196:2290–2300 http://dx.doi.org/10.1128/JB.00038-14. [PubMed]
87. Ribis JW, Ravichandran P, Putnam EE, Pishdadian K, Shen A. 2017. The conserved spore coat protein SpoVM is largely dispensable in Clostridium difficile spore formation. MSphere 2:e00315-17 http://dx.doi.org/10.1128/mSphere.00315-17. [PubMed]
88. Ribis JW, Fimlaid KA, Shen A. 2018. Differential requirements for conserved peptidoglycan remodeling enzymes during Clostridioides difficile spore formation. Mol Microbiol 110:370–389 http://dx.doi.org/10.1111/mmi.14090. [PubMed]
89. Anjuwon-Foster BR, Tamayo R. 2017. A genetic switch controls the production of flagella and toxins in Clostridium difficile. PLoS Genet 13:e1006701 http://dx.doi.org/10.1371/journal.pgen.1006701. [PubMed]
90. Ransom EM, Kaus GM, Tran PM, Ellermeier CD, Weiss DS. 2018. Multiple factors contribute to bimodal toxin gene expression in Clostridioides ( Clostridium) difficile. Mol Microbiol 110:533–549 http://dx.doi.org/10.1111/mmi.14107. [PubMed]
91. Drepper T, Eggert T, Circolone F, Heck A, Krauss U, Guterl JK, Wendorff M, Losi A, Gärtner W, Jaeger KE. 2007. Reporter proteins for in vivo fluorescence without oxygen. Nat Biotechnol 25:443–445 http://dx.doi.org/10.1038/nbt1293. [PubMed]
92. Christie JM, Gawthorne J, Young G, Fraser NJ, Roe AJ. 2012. LOV to BLUF: flavoprotein contributions to the optogenetic toolkit. Mol Plant 5:533–544 http://dx.doi.org/10.1093/mp/sss020. [PubMed]
93. Molitor B, Kirchner K, Henrich AW, Schmitz S, Rosenbaum MA. 2016. Expanding the molecular toolkit for the homoacetogen Clostridium ljungdahlii. Sci Rep 6:31518 http://dx.doi.org/10.1038/srep31518. [PubMed]
94. Serrano M, Crawshaw AD, Dembek M, Monteiro JM, Pereira FC, Pinho MG, Fairweather NF, Salgado PS, Henriques AO. 2016. The SpoIIQ-SpoIIIAH complex of Clostridium difficile controls forespore engulfment and late stages of gene expression and spore morphogenesis. Mol Microbiol 100:204–228 http://dx.doi.org/10.1111/mmi.13311. [PubMed]
95. Fimlaid KA, Jensen O, Donnelly ML, Siegrist MS, Shen A. 2015. Regulation of Clostridium difficile spore formation by the SpoIIQ and SpoIIIA proteins. PLoS Genet 11:e1005562 http://dx.doi.org/10.1371/journal.pgen.1005562. [PubMed]
96. Mani N, Dupuy B. 2001. Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci U S A 98:5844–5849 http://dx.doi.org/10.1073/pnas.101126598. [PubMed]
97. Hensbergen PJ, Klychnikov OI, Bakker D, van Winden VJ, Ras N, Kemp AC, Cordfunke RA, Dragan I, Deelder AM, Kuijper EJ, Corver J, Drijfhout JW, van Leeuwen HC. 2014. A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG cell surface proteins. Mol Cell Proteomics 13:1231–1244 http://dx.doi.org/10.1074/mcp.M113.034728. [PubMed]
98. Oliveira Paiva AM, Friggen AH, Hossein-Javaheri S, Smits WK. 2016. The signal sequence of the abundant extracellular metalloprotease PPEP-1 can be used to secrete synthetic reporter proteins in Clostridium difficile. ACS Synth Biol 5:1376–1382 http://dx.doi.org/10.1021/acssynbio.6b00104. [PubMed]
99. Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV. 2012. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7:1848–1857 http://dx.doi.org/10.1021/cb3002478. [PubMed]
100. Edwards AN, Pascual RA, Childress KO, Nawrocki KL, Woods EC, McBride SM. 2015. An alkaline phosphatase reporter for use in Clostridium difficile. Anaerobe 32:98–104 http://dx.doi.org/10.1016/j.anaerobe.2015.01.002. [PubMed]
101. Matsushita C, Matsushita O, Koyama M, Okabe A. 1994. A Clostridium perfringens vector for the selection of promoters. Plasmid 31:317–319 http://dx.doi.org/10.1006/plas.1994.1035. [PubMed]
102. Bullifent HL, Moir A, Titball RW. 1995. The construction of a reporter system and use for the investigation of Clostridium perfringens gene expression. FEMS Microbiol Lett 131:99–105 http://dx.doi.org/10.1111/j.1574-6968.1995.tb07761.x.
103. Phillips-Jones MK. 1993. Bioluminescence ( lux) expression in the anaerobe Clostridium perfringens. FEMS Microbiol Lett 106:265–270 http://dx.doi.org/10.1111/j.1574-6968.1993.tb05974.x.
104. Zhao Y, Melville SB. 1998. Identification and characterization of sporulation-dependent promoters upstream of the enterotoxin gene ( cpe) of Clostridium perfringens. J Bacteriol 180:136–142.
105. Takamizawa A, Miyata S, Matsushita O, Kaji M, Taniguchi Y, Tamai E, Shimamoto S, Okabe A. 2004. High-level expression of clostridial sialidase using a ferredoxin gene promoter-based plasmid. Protein Expr Purif 36:70–75 http://dx.doi.org/10.1016/j.pep.2004.03.004. [PubMed]
106. Faulds-Pain A, Wren BW. 2013. Improved bacterial mutagenesis by high-frequency allele exchange, demonstrated in Clostridium difficile and Streptococcus suis. Appl Environ Microbiol 79:4768–4771 http://dx.doi.org/10.1128/AEM.01195-13. [PubMed]
107. Cartman ST, Kelly ML, Heeg D, Heap JT, Minton NP. 2012. Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production. Appl Environ Microbiol 78:4683–4690 http://dx.doi.org/10.1128/AEM.00249-12. [PubMed]
108. Ng YK, Ehsaan M, Philip S, Collery MM, Janoir C, Collignon A, Cartman ST, Minton NP. 2013. Expanding the repertoire of gene tools for precise manipulation of the Clostridium difficile genome: allelic exchange using pyrE alleles. PLoS One 8:e56051 http://dx.doi.org/10.1371/journal.pone.0056051. [PubMed]
109. Peltier J, Shaw HA, Couchman EC, Dawson LF, Yu L, Choudhary JS, Kaever V, Wren BW, Fairweather NF. 2015. Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage. J Biol Chem 290:24453–24469 http://dx.doi.org/10.1074/jbc.M115.665091. [PubMed]
110. Francis MB, Sorg JA. 2016. Dipicolinic acid release by germinating Clostridium difficile spores occurs through a mechanosensing mechanism. MSphere 1:e00306-16 http://dx.doi.org/10.1128/mSphere.00306-16. [PubMed]
111. Heap JT, Ehsaan M, Cooksley CM, Ng YK, Cartman ST, Winzer K, Minton NP. 2012. Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acids Res 40:e59 http://dx.doi.org/10.1093/nar/gkr1321. [PubMed]
112. Bilverstone TW, Kinsmore NL, Minton NP, Kuehne SA. 2017. Development of Clostridium difficile R20291ΔPaLoc model strains and in vitro methodologies reveals CdtR is required for the production of CDT to cytotoxic levels. Anaerobe 44:51–54 http://dx.doi.org/10.1016/j.anaerobe.2017.01.009. [PubMed]
113. Collins J, Robinson C, Danhof H, Knetsch CW, van Leeuwen HC, Lawley TD, Auchtung JM, Britton RA. 2018. Dietary trehalose enhances virulence of epidemic Clostridium difficile. Nature 553:291–294 http://dx.doi.org/10.1038/nature25178. [PubMed]
114. Lambowitz AM, Zimmerly S. 2004. Mobile group II introns. Annu Rev Genet 38:1–35 http://dx.doi.org/10.1146/annurev.genet.38.072902.091600. [PubMed]
115. Chen Y, McClane BA, Fisher DJ, Rood JI, Gupta P. 2005. Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron. Appl Environ Microbiol 71:7542–7547 http://dx.doi.org/10.1128/AEM.71.11.7542-7547.2005. [PubMed]
116. Chen Y, Caruso L, McClane B, Fisher D, Gupta P. 2007. Disruption of a toxin gene by introduction of a foreign gene into the chromosome of Clostridium perfringens using targetron-induced mutagenesis. Plasmid 58:182–189 http://dx.doi.org/10.1016/j.plasmid.2007.04.002. [PubMed]
117. Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP. 2007. The ClosTron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods 70:452–464 http://dx.doi.org/10.1016/j.mimet.2007.05.021. [PubMed]
118. Kuehne SA, Minton NP. 2012. ClosTron-mediated engineering of Clostridium. Bioengineered 3:247–254 http://dx.doi.org/10.4161/bioe.21004. [PubMed]
119. Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, Minton NP. 2010. The role of toxin A and toxin B in Clostridium difficile infection. Nature 467:711–713 http://dx.doi.org/10.1038/nature09397. [PubMed]
120. Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, Minton NP. 2010. The ClosTron: mutagenesis in Clostridium refined and streamlined. J Microbiol Methods 80:49–55 http://dx.doi.org/10.1016/j.mimet.2009.10.018. [PubMed]
121. Lyristis M, Bryant AE, Sloan J, Awad MM, Nisbet IT, Stevens DL, Rood JI. 1994. Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens. Mol Microbiol 12:761–777 http://dx.doi.org/10.1111/j.1365-2958.1994.tb01063.x. [PubMed]
122. Awad MM, Rood JI. 1997. Isolation of alpha-toxin, theta-toxin and kappa-toxin mutants of Clostridium perfringens by Tn 916 mutagenesis. Microb Pathog 22:275–284 http://dx.doi.org/10.1006/mpat.1996.0115. [PubMed]
123. Kaufmann PLY, Meile L. 1996. Conjugative transposition of Tn 916 from Enterococcus faecalis and Escherichia coli into Clostridium perfringens. Syst Appl Microbiol 19:35–39 http://dx.doi.org/10.1016/S0723-2020(96)80006-3.
124. Briolat V, Reysset G. 2002. Identification of the Clostridium perfringens genes involved in the adaptive response to oxidative stress. J Bacteriol 184:2333–2343 http://dx.doi.org/10.1128/JB.184.9.2333-2343.2002. [PubMed]
125. Bannam TL, Teng WL, Bulach D, Lyras D, Rood JI. 2006. Functional identification of conjugation and replication regions of the tetracycline resistance plasmid pCW3 from Clostridium perfringens. J Bacteriol 188:4942–4951 http://dx.doi.org/10.1128/JB.00298-06. [PubMed]
126. Vidal JE, Chen J, Li J, McClane BA. 2009. Use of an EZ-Tn 5-based random mutagenesis system to identify a novel toxin regulatory locus in Clostridium perfringens strain 13. PLoS One 4:e6232 http://dx.doi.org/10.1371/journal.pone.0006232. [PubMed]
127. Lanckriet A, Timbermont L, Happonen LJ, Pajunen MI, Pasmans F, Haesebrouck F, Ducatelle R, Savilahti H, Van Immerseel F. 2009. Generation of single-copy transposon insertions in Clostridium perfringens by electroporation of phage mu DNA transposition complexes. Appl Environ Microbiol 75:2638–2642 http://dx.doi.org/10.1128/AEM.02214-08. [PubMed]
128. Liu H, Bouillaut L, Sonenshein AL, Melville SB. 2013. Use of a mariner-based transposon mutagenesis system to isolate Clostridium perfringens mutants deficient in gliding motility. J Bacteriol 195:629–636 http://dx.doi.org/10.1128/JB.01288-12. [PubMed]
129. Mullany P. 2014. Functional metagenomics for the investigation of antibiotic resistance. Virulence 5:443–447 http://dx.doi.org/10.4161/viru.28196. [PubMed]
130. Wang H, Smith MC, Mullany P. 2006. The conjugative transposon Tn 5397 has a strong preference for integration into its Clostridium difficile target site. J Bacteriol 188:4871–4878 http://dx.doi.org/10.1128/JB.00210-06. [PubMed]
131. Hussain HA, Roberts AP, Whalan R, Mullany P. 2010. Transposon mutagenesis in Clostridium difficile. Methods Mol Biol 646:203–211 http://dx.doi.org/10.1007/978-1-60327-365-7_13. [PubMed]
132. Cartman ST, Minton NP. 2010. A mariner-based transposon system for in vivo random mutagenesis of Clostridium difficile. Appl Environ Microbiol 76:1103–1109 http://dx.doi.org/10.1128/AEM.02525-09. [PubMed]
133. Dembek M, Barquist L, Boinett CJ, Cain AK, Mayho M, Lawley TD, Fairweather NF, Fagan RP. 2015. High-throughput analysis of gene essentiality and sporulation in Clostridium difficile. MBio 6:e02383 http://dx.doi.org/10.1128/mBio.02383-14. [PubMed]
134. Sangster W, Hegarty JP, Stewart DB Sr. 2015. Phage tail-like particles kill Clostridium difficile and represent an alternative to conventional antibiotics. Surgery 157:96–103 http://dx.doi.org/10.1016/j.surg.2014.06.015. [PubMed]
135. Meader E, Mayer MJ, Steverding D, Carding SR, Narbad A. 2013. Evaluation of bacteriophage therapy to control Clostridium difficile and toxin production in an in vitro human colon model system. Anaerobe 22:25–30 http://dx.doi.org/10.1016/j.anaerobe.2013.05.001. [PubMed]
136. Nale JY, Spencer J, Hargreaves KR, Buckley AM, Trzepińński P, Douce GR, Clokie MR. 2015. Bacteriophage combinations significantly reduce Clostridium difficile growth in vitro and proliferation in vivo. Antimicrob Agents Chemother 60:968–981 http://dx.doi.org/10.1128/AAC.01774-15. [PubMed]
137. Goh S, Hussain H, Chang BJ, Emmett W, Riley TV, Mullany P. 2013. Phage ɸC2 mediates transduction of Tn 6215, encoding erythromycin resistance, between Clostridium difficile strains. MBio 4:e00840-13 http://dx.doi.org/10.1128/mBio.00840-13. [PubMed]
138. Pyne ME, Bruder MR, Moo-Young M, Chung DA, Chou CP. 2016. Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium. Sci Rep 6:25666 http://dx.doi.org/10.1038/srep25666. [PubMed]
139. Wang Y, Zhang ZT, Seo SO, Choi K, Lu T, Jin YS, Blaschek HP. 2015. Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system. J Biotechnol 200:1–5 http://dx.doi.org/10.1016/j.jbiotec.2015.02.005. [PubMed]
140. Xu T, Li Y, Shi Z, Hemme CL, Li Y, Zhu Y, Van Nostrand JD, He Z, Zhou J. 2015. Efficient genome editing in Clostridium cellulolyticum via CRISPR-Cas9 nickase. Appl Environ Microbiol 81:4423–4431 http://dx.doi.org/10.1128/AEM.00873-15. [PubMed]
141. McAllister KN, Bouillaut L, Kahn JN, Self WT, Sorg JA. 2017. Using CRISPR-Cas9-mediated genome editing to generate C. difficile mutants defective in selenoproteins synthesis. Sci Rep 7:14672 http://dx.doi.org/10.1038/s41598-017-15236-5. [PubMed]
142. Wang S, Hong W, Dong S, Zhang ZT, Zhang J, Wang L, Wang Y. 2018. Genome engineering of Clostridium difficile using the CRISPR-Cas9 system. Clin Microbiol Infect 24:1095–1099 http://dx.doi.org/10.1016/j.cmi.2018.03.026. [PubMed]
143. Hong W, Zhang J, Cui G, Wang L, Wang Y. 2018. Multiplexed CRISPR-Cpf1-mediated genome editing in Clostridium difficile toward the understanding of pathogenesis of C. difficile infection. ACS Synth Biol 7:1588–1600 http://dx.doi.org/10.1021/acssynbio.8b00087. [PubMed]
144. Zhang N, Shao L, Jiang Y, Gu Y, Li Q, Liu J, Jiang W, Yang S. 2015. I-SceI-mediated scarless gene modification via allelic exchange in Clostridium. J Microbiol Methods 108:49–60 http://dx.doi.org/10.1016/j.mimet.2014.11.004. [PubMed]
145. Al-Hinai MA, Fast AG, Papoutsakis ET. 2012. Novel system for efficient isolation of Clostridium double-crossover allelic exchange mutants enabling markerless chromosomal gene deletions and DNA integration. Appl Environ Microbiol 78:8112–8121 http://dx.doi.org/10.1128/AEM.02214-12. [PubMed]
146. Zhang J, Ni C, Yang Z, Piontek A, Chen H, Wang S, Fan Y, Qin Z, Piontek J. 2015. Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier. Exp Dermatol 24:605–610 http://dx.doi.org/10.1111/exd.12728. [PubMed]
147. Buffie CG, Bucci V, Stein RR, McKenney PT, Ling L, Gobourne A, No D, Liu H, Kinnebrew M, Viale A, Littmann E, van den Brink MR, Jenq RR, Taur Y, Sander C, Cross JR, Toussaint NC, Xavier JB, Pamer EG. 2015. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 517:205–208 http://dx.doi.org/10.1038/nature13828. [PubMed]
148. Studer N, Desharnais L, Beutler M, Brugiroux S, Terrazos MA, Menin L, Schürch CM, McCoy KD, Kuehne SA, Minton NP, Stecher B, Bernier-Latmani R, Hapfelmeier S. 2016. Functional intestinal bile acid 7α-dehydroxylation by Clostridium scindens associated with protection from Clostridium difficile infection in a gnotobiotic mouse model. Front Cell Infect Microbiol 6:191 http://dx.doi.org/10.3389/fcimb.2016.00191. [PubMed]
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/content/journal/microbiolspec/10.1128/microbiolspec.GPP3-0040-2018
2019-06-07
2019-10-23

Abstract:

The past 10 years have been revolutionary for clostridial genetics. The rise of next-generation sequencing led to the availability of annotated whole-genome sequences of the important pathogenic clostridia: , () , and , but also () and . These sequences were a prerequisite for the development of functional, sophisticated genetic tools for the pathogenic clostridia. A breakthrough came in the early 2000s with the development of TargeTron-based technologies specific for the clostridia, such as ClosTron, an insertional gene inactivation tool. The following years saw a plethora of new technologies being developed, mostly for , but also for other members of the genus, including . A range of tools is now available, allowing researchers to precisely delete genes, change single nucleotides in the genome, complement deletions, integrate novel DNA into genomes, or overexpress genes. There are tools for forward genetics, including an inducible transposon mutagenesis system for . As the latest addition to the tool kit, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technologies have also been adopted for the construction of single and multiple gene deletions in . This article summarizes the key genetic technologies available to manipulate, study, and understand the pathogenic clostridia.

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FIGURE 1

Illustration of the pMTL80000 modular vector series. The figure highlights the four modules separated by the unique restriction sites: I, I, I, and I. The modules consist of a Gram-positive replicon module, a selectable marker, a Gram-negative replicon unit with optional transfer () genes, and an application-specific module.

Source: microbiolspec June 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0040-2018
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TABLE 1

Module choices for pMTL80000 plasmids

Source: microbiolspec June 2019 vol. 7 no. 3 doi:10.1128/microbiolspec.GPP3-0040-2018

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