Research in this laboratory is centred on the molecular genetics of pathogenic anaerobic bacteria, asking three fundamental mechanistic questions.
1. How do pathogenic anaerobes, particularly Clostridium perfringens and Dichelobacter nodosus, cause disease in humans and animals?
2. How is the expression of virulence genes regulated in these bacteria?
3. How do virulence and antibiotic resistance genes move from one bacterium to another?
This research involves four major mechanistic research themes;
- The pathogenesis of clostridial infections
- Regulatory networks in the pathogenic clostridia
- The biology of large clostridial plasmids
- Pathogenesis, regulation and genomics of the footrot pathogen, Dichelobacter nodosus
These projects involve bacterial genetics, molecular biology, genomics and transcriptomics, gene cloning, PCR technology, protein purification and other essentially molecular methods.
The research group is an integral part of the ARC Centre for Structural and Functional Microbial Genomics. Research funding is from the ARC , NHMRC , NIH, the Norwegian Research Council and the Australian Poultry CRC
Major collaborations involve the James Whisstock’s structural biology laboratory in the Department of Biochemistry and Molecular Biology , as well as the University of Pittsburgh , the University of California-Davis , the University of Sydney , CSIRO Livestock Industries , the Norwegian Veterinary Institute and the University of Warwick.
The biology of large clostridial plasmids
Understanding bacterial virulence and antibiotic resistance mechanisms and how these genes are transferred is important for fighting infectious disease. Earlier studies focussed on resistance genes but we are now primarily studying the conjugation mechanisms of the tetracycline resistance plasmid pCW3; as a model system for the analysis of conjugation in Clostridium perfringens. We have identified the genes involved in conjugation and in collaboration with James Whisstock (Department of Biochemistry and Molecular Biology) we are focussing on structure-function relationships in the conjugation apparatus. We are also studying the large toxin plasmids encoded by C. perfringens, with the objective of examining their genetic organisation, genomic variation, mechanisms of partitioning and compatibility, the mobility of the toxin genes and their role in virulence. These studies are carried out in collaboration with Bruce McClane (University of Pittsburgh), Francisco Uzal (University of California-Davis) and Rob Moore (CSIRO Livestock Industries).
- Bantwal, R., T.L. Bannam, C. J. Porter, N. Quinsey, D. Lyras, V. Adams & J. I. Rood. 2012. The peptidoglycan hydrolase TcpG is required for efficient conjugative transfer of pCW3 in Clostridium perfringens. Plasmid 67: 139-147.
- Porter, C.J., R. Bantwal, T.L. Bannam, C. J. Rosado, M.C. Pierce, V. Adams, D. Lyras, J.C. Whisstock, J.I. Rood. 2012. The conjugation protein TcpC from Clostridium perfringens is structurally related to the type IV secretion system protein VirB8 from Gram negative bacteria. Mol. Microbiol. 83: 275-288.
- Bannam, T.L., X-X. Yan, P. F. Harrison, T. Seemann, A. L. Keyburn, C. Stubenrauch, L.H. Weeramantri, J.C. Cheung, B. A. McClane, J.D. Boyce, R. J. Moore, and J. I. Rood. 2011. Necrotic enteritis-derived Clostridium perfringens strain with three closely related independently conjugative toxin and antibiotic resistance plasmids. mBio 2:e00190-11
- Lyras, D., V. Adams, S. A. Ballard, W. L. Teng, P. M. Howarth, P.K. Crellin, T. L. Bannam, J. G. Songer & J.I. Rood. 2009. tISCpe8, an IS1595-family lincomycin resistance element located on a conjugative plasmid in Clostridium perfringens. J. Bacteriol. 191:6345-6351.
- Steen, J.A, T.L. Bannam, W.L. Teng, R.J. Devenish & J.I. Rood. 2009. The putative coupling protein TcpA interacts with other pCW3-encoded proteins to form an essential part of the conjugation complex. J. Bacteriol. 191:2926-33.
- Teng, W. L., T.L. Bannam. J. A. Parsons and J.I. Rood. 2008. Functional characterisation and localisation of the TcpH conjugation protein from Clostridium perfringens. J. Bacteriol. 190: 5075-5086.
- Parsons. J.A., T.L. Bannam, R.J. Devenish and J.I. Rood. 2007. TcpA, an FtsK/SpoIIIE homolog, is essential for transfer of the conjugative plasmid pCW3 from Clostridium perfringens. J. Bacteriol. 189: 7782-7790.
- Hughes, M. L., R. Poon., V. Adams, S. Sayeed, J. Saputo, F. A. Uzal, B. A McClane and J. I Rood. 2007. Epsilon toxin plasmids of Clostridium perfringens Type D are conjugative. J. Bacteriol. 189: 7531-7538.
- 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. 188:4942-51.
Studies in this laboratory have led to the identification and characterisation of a two-component signal transduction system (VirS/VirR) that regulates the production of several toxin genes that are involved in the pathogenesis of Clostridium perfringens -mediated gas gangrene. We have identified functional regions of the VirS and VirR proteins and have analysed the DNA binding sites of VirR. These studies show that the VirR response regulator binds to two directly repeated VirR boxes located upstream of the target gene promoters. Our current objectives are to determine the precise sequence requirements at these binding sites and to analyse the phosphotransfer reactions and protein-protein interactions that are involved in the transcriptional activation of VirR-regulated genes. In addition, we are also examining the role of two-component signal transduction systems in Clostridium difficile , with a focus on the regulation of toxin production by the alternative sigma factor TcdR (prev. TxeR) .
- Cheung, J. K. and J. I. Rood . 2000. The VirR response regulator from Clostridium perfringens binds to two imperfect direct repeats located upstream of the pfoA promoter. J. Bacteriol. 182: 57-66.
- Cheung, J. K. and J. I. Rood . 2000. Glutamate residues in the putative transmembrane region are required for the function of the VirS sensor histidine kinase from Clostridium perfringens . Microbiology 146: 517-525.
- Awad, M. M. and J. I. Rood . 2002. Perfringolysin O expression in Clostridium perfringens is independent of the upstream pfoR gene. J. Bacteriol. 184:2034-8.
- Mani, N., D. Lyras, L. Barrosos, P. Howarth, T. Wilkins, J.I. Rood, A.L. Sonenshien and B. Dupuy . 2002. Environmental response and autoregulation of Clostridium difficile TxeR, a sigma factor for toxin gene expression. J. Bacteriol. 184: 5971-5978.
- McGowan, S. I. S. Lucet, J. C. Whisstock, M. M. Awad, J. K. Cheung and J.I. Rood . 2002. The FxRxHrS Motif: a conserved region essential for DNA binding of the VirR response regulator from Clostridium perfringens . J Mol Biol 22: 997-1011.
- McGowan, S., J. R. O'Connor, J.K. Cheung and J.I. Rood. 2003. The SKHR motif is required for biological function of the VirR response regulator from Clostridium perfringens . J. Bacteriol. 185: 6205-6208.
- Cheung, J. K., B. Dupuy, D. S. Deveson and J.I. Rood . 2004. The spatial organization of the VirR boxes is critical for the VirR-mediated expression of the perfringolysin O gene, pfoA , from Clostridium perfringens . J. Bacteriol 186: 3321-3330.
- O'Connor, J. R., Lyras, D., Farrow, K. A., Adams, V., Powell, D. R., Hinds, J., Cheung, J. K., and Rood, J. I. 2006. Construction and analysis of chromosomal Clostridium difficile mutants. Mol Microbiol. 61:1335-1351.
The anaerobic bacterium Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene. The organism produces at least seventeen different extracellular enzymes and toxins, many of which are believed to be important in pathogenesis. By making isogenic chromosomal mutants in C. perfringens we have shown that the alpha-toxin is essential for virulence and that perfringolysin O has synergistic effects in the disease process. Current studies are aimed at investigating the role of other extracellular toxins in disease and at the analysis of other clostridial pathogens.
- Rood, J.I . 1998. Virulence genes of Clostridium perfringens . Ann. Rev. Microbiol. 52: 333-360.
- Ellemor, D. M., R. N. Baird, M. M. Awad, J. I. Rood, J. J. Emmins and R. L. Boyd . 1999. Use of genetically manipulated strains of Clostridium perfringens reveals both alpha-toxin and theta-toxin are required for vascular leukostasis to occur in experimental gas gangrene. Infect. Immun. 67:4902-4907.
- Awad, M.M., D.M. Ellemor, R.L. Boyd, J.J. Emmins and J.I. Rood . 2001. Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens -mediated gas gangrene. Infect. Immun 69: 7904-7910.
- Sheedy, S. A., A. B. Ingham, J. I. Rood and R. J. Moore . 2004. Highly conserved alpha toxin sequences of avian isolates of Clostridium perfringens . J. Clin. Microbiol. 42: 1345-1347.
- Rupnik M., Dupuy B., Fairweather N., Gerding D., Johnson S., Just I., Lyerly D., Popoff M.R., Rood J.I., Sonenshein A. L., Thelestam M., Wren B.W., Wilkins T.D., and Eichel-Streiber C. 2005. Revised nomenclature of Clostridium difficile toxins and associated genes. J. Med. Microbiol. 54:113-117.
- Sheedy SA, Ingham AB, Rood JI, Moore RJ. 2004. Highly conserved alpha-toxin sequences of avian isolates of Clostridium perfringens. J Clin Microbiol. 42:1345-47.
- Kennedy CL, Krejany EO, Young LF, O'Connor JR, Awad MM, Boyd RL, Emmins JJ, Lyras D, Rood JI. 2005. The alpha-toxin of Clostridium septicum is essential for virulence. Mol Microbiol. 57:1357-66.
- Sayeed S, Fernandez-Miyakawa ME, Fisher DJ, Adams V, Poon R, Rood JI, Uzal FA, McClane BA. 2005. Epsilon-toxin is required for most Clostridium perfringens type D vegetative culture supernatants to cause lethality in the mouse intravenous injection model. Infect Immun. 73:7413-21.
- 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. 71:7542-7.
- Myers GS, 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. 16:1031-40.
- Fisher DJ, Fernandez-Miyakawa ME, Sayeed S, Poon R, Adams V, Rood JI, Uzal FA, McClane BA. 2006. Dissecting the Contributions of Clostridium perfringens Type C Toxins to Lethality in the Mouse Intravenous Injection Model. Infect Immun. 74:5200-10.
Ovine footrot is one of the most economically significant diseases of sheep in Australia. The causative agent is the anaerobic bacterium Dichelobacter nodosus. Research in this laboratory has involved the cloning and analysis of genes coding for two of the major virulence antigens, namely the fimbriae and proteases and the identification and analysis of pathogenicity islands in D. nodosus . Recently we have made a major breakthrough in D. nodosus genetics by developing a method for introducing DNA into this organism. This advancement has allowed genetic analysis in D.nodosus and more importantly the study of the virulence of D. nodosus possible. New areas of research involve utilizing reverse genetics to study the role of putative virulence factors in disease. Current studies focus on making knockout mutants of the protease structural genes and genes that are involved in fimbrial biogenesis.
- Johnston, J.L., S.J. Billington, V. Haring and J.I. Rood . 1998. Complementation analysis of the Dichelobacter nodosus fimN , fimO and fimP genes in Pseudomonas aeruginosa and transcriptional analysis of the fimNOP gene region. Infect. Immun. 66: 297-304.
- Kennan, R. M., S. J. Billington and J. I. Rood . 1998. Electroporation-mediated transormation of the ovine footrot pathogen Dichelobacter nodosus . FEMS Microbiol. Lett. 169: 383-389.
- Billington, S.J., A.S. Huggins, P. Johanesen, P.K. Crellin, J. Cheung, M.E. Katz, C.L. Wright, V. Haring and J.I. Rood . 1999. Complete nucleotide sequence of the 27-kb virulence related locus ( vrl ) of Dichelobacter nodosus : evidence for an extrachromosomal origin. Infect. Immun. 67: 1277-1286.
Kennan, R. M, O.P. Dhungyel, R.J. Whittington, J. R. Egerton and J. I. Rood . 2001. The type IV fimbrial subunit gene ( fimA ) of Dichelobacter nodosus is essential for virulence, protease secretion and natural competence. J. Bacteriol. 183: 4451-4458.
- Rood, J.I . 2002. Genomic islands of Dichelobacter nodosus . In Pathogenicity islands (PAIs) and the evolution of pathogenic microbes. (Hacker, J & J. Kaper, eds). Curr Top Microbiol Immunol 264(2): 47-60.
- Kennan, R. M, O.P. Dhungyel, R.J. Whittington, J. R. Egerton and J. I. Rood . 2003. Transformation-Mediated Serogroup Conversion of Dichelobacter nodosus . Vet. Microbiol. 92: 169-178.
- Parker, D., R. M. Kennan, G. S. Myers, I. T. Paulsen and J. I. Rood. 2004. Identification of a Dichelobacter nodosus ferric uptake regulator and determination of its regulatory targets. J. Bacteriol. (in press)
- Parker, D., R. M. Kennan, G. S. Myers, I. T. Paulsen and J. I. Rood. 2005. Identification of a Dichelobacter nodosus ferric uptake regulator and determination of its regulatory targets. J. Bacteriol. 187:366-375.
- Parker D, Kennan RM, Myers GS, Paulsen IT, Songer JG, Rood JI. 2006. Regulation of type IV fimbrial biogenesis in Dichelobacter nodosus. J Bacteriol. 188:4801-11.
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