Physiological roles of catalases in protection of Agrobacterium tumefaciens from hydrogen peroxide and superoxide toxicity | |
| Author | Benjaphorn Prapagdee |
| Call Number | AIT Diss. no.EV-04-01 |
| Subject(s) | Agrobacterium tumefaciens Catalase Hydrogen peroxide |
| Note | A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Technical Science, Asian Institute of Technology; Inter-University Program on Environmental Toxicology, Technology and Management, Chulabhom Research Institute and Mahidol University |
| Publisher | Asian Institute of Technology |
| Series Statement | Dissertation ; no. EV-04-01 |
| Abstract | Agrobacterium tumefaciens is a phytopathogenic bacterium causing crown gall tumor in many dicotyledonous plants. During plant-microbe interactions, one of the initial active plant defense responses against invading microorganisms involves a rapid production and accumulation of reactive oxygen species. A. tumefaciens must overcome this oxidative burst in order to proliferate in the host plant. Here, physiological r oles of c atalases and bacterial growth phases on protection of A. tumefaciens from H202 and menadione (MD), superoxide generator, toxicity were investigated. During stationary phase, cells became more resistant to H202 than during exponential phase. A. tumefaciens possesses two catalase genes namely katA encodes a bifunctional catalase-peroxidase and catE for a mono functional c atalase. Visualization o f c atalases using gel staining revealed that katA was highly expressed at exponential phase and then declined as cells entered stationary phase of growth while catE was expressed throughout growth phases and was enhanced during stationary phase. Nevertheless, total catalase activity increased 2-fold during stationary phase of growth relative to exponential phase. Them utants of k atA and c atE were constructed. A katA mutant was more sensitive to H20 2 than the parental strain at both exponential and stationary phases _indicating that K atA plays a primarily protective role against H20 2 toxicity during all phases of growth. Conversely, inactivation of catE did not cause any significant changes in the level of H202 resistance. Moreover, the katA catE double mutant was extremely susceptible to H202 during both exponential and stationary phases relative to the katA or catE single mutant suggesting the physiological function of catE in H202 protection. Furthermore, the stationary phase cells of the katA catE double mutant were still more resistance to H202 than those of other phases. The data suggested the essential function of katA and catE in stationary phase resistance to H20 2 in A. tumefaciens and also the existence of stationary phase dependent mechanisms that rendered bacterial cells highly resistant to H20 2. The physiological function of the bifunctional catalase-peroxidase KatA in the protection of A. tumefaciens from MD was evaluated in the katA mutant. Unexpectedly, katA mutant was highly sensitive to the MD. The expression of katA from a plasmid vector complemented the MD hypersensitive phenotype. A. tumefaciens possesses an additional catalase gene, a monofunctional catalase encoded by catE. Neither inactivation nor high-level expression of the catE gene altered the MD resistance level. Moreover, heterologous expression of the catalase-peroxidase from Burkholderia pseudomallei, but not the monofunctional catalase from Xanthomonas campestris could restore normal levels of MD resistance to the katA mutant. Inactivation of putative genes encoding organic hydroperoxide scavenging enzymes such as tsa (a homolog of the tsa family), bcp (bacteriofeITitin co-migratory protein) and ohr (organic hydroperoxide resistance) do not cause any detectable changes in the MD resistance level. The level of MD resistance shows a direct correlation with the level of KatA catalaseperoxidase activity. This is a novel role for KatA and suggests that resistance to MD toxicity is mediated by a new, and as yet unknown, mechanism in A. tumefaciens. |
| Year | 2004 |
| Corresponding Series Added Entry | Asian Institute of Technology. Dissertation ; no. EV-04-01 |
| Type | Dissertation |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Other Field of Studies (No Department) |
| Academic Program/FoS | Environmental Engineering and Management (EV) |
| Chairperson(s) | Skom Mongkolsuk;Preeda Parkpian; |
| Examination Committee(s) | Paiboon Vattanaviboon; Suvit Loprasert; Chongrak Polprasert;Farrand, Stephen K. ; |
| Scholarship Donor(s) | CRI-Mahidol-AIT Fellowship Program on Post-Graduate Education, Training and Research Program in Environmental Science, Technology and Management; |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology, 2004 |