Heterologous growth phase- and temperature-dependent expression and H2O2 toxicity protection of a superoxide-inducible monofunctional catalase gene from Xanthomonas oryzae pv. oryzae

Abstract

Catalase is an important protective enzyme against H 2 O 2 toxicity. Here, we report the characterization of a Xanthomonas oryzae pv. oryzae catalase gene (katX). The gene was localized and its nucleotide sequence was determined. The gene codes for a 77-kDa polypeptide. The deduced katX amino acid sequence shares regions of high identity with other monofunctional catalases in a range of organisms from bacteria to eukaryotes. The transcriptional regulation of katX was atypical of bacterial monofunctional kat genes. Northern (RNA) analysis showed that katX transcription was highly induced by treatments with low concentrations of menadione, a superoxide generator, and methyl methanesulfonate, a mutagen. It was only weakly induced by H 2 O 2 . Unlike in other bacteria, a high level of catalase in Xanthomonas spp. provided protection from the growth-inhibitory and killing effects of H 2 O 2 but not from those of organic peroxides and superoxide generators. Unexpectedly, heterologous expression of katX in Escherichia coli was both growth phase and temperature dependent. Catalase activity in E. coli kat mutants harboring katX on an expression vector was detectable only when the cells entered the stationary phase of growth and at 28؇C. The patterns of transcription regulation, heterologous expression, and physiological function of katX are different from previously studied bacterial kat genes.Catalase is a heme-containing enzyme involved in dismutation of H 2 O 2 to oxygen and water. The enzyme plays an important role in detoxifying H 2 O 2 and minimizing oxidative stress caused by highly reactive oxygen species (ROS, i.e., OH⅐) which arise from H 2 O 2 degradation in the Fenton reaction (13). Mutations in kat have always resulted in increased sensitivity to H 2 O 2 stress, and this is an indication of the important physiological role of the enzyme (10, 13). In many bacteria, there are two types of catalase enzyme, namely a monofunctional catalase and a bifunctional catalase/peroxidase. Each enzyme is encoded by a different gene (e.g., in Escherichia coli, katE and katG code for monofunctional and bifunctional catalases, respectively) (35, 45). Monofunctional catalases share regions of an amino acid sequence that is highly conserved among microbial, plant, and mammalian enzymes (5,26,47). In many bacteria, the two kat genes are regulated differently in terms of growth phase and response to oxidative stress, suggesting that they may have different physiological functions (10,23,29).Xanthomonas oryzae pv. oryzae (Xoo) is the causative agent for the most destructive bacterial disease (the bacterial leaf blight) in rice (37). Oxidative stress is an important component of normal aerobic life and in bacterial-plant interactions. Increasing production of ROS, including superoxide, H 2 O 2 , and OH⅐, is associated with an active plant defense response and with aerobic respiration (44). ROS are highly toxic to all cellular components, and their rapid detoxification is essential for microbial survival.Xoo monofunctional catalase ...

Isolation and analysis of the Xanthomonas alkyl hydroperoxide reductase gene and the peroxide sensor regulator genes ahpC and ahpF-oxyR-orfX

“…Additionally, AhpR might play a less important role than catalase in the protection against H 2 O 2 toxicity. This is consistent with our observations that increased catalase levels alone are sufficient to protect Xanthomonas from H 2 O 2 killing (17).…”

“…Ampicillin was used at 100 g/ml for both E. coli and Xanthomonas strains. Routinely, E. coli was transformed by a chemical method, while Xanthomonas was electroporated under previously described conditions (17).…”

Pseudomonas syringae Catalases Are Collectively Required for Plant Pathogenesis

“…These data demonstrate that KatB and KatG play nonredundant roles in DC3000 for the detoxification of exogenous H 2 O 2 . (11,13,31,35). To investigate the ability of catalases to remove endogenous H 2 O 2 , we compared the growth of wild-type DC3000 to that of the catalase mutants on plates containing menadione.…”

In vitro resistance of Burkholderia cepacia complex isolates to reactive oxygen species in relation to catalase and superoxide dismutase production

“…These enzymes have been found to be associated with virulence in a wide range of lung pathogens such as Mycobacterium tuberculosis (Manca et al, 1999) and Legionella pneumophila (Bandyopadhyay & Steinman 1998 (Brown et al, 1995 ;Katsuwon & Anderson, 1992 ;Mongkolsuk et al, 1996). Under our experimental conditions, however, we have found only one enzyme class in most isolates, suggesting either that B. cepacia isolates have fewer catalase forms or, alternatively, that additional catalases may be expressed under conditions of stress not examined in this study.…”