Investigation of the oxidative defense enzymes in the Burkholderia cenocepacia strain C5424.

摘要

B. cepacia is an aerobic, Gram-negative microorganism that was originally described as the cause of soft rot in onions. Over the past 20 years this bacterium has emerged as an important multi-drug resistant opportunistic pathogen, primarily in cystic fibrosis patients. Genetic studies in Burkholderia cepacia complex (Bcc) isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. In this study, we have utilized the backbone of the plasmid pME6000, to construct a set of vectors useful for gene expression in Bcc. The functionality of both vectors was assessed by cloning the e-gfp gene and determining the level of eGFP protein expression and fluorescence emission in various clinical and environmental isolates of the Bcc .; The inflammatory response against bacteria in the airways of CF individuals generates a state of oxidative stress. Bacterial catalase, catalase-peroxidase, and superoxide dismutase activities may contribute to the survival of Bcc in this highly oxidative environment. In this study we examined the production of catalase, peroxidase, and superoxide dismutase activities by isolates belonging to the Bcc and their corresponding resistance to reactive oxygen intermediates in vitro. Our results suggest that resistance to toxic oxygen metabolites from extracellular sources may be a factor involved in the persistence of B. cepacia in the airways of CF individuals.; We have identified two genes, katA and katB , encoding bi-functional catalase/peroxidases in the B. cenocepacia strain C5424. A katA-deficient strain was constructed to investigate the function of the KatA in the cell. The growth rate and resistance to H2O2 exposure of the katA mutant was severely impaired when cultured under iron limitation in the presence of non-fermentable carbon sources. These results along with promoter activity analysis, suggest that the KatA protein is a specialized catalase/peroxidase that has a novel function of maintaining the normal activity of Krebs cycle enzymes in the presence of iron limitation, while KatB is a standard catalase/peroxidase that plays a global role in cellular protection against oxidative stress.