Genetic and physiological analysis of the major OxyR-regulated katA from Xanthomonas campestris pv. phaseoli

1 Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand 2 Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Correspondence Skorn Mongkolsuk skorn{at}tubtim.cri.or.th katA encodes the major catalase that accounts for 90 % of the total catalase activity present in Xanthomonas campestris pv. phaseoli . katA is located upstream of an ORF designated ankA encoding a cytoplasmic membrane protein homologous to eukaryotic ankyrin. Transcriptional analysis of katA and ankA identified two katA transcripts: a major monocistronic katA transcript and a minor bicistronic katA–ankA transcript. KatA expression was induced in the presence of various oxidants including H 2 O 2 , organic hydroperoxides and the superoxide-generating agent menadione, in an OxyR-dependent manner. Analysis of the katA promoter region showed a putative OxyR binding site located upstream of an Escherichia coli -like 70 –35 region that is likely to be responsible for transcription activation in response to oxidant treatment. Gel mobility shift experiments confirmed that purified OxyR specifically binds to the katA promoter. A katA mutant was highly sensitive to H 2 O 2 during both the exponential and stationary phases of growth. This phenotype could be complemented by functional katA , confirming the essential role of the gene in protecting X. campestris from H 2 O 2 toxicity. Unexpectedly, inactivation of ankA also significantly reduced resistance to H 2 O 2 and the phenotype could be complemented by plasmid-borne expression of ankA . Physiological analyses showed that katA plays an important role in, but is not solely responsible for, both the adaptive and menadione-induced cross-protective responses to H 2 O 2 killing in X. campestris . Abbreviations: tBOOH, tert-butyl hydroperoxide; NEM, N -ethylmaleimide Present address: Center for Vectors and Vector-Borne Diseases, Mahidol University, Bangkok 10400, Thailand.