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Two catalases, KatA and KatB, have already been detected in developing

Two catalases, KatA and KatB, have already been detected in developing on rich moderate. superoxide radicals, hydrogen peroxide, and hydroxyl radicals (16). For protection against these reactive oxygen species, organisms contain antioxidants and enzymes that fix oxidative harm. Catalases (H2O2:H2O2 oxidoreductase; EC 1.11.1.6) are heme-containing enzymes mixed buy FG-4592 up in dismutation of H2O2 in O2 and H2O. These enzymes play an important part in reducing the formation of the highly reactive hydroxyl radical which arises from H2O2 degradation via the Fenton reaction (16). The response of bacteria to oxidative stress offers been most extensively studied in the enteric buy FG-4592 bacterium (reviewed in reference 7) which synthesizes two types of catalase enzyme, a bifunctional catalase/peroxidase (HPI) encoded by (32) and a monofunctional catalase (HPII) encoded by (34). These two buy FG-4592 genes are regulated in a different way when it comes to growth phase and response to oxidative stress (reviewed in reference 24). is definitely a soil bacterium able to establish a symbiosis with alfalfa (functional nodules, we have previously cloned the gene, which encodes the H2O2-inducible catalase KatA (18). We showed that free-living bacteria in stationary phase on rich medium create two catalases, namely a monofunctional catalase (KatA) and a bifunctional catalase/peroxidase (KatB). A mutant showed a drastic sensitivity to H2O2, and KatA appeared to be the major component of an H2O2-adaptative response. Neither nodulating capacity nor nitrogen fixing activity were impaired in the mutant, suggesting that KatA is not essential for the nodulation and nitrogen fixation processes. Cloning and analysis of the We previously required advantage of the high homology between regions of HPII and several catalases from numerous phyla to clone the gene of by nested PCR (18). Southern analysis of genomic DNA, digested with different restriction enzymes, showed a pattern of two bands (7.1- and 14-kb coding region was radiolabeled by using the Prime-a-Gene labeling system (Promega, Charbonnires, France) and used as a probe to hybridize with an genomic cosmid library (12) under low stringency (55C). Restriction analysis of five positive clones transporting a DNA place of 22 kb indicated that three cosmids represented the same genomic region but were different from chromosome previously detected (data not shown). As expected, a search of the current nonredundant DNA buy FG-4592 and protein databases Ncam1 with the BLAST algorithm (Beckman Center for Molecular and Genetic Medicine, Stanford, Calif.) exposed that the deduced amino acid sequence of had regions of high homology with monofunctional catalases from mammals, vegetation, and bacteria but not with bifunctional catalases. A multiple alignment of KatA (“type”:”entrez-nucleotide”,”attrs”:”text”:”U59271″,”term_id”:”1698549″,”term_text”:”U59271″U59271) and Kat2 amino acid sequences from was performed with KatX (“type”:”entrez-nucleotide”,”attrs”:”text”:”X97673″,”term_id”:”1310724″,”term_text”:”X97673″X97673), hyperoxidase II (“type”:”entrez-nucleotide”,”attrs”:”text”:”M55161″,”term_id”:”146532″,”term_text”:”M55161″M55161), and sp. strain SNU003 (“type”:”entrez-nucleotide”,”attrs”:”text”:”U56239″,”term_id”:”1314849″,”term_text”:”U56239″U56239), using the Genetics Computer Group programs PILEUP and PRETTY (data not shown). Considering both identical and conservative alternative of amino acids, Kat2 showed a high degree of identity with KatX (identity of 56.9%) and HPII (identity of 48.9%). Remarkably, the Kat2 sequence showed very low amino acid identity with the KatA (28.2%) and with the sp. strain SNU003 catalase (28%), since a large divergence in the C-terminal region was observed between Kat2 and these two catalases. However, the amino acid residues thought to be involved in the active-site and the proximal- and distal-heme-site ligands (23) were highly conserved in the five sequences. Induction of a new catalase during stationary phase in minimum medium. Strains and plasmids used in this study are outlined in Table ?Table1.1. Previously, we detected only KatA and KatB in protein extracts of stationary-phase cells grown at 30C in buy FG-4592 rich medium (Luria broth [LB]-MC: yeast extract, 5 g/liter; tryptone, 10 g/liter; NaCl, 10 g/liter; 2.5 mM MgSO4; and 2.5 mM CaCl2), (18). Sequence.