Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus

An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homol ogy to some eukaryotic catalases and to a bromoperoxidase-catalase from Str eptomyces violaceus, Its identity as a catalase was confirmed by analysis o f its enzymatic activity on H2O2 and by a double-staining method on a nonde naturing polyacrylamide gel with 3,3'-diaminobenzidine and ferricyanide; th e result showed only catalase activity, but no peroxidase, By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product, The amplicon was analyzed, and the nucleotide sequence was compared to the Gen Bank database with the observation of high homology to other bacterial and eukaryotic catalases, A PCR assay based on this target sequence was perform ed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N, brasiliensis strains isolated from mycetoma, The sa me assay was used to determine whether there were homologous sequences in s everal type strains from the genera Nocardia, Rhodococcus, Gordona, and Str eptomyces, All of the N. brasiliensis strains presented a positive result b ut only some of the actinomycetes species tested were positive in the PCR a ssay. In order to confirm these findings, genomic DNA was subjected to Sout hern blot analysis. A 1.7-kbp band was observed in the N. brasiliensis stra ins, and bands of different molecular weight were observed in cross-reactin g actinomycetes. Sequence analysis of the amplicons of selected actinomycet es showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria.