Affiliation:
1. Departamentos de Dermatologı́a1
2. Bureau of Microbiology, Laboratory Centre for Disease Control, Tunney’s Pasture, Ottawa, Canada3
3. e Inmunologı́a,2 Facultad de Medicina, U.A.N.L., Monterrey, N.L., México, and
Abstract
ABSTRACT
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 homology to some eukaryotic catalases and to a bromoperoxidase-catalase from
Streptomyces violaceus
. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H
2
O
2
and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3′-diaminobenzidine and ferricyanide; the 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 GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several
N. brasiliensis
strains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the genera
Nocardia
,
Rhodococcus
,
Gordona
, and
Streptomyces
. All of the
N. brasiliensis
strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in the
N. brasiliensis
strains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria.
Publisher
American Society for Microbiology
Cited by
18 articles.
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