Cloning and molecular characterization of a nprX gene of Bacillus subtilis NS15-4 encoding a neutral protease

Abstract

An nprX gene of Bacillus subtilis NS15-4 encoding a neutral protease was cloned and its molecular characteristics were analyzed. The complete nucleotide sequence indicated that there is an open reading frame (ORF) possibly encoding 521 amino acid polypeptide. The ORF used all codons except two cysteine and a proline having a codon bias index (CBI) of 0.09 in Escherichia coli. There were homologous sequences to the consensus sequence of -35 and -10 regions of E. coli promoters and to a Shine-Dalgarno (SD) sequence located 25 bp downstream of a major transcription initiation site. Moreover, there were also five minor transcription initiation sites at 6, 7, 8, 14 and 15 nt downstream of the major site. Northern blot analysis revealed the presence of about 1.8 kb mRNA transcript in E. coli having the nprX gene. The nucleotide sequence was identified in GenBank to be a gene for a neutral protease of B. subtilis with six nucleotide difference in the ORF region. The flanking regions of the NprX ORF showed much more differences from those of other neutral protease genes except the nprE gene of B. subtilis, which has the most homology to the nprX gene, and of which the flanking regions were identical to those of the nprX gene.