Cloning and nucleotide sequencing of the secA gene from coryneform bacteria

Taking advantage of highly conserved domains present in the secA gene from Escherichia coli and Bacillus subtilis, we designed degenerate oligonucleotides (oligos) corresponding to these regions. These oligos were used as primers in PCR in order to amplify DNA sequences from Brevibacterium flavum MJ233 chromosomal DNA. The PCR product was used as a probe to recover genomic fragments from a lambda library of Br. flavum MJ233. The complete nucleotide sequence (nt) of the cloned 5.3-kb EcoR1 fragment containing the secA homolog from Br. flavum MJ233 indicated that the deduced gene product of the Br. flavum secA homolog is composed of 845 amino acids (aa) with a deduced molecular weight (MW) of 95429. Comparison of this aa sequence to the corresponding sequences from E. coli and B. subtilis revealed a high degree of conservation and suggested that the Br. flavum secA homolog has putative ATP binding regions.