B-202. Cloning, Genomic Organization and Expression of secA2 gene in Listeria species

K. K. Mishra, K. M. Burkholder, S. Medina-Maldonado, A. K. Bhunia;
Purdue Univ., West Lafayette, IN.

The translocation of proteins across the bacterial cell wall is carried out by secretory SecA and other members of the secretory system. Most bacteria have a single copy of secA gene except for 11 Gram-positive bacterial species from 5 genera (Listeria, Mycobacterium, Staphylococcus, Streptococcus and Bacillus), which have an additional copy of the secA gene, designated secA2. SecA2 is capable of translocating proteins both containing and lacking a leader sequence. We sought to determine the presence of the secA2 locus in all six Listeria spp. by PCR using primers specific for the entire or partial secA2 gene. Results indicate that four listerial species (L. monocytogenes F4244, L. welshimeri ATCC 35897, L. innocua F4248, L. seeligeri SE31) have a conserved secA2 gene (2331 bp), while divergence was found in the secA2 sequence of L. grayi and L. ivanovii. Sequence analysis indicates that secA2 in L. monocytogenes, L. welshimeri, L. innocua and L. seeligeri is linked with the p60 (also known as iap or cwh) gene and is present downstream of the p60 gene locus. PCR amplification using a forward primer originating from the p60 gene and a reverse primer originating upstream of the secA2 start codon indicated that approximately 400 nucleotides are conserved upstream of the secA2 gene in all Listeria species. The secA2 gene of L. monocytogenes is being over-expressed in L. grayi to further elucidate the role of SecA2 in protein translocation and to identify the subsets of proteins that might be translocated by this system.