Cloning and functional homology of secd within the enterobacteriaceae.

Abstract

The secD gene in Escherichia coli produces a subunit of the preprotein general secretion pathway, which transports preproteins across the inner membrane into the periplasmic space. Polymerase Chain Reaction (PCR) was performed on the isolated chromosome ofSalmonella choleraesuis with primers designed from the E. coli secD gene. Cloning was confirmed \vith DNA sequencing which was performed in triplicate to construct the S. choferaesuis seeD gene. Comparison ofthe S. choleraesuis sequence to E. coli secD shows 87% homology in genes that are both 1,848bp in size and code for a protein that is 615 amino acids in length. Translated amino acid sequence comparison shows 95% identity and 98% similar residues. Analysis of the secondary structure shows strong transmembrane similarity to E. coli SecD. PCR products were then cloned, and sequences compared in various ways from other Enterobacteriaceae. They were found to have an average homology was 87%. Primary stuctures ofthese products were all above 90% homologous to E. coli. Protein expression produced a 54kDa product at the same migration position as that of E. coli SecD. The cloned S. choleraesuis secD gene was tested for functionality in a cold sensitive mutant with successful results. Conclusions drawn are the E. coli and S. choleraesuis secD genes are analogous and other sequenced secD genes are very conserved and homologous to the E. coli secD gene.