Identification of O-antigen polymerase transcription and translation start signals and visualization of the protein in Salmonella enterica serovar Typhimurium

Abstract

The wzy/rfe gene, encoding the O-antigen polymerase, of Salmonella enterica serovar Typhimurium has been previously cloned and sequenced. In the present work, the wry transcriptional startpoint was initially identified by primer extension. Next, wzy promoter strength in Escherichia coli K-12 was measured, and was found to be greater than that of the induced lac promoter. To define the Wzy translational startpoint, DNA including the wry promoter and the putative first five residues of the Wzy protein was fused to the N-terminus of glutathione-S-transferase, and the fusion protein purified by affinity chromatography. N-terminal amino acid sequencing yielded the Wzy translational startpoint. Next, the Wzy protein was C-terminally tagged with the FLAG peptide, and immunoblotting of an S. typhimurium strain expressing a low-copy wzy-FLAG gene (five copies per cell) localized the intact Wzy protein in the cytoplasmic membrane of S. typhimurium cells. The Wzy protein was not well-expressed from a multi-copy wzy-FLAG + plasmid in S. typhimurium, or in E. coli K-12.