Identification of O-antigen polymerase transcription and translation start signals and visualization of the protein in Salmonella enterica serovar Typhimurium.
The wzy/rfc gene, encoding the O-antigen polymerase, of Salmonella enterica serovar Typhimurium has been previously cloned and sequenced. In the present work, the wzy 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 wzy 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.
Wong
Danny K-H
DK
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong Special Administrative Region, China1.
Morris
Christina
C
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong Special Administrative Region, China1.
Lam
Tin L
TL
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong Special Administrative Region, China1.
Wong
Wan K R
WKR
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong Special Administrative Region, China1.
Hackett
Jim
J
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong Special Administrative Region, China1.
D001483:Base Sequence; D002458:Cell Fractionation; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000032:analysis; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014158:Transcription, Genetic
D003470:Culture Media; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide; D006602:Hexosyltransferases; C085652:O-antigen polymerase