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.
AusubelF. M., BrentR., KingstonR. E., MooreD. D., SeidmanJ. G., SmithJ. A., StruhlK.1993; Current Protocols in Molecular Biology. vol. 2 pp. 1121–1125 New York: John Wiley;
BatchelorR. A., HaraguchiG. E., HullR. A., HullS. I.1991; Regulation by a novel protein of the bimodal distribution of lipopolysaccharide in the outer membrane of Escherichia coli. J Bacteriol 173:5699–5704
BrownP. K., RomanaL. K., ReevesP. R.1992; Molecular analysis of the rfb gene cluster of Salmonella serovar muenchen (strain M67): the genetic basis of the polymorphism between groups C2 and B. Mol Microbiol 6:1385–1394[CrossRef]
CerinH., HackettJ.1989; Molecular cloning and analysis of the incompatibility and partition functions of the virulence plasmid of Salmonella typhimurium. Microb Pathog 7:85–99[CrossRef]
CollinsL. V., HackettJ.1991; Molecular cloning, characterization, and nucleotide sequence of the rfc gene, which encodes an O-antigen polymerase of Salmonella typhimurium. J Bacteriol 173:2521–2529
CoyneM. J.Jr, GoldbergJ. B.1995; Cloning and characterization of the gene (rfc) encoding O-antigen polymerase of Pseudomonas aeruginosa PAO1. Gene 167:81–86[CrossRef]
CurdH., LiuD., ReevesP. R.1998; Relationships among the O-antigen gene clusters of Salmonella enterica groups B, D1, D2, and D3. J Bacteriol 180:1002–1007
De KievitT. R., DasguptaT., SchweizerH., LamJ. S.1995; Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype 05). Mol Microbiol 16:565–574[CrossRef]
FarinhaM. A., KropinskiA. M.1990; Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters. J Bacteriol 172:3496–3499
GrosjeanH., FiersW.1982; Preferential codon usage in prokaryotic genes: the optimal codon-anti-codon energy and the selective codon usage in efficiently expressed genes. Gene 18:199–209[CrossRef]
KlenaJ. D., SchnaitmanC. A.1993; Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Mol Microbiol 9:393–402[CrossRef]
LernerC. G., InouyeM.1990; Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nucleic Acids Res 18:463
LukomskiS., HullR. A., HullS. I.1996; Identification of the O antigen polymerase (rfc) gene in Escherichia coli O4 by insertional mutagenesis using a nonpolar chloramphenicol resistance cassette. J Bacteriol 178:240–247
NeidhardtF. C., UmbargerH. E.1996; Chemical composition of Escherichia coli. In Escherichia coli and Salmonella: Cellular and Molecular Biology pp 13–16 Washington, DC: American Society for Microbiology;
NorranderJ. M., KempeT., MessingJ.1983; Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106[CrossRef]
NurminenM., HellerqvistC. E., ValtonenV. V., MäkeläP. H.1971; The smooth lipopolysaccharide character of 1,4,(5),12 and 1,9,12 transductants formed as hybrids between groups B and D of Salmonella. Eur J Biochem 22:500–505[CrossRef]
SmithD. B., JohnsonK. S.1988; Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene 67:31–40[CrossRef]
XiangS. H., HobbsM., ReevesP. R.1994; Molecular analysis of the rfb gene cluster of a group D2 Salmonella enterica strain: evidence for its origin from an insertion sequence-mediated recombination event between group E and D1 strains.. J Bacteriol 176:4357–4365
YaoZ., ValvanoM. A.1994; Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4- α.. J Bacteriol 176:4133–4143
ZhangL., ToivanenP., SkurnikM.1996; The gene cluster directing antigen biosynthesis in Yersinia enterocolitica serotype O:8: identification of the genes for mannose and galactose biosyntheses and the gene for the O-antigen polymerase. Microbiology 142:277–288[CrossRef]
Identification of O-antigen polymerase transcription and translation start signals and visualization of the protein in Salmonella enterica serovar Typhimurium