Cloning, complementation, and characterization of an rfaE homolog from Neisseria gonorrhoeae.
Neisseria gonorrhoeae WS1 is a spontaneous pyocin (a bacteriocin produced by Pseudomonas aeruginosa)-resistant mutant of N. gonorrhoeae FA19 that produces a truncated lipooligosaccharide (LOS) and is non-transformable. The LOS-specific mutation in WS1 was moved into a transformable background by transforming FA19 with chromosomal DNA from WS1 (generating strain JWS-1). A clone (pJCL2) capable of restoring JWS-1 to wild-type LOS expression, as detected by its acquisition of reactivity with monoclonal antibodies and by its complemented sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile, was isolated. Sequential unidirectional deletion and DNA sequence analysis of pJCL2 identified an open reading frame, designated lsi-7, that could complement the defect in JWS-1. Homology searches against various databases indicated that lsi-7 bad homology with several Escherichia coli genes involved in the phosphorylation of sugars. lsi-7 is adjacent to the lsi-6 gene, another gene involved in LOS biosynthesis. Complementation studies using Salmonella typhimurium lipopolysaccharide mutants showed lsi-6 and lsi-7 to be gonococcal homologs of S. typhimurium rfaD and rfaE, respectively. Reverse transcriptase PCR analysis demonstrated that lsi-6 and lsi-7 are part of the same transcriptional unit.
Levin
J C
JC
Department of Microbiology, University of Maryland, College Park, Maryland 20742, USA.
Stein
D C
DC
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D016695:Glycosyltransferases; D008070:Lipopolysaccharides / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011709:Pyocins / Q000096:biosynthesis; D012486:Salmonella typhimurium / Q000235:genetics; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D008070:Lipopolysaccharides; D011709:Pyocins; D016695:Glycosyltransferases; C092232:RfaE protein, Bacteria; C037213:ADP-L-glycero-D-mannoheptose-6-epimerase; D002238:Carbohydrate Epimerases