title: Lipopolysaccharide core phosphates are required for viability and intrinsic drug resistance in Pseudomonas aeruginosa.
abstract: Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for its intrinsic drug resistance. We have used chemical and genetic techniques to characterize three putative kinase genes that are involved in the addition of phosphate to the inner core region of P. aeruginosa lipopolysaccharide. The first gene is a waaP homologue, whereas the other two (wapP and wapQ) are unique to P. aeruginosa. Repeated attempts using a variety of membrane-stabilizing conditions to generate waaP:Gm (Gm, gentamicin) or wapP:Gm mutants were unsuccessful. We were able to generate a chromosomal waaP mutant that had a wild-type copy of either waaPPa or waaPEc in trans, but were unable to cure this plasmid-borne copy of the gene. These results are consistent with the fact that P. aeruginosa mutants lacking inner core heptose (Hep) or phosphate have never been isolated and demonstrate the requirement of Hep-linked phosphate for P. aeruginosa viability. A wapQ:Gm mutant was isolated and it had an unaltered minimum inhibitory concentration (MIC) for novobiocin and only a small decrease in the MIC for sodium dodecyl sulphate (SDS), suggesting that the loss of a phosphate group transferred by WapQ may only be having a small impact on outer-membrane permeability. Nuclear magnetic resonance and methylation linkage analysis showed that WaaPPa could add one phosphate to O4 of HepI in a Salmonella typhimurium waaP mutant. The expression of WaaPPa increased the outer-membrane integrity of these complemented mutants, as evidenced by 35-fold and 75-fold increases in the MIC for novobiocin and SDS respectively. The S. typhimurium waaP mutant transformed with both waaP and wapP had over 250-fold and 1000-fold increases, respectively, in these MICs. The inner core phosphates of P. aeruginosa appear to be playing a key role in the intrinsic drug resistance of this bacterium.
authors.affiliation: Department of Microbiology, University of Guelph, Guelph, ON N1G 2W1, Canada.
authors.forename: A G
authors.initials: AG
authors.lastname: Walsh
authors.forename: M J
authors.initials: MJ
authors.lastname: Matewish
authors.forename: L L
authors.initials: LL
authors.lastname: Burrows
authors.forename: M A
authors.initials: MA
authors.lastname: Monteiro
authors.forename: M B
authors.initials: MB
authors.lastname: Perry
authors.forename: J S
authors.initials: JS
authors.lastname: Lam
mesh_terms: D002240:Carbohydrate Sequence; D002455:Cell Division; D002463:Cell Membrane Permeability; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004352:Drug Resistance, Microbial; D005798:Genes, Bacterial / Q000235:genetics; D020043:Genes, Essential; D006539:Heptoses / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics; D011135:Polysaccharides, Bacterial / Q000737:chemistry / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D006539:Heptoses; D010710:Phosphates; D011135:Polysaccharides, Bacterial; D010770:Phosphotransferases