title: Mutation of nfxB causes global changes in the physiology and metabolism of Pseudomonas aeruginosa.
abstract: Loss-of-function mutations in nfxB lead to up-regulation of mexCD-oprJ expression and, consequently, increased resistance to fluoroquinolone antibiotics. Such nfxB mutants have also been reported to exhibit altered virulence profiles, diminished type III secretion system-dependent cytotoxicity, and impaired fitness. However, it is not clear whether these phenotypes are directly linked to NfxB activity or whether inappropriate expression of the MexCD-OprJ pump has pleiotropic effects, thereby impacting indirectly on the phenotype of the cells. The aim of the current work is to investigate which of these possibilities is correct. We isolated a novel type of nfxB mutant generated by a spontaneous polygenic deletion and show that this mutant is rapidly out-competed when grown in a mixed culture with the wild-type progenitor. This competitive fitness defect only manifested itself during the stationary phase of growth. The endoproteome of the nfxB mutant, assessed using 2D-DiGE (difference gel electrophoresis), showed major alterations compared with the wild-type. Consistent with this, we found that the nfxB mutant was impaired in all forms of motility (swimming, swarming, and twitching) as well as in the production of siderophores, rhamnolipid, secreted protease, and pyocyanin. Further investigation showed that the exoproteome, endometabolome, and exometabolome of the nfxB mutant were all globally different compared with the wild-type. The exometabolome of the nfxB mutant was enriched in a selection of long chain fatty acids raising the possibility that these might be substrates for the MexCD-OprJ pump. The nfxB mutant metabotype could be complemented by expression of nfxB in trans and was abolished in an nfxB mexD double mutant, suggesting that inappropriate overexpression of a functional MexCD-OprJ efflux pump causes pleiotropic changes. Taken together, our data suggest that many of the nfxB mutant phenotypes are not caused by the direct effects of the NfxB regulator, but instead by inappropriate mexCD-oprJ expression. Furthermore, the pleiotropic nature of the phenotypes indicate that these may simply reflect the globally dysregulated physiology of the strain.
authors.affiliation: Department of Biochemistry, Tennis Court Road, Cambridge, United Kingdom.
authors.forename: Hannah G
authors.initials: HG
authors.lastname: Stickland
authors.forename: Peter W
authors.initials: PW
authors.lastname: Davenport
authors.forename: Kathryn S
authors.initials: KS
authors.lastname: Lilley
authors.forename: Julian L
authors.initials: JL
authors.lastname: Griffin
authors.forename: Martin
authors.initials: M
authors.lastname: Welch
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015180:Electrophoresis, Gel, Two-Dimensional; D026901:Membrane Transport Proteins / Q000378:metabolism; D055442:Metabolome; D009154:Mutation; D010641:Phenotype; D025341:Principal Component Analysis; D020543:Proteome; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D026901:Membrane Transport Proteins; C077901:NfxB protein, Pseudomonas aeruginosa; D020543:Proteome; D014157:Transcription Factors