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.

Pseudomonas aeruginosa uses N-acyl-homoserine lactone (AHL)-dependent quorum sensing (QS) systems to control the expression of secreted effectors. These effectors can be crucial to the ecological fitness of the bacterium, playing roles in nutrient acquisition, microbial competition, and virulence. In this study, we investigated the metabolic consequences of AHL-dependent QS by monitoring the metabolic profile(s) of a lasI rhlI double mutant (unable to make QS signaling molecules) and its wild-type progenitor as they progressed through the growth curve. Analysis of culture supernatants by 1 H-nuclear magnetic resonance ( 1 H-NMR) spectroscopy revealed that at the point where AHL concentrations peaked in the wild type, the metabolic footprints (i.e., extracellular metabolites) of the wild-type and lasI rhlI mutant diverged. Subsequent gas chromatography-mass spectrometry (GC-MS)-based analysis of the intracellular metabolome revealed QS-dependent perturbations in around one-third of all identified metabolites, including altered concentrations of tricarboxylic acid (TCA) cycle intermediates, amino acids, and fatty acids. Further targeted fatty acid methyl ester (FAME) GC-MS-based profiling of the cellular total fatty acid pools revealed that QS leads to changes associated with decreased membrane fluidity and higher chemical stability. However, not all of the changes we observed were necessarily a direct consequence of QS; liquid chromatography (LC)-MS analyses revealed that polyamine levels were elevated in the lasI rhlI mutant, perhaps a response to the absence of QS-dependent adaptations. Our data suggest that QS leads to a global readjustment in central metabolism and provide new insight into the metabolic changes associated with QS during stationary-phase adaptation. IMPORTANCEQuorum sensing (QS) is a transcriptional regulatory mechanism that allows bacteria to coordinate their gene expression profile with the population cell density. The opportunistic human pathogen Pseudomonas aeruginosa uses QS to control the production of secreted virulence factors. In this study, we show that QS elicits a global "metabolic rewiring" in P. aeruginosa. This metabolic rerouting of fluxes is consistent with a variety of drivers, ranging from altered QS-dependent transcription of "metabolic genes" through to the effect(s) of global "metabolic readjustment" as a consequence of QS-dependent exoproduct synthesis, as well as a general stress response, among others. To our knowledge, this is the first study of its kind to assess the global impact of QS on the metabolome. T he gammaproteobacterium Pseudomonas aeruginosa is widely distributed in the environment (1, 2). The organism is a human pathogen and is responsible for a range of acute infections, urinary tract infections, and respiratory infections (3). In addition, it is also associated with certain chronic infections, such as those that often develop in the airways of cystic fibrosis patients (4, 5). Such exquisite adaptability to different niches requires a complex re...

E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site.

Many Gram-negative bacteria employ a mechanism of cell–cell communication known as quorum sensing (QS). The role of QS is to enable the cells in a culture to coordinate their gene expression profile with changes in the population cell density. The best characterized mechanisms of QS employ N-acylated homoserine lactones (AHLs) as signalling molecules. These AHLs are made by enzymes known as Luxl homologs, and accumulate in the culture supernatant at a rate proportional to the increase in cell density. Once the AHL concentration exceeds a certain threshold value, these ligands bind to intracellular receptors known as LuxR homologs. The latter are transcriptional regulators, whose activity alters upon binding the AHL ligand, thereby eliciting a change in gene transcription. Over the last five years, it has become increasingly obvious that this is a rather simplistic view of AHL-dependent QS, and that in fact, there is considerable diversity in the way in which Luxl-R homologs operate. The aim of the current review is to describe these variations on the basic theme, and to show how functional genomics is revolutionizing our understanding of QS-controlled regulons.

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