A two-component regulatory system integrates redox state and population density sensing in Pseudomonas putida.

A two-component system formed by a sensor histidine kinase and a response regulator has been identified as an element participating in cell density signal transduction in Pseudomonas putida KT2440. It is a homolog of the Pseudomonas aeruginosa RoxS/RoxR system, which in turn belongs to the RegA/RegB family, described in photosynthetic bacteria as a key regulatory element. In KT2440, the two components are encoded by PP_0887 (roxS) and PP_0888 (roxR), which are transcribed in a single unit. Characterization of this two-component system has revealed its implication in redox signaling and cytochrome oxidase activity, as well as in expression of the cell density-dependent gene ddcA, involved in bacterial colonization of plant surfaces. Whole-genome transcriptional analysis has been performed to define the P. putida RoxS/RoxR regulon. It includes genes involved in sugar and amino acid metabolism and the sulfur starvation response and elements of the respiratory chain (a cbb3 cytochrome oxidase, Fe-S clusters, and cytochrome c-related proteins) or genes participating in the maintenance of the redox balance. A putative RoxR recognition element containing a conserved hexamer (TGCCAG) has also been identified in promoters of genes regulated by this two-component system.

Pseudomonas aeruginosa LptE is crucial for LptD assembly, cell envelope integrity, antibiotic resistance and virulence.

Lipopolysaccharide (LPS) is an essential structural component of the outer membrane (OM) of most Gram-negative bacteria. In the model organism Escherichia coli, LPS transport to the OM requires seven essential proteins (LptABCDEFG) that form a continuous bridge across the cell envelope. In Pseudomonas aeruginosa the recently-demonstrated essentiality of LptD and LptH, the P. aeruginosa LptA homologue, confirmed the crucial role of the Lpt system and, thus, of LPS in OM biogenesis in this species. Surprisingly, independent high-throughput transposon mutagenesis studies identified viable P. aeruginosa insertion mutants in the lptE gene, suggesting that it might be dispensable for bacterial growth. To test this hypothesis, we generated an lptE conditional mutant in P. aeruginosa PAO1. LptE depletion only slightly impairs P. aeruginosa growth in vitro. Conversely, LptE is important for cell envelope stability, antibiotic resistance and virulence in an insect model. Interestingly, the maturation and OM localization of LPS is only marginally affected in LptE-depleted cells, while the levels of the OM component LptD are strongly reduced. This suggests that P. aeruginosa LptE might not be directly involved in LPS transport, although it is clearly essential for the maturation and/or stability of LptD. While poor functionality of LptD caused by LptE depletion is somehow tolerated by P. aeruginosa, this has a high cost in terms of cell integrity, drug resistance and virulence, highlighting LptE function(s) as an interesting target to weaken P. aeruginosa defenses and reduce its infectivity.

In vitro and in vivo screening for novel essential cell-envelope proteins in Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa represents a prototype of multi-drug resistant opportunistic pathogens for which novel therapeutic options are urgently required. In order to identify new candidates as potential drug targets, we combined large-scale transposon mutagenesis data analysis and bioinformatics predictions to retrieve a set of putative essential genes which are conserved in P. aeruginosa and predicted to encode cell envelope or secreted proteins. By generating unmarked deletion or conditional mutants, we confirmed the in vitro essentiality of two periplasmic proteins, LptH and LolA, responsible for lipopolysaccharide and lipoproteins transport to the outer membrane respectively, and confirmed that they are important for cell envelope stability. LptH was also found to be essential for P. aeruginosa ability to cause infection in different animal models. Conversely, LolA-depleted cells appeared only partially impaired in pathogenicity, indicating that this protein likely plays a less relevant role during bacterial infection. Finally, we ruled out any involvement of the other six proteins under investigation in P. aeruginosa growth, cell envelope stability and virulence. Besides proposing LptH as a very promising drug target in P. aeruginosa, this study confirms the importance of in vitro and in vivo validation of potential essential genes identified through random transposon mutagenesis.

The periplasmic protein TolB as a potential drug target in Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa is one of the most dreaded pathogens in the hospital setting, and represents a prototype of multi-drug resistant "superbug" for which effective therapeutic options are very limited. The identification and characterization of new cellular functions that are essential for P. aeruginosa viability and/or virulence could drive the development of anti-Pseudomonas compounds with novel mechanisms of action. In this study we investigated whether TolB, the periplasmic component of the Tol-Pal trans-envelope protein complex of Gram-negative bacteria, represents a potential drug target in P. aeruginosa. By combining conditional mutagenesis with the analysis of specific pathogenicity-related phenotypes, we demonstrated that TolB is essential for P. aeruginosa growth, both in laboratory and clinical strains, and that TolB-depleted P. aeruginosa cells are strongly defective in cell-envelope integrity, resistance to human serum and several antibiotics, as well as in the ability to cause infection and persist in an insect model of P. aeruginosa infection. The essentiality of TolB for P. aeruginosa growth, resistance and pathogenicity highlights the potential of TolB as a novel molecular target for anti-P. aeruginosa drug discovery.

Fatty acid-mediated signalling between two Pseudomonas species.

We report the identification of fatty acids as mediators of intercellular signalling in Pseudomonas putida, and between Pseudomonas aeruginosa and P. putida. Tetradecanoic acid and fatty acids of similar chain length are present in supernatants of these strains and activate population density-dependent expression of ddcA, a gene involved in corn seed and root colonization by P. putida KT2440. Consistently, significant amounts of these compounds were also found in corn root exudates. The signalling pathway involves the two-component regulatory system formed by RoxS and RoxR, which had been previously shown to control expression of ddcA and of a set of genes related to the redox balance of P. putida cells. Production of the fatty acid signal in P. aeruginosa is under the control of the LasI/LasR and RhlI/RhlR quorum sensing systems. Our data indicate that in terms of cell-cell communication, P. putida KT2440 employs mechanisms closer to those of plant pathogens such as Xanthomonas spp. and fungi like Candida, which also rely on fatty acid derivatives.

The Pseudomonas aeruginosa quinolone quorum sensing signal alters the multicellular behaviour of Pseudomonas putida KT2440.

The molecule 2-heptyl-3-hydroxy-4-quinolone (referred to as the Pseudomonas quinolone signal, or PQS) is produced by Pseudomonas aeruginosa as part of its quorum sensing circuit, and has been shown to influence a variety of processes in this bacterium, including the production of siderophores and secondary metabolites, virulence determinants and biofilm development. In this report we present evidence of the effect of PQS as an interspecies signal with a negative impact on the multicellular behaviour of Pseudomonas putida KT2440. PQS reduces biofilm formation and swarming motility, and interferes with iron uptake by this bacterium. Addition of PQS also causes changes in the transcription of several P. putida genes, indicating a specific response to the signal molecule, which is not produced by strain KT2440. Among the genes with increased expression in response to PQS is PP1563, which forms part of a large prophage cluster (PP1532-PP1584); consistently, phage-mediated lysis of some cells in the population was observed in the presence of PQS. Overall, these data indicate that PQS may be used by P. aeruginosa as a chemical weapon against potential competitors.

PpoR, an orphan LuxR-family protein of Pseudomonas putida KT2440, modulates competitive fitness and surface motility independently of N-acylhomoserine lactones.

Pseudomonas putida KT2440 does not produce any of the common molecules involved in quorum sensing signalling described in other bacteria. However, as is the case in other microorganisms, the genome of this strain contains an open reading frame (PP_4647) coding for a transcriptional regulator belonging to the LuxR protein family. In this article, we present evidence indicating that this protein, named PpoR, modulates swarming motility in KT2440 and plays a role in the survival of this strain in the presence of potential competitors. These functions appear to be independent of known N-acylhomoserine lactones (AHLs), since we show that P. putida KT2440 does not produce significant quantities of these molecules under any condition tested and PpoR does not influence the expression of quorum sensing-dependent promoters even in the presence of exogenous AHLs. A ppoR mutant shows increased sensitivity to the iron chelator 2,2'-dipyridyl, while iron supplementation compensates the fitness loss of the mutant in competition with other Pseudomonas. All these data suggest that PpoR participates in both inter- and intraspecific processes relevant to the fitness of P. putida related to iron acquisition, and not necessarily mediated by canonical quorum sensing signal molecules.