PpoR is a conserved unpaired LuxR solo of Pseudomonas putida which binds N-acyl homoserine lactones

Abstract Background Only a small number of Pseudomonas putida strains possess the typical N-acyl homoserine lactone quorum sensing system (AHL QS) that consists of a modular LuxR family protein and its cognate LuxI homolog that produces the AHL signal. Moreover, AHL QS systems in P. putida strains are diverse in the type of AHLs they produce and the phenotypes that they regulate. Results We identified an unpaired LuxR solo (QS luxR homolog that occurs without the corresponding luxI homolog), which is highly conserved in both the AHL producing and non-AHL producing P. putida strains that we analyzed. In this study we report the cloning and functional characterization of this unpaired LuxR homolog designated PpoR. An AHL binding assay showed that PpoR protein binds to 3-oxo-C6-HSL. Studies using a ppoR promoter-lacZ reporter fusion revealed that it exhibits stringent growth phase dependent expression. Functional interaction of PpoR with the endogenous complete AHL QS systems of P. putida WCS358 (PpuI/R system) and PpoR was also investigated. Microarray analysis of P. putida WCS358 wild type and a PpoR over-expressing strain revealed several putative target genes that may be directly or indirectly regulated by PpoR. Conclusion Our results indicate that PpoR in P. putida strains may have a conserved role in detecting an AHL signal, either self or foreign, and regulating specific target genes.</p

Negative Regulation of Violacein Biosynthesis in Chromobacterium violaceum

In Chromobacteium violaceum, the purple pigment violacein is under positive regulation by the N-acylhomoserine lactone CviI/R quorum sensing system and negative regulation by an uncharacterized putative repressor. In this study we report that the biosynthesis of violacein is negatively controlled by a novel repressor protein, VioS. The violacein operon is regulated negatively by VioS and positively by the CviI/R system in both C. violaceum and in a heterologous Escherichia coli genetic background. VioS does not regulate the CviI/R system and apart from violacein, VioS, and quorum sensing regulate other phenotypes antagonistically. Quorum sensing regulated phenotypes in C. violaceum are therefore further regulated providing an additional level of control

The afc antifungal activity cluster, which is under tight regulatory control of ShvR, is essential for transition from intracellular persistence of Burkholderia cenocepacia to acute pro-inflammatory infection.

The opportunistic pathogen Burkholderia cenocepacia is particularly life-threatening for cystic fibrosis (CF) patients. Chronic lung infections with these bacteria can rapidly develop into fatal pulmonary necrosis and septicaemia. We have recently shown that macrophages are a critical site for replication of B. cenocepacia K56-2 and the induction of fatal pro-inflammatory responses using a zebrafish infection model. Here, we show that ShvR, a LysR-type transcriptional regulator that is important for biofilm formation, rough colony morphotype and inflammation in a rat lung infection model, is also required for the induction of fatal pro-inflammatory responses in zebrafish larvae. ShvR was not essential, however, for bacterial survival and replication in macrophages. Temporal, rhamnose-induced restoration of shvR expression in the shvR mutant during intramacrophage stages unequivocally demonstrated a key role for ShvR in transition from intracellular persistence to acute fatal pro-inflammatory disease. ShvR has been previously shown to tightly control the expression of the adjacent afc gene cluster, which specifies the synthesis of a lipopeptide with antifungal activity. Mutation of afcE, encoding an acyl-CoA dehydrogenase, has been shown to give similar phenotypes as the shvR mutant. We found that, like shvR, afcE is also critical for the switch from intracellular persistence to fatal infection in zebrafish. The closely related B. cenocepacia H111 has been shown to be less virulent than K56-2 in several infection models, including Galleria mellonella and rats. Interestingly, constitutive expression of shvR in H111 increased virulence in zebrafish larvae to almost K56-2 levels in a manner that absolutely required afc. These data confirm a critical role for afc in acute virulence caused by B. cenocepacia that depends on strain-specific regulatory control by ShvR. We propose that ShvR and AFC are important virulence factors of the more virulent Bcc species, either through pro-inflammatory effects of the lipopeptide AFC, or through AFC-dependent membrane properties

Replication data for: Orfamide A and other secreted factors of Pseudomonas protegens control Pseudomonas aeruginosa biofilm dynamics

This includes the original replication data files for the submission of the manuscript "Orfamide A and other secreted factors of Pseudomonas protegens control Pseudomonas aeruginosa biofilm dynamics." It includes confocal microscopy image files, prism files for statistical analysis, and plate scan images

LuxR-family ‘solos’: bachelor sensors/regulators of signalling molecules

N-Acylhomoserine lactone (AHL) quorum-sensing (QS) signalling is the best-understood chemical language in proteobacteria. In the last 15 years a large amount of research in several bacterial species has revealed in detail the genetic, molecular and biochemical mechanisms underlying AHL signalling. These studies have revealed the role played by protein pairs of the AHL synthase belonging to the LuxI family and cognate LuxR-family AHL sensor–regulator. Proteobacteria however commonly possess a QS LuxR-family protein for which there is no obvious cognate LuxI synthase; these proteins are found in bacteria which possess a complete AHL QS system(s) as well as in bacteria that do not. Scientists are beginning to address the roles played by these proteins and it is emerging that they could allow bacteria to respond to endogenous and exogenous signals produced by their neighbours. AHL QS research thus far has mainly focused on a cell-density response involving laboratory monoculture studies. Recent findings on the role played by the unpaired LuxR-family proteins highlight the need to address bacterial behaviour and response to signals in mixed communities. Here we review recent progress with respect to these LuxR proteins, which we propose to call LuxR ‘solos’ since they act on their own without the need for a cognate signal generator. Initial investigations have revealed that LuxR solos have diverse roles in bacterial interspecies and interkingdom communication.</jats:p

Replication data for: N-Acyl homoserine lactone mediated quorum sensing regulates species interactions in multispecies biofilm communities

Microscopy images and associated dat

Quorum sensing systems influence <i>Burkholderia cenocepacia</i> virulence

Burkholderia cepacia complex strains communicate using N-acyl homoserine lactones and BDSF-dependent quorum sensing (QS) systems. Burkholderia cenocepacia QS systems include CepIR, CciIR, CepR2 and BDSF. Analysis of CepR, CciIR, CepR2 and RpfF (BDSF synthase) QS regulons revealed that these QS systems both independently regulate and coregulate many target genes, often in an opposing manner. The role of QS and several QS-regulated genes in virulence has been determined using vertebrate, invertebrate and plant infection models. Virulence phenotypes are strain and model dependent, suggesting that different QS-regulated genes are important depending on the strain and type of infection. QS inhibitors in combination with antibiotics can reduce biofilm formation and virulence in infection models. </jats:p