The BDSF quorum sensing receptor RpfR regulates Bep exopolysaccharide synthesis in Burkholderia cenocepacia via interaction with the transcriptional regulator BerB.

The polysaccharide Bep is essential for in vitro biofilm formation of the opportunistic pathogen Burkholderia cenocepacia. We found that the Burkholderia diffusible signaling factor (BDSF) quorum sensing receptor RpfR is a negative regulator of the bep gene cluster in B. cenocepacia. An rpfR mutant formed wrinkled colonies, whereas additional mutations in the bep genes or known bep regulators like berA and berB restored the wild-type smooth colony morphology. We found that there is a good correlation between intracellular c-di-GMP levels and bep expression when the c-di-GMP level is increased or decreased through ectopic expression of a diguanylate cyclase or a c-di-GMP phosphodiesterase, respectively. However, when the intracellular c-di-GMP level is changed by site directed mutagenesis of the EAL or GGDEF domain of RpfR there is no correlation between intracellular c-di-GMP levels and bep expression. Except for rpfR, deletion mutants of all 25 c-di-GMP phosphodiesterase and diguanylate cyclase genes encoded by B. cenocepacia showed no change to berA and bep gene expression. Moreover, bacterial two-hybrid assays provided evidence that RpfR and BerB physically interact and give specificity to the regulation of the bep genes. We suggest a model where RpfR binds BerB at low c-di-GMP levels to sequester this RpoN-dependent activator to an RpfR/RpfF complex. If the c-di-GMP levels rise, possibly by the enzymatic action of RpfR, BerB binds c-di-GMP and is released from the RpfR/RpfF complex and associates with RpoN to activate transcription of berA, and the BerA protein subsequently activates transcription of the bep genes.

Key Players and Individualists of Cyclic-di-GMP Signaling in Burkholderia cenocepacia.

Burkholderia cenocepacia H111 is an opportunistic pathogen associated with chronic lung infections in cystic fibrosis patients. Biofilm formation, motility and virulence of B. cenocepacia are regulated by the second messenger cyclic di-guanosine monophosphate (c-di-GMP). In the present study, we analyzed the role of all 25 putative c-di-GMP metabolizing proteins of B. cenocepacia H111 with respect to motility, colony morphology, pellicle formation, biofilm formation, and virulence. We found that RpfR is a key regulator of c-di-GMP signaling in B. cenocepacia, affecting a broad spectrum of phenotypes under various environmental conditions. In addition, we identified Bcal2449 as a regulator of B. cenocepacia virulence in Galleria mellonella larvae. While Bcal2449 consists of protein domains that may catalyze both c-di-GMP synthesis and degradation, only the latter was essential for larvae killing, suggesting that a decreased c-di-GMP level mediated by the Bcal2449 protein is required for virulence of B. cenocepacia. Finally, our work suggests that some individual proteins play a role in regulating exclusively motility (CdpA), biofilm formation (Bcam1160) or both (Bcam2836).

High intracellular c-di-GMP levels antagonize quorum sensing and virulence gene expression in Burkholderia cenocepacia H111.

The opportunistic human pathogen Burkholderia cenocepacia H111 uses two chemically distinct signal molecules for controlling gene expression in a cell density-dependent manner: N-acyl-homoserine lactones (AHLs) and cis-2-dodecenoic acid (BDSF). Binding of BDSF to its cognate receptor RpfR lowers the intracellular c-di-GMP level, which in turn leads to differential expression of target genes. In this study we analysed the transcriptional profile of B. cenocepacia H111 upon artificially altering the cellular c-di-GMP level. One hundred and eleven genes were shown to be differentially expressed, 96 of which were downregulated at a high c-di-GMP concentration. Our analysis revealed that the BDSF, AHL and c-di-GMP regulons overlap for the regulation of 24 genes and that a high c-di-GMP level suppresses expression of AHL-regulated genes. Phenotypic analyses confirmed changes in the expression of virulence factors, the production of AHL signal molecules and the biosynthesis of different biofilm matrix components upon altered c-di-GMP levels. We also demonstrate that the intracellular c-di-GMP level determines the virulence of B. cenocepacia to Caenorhabditis elegans and Galleria mellonella.

σ54-Dependent Response to Nitrogen Limitation and Virulence in Burkholderia cenocepacia Strain H111.

Members of the genus Burkholderia are versatile bacteria capable of colonizing highly diverse environmental niches. In this study, we investigated the global response of the opportunistic pathogen Burkholderia cenocepacia H111 to nitrogen limitation at the transcript and protein expression levels. In addition to a classical response to nitrogen starvation, including the activation of glutamine synthetase, PII proteins, and the two-component regulatory system NtrBC, B. cenocepacia H111 also upregulated polyhydroxybutyrate (PHB) accumulation and exopolysaccharide (EPS) production in response to nitrogen shortage. A search for consensus sequences in promoter regions of nitrogen-responsive genes identified a σ(54) consensus sequence. The mapping of the σ(54) regulon as well as the characterization of a σ(54) mutant suggests an important role of σ(54) not only in control of nitrogen metabolism but also in the virulence of this organism.

Genome Sequence of Burkholderia cenocepacia H111, a Cystic Fibrosis Airway Isolate.

The Burkholderia cepacia complex (BCC) is a group of related bacterial species that are commonly isolated from environmental samples. Members of the BCC can cause respiratory infections in cystic fibrosis patients and immunocompromised individuals. We report here the genome sequence of Burkholderia cenocepacia H111, a well-studied model strain of the BCC.

The IclR-family regulator BapR controls biofilm formation in B. cenocepacia H111.

In Burkholderia cenocepacia H111, the large surface protein BapA plays a crucial role in the formation of highly structured communities, known as biofilms. We have recently demonstrated that quorum sensing (QS) is necessary for the maximal expression of bapA. In this study we identify BapR, a protein from the IclR family of transcriptional regulators that, in conjunction with QS, controls biofilm formation by affecting the expression of bapA. We present evidence that, in addition to bapA, BapR influences the expression of extracellular proteases, swimming motility and has a profound impact in the incidence of persister cells, making this regulator an interesting target for persister cells and biofilm eradication.

Two quorum sensing systems control biofilm formation and virulence in members of the Burkholderia cepacia complex.

The Burkholderia cepacia complex (Bcc) consists of 17 closely related species that are problematic opportunistic bacterial pathogens for cystic fibrosis patients and immunocompromised individuals. These bacteria are capable of utilizing two different chemical languages: N-acyl homoserine lactones (AHLs) and cis-2-unsaturated fatty acids. Here we summarize the current knowledge of the underlying molecular architectures of these communication systems, showing how they are interlinked and discussing how they regulate overlapping as well as specific sets of genes. A particular focus is laid on the role of these signaling systems in the formation of biofilms, which are believed to be highly important for chronic infections. We review genes that have been implicated in the sessile lifestyle of this group of bacteria. The new emerging role of the intracellular second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) as a downstream regulator of the fatty acid signaling cascade and as a key factor in biofilm formation is also discussed.

The AHL- and BDSF-dependent quorum sensing systems control specific and overlapping sets of genes in Burkholderia cenocepacia H111.

Quorum sensing in Burkholderia cenocepacia H111 involves two signalling systems that depend on different signal molecules, namely N-acyl homoserine lactones (AHLs) and the diffusible signal factor cis-2-dodecenoic acid (BDSF). Previous studies have shown that AHLs and BDSF control similar phenotypic traits, including biofilm formation, proteolytic activity and pathogenicity. In this study we mapped the BDSF stimulon by RNA-Seq and shotgun proteomics analysis. We demonstrate that a set of the identified BDSF-regulated genes or proteins are also controlled by AHLs, suggesting that the two regulons partially overlap. The detailed analysis of two mutually regulated operons, one encoding three lectins and the other one encoding the large surface protein BapA and its type I secretion machinery, revealed that both AHLs and BDSF are required for full expression, suggesting that the two signalling systems operate in parallel. In accordance with this, we show that both AHLs and BDSF are required for biofilm formation and protease production.

Identification of functions linking quorum sensing with biofilm formation in Burkholderia cenocepacia H111.

Burkholderia cenocepacia has emerged as an important pathogen for patients suffering from cystic fibrosis (CF). Previous work has shown that this organism employs the CepIR quorum-sensing (QS) system to control the expression of virulence factors as well as the formation of biofilms. To date, however, very little is known about the QS-regulated virulence factors and virtually nothing about the factors that link QS and biofilm formation. Here, we have employed a combined transcriptomic and proteomic approach to precisely define the QS regulon in our model strain B. cenocepacia H111, a CF isolate. Among the identified CepR-activated loci, three were analyzed in better detail for their roles in biofilm development: (i) a gene cluster coding for the BclACB lectins, (ii) the large surface protein BapA, and (iii) a type I pilus. The analysis of defined mutants revealed that BapA plays a major role in biofilm formation on abiotic surfaces while inactivation of the type I pilus showed little effect both in a static microtitre dish-based biofilm assay and in flow-through cells. Inactivation of the bclACB lectin genes resulted in biofilms containing hollow microcolonies, suggesting that the lectins are important for biofilm structural development.

Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover.

Many bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals in modulation of virulence and biofilm formation. Previous work on Xanthomonas campestris showed that the RpfC/RpfG two-component system is involved in sensing and responding to DSF signals, but little is known in other microorganisms. Here we show that in Burkholderia cenocepacia the DSF-family signal cis-2-dodecenoic acid (BDSF) negatively controls the intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) level through a receptor protein RpfR, which contains Per/Arnt/Sim (PAS)-GGDEF-EAL domains. RpfR regulates the same phenotypes as BDSF including swarming motility, biofilm formation, and virulence. In addition, the BDSF(-) mutant phenotypes could be rescued by in trans expression of RpfR, or its EAL domain that functions as a c-di-GMP phosphodiesterase. BDSF is shown to bind to the PAS domain of RpfR with high affinity and stimulates its phosphodiesterase activity through induction of allosteric conformational changes. Our work presents a unique and widely conserved DSF-family signal receptor that directly links the signal perception to c-di-GMP turnover in regulation of bacterial physiology.