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Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In Burkholderia cenocepacia , the QS signal receptor RpfR degrades intracellular c-di-GMP when it senses the QS signal cis -2-dodecenoic acid, also called Burkholderia diffusible signal factor (BDSF), as a proxy for high cell density. However, it was unclear how this resulted in control of BDSF-regulated phenotypes. Here, we found that RpfR forms a complex with a regulator named GtrR (BCAL1536) to enhance its binding to target gene promoters under circumstances where the BDSF signal binds to RpfR to stimulate its c-di-GMP phosphodiesterase activity. In the absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and inhibits its ability to control gene expression. Mutations in rpfR and gtrR had overlapping effects on both the B. cenocepacia transcriptome and BDSF-regulated phenotypes, including motility, biofilm formation, and virulence. These results show that RpfR is a QS signal receptor that also functions as a c-di-GMP sensor. This protein thus allows B. cenocepacia to integrate information about its physical and chemical surroundings as well as its population density to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and gamma proteobacteria., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Two-component systems are important regulatory systems that allow bacteria to adjust to environmental conditions, and in some bacteria are used in pathogenesis. We identified a novel two-component system in Burkholderia cenocepacia, an opportunistic pathogen that causes pneumonia in cystic fibrosis (CF) patients. The putative operon encodes BceS, a sensor kinase, and BceR, a response regulator. Our studies indicated that the bceR mutant showed a statistically significant decrease in protease, swimming motility, and quorum sensing when compared to the wild-type, but there was no significant difference in phospholipase C activity, swarming, and biofilm formation. In addition, the mutant showed a statistically significant reduction in virulence compared to the wild-type using the alfalfa plant model. Examination of the Burkholderia cepacia complex (a group of organisms that are phenotypically similar, but genotypically distinct) revealed that this system is prevalent in B. ambifaria, B. multivorans, B. vietnamiensis and B. dolosa. Interestingly, all these organisms have been associated with CF patients. The collective results indicate that BceSR influences various activities important in Burkholderia physiology and possibly pathogenesis. This information could be important in the design of novel therapeutics for Burkholderia infections.

Burkholderia cenocepacia is notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance in B. cenocepacia can be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps in B. cenocepacia strain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia J2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strain B. cenocepacia J2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.

Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis (CF) patients. Previously, we reported that ShvR, a LysR regulator, influences colony morphology, virulence, and biofilm formation and regulates the expression of an adjacent 24-kb genomic region encoding 24 genes. In this study, we report the functional characterization of selected genes in this region. A Tn5 mutant with shiny colony morphology was identified with a polar mutation in BCAS0208, predicted to encode an acyl-coenzyme A dehydrogenase. Mutagenesis of BCAS0208 and complementation analyses revealed that BCAS0208 is required for rough colony morphology, biofilm formation, and virulence on alfalfa seedlings. It was not possible to complement with BCAS0208 containing a mutation in the catalytic site. BCAS0201, encoding a putative flavin adenine dinucleotide (FAD)-dependent oxidoreductase, and BCAS0207, encoding a putative citrate synthase, do not influence colony morphology but are required for optimum levels of biofilm formation and virulence. Both BCAS0208 and BCAS0201 contribute to pellicle formation, although individual mutations in each of these genes had no appreciable effect on pellicle formation. A mutant with a polar insertion in BCAS0208 was significantly less virulent in a rat model of chronic lung infection as well as in the alfalfa model. Genes in this region were shown to influence utilization of branched-chain fatty acids, tricarboxylic acid cycle substrates, l-arabinose, and branched-chain amino acids. Together, our data show that the ShvR-regulated genes BCAS0208 to BCAS0201 are required for the rough colony morphotype, biofilm and pellicle formation, and virulence in B. cenocepacia.