Several transmissible Burkholderia cenocepacia strains that infect multiple cystic fibrosis patients contain a genomic island designated as the cenocepacia island (cci). The cci contains a predicted N-acylhomoserine lactone (AHL) synthase gene, cciI, and a predicted response regulator gene, cciR. AHL production profiles indicated that CciI catalyzes the synthesis of N-hexanoyl-L-homoserine lactone and minor amounts of Noctanoyl-L-homoserine lactone. The cciI and cciR genes were found to be cotranscribed by reverse transcription-PCR analysis, and the expression of a cciIR::luxCDABE fusion in a cciR mutant suggested that the cciIR system negatively regulates its own expression. B. cenocepacia strains also have a cepIR quorum-sensing system. Expression of cepI::luxCDABE or cepR::luxCDABE fusions in a cciR mutant showed that CciR negatively regulates cepI but does not regulate cepR. Expression of the cciIR::luxCDABE fusion in a cepR mutant indicated that functional CepR is required for cciIR expression. Phylogenetic analysis suggested that the cciIR system was acquired by horizontal gene transfer from a distantly related organism and subsequently incorporated into the ancestral cepIR regulatory network. Mutations in cciI, cciR, cepI cciI, and cepR cciR were constructed in B. cenocepacia K56-2. The cciI mutant had greater protease activity and less swarming motility than the parent strain. The cciR mutant had less protease activity than the parent strain. The phenotypes of the cepI cciI and cepR cciR mutants were similar to cepI or cepR mutants, with less protease activity and swarming motility than the parent strain.
Background: Burkholderia cenocepacia belongs to a group of closely related organisms called the B. cepacia complex (Bcc) which are important opportunistic human pathogens. B. cenocepacia utilizes a mechanism of cell-cell communication called quorum sensing to control gene expression including genes involved in virulence. The B. cenocepacia quorum sensing network includes the CepIR and CciIR regulatory systems.
Burkholderia cenocepacia utilizes quorum sensing to control gene expression, including the expression of genes involved in virulence. In addition to CepR and CciR, a third LuxR homolog, CepR2, was found to regulate gene expression and virulence factor production. All B. cenocepacia strains examined contained this orphan LuxR homolog, which was not associated with an adjacent N-acyl-homoserine lactone synthase gene. Expression of cepR2 was negatively autoregulated and was negatively regulated by CciR in strain K56-2. Microarray analysis and quantitative reverse transcription-PCR determined that CepR2 did not influence expression of cepIR or cciIR. However, in strain K56-2, CepR2 negatively regulated expression of several known quorum-sensing-controlled genes, including genes encoding zinc metalloproteases. CepR2 exerted positive and negative regulation on genes on three chromosomes, including strong negative regulation of a gene cluster located adjacent to cepR2. In strain H111, which lacks the CciIR quorum-sensing system, CepR2 positively regulated pyochelin production by controlling transcription of one of the operons required for the biosynthesis of the siderophore in an N-acyl-homoserine lactone-independent manner. CepR2 activation of a luxI promoter was demonstrated in a heterologous Escherichia coli host, providing further evidence that CepR2 can function in the absence of signaling molecules. This study demonstrates that the orphan LuxR homolog CepR2 contributes to the quorum-sensing regulatory network in two distinct strains of B. cenocepacia.
A major challenge to clinical therapy of Burkholderia cepacia complex (Bcc) pulmonary infections is their innate resistance to a broad range of antimicrobials, including polycationic agents such as aminoglycosides, polymyxins, and cationic peptides. To identify genetic loci associated with this phenotype, a transposon mutant library was constructed in B. multivorans ATCC 17616 and screened for increased susceptibility to polymyxin B. Compared to the parent strain, mutant 26D7 exhibited 8-and 16-fold increases in susceptibility to polymyxin B and colistin, respectively. Genetic analysis of mutant 26D7 indicated that the transposon inserted into open reading frame (ORF) Bmul_2133, part of a putative hopanoid biosynthesis gene cluster. A strain with a mutation in another ORF in this cluster, Bmul_2134, was constructed and named RMI19. Mutant RMI19 also had increased polymyxin susceptibility. Hopanoids are analogues of eukaryotic sterols involved in membrane stability and barrier function. Strains with mutations in Bmul_2133 and Bmul_2134 showed increased permeability to 1-N-phenylnaphthylamine in the presence of increasing concentrations of polymyxin, suggesting that the putative hopanoid biosynthesis genes are involved in stabilizing outer membrane permeability, contributing to polymyxin resistance. Results from a dansyl-polymyxin binding assay demonstrated that polymyxin B does not bind well to the parent or mutant strains, suggesting that Bmul_2133 and Bmul_2134 contribute to polymyxin B resistance by a mechanism that is independent of lipopolysaccharide (LPS) binding. Through this work, we propose a role for hopanoid biosynthesis as part of the multiple antimicrobial resistance phenotype in Bcc bacteria.
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