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.

SummaryThe level of penicillin resistance in clinical isolates of Streptococcus pneumoniae depends not only on the reduced affinity of penicillin binding proteins (PBPs) but also on the functioning of enzymes that modify the stem peptide structure of cell wall precursors. We used mariner mutagenesis in search of additional genetic determinants that may further attenuate the level of penicillin resistance in the bacteria. A mariner mutant of the highly penicillinresistant S. pneumoniae strain Pen6 showed reduction of the penicillin minimum inhibitory concentration (MIC) from 6 to 0.75 mg ml -1 . Decrease in penicillin MIC was also observed upon introduction of the mutation (named provisionally adr, for attenuator of drug resistance) into representatives of major epidemic clones of penicillin-resistant pneumococci. Attenuation of resistance levels was specific for b-lactams. The adr mutant has retained unchanged (low affinity) PBPs, unaltered murM gene and unchanged cell wall stem peptide composition, but the mutant became hypersensitive to exogenous lysozyme and complementation experiments showed that both phenotypes -reduced resistance and lysozyme sensitivity -were linked to the defective adr gene. DNA sequence comparison and chemical analysis of the cell wall identified adr as the structural gene of the pneumococcal peptidoglycan O-acetylase.

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.

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