A putative LysR-type transcriptional regulator inhibits biofilm synthesis in Pseudomonas aeruginosa

Biofilm formation is an important virulence factor which is controlled by complex regulatory circuits in Pseudomonas aeruginosa. In this work, a biofilm hyper-producing strain, P2-7, was selected from a collection of transposon insertion mutants in which the PA2121 gene was disrupted. PA2121 was predicted as a putative LysR-type regulator. Analyses showed that it was involved in early biofilm formation, mature biofilm development, and colony morphology. Quantitative measurements revealed that PA2121 repressed biosynthesis of extracellular polysaccharides (alginate, psl and pel). Furthermore, it was observed that PA2121 was self-regulated, highly expressed in the early phase of biofilm development, and subject to the negative regulation by a biofilm synthesis regulator SrpA that binds directly to the PA2121 gene promoter. Collectively, this study proposes that PA2121 is a novel biofilm synthesis repressor (BsrA) in P. aeruginosa.     

Regulating the regulator: MicF RNA controls expression of the global regulator Lrp

Studies on the regulatory RNA MicF in Enterobacteriaceae reveal a pivotal role in gene regulation. Multiple target gene mRNAs were identified and, importantly, MicF RNA regulates the expression of the global regulatory gene lrp (Holmqvist et al., 2012; Corcoran et al., 2012). Thus MicF RNA is a central factor in a regulatory network that regulates bacterial cell physiology.     

The transcriptional regulator CzcR modulates antibiotic resistance and quorum sensing in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa responds to zinc, cadmium and cobalt by way of the CzcRS two-component system. In presence of these metals the regulatory protein CzcR induces the expression of the CzcCBA efflux pump, expelling and thereby inducing resistance to Zn, Cd and Co. Importantly, CzcR co-regulates carbapenem antibiotic resistance by repressing the expression of the OprD porin, the route of entry for these antibiotics. This unexpected co-regulation led us to address the role of CzcR in other cellular processes unrelated to the metal response. We found that CzcR affected the expression of numerous genes directly involved in the virulence of P. aeruginosa even in the absence of the inducible metals. Notably the full expression of quorum sensing 3-oxo-C12-HSL and C4-HSL autoinducer molecules is impaired in the absence of CzcR. In agreement with this, the virulence of the czcRS deletion mutant is affected in a C. elegans animal killing assay. Additionally, chromosome immunoprecipitation experiments allowed us to localize CzcR on the promoter of several regulated genes, suggesting a direct control of target genes such as oprD, phzA1 and lasI. All together our data identify CzcR as a novel regulator involved in the control of several key genes for P. aeruginosa virulence processes.     

BdlA, a chemotaxis regulator essential for biofilm dispersion in Pseudomonas aeruginosa

Multiple environmental cues have been shown to trigger biofilm detachment, the transition from surface-attached, highly organized communities known as biofilms to the motile lifestyle. The goal of this study was to identify a gene product involved in sensing environmental cues that trigger biofilm dispersion in Pseudomonas aeruginosa. To do so, we focused on novel putative chemotaxis transducer proteins that could potentially be involved in environmental sensing. We identified a locus encoding such a protein that played a role in detachment, as indicated by the observation that an isogenic mutant biofilm could not disperse in response to a variety of environmental cues. The locus was termed bdlA for biofilm dispersion locus. The BdlA protein harbors an MCP (methyl-accepting chemotaxis protein) domain and two PAS (Per-Arnt-Sint) domains that have been shown to be essential for responding to environmental signals in other proteins. The dispersion-deficient phenotype of the bdlA mutant was confirmed by treatment with the biocide H(2)O(2) and by microscopic observations. The dispersion response was independent of motility. bdlA mutant biofilms were found to have increased adherent properties and increased intracellular levels of cyclic di-GMP (c-di-GMP). Our findings suggest that BdlA may be a link between sensing environmental cues, c-di-GMP levels, and detachment. Based on our findings, a possible involvement of BdlA in a signaling cascade resulting in biofilm dispersion is discussed.     

Identification of a small RNA that directly controls the translation of the quorum sensing signal synthase gene rhlI in Pseudomonas aeruginosa

The biofilm formation by Pseudomonas aeruginosa highly increases the bacterial resistance to antimicrobial agents and host immune clearance. The biofilm formation is positively regulated by two small RNAs, RsmY and RsmZ. Previously, we reported that mutation in the polynucleotide phosphorylase (PNPase) coding gene pnp increases the levels of RsmY/Z. However, in this study, we found that the biofilm formation is decreased in the pnp mutant, which is due to a defect in rhamnolipids production. The rhamnolipids production is regulated by the RhlI-RhlR quorum sensing system. We found that PNPase influences the translation of RhlI through its 5′-untranslated region (UTR) and identified that the sRNA P27 is responsible for the translational repression. In vitro translation experiments demonstrated that P27 directly represses the translation of the rhlI mRNA through its 5′UTR in an Hfq-dependent manner. Point mutations in the rhlI 5′UTR or P27, which abolish the pairing between the two RNAs restore the rhlI expression and rhamnolipids production as well as the biofilm formation in the pnp mutant. Overall, our results reveal a novel layer of regulation of the Rhl quorum sensing system by the sRNA P27.