Susceptibility of Pseudomonas aeruginosa Dispersed Cells to Antimicrobial Agents Is Dependent on the Dispersion Cue and Class of the Antimicrobial Agent Used.

The biofilm life cycle is characterized by the transition of planktonic cells exhibiting high susceptibly to antimicrobial agents to a biofilm mode of growth characterized by high tolerance to antimicrobials, followed by dispersion of cells from the biofilm back into the environment. Dispersed cells, however, are not identical to planktonic cells but have been characterized as having a unique transitionary phenotype relative to biofilm and planktonic cells, with dispersed cells attaching in a manner similar to exponential-phase cells, but demonstrating gene expression patterns that are distinct from both exponential and stationary-phase planktonic cells. This raised the question whether dispersed cells are as susceptible as planktonic cells and whether the dispersion inducer or the antibiotic class affects the drug susceptibility of dispersed cells. Dispersed cells obtained in response to dispersion cues glutamate and nitric oxide (NO) were thus exposed to tobramycin and colistin. Although NO-induced dispersed cells were as susceptible to colistin and tobramycin as exponential-phase planktonic cells, glutamate-induced dispersed cells were susceptible to tobramycin but resistant to colistin. The difference in colistin susceptibility was independent of cellular c-di-GMP levels, with modulation of c-di-GMP failing to induce dispersion. Instead, drug susceptibility was inversely correlated with LPS modification system and the biofilm-specific transcriptional regulator BrlR. The susceptibility phenotype of glutamate-induced dispersed cells to colistin was found to be reversible, with dispersed cells being rendered as susceptible to colistin within 2 h postdispersion, though additional time was required for dispersed cells to display expression of genes indicative of exponential growth.

Detection of c-di-GMP-Responsive DNA Binding.

Modulation of signal transduction via binding of the secondary messenger molecule cyclic di-GMP to effector proteins is a near universal regulatory schema in bacteria. In particular, direct binding of c-di-GMP to transcriptional regulators has been shown to alter gene expression of a variety of processes. Here, we illustrate a pull-down-based DNA:protein binding reaction to determine the relative importance of c-di-GMP in the binding affinity of a target protein to specific DNA sequences. Specifically, the pull-down-based assay enables DNA binding to be analyzed with differing concentrations of c-di-GMP in the absence/presence of specific and nonspecific competitors.

Detection of Cyclic di-GMP Binding Proteins Utilizing a Biotinylated Cyclic di-GMP Pull-Down Assay.

Cyclic di-GMP is an important regulatory messenger molecule that often directly interacts with proteins to alter function. It is therefore important to find potential c-di-GMP binding proteins and verify a direct interaction between them. Here, we describe a pull-down assay using biotinylated-c-di-GMP to capture a specific protein of interest followed by immunoblot analysis to determine relative protein abundance. This method also allows for addition of both specific and nonspecific competitors to determine specificity of c-di-GMP-protein binding. We also discuss using densitometry analysis on resulting immunoblots to calculate the dissociation constant (KD) of the binding reaction, allowing for a determination of binding affinity.

BrlR from Pseudomonas aeruginosa is a c-di-GMP-responsive transcription factor.

The transcriptional regulator BrlR is a member of the MerR family of multidrug transport activators that contributes to the high-level drug tolerance of Pseudomonas aeruginosa biofilms. While MerR regulators are known to activate both the expression of multidrug efflux pump genes and their own transcription upon inducer binding, little is known about BrlR activation. We demonstrate using promoter reporter strains, in vivo and in vitro DNA-binding assays combined with 5'RACE, that BrlR binds to its own promoter, likely via a MerR-like palindromic sequence. Unlike known MerR multidrug transport activators, BrlR and brlR expression are not activated by multidrug transporter substrates. Instead, BrlR-DNA binding was enhanced by the secondary messenger c-di-GMP. In addition to enhanced BrlR-DNA binding, c-di-GMP levels contributed to PbrlR promoter activity in initial attached cells with elevated c-di-GMP levels correlating with increased expression of brlR. While not harbouring amino acid motifs resembling previously defined c-di-GMP-binding domains, BrlR was found to bind c-di-GMP in vitro at a ratio of one c-di-GMP per two BrlR. Cross-linking assays confirmed dimer formation to be enhanced in the presence of elevated c-di-GMP levels. Our findings demonstrate BrlR to be an unusual MerR-family member in that BrlR function and expression require the secondary messenger c-di-GMP.

The MerR-like regulator BrlR impairs Pseudomonas aeruginosa biofilm tolerance to colistin by repressing PhoPQ.

While the MerR-like transcriptional regulator BrlR has been demonstrated to contribute to Pseudomonas aeruginosa biofilm tolerance to antimicrobial agents known as multidrug efflux pump substrates, the role of BrlR in resistance to cationic antimicrobial peptides (CAP), which is based on reduced outer membrane susceptibility, is not known. Here, we demonstrate that inactivation of brlR coincided with increased resistance of P. aeruginosa to colistin, while overexpression of brlR resulted in increased susceptibility. brlR expression correlated with reduced transcript abundances of phoP, phoQ, pmrA, pmrB, and arnC. Inactivation of pmrA and pmrB had no effect on the susceptibility of P. aeruginosa biofilms to colistin, while inactivation of phoP and phoQ rendered biofilms more susceptible than the wild type. The susceptibility phenotype of ΔphoP biofilms to colistin was comparable to that of P. aeruginosa biofilms overexpressing brlR. BrlR was found to directly bind to oprH promoter DNA of the oprH-phoPQ operon. BrlR reciprocally contributed to colistin and tobramycin resistance in P. aeruginosa PAO1 and CF clinical isolates, with overexpression of brlR resulting in increased tobramycin MICs and increased tobramycin resistance but decreased colistin MICs and increased colistin susceptibility. The opposite trend was observed upon brlR inactivation. The difference in susceptibility to colistin and tobramycin was eliminated by combination treatment of biofilms with both antibiotics. Our findings establish BrlR as an unusual member of the MerR family, as it not only functions as a multidrug transport activator, but also acts as a repressor of phoPQ expression, thus suppressing colistin resistance.

Small RNAs and their role in biofilm formation.

The formation of biofilms is initiated by bacteria transitioning from the planktonic to the surface-associated mode of growth. Several regulatory systems have been described to govern the initiation and subsequent formation of biofilms. Recent evidence suggests that regulatory networks governing the decision of bacteria whether to attach and form biofilms or remain as planktonic cells are further subject to regulation by small non-coding RNAs (sRNAs). This is accomplished by sRNAs fine-tuning regulatory networks to enable concentration-specific responses by sequestering, antagonizing, or activating regulatory proteins in response to environmental cues, or by directly affecting the synthesis of proteins promoting or disfavoring the formation of biofilms. This review gives an overview of the contribution of sRNAs in regulating the switch from the planktonic to the sessile bacterial lifestyle by highlighting how sRNAs converge with known regulatory systems required for biofilm formation.

The RNA chaperone Hfq is important for growth and stress tolerance in Francisella novicida.

The RNA-binding protein Hfq is recognized as an important regulatory factor in a variety of cellular processes, including stress resistance and pathogenesis. Hfq has been shown in several bacteria to interact with small regulatory RNAs and act as a post-transcriptional regulator of mRNA stability and translation. Here we examined the impact of Hfq on growth, stress tolerance, and gene expression in the intracellular pathogen Francisella novicida. We present evidence of Hfq involvement in the ability of F. novicida to tolerate several cellular stresses, including heat-shock and oxidative stresses, and alterations in hfq gene expression under these conditions. Furthermore, expression of numerous genes, including several associated with virulence, is altered in a hfq mutant strain suggesting they are regulated directly or indirectly by Hfq. Strikingly, we observed a delayed entry into stationary phase and increased biofilm formation in the hfq mutant. Together, these data demonstrate a critical role for Hfq in F. novicida growth and survival.

Gibberellin biosynthesis in bacteria: separate ent-copalyl diphosphate and ent-kaurene synthases in Bradyrhizobium japonicum.

Gibberellins are ent-kaurene-derived diterpenoid phytohormones produced by plants, fungi, and bacteria. The distinct gibberellin biosynthetic pathways in plants and fungi are known, but not that in bacteria. Plants typically use two diterpene synthases to form ent-kaurene, while fungi use only a single bifunctional diterpene synthase. We demonstrate here that Bradyrhizobium japonicum encodes separate ent-copalyl diphosphate and ent-kaurene synthases. These are found in an operon whose enzymatic composition indicates that gibberellin biosynthesis in bacteria represents a third independently assembled pathway relative to plants and fungi. Nevertheless, sequence comparisons also suggest potential homology between diterpene synthases from bacteria, plants, and fungi.