The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities, and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa. This bacterium is frequently adopted as a model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation, and transmission.

A relevant, yet little recognized feature of antisense regulation is the possibility of switching roles between regulatory and regulated RNAs. Here we show that induction of aSalmonellagene relies on the conversion of a small RNA from effector to regulatory target. ThechiPgene (formerlyybfM), identified and characterized in the present study, encodes a conserved enterobacterial chitoporin required for uptake of chitin-derived oligosaccharides. In the absence of inducer,chiPis kept silent by the action of a constitutively made small RNA, ChiX (formerly SroB, RybC), which pairs with a sequence at the 5′ end ofchiPmRNA. Silencing is relieved in the presence of chitooligosaccharides due to the accumulation of an RNA that pairs with ChiX and promotes its degradation. Anti-ChiX RNA originates from an intercistronic region of thechboperon, which comprises genes for chitooligosaccharide metabolism and whose transcription is activated in the presence of these sugars. We present evidence suggesting that thechbRNA destabilizes ChiX sRNA by invading the stem of its transcription terminator hairpin. Overall, our findings blur the distinction between effector and target in sRNA regulation, raising the possibility that some of the currently defined targets could actually be inhibitors of sRNA function.

The intracellular signaling molecule cyclic di-GMP (c-di-GMP) regulates the lifestyle of bacteria and controls many key functions and mechanisms. In the case of bacterial pathogens, a wide variety of virulence lifestyle factors have been shown to be regulated by c-di-GMP. Evidence of the importance of this molecule for bacterial pathogenesis has become so great that new antimicrobial agents are tested for their capacity of targeting c-di-GMP signaling. This review summarizes the current knowledge on this topic and reveals its application for the development of new antivirulence intervention strategies.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism.

Pseudomonas aeruginosa utilizes preferentially C ₄ -dicarboxylates such as malate, fumarate, and succinate as carbon and energy sources. We have identified and characterized two C ₄ -dicarboxylate transport (Dct) systems in P. aeruginosa PAO1. Inactivation of the dctA (PA1183) gene caused a growth defect of the strain in minimal media supplemented with succinate, fumarate or malate, indicating that DctA has a major role in Dct. However, residual growth of the dctA mutant in these media suggested the presence of additional C ₄ -dicarboxylate transporter(s). Tn 5 insertion mutagenesis of the ΔdctA mutant led to the identification of a second Dct system, i.e., the DctPQM transporter belonging to the tr ipartite A TP-independent p eriplasmic (TRAP) family of carriers. The ΔdctA ΔdctPQM double mutant showed no growth on malate and fumarate and residual growth on succinate, suggesting that DctA and DctPQM are the only malate and fumarate transporters, whereas additional transporters for succinate are present. Using lacZ reporter fusions, we showed that the expression of the dctA gene and the dctPQM operon was enhanced in early exponential growth phase and induced by C ₄ -dicarboxylates. Competition experiments demonstrated that the DctPQM carrier was more efficient than the DctA carrier for the utilization of succinate at micromolar concentrations, whereas DctA was the major transporter at millimolar concentrations. To conclude, this is the first time that the high- and low-affinity uptake systems for succinate DctA and DctPQM have been reported to function coordinately to transport C ₄ -dicarboxylates and that the alternative sigma factor RpoN and a DctB/DctD two-component system regulates simultaneously the dctA gene and the dctPQM operon.