SummaryThe Crc protein is a translational repressor that recognizes a specific target at some mRNAs, controlling catabolite repression and co-ordinating carbon metabolism in pseudomonads. In Pseudomonas aeruginosa, the levels of free Crc protein are controlled by CrcZ, a sRNA that sequesters Crc, acting as an antagonist. We show that, in Pseudomonas putida, the levels of free Crc are controlled by CrcZ and by a novel 368 nt sRNA named CrcY. CrcZ and CrcY, which contain six potential targets for Crc, were able to bind Crc specifically in vitro. The levels of CrcZ and CrcY were low under conditions generating a strong catabolite repression, and increased strongly when catabolite repression was absent. Deletion of either crcZ or crcY had no effect on catabolite repression, but the simultaneous absence of both sRNAs led to constitutive catabolite repression that compromised growth on some carbon sources. Overproduction of CrcZ or CrcY significantly reduced repression. We propose that CrcZ and CrcY act in concert, sequestering and modulating the levels of free Crc according to metabolic conditions. The CbrA/CbrB two-component system activated crcZ transcription, but had little effect on crcY. CrcY was detected in P. putida, Pseudomonas fluorescens and Pseudomonas syringae, but not in P. aeruginosa.

The capacity of a bacterial pathogen to produce a disease in a treated host depends on the former's virulence and resistance to antibiotics. Several scattered pieces of evidence suggest that these two characteristics can be influenced by bacterial metabolism. This potential relationship is particularly important upon infection of a host, a situation that demands bacteria adapt their physiology to their new environment, making use of newly available nutrients. To explore the potential cross-talk between bacterial metabolism, antibiotic resistance and virulence, a Pseudomonas aeruginosa model was used. This species is an important opportunistic pathogen intrinsically resistant to many antibiotics. The role of Crc, a global regulator that controls the metabolism of carbon sources and catabolite repression in Pseudomonas, was analysed to determine its contribution to the intrinsic antibiotic resistance and virulence of P. aeruginosa. Using proteomic analyses, high-throughput metabolic tests and functional assays, the present work shows the virulence and antibiotic resistance of this pathogen to be linked to its physiology, and to be under the control (directly or indirectly) of Crc. A P. aeruginosa strain lacking the Crc regulator showed defects in type III secretion, motility, expression of quorum sensing-regulated virulence factors, and was less virulent in a Dictyostelium discoideum model. In addition, this mutant strain was more susceptible to beta-lactams, aminoglycosides, fosfomycin and rifampin. Crc might therefore be a good target in the search for new antibiotics.

The Crc protein is a global translational regulator involved in catabolite repression of catabolic pathways for several non-preferred carbon sources in Pseudomonads when other preferred substrates are present. Using proteomic and transcriptomic approaches, we have analyzed the influence of Crc in cells growing in a complete medium, where amino acids are the main carbon source. Inactivation of the crc gene modified the expression of at least 134 genes. Most of them were involved in the transport and assimilation of amino acids or sugars. This allowed envisioning which amino acids are preferentially used. Crc did not inhibit the pathways for proline, alanine, glutamate, glutamine and histidine. These amino acids are good carbon sources for P. putida. In the case of arginine, lysine, aspartate and asparagine, which can be assimilated through several pathways, Crc favored one particular route, inhibiting other alternatives. Finally, Crc-inhibited genes needed to assimilate valine, isoleucine, leucine, tyrosine, phenylalanine, threonine, glycine and serine, amino acids that provide a less efficient growth. Crc has therefore a key role in coordinating metabolism, controlling the sequential assimilation of amino acids when cells grow in a complete medium. Inactivation of crc reduced growth rate, suggesting that Crc optimizes metabolism.