Pseudomonas aeruginosa (PA) can thrive in anaerobic biofilms in the lungs of cystic fibrosis (CF) patients. Here, we show that CrcZ is the most abundant PA14 RNA bound to the global regulator Hfq in anoxic biofilms grown in cystic fibrosis sputum medium. Hfq was crucial for anoxic biofilm formation. This observation complied with an RNAseq based transcriptome analysis and follow up studies that implicated Hfq in regulation of a central step preceding denitrification. CrcZ is known to act as a decoy that sequesters Hfq during relief of carbon catabolite repression, which in turn alleviates Hfq-mediated translational repression of catabolic genes. We therefore inferred that CrcZ indirectly impacts on biofilm formation by competing for Hfq. This hypothesis was supported by the findings that over-production of CrcZ mirrored the biofilm phenotype of the hfq deletion mutant, and that deletion of the crcZ gene augmented biofilm formation. To our knowledge, this is the first example where competition for Hfq by CrcZ cross-regulates an Hfq-dependent physiological process unrelated to carbon metabolism.

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for ~ 10% of hospital-acquired infections worldwide. It is notorious for its high level resistance toward many antibiotics, and the number of multi-drug resistant clinical isolates is steadily increasing. A better understanding of the molecular mechanisms underlying drug resistance is crucial for the development of novel antimicrobials and alternative strategies such as enhanced sensitization of bacteria to antibiotics in use. In P. aeruginosa several uptake channels for amino-acids and carbon sources can serve simultaneously as entry ports for antibiotics. The respective genes are often controlled by carbon catabolite repression (CCR). We have recently shown that Hfq in concert with Crc acts as a translational repressor during CCR. This function is counteracted by the regulatory RNA CrcZ, which functions as a decoy to abrogate Hfq-mediated translational repression of catabolic genes. Here, we report an increased susceptibility of P. aeruginosa hfq deletion strains to different classes of antibiotics. Transcriptome analyses indicated that Hfq impacts on different mechanisms known to be involved in antibiotic susceptibility, viz import and efflux, energy metabolism, cell wall and LPS composition as well as on the c-di-GMP levels. Furthermore, we show that sequestration of Hfq by CrcZ, which was over-produced or induced by non-preferred carbon-sources, enhances the sensitivity toward antibiotics. Thus, controlled synthesis of CrcZ could provide a means to (re)sensitize P. aeruginosa to different classes of antibiotics.