Abstract
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.
Highlights
Pseudomonas aeruginosa (PA) can form persistent biofilms in the lungs of cystic fibrosis (CF) patients[1]
With the goal to identify regulatory RNAs that impact on anoxic biofilm formation, we concentrated on RNAs that interact with Hfq
The PA14 strain was grown in modified cystic fibrosis sputum medium (SCFM)[16], which approximates to the conditions of the CF lung
Summary
PA can form persistent biofilms in the lungs of CF patients[1]. Polymorphonuclear leukocytes are known to surround the biofilms in the CF lung and to consume the majority of O2 to produce reactive oxygen species, which suggested that PA biofilms may partially grow anaerobically in this environment[2,3]. Yoon et al.[4] provided first hints for anaerobic respiration in PAO1 biofilms. This has been recently supported through the quantification of compounds of the denitrification pathway in sputum of CF patients[5]. The PA protein binding RNAs RsmY and RsmZ antagonize the function of the translational regulator RsmA13. The RsmA protein is known to act as a translational repressor of psl mRNA, which prevents exopolysaccharide synthesis, and to control biofilm formation in a negative manner[14]. Up-regulation of the RsmY/Z RNAs results in titration of RsmA, and in increased biofilm formation[7,13]. In carbon catabolite repression, where CrcZ acts as a decoy to abrogate Hfq-mediated translational repression of catabolic genes[15], this study reveals a novel aspect of Hfq sequestration by CrcZ, that is cross-regulation of other Hfq-dependent physiological processes
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