Crc in Hfq-dependent catabolite repression in P. aeruginosa

Carbon assimilation in Bacteria is governed by a mechanism known as carbon catabolite repression (CCR). In contrast to several other bacterial clades CCR in Pseudomonas is primarily regulated at the post-transcriptional level. We have identified the RNA chaperone Hfq as the principle post- transcriptional repressor of CCR in P. aeruginosa. In Enterobacteriaceae the RNA chaperone Hfq has been mainly implicated in facilitating the interaction between small regulatory RNAs and target mRNAs. Its role in P. aeruginosa CCR deviates from this canonical mechanism in that Hfq acts as a translational repressor, which is trapped by the regulatory RNA CrcZ, which in turn leads to translation (de-repression) of catabolic genes. In addition, several lines of evidence suggest that the Pseudomonas catabolite repression control protein Crc co-regulates CCR by interacting with Hfq. The major goal of this proposal is to study the interaction of Hfq and Crc at the molecular level using genetic, biochemical and biophysical methods. We are not aware of any other protein factor that physically interacts with, and that was shown to impact on Hfq function in the best studied model system E. coli. Thus, on the site of basic research, the proposed studies have the potential to shed light on an as yet undiscovered facet of Hfq-mediated regulation, that is, a modulation of Hfq function by another protein factor. On the other site, as both, Hfq and Crc, have been shown to affect antibiotic resistance and biofilm formation of P. aeruginosa, the proposed studies are anticipated to increase our knowledge of metabolic regulation of antibiotic susceptibility and biofilm development, and may aid in developing novel treatment strategies for therapeutic intervention.

BACKGROUND: In Bacteria, the uptake and utilization of carbon compounds is controlled by a mechanism known as carbon catabolite repression (CCR). In contrast to several other bacterial clades CCR in Pseudomonas species is regulated at the post-transcriptional level. Our previous studies revealed that Hfq acts as a translational repressor that prevents protein biosynthesis through binding within the ribosome binding site of mRNAs, encoding enzymes required for carbon utilization. We have also shown that the catabolite repression control protein Crc somehow enhances Hfq-mediated translational repression of catabolic genes but the underlying molecular mechanism(s) remained elusive. Therefore, this study was mainly directed towards a mechanistic understanding of the Hfq/Crc interaction. As both, Hfq and Crc, had been implicated in antibiotic susceptibility of Pseudomonas species, these studies were also anticipated to permit new insights into the regulation of these processes. RESULTS: Biochemical and biophysical approaches revealed that Crc and Hfq form an assembly in the presence of RNAs, containing A-rich motifs, and that Crc physically interacts with both, Hfq and RNA. Using a single-molecule fluorescence assay, we further showed that Crc does not change the RNA-Hfq interaction lifetimes, whereas it changes the equilibrium towards more stable repressive complexes. This observation is in accord with Cryo-EM analyses, which showed an increased compactness of repressive Hfq/Crc/RNA assemblies. By using high-resolution Cryo-EM we have not only obtained structural insights on the assembly of Hfq and Crc bound to the translation initiation site of a short target RNA but also on Hfq/Crc complexes assembled on longer mRNA substrates. These studies gave new insights into the assembly steps and hints as to mRNA signatures required for Hfq/Crc assembly. Taken together, these biophysical studies did not only reveal insights into how an interacting protein can modulate Hfq function but also how the two proteins enable quaternary structure variability in repressor complexes to fold large segment(s) of a mRNA into a more compact translationally repressive structure. -We have identified PA1677 and shown that it physically interacts with Crc. PA1677 prevents that Crc cooperates with Hfq in translational repression. It is therefore possible that PA1677 "titrates" Crc after alleviation of CCR, and thus affects the formation of Hfq/Crc repressive complexes. - Crc was shown to interfere with binding of a small regulatory RNA to Hfq, which bears implications for riboregulation. -The outer membrane porins OprD and OpdP serve as entry ports for carbapenem antibiotics. We showed that the RNA chaperone Hfq governs post-transcriptional regulation of the oprD and opdP genes in a distinctive manner. Hfq together with small regulatory RNAs was shown to mediate translational repression of oprD, whereas opdP is translationally repressed by a regulatory complex consisting of Hfq and the translational co-repressor Crc.