AUTHOR=Bogacz Marek , El Abbar Faiha M. , Cox Claudia A. , Li Jiaqi , Fiedler Jarred S. , Tran Lynn K. H. , Tran Paul M. H. , Daugherty C. Luke , Blake Kate H. , Wang Zhirui , Azadi Parastoo , Thompson Stuart A. TITLE=Binding of Campylobacter jejuni FliW Adjacent to the CsrA RNA-Binding Pockets Modulates CsrA Regulatory Activity JOURNAL=Frontiers in Microbiology VOLUME=11 YEAR=2021 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.531596 DOI=10.3389/fmicb.2020.531596 ISSN=1664-302X ABSTRACT=

Campylobacter jejuni CsrA is an mRNA-binding, post-transcriptional regulator that controls many metabolic- and virulence-related characteristics of this important pathogen. In contrast to E. coli CsrA, whose activity is modulated by binding to small non-coding RNAs (sRNAs), C. jejuni CsrA activity is controlled by binding to the CsrA antagonist FliW. In this study, we identified the FliW binding site on CsrA. Deletion of the C-terminus of C. jejuni CsrA, which is extended relative to sRNA-binding CsrA proteins, abrogated FliW binding. Bacterial two-hybrid experiments were used to assess the interaction of FliW with wild-type CsrA and mutants thereof, in which every amino acid was individually mutated. Two CsrA mutations (V51A and N55A) resulted in a significant decrease in FliW binding. The V51A and N55A mutants also showed a decrease in CsrA-FliW complex formation, as assessed by size-exclusion chromatography and surface plasmon resonance. These residues were highly conserved in bacterial species containing CsrA orthologs whose activities are predicted to be regulated by FliW. The location of FliW binding was immediately adjacent to the two RNA-binding sites of the CsrA homodimer, suggesting the model that FliW binding to CsrA modulates its ability to bind to its mRNA targets either by steric hindrance, electrostatic repulsion, or by altering the overall structure of the RNA-binding sites.