Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.
Bottom Line: The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro.Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits.Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.
Affiliation: From the Molecular Microbiology Department and.Show MeSH
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Mentions: Flagella-driven motility, and hence FliI-mediated export, is ubiquitous among Gram-negative bacteria. To investigate whether cdG-binding to FliI is similarly widespread, full-length FliI homologs from several bacterial species were cloned, expressed, purified, and then tested for cdG binding using SPR. FliI homologs were selected from human and plant pathogens, as well as commensal and symbiotic plant growth-promoting organisms. The tested FliI homologs included representatives from the α- and γ-proteobacterial classes and both monotrichous and polyflagellated bacteria. Concentration-dependent cdG binding was detected for full-length FliI alleles from the phytopathogen P. syringae pv. tomato (Pto) DC3000 (FliIPto), the human pathogen S. enterica serovar typhimurium (FliISeT), and the nitrogen-fixing symbiont Sinorhizobium meliloti (FliISm) (Fig. 3 and Table 3). Despite a reasonably high degree of fliI amino acid sequence divergence (SBW25 and S. meliloti fliI share only 35.4% identity) and significant differences in flagella regulation and cdG signaling between the tested species, all four FliI homologs bound to the dinucleotide molecule with affinities well within the expected physiological range of intracellular cdG concentrations.