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Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.

Trampari E, Stevenson CE, Little RH, Wilhelm T, Lawson DM, Malone JG - J. Biol. Chem. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Microbiology Department and.

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A and B, SPR sensorgrams and resulting affinity fit for FliIPto binding to biotinylated cdG. C and D, SPR sensorgrams and affinity fit for FliISeT binding to biotinylated cdG. E and F, SPR sensorgrams and affinity fit for FliISm binding to biotinylated cdG. In all three cases, a range of protein concentrations was used (0.625, 1.25, 2.5, 5, 10, and for FliIPto/FliISeT 20 μm), and concentration replicates were included as appropriate together with buffer only and BSA controls. The protein binding and dissociation phases for all sensorgrams are shown. For the affinity fits, binding responses were measured 4 s before the end of the injection, and KD values for each protein were calculated using the BiaEvaluation software and confirmed by GraphPad Prism (Table 3).
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Figure 3: A and B, SPR sensorgrams and resulting affinity fit for FliIPto binding to biotinylated cdG. C and D, SPR sensorgrams and affinity fit for FliISeT binding to biotinylated cdG. E and F, SPR sensorgrams and affinity fit for FliISm binding to biotinylated cdG. In all three cases, a range of protein concentrations was used (0.625, 1.25, 2.5, 5, 10, and for FliIPto/FliISeT 20 μm), and concentration replicates were included as appropriate together with buffer only and BSA controls. The protein binding and dissociation phases for all sensorgrams are shown. For the affinity fits, binding responses were measured 4 s before the end of the injection, and KD values for each protein were calculated using the BiaEvaluation software and confirmed by GraphPad Prism (Table 3).

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.


Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.

Trampari E, Stevenson CE, Little RH, Wilhelm T, Lawson DM, Malone JG - J. Biol. Chem. (2015)

A and B, SPR sensorgrams and resulting affinity fit for FliIPto binding to biotinylated cdG. C and D, SPR sensorgrams and affinity fit for FliISeT binding to biotinylated cdG. E and F, SPR sensorgrams and affinity fit for FliISm binding to biotinylated cdG. In all three cases, a range of protein concentrations was used (0.625, 1.25, 2.5, 5, 10, and for FliIPto/FliISeT 20 μm), and concentration replicates were included as appropriate together with buffer only and BSA controls. The protein binding and dissociation phases for all sensorgrams are shown. For the affinity fits, binding responses were measured 4 s before the end of the injection, and KD values for each protein were calculated using the BiaEvaluation software and confirmed by GraphPad Prism (Table 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591828&req=5

Figure 3: A and B, SPR sensorgrams and resulting affinity fit for FliIPto binding to biotinylated cdG. C and D, SPR sensorgrams and affinity fit for FliISeT binding to biotinylated cdG. E and F, SPR sensorgrams and affinity fit for FliISm binding to biotinylated cdG. In all three cases, a range of protein concentrations was used (0.625, 1.25, 2.5, 5, 10, and for FliIPto/FliISeT 20 μm), and concentration replicates were included as appropriate together with buffer only and BSA controls. The protein binding and dissociation phases for all sensorgrams are shown. For the affinity fits, binding responses were measured 4 s before the end of the injection, and KD values for each protein were calculated using the BiaEvaluation software and confirmed by GraphPad Prism (Table 3).
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.

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.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Microbiology Department and.

Show MeSH
Related in: MedlinePlus