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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, ATPase activity for FliIHis ± 50 μm cdG. FliIHis specific activity (nmol ATP hydrolyzed/min/mg protein) is shown for increasing ATP concentrations. Addition of cdG causes a decrease of the Vmax of FliIHis ATPase activity. B, IC50 curve for FliIHis ATPase activity upon addition of increasing cdG concentrations. A constant concentration of ATP (1 mm) was included alongside 1 μg of FliIHis protein. C and D, ATPase activity ± 50 μm cdG, and IC50 curve upon addition of increasing cdG concentrations, for HrcN. All parameters remain the same as in A. The IC50 curve also includes results for GTP titration showing no ATPase inhibition.
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Figure 5: A, ATPase activity for FliIHis ± 50 μm cdG. FliIHis specific activity (nmol ATP hydrolyzed/min/mg protein) is shown for increasing ATP concentrations. Addition of cdG causes a decrease of the Vmax of FliIHis ATPase activity. B, IC50 curve for FliIHis ATPase activity upon addition of increasing cdG concentrations. A constant concentration of ATP (1 mm) was included alongside 1 μg of FliIHis protein. C and D, ATPase activity ± 50 μm cdG, and IC50 curve upon addition of increasing cdG concentrations, for HrcN. All parameters remain the same as in A. The IC50 curve also includes results for GTP titration showing no ATPase inhibition.

Mentions: The established model for cdG function associates increased dinucleotide levels with reduced motility and virulence (22). This has been shown to be the case for both P. fluorescens (67) and P. syringae (17, 18, 21). Consequently, we hypothesized that cdG binding may negatively affect the ATP-dependent export activity of FliI and/or HrcN. To examine the effect of cdG binding on FliI/HrcN ATPase activity, pyruvate kinase/lactate dehydrogenase-linked ATPase activity assays were conducted for the full-length protein alleles FliIHis and HrcNHis. Purified FliIHis metabolized ATP with a Km of 0.48 ± 0.03 μm and a Vmax of 1262 ± 54.46 nm ATP/min/mg. Addition of 50 μm cdG led to a noticeable drop in Vmax, to 867.2 ± 51.65 nm ATP/min/mg (Fig. 5A). The IC50 of cdG for FliIHis (36.7 ± 1.13 μm) was then determined by increasing cdG levels while maintaining a constant ATP concentration in the reaction (Fig. 5B). Similar cdG inhibitory activity was seen for FliIΔ1–18, which metabolized ATP with Km and Vmax values of 0.45 ± 0.04 μm and 691.5 ± 41.90 nm ATP/min/mg without cdG. Upon addition of 50 μm of the dinucleotide molecule, Vmax dropped to 375.4 ± 35.34 nm ATP/min/mg. FliIΔ1–18 has an IC50 value of 48.8 ± 0.159 μm. In agreement with DRaCALA results seen for FliIHis (Fig. 1B), addition of GTP produced no change in ATPase activity, supporting a specific inhibitory role for cdG toward FliI ATPase activity (data not shown). Full-length HrcN showed a similar degree of ATPase inhibition to FliI, with Vmax dropping from 1183 ± 68.2 to 832.4 ± 119.7 nm ATP/min/mg upon addition of 50 μm cdG, with an IC50 of 25.11 ± 1.14 μm (Fig. 5, C and D). Addition of GTP had no effect on HrcNHis ATPase activity (Fig. 5D), arguing once again for specific cdG inhibition of HrcN ATPase activity.


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, ATPase activity for FliIHis ± 50 μm cdG. FliIHis specific activity (nmol ATP hydrolyzed/min/mg protein) is shown for increasing ATP concentrations. Addition of cdG causes a decrease of the Vmax of FliIHis ATPase activity. B, IC50 curve for FliIHis ATPase activity upon addition of increasing cdG concentrations. A constant concentration of ATP (1 mm) was included alongside 1 μg of FliIHis protein. C and D, ATPase activity ± 50 μm cdG, and IC50 curve upon addition of increasing cdG concentrations, for HrcN. All parameters remain the same as in A. The IC50 curve also includes results for GTP titration showing no ATPase inhibition.
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Figure 5: A, ATPase activity for FliIHis ± 50 μm cdG. FliIHis specific activity (nmol ATP hydrolyzed/min/mg protein) is shown for increasing ATP concentrations. Addition of cdG causes a decrease of the Vmax of FliIHis ATPase activity. B, IC50 curve for FliIHis ATPase activity upon addition of increasing cdG concentrations. A constant concentration of ATP (1 mm) was included alongside 1 μg of FliIHis protein. C and D, ATPase activity ± 50 μm cdG, and IC50 curve upon addition of increasing cdG concentrations, for HrcN. All parameters remain the same as in A. The IC50 curve also includes results for GTP titration showing no ATPase inhibition.
Mentions: The established model for cdG function associates increased dinucleotide levels with reduced motility and virulence (22). This has been shown to be the case for both P. fluorescens (67) and P. syringae (17, 18, 21). Consequently, we hypothesized that cdG binding may negatively affect the ATP-dependent export activity of FliI and/or HrcN. To examine the effect of cdG binding on FliI/HrcN ATPase activity, pyruvate kinase/lactate dehydrogenase-linked ATPase activity assays were conducted for the full-length protein alleles FliIHis and HrcNHis. Purified FliIHis metabolized ATP with a Km of 0.48 ± 0.03 μm and a Vmax of 1262 ± 54.46 nm ATP/min/mg. Addition of 50 μm cdG led to a noticeable drop in Vmax, to 867.2 ± 51.65 nm ATP/min/mg (Fig. 5A). The IC50 of cdG for FliIHis (36.7 ± 1.13 μm) was then determined by increasing cdG levels while maintaining a constant ATP concentration in the reaction (Fig. 5B). Similar cdG inhibitory activity was seen for FliIΔ1–18, which metabolized ATP with Km and Vmax values of 0.45 ± 0.04 μm and 691.5 ± 41.90 nm ATP/min/mg without cdG. Upon addition of 50 μm of the dinucleotide molecule, Vmax dropped to 375.4 ± 35.34 nm ATP/min/mg. FliIΔ1–18 has an IC50 value of 48.8 ± 0.159 μm. In agreement with DRaCALA results seen for FliIHis (Fig. 1B), addition of GTP produced no change in ATPase activity, supporting a specific inhibitory role for cdG toward FliI ATPase activity (data not shown). Full-length HrcN showed a similar degree of ATPase inhibition to FliI, with Vmax dropping from 1183 ± 68.2 to 832.4 ± 119.7 nm ATP/min/mg upon addition of 50 μm cdG, with an IC50 of 25.11 ± 1.14 μm (Fig. 5, C and D). Addition of GTP had no effect on HrcNHis ATPase activity (Fig. 5D), arguing once again for specific cdG inhibition of HrcN ATPase activity.

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