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Adjusting the spokes of the flagellar motor with the DNA-binding protein H-NS.

ABSTRACT: The H-NS protein of bacteria is a global regulator that stimulates transcription of flagellar genes and that also acts directly to modulate flagellar motor function. H-NS is known to bind FliG, a protein of the rotor that interacts with the stator and is directly involved in rotation of the motor. Here, we find that H-NS, well known for its ability to organize DNA, acts in the flagellar motor to organize protein subunits in the rotor. It binds to a middle domain of FliG that bridges the core parts of the rotor and parts nearer the edge that interact with the stator. In the absence of H-NS the organization of FliG subunits is disrupted, whereas overexpression of H-NS enhances FliG organization as monitored by targeted disulfide cross-linking, alters the disposition of a helix joining the middle and C-terminal domains of FliG, and enhances motor performance under conditions requiring a strengthened rotor-stator interface. The H-NS homolog StpA was also shown to bind FliG and to act similarly, though less effectively, in organizing FliG. The motility-enhancing effects of H-NS contrast with those of the recently characterized motility inhibitor YcgR. The present findings provide an integrated, structurally grounded framework for understanding the roughly opposing effects of these motility regulators.

SUBMITTER: Paul K

PROVIDER: S-EPMC3194902 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Adjusting the spokes of the flagellar motor with the DNA-binding protein H-NS.

Paul Koushik K Carlquist William C WC Blair David F DF

Journal of bacteriology 20110902 21

The H-NS protein of bacteria is a global regulator that stimulates transcription of flagellar genes and that also acts directly to modulate flagellar motor function. H-NS is known to bind FliG, a protein of the rotor that interacts with the stator and is directly involved in rotation of the motor. Here, we find that H-NS, well known for its ability to organize DNA, acts in the flagellar motor to organize protein subunits in the rotor. It binds to a middle domain of FliG that bridges the core par ...[more]

PMID: 21890701

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Project description:UnlabelledA number of investigations of Escherichia coli have suggested that the DNA-binding protein H-NS, in addition to its well-known functions in chromosome organization and gene regulation, interacts directly with the flagellar motor to modulate its function. Here, in a study initially aimed at characterizing the H-NS/motor interaction further, we identify problems and limitations in the previous work that substantially weaken the case for a direct H-NS/motor interaction. Null hns mutants are immotile, largely owing to the downregulation of the flagellar master regulators FlhD and FlhC. We, and others, previously reported that an hns mutant remains poorly motile even when FlhDC are expressed constitutively. In the present work, we use better-engineered strains to show that the motility defect in a Δhns, FlhDC-constitutive strain is milder than that reported previously and does not point to a direct action of H-NS at the motor. H-NS regulates numerous genes and might influence motility via a number of regulatory molecules besides FlhDC. To examine the sources of the motility defect that persists in an FlhDC-constitutive Δhns mutant, we measured transcript levels and overexpression effects of a number of genes in candidate regulatory pathways. The results indicate that H-NS influences motility via multiple regulatory linkages that include, minimally, the messenger molecule cyclic di-GMP, the biofilm regulatory protein CsgD, and the sigma factors σ(S) and σ(F). The results are in accordance with the more standard view of H-NS as a regulator of gene expression rather than a direct modulator of flagellar motor performance.ImportanceData from a number of previous studies have been taken to indicate that the nucleoid-organizing protein H-NS influences motility not only by its well-known DNA-based mechanisms but also by binding directly to the flagellar motor to alter function. In this study, H-NS is shown to influence motility through diverse regulatory pathways, but a direct interaction with the motor is not supported. Previous indications of a direct action at the motor appear to be related to the use of nonnull strains and, in some cases, a failure to effectively bypass the requirement for H-NS in the expression of the flagellar regulon. These findings call for a substantially revised interpretation of the literature concerning H-NS and flagellar motility and highlight the importance of H-NS in diverse regulatory processes involved in the motile-sessile transition.

Project description:Phosphorylation of Escherichia coli CheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal peptide of the FliM motor protein (FliMN). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY ⋅ FliMN with and without D13K-Y106W have shown FliMN-bound CheY contains features of both active and inactive states. We used molecular dynamics (MD) simulations to characterize the CheY conformational landscape accessed by FliMN and D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D57 phosphorylation site and the FliMN interface, namely the closure of the α4-β4 hinge and inward rotation of Y- or W106 with W58. We used hydroxy-radical foot printing with mass spectroscopy (XFMS) to track the solvent accessibility of these and other side chains. The solution XFMS oxidation rate correlated with the solvent-accessible area of the crystal structures. The protection of allosteric relay side chains reported by XFMS confirmed the intermediate conformation of the native CheY ⋅ FliMN complex, the inactive state of free D13K-Y106W CheY, and the MD-based network architecture. We extended the MD analysis to determine temporal coupling and energetics during activation. Coupled aromatic residue rotation was a graded rather than a binary switch, with Y- or W106 side-chain burial correlated with increased FliMN affinity. Activation entrained CheY fold stabilization to FliMN affinity. The CheY network could be partitioned into four dynamically coordinated sectors. Residue substitutions mapped to sectors around D57 or the FliMN interface according to phenotype. FliMN increased sector size and interactions. These sectors fused between the substituted K13-W106 residues to organize a tightly packed core and novel surfaces that may bind additional sites to explain the cooperative motor response. The community maps provide a more complete description of CheY priming than proposed thus far.

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Adjusting the spokes of the flagellar motor with the DNA-binding protein H-NS.

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Adjusting the spokes of the flagellar motor with the DNA-binding protein H-NS.

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