INTRODUCTION

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Motility in many bacterial species is achieved by rotating surface-exposed organelles called flagella. In Escherichia coli and Salmonella typhimurium about 50 genes are required for the biosynthesis and operation of these peritrichous, multicomponent structures. Gene expression is hierarchical, whereby one class of genes must be turned on before the next class can be expressed. At the top of this cascade lies the flhCD master operon, whose expression is required for the expression of all other flagellar genes (reviewed in Refs. 1 and 2).

Flagellar filament rotation is controlled by a motor embedded in the inner membrane (reviewed in Refs. 1 and 3). Energy to drive this motor is derived from the transmembrane gradient of protons, or proton-motive force (4, 5). Bacteria swim by a random walk consisting of a series of runs and tumbles (6). When the motor rotates counterclockwise (CCW),¹ flagella filaments form a tight bundle that propels the bacterium into a smooth swimming pattern. Conversely, during clockwise (CW) rotation, flagellar filaments separate, causing tumbling and directional reorientation (7).

The E. coli flagellar motor consists of a MotA-MotB stator complex which forms a transmembrane proton channel (8-10), and a rotor of three interacting proteins, FliG, FliM, and FliN (11, 12). All three rotor proteins are involved in the processes of flagellar assembly, switching, and rotation (13, 14). However, FliG is predominately involved in torque generation (12, 14, 15), whereas FliM mainly functions in switching rotor direction (16). The precise role of FliN is the least well defined, but it may participate in flagella protein-specific export and assembly (17, 18).

It has been shown that hns insertion mutations render bacteria non-motile, suggesting that H-NS is a positive regulator of flagellar gene expression (19, 20). H-NS is a 15.4-kDa nucleoid-associated DNA-binding protein (21-23) that modulates the expression of many unrelated genes in E. coli (24-26). In most instances, such as fimB (27) and proU (28) expression, H-NS acts as a direct transcriptional repressor.

Here we characterized two independent hns point mutations, hnsT108I and hnsA18E, in relation to E. coli motility. Rather than displaying a non-motile behavior, strains carrying these mutations exhibited an unprecedented hypermotility. This novel hypermotile phenotype was a result of enhanced binding of mutant protein H-NST108I to the flagellar rotor, causing increased flagellar rotational speed.

	EXPERIMENTAL PROCEDURES

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Bacterial Strains, Plasmids, Phage, Media, and Genetic Techniques-- Table I lists all bacterial strains, plasmids, and phage used. Media consisted of Luria-Bertani (LB) broth, LB agar, T broth (1% tryptone, 0.5% NaCl), and soft-agar (T broth, 0.3% agar) (Difco). Antibiotics were added to a final concentration of 100 µg (ampicillin), 20 µg (tetracycline), 20 µg (chloramphenicol), or 50 µg (kanamycin) per ml of medium. Isopropylthiogalactose was used at a final concentration of 1 mM. P1 vir generalized transductions were carried out as described previously (29). Cultures were grown at 37 °C for protein purification and 30 °C for motility and flagella assays.

Swarming and Motility-- AL127 was transformed with various plasmids. Fresh colonies were picked, inoculated onto low agar (0.3%) tryptone plates, and incubated at 30 °C. Swarm diameters were measured every 2-3 h, beginning 9 h post-inoculation. Differences in motility rates were calculated by comparing the swarm diameter of AL127 expressing an hns mutant plasmid relative to the same strain expressing a wild-type hns gene, on the same plate for three individual experiments.

Flagellation-- Strains were grown at 30 °C overnight in T broth with the appropriate antibiotics and then diluted 25-fold into fresh media and incubated until the A₆₀₀ reached 0.3-0.5. Cells from these cultures were incubated on polylysine-coated coverslips for 15 min at room temperature, rinsed twice with phosphate-buffered saline, and fixed in 3% glutaraldehyde, 0.1 M sodium phosphate buffer (pH 7.4). Bacteria-coated coverslips were dehydrated, critical point dried, attached to aluminum stubs, and sputter coated with gold:palladium. Bacterial flagella were visualized by scanning electron microscopy (Cambridge Stereo Scan S200, LEO Electron Microscopy, Inc.). Flagella were counted on at least 10 fields per strain.

Tethering Experiments-- Tethering for the flagellar rotational bias and speed experiments was performed as described previously (30), with the following modifications: diluted cultures were grown without inducer until the A₆₀₀ reached 0.4-0.8; flagella were sheared with a Tissue Tearor model 985-370 (Biospec Products, Inc.); and a 1:100 dilution of anti-flagella antibody (36) was used. Bacteria were observed by dark field microscopy, recorded to videotape, played back at 1/5 the speed, and scored using The Observer 3.1 videotape analysis system software (Noldus Information Technology). Typically, 30 individual cells were quantitated for 30 s each for CCW bias or 1 min each for flagellum rotational speed.

Protein Purifications-- E. coli wild-type H-NS and H-NST108I were each purified as described before (27) except the mutant protein lysate was washed from the double stranded DNA-cellulose column at a lower salt concentration (125 versus 250 mM NaCl) than the wild-type cell lysate. His-tagged FliG was nickel-affinity purified basically by the method of Toker and Macnab (31) from BL21(DE3) cultures harboring pET28NdefliG. Protein solutions were concentrated with Centricon-10 columns as instructed by the manufacturer (Amicon). Protein concentrations were measured with the Bio-Rad Dc protein assay kit (Bio-Rad).

Fluorescence Anisotropy-- H-NS was labeled with the thiol-reactive probe, fluorescein 5-maleimide, after reduction with tris-(2-carboxyethyl)phosphine hydrochloride, according to the manufacturer's instructions (Molecular Probes). Labeled protein was separated from excess probe by G-25 Sephadex protein spin column purification (Boehringer Mannheim). Anisotropy was measured at excitation and emission wavelengths of 490 and 515 nm, respectively. Data was collected on a LS 50B Luminescence Spectrometer with FL Data Manager software (Perkin-Elmer). The K_d was calculated as the FliG concentration corresponding to one-half the maximum anisotropy value on the H-NS-FliG binding curve.

Chemical Cross-linking-- FliG was first attached to the heterobifunctional cross-linker, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Pierce) by incubating for 1 h at room temperature. Reactions were quenched by the addition of 1 M Tris (pH 7.5), H-NS (wild-type or T108I) was added, and the entire reaction was incubated another hour. The cross-linking reactions were terminated by the addition of 5× Laemmli denaturing sample buffer. Reactions containing equal amounts of each protein (5 µM) were electrophoresed on a 4-20% denaturing gradient gel (Jule Biotechnologies, Inc.) and visualized by Coomassie or Sypro-Orange (Molecular Probes) staining. Complexes were quantitated by volumetric integration on a Gel-Doc 1000 with Molecular Analyst 2.1 software (Bio-Rad). To ensure that we added equal amounts of wild-type H-NS and H-NST108I to FliG, we also quantitated free, uncomplexed H-NS protein.

Antibody Production and Western Blotting-- A C-terminal 6xHis-H-NS fusion protein was expressed from pDMG1 and purified under denaturing conditions according to the manufacturer's instructions (Qiagen). An emulsion of TiterMax (Vaxcel) and 300 µg of 6xHis-H-NS was used to inoculate and boost New Zealand White rabbits. Anti-H-NS antiserum obtained was used at a 1:5000 dilution. Anti-FliG antiserum was kindly provided by David Blair (University of Utah, Salt Lake City, UT) and used at a 1:7000 dilution. H-NS-FliG cross-linked complex reactions were run on 12% SDS-polyacrylamide gel electrophoresis, transferred, probed with antiserum, and detected as described previously (27).

	RESULTS

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Effect of hns Mutations on E. coli Motility-- H-NS is a global regulator of E. coli gene expression (reviewed in Refs. 32 and 33). Mutations in hns have pleiotropic effects on the cell altering synthesis of a variety of gene products involved in numerous, diverse biological pathways (24-26). In particular, transposon insertions in hns result in the loss of bacterial motility due to the lack of master operon flhCD expression (20). We isolated a set of random hns mutations² and determined their effect on motility. Plasmids carrying either no insertion (pACYC vector alone), a wild-type hns gene (pTHK116), or hns point mutations T108I (pMASS46-1), or A18E (pMASS73-4) were separately introduced into AL127, an E. coli hns2-tetR (34) insertion mutant background strain (Table I). Transformants were inoculated onto semi-solid agar plates and motility was determined by measuring the diameter of the bacterial swarm over time. Motility was classified into three distinct categories based on swarm size (Fig. 1). Strains lacking H-NS were non-motile. Motility was restored in these strains upon the addition of a wild-type hns clone in trans. However, the hnsT108I and hnsA18E mutations each conferred a greater than 2-fold increase (2.1 ± .21 and 2.5 ± .17, respectively) in swarm rate as compared with the wild-type strain. These data represent the first instance in which any E. coli mutation, hns or otherwise, resulted in a hypermotile phenotype.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Swarm plate assay. Fresh colonies from strains carrying the indicated alleles were inoculated onto semi-solid agar plates and grown at 30 °C. Growth was measured as the diameter of the bacterial swarm over several time points. Bacteria with swarm diameters under 10 mm at the end of 17 h incubation were considered non-motile. Data representative of three individual experiments. *, vector; , hns2-tetR; , wild-type hns; , hnsT108I; , hnsA18E.

Flagellation-- H-NS is a positive transcriptional regulator of the flhCD operon, the master operon which controls expression of all other flagellar components (1). In the absence of H-NS, flagellar genes are not expressed resulting in the loss of intact flagella and motility (20). One explanation for the hypermotile phenotype that we observed with specific hns point mutations was that these alleles affected flagella biosynthesis and/or assembly. To test this hypothesis, we examined strains expressing different hns alleles by scanning electron microscopy (SEM). Visually, there was no discernible difference in cell size or shape or flagellum length or distribution between strains carrying a wild-type hns allele (Fig. 2A) or either hns mutation (Fig. 2, B and C). There was also no statistical difference in the average number of flagella/cell between the control and hnsA18E strains and only a very slight increase expressed from the hnsT108I allele (Table II). We concluded that the increased motility rates exhibited by the hnsT108I and hnsA18E alleles were not due to an alteration in the number or construction of functional flagella.

View larger version (103K): [in this window] [in a new window]   Fig. 2.   Scanning electron micrographs. AL127 (hns2-tetR) carrying (A) wild-type hns (pTHK116), (B) hnsT108I (pMASS46-1), or (C) hnsA18E (pMASS73-4). Magnification, × 5000; scale bar, 10 µm.

Flagellar Rotational Behavior-- E. coli swim by rotating their flagella filaments (35, 36) either CCW or CW (7). Motion is an alternating series of smooth runs (CCW rotation) and abrupt directional changes called tumbles (CW rotation) (6). It is possible that the hnsT108I and hnsA18E mutations caused a shift in flagellar rotational bias resulting in hypermotility. Strains bearing hns mutant alleles may "run" (versus tumble) a higher percentage of the time than their wild-type counterparts. To address this issue, we performed tethering experiments (30) to compare the rotational bias of strains containing wild-type or mutant hns genes. In this assay, individual cells harboring plasmid-based hns alleles were tethered to a microscope slide via an anti-flagella antibody. The spinning direction of cell bodies was observed and bias was calculated to be the proportion of the time cells spent rotating CCW (Table III).

Flagellar Speed-- Flagella propel bacteria by rotating motor-driven helical filaments (35, 36) whereby swimming speed is directly related to flagellar rotational speed (39). Free-swimming bacteria can transverse 20-60 µm/s in liquid media (40), whereas tethered bacteria can rotate their cell bodies 2-9 revolutions/s (36). We postulated that the hnsT108I and hnsA18E mutations caused hypermotile bacterial swimming behavior by directly increasing the speed at which individual flagella rotated. Once again, we employed bacterial tethering to measure rotational speed using a host strain (AL128) containing a cheA-cheZ deletion as well as an hns2-tetR mutation. This strain enabled us to survey cells that were all rotating in one direction, CCW, without the complications of switching. Rotational speed data (Table IV) were accumulated from cells of the same size and tethered at the same point to maintain similar drag coefficients, overall cell geometry, and load. The controls (Table IV, lines 1 and 2) displayed similar rotational speeds regardless of the location of hns (chromosome- or plasmid-based). In contrast, the addition of either hns point mutation in trans (lines 3 and 4) resulted in significant (p < .005 and p < .025, respectively) increases in the speed of the tethered cell body. We concluded that hnsA18E and hnsT108I accelerated flagellar speeds 44-62% (Table IV) over wild-type levels without affecting flagellar assembly (Fig. 2 and Table II) or switching (Table III). This direct effect on flagellum rotational speed likely caused the original swarm plate hypermotile phenotype displayed by these hns mutant alleles.

H-NS-FliG Binding via Fluorescence Anisotropy-- Flagellar rotation is driven by a reversible rotary motor anchored in the inner membrane at the base of the flagellum (reviewed in Refs. 1 and 3). The motor consists of a stator (MotA and MotB) and a rotor (FliG, FliM, and FliN). The three rotor proteins interact with each other (11, 12) to form a switch complex peripherally attached to the inner membrane, facing the cytoplasm (41-43). All three proteins are involved in flagellar assembly, switching, and rotation (13, 14). Since FliG is the motor protein most involved in speed and torque generation (12, 14, 15), we examined the binding between H-NS and FliG in fluorescence anisotropy assays. This technique quantitates protein-protein interactions by measuring the change in anisotropy of a fluorescently labeled protein upon the addition of a second unlabeled protein (44).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Fluorescence anisotropy of H-NS-FliG interactions. Increasing amounts of FliG were added to fluorescein-labeled H-NS. Ten anisotropy values were measured at each FliG concentration and averaged. Graph is representative of two separate experiments.

H-NS-FliG Binding via Chemical Cross-linking-- Since wild-type H-NS bound FliG in the anisotropy studies (Fig. 3), we wanted to determine if the mutant H-NST108I also bound FliG. A difference in binding affinities between FliG and wild-type or mutant H-NS protein might account for the hypermotile swarm phenotype seen with the mutant. We compared binding capabilities of each of these proteins to FliG by chemical cross-linking.

View larger version (54K): [in this window] [in a new window]   Fig. 4.   H-NS-FliG cross-linked complexes. Reactions were incubated at room temperature and equal amounts were electrophoresed on denaturing polyacrylamide gels. A, Coomassie-stained 4-20% SDS-polyacrylamide gradient gel; B, 12% SDS-polyacrylamide gel transferred to nitrocellulose, and probed with H-NS antiserum; C, second half of gel in B probed with FliG antiserum. Protein standard sizes are indicated by lines; lmw, low molecular weight markers; hmw, high molecular weight markers; protein monomers, dimers, and H-NS-FliG complexes are indicated by arrows. Reactions for all panels: 1, wild-type H-NS only; 2, H-NST108I only; 3, FliG only; 4, wild-type H-NS with cross-linker; 5, H-NST108I with cross-linker; 6, FliG with cross-linker; 7, 1:1 molar ratio of wild-type H-NS to FliG with cross-linker; 8, 1:1 molar ratio of H-NST108I to FliG with cross-linker.

	DISCUSSION

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Hypermotility-- We studied the effect of two hns point mutations, hnsT108I and hnsA18E, on E. coli motility. In swarm plate assays, we observed that each of these hns mutations bestowed a hypermotile phenotype (Fig. 1). Swarm rates for strains carrying the hns mutant alleles were approximately twice as fast as strains with the wild-type hns allele in trans. Previously characterized mutations in genes involved in flagella functions have led to three basic mutant phenotype classifications: non-flagellated (Fla) (15), non-rotating flagella ("paralyzed", Mot) (14), and skewed chemotaxis, either CCW-biased (14) or CW-biased (46). In terms of swarming, mutations typically cause a decrease in motility (47). To the best of our knowledge, hnsT108I and hnsA18E are the first documented mutations that cause a 2-fold increase in swarm rates.

H-NS-FliG Interactions-- Since our hns mutations seemed to be specifically affecting flagellar speed, we investigated the possible interaction between H-NS and the protein complex that controls rotation. Of the three proteins that make up the rotor portion of the motor, we chose to test FliG for the following reasons. First, FliG is situated at the cytoplasmic face of the inner membrane attached to the embedded MS ring through interactions with FliF (41-43). This location allows free access to cytoplasmic H-NS. Second, FliG is presumably part of the active flagellar rotating unit along with the MS ring, rod, hook, and filament (3, 12, 51). Thus, H-NS binding directly to a portion of the moving apparatus could potentially alter speed. Furthermore, FliG functions primarily in flagella rotation and torque generation (12, 14, 15) rather than assembly or switching. Finally, there is previous genetic data indicating that fliG- and hns-encoded fusion proteins interacted in a yeast two-hybrid system (11).

Models-- No existing model sufficiently accounts for the unexpected effects of H-NS on flagella rotation. Thus, we present a speculative hypothesis of H-NS action (Fig. 5) based on the data presented here as well as previous investigators results (10-12, 31, 41). We postulate that H-NS is involved in torque generation through its interactions with FliG. Given the fact that (i) hns mutations resulting in the hypermotile phenotype specifically affected flagellar rotational speed, (ii) speed, torque, and presumably proton-motive force are all directly related (39, 55), and (iii) the C-terminal domain of FliG functions specifically in torque generation (15), we position H-NS at the interface between the rotor and stator, directly linked to the C terminus of FliG (Fig. 5A). Tighter binding of mutant H-NST108I to FliG (Fig. 5B) may cause increases in flagellar speed by altering the conformation of FliG relative to the other rotor proteins and/or the MotA·B complex, thus, compacting the motor complex and allowing faster rotation by creating less friction within the surrounding stationary MotA·B ring complex (56). Alternatively, the movement or rate of proton flux through this channel could also be affected, or the H-NS-FliG complex may play a regulatory role by altering expression of genes downstream of fliG in the flagellar assembly cascade.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Model of H-NS activity at the flagellar rotor. A, wild-type H-NS interactions with FliG; B, proposed conformational changes induced by the binding of H-NST108I to FliG. PD, peptidoglycan; IM, inner membrane. See "Discussion" for details.