Vol. 173, No. 23JOURNAL OF BACTERIOLOGY, Dec. 1991, p. 7615-76240021-9193/91/237615-10$02.00/0Copyright C 1991, American Society for Microbiology

Function of MglA, a 22-Kilodalton Protein Essential forGliding in Myxococcus xanthus

PATRICIA HARTZELLt AND DALE KAISER*Department ofBiochemistry and Department of Developmental Biology, Stanford University

School of Medicine, Stanford, California 94305-5307

Received 24 April 1991/Accepted 25 September 1991

Single mutations in the mgLA gene in Myxococcus xanthus render cells incapable of gliding. The mglA strainsare unique in that all other nonmotile strains of M. xanthus isolated are the result of at least two independentmutations in separate motility system genes. Translational fusions of trpE, or of lacZ, to mgLA were constructed,and the resulting fusion polypeptides were used to generate antibodies. Antibodies specific to MglA proteinwere purified. Antibody-tagged MglA was found localized to the cytoplasm of M. xanthus cells both byfractionation of cell extracts and by electron microscopy of thin sections of whole cells. Four of the five mglAmissense mutants tested failed to produce detectable levels of the MglA antigen in whole cell extracts.Nonmotile double mutants (A- S-), which have one mutation in a gene of system A and one mutation in a geneof system S, have the same phenotype as null mglA mutants but produce wild-type levels ofMglA protein. MgIAprotein is conserved in all strains of myxobacteria tested. The amino acid sequence of MglA protein includesthree sequence motifs characteristic of GDP/GTP-binding proteins. On the basis of its genetic properties,intracellular location, and amino acid sequence, it is argued that MglA protein is a regulator in the sequenceof functions leading to cell movement.

Gliding myxobacterial cells move smoothly along theirmajor axis and over a surface without employing flagella orother visible motor organelles (7, 11). Cell movements havebeen recorded in detailed time lapse motion pictures byReichenbach (26) and later in photographic studies (1, 16).Individual cells are seen to start, stop, restart, or reversedirection. Despite their ability to move as individuals, myxo-bacterial cells tend to move as groups. A raft of 5 to 10 cells,with their long axes parallel and in side-by-side contact,moves as a cohort (26). Circular swarms of several thousandcells migrate as loosely coherent masses over an agar surface(27). Masses of cells can emerge from the edge of a colonyeither as loose flares (13) or as dense elongate peninsulaswith no gaps between cells and often several cell layers thick(16). Parallel, ridgelike heaps of cells move as travellingwaves or ripples (23, 30).The myxobacteria, when starved for nutrients, enter a

developmental cycle in which a multicellular fruiting bodycontaining myxospores is constructed (31). To form thisfruiting body, nascent aggregates of many thousand cellsmust migrate to a new site (26). Cells must be motile toconstruct a fruiting body and to differentiate into myxos-pores within it (17-19). The organization evident withinthese group movements suggest that myxobacterial gliding iscoordinately regulated.

Organized gliding is controlled by at least 37 genes inMyxococcus xanthus. Mutations in six genes,frzA, -B, -CID,-E, -F, and -G, alter the frequency at which individual cellsreverse their direction of gliding (1). These genes specifyproteins that are related in amino acid sequence to thechemotaxis proteins (encoded by the che genes) of Esche-richia coli (21, 22). Another 31 loci belong to the A (adven-turous) and S (social) motility systems and appear to coor-

* Corresponding author.t Present address: Department of Microbiology and Molecular

Genetics, University of California, Los Angeles, CA 90024-1489.

dinate cell movements. Adventurous motile cells can movewhen they are far apart, whereas socially motile cells do notmove under these conditions (13, 14). Although a mutation inany gene of system A inactivates A motility, S motilityremains. Similarly, a mutation in any gene of system Sinactivates S motility but A motility remains (14). The rate ofexpansion of a circular swarm is highly dependent on itsinitial cell density, and the A- and S-motility systems re-spond to cell density in different ways (16). The rate ofexpansion of A-motile swarms responds to changes at rela-tively low cell densities. In S-motile swarms, the expansionrate responds to changes at low cell density and continues torespond to changes at densities that are 10-fold higher thanthe saturating density for A-motile swarms. Wild-type (A'S+) cells combine the cell density responses of the A and Ssystems, but nonadditively: (i) the rate of swarm expansionof wild-type cells responds to low and moderate cell densi-ties, but no further changes occur at higher densities; and (ii)the maximum rate of expansion in wild-type cells exceedsthe sum of the maximum rates of A and S motility. Further-more, A- S- double mutants are nonmotile and do notrespond to changes at any cell density.One gene is required for both A and S motility in M.

xanthus. Mutations in mglA (for mutual gliding function) arethe only single mutations yet isolated in M. xanthus that giverise to totally nonmotile cells (13, 14). Null mglA mutants arenonmotile during growth, and they fail to carry out any of theaggregation movements required for fruiting body develop-ment (19, 33). A' S+ mglA mutants are nonmotile as are A-S- mglA+ cells. The absolute requirement of mglA formotility has prompted this investigation into the function ofits protein product. We have isolated and purified MglApolypeptide fusions and induced an immune response inrabbits to the fusion proteins. Antibodies shown to bedirected against MglA protein have been used to determinethe distribution of MglA protein among motility mutants ofM. xanthus, other species of myxobacteria, and other gliding

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TABLE 1. Strains and plasmids used

Strain or plasmid Relevant genotype Source or reference

StrainsCytophaga johnsonae U67 Fully motile J. PateFlexibacter columnaris IR43 Fully motile J. PateMicroscilla sp. Fully motile J. PateStigmatella aurantiaca Fully motile S. InouyeDictyostelium discoideum Wild type J. SpudichEscherichia coli MC1061 Maniatis et al. (20)Myxococcus virescens Fully motile H. ReichenbachMyxococcus xanthusDK1622 A+ S+ Kaiser (15)DK6204 AmglB AmglA Hartzell and Kaiser (10)DK7501 A+ S+ TnS-lac H. KimseyDK4052 mglAl Stephens and Kaiser (33)DK4050 mglA4 Stephens and Kaiser (33)DK4154 mglA5 Stephens and Kaiser (33)DK4155 mglA7 Stephens and Kaiser (33)DK4141 mglA9 Stephens and Kaiser (33)DK4140 mglA10 Stephens and Kaiser (33)DK4156 mglAll Stephens and Kaiser (33)DK4166 A+ S+ mglA:TnSf26 Stephens and Kaiser (33)DK1245 agiGI sgl-45 Hodgkin and Kaiser (13, 14)DK1250 agiBi tgl-l Hodgkin and Kaiser (13, 14)DK1251 agiJI tgl-2 Hodgkin and Kaiser (13, 14)DK1252 cglFl tgl-3 Hodgkin and Kaiser (13, 14)DK1259 aglBI sglGl Hodgkin and Kaiser (13, 14)DK1260 aglR4 sgl-60 Hodgkin and Kaiser (13, 14)

PlasmidspKNS281 2.8-kb BamHI-PvuII mgl in pUC18 from pKNS264 Karen Stephens

(Stephens et al. [32])pPLH300 1.75-kb BamHI-SalI from pKNS281 into pUC18 This workpUR289 lacZ Ruther and Muller-Hill (28)pPLH401 mglA-lacZ gene fusion This workpATH2 trpE Dieckmann and Tzagoloff (8)pPLH415 mglA-trpE gene fusion This work

organisms and the location of MglA protein within M.xanthus.

MATERIALS AND METHODS

Amino acid abbreviations. Standard three-letter and one-letter abbreviations for the amino acids are used: A, Ala,alanine; C, Cys, cysteine; D, Asp, aspartate; E, Glu, gluta-mate; F, Phe, phenylalanine; G, Gly, glycine; H, His,histidine; I, Ile, isoleucine; K, Lys, lysine; L, Leu, leucine;M, Met, methionine; N, Asn, asparagine; P, Pro, proline; Q,Gln, glutamine; R, Arg, arginine; S, Ser, serine; T, Thr,threonine; V, Val, valine; W, Trp, tryptophan; Y, Tyr,tyrosine.

Strains and culture conditions. Strains and plasmids arelisted in Table 1. Cells were cultured in CTT broth or on agarplates (12). The Cytophaga, Flexibacter, and Microscillacells were cultured in HTC broth containing, per liter, 4.0 gof tryptone, 0.5 g of yeast extract, 0.5 g of beef extract, and0.2 g of sodium acetate (pH 7.3). For plate-grown cultures,HTC was supplemented with 1.5% Difco agar.pKNS281 contains the BamHI-PvuII mgl fragment from

pKNS264 described by Stephens et al. (32). pPLH300 wasgenerated by ligation of a 1.75-kb BamHI-SaIl fragment frompKNS281 into a BamHI-SalI fragment of pUC18.A translational fusion of mglA to lacZ was constructed

by inserting a 537-bp XhoI-HindIII mglA fragment frompPLH300, eluted from 0.7% low-melting-point agarose (Sea-Plaque) after electrophoretic separation, into the Sal-

HindIII site within the lacZ gene of the pUR289 vector (a giftfrom Randy Scheckman, at University of California, Berke-ley [28]). This construction generated a fusion gene in whichmglA comprised the 3' end and lacZ was at the 5' end (Fig.1). The original E. coli promoter and ribosome binding site ofthe pUR289 vector remained intact. The resulting plasmid,pPLH401, was transformed into E. coli LC137 and main-tained at 30°C.

pPLH300706 mgM Ius

Hrn

120 17

FIG. 1. Construction of plasmids for lacZ-mglA (right) and trpE-mglA (left) translational fusions. B-gal, 3-galactosidase.

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A second translational fusion was constructed by intro-ducing the 3' end of the mglA gene into the trpE gene in thepATH2 vector (8). A 531-bp XmaI-HindIII mglA fragmentfrom pPLH300 was inserted into the Sma-HindIII region ofthe trpE gene in the pATH2 vector (Fig. 1). The trpE-mgIA-containing plasmid, pPLH415, was used to transform E. coliHB101.

Expression and purification of fusion polypeptides.pPLH401 was grown in E. coli LC137 at 30°C in LB brothwith carbenicillin (100 ,ug/ml). To allow overproduction ofthe LacZ-MglA fusion polypeptide, gene expression wasinduced by addition of isopropyl-3-D-thiogalactopyranoside(IPTG; Boehringer Mannheim) to a final concentration of 2mM. 5-Bromo-4-chloro-3-indolyl-p-D-galactopyranoside (X-Gal; Boehringer Mannheim) was added to a final concentra-tion of 0.02% to monitor production of ,B-galactosidase. Cellswere grown for 3 to 5 h, until the cultures were blue (opticaldensity at 550 nm = 2.0) in the presence of IPTG, andsubsequently were harvested by centrifugation at 12,000 x gat 4°C. The P-galactosidase-MglA fusion polypeptide wascompared with the 3-galactosidase protein from the religatedparent vector, pUR289, on 7% sodium dodecyl sulfate(SDS)-polyacrylamide gels to determine that a fusion poly-peptide of the predicted molecular mass was produced.

Overexpression of the TrpE-MglA fusion polypeptidefrom E. coli HB101 harboring plasmid pPLH415 wasachieved by addition of 33-indoleacrylic acid (final concen-tration, 20 ,ug/ml; Sigma) to an exponential-phase culture(optical density at 660 nm = 0.2) growing in M9 salts medium(20) containing carbenicillin (100 ,ug/ml). At 5 h after induc-tion, the cells were harvested and the pellet was suspendedin 0.1 volume of TEN buffer (50 mM Tris-HCl [pH 7.5], 0.5mM EDTA, 0.3 M NaCl). Lysozyme was added to a finalconcentration of 1 mg/ml, and the mixture was incubated onice for 15 min. Nonidet P-40 (Sigma) was added to a finalconcentration of 0.2%, and the mixture was left on ice anadditional 10 min. DNase I buffer was added to a finalconcentration of 0.15 M NaCl-1.2 mM MgCl2, and DNase I(Sigma) was added to a final concentration of 50 ,ug/ml. Afterincubation on ice for 1 h, the insoluble material, approxi-mately 90% of which was the fusion polypeptide, wascollected by centrifugation at 5,000 x g at 4°C. The pelletwas washed in TEN buffer and solubilized upon addition of2% SDS and 4 M urea. An aliquot was added to an equalvolume of SDS-sample buffer and boiled for 5 min. Thismaterial was separated by electrophoresis in 12% SDS-polyacrylamide gels (2).The fusion protein bands were visualized by staining with

cold 0.25 M KCl and 1 mM dithiothreitol for 1 h and thendestaining with 1 mM dithiothreitol. Bands corresponding tothe fusion polypeptides were excised from the gels and dicedinto small pieces. The gel slices were lyophilized and subse-quently ground to a powder with a mortar and pestle. Thefinely ground material was dissolved in phosphate-bufferedsaline and mixed, by sonication, on ice for 2 min withadjuvant (monophosphoryl lipid A plus trehalose dimycolateemulsion; RIBI Immunochemicals, Hamilton, Mont.) andused to immunize rabbits.Western immunoblot analysis. Proteins, after SDS-poly-

acrylamide gel electrophoresis (PAGE) separation, weretransferred to nitrocellulose (0.45-,um pore size; Bio-Rad,Richmond, Calif.) by electrophoresis, using the Transblotapparatus (Bio-Rad) equipped with a cooling coil, at 90 V for2 h. The transfer buffer, prepared according to the manufac-turer's instructions, contained 20% methanol, 20 mM Trismabase, and 0.15 M glycine. The transferred proteins were

visualized by staining with 0.2% Ponceau S in 3% trichloro-acetic acid and photocopied for future reference. The nitro-cellulose blot was blocked in 3% gelatin in TBS buffer (500mM NaCl, 20 mM Tris, pH 7.5) for 1 to 2 h at roomtemperature (approximately 25°C) with gentle rotation. Theblot was washed three times with TTBS (TBS with 0.05%Tween 20) for 5 min each. The blot was then incubated witha 1:1,200 dilution of primary antibody (anti-MglA immuno-globulin G [IgG]) in 1% gelatin with TTBS for 2 to 6 h atroom temperature. This was followed by three 5-min washeswith TTBS prior to addition of a 1:2,000 dilution of second-ary antibody (goat anti-rabbit conjugated to alkaline phos-phatase; Bio-Rad) in TTBS with 0.4% gelatin and 0.1%bovine serum albumin. Secondary antibody was incubatedwith the blot at room temperature for 2 to 4 h. The blot waswashed three times with TTBS buffer (5 min each) and thenthree times with 0.15 M Tris-HCl (pH 9.6) (5 min each). Thealkaline phosphatase was visualized by development at 37°Cwith a solution containing 0.15 M Tris-HCl (pH 9.6), 4 mMMgCl2, 0.1 mg of p-nitroblue tetrazolium chloride per ml,and 50 mg of 5-bromo-4-chloro-3-indolylphosphate (U.S.Biochemical) (3) per ml.

Cell fractionation. M. xanthus DK7501, a strain that pro-duces P-galactosidase during vegetative growth, was used sothat P-galactosidase would be available as a protein andenzymatic marker of cell fractions. For comparison, cellfractions were prepared from both liquid and plate-growncultures of M. xanthus. After the initial harvest of cells,fractionations were done in parallel with liquid-grown andplate-grown cultures. Liquid-grown cultures were harvestedat a cell density of 4 x 108 cell per ml and centrifuged at8,000 x g for 10 min at 15°C. To obtain a more homogeneoussuspension of cells grown on plates, 0.5 ml of M. xanthuscells grown in liquid CTT to a density of 5 x 108 cells per mlwas spread on CTT agar plates and allowed to grow at 33°Cfor 24 h. The cells were harvested by scraping the growthfrom plates and washing the cells in TPM (10 mM Tris, 1 mMpotassium phosphate, 8 mM magnesium sulfate, pH 7.5).The cell fractionation procedure of Orndorff and Dworkin(24) was then used for liquid- or plate-grown cells with thefollowing modifications. Cells were harvested in 50 mM Tris(pH 7.5) and centrifuged at 16,000 x g for 15 min. The pelletwas suspended in 0.25 volume of 0.75 M sucrose-10mM Trisacetate (pH 7.9). Lysozyme and EDTA were added to finalconcentrations of 2 mg/ml and 0.5 mM, respectively. Allbuffers contained a mixture of the following protease inhib-itors: leupeptin (0.7 ,ug/ml), pepstatin (0.5 ,ug/ml), phenazinemethyl sulfonyl fluoride (0.2 mM), benzamidine-HCl (0.5mM), and EDTA (1 mM) (mixed protease inhibitor). Thereaction was incubated on ice for 1 h and then centrifuged at21,000 x g for 10 min at 5°C. The pellet was suspended in 2volumes of cold water and incubated on ice. After 1 to 2 h,greater than 95% of the cells had formed phase-dark sphero-plasts and the preparation was centrifuged at 16,000 x g for10 min. The pellet was suspended in 1 volume of 50 mMEDTA (pH 8.0) and incubated on ice. Lysis usually occurredwithin 10 to 30 min. When lysis was complete, as determinedby microscopic examination, the material was centrifuged at280,000 x g for 10 min at 5°C. The supernatant containedcytoplasmic components, and the pellet contained inner andouter membrane components. The membrane fraction was

solubilized in 0.5 volume of 5% SDS-4 M urea.Purification of anti-MglA antibody. Serum from an immu-

nized rabbit was treated as follows to purify the anti-MglAIgG component. Serum was first treated with a saturatedammonium sulfate solution at pH 7 to give a final concen-

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tration of 40% ammonium sulfate. Precipitated material wasdialyzed and passed through a protein A-Sepharose CL-4Bcolumn (Sigma) equilibrated with 50 mM Tris (pH 8.6) and150 mM NaCl. The IgG fraction of interest eluted with buffercontaining 50 mM glycine (pH 2.3) and 150 mM NaCI andwas immediately treated with 3 M NaOH aliquots until thepH was 7.2. The IgG material was passed through twoaffinity columns to separate the anti-TrpE (or anti-3-galac-tosidase) IgG components from the anti-MglA IgG. The firstaffinity column contained a Reacti-Gel HW-65 (Pierce Chem-ical Co., Rockford, Ill.) resin, coupled according to themanufacturer's instructions, to a fraction highly enriched forTrpE protein (or j-galactosidase). A second Reacti-Gelcolumn was prepared in the same fashion but was coupled tothe partially purified TrpE-MglA (or ,-galactosidase-MglA)fusion polypeptide. Material for both columns, TrpE orTrpE-MglA, was isolated from the insoluble fraction ob-tained after overexpression of the TrpE protein or theTrpE-MglA fusion polypeptide in E. coli as described ear-lier. Mixed protease inhibitor was added as described ear-lier. Both columns were equilibrated with 50 mM sodium N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES;pH 7.5) with 50 mM NaCl and 1 mM EDTA (buffer A).Antibody elicited against the fusion protein was first passedthrough the affinity column conjugated to the TrpE proteinonly to remove IgG molecules which recognize the TrpEregion of the fusion. Anti-MgIA IgG was detected in thematerial which failed to bind to the TrpE affinity column.This material was pooled and subsequently loaded on thesecond column containing the fusion polypeptide. Anti-MgIA IgG molecules, of different affinities, were eluted fromthe fusion affinity column with three washes: (i) buffer Awith 4.5 M MgCl2, (ii) 100 mM diethylamine (pH 11.5) with150 mM NaCl and 1 mM EDTA, and (iii) 200 mM glycine(pH 2.5) with 150 mM NaCl and 1 mM EDTA. Material inthese three fractions was pooled and dialyzed against 50 mMTris (pH 7.5) with 150 mM NaCl. An additional chromato-graphic step was used to remove material which reacted withcontaminating M. xanthus proteins and contributed to back-ground. For this purpose, the anti-MgiA fraction was passedthrough a Reacti-Gel column which had been coupled toproteins contained in a solubilized extract of M. xanthusDK6204, a strain in which the mgl coding region has beendeleted.Immunocytochemistry. DK7501 and DK6204 were grown

on CTT plates and harvested after 2 days to prepare samplesfor embedding in the hydrophilic resin LRWhite as describedby Wright and Rine (34), with the following modifications.The collected cells were centrifuged at 12,000 x g, and thepellet was suspended in 0.2 volume of 5x fixative (lxfixative is 1% glutaraldehyde plus 1% p-formaldehyde) for 30min at room temperature. The cells were centrifuged againand suspended in 0.1 volume of ice-cold 1 x fixative andwere incubated on ice for 30 min. Excess fixative wasremoved by three washes with 50 mM potassium phosphatebuffer (pH 7.5). A 15-min 1% meta-periodate oxidationreaction at room temperature was added to promote forma-tion of clumps of cells. Cells were then washed with 50 mMNH4Cl for 15 min at room temperature and subsequentlywashed twice with ultrapure water. Samples were dehy-drated and infiltrated with LRWhite resin (Ted Pella, LaJolla, Calif.) at room temperature with successive changes(10 ml each) of ethanol solutions as follows: 50%, 70%, 90%,95% (twice), and 100% (twice) (all 30 min each), ethanol-LRWhite (50:50) overnight, and 100% LRWhite (twice).During the dehydration steps, cells were centrifuged at 1,500

x g, and small clumps of cells were generated during thissequence. Clumps were placed in gelatin capsules, and thecapsules were filled with 100% LRWhite. Polymerizationwas accomplished by placing the capsules in a fitted temper-ature block at 45°C overnight with continued incubation at50°C for at least 48 h. Ultrathin sections were obtained byusing a Sorvall Porter-Blum ultramicrotome MT-2. Sectionswere placed on 400-mesh nickel grids (Ted Pella) which hadbeen coated with 0.25% Formvar in ethylene dichloride. Thethin sections were picked up on grids that had just beenexposed to glow discharge, using a Denton DV502 evapora-tor.MgIA antigen was stained in thin sections with the follow-

ing steps (at 25°C): (i) blocking with 2% albumin (fraction V)in PBST (140 mM NaCl, 3 mM KCI, 8 mM Na2HPO4, 1.5mM KH2PO4, 0.05% Tween 20) for 1 to 2 h at roomtemperature, (ii) primary antibody binding from a 10- to100-fold dilution in blocking solution for 2 h (or 12 h at 5°C),(iii) washing three times in PBST, (iv) secondary antibodybinding with goat anti-rabbit IgG conjugated to 10-nm goldparticles (Janssen, Piscataway, N.J.) diluted in blockingsolution to give A520 = 0.13, (v) washing three times inPBST, (vi) washing 10 times in ultrapure water, (vii) stainingwith 2% uranyl acetate for 10 min, and (viii) washing 10times in ultrapure water. Specimens were photographed atan electron-optical magnification of x23,000. Gold particleswere counted after placing a 1-cm2 grid over 4- by 5-in. plateimages.Sample preparation, protein assays, and PAGE. Protein

concentrations were determined by the Coomassie dye bind-ing assay (6); for dilute samples, the BCA assay was used asinstructed by the manufacturer (Pierce). Native and dena-turing gels for electrophoresis were prepared according tothe method of Blackshear (2). To reduce loss of protein byproteolysis, M. xanthus samples, to be used for Westernanalysis, were harvested from CTT agar plates in cold 20mM Tris (pH 7.5) and were sonicated for 10 s at 80% outputwith a Microson ultrasonic disruptor (Heat Systems,Farmindale, N.Y.) to break up clumps of cells. Aliquotswere immediately mixed with SDS-sample buffer (2 volumesof cell extract:1 volume of buffer) and placed in a boilingwater bath for 5 min.

RESULTS

Construction of fusion polypeptides and induction of mgUl-specific antibodies. To facilitate preparation of Mg1A proteinantigen, translational fusions of mglA to trpE and of mglA tolacZ were constructed as depicted in Fig. 1. Expression ofthese fusions can be induced in E. coli by the addition ofIPTG and indoleacrylic acid, respectively. (The mglA geneproduct will be referred to as MglA.) The molecular mass ofthe ,-galactosidase-MglA fusion product was predictedfrom the DNA sequence to be 138,000 Da, and an Mr-135,000polypeptide was found by PAGE (Fig. 2A). This fusionpolypeptide should contain 38% of the MglA protein startingfrom its carboxyl terminus. Two larger fusion polypeptidesthat contained 55 and 87% starting from the carboxyl end ofthe MglA protein fused to 3-galactosidase were found to beless stable, as judged by the amount of fusion polypeptidedetected in gel electrophoresis. However, a fusion polypep-tide to TrpE that contained 82% of the MglA protein(measured from its carboxyl end) was obtained. This fusionpolypeptide had a molecular mass of 54,000 Da, as estimatedfrom gel electrophoresis (Fig. 2B), close to the expectedvalue.

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A'.

Bgal-MgIA fusion _B-galactosldase _

_mw

; 205

TrpE-MgIA116

66

1 45

Lane 1 2 3

B: 6648

29& - il-.

17 LU'

Lane 1 2 3

_ TrpE-Mg9A fusion_ TrpE

FIG. 2. Expression of fusion polypeptides in E. coli. (A) 7%SDS-polyacrylamide gel separation of 3-galactosidase and the p-ga-lactosidase (Bgal)-MgIA fusion polypeptide from insoluble materialafter induction with IPTG. Lanes: 1, E. coli LC137 with pPLH401;2, E. coli LC137 with pUR289; 3, molecular weight (mw) standards(myosin, P-galactosidase, bovine serum albumin, and egg albumin;positions indicated in thousands). Arrowheads indicate positions of,B-galactosidase and the ,B-galactosidase-MgIA fusion polypeptide.(B) Separation of insoluble fraction containing the TrpE-MglAfusion polypeptide on a 12% SDS-polyacrylamide gel. Lanes: 1,molecular weight (mw) standards (bovine serum albumin, ovalbu-min, carbonic anhydrase, and lysozyme; positions indicated inthousands); 2, E. coli HB101 with pPLH415 after induction with3p-indoleacrylic acid; 3, E. coli HB101 with pATH2 after inductionwith 3p-indoleacrylic acid. Arrowheads indicate positions of theTrpE protein and the TrpE-MglA fusion protein.

To ensure that the fusion protein maintained a readingframe appropriate for translation of mglA, different trpE-mglA DNA fusions were constructed, and their proteinproducts were compared. Three mglA fragments were cut togenerate each of the reading frames and ligated into the SmaIrestriction site of pATH2 (8). Three different TrpE-MglAfusion polypeptides were observed. The observed and pre-dicted sizes (based on the trpE and mglA base sequences[32]) were Mr 59,500 (predicted = 60,298), 54,000 (predicted= 56,380), and 41,000 (predicted = 42,207). In parallel, asecond set of three fusions was generated by inserting aSmaI-HindIIl fragment of mglA into the SmaI-HindIII siteof pATH2, pATH11, and pATH23. The SmaI site in thesethree related vectors is offset by one nucleotide to yieldTrpE-MglA fusion polypeptides in each of the three readingframes. It was possible to choose the SmaI-HindIII fragmentof mglA to generate fusion polypeptides for the second setthat were predicted to be the same size as the first set, andindeed fusion polypeptides of the same electrophoretic mo-bility as set one were observed. Agreement between pre-dicted and observed in both sets of polypeptide fusionssupports the integrity of the published mglA reading frame(32) and ensures appropriate translation of mglA.The TrpE-MglA (82%) and ,3-galactosidase-MglA (38%)

fusion polypeptides of correct reading frame were excisedfrom polyacrylamide gels and each injected into rabbits to

Lane 1 2FIG. 3. Evidence that antibody to the p-galactosidase-MgIA

fusion polypeptide specifically recognizes the MglA portion of theTrpE-MglA fusion polypeptide. A Western immunoblot was probedwith affinity-purified antibody against the ,B-galactosidase-MglAfusion polypeptide. Lanes: 1, E. coli extract containing the TrpEpolypeptide from E. coli(pATH2); 2, extract containing the TrpE-MglA fusion polypeptide. The arrow indicates the position of theTrpE-MglA polypeptide.

elicit antibody production. The resulting polyclonal serawere affinity purified as described in Materials and Methods.To test whether antibodies elicited by the ,-galactosidase-MglA and TrpE-MglA fusion polypeptides were directedagainst MglA, each antibody preparation was also testedagainst the other fusion protein. Antibody to the ,-galactosi-dase-MglA fusion polypeptide was found to react with theTrpE-MglA fusion polypeptide and not with TrpE alone(Fig. 3). Similarly, the anti-TrpE-MglA antibody reactedwith the P-galactosidase-MglA fusion polypeptide and notwith P-galactosidase alone (data not shown).

Strain distribution of MgIA antigen. Extracts of M. xan-thus cells and of several motility mutants were separated onSDS-PAGE, blotted to nitroceilulose, and then exposed toaffinity-purified antibody. Antibody directed against theTrpE-MglA fusion reacted with a protein in extracts of thefully motile reference type M. xanthus strain DK1622,estimated by its electrophoretic mobility to have an Mr of-21,500 (Fig. 4). The molecular mass of the Mg1A protein,deduced from the DNA sequence, is 21,999 Da. No MglAantigen was detected in DK6204, an mglA deletion mutant(10).

Stephens et al. (32) have reported the nucleotide sequencealterations in a series of mglA mutants, and the mglAantigenic activity in these mutants was tested (Fig. 4). NoMglA antigen was detected in DK4166, containing a TnSinsertion at fQ26 in mglA, or in DK4050, carrying mglA4, amutation which results in termination after amino acid 6 dueto a C-to-A transversion yielding an ochre codon. DK4155mgIA7, with a missense mutation that changes cysteine 23 tophenylalanine, lacked the 22-kDa protein. The 22-kDa bandwas also absent from DK4154 mglA5, with a mutation thatchanges alanine 170 to glutamine. No MglA protein wasdetected in strains carrying mglAl, mglA9, and mglA1O,whose sequence changes remain to be determined. DK4156mglAII carries a missense mutation changing glycine 81 tovaline. It produces MglA protein in low but detectableamounts when cultures are incubated at 18°C instead of thetypical 33°C. Tiny projections (flares) of motile cells areoccasionally observed at the periphery of an otherwisesmooth colony edge produced by an mglAlI strain afterseveral days at 18°C. These flares are not due to geneticreversion because cells picked from the flares give rise to

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7620 HARTZELL AND KAISER

FIG. 4. Western immunoblot analysis of M. xanthus strains with anti-MgIA IgG. Lanes: 1, M. xanthus DK1622 (wild type); 2, DK6204(mgl null); 3, E. coli MC1061; 4, DK1252 (A- S-); 5, DK4052 (mglAl); 6, DK4050 (mglA4); 7, DK4154 (mglA5); 8, DK4155 (mglA7); 9,DK4141 (mglA9); 10, DK4140 (mglA1O); 11, DK4156 (mglAll); 12, f126, Tn5 insertion in mglA; 13, DK1622 (wild type); 14, prestainedmolecular weight (MW) standards (P-galactosidase, fructose-6-phosphate kinase, pyruvate kinase, fumarase, lactate dehydrogenase, andtriosephosphate isomerase; positions indicated in thousands).

nonmotile colonies (at 33°C) as does the original mglAJlstrain.Nonmotile strains that exhibit the same single cell behav-

ior, colony morphology, and developmental phenotype as domglA mutants result from a combination of two mutations ofthe A- S- type (13, 14, 19). Strains with a mutation in any Alocus (aglA-H or cglB-F) plus a mutation in any S locus (sglor tgl) are nonmotile (14), while single mutants retain eithersocial motility (A- S+) or adventurous motility (A' S-). Totest whether loss of motility in an A- S- strain might be dueto the lack of MglA protein, extracts of six geneticallydifferent A- S- strains were examined. Approximatelyequivalent amounts of MgIA antigen, relative to the A' S+mgl+ strain DK1622, were detected by Western immuno-blots (Fig. 4) and by enzyme-linked immunosorbent assay(ELISA) (Table 2).

If Mgl protein plays a general role in myxobacterialgliding, it should also be present in other species. Extracts oftwo other species of myxobacteria were examined, and a22-kDa protein antigen was detected in both of them, Myxo-coccus virescens and Stigmatella aurantiaca (Fig. 5).Among the large group of cytophagas, also gram-negativegliding bacteria, a cross-reacting band of Mr 42,000 wasdetected in Microscilla sp. and Flexibacter columnaris. Nocross-reacting material was detected in Cytophaga johnso-nae U67 or in amoebae of Dictyostelium discoideum.

Intracellular location of MgIA protein in M. xanthus. Be-cause M. xanthus moves on solid medium but cannot swimin cell suspension, cells were typically harvested from solidmedium in which they would have been able to glide. In suchcells, the bulk of MglA protein was found, by severalmethods, to fractionate as a soluble, cytoplasmic protein.When broken DK7501 cells, which had been disrupted bysonication or by passage through a French pressure cell,

TABLE 2. ELISA of MglA antigen in A- S- M. xanthus strains

Strain A420 %

DK1622 (A' S+ mgl+) 0.231 l0aDK1245 (agIGi sgl-45) 0.256 111DK1250 (aglBI tgl-J) 0.195 84DK1252 (cglFJ tgl-3) 0.221 96DK1259 (agiBi sglGJ) 0.206 89DK6204 (mgl deletion) 0.010 2

a Value set at 100% for comparison.

were sedimented for 15 min at 280,000 x g to bring downmembranes and their associated proteins, 85 to 95% of theMglA antigen was detected in the supernatant fluid. Todistinguish a cytoplasmic location from a periplasmic site,the cells were converted to spheroplasts by the method ofOrndorff and Dworkin (24) before lysis. ,-Galactosidase wasused as a protein marker for the cytoplasm. As proteindegradation was observed during this procedure, proteaseinhibitors were added at each step and the time of eachincubation was shortened from the published protocol. Byincreasing the concentration of lysozyme to 1 mg/ml, it waspossible to shorten the initial incubation from 12 h to lessthan 1 h. When cells were grown in liquid medium, no MglAprotein was detected in the culture medium after the cellshad been removed by centrifugation or in the first cell washfluid, even after these samples had been concentrated byAmicon ultrafiltration with a PM-10 filter (molecular weightcutoff of 10,000) (Fig. 6). Upon suspension of lysozyme-treated cells in water, spheroplasts were formed and approx-imately 10% of the total cellular protein (presumably theperiplasmic fraction) was released. MglA protein was notdetected in the released (periplasmic) material, and 5% of thetotal ,-galactosidase activity was detected in this material.To lyse the spheroplasts and to release their cytoplasmic

FIG. 5. Western immunoblot of anti-MgIA cross-reacting mate-rial from extracts of other gliding organisms. Lanes: 1, M. xanthusDK4141 (mglA9); 2, M. virescens; 3, F. columnaris; 4, Microscillasp.; 5, C. johnsonae U67; 6, S. aurantiaca; 7, D. discoideum; 8, M.xanthus DK1622 (wild type); 9, prestained molecular weight (MW)standards (see legend to Fig. 4). Twenty micrograms of protein wasloaded per lane. Positions of the 22-kDa MgIA protein (lower arrow)and 42-kDa cross-reacting material (upper arrow) are indicated. Theblot was probed with anti-MglA IgG directed against the p-galac-tosidase-MglA fusion polypeptide.

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FUNCTION OF MgIA IN M. XANTHUS 7621

MW

;. 116

.. ~~~~~ ~~~~~~~~~~~~~~..... _.........

-116

....... ......

id *t_ -26

Lane 1 --- 2 3 4 56 7 8

FIG. 6. Western analysis of cellular fractions prepared from M.xanthus DK7501. (A) 10%o SDS-polyacrylamide Coomassie-stainedgel of cellular fractions. (B) Immunoblot from a 7% SDS-polyacryl-amide gel probed with anti-l3-galactosidase (Janssen). The arrowindicates the position of P-galactosidase. (C) Immunoblot from a12.5% SDS-polyacrylamide gel probed with anti-MglA IgG. Thearrow indicates the position of MglA protein at 22 kDa. Positions ofmolecular weight (MW) standards are indicated in thousands. Lanes(same for all three panels): 1, whole cell extract of DK7501; 2, wholecell preparation after sucrose wash; 3, periplasmic material released(lower band appears to comigrate with lysozyme); 4, spheroplastpreparation; 5, 280,000 x g supernatant (cytoplasmic fraction); 6,280,000 x g pellet (membrane); 7, DK1622 whole cell control(wild-type M. xanthus lacking 3-galactosidase); 8, culture mediumfrom liquid-grown cells.

contents, the spheroplasts were suspended in a solution oflow osmolarity, 50 mM EDTA (pH 8.0). Greater than 80% ofthe total MglA protein was released by the ensuing lysis;92% of the total cellular 3-galactosidase activity was foundin this fraction. In the event that the EDTA treatment failedto lyse all of the spheroplasts, we subjected the 50 mMEDTA-treated samples either to sonication or to passagethrough a French pressure cell. No MglA protein was

detected in the material pelleted by high-speed centrifuga-tion after sonication or French press treatment.

Intracellular distribution of the MglA protein was exam-ined by immunoelectron microscopy of thin sections.DK7501, which expresses P-galactosidase in M. xanthusduring vegetative growth, was used as the wild-type strain sothat antibody to 3-galactosidase could be used as a control.Similarly, DK6204, a strain which is deleted for the mglAgene, was used as a negative control. As illustrated in Fig.7A, anti-MglA IgG reacted with antigen distributed in roughuniformity across the cell s,ctions. In 40 equivalent areas

from sections of DK7501, 290 gold particles were found.Without antibody to Mg1A, fewer than 45 gold particles werefound per 40 equivalent areas (Fig. 7C). Sections of DK6204,an mgl deletion mutant, had 68 particles per 40 equivalentareas (Fig. 7D). Gold-tagged anti-,-galactosidase labeling of

DK7501 thin sections also showed a roughly uniform distri-bution of gold particles across the cells (Fig. 7B); 418 goldparticles were observed per 40 equivalent regions.

DISCUSSION

Amino acid sequence comparison of MgIA and small GDP/GTP-binding proteins. Searching of a protein sequence database with the MglA sequence uncovered a 32% match over108 amino acid residues with the Sarl protein of Saccharo-myces cerevisiae described by Nakano and Muramatsu (23).The two polypeptides are of similar length, 190 residues forthe Sarl protein and 195 residues for MglA. The Sarl proteinbelongs to the class of small GDP/GTP-binding proteins, anda comparison of MglA with other members of this classrevealed G-1, G-3, and G-4 sequence motifs characteristic ofthese proteins (4, 5, 9) (Fig. 8). X-ray crystallography ofprotein p2lras, a member of the small G-protein family that isencoded by the c-H-ras gene, has shown that the G-1, G-3,and G-4 sequence motifs are parts of its guanine nucleotide-binding site (25). In the G-1 motif, MglA matches theconsensus. In the G-3 motif, Mg1A matches six of eightpositions. A highly conserved Gln (5) which occupies posi-tion 61 in p2lras and which would play a catalytic role in GTPhydrolysis, according to Pai et al. (25), occupies the equiv-alent position in MglA (Gln-82). Although the highly con-served aspartate residue of the G-3 consensus is absent fromMglA, a threonine residue, which is frequently found tofollow the missing aspartate (5), is present. In G-4, all eightresidues match the consensus (Fig. 8). Significantly, thespacing of the G-1, G-3, and G-4 motifs within the MglAsequence are the same as in established members of theGDP/GTP-binding superfamily (Fig. 8), consistent with ele-ments of secondary structure bringing components of thenucleotide-binding site into their proper spatial relations (4,5). When the assignments of G-1, G-3, and G-4 segments inMg1A are made as shown in Fig. 8, the three missense MglAmutants whose sequence alterations have been determined(mglA5, mglA7, and mglAJJ) fall within the putative nucle-otide-binding site of MglA.

Intracellular location of MgIA protein. Important for thedata presented here is the specificity of a polyclonal antibodydirected against MglA protein. The antiserum reacts withboth TrpE and ,-galactosidase fusions to MglA and identi-fies fusion polypeptides having the predicted sizes. Thisantiserum reacts with a 22-kDa protein in extracts of mglA+Myxococcus strains; 22 kDa is the size predicted by thenucleotide sequence of the mglA gene (32). The antiserumfails to react with extracts of a deletion mutant of mglA.MglA is highly conserved among myxobacteria, since allother motile strains of myxobacteria tested produced a22-kDa protein with MglA antigenic specificity.The antigen that binds this MgIA antibody is found in the

cytoplasmic fraction of Myxococcus cell extracts. Electronmicroscopy of immunogold-stained thin sections shows goldparticles scattered about the cytoplasm. Although smallportions of the mglA protein (up to 15%) were found innoncytoplasmic cell fractions, there was no indication ofassociation with an organized structure. MglA antigen wasfound in the same cell fractions as E. coli 3-galactosidase,which marks the cytoplasmic fraction of E. coli cells. Thepossibility raised by Stephens et al. (32) that Mg1A protein ispart of the structure that generates a translational glidingforce against the substratum now seems remote because ofthe observed dispersed cytoplasmic location of its antigen.

Proposed function of Mg1A. A more likely role for MgIA

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7622 HARTZELL AND KAISER

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FUNCTION OF MgIA IN M. XANTHUS 7623

FIG. 7. Immunocytochemical localization of the MglA protein in ultrathin sections of M. xanthus DK7501. (A) M. xanthus DK7501(x34,200) stained with anti-MglA primary antibody and goat anti-rabbit conjugated to 10-nm gold particles as secondary antibody; (B) A.xanthus DK7501 (x35,000) stained with anti-p-galactosidase primary antibody and goat anti-rabbit conjugated to 10-nm gold particles assecondary antibody; (C) M. xanthus DK7501 (x 35,000) stained with goat anti-rabbit conjugated to 10-nm gold particles only; (D) M. xanthusDK6204 (mgl null) (x35,000) stained with anti-MglA primary antibody and goat anti-rabbit conjugated to 10-nm gold particles as secondaryantibody.

that is consistent with a dispersed cytoplasmic location issuggested by the similarity of its amino acid sequence tosequences of the class of small GDP/GTP-binding proteins.The amino acid sequence of MglA includes three sequencemotifs separated from each other by a characteristic numberof amino acid residues. Key residues are present that woulddistinguish GTP from ATP: Asp-144 and Ala-170 of MgIAoccupy positions that are the functional equivalents ofAsp-119 and Ala-146 in p2lras. Present also is a phosphate-binding loop that includes MglA residues Gly-81 and Gln-82,which occupy positions functionally equivalent to Gly-60and Gln-61 of p2lras.The hypothesis that MglA protein is a GDP/GTP-binding

protein may help to rationalize the finding that mutantscarrying mgIA5, mglA7, and mglAJJ are missense yet pro-duce no detectable MglA antigen. Since the MglA antibodywas raised against denatured protein excised from an SDS-polyacrylamide gel and since the proteins tested with theantiserum were separated by SDS-PAGE, missense mutantproteins, if present, would have been expected to react evenif they were improperly folded. The mglAll mutant pro-duced a low level of antigen after growth at low temperature(18°C as opposed to 33°C), suggesting that this missenseprotein might be unstable; mglA7 might also be unstable. Ifnative MglA protein normally has guanine nucleotide boundto it, then the mglA7 mutation, which changes Cys-23 to Phe(C23F), would have altered the phosphate-binding loop; C23is the functional replacement in MglA of Val-14 in p21r"swhich provides its main chain amide to the phosphate-binding site (5). Similarly, mglA5 (A170E) changes theresidue corresponding to Ala-146 in p21ras, a conservedresidue in its guanine-binding site. Finally, in mglAJJ(G81V) corresponds to a change in the highly conservedGly-60 that is part of the DxAG consensus in G-3 and bindsthe y-phosphate of GTP in p21ras. Mutants in Gly-60 ofp2lras cannot switch between GDP and GTP states (25).Assuming that MglA is a GDP/GTP-binding protein, themglAS, mglA7, and mglAJJ alleles might have lost some orall of the capacity for nucleotide binding or exchange, which

Motif G-1

Consensus 4-15xooooGxxxxGKTx

MglA 14

NA KIVYYGPGLCGKTT

G-3 G-4

53-79 111-138xoJooDxAGjx xooooNKxDx A

73 136 170RFHLYTVPGQV YVIQYNKRDL A -C

FIG. 8. Comparison of the GTP-binding consensus site withcorresponding residues of MglA protein. x, any amino acid; o,hydrophobic amino acid (F, V, W, P, M, L, I, or A); j, hydrophilicamino acid (E, D, K, H, R, Y, S, G, Q, C, or N); *, location of a

missense mutation which produces the mglA phenotype. Consensussequences are from Bourne et al. (4), and the range is shown for thefirst residue position that corresponds to the consensus sequencesfor small GTP-binding proteins compiled from Bourne et al. (4). TheMgIA sequence is from Stephens et al. (32). Residue A146 in p2lrascorresponds to A170 in mglA.

might thereby create inactive and unstable protein mole-cules.Among more than 50 nonmotile mutants isolated by

Hodgkin and Kaiser (13), those altered in mglA were theonly ones that arrested gliding in one mutational step. Allother nonmotile mutants resulted from two mutations, onedefect in any gene of system A and a second in any gene ofsystem S. mglA is not a vital gene under laboratory growthconditions, since a TnS insertion mutation within it (32) or adeletion of it (10) is compatible with viability. Apart fromloss of motility and consequences secondary to that loss(19), no other change is evident in mglA mutants. The A- S-mutants produce wild-type levels of MglA protein (Table 2),but all have the same phenotype as mglA mutants (19). Thephenotypes of mglA and A- S- strains are identical not onlyin vegetative growth but also during fruiting body develop-ment. This identity suggests the possibility of a close func-tional relationship between mglA and both motility systems:mglA might act immediately before A and S in a way thatchanges input to both systems or immediately after A and Sin a way that takes output from both systems. p2lras has twoconformations associated with bound GDP and bound GTP(5). G proteins as a class are believed to function byswitching between these two conformations and possibly athird nucleotide-free state (4, 5). Many G proteins are knownto interact with proteins called GTPase-activating proteinsand guanine nucleotide release proteins which regulate andrespond to transitions between the two conformations (9).Constituents of the A and S systems might function asGTPase-activating or guanine nucleotide release proteins toMgIA.

It is remarkable that three missense mglA mutants wouldhave alterations around a putative nucleotide-binding siteeven though that site is constructed from residues drawnfrom widely separated regions of the polypeptide chain (Fig.8 [25]). Given the similarity of MglA to GDP/GTP-bindingproteins, this constellation of mutations suggests that gua-nine nucleotide binding to MglA protein is necessary forMgIA function and that, like other proteins of this class,MglA alternates between GTP- and GDP-bound states.These states could transfer energy to the gliding motors, orthey could be elements in a sensory transduction pathway,as suggested above. If the function of MglA immediatelyprecedes the functions of the A- and S-motility systems, thetwo states of MglA could trigger A or S motility. Alterna-tively, A and S could be two sensory pathways that respondto the presence of other cells as described in the introduc-tion. In this case, MglA could integrate the output from twosensory systems. Experimental attempts to distinguish thesepossibilities are in progress.

ACKNOWLEDGMENTS

We thank Paul Evans (Oregon State University) for suggestingthat the MglA protein might have a GTP-binding consensus site. Wealso thank Chris Kaiser (Massachusetts Institute of Technology) foridentifying MglA similarity to Sarl protein and Georganna Barnes(Stanford University) for technical assistance. We thank Yoshito

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7624 HARTZELL AND KAISER J. BACTERIOL.

Kaziro (DNAX, Palo Alto, Calif.) for help with the sequencealignment.

This work was supported by National Science Foundation grantDCB8903705 to D. Kaiser. P.H. was supported by a postdoctoralfellowship from the American Cancer Society.

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6. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

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