The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait . MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation . Genetic studies showed thataglZ is part of the A motility system, because disruption ordeletion of aglZ abolished movement of isolated cells and aglZsglK double mutants were nonmotile . The aglZ gene encodes a153-kDa protein that interacts with purified MglA in vitro.The N terminus of AglZ shows similarity to the receiver domainof two-component response regulator proteins, while the C terminuscontains heptad repeats characteristic of coiled-coil proteins,such as myosin . Consistent with this motif, expression of AglZin Escherichia coli resulted in production of striated latticestructures . Similar to the myosin heavy chain, the purifiedC-terminal coiled-coil domain of AglZ forms filament structuresin vitro.

 
Myxococcus xanthus has a complex life cycle . In the presence of adequate nutrients, the cells undergo vegetative growth and divide, but when the cells are starved of nutrients they aggregate and form fruiting bodies containing myxospores . When nutrientsbecome available, myxospores can germinate into vegetative cells.

Gliding motility of M . xanthus requires a solid surface and is controlled by two sets of genes: S [social] genes, which predominantly control movement of groups of cells [47], and A [adventurous] genes, which predominantly control movementof single cells [17] . Both S and A motility are involved in vegetative swarming and developmental aggregation . A mutation that inactivates either an A gene or an S gene reduces, butdoes not abolish, gliding [18] . However any combination of A^– and S^– mutations abolishes gliding, revealing that A and S mechanisms of motility are not only different but also independent.

Cells lacking S motility can still move as single cells by using the A system and form colonies with lace-like flares of individual cells at the edges . Various studies have shown that M . xanthus social motility requires type IV pili, extracellular matrixfibrils, lipopolysaccharide [LPS], and FrzS, a response regulator-coiled-coil hybrid protein [6, 9, 42, 45] . Cells lacking A motility canstill move in groups using the S system and form colonies withruffles of closely grouped cells at their edges . Recent studieshave shown that the A motility system may be powered by secretionof a polyelectrolyte through polar nozzle-like structures [46].

The A and S motility systems are adapted for optimal movementin different environments . Mutants with only A motility [A⁺ S^–] glide like the wild-type strain over 1.5% agar surfacesbut move more poorly than the wild-type over 0.3% agar . Theopposite is true for A^– S⁺ strains, which move more poorlyover 1.5% agar but behave like the wild type on 0.3% agar . Whenmutant cells are viewed by videomicroscopy, A⁺ S^– cellscan move as single cells, but A^– S⁺ cells need to be near other cells to move.

Single mutations in only one known M . xanthus gene, mglA, preventboth A and S motility [16] . Colonies formed by mglA mutants,like those of A^– S^– double mutants, do not spreadand have a sharp edge without ruffles or flares . The mglA geneencodes a 22-kDa Ras-like GTPase that is essential for glidingand development, but not for growth [15, 38].

MglA does not appear to be involved directly in the mechanism[s] of gliding . Although colonies formed by mglA mutants are indistinguishablefrom the colonies formed by A^– S^– double mutants,time-lapse studies of individual cells have shown that mglAmutants reverse direction 17 times more often than cells ofthe wild-type strain [2.9/min for mgl versus 0.17/min for DK1622][36] . Hence, they are capable of gliding but are incapable ofmaking net movement . mglA mutants appear to produce the components,such as pili, fibrils, and polyelectrolyte, that are requiredfor A and S motility [39; R . Otto and P . Hartzell, unpublisheddata] . Hence, the role of MglA may be to coordinate the twogliding motility systems or regulate the frequency with whichcells reverse direction while gliding.

If MglA regulates the two motility systems, it is likely to interact with a component of each system to undertake differentroles in cell motility . Recently, our investigators showed thatMglA interacts with a tyrosine kinase that is required for Smotility and development in M . xanthus [39] . In this study, we report on another protein, AglZ, that interacts with MglA and is required for A motility.

 
Strains, plasmids, and media. Strains, plasmids, and oligonucleotides used in this study arelisted in Tables 1 and 2 . Escherichia coli DH10B and JM109 wereused for the construction and maintenance of plasmid DNA andfor the amplification of library DNA . E . coli cells were grownat 37°C in Luria broth supplemented with ampicillin [100 µg/ml] or kanamycin [40 µg/ml] where applicable.M . xanthus strains were grown at 32°C in medium containing1% Casitone, 10 mM Tris, 1 mM potassium phosphate, and 5 mMMgSO₄ [final pH, 7.5; CTPM medium] [43] supplemented with 40µg of kanamycin/ml where applicable . M . xanthus developmentalassays were performed on TPM medium . Restriction enzymes werepurchased from Gibco BRL, Promega, and New England BioLabs.Saccharomyces cerevisiae strain PJ69-4A was grown in yeast extract-peptone-adenine-dextroseand synthetic complete [SC] minus medium . Chemicals were purchasedfrom Sigma-Aldrich.

 
Yeast two-hybrid screen. A library containing chromosomal DNA from M . xanthus was preparedin pGAD-C1, pGAD-C2, and pGAD-C3 vectors as described elsewhere[39] . Yeast transformations were performed using the quick andeasy method [13] . For transformations of the library, plasmidDNA [0.1 µg for 1-fold transformation, 1.0 µg for10-fold transformation] was used to transform yeast using thehigh-efficiency method [1] . Efficiencies were determined onSC minus Leu and Trp and varied from 500,000 to 1,100,000 colonies/µgof DNA . The three different translational reading frames ofthe library were transformed separately, in triplicate, intoPJ69-4A containing pAGS145 [GBD-mglA] . Colonies that grew onSC minus Leu, Trp, and His were replica plated onto SC minusLeu, Trp, and Ade medium, as described elsewhere [22] . Coloniesthat grew on medium lacking adenine were tested further for ß-galactosidase activity . Plasmid DNA was recoveredfrom colonies that satisfied three conditions: [i] growth onSC minus Leu, Trp, and His; [ii] growth on SC minus Leu, Trp,and Ade; and [iii] ß-galactosidase activity.

Recovery of plasmid DNA from yeast. Colonies that contained the pGAD plasmid and a gene encodinga protein that interacted with pGBD-mglA were isolated, andplasmid DNA, named pAGS152, was recovered . The isolated plasmidswere electroporated into E . coli DH10B cells and selected onLuria broth-ampicillin . Sequence analysis showed that pAGS152contained a 784-bp insert of M . xanthus DNA in pGAD . To confirmthe protein-protein interaction, the unknown gene in pGAD wasreintroduced along with pGBD-mglA into PJ69-4A and plated onSC minus Leu, Trp, His, and Ade . The M . xanthus DNA insert inpGAD plasmids, which were confirmed to carry fusions that interactwith GBD-MglA, was recovered by PCR using primers GADF and GADR.The PCR product was cloned into pCR2.1 and sequenced . Sequencewas obtained using an ABI Prism sequencing apparatus with primersthat complemented the forward and reverse regions flanking theinsert.

Because the 784-bp insert was recovered from a reaction thatused the pGAD-C2 library, we could predict the reading framefor the gene . Based on this, we concluded that the 784-bp fragmentwas missing the 5' and 3' ends of the gene . Sequence upstreamof the 784-bp fragment was recovered from the Monsanto MicrobialGenome database of the M . xanthus genome . The 5' end of thefragment aligned with the end of the 6,633-bp Monsanto contigMYX10C862, but no match was found for the 3' end of the fragment.

To obtain sequence downstream of the 784-bp fragment, the scheme shown in Fig . 1 was employed . Plasmid pAGS152 was digested withHindIII and EcoRI to liberate the 784-bp fragment, which was ligated with HindIII- and EcoRI-cut pBGS18 to generate pAGS164. Plasmid pAGS164 was introduced into the chromosome of M . xanthus DK1622 [wild type] by electroporation . Of the 78 Kan^r colonies that were isolated, all exhibited an A^– S⁺ colony phenotype.One representative colony was selected and named M . xanthusMxH2223 . Genomic DNA was isolated from strain MxH2223 by usingthe Easy DNA protocol [Invitrogen] and digested with SacI at 37°C for 12 h . The enzyme was heat inactivated at 65°Cfor 30 min and then dialyzed against 1,000 volumes of distilledwater on a 0.025-µm-pore-size filter [Millipore] for 30min . The DNA was then diluted 1:20, incubated with T4 DNA ligaseat 25°C for 12 h, dialyzed, and introduced into E . coliDH10B by electroporation . A plasmid, pAGS160, containing about15 kb of chromosomal DNA flanking the original fragment wasrecovered from Kan^r colonies . A 2-kb fragment predicted by restrictionmapping to contain DNA upstream and downstream of the knownsequence was subcloned into pBSG18 to yield pAGS163 . pAGS163was used as probe to recover a larger clone, named pMR, froma -ZAP library of M . xanthus DNA . Plasmid pMR contained 6,000 bp of sequence downstream of the 784-bp fragment . When compared with the Monsanto Microbial Genome [Cereon] database, we founda match with contig MYX10C1267 . The sequences of contig MYX10C862 [partial], pAGS163, pMR, and MYX10C1267 [partial sequence] wereassembled as a 10,581-bp fragment by using Vector NTI software.The fragment recovered from the original two-hybrid clone correspondedto an internal region of a 4,188-bp gene . Because disruptionof this gene abolished A motility, the gene was named aglZ.The sequence and predicted products of aglZ and flanking regionswere analyzed using Frameplot 3.0ß [21] and BLAST[2].

 
Transductions. The generalized transducing phage Mx8 was used to transfer theaglZ disruption into different genetic backgrounds [27] . Phagelysates of M . xanthus MxH2224 cells were prepared as describedelsewhere [27] . Immediately prior to transduction, an aliquotof the phage lysate was irradiated with UV light for 1 min ina UV Stratalinker 1800 [Stratagene] and then mixed with therecipient strain [wild-type and aglU and sglK mutant strains]for 20 min at 25°C and plated on CTPM supplemented withkanamycin . Plates were incubated for 4 to 6 days at 32°Cuntil colonies arose.

Construction of an aglZ deletion mutation in M . xanthus. Strain MxH2265 was constructed to carry a deletion in the coding region for aglZ . Plasmid pMR26 was digested with NruI, which cleaves at bp 5904 and 8563 in AY487937 [bp 1108 and 3767 ofaglZ], to eliminate a 2,659-bp internal fragment of aglZ . Thelarge fragment, containing vector sequence plus 1,107 bp ofthe 5' end of aglZ and 418 bp of the 3' end of aglZ, was purifiedand ligated to yield pRY3 . The 1.6-kb EcoRI fragment from pRY3was purified and cloned into EcoRI-digested pBJ114, which carriesgalK and nptII genes, to make pRY4 . pRY4 was introduced intoDK1622 by electroporation, and Kan^r colonies were selected. Recombination of plasmid pRY4, which carries galK, nptII, and aglZ, with chromosomal aglZ in the motile strain DK1622 wasused to generate a Kan^r merodiploid, MxH2254 . MxH2254 was grownon CTPM medium without selection to 5 x 10⁸ cell/ml and then diluted 1:50 in fresh medium every other day for a period of2 weeks . After >10 generations, 100-µl aliquots wereplated on CTPM medium with 2% galactose to promote excisionof the integrated plasmid caring galK and allele exchange [40].About 30% [121 of 400] of the colonies exhibited a defect inA motility . These colonies were transferred to CTPM plates withoutantibiotic and incubated at 32°C for 3 days . ChromosomalDNA from four Kan^s A^– S⁺ colonies was prepared for further analysis . The PCR was performed using oligonucleotide primersA and B [Table 2] to confirm the presence of aglZ deletion. Amplification of the aglZ gene from the wild-type strain yielded a 4.2-kb product, whereas amplification of the aglZ gene from two of the four A^– S⁺ strains yielded only a 1.6-kb product,showing that 2.6 kb of the aglZ fragment had been removed [datanot shown] . Southern analysis was used to confirm these results[32] . Primer 319, which anneals with a region 529 bp upstreamof the ATG start in aglZ, and primer 320, which anneals witha region 171 bp downstream of the ATG start of aglZ, were usedin the PCR to make a 700-bp hybridization probe by using theNEBlot Phototope kit [New England BioLabs] . Chromosomal DNAfrom the wild-type parent and A^– mutants, digested withBspEI, confirmed the presence of a 2.66-kb aglZ deletion inMxH2265.

Phenotypic analysis. The spreading rate was determined by the method of Shi and Zusman[34] as follows . M . xanthus strains were grown to a densityof 5 x 10⁸ cells/ml in CTPM medium at 32°C, harvested bycentrifugation, and suspended in TPM buffer to 5 x 10⁹ cells/ml.In triplicate, 5 µl of concentrated cells was spottedonto CTPM plates containing either 1.5 or 0.3% agar, and colonydiameters were measured at 24-h intervals.

Time-lapse analysis of cell motility was performed by photographing cells spread on a thin layer of 1.5% CTPM . In each case, roughly 10 µl of a dilute cell suspension [10⁷ cells/ml] was placed on the thin layer of agar in the middle of the chamber slide. Motility also was assayed for cells treated with methylcellulose, which has been shown to suppress certain motility defect phenotypes[19] . Cells on the chamber slide were overlaid with 150 µlof a solution containing 1% methylcellulose and 0.1% pyruvate.Images were collected at 1-min intervals for 20 min with a KodakDC-290 digital camera attached to a Nikon FXA labphot-2 microscopeand assembled to video using Quicktime 6.0 and NIH Image programs.

The aglZ mutant was compared with the wild-type strain to ascertain the presence of cellular components known to be critical for gliding . The production of extracellular fibrils and pili bythe aglZ mutant was examined by immunoblot analysis using monoclonalantibody 2105, which reacts with the zinc metalloprotease FibAin fibril material [7, 23], and polyclonal antibody againstPilA [48], respectively . LPS [O-antigen] and fibrils also wereassayed by gel electrophoresis and Congo red binding, respectively,as described elsewhere [3, 9].

Development assays were performed by plating several spots containing 10⁷ cells on TPM starvation agar plates and incubating at 32°C for 120 h [24] . After 5 days, the plates were incubated at 50°Cfor 2 h to destroy vegetative cells, harvested in buffer, and sonicated briefly to break clumps . The number of heat-resistant spores was determined by counting the number of colonies produced after allowing spores to germinate on rich medium [CTPM agar].

Expression of aglZ. The aglZ gene was cloned into the expression vector pET24b toyield an aglZ-his6 fusion as follows . The aglZ gene was amplifiedfrom pMR26 with primers 302 and 303, and the 4.2-kb productwas cloned into pCR2.1 to yield pRY1 . The aglZ gene was removedby digesting pRY1 with BamHI and HindIII and cloned into pET24bto yield pSMB1 . The plasmid pSMB1 was subsequently electroporatedinto the induction host E . coli BL21-A1 and into wild-type strainDK1662 and aglZ strain MxH2265 to yield MxH2274 and MxH2275,respectively.

Expression of aglZ was induced upon addition of isopropyl-ß-D-thiogalactopyranoside [IPTG] and arabinose to 1 mM and 1%, respectively, to 200 mlof E . coli BL21-A1 cells at an optical density at 600 nm of0.4 . After 3 h, cells were harvested by centrifugation, suspendedin 1 ml of 10 mM Tris [pH 7.4], and passed twice through a Frenchpressure cell at 18,000 lb/in² . Cell extract was prepared by centrifuging the lysed material at 20,000 x g for 30 min . Thesupernatant was passed over a Talon resin that had been equilibratedwith phosphate-buffered saline [PBS] . After washing the columnwith PBS buffer, AglZ-His was eluted with 150 mM imidazole inPBS buffer . All buffers were at pH 7.

To amplify the coiled-coil region of aglZ, primer 335, which anneals with DNA at a site 732 bp downstream from the ATG start in aglZ, and primer 303, which anneals with DNA at the 3' end of aglZ, were used in the PCR . The PCR product was cloned into pCR2.1 and subcloned into pET24b to yield pRY15 . Expressionof this truncated version of aglZ, called aglZ-coil, in E . coli DE3 cells was induced with 1 mM IPTG . The 130-kDa AglZ-coilwas purified by the same method used to purify the full-lengthprotein described above.

Complementation analysis. To determine if the aglZ gene is able to complement the A^– phenotype of strain MxH2265 [aglZ], plasmid pSMB1, which carriesan aglZ-his6 fusion, was introduced into MxH2265 by electroporationto make strain MxH2275 . Because MxH2265 retains about 1,108bp of the 5' end of the chromosomal copy of the aglZ gene, homologousrecombination can occur between the chromosome and the aglZgene on pSMB1 . Transformants were selected on CTPM plates containing40 µg of kanamycin/ml . Production of AglZ-His was confirmedby immunoblot analysis using anti-His antibody.

Electron microscopy. To determine if AglZ might form a structure in vivo, E . colicells expressing AglZ-coil were fixed with 2% [vol/vol] glutaraldehydeand 2% paraformaldehyde in 0.1 M sodium cacodylate buffer [pH7.2] for 2 h at room temperature and subsequently washed withthe same buffer . The samples were then posttreated in 2% [wt/vol]osmium tetroxide overnight at 4°C, dehydrated in acetone,embedded in Spurrs resin [Polyscience], and cut into thin sectionsusing a ultramicrotome.

A sample of purified AglZ-coil was stored at 4°C for severaldays, during which time a precipitate formed . The precipitatewas removed carefully and diluted in distilled water to a proteinconcentration of about 30 µg/ml . A 3-µl aliquotwas applied to carbon-coated grids, left for 1 min, and blottedto near dryness before staining for 1 min with 0.5% uranyl acetateat pH 5.0 . All samples were examined using a JEOL JEM-1200 transmissionelectron microscope operating at an accelerating voltage of100 keV.

Immunoblot analysis. To examine expression of aglZ-his in M . xanthus, immunoblotsof extracts from M . xanthus strains MxH2274 and MxH2275 bearingaglZ-his fusions were probed with antibody . AglZ-His fusionprotein was detected by primary anti-His mouse monoclonal antibodyand goat anti-mouse secondary antibody conjugated with horseradishperoxidase [HRP] . The samples were developed using the ECL detectionkit [Perkin-Elmer] according to the manufacturer's instructions.

Protein cross-linking. Protein cross-linking was performed based on the proceduresdescribed previously, with modifications [14, 35] . A 10-µlaliquot of 3 µM affinity-purified AglZ-His fusion proteinand 10 µl of 3.8 µM affinity-purified MglA-His proteinwere mixed with 1% formaldehyde or 10 mM dimethyl pimelimidate[Pierce] . Controls contained 3 µM bovine serum albumin [BSA; Sigma] in place of MglA-His . Following a 60-min incubation at 23°C, the samples were mixed with sodium dodecyl sulfate[SDS] sample buffer and separated by SDS-10% polyacrylamidegel electrophoresis [SDS-PAGE] . Proteins were transferred topolyvinylidene difluoride membranes and probed with anti-MglAprimary antibody and anti-rabbit secondary antibody conjugatedwith HRP . HRP was assayed as described above.

Dot-far Western blotting analysis. The dot-far Western blotting method of Ohba et al . [30] wasperformed with the following modifications . Aliquots containing3 and 6 pmol of purified MglA-His fusion protein [or BSA forcontrols] were absorbed to nitrocellulose . A small amount ofAglZ-His fusion protein [0.3 pmol] was also spotted on the membraneas a positive control . When the samples were dry, the membranewas blocked for 1 h at 23°C with 5% [wt/vol] skim milk,washed in PBS-T [10 mM Na-PO₄, 150 mM NaCl, 0.05% Tween 20;pH 7.5], and then incubated with 2 ml of PBS-T containing 1.5µM AglZ-His fusion protein at 23°C for 1 h . The membranewas washed three times with PBS-T and probed with anti-AglZ diluted 1:10,000 in 5% skim milk-PBS buffer for 1 h at 23°C. After washing three times with PBS, the membrane was probedwith anti-rabbit secondary antibody conjugated with HRP anddeveloped as described above.

Nucleotide sequence accession number. The 10,581-bp sequence of the aglZ gene and flanking DNA hasbeen deposited in GenBank as accession number AY487937.

 
Isolation of the aglZ gene, which encodes a protein that interacts with MglA. To find proteins that interact with MglA, mglA was fused withthe GAL4 binding domain to generate a translational fusion andused as bait with a pGAD library containing random fragmentsfrom M . xanthus in the yeast two-hybrid system . Yeast that carriedGBD-MglA and an interacting clone from the GAD-C2 library expressingGAD-X were identified as colonies that grew on medium lackinghistidine and adenine in a strain engineered to express HISand ADE from GAL4 promoters . Plasmids pGAD-X and pGBD-mglA wereisolated from yeast and recovered in E . coli . Plasmids pGAD-Xand pGBD-mglA were used to retransform a naïve PJ69-4Ayeast host together and with controls pGBD and pGAD, respectively.Growth on medium lacking His or Ade occurred only when GAD-Xwas paired with GBD-mglA . Plasmid pGAD-X was named pAGS152.Confirmation of the interaction was shown by assaying the reciprocalpair of plasmids . Yeast cells carrying pGAD-mglA and pGBD-Xgrew on medium lacking adenine, whereas cells with pGAD-mglAplus pGBD or pGAD plus pGBD-X failed to grow.

The sequence of pAGS152 contained a 784-bp insert, but the gene that was in frame with GAD lacked an apparent stop codon . Torecover additional sequence, the scheme shown in Fig . 1 was employed . A derivative of pAGS152, pAGS164, was introduced intoM . xanthus and integrated onto the chromosome . Yellow, Kan^r colonies carrying pAGS164 exhibited S motility, but not A motility[Fig. 2C], suggesting that the gene recovered from the yeast two-hybrid is part of the A motility system . Hence, the name aglZ [for adventurous gliding Z] was assigned to this gene. Sequence analysis of plasmid pAGS163, which carries sequenceflanking the pAGS164 chromosomal insertion, yielded the 5' endof the aglZ gene and additional sequence downstream but appearedto lack the 3' end of the gene . To obtain the 3' end of aglZ,a 2-kb fragment of aglZ from pAGS163 was used as probe to isolatepMR from an M . xanthus -ZAP library . Sequence analysis of pMR26,which was subcloned from pMR, yielded the 3' end of aglZ . ContigsMYX10C862 and MYX10C1267 from the Cereon Microbial Genome database[Monsanto] each aligned with a part of the sequence . The assembledsequence has been deposited in GenBank.

 
aglZ is essential for A gliding. To confirm that aglZ is a gene of the A motility system, theaglZ disruption in MxH2224 was transduced into strains thatcarry mutations in known A and S motility genes and the phenotypesof double mutants were analyzed . As shown in Fig . 2D, the aglZaglU double mutant had an A^– S⁺ gliding phenotype like that of its aglU parent, whereas the aglZ sglK double mutant,MxH1139 pAGS164, had an A^– S^– [nonmotile] glidingphenotype [Fig . 2F] . These results confirmed that aglZ is partof the A motility system.

Because of concern that the phenotype might reflect polar effects on downstream genes due to the integration of pAGS164, a strain carrying a markerless deletion of aglZ was made . Recovered colonies exhibited either a normal or a small-colony phenotype . DNA from two colonies with the small-colony phenotype was used as templatein the PCR and for Southern analysis to confirm the presenceof the engineered aglZ deletion and the absence of the parentalcopy of aglZ . Southern analysis revealed bands at 2.3 and 6.5kb, which hybridized with the probe in the wild-type strain[DK1622], and bands at 2.3 and 3.9 kb in the deletion mutant,which subsequently was named MxH2265 . These data showed thatMxH2265 is a null mutant for aglZ.

The colony phenotype of the MxH2265 mutant was identical withthat of strain MxH2223, which carries an insertion in aglZ inthe wild-type background . These data suggested that the lossof A motility is not due to an effect on genes downstream ofaglZ . Furthermore, there is a 650-bp gap between the 3' endof aglZ and the next gene, making the notion of a second genein the operon unlikely . Strain MxH2265 was used for more detailedmotility and complementation assays.

Although the aglZ mutant possessed S motility, as evidenced by the groups of motile cells at the edge of the colony shownin Fig. 3B, the mutant showed defects in spreading on both 0.3 and 1.5% agar surfaces [Fig . 3D] . This was surprising, becausemutants that lack A motility typically show reduced spreading on 1.5% agar but are able to spread on 0.3% agar, as S motility is functional under these conditions . The motility defect of aglZ was characterized further by time-lapse video microscopy.During a 30-min period, isolated cells of the wild-type parentalstrain DK1622 moved two to three cell lengths [about 25 to 30µm] in the same direction . In contrast, isolated cellsof the aglZ mutant MxH2265 did not show any movement under theseconditions, even on 1% methylcellulose . Hence, although aglZis essential for A motility, loss of aglZ also reduces motilityon soft agar, where S motility predominates.

 
To determine if disruption of aglZ alters the production of components known to be required for S motility, extracts ofwild-type and mutant cells were assayed for PilA and FibA, indicatorsof pili and fibrils, respectively . Wild-type and aglZ cellshave similar amounts of the 27-kDa PilA in whole-cell extracts and in sheared pilus preparations [data not shown] . In contrast, aglZ cells produce an elevated amount of FibA in the whole-cellextract compared to the wild type . Profiles of the aglZ mutant resembled those of the wild-type strain on acrylamide gel assays[for O-antigen, LPS] and Congo red binding for fibril material[data not shown] . The slight increase in FibA may account forthe reduced capacity for movement on soft [0.3%] agar.

The aglZ mutant [MxH2265] was assayed for developmental defectsin nutrient-free TPM agar plates as described in Materials andMethods . The mutant strain produced dark fruiting bodies similarto those of the wild-type strain . After 5 days, spore sampleswere heated at 50°C for 2 h and then transferred to CTPMrich medium plates to stimulate the germination of heat-resistantspores . The aglZ mutant produced a wild-type complement of heat-resistantspores.

The motility defect of aglZ can be complemented by an aglZ-his6 fusion. The plasmid pSMB1, which carries aglZ-his6, was electroporatedinto the M . xanthus MxH2265 [aglZ] mutant strain, and Kan^r colonieswere selected to yield MxH2275 . Plasmid pSMB1 contains onlythe aglZ gene and can integrate onto the chromosome by homologousrecombination with the 5' end of aglZ or the 3' end of the aglZ gene on the chromosome . Only recombination between the 5' end of aglZ and the plasmid-borne aglZ could generate a functionalcopy of aglZ with its promoter . Since a larger portion of the5' region of aglZ remains on the chromosome than the 3' end,we anticipated that a greater number of recombinants would resultfrom recombination with the 5' end of aglZ . Consistent withthis, two different motility phenotypes were found among theKan^r colonies derived from electroporation of pSMB1 into the aglZ mutant . Forty-nine of the 74 transformants produced coloniesthat were identical to the fully motile wild-type strain, andthe remaining transformants were similar to the aglZ parent.Motility and development assays of the fully motile transformantswere performed alongside the wild-type strain and the aglZ parent.As shown in Fig . 3C, numerous single cells could be seen emanatingfrom the edge of the colony, in contrast with the aglZ parentin Fig . 3B, which was devoid of single cells . Hence, the aglZ-his6fusion complemented the A motility defect of the aglZ mutant.Moreover, as shown in Fig . 3D, the ability of MxH2275 to spreadon 0.3 and 1.5% CTPM motility agar surfaces was identical tothat of the wild type.

aglZ encodes a protein related to type 2 myosin. The deduced aglZ gene product, AglZ, is predicted to be a 1,395-amino-acid protein with an expected molecular weight of about 153,600.The N-terminal 120 amino acids contain residues that match theconsensus for the REC signal receiver domain family [cd00156;9e–11] of proteins that includes FrzS, CheY, and OmpR[Fig . 4C] . FrzS, like AglZ, is a protein required for motilityof M . xanthus [42] . The strongest match was between AglZ andPhoB of Vibrio cholera [AAC25063.1] . The N termini of thesetwo proteins share 34% identity and 49% similarity [4e–07].AglZ lacks the Asp53 residue that typically is the site of phosphorylationin response regulators . However, like Mycobacterium tuberculosisresponse regulator MprA [52], the aspartate residue at position48 of AglZ may be a target for phosphorylation.

 
Following the N-terminal response regulator domain is a 120-residue proline-rich [15 proline residues of 120] linker domain . The isoelectric point for this proline-rich region is predictedto be 10, while the regions before and after this region havea pI of about 4.5 . The linker precedes a 1,048-amino-acid domainthat is devoid of proline residues . Analysis of this regionusing the algorithm MULTICOIL [45] indicated a strong tendencyto form a coiled-coil structure, with a propensity for dimerformation [Fig. 4D] . As shown in Fig . 4B, the predicted coil structures extended over greater than 80% of the protein . This domain included multiple matches with the myosin class II heavychain [KOG0161; 1e–11].

The aglZ fragment originally recovered from the yeast two-hybrid clone encodes a part of AglZ that corresponds to these two domains. When translated, the two-hybrid fragment yielded 193 amino acids, of which 73 residues overlapped with the proline-rich regionand 120 residues overlapped with the beginning of the coiled-coilregion.

A comparison of the translated 1,395-amino-acid sequence of aglZ with known proteins using BLAST [2] revealed 21% identityand 43% similarity with 467 residues of myosin heavy chain isoformA of Loligo pealei [AAC24207.1] and 27% identity and 49% similaritywith 226 residues of myosin heavy chain, nonmuscle type A [MYH9_Human]of Homo sapiens . Although most matches [85%] were with myosinproteins, AglZ also shared some identity with the 170-kDa Golgiperipheral membrane protein . These proteins share a common structuralmotif that enables them to form coiled-coil structures, andmany of them play essential roles in cytoskeleton structureand motility.

Coiled-coil proteins are a mixture of hydrophobic and hydrophilic residues repeated every seven amino acids, forming parallel -helices that intertwine [12] . A minimum of four heptad repeats yields a stable coiled-coil structure [29, 31] . Amino acids248 to 1292 of AglZ, which form the coiled-coil domain, containthe requisite heptad repeats [a, b, c, d, e, f, g]_n . The sequenceshowed both four- and seven-heptad repeats . In each heptad,amino acids at positions a and d are comprised of mainly hydrophobicresidues, such as leucine, whereas amino acids at positionse and g are comprised mainly of hydrophilic residues, such asglutamate [12] . The coiled-coil domain of AglZ also contains21 repetitive structures that range in size from 5 to 33 aminoacid repeats . While the significance of these repeats is notunderstood, similar patterns of repeats are found in proteinssuch as fibronectin . Immediately following the coiled-coil domainis a second proline-rich [15% proline] domain . In contrast withthe linker domain, the terminal proline-rich tail has a pI of4.7.

Coiled-coil proteins typically migrate more slowly than expected from their primary sequence predictions . As shown in Fig . 5A, lane 2, two high-molecular-mass bands were detected when full-length AglZ-His6 was expressed in E . coli BL21-A1 . In contrast, these bands were missing in the uninduced E . coli sample and the E. coli strain carrying the parent plasmid, pET24b [data not shown]. Two protein bands also were detected in M . xanthus cells expressing AglZ-His [Fig . 5A, lane 3] . The AglZ proteins in M . xanthusmigrated more slowly than the AglZ proteins in E . coli, whichhinted that AglZ is modified in M . xanthus.

 
The C-terminal domain of AglZ forms a structure with a striated pattern in E . coli. In extracts of E . coli bearing pRY15, the truncated versionof AglZ-His, which lacks the N-terminal response regulator domain,two high-molecular-weight protein bands were seen on Coomassie-stainedgels . As shown in Fig . 5B, both bands were greater than the220-kDa standard, indicating that AglZ-coil forms a structurethat is significantly larger than its predicted size [130 kDa].This aberrant shift in mobility under denaturing conditions was consistent with proteins that can adopt a coiled-coil structure. FrzS, an M . xanthus protein with a domain structure similar to that of AglZ, also shows aberrant migration on SDS-PAGE [42]. To confirm that these high-molecular-weight proteins were forms of AglZ, extracts were probed with anti-His6 antibody . Immunoblotting confirmed that the two large bands were forms of AglZ and revealed the presence of a third smaller band estimated to be about 150kDa [Fig . 5C] . The higher-molecular-weight bands were discrete and their sizes were consistent with dimer and trimer formsof AglZ, which can remain associated under SDS-PAGE denaturing conditions but not in the presence of urea [data not shown].

Analysis of cells of E . coli producing AglZ-coil by transmission electron microscopy revealed a repeating pattern in the cells due to formation of a higher-order structure [Fig . 6B] . This pattern was not present in the induced cells containing only the expression vector pET24b [Fig . 6A] . As rod regions of knowncoiled-coil proteins are predicted to extend 15 nm per 100 aminoacids [8], the theoretical length of AglZ-coil [1,045 residueswith a 102-residue tail] is expected to be approximately 157nm . Measurement of the repeating pattern formed by AglZ-coilshowed a periodicity of 74 nm [Fig . 6C], which is about halfof the length predicted for AglZ-coil . This is in contrast withprevious reports for other proteins that form striated latticestructures, in which the theoretical periodicity is close to the actual periodicity of the coiled-coil region [20] . We interpretthis to mean that the light and dark patterns formed by AglZare caused by the substantial overlap between AglZ molecules, as shown in Fig . 6D.

 
Biochemical evidence that AglZ interacts with MglA. With purified proteins and antibodies in hand, we were ableto use more refined methods to confirm the interaction betweenMglA and AglZ that was predicted from the yeast two-hybrid results.Two biochemical approaches were taken to probe the protein-proteininteractions . First, mixtures of purified AglZ and MglA or controlprotein were treated with various cross-linking agents and thenseparated on denaturing gels . The proteins were transferredto polyvinylidene difluoride and probed with anti-MglA antibody.As shown in Fig. 7A, antibody reacted with the high-molecular-weight species only in samples that contained MglA, AglZ, and cross-linker. Anti-MglA did not react with AglZ in samples that lacked MglA. Second, the dot-far Western technique [30] was performed . Differentamounts of MglA-His, AglZ-His, and BSA were spotted onto nitrocellulosemembranes and incubated with and without AglZ . Samples werewashed and probed with anti-AglZ antibody and secondary antibody.As shown in Fig . 7B, AglZ selectively bound to different amountsof MglA [areas 3 and 5] and to itself [area 1], but not to BSA[areas 2 and 4].

 
AglZ forms a filament structure in vitro. The formation of striated patterned structures in E . coli wasreminiscent of striated muscle, and the similarities betweenAglZ and myosin hinted that AglZ might form a filament structure.To test this idea, AglZ and AglZ-coil were purified to apparenthomogeneity and incubated at 4°C . In both samples, a precipitatecould be seen after several days . An aliquot of each precipitatewas diluted in distilled water, stained with uranyl acetate,and placed on grids . As shown in Fig. 8, electron microscopyrevealed the presence of 1- to 3-µm-long filaments havingan average diameter of 50 nm . Upon close examination, some ofthe filaments appeared to be hollow, suggesting that the helicesof AglZ can rope together by multiple interactions . Filamentstructures were not detected in the sample prepared from thefull-length AglZ protein.

 
M . xanthus uses two distinct processes to gliding over surfaces. One process, known as S gliding, involves extension and retraction of pili that emanate from one pole of the cell . Mutations that affect the genes for type IV pili [48], the exopolysaccharide-proteinmatrix that constitutes fibrils [6, 22], and the O-antigen ofLPS [9] abolish S motility . The other gliding process is calledA motility . Secretion of a polyelectrolyte through polar nozzlesis postulated to be the driving force for A motility [46] . In support of this idea, genetic studies have shown that two setsof proteins related to the E . coli TolABQR proteins are required for A motility [44, 51] . In E . coli, the Tol proteins form acomplex that links the inner and outer membranes and facilitatestransport of macromolecules [25] . The M . xanthus Tol counterpartsmay be functionally analogous, which makes them prime candidatesfor the nozzle structure.

The aglZ mutants showed a primary defect in A motility, as demonstratedby the lack of isolated cells at the colony edge and reducedspreading on a hard agar [1.5%] surface . When viewed by time-lapsevideomicroscopy, aglZ showed no single-cell movement on 1.5%agar or on 1% methylcellulose, which can suppress the nonmotilephenotype of particular mutants . Genetically, aglZ is part ofthe A motility system, because when paired with a mutation inan S motility gene, the resulting double mutant was nonmotile.

The aglZ mutant exhibited a phenotype that was more severe than most A motility mutants . The mutant showed reduced spreadingon soft [0.3%] agar even though flares, or groupings, of cellsthat are characteristic of S motility were visible at the colonyedge, and the mutant appeared to make components necessary forS gliding . Despite the fact that the aglZ mutation affectedmotility, fruiting body development and spore formation wereunaffected in the mutant.

The aglZ gene appears to be a single gene operon . This is supported by the fact that a disruption mutant has the same phenotypeas the deletion mutant, which argues against a polar effecton a downstream gene . Moreover, there is a 103-bp gap betweenaglZ and its upstream gene and a 650-bp gap between aglZ andits downstream gene.

The domain organization of AglZ is very similar to that of another M . xanthus protein, FrzS, which is required for S motility [42]. Both AglZ and FrzS have a large C-terminal coiled-coil domain that is similar to myosin, although the two proteins do notshow any significant identity with one another in this regionby direct comparison . In place of the N-terminal ATPase headof myosin, these bacterial counterparts have an N-terminal responseregulator domain . The N-terminal domain lacks the conservedaspartate at position 53 that is a potential target for modification,but it has an aspartate at position 48 which may be phosphorylated.If phosphorylation of AglZ occurs, it might furnish a primitiveregulatory equivalent to ATP hydrolysis in myosin.

The aglZ gene first was identified from a clone recovered from the yeast two-hybrid system because it produced a fusion with the GAL4 activation domain that could interact with a bindingdomain fused with MglA . MglA is a small GTPase that is requiredfor both A and S motility . Previously, our group had shown bysuppressor analysis and the yeast two-hybrid assay that MglAinteracts with MasK, a membrane-bound tyrosine kinase [39].A mutation in masK suppressed a mutation in mglA to restore S motility to the mglA mutant . The MasK study showed that MglA interacts with a component of the S gliding system . The data presented in this paper confirm that MglA also interacts witha protein that is part of the A motility system . Establishinga link between MglA, AglZ, and MasK is a major step toward understandinghow MglA might regulate the two gliding systems . The abilityof MglA to interact with a component of each motility systemmight provide a switch that enables A and S gliding systemsto operate simultaneously in the wild-type strain . Because AglZand MasK are both potential targets for regulation by phosphorylation,it is conceivable that a change in the phosphorylation stateof either AglZ or MasK could alter their interaction with MglA.The putative target for phosphorylation in AglZ is in the N-terminaldomain near the proline-rich linker region to which MglA binds.

Although the discovery of an interaction between a small GTPase and a myosin-like protein has not previously been reported ina prokaryote, it is well documented in eukaryotic systems . Forexample, the GTPase Rab6 interacts with the coiled-coil regionof several golgins, which are involved in vesicular transport[5], and Ras3A interacts with the coiled-coil domain of Rabin3[10] . Sec4b interacts with the coiled-coil domain of Sec2b,which is essential for vesicular transport in S . cerevisiae[4] . In these cases, GTPases interact with coiled-coil proteinsto transport intermediates . If AglZ functions in an analogousfashion, it may participate in transporting substances thatare involved in gliding, subject to regulation by the GTPaseMglA.

The finding that AglZ is a filament-forming protein that is structurally related to myosin raises the possibility that the mechanism of A motility is functionally related to myosin-mediated movement . If so, A gliding is more complex than previously thought, and secretion of material through a nozzle as described by Wolgemuth et al . [46] may be only part of the A gliding mechanism . Thereare numerous historical accounts of filaments in M . xanthus that are thought to be involved in movement . AglZ may form the 12-nm filament structures that were described in M . xanthus25 years ago [11] . Tubules that were estimated to be 13 to 17nm in diameter were found in the debris of disrupted cells, and 7.5- to 10-nm filaments arranged in a herringbone pattern were described previously [11] . Reports of tubules that traversethe length of the cells also have been described [26, 33] . Workis under way to ascertain if the filaments described by thesethree different groups are related to the A motility proteinAglZ.

 
We thank Christine Davitt at WSU for expert help with electron microscopy and Ann Norton for help with microscopy used formotility analysis.

This work was supported by grant MCB0242191 from the National Science Foundation [NSF], NIH-Idaho BRIN to P.L.H., and NSFREU 0245188 to R.O.

 
* Corresponding author . Mailing address: Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83844 . Phone: [208] 885-0572 . Fax: [208] 885-6518 . E-mail: hartzell@uidaho.edu .

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