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Journal of Bacteriology, October 2002, p. 5654-5660, Vol. 184, No. 20
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.20.5654-5660.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A CheW Homologue Is Required for Myxococcus xanthus Fruiting Body Development, Social Gliding Motility, and Fibril Biogenesis

Kristen Bellenger,1 Xiaoyuan Ma,2 Wenyuan Shi,2 and Zhaomin Yang1*

Department of Biological Sciences, Auburn University, Auburn, Alabama 36849-5407,1 School of Dentistry, Molecular Biology Institute, and Dental Research Institute, University of California—Los Angeles, Los Angeles, California 90095-16682

Received 11 February 2002/ Accepted 7 June 2002


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ABSTRACT
 
In bacteria with multiple sets of chemotaxis genes, the deletion of homologous genes or even different genes in the same operon can result in disparate phenotypes. Myxococcus xanthus is a bacterium with multiple sets of chemotaxis genes and/or homologues. It was shown previously that difA and difE, encoding homologues of the methyl-accepting chemoreceptor protein (MCP) and the CheA kinase, respectively, are required for M. xanthus social gliding (S) motility and development. Both difA and difE mutants were also defective in the biogenesis of the cell surface appendages known as extracellular matrix fibrils. In this study, we investigated the roles of the CheW homologue encoded by difC, a gene at the same locus as difA and difE. We showed that difC mutations resulted in defects in M. xanthus developmental aggregation, sporulation, and S motility. We demonstrated that difC is indispensable for wild-type cellular cohesion and fibril biogenesis but not for pilus production. We further illustrated the ectopic complementation of a difC in-frame deletion by a wild-type difC. The identical phenotypes of difA, difC, and difE mutants are consistent and supportive of the hypothesis that the Dif chemotaxis homologues constitute a chemotaxis-like signal transduction pathway that regulates M. xanthus fibril biogenesis and S motility.


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INTRODUCTION
 
Myxococcus xanthus is a gram-negative bacterium that exhibits a complex multicellular developmental cycle and coordinated cell movement (13). Under starvation conditions, hundreds of thousands of vegetative cells aggregate and form mature fruiting bodies containing differentiated myxospores (13). The desiccation- and heat-resistant spores can germinate and reenter the vegetative cell cycle under conditions favorable for growth. In addition to developmental aggregation, M. xanthus cells display another form of coordinated cell movement in which vegetative cells move cooperatively over solid surfaces as large groups. This group behavior is important for the predatory lifestyle of M. xanthus because groups of cells can collectively produce sufficiently large amounts of antimicrobial and hydrolytic activities for the effective killing and digestion of other microbes as their prey (11, 41).

The developmental aggregation and cooperative movement of M. xanthus is supported by a form of bacterial surface motility known as gliding (17, 35). Despite recent advances, bacterial gliding is still one of the most mysterious modes of bacterial locomotion (34). Furthermore, there are likely multiple evolutionary origins for bacterial gliding because gliding bacteria, which are widespread in nature, can be found in many branches of the bacterial phylogenetic tree, and multiple mechanisms possibly exist for bacterial gliding (34). In fact, M. xanthus, one of the best-studied bacterial gliders, possesses two genetically separable gliding systems, the adventurous (A) and the social (S) motility systems (18, 19). These two systems appear to function independently such that mutations in either A or S motility genes rarely cause any obvious defects in the other gliding system. While A motility, as seen in A+ S- strains, is described as motility of well-isolated cells, S motility, as seen in A- S+ strains, is manifested only as movement of multicellular groups (18, 19). It has been proposed that the A-motility gene system regulates the movement of single cells and S-motility genes control the movement of cell groups (19). S motility is crucial for M. xanthus development because all S-motility mutants are defective in fruiting body development to various degrees (19, 31, 58). In addition, it was discovered that A motility is essentially nonfunctional on soft-agar plates and that S motility is absolutely required for effective translocation by vegetative cells on such highly hydrated surfaces (43).

It has become clear from various studies that M. xanthus social gliding requires at least two cell surface appendages: the polarly localized type IV pili (21, 54) and the peritrichous extracellular matrix fibrils (2, 3, 12, 45, 52, 58, 60). It has been hypothesized that type IV pili are the motors for M. xanthus S motility and for another form of bacterial surface locomotion known as twitching and that the retraction of pili powers both of these forms of bacterial surface motility (20). Recent studies with innovative microscopy have indeed lent support to the above hypothesis (37, 46, 48). Studies with various types of microscopy showed that M. xanthus fibrils can form a cell surface layer and/or filaments that are variable in dimensions and polymorphic in appearance (2, 12, 25). They have been observed to form connections between neighboring cells and between cells and their gliding surfaces. Preliminary biochemical analysis has indicated that they are composed of about equal amounts of protein and carbohydrate (4). Disruption or elimination of fibrils by chemicals or by mutations results in defects in M. xanthus S motility and cellular cohesion (2, 3, 12, 45, 52, 58, 60).

It was discovered that a wild-type dif (for defective in fruiting) locus is required for fruiting body development, S motility, and fibril biogenesis in M. xanthus (58, 60). The dif locus contains five genes, four of which encode homologues of chemotaxis proteins. DifA is homologous to methyl-accepting chemoreceptor proteins (MCPs): DifC is homologous to CheW, DifD is homologous to CheY, and DifE is homologous to CheA. These findings strongly suggest that M. xanthus S motility is a dynamic process regulated by sensory inputs.

Previous work has demonstrated that both difA and difE are essential for M. xanthus development, S motility, and fibril biogenesis (58, 60). It was proposed that the Dif proteins constitute a signal transduction pathway required for fibril biogenesis and S motility. It follows that difC, encoding the CheW homologue, would be required for the same processes. On the other hand, it is clear that multiple CheW homologues are present in M. xanthus (36, 58; J. Kirby, personal communication). Another bacterium with multiple CheWs is Rhodobacter sphaeroides, which has three CheW homologues (33). The deletions of these three cheW genes in R. sphaeroides resulted in very distinct phenotypes (33). Furthermore, although cheW3 and cheA2 are in the same operon in R. sphaeroides, mutations in these two genes resulted in different chemotaxis phenotypes (33). It is therefore not certain whether difC functions in the same pathway as difA and difE. In this work, we investigate the roles of difC in M. xanthus motility and development. Our results demonstrate that difC, like difA and difE, is essential for M. xanthus fruiting body development, S motility, and fibril biogenesis. We propose that the S-motility regulatory pathway defined by the dif genes shares certain characteristics and properties with the classical bacterial chemotaxis signal transduction pathways in enteric bacteria.


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MATERIALS AND METHODS
 
Bacterial strains and culture conditions. The bacterial strains used in this study are listed in Table 1. For maintenance, M. xanthus strains were grown at 32°C on CYE (9) plates and stored at room temperature for up to 10 days. Liquid cultures of M. xanthus were grown in CYE medium at 32°C on a rotary shaker at 250 rpm. Escherichia coli strains were grown and maintained at 37°C in Luria-Bertani medium (38). The media were supplemented with ampicillin (100 µg/ml) or kanamycin (100 µg/ml) when necessary.


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  		TABLE 1. Bacterial strains and plasmids

Construction of plasmids and M. xanthus strains. The difC in-frame deletion mutant SW403 was constructed by gene replacement using the positive and negative KG cassettes described by Ueki et al. (49). A 1.27-kb SalI fragment containing difC (GenBank accession no. AF076485) was cloned into pBluescript to generate pYZ301L. pYZ301L was digested with the restriction enzymes MscI and NarI, both of which recognize sites within the open reading frame of DifC but not anywhere else on the plasmid. The restricted plasmid was blunt ended with T4 polymerase and religated to generate pYZ677. This construct resulted in the deletion of 62 amino acids, from the 47th to the 108th residues of the predicted DifC. The fragment containing this difC in-frame deletion was cloned into the SalI site of the KG cassette-containing vector pBJ113 (58) to construct pYZ678, which was then used to construct SW403 by gene replacement through a two-step homologous recombination. The replacement of wild-type difC by the constructed mutation was confirmed by PCR and Southern analysis. The double mutants YZ144 ({Delta}difC S-) and YZ145 ({Delta}difC A-) were constructed by Mx4-mediated transduction using SW403 as the recipient strain and DK3473 and MXH1216 as donor strains, respectively. pKB106, the plasmid used for ectopic complementation, was constructed by cloning a 2.76-kb PstI-BamHI fragment containing the difC gene into pYC274, an M. xanthus integrative vector with the Mx8 attachment site (15). This PstI-BamHI fragment contains sequences immediately downstream of difA to the 5' portion of difE. YZ143 was constructed by electroporation of pKB106 into SW403.

Assays of development, motility, cellular cohesion, and binding of calcofluor white. For the examination of fruiting body development, 10 µl of cells at 5 x 109/ml were spotted onto MOPS (morpholinepropanesulfonic acid) plates (10 mM MOPS [pH 7.6], 2 mM MgSO4, 1.5% agar) (16) and incubated at 32°C for 4 days. Developmental aggregation was examined and documented with a dissecting microscope. For the examination of the formation of developmental spores, samples were scraped from developmental plates, suspended in MOPS buffer (10 mM MOPS [pH 7.6], 2 mM MgSO4), and observed by phase-contrast microscopy. For examination of motility on hard agar, 3 µl of cells at 5 x 109/ml were spotted onto the centers of CYE plates with 1.5% agar and incubated at 32°C for 6 days.

Cellular cohesion was examined by the agglutination assay as described by Wu et al. (56). M. xanthus cells from exponential cultures were adjusted to a cell density of ~2.5 x 108/ml with CYE medium, and the optical density (OD) at 600 nm was measured with a Shimadzu UV-1601 spectrophotometer every 10 min for 1.5 h. Samples were kept at room temperature in the dark between OD readings. The relative absorbance was calculated by dividing the OD value at a given time by the initial OD of the sample.

Procedures similar to those described previously (40, 60) were followed for the examination of the binding of the fluorescent dye calcofluor white by polysaccharides on the cell surfaces. To briefly summarize, aliquots of 10 µl of cell suspensions at 5 x 107/ml were spotted onto CYE plates with 1.5% agar containing calcofluor white at 50 µg/ml. After 7 days of incubation at 32°C, the plates were examined and documented under 365-nm-wavelength UV illumination from a handheld UV lamp.

Analysis of pili and fibrils by immunoblotting. Detection of pili and fibrils was conducted as described previously (60). M. xanthus cell surface pili were prepared from liquid-grown cells by vortexing and differential precipitation (50). For the analysis of fibril-specific protein antigens, whole-cell lysates were prepared from overnight liquid cultures. Various samples were separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-10% PAGE) and analyzed by immunoblotting (42). For the detection of surface pili or pilin, the polyclonal anti-PilA antibodies were used as the primary antibody and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin M (IgM) and IgG were used as the secondary antibody (55). For the detection of the fibril-specific proteins, the monoclonal antibody MAb2105 was used as the primary antibody and alkaline phosphatase-conjugated goat anti-mouse IgG was used as the secondary antibody.


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RESULTS
 
Homology of DifC to CheW and CheW-like domains of CheA kinases. There are five predicted open reading frames at the dif locus (58). The deduced polypeptide sequence of DifC is 147 amino acids long and shows homology with the bacterial chemotaxis protein CheW and the CheW-like domain at the C terminus of the CheA protein kinase (58). Our alignment indicated that DifC shows about 21% identity with Thermotoga maritima CheW and the CheW-like domain of M. xanthus DifE (Fig. 1). In the chemotaxis signal transduction pathway in enteric bacteria, CheW functions to couple the signal from MCPs to CheA kinases (8, 14, 47). The CheW-like domain of CheA structurally resembles two SH3 domains which function as scaffolds to mediate protein-protein interactions (6). In E. coli and Salmonella enterica serovar Typhimurium, mutations in CheW result in defects in chemotaxis similar to those in cheA and chemoreceptor genes (26, 39). The homology of DifC to CheW and CheW-like domains suggests that difC may function in the putative dif signal transduction pathway in a capacity similar to that of cheW in classical chemotaxis signal transduction pathways. This idea is further strengthened by the observation that the residues known to be crucial for the function of E. coli CheW (27) are all conserved in M. xanthus DifC (not shown).



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  		FIG. 1. Homology among the deduced polypeptide sequence of M. xanthus DifC (Mx_DifC), the CheW domain of M. xanthus DifE (Mx_DifE), and T. maritima CheW (Tm_CheW). Identical residues in M. xanthus DifC and DifE and those in M. xanthus DifC and T. maritima CheW are highlighted in boldface. DifC has 21.7% (30 of 138) amino acid identity with DifE and 20.3% (28 of 138) identity with T. maritima CheW over the aligned region.

Construction of SW403, a difC in-frame deletion mutant. To examine the role of difC in M. xanthus, a difC in-frame deletion mutant, SW403, was constructed. This mutant contains a deletion of 186 bp internal to difC, which resulted in the deletion of the 47th to 108th amino acids from DifC. The construction and confirmation of this mutant were performed as described in Materials and Methods. SW403 was used to examine the possible involvement of difC in the same processes as difA and difE.

Ectopic complementation of the difC deletion mutant. The in-frame deletion of difC may minimize any polar effects on the transcription of downstream genes. However, since difE is downstream of difC and has been shown to be necessary for M. xanthus fruiting, S motility, and fibril biogenesis, it is possible that the deletion of difC might affect the secondary structure of the mRNA and thus the transcription, stability, or translation of the mRNA encoding difE. In addition, the difC mutation in SW403 resulted in the deletion of 62 residues from DifC, with a predicted total of 147 amino acids. The in-frame deletion could create a gain-of-function mutation, which could be responsible for any altered phenotype of SW403. If either or both of the above scenarios are true, the difC mutant should not be complemented by a copy of a wild-type difC gene ectopically. As described in Materials and Methods, a fragment containing the entire difC gene but less than half of difE was cloned into pYC274, an M. xanthus integrative vector with the Mx8 attachment (att) site (15). The resulting plasmid, pKB106, was electroporated (22) into SW403 to generate YZ143, a difC+ strain. Whenever possible, YZ143 was examined along with SW403 (the difC mutant) to ensure that any observed defects of SW403 were the consequences of the lack of difC products, not the results of any polar effects on downstream genes or a difC gain-of-function mutation. From the results described below, it is apparent that the fragment in pKB106 can complement the difC deletion mutant, as can the same fragment cloned into the same vector in the opposite orientation (data not shown). The integrative plasmid pYC274 is not designed as an expression vector for M. xanthus (15), and the fragment in pKB106 contains only sequences downstream of difA. The complementation results may suggest that difC can be expressed from a promoter other than the one for difA in M. xanthus.

Developmental defects of the difC deletion mutant. The difC deletion mutant SW403 was examined for fruiting body development as previously described (58). As shown in Fig. 2, the wild-type strain, DK1622, and the difC+ strain, YZ143, both formed visible fruiting bodies after 4 days of incubation on MOPS plates, and their fruiting bodies contained spherical myxospores as expected. In contrast, SW403 showed little aggregation under the same condition, and the cells remained as rod-shaped vegetative cells, indicating that difC, like difA and difE, is required for M. xanthus fruiting body development, including aggregation and sporulation, and that pKB106 can complement the developmental defects resulting from difC deletion.



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  		FIG. 2. Development of fruiting bodies and sporulation. Developmental phenotypes of various strains were examined and photographed using a dissecting microscope as shown in the upper panels, where the scale bar represents 1 mm. The bright or light spots are top views of fruiting bodies. Spore formation was examined and documented using phase-contrast microscopy as shown in the lower panels, where the scale bar represents approximately 50 µm. Myxospores appear reflective and spherical, and vegetative cells appear rod shaped. (A and D) DK1622 (wild type); (B and E) SW403 ({Delta}difC); (C and F) YZ143 ({Delta}difC att::difC).

Defects of difC mutants in M. xanthus S motility. Developmental defects in M. xanthus may result from defects in gliding motility (19, 31). Furthermore, both difA and difE mutants were shown to be defective in S motility (58). Hence, there was a strong possibility that the difC deletion mutant could be defective in S motility, which in turn could result in the observed defects in developmental aggregation and sporulation. The colony morphology of SW403 on 1.5% agar was first examined in comparison with that of a wild-type strain (Fig. 3A and D). The more compact and glossy colony morphology of the difC mutant was reminiscent of those of difA and difE mutants and was suggestive of defects in S motility.



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  		FIG. 3. Colony spreading of different M. xanthus strains on hard agar. (A) DK1622 (wild type); (B) DK3473 (S- strain); (C) MXH1216 (A- strain); (D) SW403 ({Delta}difC); (E) YZ144 ({Delta}difC S-); and (F) YZ145 ({Delta}difC A-). The scale bar represents 1 cm.

The two motility systems in M. xanthus, A motility and S motility, are defined genetically (19). That is, if a mutation occurs in a gene of one motility system, when it is combined with a mutation in the other system, the double mutant should show no or little motility. To further examine and define the possible motility defects of the difC mutant, {Delta}difC A- and {Delta}difC S- double mutants were constructed as described in Materials and Methods. The colony spreading of the double mutants and that of their parental strains on 1.5% agar plates were examined (Fig. 3). The {Delta}difC S- double mutant, YZ144 (Fig. 3E), showed spreading similar to that of its S- mutant parent (Fig. 3B), indicating that the difC deletion resulted in no apparent defects in the M. xanthus A-motility system. In contrast, the {Delta}difC A- double mutant, YZ145 (Fig. 3F), displayed no significant colony expansion after several days of incubation, indicating that the double mutant possesses little motility. The S motility of difC mutants was additionally examined on soft-agar plates as described previously (57). The difC mutant (SW403) was severely defective in locomotion on soft-agar plates, as expected for S- mutants, but YZ143 with the difC gene integrated at the Mx8 att site showed motility behavior on soft agar similar to that of the wild type (data not shown). These results indicate that difC, like the closely linked difA and difE, is required for M. xanthus S motility.

Examination of cellular cohesion. There is a close correlation between S-motility defects and defects in cellular cohesion (45, 52, 56-58, 60). All of the known S-motility genes, with the exception of pilT (56), are required for wild-type cellular cohesion. This correlation suggests that physical cell-cell interactions are an integral part of the S-motility system in M. xanthus. The cohesion of the difC mutant SW403 was analyzed by agglutination assays (56) to examine possible defects in cell-cell interactions. As shown in Fig. 4, wild-type cells agglutinated as expected, as did YZ143 with the difC gene integrated at the Mx8 att site. On the other hand, the difC mutant, similar to the difE mutant (SW501), showed no obvious signs of agglutination. Like those of most known S- mutants, the defects of the difC mutant in S motility are therefore correlated with defects in cellular cohesion, which in turn indicate defects in physical cell-cell interactions and possibly alteration of cell surface properties.



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  		FIG. 4. Agglutination assays of M. xanthus strains. Cells were grown at 32°C overnight in CYE medium, and the OD at 600 nm was adjusted to ~0.5 with CYE. Except during measurements, the samples were kept in the dark at room temperature. The relative absorbance was calculated by dividing the OD reading at a given time by the OD reading at time zero. The experiments were repeated five times with similar results. The strains shown here are the wild-type DK1622, the difC deletion mutant SW403, the complemented difC mutant YZ143, and the difE mutant SW501 as a negative control.

Production of surface pili. The agglutination of M. xanthus cells under the assay conditions described above has been shown to require the presence of the cell surface components pili and fibrils (56, 60). The lack of cellular cohesion of the difC mutant could result from defects in the biogenesis of pili, fibrils, or both. The presence of pili on cell surfaces was examined by a procedure described previously (50, 60). Fractions containing cell surface pilin were prepared and analyzed by immunoblotting with polyclonal antibodies against PilA, the subunit of M. xanthus pili (Fig. 5). A band of about 25 kDa was detected in the wild type, the difC mutant, and the difC+ strain, but not in the pilA mutant DK10407, indicating that, similar to difA and difE mutants, the difC mutant is not defective in the production of surface pili. Like difA, difE, and pilT mutants (56, 60), the difC mutant also appears to be hyperpiliated.



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  		FIG. 5. Immunoblot analysis using polyclonal antibodies against M. xanthus PilA protein. The fraction containing cell surface pili was prepared as described in Materials and Methods. Samples were separated by SDS-10% PAGE and analyzed by immunoblotting with anti-PilA antibodies. Lanes A, C, and E contain surface pili from 2.5 x 109 cells. Lanes B and D contain surface pili from 5 x 108 cells. Lanes: A, DK1622 (wild type); B, DK10409 (pilT); C, YZ143 (difC complemented); D, SW403 (difC); and E, DK10407 (pilA).

Defects of the difC mutant in fibril biogenesis. Since no defects were found in its production of surface pili, the difC deletion mutant was examined for defects in production of the protein and the carbohydrate moieties of M. xanthus extracellular matrix fibrils. The presence of a fibril-specific protein antigen on difC mutant cells was examined by immunoblotting with the monoclonal antibody MAb2105 (5, 60). As shown in Fig. 6, the difC mutant SW403 and the negative control, SW501, showed no or little reactivity with the antibody, whereas the pilT mutant DK10409, which is S- but still produces pili (56), the difC+ strain YZ143, and the wild-type DK1622 showed comparable levels of reactivity with MAb2105. These results indicate that the difC mutant is defective in fibril biogenesis, at least in the production or assembly of a fibril-associated protein antigen. In addition, the results with the pilT mutant showed that pili-producing S- mutants do not necessarily have defects in fibril biogenesis.



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  		FIG. 6. Immunoblot analysis of fibril-specific protein antigens with MAb2105. Whole-cell lysates from 1.25 x 107 cells were separated by SDS-10% PAGE and analyzed by immunoblotting with MAb2105. Lanes: A, DK1622 (wild type); B, DK10409 (pilT); C, YZ143 (difC complemented); D, SW403 (difC); and E, SW501 (difE).

The other major constituents of M. xanthus fibrils are the carbohydrates or exopolysaccharides (4). The presence of these polysaccharides on the cell surface was examined in vivo by the binding of the fluorescent dye calcofluor white (Fig. 7). The colonies of the wild type (DK1622) and the complemented difC strain (YZ143) showed binding of the dye, as indicated by fluorescence when illuminated by UV light. The difC mutant showed no detectable binding of the fluorescent dye in this assay. The lack of calcofluor white binding by the mutant cells indicates that the M. xanthus difC mutant is defective in the synthesis or assembly of the exopolysaccharide moiety of extracellular matrix fibrils. Together with the immunoblotting analysis with MAb2105, these results demonstrate that the difC gene is essential for the biogenesis of fibrils in M. xanthus.



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  		FIG. 7. Binding of calcofluor white (fluorescent dye) by M. xanthus. Aliquots of 10 µl of cell suspensions at 5 x 107 cells/ml were spotted onto CYE plates containing calcofluor white at 50 µg/ml. After 7 days of incubation at 32°C, the plates were examined and photographed under the illumination of 365-nm-wavelength UV light. The diameter of the plate shown is 9 cm. (A) SW403 (difC); (B) DK1622 (wild type); (C) SW501 (difE); and (D) YZ143 (complemented difC).


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DISCUSSION
 
We investigated the roles of difC, the gene encoding a CheW homologue, and provided direct evidence for its involvement in M. xanthus development, S motility, and fibril biogenesis. It was demonstrated that a wild-type difC integrated ectopically complemented the difC deletion mutation, restoring development, S motility, and fibril production. These results are consistent with and provide further support for the hypothesis that the Dif proteins constitute a signal transduction pathway that regulates S motility in M. xanthus. In addition, the defects of difC mutants in fibril biogenesis support the possibility that the regulation of S motility by the dif genes is exerted through extracellular matrix fibrils. We submit that the inability to produce fibrils may be the key to explaining all of the observed defects of the difC mutants.

The clues for the requirement of a CheW homologue for fibril biogenesis may come from the well-studied bacterial chemotaxis pathway (1, 8, 14, 47). In chemotaxis regulation in E. coli, for example, chemotactic stimuli are detected by membrane chemoreceptors, or MCPs. Many of these proteins sense signals through their periplasmic domains either by direct ligand binding or by interactions with ligand binding proteins. Detection of signals results in conformational changes in MCPs, which transduce the signal to the CheA kinase for the regulation of flagellar-motor rotation. The transduction of signals from MCPs to the CheA kinase requires the chemotaxis protein CheW, which physically interacts with both MCPs and CheA kinases for the relay of signals. The requirement for DifA (MCP-like), DifC (CheW-like), and DifE (CheA-like) proteins for the same processes suggests that, for the regulation of fibril biogenesis and S motility, the dif gene products may interact with one another in a fashion similar to those in a chemotaxis signal transduction pathway in enteric bacteria. In particular, it is known that MCPs, CheAs, and CheWs assemble into polarly localized clusters which are essential for their functions (28, 29, 32, 44). Although the Dif proteins of interest have not yet been localized within the cell, their homology to chemotaxis proteins and the absolute requirement for difA, difC, and difE in the same processes examined so far have led us to propose that the dif genes define an S-motility regulatory-signal transduction pathway with chemotaxis-like interactions and characteristics.

How may the putative dif chemotaxis-like signal transduction pathway regulate M. xanthus S motility? In flagellated bacteria such as E. coli, the output of the chemotaxis pathway directly interacts with flagellar switches to achieve chemotaxis. Likewise, it is plausible that the dif gene products may regulate M. xanthus S motility by interactions with the elusive S-motility motor. However, the available evidence points to a regulatory mechanism through the mediation of the cell surface component fibrils instead. It has been known for some time that there is a tight correlation between the presence of fibrils and S motility. The disruption or elimination of fibrils, either by chemicals or by genetic mutations, is associated with S-motility defects (45, 52, 58, 60). Arnold and Shimkets first demonstrated that there is a correlation between energy-dependent cell cohesion and social motility and that cell cohesion requires extracellular matrix fibrils (2, 3, 45). They discovered that dsp mutants, which are defective in cell cohesion and fibril biogenesis, are also defective in S motility and development. In addition, sglK and dif mutants were shown to have simultaneous defects in S motility and fibril biogenesis (52, 60). It has also been demonstrated that the addition of isolated fibril materials can partially restore cellular cohesion, developmental aggregation, and sporulation to dsp and dif mutants (10, 60). Although the available experimental evidence does not rule out the possibility that Dif proteins are components of the S-motility motor, we favor a model where the dif genes regulate M. xanthus S motility through the regulation of fibril biogenesis.

Many questions remain concerning the functionality of the dif pathway. Concerning the way in which a chemotaxis-like signal transduction pathway may regulate the biogenesis of a complex cell surface structure composed of proteins and polysaccharides, there are at least two possibilities that should be considered. One possibility is that the putative Dif pathway may regulate the expression of genes essential for the synthesis, export, or assembly of fibrils. The other possibility is that the dif gene products may themselves play essential and direct roles in the export or assembly of fibrils. We have no evidence at present to favor one or the other possibility. In addition, the O antigens of M. xanthus lipopolysaccharides have also been found to affect gliding motility (7, 59). Since both lipopolysaccharide O antigens and matrix fibrils are associated with cell surfaces, they may interact with each other in ways we do not yet understand.

In addition to their involvement in fibril biogenesis and S motility, dif genes have also been implicated in the chemotactic responses of M. xanthus to phosphatidylethanolamine (PE) (23). It should be noted that the dif genes were the second set of chemotaxis-like genes discovered in M. xanthus. The first set of chemotaxis homologues are encoded by the frz genes (51). frz null mutants show defects in the regulation of reversal in their gliding direction. Cells of such mutants display considerably longer intervals between directional changes in movements. The frz genes have also been implicated in PE responses (24). A major difference is that frz mutants appeared to have normal excitation in response to PE, but they were defective in adaptation. In contrast, the dif mutants appeared to be defective in their excitation responses (23). The lack of excitation of dif mutants in response to PE cannot be attributed to their defects in fibril biogenesis alone because excitation responses to PE can be restored by the addition of isolated fibrils to certain dsp mutants but not to the dif mutants (23). How M. xanthus cells coordinate their chemotactic responses to PE using at least two sets of chemotaxis genes remains a mystery. How dif genes play roles in two seemingly distinct regulatory processes, fibril biogenesis and chemotaxis, has yet to be elucidated.


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ADDENDUM
 
During the review of this article, two papers relevant to this work were published by Lancero et al. (26a) and Kearns et al. (24a). The first paper demonstrated that most of the dsp mutations, a known group of mutations affecting cellular cohesion and S motility, mapped at the dif locus. The second reported the identification of the fibril protein (FibA) recognized by the monoclonal antibody MAb2105, which was used in this work for the analysis of fibril biogenesis. FibA was found to be homologous to metalloproteases, and fibA mutants were found to have lost the ability to respond to dilauroyl PE, a known chemoattractant for M. xanthus. fibA mutants did not otherwise display any obvious defects in cellular cohesion or fibril biogenesis.


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ACKNOWLEDGMENTS
 
We are grateful to the laboratories of D. Kaiser, P. Hartzell, and H. Kaplan for providing bacterial strains, plasmids, and/or antibodies. We thank Wesley Black and Zhuo Li for critical reading of the manuscript. Brandi Stansell, an Auburn University undergraduate, assisted in this study.

This work was supported by NSF grant MCB-0135434 and institutional grants from Auburn University to Z. Yang and NIH grant GM54666 to W. Shi.


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FOOTNOTES
 
* Corresponding author. Present address: 2119 Derring Hall, Virginia Tech, Blacksburg, VA 24061. Phone: (540) 231-1350. Fax: (540) 231-9307. E-mail: zmyang{at}vt.edu. Back


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Journal of Bacteriology, October 2002, p. 5654-5660, Vol. 184, No. 20
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.20.5654-5660.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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