Biochemical and structural analyses of the extracellular matrix fibrils of Myxococcus xanthus.

It is characteristic of myxobacteria to produce large amounts of extracellular material. This report demonstrates that this material in Myxococcus xanthus is fibrillar and describes the structure and chemical composition of the fibrils. The extracellular matrix fibrils are the mediators of cell-cell cohesion in M. xanthus. As such, the fibrils play an important role in the cell-cell interactions that form the basis for the social and developmental lifestyle of this organism. The fibrils are composed of protein and carbohydrate in a 1.0:1.2 ratio. Combined, the two fractions accounted for greater than 85% of the mass of isolated fibrils, and the fibrils were found to compose up to 10% of the dry weight of cells grown at high density on a solid surface. The polysaccharide portion of the fibrils was shown to be composed of five different monosaccharides: galactose, glucosamine, glucose, rhamnose, and xylose. Glucosamine, one of the component monosaccharides of the fibrils and a known morphogen for M. xanthus, inhibited cohesion to a level near that of Congo red (the positive control for cohesion inhibition). Glucose and xylose also inhibited cohesion but less than did glucosamine. Analysis of the morphology of the fibrils, the periodicities within the distribution of fibril diameters observed by field emission scanning electron microscopy, and the observation of fibrils on hydrated cells strongly suggested that the extracellular matrix of M. xanthus was indeed arranged as fibrils. Furthermore, results suggested that the fibrils were constructed as carbohydrate structures with associated proteins.     

Myxococcus xanthus dif genes are required for biogenesis of cell surface fibrils essential for social gliding motility

Myxococcus xanthus social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the dif genes. In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to M. xanthus S motility. We have demonstrated here that M. xanthus dif genes are required for the biogenesis of fibrils but not for that of type IV pili. Furthermore, the developmental defects of dif mutants can be partially rescued by the addition of isolated fibril materials. Along with the chemotaxis genes of various swarming bacteria and the pilGHIJ genes of the twitching bacterium Pseudomonas aeruginosa, the M. xanthus dif genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion. Genetic studies indicate that the dif genes are linked to the M. xanthus dsp region, a locus known to be crucial for M. xanthus fibril biogenesis and S gliding.     

Extracellular fibrils and contact-mediated cell interactions in Myxococcus xanthus.

Contact-mediated cell-cell interactions play an important role in the social life-style of Myxococcus xanthus. Previous investigations have demonstrated that fimbriae (also referred to as pili) and extracellular fibrils are involved in these social interactions (L. J. Shimkets, Microbiol. Rev. 54:473-501, 1990). We have used the relatively new technique of low-voltage scanning electron microscopy (an ultra-high-resolution scanning technique that allows for the nanometer resolution of biological materials) to observe the topological details of cell-cell interactions in M. xanthus. Our observations indicated that the fibrils (which measure approximately 30 nm in diameter) are produced most extensively by cells that are in close contact with each other and are aberrantly produced by the cohesion-deficient dsp mutants. Immunogold analysis identified an antigen which is located exclusively on the extracellular fibrils. Western blots (immunoblots) of this antigen (designated FA-1 for fibrillar antigen 1) indicated that it is composed of several immunoreactive bands (molecular size range, 90 to 14 kDa), all of which are sensitive to protease digestion. A technique for fibril isolation was developed by using FA-1 as a fibril-specific marker. Low-voltage scanning electron microscope observations of swarming cells demonstrated that the expression of fibrils is differentially regulated between adventurous (individual) and socially (group) motile cells. The differential expression of fibrils suggests the existence of a mechanism for the regulation of fibril biosynthesis that functions within the overall system governing social interactions in M. xanthus.     

A CheW homologue is required for Myxococcus xanthus fruiting body development,social gliding motility,and fibril biogenesis

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.     

Mutants of Myxococcus xanthus dsp defective in fibril binding.

The dsp mutant of Myxococcus xanthus lacks extracellular fibrils and as a result is unable to undergo cohesion, group motility, or development (J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5765-5770, 1983; J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5771-5777, 1983; R. M. Behmlander and M. Dworkin, J. Bacteriol. 173:7810-7821, 1991; L. J. Shimkets, J. Bacteriol. 166:837-841, 1986; L. J. Shimkets, J. Bacteriol. 166:842-848, 1986). However, cohesion and development can be phenotypically restored by the addition of isolated fibrils (R. M. Behmlander, Ph.D. thesis, University of Minnesota, Minneapolis, 1994; B.-Y. Chang and M. Dworkin, J. Bacteriol. 176:7190-7196, 1994). As part of our attempts to examine the interaction of fibrils and cells of M. xanthus, we have isolated a series of secondary mutants of M. xanthus dsp in which cohesion, unlike that of the parent strain, could not be rescued by the addition of isolated fibrils. Cells of M. xanthus dsp were mutagenized either by ethyl methanesulfonate or by Tn5 insertions. Mutagenized cultures were enriched by selection of those cells that could not be rescued, i.e., that failed to cohere in the presence of isolated fibrils. Seven mutants of M. xanthus dsp, designated fbd mutants, were isolated from 6,983 colonies; these represent putative fibril receptor-minus mutants. The fbd mutants, like the parent dsp mutant, still lacked fibrils, but displayed a number of unexpected properties. They regained group motility and the ability to aggregate but not the ability to form mature fruiting bodies. In addition, they partially regained the ability to form myxospores. The fbd mutant was backcrossed into the dsp mutant by Mx4 transduction. Three independently isolated transconjugants showed essentially the same properties as the fbd mutants--loss of fibril rescue of cohesion, partial restoration of myxospore morphogenesis, and restoration of group motility. These results suggest that the physical presence of fibrils is not necessary for group motility, myxospore formation, or the early aggregative stage of development. We propose, however, that the perception of fibril binding is required for normal social behavior and development. The dsp fbd mutants (from here on referred to as fbd mutants) open the possibility of isolating and characterizing a putative fibril receptor gene.     

Myxococcus xanthus chemotaxis homologs DifD and DifG negatively regulate fibril polysaccharide production

The extracellular matrix fibrils of Myxococcus xanthus are essential for the social lifestyle of this unusual bacterium. These fibrils form networks linking or encasing cells and are tightly correlated with cellular cohesion, development, and social (S) gliding motility. Previous studies identified a set of bacterial chemotaxis homologs encoded by the dif locus. It was determined that difA, difC, and difE, encoding respective homologs of a methyl-accepting chemotaxis protein, CheW, and CheA, are required for fibril production and therefore S motility and development. Here we report the studies of three additional genes residing at the dif locus, difB, difD, and difG. difD and difG encode homologs of chemotaxis proteins CheY and CheC, respectively. difB encodes a positively charged protein with limited homology at its N terminus to conserved bacterial proteins with unknown functions. Unlike the previously characterized dif genes, none of these three newly studied dif genes are essential for fibril production, S motility, or development. The difB mutant showed no obvious defects in any of the processes examined. In contrast, the difD and the difG mutants were observed to overproduce fibril polysaccharides in comparison with production by the wild type. The observation that DifD and DifG negatively regulate fibril polysaccharide production strengthens our hypothesis that the M. xanthus dif genes define a chemotaxis-like signal transduction pathway which regulates fibril biogenesis. To our knowledge, this is the first report of functional studies of a CheC homolog in proteobacteria. In addition, during this study, we slightly modified previously developed assays to easily quantify fibril polysaccharide production in M. xanthus.     

Isolated fibrils rescue cohesion and development in the Dsp mutant of Myxococcus xanthus.

Extracellular fibrils are involved in cell cohesion and cell development in Myxococcus xanthus. One group of social motility mutants, Dsp, is unable to produce extracellular fibrils; these mutants also lose the abilities to cohere and to develop. Extracellular fibrils isolated from vegetative wild-type cells and added to Dsp cells fully restored the abilities of these cells to cohere and to undergo normal morphological development. The fibrils thus mimic the ability of intact, wild-type cells to carry out the same rescue. Optimal cohesion rescue by fibrils required calcium and magnesium ions, did not require protein synthesis, but was energy dependent, i.e., sodium azide and sodium cyanide blocked rescue. Cohesion rescue was also blocked by the diazo dye Congo red. Cohesion rescue is genus specific, i.e., isolated fibrils did not cause the cohesion of Pseudomonas aeruginosa, Bacillus subtilis, Proteus mirabilis, Escherichia coli, or the related myxobacterium Stigmatella aurantiaca. Developmental rescue of Dsp by isolated fibrils included aggregation, fruiting body formation, and myxospore morphogenesis. Developmental gene expression in the Dsp mutant was only partially rescued by the isolated fibrils.     

Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding.

Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates.     

Effect of dsp mutations on the cell-to-cell transmission of CsgA in Myxococcus xanthus.

The dsp locus contains genes involved in the subunit synthesis and/or assembly of fibrils that radiate outward from the Myxococcus xanthus cell surface and attach to other cells. The csgA gene encodes an extracellular protein morphogen which is essential for fruiting body development. The question of whether fibrils are involved in the transmission of CsgA to adjacent cells was investigated in three ways. First, the dsp and csgA mutants were mixed in a ratio of 1:1 and allowed to develop; fruiting bodies containing spores derived from the csgA mutant were formed, suggesting efficient CsgA transfer. Second, the csgA mutation affected expression of many developmentally regulated genes differently from the way dsp affected their expression. Third, the expression of one developmentally regulated gene, which was partially expressed in csgA and dsp backgrounds, was almost completely inhibited in the presence of both mutations, suggesting that its promoter is regulated independently by two distinct stimuli, one that is csgA dependent and one that is dsp dependent. Together these results argue that fibrils are not necessary for cell-to-cell transmission or perception of CsgA, and their precise function remains unknown.     

An extracellular matrix-associated zinc metalloprotease is required for dilauroyl phosphatidylethanolamine chemotactic excitation in Myxococcus xanthus

An extracellular matrix connects bacteria that live in organized assemblages called biofilms. While the role of the matrix in the regulation of cell behavior has not been extensively examined in bacteria, we suggest that, like mammalian cells, the matrix facilitates cell-cell interactions involved with regulation of cohesion, motility, and sensory transduction. The extracellular matrix of the soil bacterium Myxococcus xanthus is essential for biofilm formation and fruiting body development. The matrix material is extruded as long, thin fibrils that mediate adhesion to surfaces, cohesion to other cells, and excitation by the chemoattractant dilauroyl phosphatidylethanolamine. We report the identification of a putative matrix-associated zinc metalloprotease called FibA (fibril protein A). Western blotting with FibA-specific monoclonal antibody 2105 suggests extensive proteolytic processing of FibA during assembly into fibrils, consistent with the autoprocessing observed with other members of the M4 metalloprotease family. Disruption of fibA had no obvious effect on the structure of the fibrils and did not inhibit cell cohesion, excitation by dioleoyl phosphatidylethanolamine, or activity of the A- or S-motility motors. However, the cells lost the ability to respond to dilauroyl phosphatidylethanolamine and to form well-spaced fruiting bodies, though substantial aggregation was observed. Chemotactic excitation of the fibA mutant was restored by incubation with purified wild-type fibrils. The results suggest that this metalloprotease is involved in sensory transduction.     

A Clp/Hsp100 chaperone functions in Myxococcus xanthus sporulation and self-organization

The Clp/Hsp100 proteins are chaperones that play a role in protein degradation and reactivation. In bacteria, they exhibit a high degree of pleiotropy, affecting both individual and multicellular phenotypes. In this article, we present the first characterization of a Clp/Hsp100 homolog in Myxococcus xanthus (MXAN_4832 gene locus). Deletion of MXAN_4832 causes defects in both swarming and aggregation related to cell motility and the production of fibrils, which are an important component of the extracellular matrix of a swarm. The deletion also affects the formation of myxospores during development, causing them to become sensitive to heat. The protein product of MXAN_4832 can act as a chaperone in vitro, providing biochemical evidence in support of our hypothesis that MXAN_4832 is a functional Clp/Hsp100 homolog. There are a total of 12 Clp/Hsp100 homologs in M. xanthus, including MXAN_4832, and, based on its mutational and biochemical characterization, they may well represent an important group.     

Cell surface properties correlated with cohesion in Myxococcus xanthus.

The gliding behavior of Myxococcus xanthus cells is controlled by two multigene systems, A and S, which encode information for adventurous and social behaviors, respectively. The S system can be genetically disrupted through mutation, such as a dsp mutation, or phenotypically disrupted by treating cells with the diazo dye Congo red (Arnold and Shimkets, J. Bacteriol. 170:5765-5770, 1988). One of the functions controlled by the S system is cell agglutination. Immediately after the induction of agglutination, wild-type cells begin to form aggregates, and within 30 min the cells are packed side-to-side in clumps containing thousands of cells. Changes in the cohesive properties of S+ cells are correlated with changes in the topology of the cell surface observed by electron microscopy. Two types of cell-associated appendages were observed on wild-type cells: thin filaments (ca. 5 nm in diameter), which have been called fimbriae or pili, at one cell pole, and thick, flaccid filaments (ca. 50 nm in diameter), referred to as fibrils, at both the sides and tips of cells. Cohesion was correlated with the secretion of the thick fibrils, which coat the cell surface and form an extracellular matrix in which the cells are interconnected. Several lines of evidence suggest that these thick fibrils are involved in cohesion. First, Dsp cells were unable to agglutinate or secrete this extracellular material. Second, wild-type cells which were treated with Congo red neither agglutinated nor secreted the extracellular fibrils. Finally, removal of the Congo red from wild-type cells restored cohesion and also restored production of the thick fibrils. Attempts to estimate the efficiency with which two cells cohered following collision suggested that under optimal conditions, one in three collisions resulted in stable contact. The collision efficiency decreased linearly as the cell density increased, suggesting a cell density-dependent regulation of cohesion. Some aspects of gliding behavior can be explained in terms of an inducer and an inhibitor of S motility.     

Cell surface modifications induced by calcium ion in the myxobacterium Stigmatella aurantiaca.

Calcium ion induces in the myxobacterium Stigmatella aurantiaca the ability to glide on solid surfaces and to become cohesive (D. F. Gilmore and D. White, J. Bacteriol. 161:113-117, 1985; B. J. Womack, D. F. Gilmore, and D. White, J. Bacteriol. 171:6093-6096, 1989). The addition of calcium ion to the growth medium resulted in the formation of extracellular fibrils, the appearance in the membrane fractions of a 30-kDa protein, and the accumulation in a low-speed centrifugal pellet of 10 polypeptides that cross-reacted with affinity-purified antibody to one of the polypeptides. One of the polypeptides, a 55-kDa protein, was present in the membrane fraction of control cells not incubated with calcium ion and was apparently translocated to the extracellular matrix during incubation in medium containing calcium ion. The 55-kDa protein was immunologically related to a 65-kDa protein located on the fibrils of another myxobacterium, Myxococcus xanthus.     

The Dif chemosensory pathway is directly involved in phosphatidylethanolamine sensory transduction in Myxococcus xanthus

Myxococcus xanthus cells glide on solid surfaces and are chemotactically stimulated by certain phosphatidylethanolamine species. The dif gene cluster consists of six genes, difABCDEG, five of which encode proteins homologous to known chemotaxis proteins. DifA and DifE are required for the biosynthesis of fibrils, an extracellular matrix comprised of polysaccharide and protein. Chemotactic stimulation by 1,2-O-Bis[11-(Z)-hexadecenoyl]-sn-glycero-3-phosphatidylethanolamine (16:1 PE) and dilauroyl PE (12:0 PE) requires fibrils. Although previous work has shown that difA and difE mutants are not stimulated by 12:0 PE, these results do not distinguish between a dependence on fibrils or a direct role in chemosensory transduction. Here we provide evidence that the Dif chemosensory pathway directly mediates PE sensory transduction. First, stimulation by and adaptation to 16:1 PE requires all of the dif genes, including difBDG, which are not essential for fibril biogenesis. Second, a specific residue within the first putative methylation domain of DifA is required for stimulation by 16:1 PE but not fibril biogenesis. Transmembrane signalling through a chimeric NarX-DifA chemoreceptor is required for fibril formation but not for stimulation by or adaptation to 16:1 PE. Third, difD and difE are required for stimulation by dioleoyl PE (18:1 PE) although the response does not require fibrils. Taken together these results argue that the Dif pathway mediates both matrix formation and lipid chemotaxis.     

An extracellular monoADP-ribosyl transferase activity in Entamoeba histolytica trophozoites

Due to the important role of monoADP-ribosyl transferases in physiological and pathological events, we investigated whether the protozoan parasite Entamoeba histolytica had monoADP-ribosyl transferase activity. Reactions were initiated using ameba-free medium as the source of both enzyme and ADP-ribosylation substrate(s) and [32P]NAD+ as source of ADP-ribose. Proteins were analyzed by electrophoresis, and [32P]-labeled proteins were detected by autoradiography. Using the crude extracellular medium, a major labeled product of Mr 37.000 was observed. The yield of this product was reduced markedly using medium from Brefeldin A-treated trophozoites, indicating that the extracellular monoADP-ribosyl transferase and/or its substrate depended on vesicular transport. The labeling of the 37-kDa substrate was dependent on reaction time, temperature, pH, and the ratio of unlabeled NAD+ to [32P]NAD+. After two purification steps, several new substrates were observed, perhaps due to their enrichment. The reaction measured ADP-ribosylation since [14C-carbonyl]NAD+ was not incorporated into ameba substrates and a 75-fold molar excess of ADP-ribose caused no detectable inhibition of the monoADP-ribosyl transferase reaction. On the basis of sensitivity to NH2OH, the extracellular monoADP-ribosyl transferase of E. histolytica may be an arginine-specific enzyme. These results demonstrate the existence in E. histolytica of at least one extracellular monoADP-ribosyl transferase, whose localization depends upon a secretion process.     

MglA,a small GTPase,interacts with a tyrosine kinase to control type IV pili-mediated motility and development of Myxococcus xanthus

The mglA gene encodes a 22 kDa GTPase that is critical for single-cell (A) gliding, type IV pili-mediated (S) gliding and development of Myxococcus xanthus. To identify components that interact with MglA to control these processes, second-site mutations that restore movement to non-motile mglA mutants were sought. An allele-specific extragenic suppressor of mglA8, named mas815 (mglA8 suppressor 15), was obtained. mas815 does not bypass the requirement for MglA, yet it restores type IV pili-mediated motility and starvation-induced development. Single-cell (A) motility is not restored. The suppressing mutation maps to the 3' end of a gene, masK, in an operon immediately upstream of the mglBA operon. masK encodes a protein of the STY kinase family. When the masK gene was used as bait against a library carrying M. xanthus DNA in the yeast two-hybrid system, eight positive, independent clones containing fusions of mglA to GAL4 were obtained, thus confirming the interaction between MglA and MasK. MasK, expressed in Escherichia coli, was shown to phosphorylate at a tyrosine residue(s). The gain-of-function in the masK815 mutant was correlated with increased production of extracellular fibrils, which are required for adhesion, cell-cell contact and sensing phosphatidylethanolamine chemoattractants. These data suggest that the interaction between MasK and MglA is an essential part of a signal transduction pathway controlling motility and development.     

Identification of esg, a genetic locus involved in cell-cell signaling during Myxococcus xanthus development.

JD258, a Tn5 insertion mutant of Myxococcus xanthus, was shown to have major defects in three development-associated properties: expression of the developmentally regulated tps gene, spore formation, and production of multicellular fruiting bodies. The defects in tps gene expression and sporulation could be substantially corrected, at the phenotypic level, by mixing JD258 with wild-type cells (extracellular complementation). By this criterion, JD258 appeared to be a new member of a group of conditional developmental mutants that were previously characterized and placed in four extracellular complementation groups (A to D) based on the ability of mutants in one group to stimulate development in mutants belonging to a different group (D. C. Hagen, A. P. Bretscher, and D. Kaiser, Dev. Biol. 64:284-296, 1978). Mutants from groups A, B, C, and D all displayed extracellular complementation activity when mixed with JD258. These results, and other aspects of the phenotype of JD258, indicate that this mutant defines a fifth extracellular complementation group, group E. The M. xanthus esg locus identified by the Tn5 insertion in JD258 was cloned in Escherichia coli and used for further genetic analysis of the locus. These studies indicated that the esg locus resides within a 2.5-kb region of the M. xanthus chromosome and that the locus contains at least two genetic complementation groups. Our results are consistent with a model in which the esg locus controls the production of a previously unrecognized extracellular signal that must be transmitted between cells for the completion of M. xanthus development.     

Separation of the 24 kDa substrate for botulinum C3 ADP-ribosyltransferase and the cholera toxin ADP-ribosylation factor

Botulinum C3 ADP-ribosyltransferase modifies a approximately 24 kDa membrane protein believed to bind guanine nucleotides. Cholera toxin ADP-ribosylation factors are approximately 19 kDa GTP-binding proteins that directly activate the toxin. To evaluate a possible relationship between C3 ADP-ribosyltransferase substrate and ADP-ribosylation factor, they were partially purified from bovine brain. ADP-ribosylation factor, but not C3 ADP-ribosyltransferase substrate, stimulated auto-ADP-ribosylation of the choleragen A1 subunit whereas C3 ADP-ribosyltransferase substrate, but not ADP-ribosylation factor, was ADP-ribosylated by C3 ADP-ribosyltransferase. Thus, although both may be GTP-binding proteins, no functional similarity between ADP-ribosylation factor and C3 ADP-ribosyltransferase substrate was found.     

Exopolysaccharide biosynthesis genes required for social motility in Myxococcus xanthus

Social (S)-motility in Myxococcus xanthus is a flagellum-independent gliding motility system that allows bacteria to move in groups on solid surfaces. S-motility has been shown to require type IV pili (TFP), exopolysaccharide (EPS; a component of fibrils) and lipopolysaccharide (LPS). Previously, information concerning EPS biogenesis in M. xanthus was lacking. In this study, we screened 5000 randomly mutagenized colonies for defects in S-motility and EPS and identified two genetic regions essential for EPS biogenesis: the EPS synthesis (eps) region and the EPS-associated (eas) region. Mutants with insertions in the eps and eas regions were defective in S-motility and fruiting body formation. These mutants failed to bind the dye calcofluor white, indicating that they lacked EPS; however, they retained normal TFP and LPS. Analysis of the eps locus showed several open reading frames (ORFs) that encode homologues to glycosyltransferases, glucanases and EPS transporters as well as regulatory proteins; the eas locus contains two ORFs: one exhibits homology to hypothetical proteins with a conserved domain of unknown function and the other displays no apparent homology to other proteins in the database. Further genetic mutagenesis analysis indicates that the whole eps region is involved in the biosynthesis of fibrils and fibril EPS. The operon at the proximal end of the eps region was analysed by generating in-frame deletion mutations. These mutants showed varying degrees of defects in the bacterium's ability to produce EPS or perform EPS-related functions, confirming the involvement of these genes in M. xanthus EPS biogenesis.     

Regulated exopolysaccharide production in Myxococcus xanthus

Myxococcus xanthus fibrils are cell surface-associated structures composed of roughly equal amounts of polysaccharide and protein. The level of M. xanthus polysaccharide production under different conditions in the wild type and in several mutants known to have alterations in fibril production was investigated. Wild-type exopolysaccharide increased significantly as cells entered the stationary phase of growth or upon addition of Ca2+ to growing cells, and the polysaccharide-induced cells exhibited an enhanced capacity for cell-cell agglutination. The activity of the key gluconeogenic pathway enzyme phosphoenolpyruvate carboxykinase (Pck) also increased under these conditions. Most fibril-deficient mutants failed to produce polysaccharide in a stationary-phase- or Ca2+-dependent fashion. However, regulation of Pck activity was generally unimpaired in these mutant strains. In an stk mutant, which overproduces fibrils, polysaccharide production and Pck activity were constitutively high under the conditions tested. Polysaccharide production increased in most fibril-deficient strains when an stk mutant allele was present, indicating that these fibril-deficient mutants retained the basic cellular components required for fibril polysaccharide production. In contrast to other divalent cations tested, Sr2+ effectively replaced Ca2+ in stimulating polysaccharide production, and either Ca2+ or Sr2+ was required for fruiting-body formation by wild-type cells. By using transmission electron microscopy of freeze-substituted log-phase wild-type cells, fibril material was observed as a cell surface-associated layer of uniform thickness composed of filaments with an ordered structure.