Xanthus Cells Research Articles

A new set of chemotaxis homologues is essential for Myxococcus xanthus social motility.

Myxococcus xanthus cells aggregate and develop into multicellular fruiting bodies in response to starvation. A new M. xanthus locus, designated diffor defective in fruiting, was identified by the characterization of a mutant defective in fruiting body formation. Molecular cloning, DNA sequencing and sequence analysis indicate that the dif locus encodes a new set of chemotaxis homologues of the bacterial chemotaxis proteins MCPs (methyl-accepting chemotaxis proteins), CheW, CheY and CheA. The dif genes are distinct genetically and functionally from the previously identified M. xanthus frz chemotaxis genes, suggesting that multiple chemotaxis-like systems are required for the developmental process of M. xanthus fruiting body formation. Genetic analysis and phenotypical characterization indicate that the M. xanthus dif locus is required for social (S) motility. This is the first report of a M. xanthus chemotaxis-like signal transduction pathway that could regulate or co-ordinate the movement of M. xanthus cells to bring about S motility.

Struvite crystallization on Myxococcus cells

It is an already well established and experimentally proven fact that bacterial structures can participate in extra or intracellular accumulation of inorganic material. Myxoccocus coralloides and M. xanthus cells from cultures of 48 h or more presented signs of globular deformities. Further study of these deformities through transmission electron microscopy indicated that small crystalline forms of struvite were present but were sometimes destroyed by the mere impact of the electron beam.

Penicillin and D-Alanyl-D-alanine Accelerate Spore Formation of Myxococcus xanthus Subcultured Cells.

In repetitive subcultures of Myxococcus xanthus cells, addition of penicillin to the developmental medium accelerated the appearance of the fruiting bodies and spore formation. During development of M. xanthus, DD-carboxypeptidase, which functions in the regulation of the level of cross-linkage, was also induced by penicillin. When D-alanyl-D-alanine, the structure of which is similar to the β-lactam structure of penicillin, was added to the developmental medium, 0.3 to 0.6 mM D-alanyl-D-alanine accelerated spore formation strongly. The activity of D-alanyl-D-alanine ligase, which synthesizes D-alanyl-D-alanine from D-alanine, increased gradually with maximum activity observed at the initial stage of sporulation. These results raise the hypothesis that M. xanthus produces and releases D-alanyl-D-alanine during development, and the released D-alanyl-D-alanine may function as an inducer of sporulation.

In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing sigmaA, the major sigma factor in growing cells.

Myxococcus xanthus is a Gram-negative bacterium that undergoes multicellular development upon starvation. We have developed a simple and rapid procedure for partial purification of RNA polymerase from growing M. xanthus cells, using heparin-agarose and DNA-cellulose chromatographies. In addition to core subunits, the enzyme contains one fairly abundant polypeptide of approximately 105 kDa. We have shown by Western blot analysis and protein sequencing that the 105-kDa polypeptide is sigmaA, the product of the M. xanthus sigA gene. Partially purified sigmaA RNA polymerase, or holoenzyme reconstituted from sigmaA and core RNA polymerase, transcribed in vitro the vegA and aphII genes that are known to be expressed in growing M. xanthus cells. Reconstituted sigmaA RNA polymerase produced vegA mRNA in vitro with the same 5' end as vegA mRNA produced in vivo, demonstrating that initiation of transcription was accurate in vitro. These results provide biochemical evidence that sigmaA is the major vegetative sigma factor of M. xanthus. To our knowledge, this is the first report of in vitro transcription of M. xanthus chromosomal genes, providing a foundation for further biochemical analysis of transcriptional regulatory mechanisms in a microbe that relies extensively on cell-cell interactions.

Identification and localization of the Tgl protein, which is required for Myxococcus xanthus social motility.

Tgl protein is required for the production of the type IV pili found at a pole of the Myxococcus xanthus cell. These pili are essential for social motility. Evidence is presented that Tgl is a membrane protein, based on experiments with polyclonal antibody specific for Tgl that was raised against the fusion proteins beta-galactosidase-Tgl and TrpE-Tgl. Immunoaffiity-purified antibody reacted with a protein in M. xanthus having an apparent molecular mass of 27.5 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sequence of the tgl gene translates into a polypeptide of 27 kDa. Although these numbers are close, it is likely that the primary tgl translation product is processed and modified in M. xanthus. The N terminus has a signal peptidase II recognition sequence, cleavage of which is expected to remove 19 amino acid residues. When the tgl gene is expressed in Escherichia coli, the protein product consistently migrates faster in the gel than mature Tgl expressed in M. xanthus, suggesting a second modification by addition which slows migration of the protein from M. xanthus. Tgl, as detected by its specific antibody, sediments with the membrane fraction of cells. It can be extracted with detergents but not with salt or by the addition of chelators for divalent cations. In an equilibrium gradient, Tgl bands at the buoyant density of membranes and with the NADH-oxidase activity. Intact cells failed to bind anti-Tgl antibody, and less than 2% of the total Tgl is released in soluble form from the periplasm. Yet, cells that had been osmotically shocked and treated with paraformaldehyde were able to react with the specific antibody--a reaction absent from cells with a deletion of the tgl transcription unit. Assuming that osmotic shock disrupts the outer membrane, the fractionation and localization data imply that Tgl is attached to the inner or outer membranes, from which it is exposed to the intermembranous space. Tgl is necessary for synthesis of pili in M. xanthus and is the only pilus protein that can be donated by other cells (stimulation). Tgl contains six tandem copies of the tetratrico peptide repeat structural motif. Its membrane localization, capacity for stimulation, and content of tetratrico structural repeats together suggest that Tgl may be necessary for the assembly of pilin subunits into filaments.

FruA, a putative transcription factor essential for the development of Myxococcus xanthus.

A new developmental gene, fruA, of Myxococcus xanthus was cloned using a one-step cloning vector, TnV. DNA sequencing of the wild-type allele of the fruA gene indicated that the fruA gene encodes a protein of 229 amino acid residues with a calculated molecular weight of 24672. The deduced amino acid sequence of FruA protein showed similarity to those of many bacterial regulatory proteins carrying a DNA-binding helix-turn-helix motif. The transcription-initiation site of the fruA gene was determined by a primer-extension experiment. Development of M. xanthus cells with a disrupted fruA gene stopped at the stage of mound formation. Although cells were able to aggregate to form mounds, myxospores were not formed. By Northern and Western blot analysis, it was found that the fruA expression was not detected during vegetative growth but initiated at around 6 h and reached the highest level at 12 h after the onset of development. Expression of the fruA gene was dependent on the expression of asg, bsg, csg, dsg, and esg genes, indicating that a series of intercellular signalling is necessary for the expression of the fruA gene. The effects of the fruA mutation on beta-galactosidase expression of various developmentally regulated genes fused with the lacZ gene were analysed; three developmental lacZ fusions (omega 4469, omega 4273 and omega 4500) were either poorly induced or not induced at all, while three other lacZ fusions (omega 4408, omega 4521 and omega 4455) expressed at the early stage of development were normally induced but were unable to be repressed at a later stage of development as in the wild-type strain. Interestingly, in the fruA mutant, tps (the gene for protein S) was not activated. From these results together with analysis of the amino acid sequence of FruA, we propose that FruA is a putative transcription factor required for the development of M.xanthus.

Cell density regulates cellular reversal frequency in Myxococcus xanthus.

Myxococcus xanthus is a Gram-negative bacterium that aggregates to form fruiting bodies when nutrients are limiting. Previous studies showed that the frz mutants that are defective in chemotaxis exhibited irregular and infrequent patterns of cellular reversal. In contrast, wild-type cells, when examined individually, reverse relatively frequently, about once every 6 min. It is not known how the change of reversal frequency effects cellular aggregation during fruiting body formation in M. xanthus. In this study, we stained cells with a tetrazolium dye so that we could track the reversal frequencies of single cells and cells in groups. We found that developmental cells in large groups reverse much less than cells in small groups or as single cells. This reduced cellular reversal frequency is related to the frz signal transduction system and correlated with the methylation of FrzCD (a methyl-accepting chemotaxis protein). Cells containing a mutation in the frz genes or in the genes required for social motility do not respond in this way. The reduction in cellular reversals as developmental cells accumulate in groups suggests a simple hypothesis for the aggregation of cells into discrete mounds during fruiting body formation. We also found that M. xanthus cells glide with equal frequency in the forward or reverse directions, indicating that cells do not contain a "head" or "tail."

Behavioral analysis of single cells of Myxococcus xanthus in response to prey cells of Escherichia coli.

Myxococcus xanthus cells move over surfaces by gliding motility. The frz signal transduction system is used to control the reversal frequency, and thus the overall direction of movement of M. xanthus cells. We analyzed the behavior of wild-type and frz mutant cells in response to prey bacteria (Escherichia coli). Wild-type cells of M. xanthus did not respond to microcolonies of E. coli until they made physical contact. Cells which penetrated a colony remained in the colony until all of the prey cells were digested. Cells of frz mutants also penetrated E. coli microcolonies and digested some of the E. coli cells, but they invariably abandoned the microcolony leaving their food source behind. These observations illustrate the importance of the frz system of signal transduction for the feeding behavior of M. xanthus cells.

Behavioral analysis of single cells of Myxococcus xanthus in response to prey cells of Escherichia coli

Myxococcus xanthus cells move over surfaces by gliding motility. The frz signal transduction system is used to control the reversal frequency, and thus the overall direction of movement of M. xanthus cells. We analyzed the behavior of wild-type and frz mutant cells in response to prey bacteria ( Escherichia coli). Wild-type cells of M. xanthus did not respond to microcolonies of E. coli until they made physical contact. Cells which penetrated a colony remained in the colony until all of the prey cells were digested. Cells of frz mutants also penetrated E. coli microcolonies and digested some of the E. coli cells, but they invariably abandoned the microcolony leaving their food source behind. These observations illustrate the importance of the frz system of signal transduction for the feeding behavior of M. xanthus cells.

Gliding movements in Myxococcus xanthus.

Prokaryotic gliding motility is described as the movement of a cell on a solid surface in the direction of the cell's long axis, but its mechanics are unknown. To investigate the basis of gliding, movements of individual Myxococcus xanthus cells were monitored by employing a video microscopy method by which displacements as small as 0.03 micron could be detected and speeds as low as 1 micron/min could be resolved. Single cells were observed to glide with speeds varying between 1 and 20 microns/min. We found that speed variation was due to differences in distance between the moving cell and the nearest cell. Cells separated by less than one cell diameter (0.5 micron) moved with an average speed of 5.0 micron/min, whereas cells separated by more than 0.5 micron glided with an average speed of 3.8 microns/min. The power to glide was found to be carried separately at both ends of a cell.

Branched‐chain fatty acids: the case for a novel form of cell‐cell signalling during Myxococcus xanthus development

The esg locus is required for the formation of multicellular fruiting bodies and spores by the developmental bacterium Myxococcus xanthus. Studies have suggested that esg mutants are defective in the production of an essential signal (E-signal) used in cell-cell communication and that E-signalling is required for the expression of many developmental genes. Recently we have determined that the esg locus encodes components of a branched-chain keto acid dehydrogenase, a multienzyme complex involved in branched-chain amino acid metabolism in many bacteria and higher organisms. During vegetative growth in M. xanthus, this enzyme complex appears to participate in the production of the branched-chain fatty acids found in this organism. M. xanthus fatty acids (including the branched-chain fatty acids) have been observed to have a variety of effects on developing cells. These effects include: (i) the lysis of M. xanthus cells (autocide activity), (ii) acceleration of the rate of sporulation and (iii) rescue of sporulation by certain development-defective mutants. These and other results suggest a model in which the branched-chain fatty acids, synthesized during growth, are released from cellular phospholipid by a developmentally regulated phospholipase during fruiting-body formation. This model proposes that one or more of the branched-chain fatty acids that are released constitutes the E-signal which must be transmitted between cells to complete M. xanthus development.

Intercellular C-signaling and the traveling waves of Myxococcus.

Early in their development into fruiting bodies, Myxococcus xanthus cells organize themselves into dense bands that move as trains of traveling waves. C-factor, a 20-kD cell-surface bound protein, is a short-range developmental signal molecule required for these waves. What is the role of C-factor in the wave pattern? It is proposed that oriented collisions between cells initiate C-signaling, which, in turn, causes individual cells to reverse their direction of gliding. Cells would move about one wavelength and then reverse. Several lines of experimental evidence support these proposals: (1) Cells that suffered a mutation in the signal transduction pathway that controls the spontaneous reversal frequency lost the ability to form waves; (2) presentation of developing cells with detergent-solubilized C-factor increased the mean frequency of single cell reversal by three-fold; and (3) fluorescently labeled cells in the waves were tracked, and it was found that they moved and reversed on linear paths along the axis of wave propagation. Similar numbers of cells were found moving in the direction of ripple propagation, and in the reverse direction, as expected. (4) Dilution of C-signaling-competent cells with C-factor-deficient cells increased the wavelength as the probability of productive collision decreased. The waves exemplify a way that a multicellular pattern of stripes can be produced de novo, one that maintains a uniform 50-microns separation between stripes over a distance as large as 1 cm.

Endogenous ADP-ribosylation during development of the prokaryote Myxococcus xanthus.

We examined endogenous ADP-ribosylation of proteins during the development of the prokaryote Myxococcus xanthus. In vivo and in vitro endogenous ADP-ribosylation of M. xanthus proteins was detected and the profile of modified proteins changed during development. Adenosine and nicotinamide inhibited ADP-ribosylation. Nicotinamide stimulated cells at low density to develop, in a manner similar to that previously observed with adenosine. Higher concentrations of nicotinamide inhibited aggregation. The in vivo effects of nicotinamide on developing M. xanthus cells correlate with its in vitro effects on ADP-ribosylation and the developmental profile of putative ADP-ribosylation substrates. These results suggest that ADP-ribosylation may regulate developmental proteins in M. xanthus.

Genes required for both gliding motility and development in Myxococcus xanthus

Myxococcus xanthus cells can glide both as individual cells, dependent on Adventurous motility (A motility), and as groups of cells, dependent upon Social motility (S motility). Tn5-lac mutagenesis was used to generate 16 new A- and nine new S- mutations. In contrast with previous results, we find that subsets of A- mutants are defective in fruiting body morphogenesis and/or myxospore differentiation. All S- mutants are defective in fruiting body morphogenesis, consistent with previous results. Whereas some S- mutants produce a wild-type complement of spores, others are defective in the differentiation of myxospores. Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis. Subsets of both A and S genes are essential for sporulation. Three S::Tn5-lac insertions result in surprising phenotypes. Colonies of two S- mutants glide on 'swim' (0.35% agar) plates to form fractal patterns. These S- mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading. An otherwise wild-type strain with one S- insertion resembles the frz- sglA1- mutants upon development, suggesting that this S- gene defines a new chemotaxis component in M. xanthus.

A genetic link between light response and multicellular development in the bacterium Myxococcus xanthus.

The Gram-negative bacterium Myxococcus xanthus responds to blue light by producing carotenoid pigments (Car+ phenotype). Genes for carotenoid synthesis lie at two unlinked chromosomal sites, the carC and the carBA operon, but are integrated in a single "light regulon" by the action of common trans-acting regulatory elements. Three known regulatory genes are grouped together at the (light-inducible) carQRS operon. By screening the Car phenotype of a large collection of transposon-induced mutants, we have identified a new car locus that has been named carD (carD1 for the mutant allele). The carD gene product plays a critical role in the light regulon, as it is required for activation of the carQRS and carC promoters by blue light. The carD1 mutant is impaired in the (starvation-induced) developmental process that allows M. xanthus cells both to form multicellular fruiting bodies and to sporulate. Our results indicate that the carD gene product is also required for the expression of a particular set of development-specific genes that are normally activated through the action of intercellular signals.

Sensory adaptation during negative chemotaxis in Myxococcus xanthus

Myxococcus xanthus exhibits many tactic movements that require the frz signal transduction system, such as colony swarming and cellular aggregation during fruiting body formation. Previously we demonstrated that the Frz proteins control the chemotactic movements of M. xanthus (W. Shi, T. Köhler, and D. R. Zusman, Mol. Microbiol. 9:601-611, 1993). However it was unclear from that study how chemotaxis might be achieved at the cellular level. In this study, we showed that M. xanthus cells not only modulate the reversal frequency of cell movement in response to repellent stimuli but also exhibit sensory adaptation in response to the continuous presence of nonsaturating repellent stimuli. The sensory adaptation behavior requires FrzF (a putative methyltransferase) and is correlated with the methylation-demethylation of FrzCD, a methyl-accepting chemotaxis protein. These results indicate that negative chemotaxis in M. xanthus is achieved by chemokinesis plus sensory adaptation in a manner analogous to that of the free-swimming enteric bacteria.

Beta-galactosidase activity in single differentiating bacterial cells.

Myxococcus xanthus strains containing transcriptional fusions to lacZ were analyzed and fractionated by differences in their levels of beta-galactosidase expression. The fluorogenic substrate for beta-galactosidase, fluorescein di-beta-galactopyranoside, was introduced into M. xanthus cells during a rapid decrease in osmolarity of the medium followed by a return to isoosmolarity. Fluorescein, the product of hydrolysis, was retained within the cells and their viability was preserved. Fluorescence increased linearly with time and was proportional to beta-galactosidase activity. beta-Galactosidase expression in most fusion strains, though beginning at different phases of growth or development, was distributed unimodally amongst cells. However, fusion strain Tn5 lac omega 4473 was shown to be heterogeneous at 9 hr of development. It was possible to separate physically cells that expressed beta-galactosidase at a high level from other, still viable, cells with no expression. The approach described here could be adapted to study differentiation in plants and animals as well, where transcriptional fusions and fluorogenic substrates for enzyme probes of gene expression also can be used.

Spatial restriction of cellular differentiation.

Myxococcus xanthus cells differentiate into myxospores within a fruiting body, an aggregate of approximately 10(5) cells. Previous work had discerned an inner and outer domain within the fruiting body differentiated by cell density and cell alignment. To test whether the two domains might play different roles in spore differentiation, developmentally regulated gene fusions were screened for expression restricted to one domain or the other. Transcriptional lacZ fusions to 80 developmentally regulated genes were examined and eight fusions were found that restricted expression to the inner domain, while one fusion, omega 7621, showed initial expression in the outer domain. Initial omega 7621 expression coincided with patches of spore precursors evident in bright-field microscopy. Later in development, both omega 7621 expression and the patches expanded inward, eventually filling both the inner and outer domains. Previous work had also shown that high cell density and cell alignment are required for transmission of the C-signal, which is needed to initiate spore differentiation. Evidence is presented for a novel morphogenetic mechanism in which C-signaling in the outer (high density) domain initiates spore differentiation. It is proposed that spore precursors are passively transported from the outer to the inner domain by the movements of undifferentiated rod cells. Reconstruction experiments showed that developing rod cells move with sufficient force to displace spores. Spore precursors thus accumulate in the inner domain where they express spore-specific genes at high levels and account for inner domain specific expression.

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.

CONTROL OF CELL DENSITY AND PATTERN BY INTERCELLULAR SIGNALING IN MYXOCOCCUS DEVELOPMENT

Myxococcus xanthus cells feed, move, and develop cooperatively. Genetic, biochemical, and cell mosaic studies demonstrate that cells coordinate their multicellular behavior by transmission of intercellular signals. Starvation for amino acids at sufficiently high density on a solid surface initiates a series of events culminating in the formation of a multicellular structure called a fruiting body filled with dormant, environmentally resistant spores. This review discusses how myxobacteria use extracellular signals to sequentially check the density and arrangement of cells at different stages during development. For at least one early and one late developmental signal, cell density determines the efficiency of intercellular signaling. In turn, proper signaling insures that the appropriate cell density exists, thus controlling the progress of multicellular development in M. xanthus.