Reexamination of the role of autolysis in the development of Myxococcus xanthus.

It has been widely reported that 80 to 90% of the cell population undergoes autolysis during sporulation in Myxococcus xanthus. A re-evaluation of the techniques used to measure autolysis in M. xanthus showed that the methods previously used to draw this conclusion are subject to artifacts, which result in a substantial underestimation of the number of cells present during development. We found that at least 80% of the cells that enter development survive throughout fruiting body formation. The cell loss that did occur appeared to be gradual over a period of at least 7 days. Our results suggest that autolysis is not an obligate stage in the development of M. xanthus. The data also showed that sporulating cells pass through a prespore stage in which they become osmotically and physically fragile and therefore difficult to harvest intact. The fragility was correlated with the change from a rod to a spherical shape. As the prespores differentiated into refractile spores, they lost fragility and became amenable to harvesting by standard protocols.     

Iodination of Myxococcus xanthus during development

Intact cells of Myxococcus xanthus were iodinated with [125I]lactoperoxidase to permit examination of the surface components accessible to labeling during cell development. Vegetative cells, starved on a defined solid medium, aggregated, formed fruiting bodies, and produced myxospores. Cells collected at different stages were iodinated, and their proteins were analyzed by one- and two-dimensional electrophoresis and autoradiography. One-dimensional electrophoresis revealed six iodinated bands in vegetative cell extracts. During development, 10 radioactive bands were detected, 4 of which migrated to the same positions as those of vegetative cells. Only six bands were detected in purified, labeled myxospores. Of these, one band possessed mobility similar to that of labeled vegetative cell proteins, whereas the other bands possessed mobility similar to that detected in developing cells. Analysis of two-dimensional gels indicated that at least 14 proteins were iodinated in vegetative cells, one of which was intensely labeled (protein b). Another of the proteins (protein a) was labeled throughout development. During development, about 30 proteins were iodinated and the prominently labeled ones were designated c, d, e, f, and g. The latter two (proteins f and g) were not detected in purified, iodinated myxospores. The data indicated a pronounced change in surface structure during development; some of the change may be involved in cellular interaction during aggregation.     

BrgE is a regulator of Myxococcus xanthus development

We report here the identification and characterization of a member of the Myxococcus xanthus SdeK signal transduction pathway, BrgE. This protein was identified as an SdeK-interacting component using a yeast two-hybrid screen, and we further confirmed this interaction by the glutathione S-transferase (GST) pulldown assay. Additional yeast two-hybrid analyses revealed that BrgE preferentially interacts with the putative amino-terminal sensor domain of SdeK, but not with the carboxy-terminal kinase domain. A brgE insertion strain was shown to be blocked in development between aggregation and mound formation, and decreased by 50-fold in viable spore production compared with the parental wild type. These phenotypes are similar to those of sdeK mutants. The brgE mutation also altered expression of a sample of Tn5 lac developmental markers that are also SdeK regulated. Finally, we demonstrated that a brgE sdeK double mutant has a more severe sporulation defect than either of the two single mutants, suggesting that BrgE and SdeK act synergistically to regulate wild-type levels of sporulation. In sum, these data suggest that BrgE operates as an auxiliary factor to stimulate the SdeK signal transduction pathway by directly binding to the amino-terminal sensor domain of SdeK.     

Chemotaxis plays a role in the social behaviour of Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that glides on a solid surface and displays a wide range of social behaviour including microbial development. The frz genes are homologues to the chemotaxis genes of Escherichia coli and Salmonella typhimurium and have been shown to be involved in microbial development. However, chemotaxis has never been clearly demonstrated in Myxococcus. In this study, we showed that M. xanthus exhibited tactic movements to many chemicals when they were subjected to steep and stable chemical gradients. M. xanthus was observed to spread into areas with abundant nutrients like yeast extract or Casitone and avoid areas with no nutrients or repellents (short-chain alcohols or DMSO. Responses to attractants and repellents were additive. Movement towards attractants or away from repellents required the frz genes and was correlated with methylation or demethylation of FrzCD, a methyl-accepting taxis protein. Furthermore, the frz genes were found to be required for both fruiting body formation during starvation and swarming in nutrient-rich medium. In wild-type strains, cells near the colony edge were observed to swarm towards the surrounding growth medium and to contain highly methylated FrzCD; cells near the colony centre contained mainly demethylated FrzCD and showed directed movement towards the colony edge. FrzCD was also found to be methylated during the aggregation stage of fruiting body formation on agar but largely demethylated in cells shaken in liquid starvation media. An frzf mutant failed to exhibit directed cell movements and no longer showed modification of FrzCD under these conditions. These observations suggest that M. xanthus does show chemotactic movements, that these movements require the frz genes, and that chemotaxis plays a very important role in the social behaviour of this organism.     

Quantifying aggregation dynamics during Myxococcus xanthus development

Under starvation conditions, a swarm of Myxococcus xanthus cells will undergo development, a multicellular process culminating in the formation of many aggregates called fruiting bodies, each of which contains up to 100,000 spores. The mechanics of symmetry breaking and the self-organization of cells into fruiting bodies is an active area of research. Here we use microcinematography and automated image processing to quantify several transient features of developmental dynamics. An analysis of experimental data indicates that aggregation reaches its steady state in a highly nonmonotonic fashion. The number of aggregates rapidly peaks at a value 2- to 3-fold higher than the final value and then decreases before reaching a steady state. The time dependence of aggregate size is also nonmonotonic, but to a lesser extent: average aggregate size increases from the onset of aggregation to between 10 and 15 h and then gradually decreases thereafter. During this process, the distribution of aggregates transitions from a nearly random state early in development to a more ordered state later in development. A comparison of experimental results to a mathematical model based on the traffic jam hypothesis indicates that the model fails to reproduce these dynamic features of aggregation, even though it accurately describes its final outcome. The dynamic features of M. xanthus aggregation uncovered in this study impose severe constraints on its underlying mechanisms.     

Multicellular development and gliding motility in Myxococcus xanthus

A great deal of progress has been made in the studies of fruiting body development and social gliding in Myxocococcus xanthus in the past few years. This includes identification of the bone fide C-signal and a receptor for type IV pili, and development of a model for the mechanism of adventurous gliding motility. It is anticipated that the next few years will see even more progress as the complete genome sequence is available and genomic and proteomic tools are applied to the study of M. xanthus social behaviors.     

Using a phase-locked mutant of Myxococcus xanthus to study the role of phase variation in development.

The bacterium Myxococcus xanthus undergoes a primitive developmental cycle in response to nutrient deprivation. The cells aggregate to form fruiting bodies in which a portion of the cells differentiate into environmentally resistant myxospores. During the growth portion of the M. xanthus life cycle, the organism also undergoes a phase variation, in which cells alternate between yellow and tan colony-forming variants. Phase variation occurs in our laboratory strain (M102, a derivative of DK1622) at a frequency high enough that a single colony of either the yellow or the tan phase already contains cells of the alternate phase. In this study we demonstrate that tan cells within a predominantly yellow population of phase variation-proficient cells are preferentially recovered as heat- and sonication-resistant spores. To further investigate the possibility of a differential role of tan and yellow cells during development, a tan-phase-locked mutant was used to compare the developmental phenotypes of a pure tan population with a predominantly yellow, phase variation-proficient population. Pure tan-phase populations did not produce fruiting bodies or mature spores under conditions in which predominantly yellow wild-type populations did so efficiently. Pure populations of tan-phase cells responded to developmental induction by changing from vegetative rod-shaped cells to round forms but were unable to complete the maturation to heat- and sonication-resistant, refractile spores. The developmental defect of a tan-phase-locked mutant was rescued by the addition of phase variation-proficient cells from a predominantly yellow culture. In such mixtures the tan-phase-locked mutant not only completed the process of forming spores but also was again preferentially represented among the viable spores. These findings suggest the intriguing possibility that the tan-phase cells within the vegetative population entering development are the progenitors of spores and implicate a requirement for yellow-phase cells in spore maturation.     

Spatial organization of Myxococcus xanthus during fruiting body formation

Microcinematography was used to examine fruiting body development of Myxococcus xanthus. Wild-type cells progress through three distinct phases: a quiescent phase with some motility but little aggregation (0 to 8 h), a period of vigorous motility leading to raised fruiting bodies (8 to 16 h), and a period of maturation during which sporulation is initiated (16 to 48 h). Fruiting bodies are extended vertically in a series of tiers, each involving the addition of a cell monolayer on top of the uppermost layer. A pilA (MXAN_5783) mutant produced less extracellular matrix material and thus allowed closer examination of tiered aggregate formation. A csgA (MXAN_1294) mutant exhibited no quiescent phase, aberrant aggregation in phase 2, and disintegration of the fruiting bodies in the third phase.     

Rifampin-resistant mutants of Myxococcus xanthus defective in development.

Rifampin, an antibiotic which is known to bind to and inhibit RNA polymerase, was used to probe the molecular regulation of development in Myxococcus xanthus. Rifampin-resistant mutants were screened for defects in fruiting-body formation. About 20% of the isolates in the initial screenings showed major defects in developmental aggregation or sporulation. Eleven independent mutants with wild-type growth rates and stable phenotypes were analyzed by transduction. In these strains, the rifampin-resistant and nonfruiting phenotypes showed cotransduction frequencies equal to or greater than 99.0 to 99.9%. The RNA polymerase activities were resistant to rifampin in vitro, indicating that the RNA polymerase is altered in these strains. Although their fruiting phenotypes are heterogeneous, these strains can be divided into two classes based on the level of aggregation. The results suggest that RNA polymerase plays a significant role in the regulation of development in M. xanthus since mutations which cause no apparent changes in vegetative growth result in striking defects in fruiting-body formation.     

Methylation of macromolecules during development in Myxococcus xanthus.

Covalent modification of macromolecules can serve to alter their biological activities and is therefore frequently involved in regulation. I examined methylation of proteins and carbohydrates during development and vegetative growth in the procaryote Myxococcus xanthus. Striking differences in the patterns of protein methylation occurred when cell development was induced by nutrient deprivation on solid media and when cells were starved in liquid. In addition, a methylated, protease-resistant macromolecule which contained carbohydrate and which may have been an unusual type of lipopolysaccharide was observed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of methylation patterns in various media and an analysis of the time course of methylation indicated that changes in methylation were part of the developmental pathway which includes aggregation. Induction of development in liquid by glycerol produced no changes in methylation.     

Chemosensory pathways, motility and development in Myxococcus xanthus

The complex life cycle of Myxococcus xanthus includes predation, swarming, fruiting-body formation and sporulation. The genome of M. xanthus is large and comprises an estimated 7,400 open reading frames, of which approximately 605 code for regulatory genes. These include eight clusters of chemotaxis-like genes that define eight chemosensory pathways, most of which have dedicated functions. Although many of these chemosensory pathways have a role in controlling motility, at least two of these pathways control gene expression during development.     

Motility in Myxococcus xanthus and its role in developmental aggregation

The Frz signal transduction system of Myxococcus xanthus was originally thought to be a simple variation of the well-characterized Che system of the enteric bacteria. Recently, however, many additional Frz proteins, along with alternative signal transduction systems, have been discovered. Together these signal transduction pathways coordinate cell-cell behavior, permitting the complex interactions required for developmental aggregation and fruiting body formation.     

Novel lipids in Myxococcus xanthus and their role in chemotaxis

Organisms that colonize solid surfaces, like Myxococcus xanthus, use novel signalling systems to organize multicellular behaviour. Phosphatidylethanolamine (PE) containing the fatty acid 16:1omega5 (Delta11) elicits a chemotactic response. The phenomenon was examined by observing the effects of PE species with varying fatty acid pairings. Wild-type M. xanthus contains 17 different PE species under vegetative conditions and 19 at the midpoint of development; 13 of the 17 have an unsaturated fatty acid at the sn-1 position, a novelty among Proteobacteria. Myxococcus xanthus has two glycerol-3-phosphate acyltransferase (PlsB) homologues which add the sn-1 fatty acid. Each produces PE with 16:1 at the sn-1 position and supports growth and fruiting body development. Deletion of plsB1 (MXAN3288) results in more dramatic changes in PE species distribution than deletion of plsB2 (MXAN1675). PlsB2 has a putative N-terminal eukaryotic fatty acid reductase domain and may support both ether lipid synthesis and PE synthesis. Disruption of a single sn-2 acyltransferase homologue (PlsC, of which M. xanthus contains five) results in minor changes in membrane PE. Derivatization of purified PE extracts with dimethyldisulfide was used to determine the position of the double bonds in unsaturated fatty acids. The results suggest that Delta5 and Delta11 desaturases may create the double bonds after synthesis of the fatty acid. Phosphatidylethanolamine enriched for 16:1 at the sn-1 position stimulates chemotaxis more strongly than PE with 16:1 enriched at the sn-2 position. It appears that the deployment of a rare fatty acid (16:1omega5) at an unusual position (sn-1) has facilitated the evolution of a novel cell signal.     

Autocide AMI rescues development in dsg mutants of Myxococcus xanthus

Low concentrations of autocide AMI rescued aggregation and sporulation in the dsg mutant class of Myxococcus xanthus but were incapable of rescuing asg, bsg, or csg mutants. AMI-induced spores of dsg mutants were resistant to heat and sonication and germinated when plated on nutrient-rich agar. AMI accelerated aggregation and sporulation and increased the final spore number in submerged cultures of a wild-type strain of M. xanthus. Development of M. xanthus was accompanied by release of a fluorescent material (emission maximum, 438 nm) into the supernatant fluid. The release of this material began early and continued throughout development. All Spo- mutant strains tested released significantly reduced levels of this material. These levels were increased in the presence of AMI in all Spo- mutant classes, most dramatically in the dsg mutants.     

Role of autocide AMI in development of Myxococcus xanthus.

A new developmental mutant of Myxococcus xanthus has been isolated by screening TnV insertion mutants for AMI-dependent development in submerged culture. This mutant (ER304) aggregated and sporulated on agar surfaces but required at least 3.8 micrograms of autocide AMI per ml for development in submerged cultures. Spore rescue of ER304 was obtained with the saturated, monounsaturated, and diunsaturated fatty acid fractions of AMI, with specific activities of 68, 115, and 700 U/mg, respectively. In addition, several model fatty acids were capable of rescuing sporulation of ER304; however, there was no correlation between specific lytic activity observed in vegetative cultures and specific rescue activity. Rescue of ER304 was effected during the first ca. 12 h after the initiation of starvation conditions; after this time, addition of AMI or model fatty acids killed the cells. Supernatant fluids of ER304 rescued development in dsg mutants (e.g., DK3260) in submerged cultures, but dsg mutant supernatant fluids were incapable of rescuing ER304 development. The data presented in this article support the idea that the primary mechanism of rescue by AMI is not via lysis, although developmental lysis may be an indirect result of the rescue event. A membrane permeability model is presented to explain the role of autocides in early developmental events in wild-type strains and in the aggregation and sporulation rescue of developmental mutants ER304 and DK3260.