Developmentally induced autolysis during fruiting body formation by Myxococcus xanthus.

The developmental events during fruiting body construction by the myxobacterium M. xanthus is an orderly process characterized by several sequential stages: growth leads to aggregation leads to formation of raised, darkened mounds of cells leads to autolysis leads to myxospore induction. The temporal sequence of autolysis followed by myxospore induction is consistent with the interpretation that developmental autolysis provides essential requirements for the surviving cells to induce to myxospores. At intermediate developmental times on agar plates a fraction of the cell population is irreversibly committed to lyse; i.e., lysis continues in liquid growth medium or in magnesium-phosphate buffer. Lysis is cell concentration independent and is therefore likely to be by an autolytic mechanism. The lysis sequence can be preliminarily characterized as having an early stage during which deoxyribonucleic acid synthesis continues and a later irreversible stage during which deoxyribonucleic acid synthesis does not occur. Irreversible lysis in liquid growth medium or in magnesium-phosphate buffer is initiated on agar plates during nutrient deprivation and such lysis results in the induction of a fraction of the population to myxospores. This induction is dependent upon the concentration of lysis products, thus providing evidence that developmentally induced autolysis is required for myxospore induction.     

Rippling is a predatory behavior in Myxococcus xanthus

Cells of Myxococcus xanthus will, at times, organize their movement such that macroscopic traveling waves, termed ripples, are formed as groups of cells glide together on a solid surface. The reason for this behavior has long been a mystery, but we demonstrate here that rippling is a feeding behavior which occurs when M. xanthus cells make direct contact with either prey or large macromolecules. Rippling has been observed during two fundamentally distinct environmental conditions: (i) starvation-induced fruiting body development and (ii) predation of other organisms. Our results indicate that case (i) does not occur in all wild-type strains and is dependent on the intrinsic level of autolysis. Analysis of predatory rippling indicates that rippling behavior is inducible during predation on proteobacteria, gram-positive bacteria, yeast (such as Saccharomyces cerevisiae), and phage. Predatory efficiency decreases under genetic and physiological conditions in which rippling is inhibited. Rippling will also occur in the presence of purified macromolecules such as peptidoglycan, protein, and nucleic acid but does not occur in the presence of the respective monomeric components and also does not occur when the macromolecules are physically separated from M. xanthus cells. We conclude that rippling behavior is a mechanism utilized to efficiently consume nondiffusing growth substrates and that developmental rippling is a result of scavenging lysed cell debris.     

Autocides produced by Myxococcus xanthus.

Ethanol extracts of Myxococcus xanthus contained several substances, referred to as autocides, which were bactericidal to the producing strain but showed no activity against other bacteria. The autocides were produced by growing cells and remained largely cell bound throughout the growth cycle; ca. 5% of the autocidal activity was found in the supernatant fluid at the time cell lysis began. The autocides were separated by sequential-column and thin-layer chromatography into five active fractions (AM I through AM V). Each of the fractions was at least 20 times more active against M. xanthus than against the other gram-negative or gram-positive bacteria tested. AM I, AM IV, and AM V were inactive against yeasts, whereas a mixture of fractions AM II and AM III was active against Rhodotorula sp. At low concentrations, AM I reversibly inhibited the growth of M. xanthus; at higher concentrations of AM I, the cells lysed within 1 h. The lowest concentration of AM IV that showed any activity caused rapid cell death and lysis. The mode of action of the major autocide, AM V, was different from that of AM I and AM IV. During the initial 2 h of treatment, the viable count of M. xanthus cells remained constant; during the next few hours killing occurred without lysis; within 24 h lysis was complete. The autocidal activity of each of the fractions was expressed when the cells were suspended in buffer, as well as in growth medium. The possible role of autocides in developmental lysis of M. xanthus is discussed.     

Gene expression during development of Myxococcus xanthus. Analysis of the genes for protein S

Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10(3) base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of beta-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.     

A-signalling and the cell density requirement for Myxococcus xanthus development.

Mutations in any of three asg (A-signalling) loci cause fruiting body development of Myxococcus xanthus to arrest at about the 2-h stage. Development can be restored to asg mutants by the addition of conditioned buffer in which wild-type cells have been developing or of A-factor purified from the conditioned buffer. Two forms of A-factor have been identified: heat-stable A-factor, which is composed of amino acids and peptides, and heat-labile A-factor, which consists of at least two proteases. A-factor is found in conditioned buffer in rough proportion to the cell density. As decreasing amounts of either form of A-factor are added, the developmental response of asg cells decreases until a threshold concentration is reached, below which no response is detected. In addition, wild-type cells fail to develop when their density is decreased below the point at which the level of A-factor is predicted to fall short of this threshold. The development of low-density asg+ cells can, however, be restored by the addition of either form of A-factor. These experiments show that A-factor is important for the development of wild-type cells. Moreover, the development of an asgB mutant that produces 5 to 10% the wild-type level of A-factor can be restored when the cell density is increased 10-fold above the standard density. We propose that the A-signal is used by M. xanthus to specify the minimum cell density required for the initiation of development. Differences in the response to A-factor between different asg mutants suggest that the different asg loci govern A-factor production in diverse ways.     

Induction of beta-lactamase influences the course of development in Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium that develops in response to starvation on a solid surface. The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores. Previously, we have shown that the induction of beta-lactamase is associated with starvation-independent sporulation in liquid culture (K. A. O'Connor and D. R. Zusman, Mol. Microbiol. 24:839-850, 1997). In this paper, we show that the chromosomally encoded beta-lactamase of M. xanthus is autogenously induced during development. The specific activity of the enzyme begins to increase during aggregation, before spores are detectable. The addition of inducers of beta-lactamase in M. xanthus, such as ampicillin, D-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells. In addition, the exogenous induction of beta-lactamase allows M. xanthus to fruit on media containing concentrations of nutrients that are normally too high to support development. We propose that the induction of beta-lactamase is an integral step in the development of M. xanthus and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores. In support of this hypothesis, we show that exogenous induction of beta-lactamase can rescue aggregation and sporulation of certain mutants. Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy. These results show that the signal transduction pathway leading to the induction of beta-lactamase plays an important role in aggregation and sporulation in M. xanthus.     

The lonD gene is homologous to the lon gene encoding an ATP-dependent protease and is essential for the development of Myxococcus xanthus.

Myxococcus xanthus contains two genes (lonV and lonD) homologous to the Escherichia coli lon gene for an ATP-dependent protease. We found that the lonD gene encodes a 90-kDa protein consisting of 827 amino acid residues. The lonD gene product shows 49, 48, and 52% sequence identity to the products of the M. xanthus lonV, E. coli lon, and Bacillus brevis lon genes, respectively. When a lonD-lacZ fusion was used, lonD was expressed during both vegetative growth and development. However, while lonD-disrupted strains were able to grow normally vegetatively, the development of M. xanthus was found to be arrested at an early stage in these strains. The mutant strains were able to form neither fruiting bodies nor myxospores.     

Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth.

The lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation. A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E. coli as a probe. The nucleotide sequence of the M. xanthus lon gene was determined. It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues. The deduced amino acid sequence of the M. xanthus lon gene product showed 60 and 56% identity with those of the E. coli and Bacillus brevis lon gene products, respectively. Analysis of an M. xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M. xanthus. In contrast to that of E. coli, the M. xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.     

Experimentally guided computational model discovers important elements for social behavior in myxobacteria

Identifying essential factors in cellular interactions and organized movement of cells is important in predicting behavioral phenotypes exhibited by many bacterial cells. We chose to study Myxococcus xanthus, a soil bacterium whose individual cell behavior changes while in groups, leading to spontaneous formation of aggregation center during the early stage of fruiting body development. In this paper, we develop a cell-based computational model that solely relies on experimentally determined parameters to investigate minimal elements required to produce the observed social behaviors in M. xanthus. The model verifies previously known essential parameters and identifies one novel parameter, the active turning, which we define as the ability and tendency of a cell to turn to a certain angle without the presence of any obvious external factors. The simulation is able to produce both gliding pattern and spontaneous aggregation center formation as observed in experiments. The model is tested against several known M. xanthus mutants and our modification of parameter values relevant for the individual mutants produces good phenotypic agreements. This outcome indicates the strong predictive potential of our model for the social behaviors of uncharacterized mutants and their expected phenotypes during development.     

Site-specific integration and expression of a developmental promoter in Myxococcus xanthus.

A series of intercellular signals are involved in the regulation of gene expression during fruiting body formation of Myxococcus xanthus. Mutations which block cell interactions, such as csgA (formerly known as spoC), also prevent expression of certain developmentally regulated promoters. csgA+ cells containing Tn5 lac omega DK4435, a developmentally regulated promoter fused to lacZ, began synthesizing lacZ mRNA 12 to 18 h into the developmental cycle. beta-Galactosidase specific activity increased about 12 h later. Neither lacZ mRNA nor beta-galactosidase activity was detected in a developing csgA mutant containing omega DK4435. The developmental promoter and its fused lacZ reporter gene were cloned into a pBR322-derived plasmid vector containing a portion of bacteriophage Mx8. These plasmids preferentially integrated into the M. xanthus chromosome by site-specific recombination at the bacteriophage Mx8 attachment site and maintained a copy number of 1 per chromosome. The integrated plasmids were relatively stable, segregating at a frequency of 0.0007% per generation in the absence of selection. The cloned and integrated promoter behaved like the native promoter, expressing beta-galactosidase at the proper time during wild-type development and failing to express the enzyme during development of a csgA mutant. The overall level of beta-galactosidase expression in merodiploid cells containing one native promoter and one promoter fused to lacZ was about half that of cells containing a single promoter fused to lacZ. These results suggest that the timing of developmentally regulated gene expression is largely independent of the location of this gene within the chromosome. Furthermore, they show that site-specific recombination can be a useful tool for establishing assays for promoter or gene function in M. xanthus.     

Lipid body formation plays a central role in cell fate determination during developmental differentiation of Myxococcus xanthus

Cell differentiation is widespread during the development of multicellular organisms, but rarely observed in prokaryotes. One example of prokaryotic differentiation is the Gram-negative bacterium Myxococcus xanthus . In response to starvation, this gliding bacterium initiates a complex developmental programme that results in the formation of spore-filled fruiting bodies. How the cells metabolically support the necessary complex cellular differentiation from rod-shaped vegetative cells into spherical spores is unknown. Here, we present evidence that intracellular lipid bodies provide the necessary metabolic fuel for the development of spores. Formed at the onset of starvation, these lipid bodies gradually disappear until they are completely used up by the time the cells have become mature spores. Moreover, it appears that lipid body formation in M. xanthus is an important initial step indicating cell fate during differentiation. Upon starvation, two subpopulations of cells occur: cells that form lipid bodies invariably develop into spores, while cells that do not form lipid bodies end up becoming peripheral rods, which are cells that lack signs of morphological differentiation and stay in a vegetative-like state. These data indicate that lipid bodies not only fuel cellular differentiation but that their formation represents the first known morphological sign indicating cell fate during differentiation.     

Behavior of peripheral rods and their role in the life cycle of Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium with a complex life cycle including a developmental phase in which cells aggregate and sporulate in response to starvation. In previous papers, we have described a heretofore unsuspected layer of complexity in the development of M. xanthus: vegetatively growing cells differentiate into two cell types during development. In addition to the differentiation of spores within fruiting bodies, a second cell type, peripheral rods, arises outside fruiting bodies. The pattern of expression of proteins in peripheral rods is different from that of either vegetatively growing cells or spores, and peripheral rods express a number of recognized developmental markers. In this report, we examine four aspects of the biology of peripheral rods: (i) the influence of nutrients on the proportion of peripheral rods in a population of developing cells, (ii) the capacity of peripheral rods to recapitulate development, (iii) the development of peripheral rods on conditioned medium, and (iv) the ability of peripheral rods to resume growth on low amounts of exogenously added nutrients. The results of these studies suggest that peripheral rods play a significant role in the life cycle of M. xanthus by allowing the exploitation of low amounts or transient influxes of nutrients without the investment of energy in spore germination. The differentiation of vegetatively growing cells into two cell types that differ significantly in biology, shape, and localization within the population has been incorporated into a model of the life cycle of M. xanthus.     

The peptidoglycan sacculus of Myxococcus xanthus has unusual structural features and is degraded during glycerol-induced myxospore development

Upon nutrient limitation cells of the swarming soil bacterium Myxococcus xanthus form a multicellular fruiting body in which a fraction of the cells develop into myxospores. Spore development includes the transition from a rod-shaped vegetative cell to a spherical myxospore and so is expected to be accompanied by changes in the bacterial cell envelope. Peptidoglycan is the shape-determining structure in the cell envelope of most bacteria, including myxobacteria. We analyzed the composition of peptidoglycan isolated from M. xanthus. While the basic structural elements of peptidoglycan in myxobacteria were identical to those in other gram-negative bacteria, the peptidoglycan of M. xanthus had unique structural features. meso- or LL-diaminopimelic acid was present in the stem peptides, and a new modification of N-acetylmuramic acid was detected in a fraction of the muropeptides. Peptidoglycan formed a continuous, bag-shaped sacculus in vegetative cells. The sacculus was degraded during the transition from vegetative cells to glycerol-induced myxospores. The spherical, bag-shaped coats isolated from glycerol-induced spores contained no detectable muropeptides, but they contained small amounts of N-acetylmuramic acid and meso-diaminopimelic acid.     

Genetic circuitry controlling motility behaviors of Myxococcus xanthus

M. xanthus has a complex multicellular lifestyle including swarming, predation and development. These behaviors depend on the ability of the cells to achieve directed motility across solid surfaces. M. xanthus cells have evolved two motility systems including Type-IV pili that act as grappling hooks and a controversial engine involving mucus secretion and fixed focal adhesion sites. The necessity for cells to coordinate the motility systems and to respond rapidly to environmental cues is reflected by a complex genetic network involving at least three complete sets of chemosensory systems and eukaryotic-like signaling proteins. In this review, we discuss recent advances suggesting that motor synchronization results from spatial oscillations of motility proteins. We further propose that these dynamics are modulated by the action of multiple upstream complementary signaling systems. M. xanthus is thus an exciting emerging model system to study the intricate processes of directed cell migration.     

Role of cell cohesion in Myxococcus xanthus fruiting body formation.

Dsp mutants of Myxococcus xanthus have a complex phenotype with abnormal cell cohesion, social motility, and development. All three defects are the result of a single mutation in the dsp locus, a region of DNA about 14 kilobases long. Cohesion appears to play a central role in social motility, since nonsocial mutants exhibit weak agglutination or, in the case of Dsp cells, no agglutination (L. J. Shimkets, J. Bacteriol. 166:837-841, 1986). However, Dsp cells can be agglutinated by cohesive strains of M. xanthus. This provided the opportunity to examine the role of cohesion during development by comparing the developmental phenotype of Dsp cells with that of Dsp cells mixed with cohesive strains. Dsp mutants were unable to complete any of the developmental behaviors: aggregation, fruiting body formation, developmental autolysis, and sporulation. Contact with cohesive strains seemed to restore some developmental characteristics to the Dsp cells. When allowed to develop with wild-type cells, Dsp cells accumulated in fruiting bodies and underwent developmental autolysis, but did not form a significant portion of the spore population. Igl mutants, which may be similar to the previously described frizzy mutants, are cohesive strains that are unable to form fruiting bodies. Mixing Igl cells with Dsp cells under developmental conditions resulted in fruiting body formation, although the Dsp cells were unable to form significant levels of myxospores. In spite of their inability to sporulate under developmental conditions, Dsp mutants did not appear to be defective in the sporulation process. In fact, they formed normal levels of myxospores in response to the chemical inducer glycerol.     

Myxococcus xanthus developmental cell fate production: heterogeneous accumulation of developmental regulatory proteins and reexamination of the role of MazF in developmental lysis

Myxococcus xanthus undergoes a starvation-induced multicellular developmental program during which cells partition into three known fates: (i) aggregation into fruiting bodies followed by differentiation into spores, (ii) lysis, or (iii) differentiation into nonaggregating persister-like cells, termed peripheral rods. As a first step to characterize cell fate segregation, we enumerated total, aggregating, and nonaggregating cells throughout the developmental program. We demonstrate that both cell lysis and cell aggregation begin with similar timing at approximately 24 h after induction of development. Examination of several known regulatory proteins in the separated aggregated and nonaggregated cell fractions revealed previously unknown heterogeneity in the accumulation patterns of proteins involved in type IV pilus (T4P)-mediated motility (PilC and PilA) and regulation of development (MrpC, FruA, and C-signal). As part of our characterization of the cell lysis fate, we set out to investigate the unorthodox MazF-MrpC toxin-antitoxin system which was previously proposed to induce programmed cell death (PCD). We demonstrate that deletion of mazF in two different wild-type M. xanthus laboratory strains does not significantly reduce developmental cell lysis, suggesting that MazF's role in promoting PCD is an adaption to the mutant background strain used previously.     

Biochemical characterization of a mutant of the yeast Pichia anomala derepressed for malic acid utilization in the presence of glucose

Abstract 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.