Pph1 from Myxococcus xanthus is a protein phosphatase involved in vegetative growth and development.

Myxococcus xanthus is a Gram-negative bacterium with a complex life cycle that includes vegetative swarming on rich medium and, upon starvation, aggregation to form fruiting bodies containing spores. Both of these behaviours require multiple Ser/Thr protein kinases. In this paper, we report the first Ser/Thr protein phosphatase gene, pph1, from M. xanthus. DNA sequence analysis of pph1 indicates that it encodes a protein of 254 residues (Mr = 28 308) with strong homology to eukaryotic PP2C phosphatases and that it belongs to a new group of bacterial protein phosphatases that are distinct from bacterial PP2C phosphatases such as RsbU, RsbX and SpoIIE. Recombinant His-tagged Pph1 was purified from Escherichia coli and shown to have Mn2+ or Mg2+ dependent, okadaic acid-resistant phosphatase activity on a synthetic phosphorylated peptide, RRA(pT)VA, indicating that Pph1 is a PP2C phosphatase. Pph1-expression was observed under both vegetative and developmental conditions, but peaked during early aggregation. A pph1 null mutant showed defects during late vegetative growth, swarming and glycerol spore formation. Under starvation-induced developmental conditions, the mutant showed reduced aggregation and failure to form fruiting bodies with viable spores. Using the yeast two-hybrid system, we have observed a strong interaction between Pph1 and the M. xanthus protein kinase Pkn5, a negative effector of development. These results suggest a functional link between a Pkn2-type protein kinase and a PP2C phosphatase.

Developmental aggregation of Myxococcus xanthus requires frgA, an frz-related gene.

Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle that includes multicellular fruiting body formation. Frizzy mutants are characterized by the formation of tangled filaments instead of hemispherical fruiting bodies on fruiting agar. Mutations in the frz genes have been shown to cause defects in directed motility, which is essential for both vegetative swarming and fruiting body formation. In this paper, we report the discovery of a new gene, called frgA (for frz-related gene), which confers a subset of the frizzy phenotype when mutated. The frgA null mutant showed reduced swarming and the formation of frizzy aggregates on fruiting agar. However, this mutant still displayed directed motility in a spatial chemotaxis assay, whereas the majority of frz mutants fail to show directed movements in this assay. Furthermore, the frizzy phenotype of the frgA mutant could be complemented extracellularly by wild-type cells or strains carrying non-frz mutations. The phenotype of the frgA mutant is similar to that of the abcA mutant and suggests that both of these mutants could be defective in the production or export of extracellular signals required for fruiting body formation rather than in the sensing of such extracellular signals. The frgA gene encodes a large protein of 883 amino acids which lacks homologues in the databases. The frgA gene is part of an operon which includes two additional genes, frgB and frgC. The frgB gene encodes a putative histidine protein kinase, and the frgC gene encodes a putative response regulator. The frgB and frgC null mutants, however, formed wild-type fruiting bodies.

Type IV pilus of Myxococcus xanthus is a motility apparatus controlled by the frz chemosensory system.

Although flagella are the best-understood means of locomotion in bacteria [1], other bacterial motility mechanisms must exist as many diverse groups of bacteria move without the aid of flagella [2-4]. One unusual structure that may contribute to motility is the type IV pilus [5,6]. Genetic evidence indicates that type IV pili are required for social gliding motility (S-motility) in Myxococcus, and twitching motility in Pseudomonas and Neisseria [6,7]. It is thought that type IV pili may retract or rotate to bring about cellular motility [6,8], but there is no direct evidence for the role of pili in cell movements. Here, using a tethering assay, we obtained evidence that the type IV pilus of Myxococcus xanthus functions as a motility apparatus. Pili were required for M. xanthus cells to adhere to solid surfaces and to generate cellular movement using S-motility. Tethered cells were released from the surface at intervals corresponding to the reversal frequency of wild-type cells when gliding on a solid surface. Mutants defective in the control of directional movements and cellular reversals (frz mutants) showed altered patterns of adherence that correlate reversal frequencies with tethering. The behavior of the tethered cells was consistent with a model in which the pili are extruded from one cell pole, adhere to a surface, and then retract, pulling the cell in the direction of the adhering pili. Cellular reversals would result from the sites of pili extrusion switching from one cell pole to another and are controlled by the frz chemosensory system.

Social motility in Myxococcus xanthus requires FrzS, a protein with an extensive coiled-coil domain.

Gliding motility in the developmental bacterium Myxococcus xanthus involves two genetically distinct motility systems, designated adventurous (A) and social (S). Directed motility responses, which facilitate both vegetative swarming and developmental aggregation, additionally require the 'frizzy' (Frz) signal transduction pathway. In this study, we have analysed a new gene (frzS), which is positioned upstream of the frzA-F operon. Insertion mutations in frzS caused both vegetative spreading and developmental defects, including 'frizzy' aggregates in the FB strain background. The 'frizzy' phenotype was previously considered to result only from defective directed motility responses. However, deletion of the frzS gene in an A-S+ motility background demonstrated that FrzS is a new component of the S-motility system, as the A-frzS double mutant was non-spreading (A-S-). Compared with known S-motility mutants, the frzS mutants appear similar to pilT mutants, in that both produce type IV pili, extracellular fibrils and lipopolysaccharide (LPS) O-antigen, and both agglutinate rapidly in a cohesion assay. The FrzS protein has an unusual domain composition for a bacterial protein. The N-terminal domain shows similarity to the receiver domains of the two-component response regulator proteins. The C-terminal domain is composed of up to 38 heptad repeats (a b c d e f g)38, in which residues at positions a and d are predominantly hydrophobic, whereas residues at positions e and g are predominantly charged. This periodic disposition of specific residues suggests that the domain forms a long coiled-coil structure, similar to those found in the alpha-fibrous proteins, such as myosin. Overexpression of this domain in Escherichia coli resulted in the formation of an unusual striated protein lattice that filled the cells. We speculate on the role that this novel protein could play in gliding motility.

Disruption of aldA influences the developmental process in Myxococcus xanthus.

Previously, we identified a gene (aldA) from Myxococcus xanthus, which we suggested encoded the enzyme alanine dehydrogenase on the basis of similarity to known Ald protein sequences (M. J. Ward, H. Lew, A. Treuner-Lange, and D. R. Zusman, J. Bacteriol. 180:5668-5675, 1998). In this study, we have confirmed that aldA does encode a functional alanine dehydrogenase, since it catalyzes the reversible conversion of alanine to pyruvate and ammonia. Whereas an aldA gene disruption mutation did not significantly influence the rate of growth or spreading on a rich medium, AldA was required for growth on a minimal medium containing L-alanine as the major source of carbon. Under developmental conditions, the aldA mutation caused delayed aggregation in both wild-type (DZ2) and FB (DZF1) strains. Poorly formed aggregates and reduced levels of spores were apparent in the DZ2 aldA mutant, even after prolonged 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.

AsgD, a new two-component regulator required for A-signalling and nutrient sensing during early development of Myxococcus xanthus.

Myxococcus xanthus has a complex life cycle that includes fruiting body formation. One of the first stages in development has been called A-signalling. The asg (A-signalling) mutants have been proposed to be deficient in producing A-signal, resulting in development arresting at an early stage. In this paper, we report the identification of a new asg locus asgD. This locus appears to be involved in both environmental sensing and intercellular signalling. Expression of asgD was undetected during vegetative growth, but increased dramatically within 1 h of starvation. The AsgD protein is predicted to contain 773 amino acids and to be part of a two-component regulatory system because it has a receiver domain located at the N-terminus and a histidine protein kinase at the C-terminus. An asgD null mutant was defective in fruiting body formation and sporulation on CF medium. However, the defects of the mutant were complemented extracellularly when cells were mixed with wild-type strains or with bsgA, csgA, dsgA or esgA mutants, but were not complemented extracellularly by asgA, asgB or asgC mutants. In addition, the mutant was rescued by a subset of A-factor amino acids. Surprisingly, when the mutant was plated on stringent starvation medium rather than CF, cells were able to form fruiting bodies. Thus, it appears that AsgD is directly or indirectly involved in sensing nutritionally limiting conditions. The discovery of the asgD locus provides an important sensory transduction component of early development in M. xanthus.

Sporulation timing in Myxococcus xanthus is controlled by the espAB locus.

The fruiting body development of Myxococcus xanthus consists of two separate but interacting pathways: one for aggregation of many cells to form raised mounds and the other for sporulation of individual cells into myxospores. Sporulation of individual cells normally occurs after mound formation, and is delayed at least 30 h after starvation under our laboratory conditions. This suggests that M. xanthus has a mechanism that monitors progress towards aggregation prior to triggering sporulation. A null mutation in a newly identified gene, espA (early sporulation), causes sporulation to occur much earlier compared with the wild type (16 h earlier). In contrast, a null mutation in an adjacent gene, espB, delays sporulation by about 16 h compared with the wild type. Interestingly, it appears that the espA mutant does not require raised mounds for sporulation. Many mutant cells sporulate outside the fruiting bodies. In addition, the mutant can sporulate, without aggregation into raised mounds, under some conditions in which cells normally do not form fruiting bodies. Based on these observations, it is hypothesized that EspA functions as an inhibitor of sporulation during early fruiting body development while cells are aggregating into raised mounds. The aggregation-independent sporulation of the espA mutant still requires starvation and high cell density. The espA and espB genes are expressed as an operon and their translations appear to be coupled. Expression occurs only under developmental conditions and does not occur during vegetative growth or during glycerol-induced sporulation. Sequence analysis of EspA indicates that it is a histidine protein kinase with a fork head-associated (FHA) domain at the N-terminus and a receiver domain at the C-terminus. This suggests that EspA is part of a two-component signal transduction system that regulates the timing of sporulation initiation.

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.

Regulation of motility behavior in Myxococcus xanthus may require an extracytoplasmic-function sigma factor.

Using interaction trap technology, we identified a putative extracytoplasmic-function (ECF) sigma factor (RpoE1) in Myxococcus xanthus, a bacterium which has a complex life cycle that includes fruiting body formation. The first domain of the response regulator protein FrzZ, a component of the Frz signal transduction system, was used as bait. Although the RpoE1 protein displayed no interactions with control proteins presented as bait, a weak interaction with a second M. xanthus response regulator (AsgA) was observed. While the specificity of the FrzZ-RpoE1 interaction therefore remains speculative, cloning and sequencing of the region surrounding rpoE1 localized it to a position downstream of the frzZ gene. A potential promoter site for binding of an ECF sigma factor was identified upstream of rpoE1, suggesting the gene may be autoregulated. However, primer extension studies suggested that transcription of rpoE1 occurs under both vegetative and developmental conditions from a sigma70-like promoter. Dot blot analysis of RNA preparations confirmed the low-level, constitutive expression of rpoE1 during both stages of the life cycle. Analysis of an insertion mutant also indicated a role for RpoE1 under both vegetative and developmental conditions, since swarming was reduced on nutrient-rich agar and developmental aggregation was effected under starvation conditions, especially at high cell densities. An insertion mutation introduced into the gene directly downstream of rpoE1 (orf5) did not result in either swarming or developmental aggregation defects, even though the gene is transcribed as part of the same operon. Therefore, we propose that this new ECF sigma factor could play a role in the transcriptional regulation of genes involved in motility behavior during both stages of the complex M. xanthus life cycle.

An ABC transporter plays a developmental aggregation role in Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle. Autochemotaxis, a process whereby cells release a self-generated signaling molecule, may be the principal mechanism facilitating directed motility in both the vegetative swarming and developmental aggregation stages of this life cycle. The process requires the Frz signal transduction system, including FrzZ, a protein which is composed of two domains, both showing homology to the enteric chemotaxis response regulator CheY. The first domain of FrzZ (FrzZ1), when expressed as bait in the yeast two-hybrid system and screened against a library, was shown to potentially interact with the C-terminal portion of a protein encoding an ATP-binding cassette (AbcA). The activation domain-AbcA fusion protein did not interact with the second domain of FrzZ (FrzZ2) or with two other M. xanthus response regulator-containing proteins presented as bait, suggesting that the FrzZ1-AbcA interaction may be specific. Cloning and sequencing of the upstream region of the abcA gene showed the ATP-binding cassette to be linked to a large hydrophobic, potentially membrane-spanning domain. This domain organization is characteristic of a subgroup of ABC transporters which perform export functions. Cloning and sequencing downstream of abcA indicated that the ABC transporter is at the start of an operon containing three open reading frames. An insertion mutation in the abcA gene resulted in cells displaying the frizzy aggregation phenotype, providing additional evidence that FrzZ and AbcA may be part of the same signal transduction pathway. Cells with mutations in genes downstream of abcA showed no developmental defects. Analysis of the proposed exporter role of AbcA in cell mixing experiments showed that the ABC transporter mutant could be rescued by extracellular complementation. We speculate that the AbcA protein may be involved in the export of a molecule required for the autochemotactic process.

Methylation of FrzCD defines a discrete step in the developmental program of Myxococcus xanthus.

Myxococcus xanthus is a gram-negative soil bacterium which undergoes fruiting body formation during starvation. The frz signal transduction system has been found to play an important role in this process. FrzCD, a methyl-accepting taxis protein homologue, shows modulated methylation during cellular aggregation, which is thought to be part of an adaptation response to an aggregation signal. In this study, we assayed FrzCD methylation in many known and newly isolated mutants defective in fruiting body formation to determine a possible relationship between the methylation response and fruiting morphology. The results of our analysis indicated that the developmental mutants could be divided into two groups based on their ability to show normal FrzCD methylation during development. Many mutants blocked early in development, i.e., nonaggregating or abnormally aggregating mutants, showed poor FrzCD methylation. The well-characterized asg, bsg, csg, and esg mutants were found to be of this type. The defects in FrzCD methylation of these signaling mutants could be partially rescued by extracellular complementation with wild-type cells or addition of chemicals which restore their fruiting body formation. Mutants blocked in late development, i.e., translucent mounds, showed normal FrzCD methylation. Surprisingly, some mutants blocked in early development also exhibited a normal level of FrzCD methylation. The characterized mutants in this group were found to be defective in social motility. This indicates that FrzCD methylation defines a discrete step in the development of M. xanthus and that social motility mutants are not blocked in these early developmental steps.

Myxococcus xanthus displays Frz-dependent chemokinetic behavior during vegetative swarming.

Myxococcus xanthus has been shown to utilize both directed (tactic) and undirected (kinetic) movements during different stages of its complex life cycle. We have used time-lapse video microscopic analysis to separate tactic and kinetic behaviors associated specifically with vegetatively swarming cells. Isolated individual cells separated by a thin agar barrier from mature swarms showed significant increases in gliding velocity compared to that of similar cells some distance from the swarm. This orthokinetic behavior was independent of the frequency of reversals of gliding direction (klinokinesis) but did require both the Frz signal transduction system and S-motility. We propose that M. xanthus uses Frz-dependent, auto-orthokinetic behavior to facilitate the dispersal of cells under conditions where both cell density and nutrient levels are high.

Regulation of directed motility in Myxococcus xanthus.

Myxococcus xanthus is a Gram-negative bacterium that exhibits a complex life cycle. During vegetative growth, cells move as large swarms. However, when starved, cells aggregate into fruiting bodies and sporulate. Both vegetative swarming and developmental aggregation require gliding motility, which involves the slow movement of cells on a solid surface in the absence of flagella. The frequency of cell reversals controls the direction of movement and is regulated by the frz genes, which encode the 'frizzy' signal-transduction proteins. These proteins contain domains which bear striking similarities to the major chemotaxis proteins of the enteric bacteria: CheA, CheY, CheW, CheR, CheB and Tar. However, significant differences exist between the Myxococcus Frz proteins and the enteric Che/MCP proteins. For example, the Frz system contains three CheY-like response-regulator domains: one is present on FrzE, which also contains a CheA-like domain, and two are present on FrzZ, which is a novel protein required for attractant, but not for repellent, responses. The identification of multiple CheY homologues in this system indicates a more complex regulatory pathway than that found in the enteric bacteria. While responses to repellent stimuli appear to follow the enteric paradigm, responses to attractants during vegetative swarming and development are more complex and may involve self-generated autoattractants. The Frz signal-transduction system regulates directed motility in M. xanthus and is essential for controlling both fruiting-body development and vegetative swarming.

Starvation-independent sporulation in Myxococcus xanthus involves the pathway for beta-lactamase induction and provides a mechanism for competitive cell survival.

Myxococcus xanthus is a Gram-negative, soil-dwelling bacterium with a complex life cycle which includes fruiting body formation and sporulation in response to starvation. This developmental process is slow, requiring a minimum of 24-48 h, and requires cells to be at high cell density on a solid surface. It is known that, in the absence of starvation, vegetatively growing cell suspensions can form 'glycerol spores' when exposed to high levels of glycerol, usually 0.5 M. The cells differentiate from rods to resistant spheres rapidly (2-4 h) and synchronously. We have found that the chromosomally encoded beta-lactamase of M. xanthus can be induced by numerous beta-lactam antibiotics as well as by non-specific inducers including glycine and many D-amino acids. In addition, D-cycloserine, phosphomycin, and hen egg-white lysozyme also induce beta-lactamase in this bacterium. Unexpectedly, agents which induce beta-lactamase can induce 'glycerol spores'; all of the agents tested which induce glycerol spores (glycerol, DMSO, ethylene glycol) also induce beta-lactamase. During the induction of sporulation, beta-lactamase activity increases, reaching a peak during the morphological transition from rod-shaped cells to spherical spores. These spores are viable and resistant to many treatments which disrupt vegetatively growing rods but are not as resistant as fruiting body spores. The concomitant induction of beta-lactamase and starvation-independent sporulation suggests that these processes share a common signal-transduction pathway. These results also suggest that starvation-independent sporulation may be an adaptation of cells in order to resist agents that damage peptidoglycan structure and therefore threaten cell survival.

Identification and characterization of FrzZ, a novel response regulator necessary for swarming and fruiting-body formation in Myxococcus xanthus.

The frz genes of Myxococcus xanthus constitute a signal-transduction pathway that processes chemotactic information in a manner analogous to that found in enteric bacteria. Ultimately, these genes regulate the frequency of individual cell reversal. We report here the identification of a novel component of this signal-transduction pathway, designated frzZ, which was discovered as an open reading frame located 5' to the frz operon but transcribed in the opposite orientation. The translational start site of frzZ is 170 base pairs from that of frzA.frzZ utilizes a promoter similar to the sigma 70 promoters of Escherichia coli, and encodes a 290-amino-acid soluble protein, FrzZ (M(r) 30,500). FrzZ contains two domains, both of which show strong homology to CheY and other members of the response-regulator family. Linking these domains is a 39-amino-acid region that is very rich in alanine and proline (38% Ala and 33% Pro). A frzZ null mutant showed abnormally low reversal rates when compared to the wild-type control and was unable to form fruiting bodies on starvation medium, but it did form 'frizzy' aggregates. In addition, the frzZ mutant was defective in swarming, particularly on soft agar (0.3% w/v). However, unlike most frz mutants, the frzZ mutant was able to respond to attractants and repellents in the spatial chemotaxis assay. The discovery of FrzZ demonstrates that the M. xanthus frz signal-transduction pathway utilizes multiple response-regulator (CheY-like) proteins.

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.

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."

Photolyase of Myxococcus xanthus, a Gram-negative eubacterium, is more similar to photolyases found in Archaea and "higher" eukaryotes than to photolyases of other eubacteria.

We report the identification of the gene encoding a DNA photolyase (phrA) from the Gram-negative eubacterium Myxococcus xanthus. The deduced amino acid sequence of M. xanthus photolyase indicates that the protein contains 401 amino acids (Mr 45,071). By comparison of the amino acid and DNA sequences with those of other known photolyases, it has been found that it is more similar to the deduced amino acid sequences of the photolyases of "higher" eukaryotes than to the photolyases of other eubacteria. Recombinant plasmids carrying M. xanthus phrA rescue the photoreactivation activity of an irradiated strain of Escherichia coli with a deletion in phrA. This rescue is light-dependent.

Methionine inhibits developmental aggregation of Myxococcus xanthus by blocking the biosynthesis of S-adenosyl methionine.

Previous studies showed that high concentrations of methionine (> 1 mM) inhibited aggregation and fruiting body formation in Myxococcus xanthus (E. Rosenberg, D. Filer, D. Zafriti, and S. H. Kindler, J. Bacteriol. 115: 29-34, 1973, and J. M. Campos and D. R. Zusman, Proc. Natl. Acad. Sci. USA 72:518-522, 1975). However, the mechanism for the inhibition was unclear. In this study, we found that high levels of methionine inhibited the biosynthesis of S-adenosylmethionine (SAM) and that reduced intracellular levels of SAM are correlated with defective chemotactic movements and reduced developmental gene expression. In addition, we found that methionine analogs and high concentrations of amino acids which are known to affect SAM synthesis in other bacteria, such as threonine, lysine, and isoleucine, also caused reduced cellular levels of SAM and blocked fruiting body formation in M. xanthus. These results indicate that SAM is required for development of M. xanthus and the inhibitory effect of methionine on development results, at least in part, from its blocking of the biosynthesis of SAM.