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 dif for 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.     

Effects of heat shock upon the expression of developmentally regulated genes in Myxococcus xanthus

Abstract The effects of heat shock upon the expression of several developmentally regulated genes of Myxococcus xanthus were examined. No effects were observed on levels or timing of developmentally regulated β-galactosidase expression in eight randomly selected Tn5lac insertion mutants. However, heat shock significantly affected the fruiting behavior of temperature-sensitive aggregation ( tag ) mutants of M. xanthus . The tag mutant phenotype exhibits the normal aggregation of cells to form fruiting bodies at temperatures < 34°C, but cells fail to aggregate at temperatures ⩾ 34°C. Heat shock administered to tag mutant strains prior to starvation prohibited fruiting body formation at permissive temperatures. Additionally, tag mutant strains were found to be extremely sensitive to killing at 40°C. Heat shock was also found to increase tagA and tagE expression by 22 and 47%, respectively. Mutations in tagA blocked heat shock induced expression of tagE .     

Aspartokinase Activity and the Developmental Cycle of Myxococcus xanthus

The relationship between aspartokinase activity and fruiting body formation in Myxococcus xanthus was investigated. Two required amino acids, methionine and isoleucine, which stimulated the enzyme in vitro also inhibited fruiting body formation when added to 0.1% Casitone agar. Threonine, a potent feedback inhibitor of the aspartokinase, completely reversed the effects of methionine and isoleucine both on enzyme activity and fruiting body formation. A mutant, M. xanthus FB-S, which had the unusual property of forming fruiting bodies on 1.0% Casitone agar, also exhibited an altered regulation of aspartokinase activity. Spermidine, which is a strong stimulator of the enzyme in vitro, interfered with the developmental cycle of both M. xanthus FB and FS-S. During glycerol induction of myxospores the level of aspartokinase dropped more than 75% during the first hour. These data indicate a strong correlation between aspartokinase activity and the induction of the developmental cycle in M. xanthus. It is suggested that the decrease in aspartokinase activity results in diaminopimelic acid starvation, blockage of cell wall growth, and subsequent induction of the developmental cycle.     

Effects of deletion of the gene for the development-specific protein S on differentiation in Myxococcus xanthus

A deletion mutation of the gene for protein S (tps), a development-specific protein of Myxococcus xanthus, was constructed. No significant differences in the process of fruiting body formation or the yield of myxospores were observed between mutant and wild-type cells. On the other hand, when the tps gene was deleted together with a 2.0-kilobase sequence including the ops gene immediately upstream of the tps gene, fruiting body formation was substantially delayed, and the yield of myxospores was reduced. These results indicate that protein S is not essential for differentiation of M. xanthus, whereas a gene product(s) coded from the sequence upstream of the tps gene appears to be required for normal fruiting body formation.     

Purine-containing compounds, including cyclic adenosine 3'',5''-monophosphate, induce fruiting of Myxococcus xanthus by nutritional imbalance.

Induction of Myxococcus xanthus fruiting by a number of different purine-containing compounds, including cyclic adenosine 3',5'-monophosphate, is defective in a mutant resistant to 2,6-diaminopurine. Furthermore, the purine-induced fruiting of wild-type cultures is uniquely blocked by a low concentration of added glycine. These results imply that different purine-containing compounds induce fruiting through a single mechanism involving nutritional imbalance.     

Intra- and interprotein phosphorylation between two-hybrid histidine kinases controls Myxococcus xanthus developmental progression

Histidine-aspartate phosphorelay signaling systems are used to couple stimuli to cellular responses. A hallmark feature is the highly modular signal transmission modules that can form both simple "two-component" systems and sophisticated multicomponent systems that integrate stimuli over time and space to generate coordinated and fine-tuned responses. The deltaproteobacterium Myxococcus xanthus contains a large repertoire of signaling proteins, many of which regulate its multicellular developmental program. Here, we assign an orphan hybrid histidine protein kinase, EspC, to the Esp signaling system that negatively regulates progression through the M. xanthus developmental program. The Esp signal system consists of the hybrid histidine protein kinase, EspA, two serine/threonine protein kinases, and a putative transport protein. We demonstrate that EspC is an essential component of this system because ΔespA, ΔespC, and ΔespA ΔespC double mutants share an identical developmental phenotype. Neither substitution of the phosphoaccepting histidine residue nor deletion of the entire catalytic ATPase domain in EspC produces an in vivo mutant developmental phenotype. In contrast, substitution of the receiver phosphoaccepting residue yields the null phenotype. Although the EspC histidine kinase can efficiently autophosphorylate in vitro, it does not act as a phosphodonor to its own receiver domain. Our in vitro and in vivo analyses suggest the phosphodonor is instead the EspA histidine kinase. We propose EspA and EspC participate in a novel hybrid histidine protein kinase signaling mechanism involving both inter- and intraprotein phosphotransfer. The output of this signaling system appears to be the combined phosphorylated state of the EspA and EspC receiver modules. This system regulates the proteolytic turnover of MrpC, an important regulator of the developmental program.     

Identification of major sporulation proteins of Myxococcus xanthus using a proteomic approach

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.     

Propionyl coenzyme A carboxylase is required for development of Myxococcus xanthus.

A dcm-1 mutant, obtained by transposon mutagenesis of Myxococcus xanthus, could aggregate and form mounds but was unable to sporulate under nutrient starvation. A sequence analysis of the site of insertion of the transposon showed that the insertion lies within the 3' end of a 1,572-bp open reading frame (ORF) designated the M. xanthus pccB ORF. The wild-type form of the M. xanthus pccB gene, obtained from a lambdaEMBL library of M. xanthus, shows extensive similarity to a beta subunit of propionyl coenzyme A (CoA) carboxylase, an alpha subunit of methylmalonyl-CoA decarboxylase, and a 12S subunit of transcarboxylase. In enzyme assays, extracts of the dcm-1 mutant were deficient in propionyl-CoA carboxylase activity. This enzyme catalyzes the ATP-dependent carboxylation of propionyl-CoA to yield methylmalonyl-CoA. The methylmalonyl-CoA rescued the dcm-1 mutant fruiting body and spore development. During development, the dcm-1 mutant cells also had reduced levels of long-chain fatty acids (C16 to C18) compared to wild-type cells.     

A new putative sigma factor of Myxococcus xanthus.

A third putative sigma factor gene, sigC, has been isolated from Myxococcus xanthus by using the sigA gene (formerly rpoD of M. xanthus) as a probe. The nucleotide sequence of sigC has been determined, and an open reading frame of 295 residues (M(r) = 33,430) has been identified. The deduced amino acid sequence of sigC exhibits the features which are characteristic of other bacterial sigma factors. The characterization of a sigC-lacZ strain has demonstrated that sigC expression is induced immediately after cells enter into the developmental cycle and is dramatically reduced at the onset of sporulation. A deletion mutant of sigC grows normally in vegetative culture and is able to develop normally. However, in contrast to the wild-type cells, the sigC deletion mutant cells became capable of forming fruiting bodies and myxospores on semirich agar plates. This suggests that sigC may play a role in expression of genes involved in negatively regulating the initiation of fruiting body formation.     

Bypass of A- and B-signaling requirements for Myxococcus xanthus development by mutations in spdR

Mutations in spdR, previously reported to bypass the developmental requirement for B-signaling in Myxococcus xanthus, also bypass the requirement for A-signaling but not C-, D-, or E-signaling. Mutations in spdR restored nearly wild-type levels of sporulation to representative A-signal-deficient mutants carrying asgA476, asgB480, and asgC767 and improved the quality of fruiting body formation in the asgB480 mutant. The defect in A-factor production by the asgB480 mutant was not restored in the spdR2134 asgB480 double mutant.     

SigB, SigC, and SigE from Myxococcus xanthus homologous to sigma32 are not required for heat shock response but for multicellular differentiation

Myxococcus xanthus has been known to have multiple sigma factors which are considered to play important roles in regulation of gene expression in development. A new gene encoding a putative sigma factor, sigE, was cloned by using a degenerate oligonucleotide corresponding to the conserved region 2.2 of M. xanthus SigA. In the 2.0-kb nucleotide sequence, an open reading frame consisting of 280 amino acid residues was identified. The amino acid sequence of SigE shows high similarity to heat shock sigma factors in bacteria. However, the sigE gene is not induced by heat shock and deletion of sigE does not affect production of heat shock proteins. SigE is expressed during both vegetative growth and fruiting body development. In the deletion mutant of the sigE gene fruiting body formation is initiated earlier and fewer spores are produced than in the parent strain. Interestingly, the deltasigE mutant shows defects in fruiting body formation at 37 degrees C. In addition to SigE, SigB and SigC show high sequence similarity to heat shock sigma factors. However, even if all three sigma factor genes are disrupted, heat shock proteins are still normally induced. A deltasigBdeltasigCdeltasigE triple deletion strain forms fruiting bodies earlier, but sporulats later than the parent strain. Spores from the triple deletion mutant are aberrant and their viability is less than 0.001% compared with that of the parent strain, suggesting that these sigma factors may have redundant functions in multicellular differentiation of M. xanthus.     

Multicellular development in Myxococcus xanthus is stimulated by predator-prey interactions

Myxococcus xanthus is a predatory bacterium that exhibits complex social behavior. The most pronounced behavior is the aggregation of cells into raised fruiting body structures in which cells differentiate into stress-resistant spores. In the laboratory, monocultures of M. xanthus at a very high density will reproducibly induce hundreds of randomly localized fruiting bodies when exposed to low nutrient availability and a solid surface. In this report, we analyze how M. xanthus fruiting body development proceeds in a coculture with suitable prey. Our analysis indicates that when prey bacteria are provided as a nutrient source, fruiting body aggregation is more organized, such that fruiting bodies form specifically after a step-down or loss of prey availability, whereas a step-up in prey availability inhibits fruiting body formation. This localization of aggregates occurs independently of the basal nutrient levels tested, indicating that starvation is not required for this process. Analysis of early developmental signaling relA and asgD mutants indicates that they are capable of forming fruiting body aggregates in the presence of prey, demonstrating that the stringent response and A-signal production are surprisingly not required for the initiation of fruiting behavior. However, these strains are still defective in differentiating to spores. We conclude that fruiting body formation does not occur exclusively in response to starvation and propose an alternative model in which multicellular development is driven by the interactions between M. xanthus cells and their cognate prey.     

Role of phase variation in the resistance of Myxococcus xanthus fruiting bodies to Caenorhabditis elegans predation

The phenomenon of phase variation between yellow and tan forms of Myxococcus xanthus has been recognized for several decades, but it is not known what role this variation may play in the ecology of myxobacteria. We confirm an earlier report that tan variants are disproportionately more numerous in the resulting spore population of a M. xanthus fruiting body than the tan vegetative cells that contributed to fruiting body formation. However, we found that tan cells may not require yellow cells for fruiting body formation or starvation-induced sporulation of tan cells. Here we report three differences between the yellow and tan variants that may play important roles in the soil ecology of M. xanthus. Specifically, the yellow variant is more capable of forming biofilms, is more sensitive to lysozyme, and is more resistant to ingestion by bacteriophagous nematodes. We also show that the myxobacterial fruiting body is more resistant to predation by worms than are dispersed M. xanthus cells.     

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.     

Enzymatic and functional analysis of a protein phosphatase, Pph3, from Myxococcus xanthus

A protein phosphatase, designated Pph3, from Myxococcus xanthus showed the enzymatic characteristics of PP2C-type serine/threonine protein phosphatases, which are metal ion-dependent, okadaic acid-insensitive protein phosphatases. The pph3 mutant under starvation conditions formed immature fruiting bodies and reduced sporulation.     

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.