Development Of Myxococcus Xanthus Research Articles

Role of fruA and csgA Genes in Gene Expression during Development of Myxococcus xanthus: ANALYSIS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS

Two genes, fruA and csgA, encoding a putative transcription factor and C-factor, respectively, are essential for fruiting body formation of Myxococcus xanthus. To investigate the role of fruA and csgA genes in developmental gene expression, developing cells as well as vegetative cells of M. xanthus wild-type, fruA::Tc, and csgA731 strains were pulse-labeled with [(35)S]methionine, and the whole cell proteins were analyzed using two-dimensional immobilized pH gradient/SDS-PAGE. Differences in protein synthesis patterns among more than 700 protein spots were detected during development of the three strains. Fourteen proteins showing distinctly different expression patterns in mutant cells were analyzed in more detail. Five of the 14 proteins were identified as elongation factor Tu (EF-Tu), Dru, DofA, FruA, and protein S by immunoblot analysis and mass spectroscopy. A gene encoding DofA was cloned and sequenced. Although both fruA and csgA genes regulate early development of M. xanthus, they were found to differently regulate expression of several developmental genes. The production of six proteins, including DofA and protein S, was dependent on fruA, whereas the production of two proteins was dependent on csgA, and one protein was dependent on both fruA and csgA. To explain the present findings, a new model was presented in which different levels of FruA phosphorylation may distinctively regulate the expression of two groups of developmental genes.

GidA is an FAD‐binding protein involved in development of Myxococcus xanthus

A gene encoding a homologue of the Escherichia coli GidA protein (glucose-inhibited division protein A) lies immediately upstream of aglU, a gene encoding a WD-repeat protein required for motility and development in Myxococcus xanthus. The GidA protein of M. xanthus shares about 48% identity overall with the small (approximately equal to 450 amino acid) form of GidA from eubacteria and about 24% identity overall with the large (approximately equal to 620 amino acid) form of GidA from eubacteria and eukaryotes. Each of these proteins has a conserved dinucleotide-binding motif at the N-terminus. To determine if GidA binds dinucleotide, the M. xanthus gene was expressed with a His6 tag in E. coli cells. Purified rGidA is a yellow protein that absorbs maximally at 374 and 450 nm, consistent with FAD or FMN. Thin-layer chromatography (TLC) showed that rGidA contains an FAD cofactor. Fractionation and immunocytochemical localization show that full length GidA protein is present in the cytoplasm and transported to the periplasm of vegetative-grown M. xanthus cells. In cells that have been starved for nutrients, GidA is found in the cytoplasm. Although GidA lacks an obvious signal sequence, it contains a twin arginine transport (Tat) motif, which is conserved among proteins that bind cofactors in the cytoplasm and are transported to the periplasm as folded proteins. To determine if GidA, like AglU, is involved in motility and development, the gidA gene was disrupted. The gidA- mutant has wild-type gliding motility and initially is able to form fruiting bodies like the wild type when starved for nutrients. However, after several generations, a stable derivative arises, gidA*, which is indistinguishable from the gidA- parent on vegetative medium, but is no longer able to form fruiting bodies. The gidA* mutant releases a heat-stable, protease-resistant, small molecular weight molecule that acts in trans to inhibit aggregation and gene expression of wild-type cells during development.

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.

Genetic suppression analysis of an asgA missense mutation in Myxococcus xanthus.

The asgA gene is required for generation of extracellular A signal, which serves as a cell-density signal for fruiting body development in Myxococcus xanthus. The AsgA protein is a histidine protein kinase and consists of a receiver domain that is conserved among response regulators of two-component signal transduction systems, followed by a histidine protein kinase domain that is conserved among sensor proteins of two-component systems. AsgA is thought to function in a signal transduction pathway that leads to expression of genes required for A-signal generation. A genetic suppression analysis of an asgA missense mutation was undertaken in order to identify genes that may provide information regarding the role of AsgA in A-signal generation and fruiting body formation. Twenty-two independent strains containing mutations that suppress asgA473 were isolated by selecting for production of heat-resistant spores under conditions that promote fruiting body development in wild-type cells. Ten of the 22 suppressor strains contained bypass suppressors. All the suppressor strains had direct spore counts at least three to four times greater than the original asgA473 mutant, and three strains had viable counts that exceeded wild-type by more than one order of magnitude. Surprisingly, none of the suppressor strains produced wild-type levels of extracellular A-signal.

Mutations that confer resistance to 2-deoxyglucose reduce the specific activity of hexokinase from Myxococcus xanthus.

The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus. Mutants of M. xanthus resistant to 2dGlc, designated hex mutants, arise at a low spontaneous frequency. Expression of the Escherichia coli glk (glucokinase) gene in M. xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of M. xanthus extracts reveal a soluble hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) activity but no phosphotransferase system activities. The hex mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hex mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and N-acetylglucosamine act as inhibitors of glucose turnover by the M. xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.

The guanosine nucleotide (p)ppGpp initiates development and A-factor production in myxococcus xanthus.

Guanosine 3'-di-5'-(tri)di-phosphate nucleotides [(p)ppGpp], synthesized in response to amino acid limitation, induce early gene expression leading to multicellular fruiting body formation in Myxococcus xanthus. A mutant (DK527) that fails to accumulate (p)ppGpp in response to starvation was found to be blocked in development prior to aggregation. By use of a series of developmentally regulated Tn5lac transcriptional fusion reporters, the time of developmental arrest in DK527 was narrowed to within the few hours of development, the period of starvation recognition. The mutant is also defective in the production of A-factor, an early extracellular cell-density signal. The relA gene from Escherichia coli, which encodes a ribosome-dependent (p)ppGpp synthetase, rescues this mutant. We also demonstrate that inactivation of the M. xanthus relA homolog blocks development and the accumulation of (p)ppGpp. Moreover, the wild-type allele of Myxococcus relA rescues DK527. These observations support a model in which accumulation of (p)ppGpp, in response to starvation, initiates the program of fruiting body development, including the production of A-factor.

A mutation that affects fibril protein, development, cohesion and gene expression in Myxococcus xanthus.

Extracellular matrix fibrils are involved in the cell-cell interactions of the social prokaryote, Myxococcus xanthus. The fibrils are composed of a carbohydrate backbone and a set of five integral fibrillar proteins (IFPs) ranging from 14 to 66 kDa. As part of an attempt to understand the function(s) of the IFPs, a mutant (ifp-1:20) was generated that lacks IFP-1:20, one of the fibril proteins, as shown by Western blot analysis of both whole cells and isolated fibrils. Unlike those of the parent strain, the fibrils of the mutant were removed easily from the cells by shear forces. Development in ifp-1:20 was aberrant--aggregation and early mound formation were delayed by 6-10 h and mature fruiting bodies never formed. Myxospore production was also greatly reduced. Additionally, fibril-mediated cohesion in ifp-1:20 was changed. Cohesion resulted in chains of cells rather than the characteristic clumps of cells seen for the parent strain. Isolated ifp-1:20 fibrils, unlike wild-type fibrils, could not rescue cohesion of non-cohesive, fibril-negative dsp cells, supporting the notion that the fibrils were functionally altered. The mutation also reduced developmental gene expression by three- to fourfold in omega 4521, a transposon insertion mutant expressed early in development. Expression of a later developmental gene fusion was not affected, suggesting that the fibrils may not be required for later developmental gene expression. These data suggest that intact fibrils may function early in development to facilitate close cell proximity for signal exchange.

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.

Effects of overexpression of Pkn2, a transmembrane protein serine/threonine kinase, on development of Myxococcus xanthus.

Pkn2 is a putative transmembrane protein serine/threonine kinase required for normal development of Myxococcus xanthus. The effect of Pkn2 overexpression on development of M. xanthus was examined by expressing pkn2 under the control of a kanamycin promoter. Pkn2 was clearly detected by Western blot (immunoblot) analysis in the overexpression strain (the PKm/pkn2 strain) but could not be detected in the wild-type strain. Overexpressed Pkn2 was located almost exclusively in the membrane fraction, suggesting that Pkn2 is a transmembrane receptor-type protein Ser/Thr kinase. The PKm/pkn2 strain formed fruiting bodies more slowly than the wild-type strain, in contrast to a Pkn2 deletion strain, the delta pkn2 strain, which developed faster than the wild-type strain. However, spore production was reduced in both the PKm/pkn2 and delta pkn2 strains. These data suggest that Pkn2 functions as a negative regulator for fruiting-body formation and that the proper level of Pkn2 is necessary for maximum myxospore yield.

Genetics of gliding motility and development in Myxococcus xanthus

Genetics of gliding motility and development in Myxococcus xanthus

Genetics of gliding motility and development inMyxococcus xanthus

Successful development in multicellular eukaryotes requires cell-cell communication and the coordinated spatial and temporal movements of cells. The complex array of networks required to bring eukaryotic development to fruition can be modeled by the development of the simpler prokaryote Myxococcus xanthus. As part of its life cycle, M. xanthus forms multicellular fruiting bodies containing differentiated cells. Analysis of the genes essential for M. xanthus development is possible because strains with mutations that block development can be maintained in the vegetative state. Development in M. xanthus is induced by starvation, and early events in development suggest that signaling stages have evolved to monitor the metabolic state of the developing cell. In the absence of these signals, which include amino acids, alpha-keto acids, and other intermediary metabolites, the ability of cells to differentiate into myxospores is impaired. Mutations that block genes controlling gliding motility disrupt the morphogenesis of fruiting bodies and sporogenesis in surprising ways. In this review, we present data that encourage future genetic and biochemical studies of the relationships between motility, cell-cell signaling, and development in M. xanthus.

Isolated fibrils rescue cohesion and development in the Dsp mutant of Myxococcus xanthus.

Extracellular fibrils are involved in cell cohesion and cell development in Myxococcus xanthus. One group of social motility mutants, Dsp, is unable to produce extracellular fibrils; these mutants also lose the abilities to cohere and to develop. Extracellular fibrils isolated from vegetative wild-type cells and added to Dsp cells fully restored the abilities of these cells to cohere and to undergo normal morphological development. The fibrils thus mimic the ability of intact, wild-type cells to carry out the same rescue. Optimal cohesion rescue by fibrils required calcium and magnesium ions, did not require protein synthesis, but was energy dependent, i.e., sodium azide and sodium cyanide blocked rescue. Cohesion rescue was also blocked by the diazo dye Congo red. Cohesion rescue is genus specific, i.e., isolated fibrils did not cause the cohesion of Pseudomonas aeruginosa, Bacillus subtilis, Proteus mirabilis, Escherichia coli, or the related myxobacterium Stigmatella aurantiaca. Developmental rescue of Dsp by isolated fibrils included aggregation, fruiting body formation, and myxospore morphogenesis. Developmental gene expression in the Dsp mutant was only partially rescued by the isolated fibrils.

Increases in the intracellular concentration of glycerol during development in Myxococcus xanthus S. Courtney Frasch

The role of glycerol as a natural morphogen of myxospore formation in Myxococcus xanthus was examined. Glycerol was extracted from cells undergoing development and analyzed by gas chromatography. Glycerol is present in cells, and the intracellular level undergoes a series of transient increases during development. The data suggest a role for glycerol in myxosporulation and fruiting body morphogenesis supporting the notion that this chemical induction of sporulation may represent a physiological pathway in development.

Increases in the intracellular concentration of glycerol during development in Myxococcus xanthus S. Courtney Frasch.

The role of glycerol as a natural morphogen of myxospore formation in Myxococcus xanthus was examined. Glycerol was extracted from cells undergoing development and analyzed by gas chromatography. Glycerol is present in cells, and the intracellular level undergoes a series of transient increases during development. The data suggest a role for glycerol in myxosporulation and fruiting body morphogenesis supporting the notion that this chemical induction of sporulation may represent a physiological pathway in development.

Evidence that asgB encodes a DNA-binding protein essential for growth and development of Myxococcus xanthus

The asg mutants of Myxococcus xanthus are defective in production of extracellular A-signal, which serves as a cell density signal for fruiting-body development. The DNA sequence of asgB, one of the three asg genes, was determined. The deduced amino acid sequence of AsgB contains a DNA-binding helix-turn-helix motif near the C terminus. This putative helix-turn-helix is highly similar to the helix-turn-helix in region 4.2 of major sigma factors, which is the region that recognizes and interacts with -35 sequences of promoters. We propose that AsgB is a transcription factor that binds to DNA sequences similar to the -35 hexamer, TTGACA. Analyses of asgB RNA levels and expression of an asgB-lacZ translational fusion indicate that expression of asgB remains fairly constant during the transition from growth into early development. The mutation within the asgB480 allele was identified as an A-to-G transition that results in a threonine-to-alanine substitution in the predicted protein product. Attempts to replace the wild-type copy of asgB with a null allele failed, indicating that asgB may be essential for growth.

Identification of esg, a genetic locus involved in cell-cell signaling during Myxococcus xanthus development

JD258, a Tn5 insertion mutant of Myxococcus xanthus, was shown to have major defects in three development-associated properties: expression of the developmentally regulated tps gene, spore formation, and production of multicellular fruiting bodies. The defects in tps gene expression and sporulation could be substantially corrected, at the phenotypic level, by mixing JD258 with wild-type cells (extracellular complementation). By this criterion, JD258 appeared to be a new member of a group of conditional developmental mutants that were previously characterized and placed in four extracellular complementation groups (A to D) based on the ability of mutants in one group to stimulate development in mutants belonging to a different group (D. C. Hagen, A. P. Bretscher, and D. Kaiser, Dev. Biol. 64:284-296, 1978). Mutants from groups A, B, C, and D all displayed extracellular complementation activity when mixed with JD258. These results, and other aspects of the phenotype of JD258, indicate that this mutant defines a fifth extracellular complementation group, group E. The M. xanthus esg locus identified by the Tn5 insertion in JD258 was cloned in Escherichia coli and used for further genetic analysis of the locus. These studies indicated that the esg locus resides within a 2.5-kb region of the M. xanthus chromosome and that the locus contains at least two genetic complementation groups. Our results are consistent with a model in which the esg locus controls the production of a previously unrecognized extracellular signal that must be transmitted between cells for the completion of M. xanthus development.

DevRS, an autoregulated and essential genetic locus for fruiting body development in Myxococcus xanthus.

Two Tn5 lac insertions into the Myxococcus genome at sites omega 4414 and omega 4473, which are separated by 550 nucleotides, inactivate fruiting body development. Sporulation is decreased 100- to 10,000-fold. At least two genes, devR and devS, are transcribed in this region, probably as an operon. Expression of devR begins by 6 h after starvation has initiated development. On the basis of their nucleotide sequences, devR and devS are expected to encode proteins of 302 and 214 amino acids, respectively. Dev+ function can be restored by a segment of 7.8 kb cloned from the devRS region of wild-type cells. Two experiments show that devR expression is under strong negative autoregulation. beta-Galactosidase is expressed at a higher level from a transcriptional devR::lacZ fusion when the fused operon is in a dev strain than when it is in the dev/dev+ genetic background of a partial diploid. There is more mRNA accumulation from the devRS region in the dev strain than in a rescued dev/dev+ tandem duplication strain. Sporulation rescue is correlated with some degree of negative autoregulation, even though sporulation is not inversely proportional to beta-galactosidase expression from omega 4414. A second level of regulation is suggested by complementation of dev by dev+ in duplication strains. The expression of devRS, measured by sporulation levels, differs 1,000-fold when devRS+ is moved from a distance of 20 kb to 3 Mb from the mutant devRS locus. Expression of devR is also dependent on the cell density at which development is initiated, a third level of regulation. Multiple levels of regulation suggest that devRS is a switch required to activate completion of aggregation and sporulation.

Mutations in two new loci that impair both extracellular protein production and development in Myxococcus xanthus.

Two transposon insertion mutants of Myxococcus xanthus altered in the secretion of protein as determined by the hydrolytic activities of several enzymes during vegetative growth were also unable to complete fruiting body formation and were severely impaired in sporulation. The insertions were located in the same part of the M. xanthus chromosome but were unlinked by transduction and therefore define two distinct loci, called excA and excB. Since both Exc +/- mutants were able to rescue development of an asgB mutation, they do not belong to the Asg- group, despite of the fact that asg mutants are also Exc +/-. Our results sustain the hypothesis of a possible relationship between protein secretion during vegetative growth and development or 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.

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.