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PubMed1990

Role of autocide AMI in development of Myxococcus xanthus.

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

DNA Transposable Elements↗
PubMed1990

Developmental sensory transduction in Myxococcus xanthus involves methylation and demethylation of FrzCD.

Myxococcus xanthus is a bacterium that moves by gliding motility and exhibits multicellular development (fruiting body formation). The frizzy (frz) mutants aggregate aberrantly and therefore fail to form fruiting bodies. Individual frz cells cannot control the frequency at which they reverse direction while gliding. Previously, FrzCD was shown to exhibit significant sequence similarity to the enteric methyl-accepting chemotaxis proteins. In this report, we show that FrzCD is modified by methylation and that frzF encodes the methyltransferase. We also identify a new gene, frzG, whose predicted product is homologous to that of the cheB (methylesterase) gene from Escherichia coli. Thus, although M. xanthus is unflagellated, it appears to have a sensory transduction system which is similar in many of its components to those found in flagellated bacteria.

Amino Acid Sequence↗
PubMed1990

CsgA, an extracellular protein essential for Myxococcus xanthus development.

CsgA mutants of Myxococcus xanthus appear to be defective in producing an extracellular molecule essential for the developmental behaviors of this bacterium. The csgA gene encodes a 17.7-kilodalton polypeptide whose function and cellular location were investigated with immunological probes. Large quantities of the CsgA gene product were obtained from a lacZ-csgA translational gene fusion expressed in Escherichia coli. The chimeric 21-kilodalton protein was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity-purified polyclonal antibodies raised against the fusion protein were used to determine the cellular location of the native CsgA protein by colloidal gold labeling and transmission electron microscopy. Between 1,100 and 2,200 extracellular molecules of CsgA per developing M. xanthus cell were detected, most of which were associated with the extracellular matrix. The anti-CsgA antibodies inhibited wild-type development unless they were first neutralized with the fusion protein. Together these results suggest that the CsgA gene product has an essential, extracellular function during development, possibly as a pheromone.

Bacterial Proteins↗
PubMed1991

Development in Myxococcus xanthus involves differentiation into two cell types, peripheral rods and spores.

Myxococcus xanthus, a gram-negative bacterium, has a complex life cycle. In response to starvation, most cells in a population participate in the formation of multicellular aggregates (i.e., fruiting bodies) in which cells differentiate into spores. However, some cells do not enter aggregates. In this and the two accompanying reports, the biology and physiology of these nonaggregated cells is examined. A technique to separate aggregated cells from nonaggregated cells was developed; then differentiating cells at stages throughout the course of development were isolated. In this report we (i) describe peripheral rods, those cells which remain outside aggregates after aggregation has ceased in the rest of the population; (ii) document the occurrence of peripheral rods in several wild-type strains; and (iii) characterize the expression of developmentally regulated genes in both aggregated and nonaggregated cells. These studies have shown that myxobacterial hemagglutinin, protein S (Tps), protein S1 (Ops), protein C, and several phosphatase activities are expressed in cell-type-specific patterns. These data demonstrate that peripheral rods constitute a cell type distinct from either vegetatively growing cells or spores. The description of a second, late developmental cell type (in addition to spores) opens an entirely new line of investigation in M. xanthus, i.e., the regulation of the differentiation of vegetatively growing cells into two cell types that differ significantly in biology, shape, and localization within the population.

Bacterial Outer Membrane Proteins↗
PubMed1991

Analysis of Myxococcus xanthus cell types by two-dimensional polyacrylamide gel electrophoresis.

Myxococcus xanthus is a gram-negative, soil-dwelling bacterium that undergoes development in response to depletion of nutrients. Whereas most cells aggregate into multicellular mounds in which they differentiate into spores, 10 to 20% of the developing cells remain outside fruiting bodies as peripheral rods. We used two-dimensional polyacrylamide gel electrophoresis to analyze the global expression of polypeptides in cells taken from six stages in the life cycle: vegetatively growing cells, cells 15 h after the induction of development, peripheral rods, prespores (sonication-sensitive, aggregated cells), fruiting-body spores (sonication-resistant, aggregated cells) 96 h after the induction of development, and glycerol-induced spores 15 h after induction. Seven hundred sixty-one discrete sample spots (SSPs) were identified among the six gels. Comparisons among the samples revealed that each sample had some unique SSPs, ranging from 0.3% of the 15-h developing cell SSPs to 17.9% of 96-h peripheral rod SSPs. Sixty-eight SSPs were ubiquitously distributed, but the relative amounts of these SSPs varied among the samples. Statistical analyses of the distribution and relative quantities of the SSPs indicate that, within a confidence level of greater than 99.99%, peripheral rods are significantly different from vegetatively growing cells, 15-h developing cells, prespores, fruiting-body spores, and glycerol-induced spores. In fact, among the six samples studied, only 15-h developing cells and glycerol-induced spores were similar to each other within a confidence level of P greater than or equal to 0.05. These results are consistent with the description of peripheral rods as a distinct developmental cell type.

Bacterial Proteins↗
PubMed1991

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.

Cell Adhesion↗
PubMed1991

Protein U, a late-developmental spore coat protein of Myxococcus xanthus, is a secretory protein.

Protein U is a spore coat protein produced at the late stage of development of Myxococcus xanthus. This protein was isolated from developmental cells, and its amino-terminal sequence was determined. On the basis of this sequence, the gene for protein U (pru) was cloned and its DNA sequence was determined, revealing an open reading frame of 179 codons. The product from this open reading frame has a typical signal peptide of 25 amino acid residues at the amino terminal end, followed by protein U of 154 residues. This result indicates that protein U is produced as a secretory precursor, pro-protein U, which is then secreted across the membrane to assemble on the spore surface. This is in sharp contrast to protein S, a major spore coat protein produced early in development, which has no signal peptide, indicating that there are two distinct pathways for trafficking of spore coat proteins during the differentiation of M. xanthus.

Amino Acid Sequence↗
PubMed1991

Survey of multicopy single-stranded DNAs and reverse transcriptase genes among natural isolates of Myxococcus xanthus.

Twenty different isolates of the soil bacterium Myxococcus xanthus were examined for the presence of multicopy single-stranded DNA (msDNA)-producing retroelements, or retrons. Each strain was analyzed by ethidium bromide staining for msDNA, 32P labeling of the msDNA molecule by the reverse transcriptase (RT) extension method, and DNA hybridization experiments with probes derived from two retrons, Mx162 and Mx65, previously cloned from M. xanthus DZF1. These analyses revealed that all M. xanthus strains contain an msDNA very similar to Mx162 msDNA, and 13 strains also contain a second smaller msDNA very similar to Mx65 msDNA. In addition, the strains contained retron-encoded genes msr and msd, which code for msDNA, and a gene for RT responsible for the synthesis of msDNA. These genes show greater than 80% nucleotide sequence similarity to retrons Mx162 or Mx65. The near-ubiquitous occurrence of msDNA retrons among M. xanthus strains and their homogeneous nature are in marked contrast to the highly diverse but rarely occurring msDNA-producing elements of Escherichia coli. The possible origin and evolution of RT and retron elements is discussed in view of these findings.

Bacteria, Aerobic↗
PubMed1991

Suppressors that permit A-signal-independent developmental gene expression in Myxococcus xanthus.

Progression through the early stages of Myxococcus xanthus fruiting body development requires the cell-to-cell transmission of soluble material called A signal. During these early stages, expression from the gene identified by Tn5 lac insertion omega 4521 increases. A DNA probe of the omega 4521 gene was constructed. Use of this probe showed that accumulation of mRNA corresponding to the omega 4521 gene depends upon A signal. A-signal-deficient (asg) mutants fail to accumulate this RNA, and the external addition of A signal restores accumulation. To identify links between A signal and its responsive gene, omega 4521, suppressors of an asg mutation were generated. All of the suppressor alleles restored lacZ expression from omega 4521 in the absence of A signal, and they were demonstrated to be neither reversions of the asgB mutation nor mutations in the promoter of omega 4521. Fifteen suppressor mutations map to two loci, sasA and sasB (for suppressor of asg). sasA and sasB mutants differ phenotypically during growth and development. Mid-logarithmic-phase sasA asgB double mutants, like sas+ asg+ strains, express low levels of lacZ, whereas sasB asgB double mutants express high levels. sasA asg+ mutants form abnormal colonies, are less cohesive than wild type, and are defective in fruiting body formation and sporulation. In contrast, sasB asg+ mutants form normal colonies, are as cohesive as wild type, and appear to develop normally. The characteristics of sasA suppressors implicate the sasA+ product as a negative regulator in the A-signal-dependent regulation of omega 4521.

Blotting, Northern↗
PubMed1991

C-factor has distinct aggregation and sporulation thresholds during Myxococcus development.

C-factor, the protein product of the csgA gene, acts as a short-range morphogenetic signal. It is required for fruiting body development of the gram-negative bacterium Myxococcus xanthus. Aggregation, sporulation, and expression of a set of genes that are C-factor dependent, all of which fail in csgA mutant cells, are completely restored by addition of purified C-factor. We report here that, depending on its concentration, C-factor can elicit two distinct morphogenetic and transcriptional responses from csgA cells. Low levels of C-factor bring about aggregation and expression of an early C-dependent gene, whereas higher levels lead to the same effects plus expression of a late C-dependent gene and spore formation. C-factor positively regulates its own transcription. An approximately fourfold net increase in csgA transcription and C-factor levels during development was measured. We propose that autoregulation and the two distinct activity thresholds allow C-factor to act as a timer, first triggering aggregation, then sporulation, thereby producing the appropriate developmental order.

Bacterial Proteins↗
PubMed1991

Physical map of the Myxococcus xanthus chromosome.

The genome of Myxococcus xanthus, which is 9,454 kbp, is one of the largest bacterial genomes. The organization of the DNA and the distribution of genes encoding social and developmental behaviors were examined by using pulsed field gel electrophoresis. Intact genomic DNA was digested with AseI into 16 restriction fragments, which were separated by contour-clamped homogeneous electric field electrophoresis, purified, and radiolabeled. Each AseI fragment was hybridized to SpeI-digested DNA and to an M. xanthus genomic library contained in yeast artificial chromosomes. Some SpeI restriction fragments and yeast artificial chromosome clones contained AseI sites and hybridized with two different AseI restriction fragments, providing evidence for the juxtaposition of these AseI restriction fragments in the chromosome. The deduced AseI physical map is circular, suggesting that this bacterium contains a single, circular chromosome. Transposable elements shown by transduction to be in or near genes of interest were located on specific AseI restriction fragments by restriction analysis and Southern hybridization. Most AseI restriction fragments contained genes involved in social and developmental behaviors.

Blotting, Southern↗
PubMed1991

A unique repetitive DNA sequence in the Myxococcus xanthus genome.

We found a novel type of repetitive DNA sequence in the Myxococcus xanthus genome. The first repetitive sequence is located in the spacer region between the ops and tps genes. We cloned five other repetitive sequences using the first repetitive sequence as a probe and determined their nucleotide sequences. Comparison of these sequences revealed that the repetitive sequences consist of a 87-bp core sequence and that some clones share additional homology on their flanking regions.

Base Sequence↗
PubMed1991

Myxococcus xanthus protein C is a major spore surface protein.

Fruiting body formation in Myxococcus xanthus involves the aggregation of cells to form mounds and the differentiation of rod-shaped cells into spherical myxospores. The surface of the myxospore is composed of several sodium dodecyl sulfate (SDS)-soluble proteins, the best characterized of which is protein S (Mr, 19,000). We have identified a new major spore surface protein called protein C (Mr, 30,000). Protein C is not present in extracts of vegetative cells but appears in extracts of developing cells by 6 h. Protein C, like protein S, is produced during starvation in liquid medium but is not made during glycerol-induced sporulation. Its synthesis is blocked in certain developmental mutants but not others. When examined by SDS-polyacrylamide gel electrophoresis, two forms of protein C are observed. Protein C is quantitatively released from spores by treatment with 0.1 N NaOH or by boiling in 1% SDS. It is slowly washed from the spore surface in water but is stabilized by the presence of magnesium. Protein C binds to the surface of spores depleted of protein C and protein S. Protein C is a useful new marker for development in M. xanthus because it is developmentally regulated, spore associated, abundant, and easily purified.

Antigens, Surface↗
PubMed1991

Low-temperature induction of Myxococcus xanthus developmental gene expression in wild-type and csgA suppressor cells.

The csgA gene encodes an extracellular protein that plays an essential role in the regulation of fruiting-body formation and sporulation of Myxococcus xanthus. The csgA suppressor allele soc-500 (formerly referred to as csp-500) was selected based on its ability to restore sporulation to csgA cells under developmental conditions at 32 degrees C. The soc-500 allele was subsequently found to induce sporulation of csgA+ or csgA cells simply by shifting the temperature of vegetatively growing cells to 15 degrees C. Low-temperature-induced sporulation of soc-500 strains occurred in the absence of two requirements for fruiting-body sporulation: low nutrient levels and a high temperature. Low temperature alone caused the expression of many developmentally regulated genes but did not support the development of wild-type cells. The soc-500 allele appears to activate genes involved with sensing nutritional stress. At low temperature on a nutritionally rich medium, soc-500 induced expression of the tps gene which is normally expressed following nutritional shiftdown. The soc-500 allele was cloned and integrated into the wild-type chromosome by site-specific recombination. It was dominant over the wild-type allele in merodiploids and is contained on a 3-kbp DraI-ClaI restriction fragment. The soc-500 transcriptional unit spans a 300-bp PstI-PstI restriction fragment, since deletion of the PstI restriction fragment inhibits both csgA suppression and low-temperature induction. These results suggest that the soc-500 mutation lies in a gene that is involved in nutrient sensing.

Alleles↗
PubMed1967

Deoxyribonucleic acid synthesis during exponential growth and microcyst formation in Myxococcus xanthus.

Myxococcus xanthus in exponential phase with a generation time of 270 min contained a period of 50 min during which deoxyribonucleic acid (DNA) synthesis did not take place. After induction of microcysts by the glycerol technique, the DNA content increased 19%. Autoradiographic experiments demonstrated that the DNA made after glycerol induction was not evenly distributed among the microcysts. The distribution of grains per microcyst fits the following model of chromosome replication: in exponential phase, each daughter cell receives two chromosomes which are replicated sequentially during 80% of the divison cycle; after microcyst induction, no chromosomes are initiated. Mathematical formulas were derived which predict the kinetics and discrete probability distribution for several chromosome models.

Autoradiography↗
PubMed1967

Ribonucleic acid synthesis during morphogenesis in Myxococcus xanthus.

Ribonucleic acid synthesis was measured during the morphogenesis of Myxococcus xanthus. After induction of microcyst formation by the addition of glycerol to an exponential culture, net ribonucleic acid (RNA) synthesis was immediately terminated (measured either chemically or by the accumulation of acid-insoluble radioactivity). Extensive RNA turnover did take place, however, including RNA made both before and after induction. Sucrose gradient centrifugation revealed that ribosomes and ribosomal RNA were synthesized during microcyst formation even though there was no net RNA synthesis. Base analyses of the total RNA of vegetative cells and 120-min microcysts were indistinguishable.

Adenine↗
PubMed1968

Nutrition of Myxococcus xanthus FBa and some of its auxotrophic mutants.

A defined medium containing 15 amino acids plus salts was used to study the nutrition of Myxococcus xanthus FBa. The amino acids phenylalanine, leucine, isoleucine, valine, and methionine were essential for growth, whereas glycine, proline, asparagine, alanine, lysine, and threonine stimulated growth. An unusual pattern of requirement was found in the aromatic amino acids. Phenylalanine was essential and served as the precursor of tyrosine. Growth in the absence of tryptophan was adaptive, with cells reaching a growth rate equal to that of controls after a lag of about a week. (14)C-labeled ribose and glucose were not appreciably metabolized. Auxotrophs requiring purines and pyrimidines were isolated and were used to study the fate of externally supplied nucleic acid derivatives. Appropriate mutants could satisfy their requirements with free bases, nucleosides, and nucleotides, and could hydrolyze nucleic acids and use the products. However, studies using (14)C-ribose-labeled uridine (isolated from a Salmonella typhimurium pyrimidine auxotroph) showed that externally supplied nucleic acid derivatives were incorporated almost solely into the nucleic acids of the myxobacters, with little used either for energy-yielding oxidations or other cell anabolism.

Amino Acids↗
PubMed1968

Nutritional induction and suppression of fruiting in Myxococcus xanthus FBa.

A defined agar medium (A agar) containing 15 amino acids in concentrations between 0.5 and 2 mm was developed for studying the fruiting cycle of Myxococcus xanthus FBa. Cells grew only vegetatively in this medium unless the initial concentration of one of nine required or stimulatory amino acids was lowered about 50-fold. In the latter circumstance, fruiting bodies developed after several days of vegetative growth. The conclusion was that fruiting occurred when any amino acid required for normal growth became limiting in the environment. High concentrations (10 mm) of phenylalanine, tryptophan, or methionine prevented fruiting without affecting growth. Mutants requiring arginine, thymidine, or adenine could not be induced to fruit by limiting their unique requirement although they responded to the same deprivations which brought about fruiting of the wild type. A histidine auxotroph formed fruiting bodies when histidine was lowered to growth-limiting concentrations, provided that the medium was supplemented with purines. A uracil auxotroph was isolated that, perhaps secondarily, had lost some of the mechanisms which control the formation of fruiting bodies; if uracil was present, it formed fruits even when no amino acid was limiting. No concentration of uracil was sufficient to prevent fruiting. Fruiting bodies were formed when mixtures of the uracil auxotroph and wild-type cells were inoculated on A agar plus uracil, even when 75% of the cells were wild type. Microcysts of both strains were present in the fruiting bodies.

Amino Acids↗