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PubMed1984

Abnormal motility and fruiting behavior of Myxococcus xanthus bacteriophage-resistant strains induced by a clear-plaque mutant of bacteriophage Mx8.

Myxococcus xanthus mutants resistant to a clear-plaque derivative of phage Mx8 were isolated. A significant fraction of the mutants, easily recognizable by their colony morphology, were induced by the presence of the phage and may correspond to low-frequency lysogens. They were all defective in cell motility and showed the same nonfruiting phenotype under starvation conditions.

Bacteriophages↗
PubMed1984

Autocides produced by Myxococcus xanthus.

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

Anti-Bacterial Agents↗
PubMed1985

Transfer of plasmid RP4 to Myxococcus xanthus and evidence for its integration into the chromosome.

The broad-host-range plasmid RP4 and its derivative R68.45 were transferred to Myxococcus xanthus DK101 and DZ1; RP4 was maintained integrated in the chromosome. Loss of plasmid markers occurred during the growth of the transconjugants, which could be prevented by selective pressure with oxytetracycline. The integrated plasmid was transferred back to Escherichia coli often as RP4-prime plasmids carrying various segments of the M. xanthus chromosome. It also mediated chromosomal transfer between M. xanthus strains.

Chromosomes, Bacterial↗
PubMed1985

Differential expression of protein S genes during Myxococcus xanthus development.

Protein S, the most abundant protein synthesized during development of the fruiting bacterium Myxococcus xanthus, is coded by two highly homologous genes called protein S gene 1 (ops) and protein S gene 2 (tps). The expression of these genes was studied with fusions of the protein S genes to the lacZ gene of Escherichia coli. The gene fusions were constructed so that expression of beta-galactosidase activity was dependent on protein S gene regulatory sequences. Both the gene 1-lacZ fusion and the gene 2-lacZ fusion were expressed exclusively during fruiting body formation (development) in M. xanthus. However, distinct patterns of induction of fusion protein activity were observed for the two genes. Gene 2 fusion activity was detected early during development on an agar surface and could also be observed during nutritional downshift in dispersed liquid culture. Gene 1 fusion activity was not detected until much later in development and was not observed after downshift in liquid culture. The time of induction of gene 1 fusion activity was correlated with the onset of sporulation, and most of the activity was spore associated. This gene fusion was expressed during glycerol-induced sporulation when gene 2 fusion activity could not be detected. The protein S genes appear to be members of distinct regulatory classes of developmental genes in M. xanthus.

Bacterial Proteins↗
PubMed1985

Physical mapping of a 330 X 10(3)-base-pair region of the Myxococcus xanthus chromosome that is preferentially labeled during spore germination.

Myxococcus xanthus was pulse-labeled with [3H]thymidine immediately after germination of dimethyl sulfoxide-induced spores. The restriction enzyme digests of the total chromosomal DNA from the pulse-labeled cells were analyzed by one-dimensional as well as two-dimensional agarose gel electrophoresis. Four PstI fragments preferentially labeled at a very early stage of germination were cloned into the unique PstI site of pBR322. By using these clones as probes, a restriction enzyme map was established covering approximately 6% of the total M. xanthus genome (330 X 10(3) base pairs). The distribution of the specific activities of the restriction fragments pulse-labeled after germination suggests a bidirectional mode of DNA replication from a fixed origin.

Chromosome Mapping↗
PubMed1985

Two homologous genes coding for spore-specific proteins are expressed at different times during development of Myxococcus xanthus.

The ops and tps genes of Myxococcus xanthus have ca. 90% DNA and amino acid sequence homology and are in the same orientation separated by a spacer region of only 1.4 kilobases. The products of the two genes were found to cross-react immunologically, and both were capable of Ca2+-dependent self-assembly on the surface of myxospores. However, the ops and tps genes were expressed very differently during the developmental cycle of M. xanthus. The tps gene is induced early during fruiting body formation on a solid surface, and its product, protein S, is made in large quantities (up to 15% of total protein synthesis). When the cells turn into myxospores, protein S is assembled on the outer surface of the spore. We have now also found it in much smaller quantities inside the spores. The ops gene, on the other hand, appears to be induced later in development, after the cells have sporulated, since the ops gene product was found only inside the spores. When an ops gene under the control of a tps gene promoter was inserted into a wild-type strain, the ops gene product was synthesized at the same time as protein S and assembled onto the spore surface.

Bacterial Proteins↗
PubMed1985

Novel one-step cloning vector with a transposable element: application to the Myxococcus xanthus genome.

A new strategy was developed for rapid cloning of genes with a transposon mutation library. We constructed a transposon designated TnV that was derived from Tn5 and consists of the gene coding for neomycin phosphotransferase II as well as the replication origin of an Escherichia coli plasmid, pSC101, flanked by Tn5 inverted repeats (IS50L and IS50R). TnV can transpose to many different sites of DNA in E. coli and Myxococcus xanthus and confers kanamycin resistance (Kmr) to the cells. From the Kmr cells, one-step cloning of a gene which is mutated as a result of TnV insertion can be achieved as follows. Chromosomal DNA isolated from TnV-mutagenized cells is digested with an appropriate restriction enzyme, ligated, and transformed into E. coli cells with selection for Kmr. The plasmids isolated contain TnV in the target gene. The plasmid DNA can then be used as a probe for characterization of the gene and screening of clones from a genomic library. We used this vector to clone DNA fragments containing genes involved in the development of M. xanthus.

Cloning, Molecular↗
PubMed1985

Methylation of macromolecules during development in Myxococcus xanthus.

Covalent modification of macromolecules can serve to alter their biological activities and is therefore frequently involved in regulation. I examined methylation of proteins and carbohydrates during development and vegetative growth in the procaryote Myxococcus xanthus. Striking differences in the patterns of protein methylation occurred when cell development was induced by nutrient deprivation on solid media and when cells were starved in liquid. In addition, a methylated, protease-resistant macromolecule which contained carbohydrate and which may have been an unusual type of lipopolysaccharide was observed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of methylation patterns in various media and an analysis of the time course of methylation indicated that changes in methylation were part of the developmental pathway which includes aggregation. Induction of development in liquid by glycerol produced no changes in methylation.

Bacterial Proteins↗
PubMed1986

Isolation of a surface glycoprotein from Myxococcus xanthus.

The isolation of a glycoprotein from vegetative cells of Myxococcus xanthus is reported. The protein, abbreviated VGP, was first identified during a survey of surface proteins as a major protein that could be radioiodinated in vegetative, but not developing, cells (P.Y. Maeba, J. Bacteriol. 155:1033-1041, 1983). The protein was extracted from membranes with Triton X-100 and subsequently purified by DEAE-cellulose chromatography, chromatofocusing, and gel filtration. The protein has an Mr of approximately 74,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.2 to 3.3. The carbohydrate moiety which made up approximately 13.5% of the weight of the VGP comprised primarily neutral sugars and smaller amounts of hexosamines and uronic acids. The amino acid content revealed no unusual features, but analysis by the method of Barrantes (F. Barrantes, Biochem. Biophys. Res. Commun. 62:407-414, 1975) indicated it is likely a peripheral membrane protein. The protein makes up approximately 1% of the total cell protein and is a prominent surface structure. Because glycoproteins have been implicated in cellular interactions in a number of systems, the VGP may play an important role in the social behavior exhibited by M. xanthus.

Amino Acids↗
PubMed1986

Analysis of the products of the Myxococcus xanthus frz genes.

The frizzy (frz) genes of Myxococcus xanthus control the ability of cells to reverse direction of gliding motility. The orientation of the frz genes was studied by isolating transcriptional fusions with the transposon derivative Tn5-lac. The frz genes were then cloned in the proper orientation in an expression vector. By using maxicell experiments, we were able to identify several labeled bands which were plasmid encoded. To identify the labeled proteins and their respective genes, we constructed deletion plasmids in which various regions of the insert DNA had been removed. The plasmid-encoded proteins were then labeled in maxicell experiments, and the bands which correspond to the frzCD, frzE, and frzF gene products were identified. The sizes of the gene products agreed with the genetic and physical map of the cloned DNA.

Bacterial Proteins↗
PubMed1986

Correlation of energy-dependent cell cohesion with social motility in Myxococcus xanthus.

An agglutination assay was used to study cell cohesion in the myxobacterium Myxococcus xanthus. Vegetative cells agglutinated in the presence of the divalent cations Mg2+ and Ca2+. Agglutination was blocked by energy poisons that inhibit electron transport, uncouple oxidative phosphorylation, or inhibit the membrane-bound ATPase. However, energy was not required for the maintenance of cells in the multicellular aggregate. Cyanide, a strong inhibitor of agglutination, did not cause cells to dissociate from the aggregate even when shear forces were applied. While gliding motility was not necessary for agglutination, some gliding mutants exhibited aberrant agglutination that was generally correlated with cell behavior. Cells with an intact social motility system were cohesive and glided in large multicellular swarms. Cells with a mutation in their social motility system were 5- to 10-fold less cohesive and tended to glide as single cells. One group of social motility mutants, known as Dsp, did not agglutinate.

Adenosine Triphosphatases↗
PubMed1986

Role of cell cohesion in Myxococcus xanthus fruiting body formation.

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

Agglutination↗
PubMed1986

Myxococcus xanthus autocide AMI.

Autocide AMI of Myxococcus xanthus was purified and shown to be a mixture of fatty acids: 46.4% saturated, 49.3% monounsaturated, and 4.3% diunsaturated. The specific autocidal activities (units per milligram) were as follows: purified AMI, 1,000; saturated fraction, 100; monounsaturated fraction, 800; diunsaturated fraction, 2,200. Model fatty acids mimicked to some extent the activity of AMI, although none of the fatty acids tested were as active as purified AMI. Spontaneous and induced mutants of M. xanthus were selected for resistance to AMI and to fatty acids. The AMI-resistant mutants were also resistant to the model fatty acids, whereas resistance to fatty acids was specific to the compound used for mutant selection. All AMI- and fatty acid-resistant mutants examined were found to be blocked in fruiting body formation. Some of these mutants were able to form normal fruiting bodies when mixed with the extracellular fluid of the parental strain. The data suggest that AMI plays a role in developmental lysis of M. xanthus.

Bacteriolysis↗
PubMed1986

Genetic analysis of Myxococcus xanthus and isolation of gene replacements after transduction under conditions of limited homology.

Genetic analysis of Myxococcus xanthus is greatly facilitated by the ability to introduce cloned DNA into M. xanthus to generate gene replacement and merodiploid strains. However, gene replacement strains are difficult to obtain when the region(s) of homology between the cloned DNA and the M. xanthus chromosome is limited (less than 1 kilobase). We found that gene replacements can be obtained at an increased frequency by a two-step procedure involving the use of bacteriophage P1 to isolate merodiploid strains followed by generalized transduction to another M. xanthus strain by using phage Mx4.

Anti-Bacterial Agents↗
PubMed1986

Control of developmental gene expression by cell-to-cell interactions in Myxococcus xanthus.

The ssbA mutants of Myxococcus xanthus behave as if they are unable to produce a cell-to-cell signal required for normal development. They are unable to form fruiting bodies or spores on developmental medium. They do sporulate, however, if allowed to develop in mixtures with wild-type cells. Fusions of developmentally induced promoters of M. xanthus to the Escherichia coli lacZ gene were used to characterize the effect of the ssbA mutations on developmental gene expression. Each of the five independent fusions tested was found to be dependent upon the ssbA+ allele for full expression. The ssbA mutants were able to express each of these fusions if the mutants were allowed to develop in mixtures with wild-type (Lac-) cells. These results cannot be explained on the basis of genetic exchange. The data are consistent with regulation of gene expression mediated by cell-to-cell interactions.

DNA, Recombinant↗
PubMed1986

Cell surface antigens during submerged development of Myxococcus xanthus examined with monoclonal antibodies.

Eighteen monoclonal antibodies directed against cell surface antigens of Myxococcus xanthus were followed by enzyme-linked immunosorbent assay. Three of the monoclonal antibodies were specifically directed against antigens present only on cells undergoing fruiting body development. These cell surface antigens became detectable by the early preaggregation stage (2 to 4 h) of development and increased until early aggregation (9 to 10 h), after which the concentrations of two of the cell surface antigens remained constant and the concentration of the third decreased. The remaining 15 monoclonal antibodies recognized cell surface antigens that were shared by vegetative and developing cells. Based on their relative concentrations during development, these shared antigens can be grouped into three classes. In the first class antigen concentration remained constant, in the second it decreased, and in the third it increased. Western blots of cell surface antigens were probed with monoclonal antibodies. Five monoclonal antibodies reacted with material in distinct bands, five monoclonal antibodies reacted with multiple, diffuse bands, and eight monoclonal antibodies were not reactive in Western blots.

Antibodies, Bacterial↗
PubMed1986

Heat shock proteins of vegetative and fruiting Myxococcus xanthus cells.

The heat shock response of Myxococcus xanthus was investigated and characterized. When shifted from 28 to 40 degrees C, log-phase cells rapidly ceased growth, exhibited a 50% reduction in CFU, and initiated the synthesis of heat shock proteins (HTPs). Heat-shocked log-phase M. xanthus cells labeled with [35S]methionine were found to produce 18 major HTPs. The HTPs, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, were characterized with regard to molecular mass, subcellular location (periplasm, membrane, or cytoplasm), and temperature required for expression. Most HTPs were expressed at 36 degrees C, the optimum growth temperature of M. xanthus. Cells preincubated at 36 degrees C for 1 h before being shifted to 40 degrees C demonstrated increased thermotolerance compared with cells shifted directly from 28 to 40 degrees C. The HTPs produced by heat-shocked starvation-induced fruiting cells and glycerol-induced sporulating cells were also analyzed and characterized. Thirteen HTPs were detected in fruiting cells shifted from 28 to 40 degrees C. Six of these HTPs were not seen in vegetative M. xanthus cells. Log-phase cells induced to sporulate by the addition of glycerol produced 17 HTPs after being shifted to 40 degrees C. These HTPs were found to be a mixture of HTPs detected in heat-shocked log-phase cells and heat-shocked fruiting cells.

Bacterial Proteins↗
PubMed1987

Cell-density-dependent killing of Myxococcus xanthus by autocide AMV.

Autocide AMV of Myxococcus xanthus was purified and identified as phosphatidylethanolamine. Alkaline hydrolysis of AMV yielded a high proportion of mono- and diunsaturated fatty acids. The bactericidal activity of AMV on M. xanthus depended upon the density of target cells: the greater the cell density, the greater the killing by AMV. For example, at 2 U of AMV per ml, 0, 50, and 99% killing was measured with 2 X 10(4), 2 X 10(5), and 2 X 10(7) target cells per ml, respectively. The cell-density-dependent activity of AMV was also observed on solid medium. Studies with model lipid compounds suggest that the inhibitory activity of AMV is due to the fatty acid moiety, released from phosphatidylethanolamine by the concerted (enzymatic) activity of many cells. Mutants of M. xanthus selected for resistance to AMI (a mixture of fatty acids) were also resistant to AMV. The possible role of AMV in developmental lysis is discussed.

Chromatography, Thin Layer↗