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.     

Enhancement of extracellular enzymatic activities produced by immobilized growing cells of Myxococcus xanthus

Summary The gram-negative soil bacterium Myxococcus xanthus was immobilized by entrapping into carrageenan gel beads. Unexpectedly, the growth rate was hardly increased, and the released free cell concentration remained low. However, extracellular proteolytic and bacteriolytic activities produced in the medium or inside the beads was greatly increased and (or) stabilized as compared to the control. These properties might be quite useful in view of using Myxococcus xanthus as a cloning vehicle for secretion of foreign proteins.     

Immunomodulation by myxospores of Myxococcus xanthus

Glycerol-induced myxospores of Myxococcus xanthus caused non-specific modulation of humoral and cellular immune responses in laboratory animals. The number of cells which formed specific haemolysins in spleens of mice immunized with sheep erythrocytes was increased when 0.5 X 10(8) myxospores were inoculated 2 d after the erythrocytes, and decreased when myxospores were injected 2 d before or at the same time as the erythrocytes. Both the IgG primary response and the secondary response to erythrocytes were decreased in rabbits after pretreatment with 2 X 10(8) myxospores per rabbit. Delayed-type hypersensitivity to sheep erythrocytes was also suppressed in mice after intraperitoneal (i.p.) injection of 0.3 X 10(8) myxospores. One day after i.p. injection of myxospores, neither an inflammatory response nor bone marrow cell depletion was observed in mice. These results support the idea that M. xanthus myxospores possess diverse immunomodulation properties apparently due to factors different from the classical LPS of Gram-negative bacteria.     

Untersuchungen an Myxococcus xanthus

Ohne Zusammenfassung     

Natural transformation of Myxococcus xanthus

Myxococcus xanthus belongs to the delta class of the proteobacteria and is notable for its complex life-style with social behaviors and relatively large genome. Although previous observations have suggested the existence of horizontal gene transfer in M. xanthus, its ability to take up exogenous DNA via natural transformation has not been experimentally demonstrated. In this study, we achieved natural transformation in M. xanthus using the autonomously replicating myxobacterial plasmid pZJY41 as donor DNA. M. xanthus exopolysaccharide (EPS) was shown to be an extracellular barrier for transformation. Cells deficient in EPS production, e.g., mutant strains carrying ΔdifA or ΔepsA, became naturally transformable. Among the inner barriers to transformation were restriction-modification systems in M. xanthus, which could be partially overcome by methylating DNA in vitro using cell extracts of M. xanthus prior to transformation. In addition, the incubation time of DNA with cells and the presence of divalent magnesium ion affected transformation frequency of M. xanthus. Furthermore, we also observed a potential involvement of the type IV pilus system in the DNA uptake machinery of M. xanthus. The natural transformation was totally eliminated in the ΔpilQ/epsA and Δtgl/epsA mutants, and null mutation of pilB or pilC in an ΔepsA background diminished the transformation rate. Our study, to the best of our knowledge, provides the first example of a naturally transformable species among deltaproteobacteria.     

Fatty Acids of Myxococcus xanthus

Fatty acids were extracted from saponified vegetative cells and myxospores of Myxococcus xanthus and examined as the methyl esters by gas-liquid chromatography. The acids consisted mainly of C₁₄ to C₁₇ species. Branched acids predominated, and iso-pentadecanoic acid constituted half or more of the mixture. The other leading component (11–28%) was found to be 11-n-hexadecenoic acid. Among the unsaturated acids were two diunsaturated ones, an n-hexadecadienoic acid and an iso-heptadecadienoic acid. No significant differences between the fatty acid compositions of the vegetative cells and myxospores could be detected. The fatty acid composition of M. xanthus was found to be markedly similar to that of Stigmatella aurantiaca. It is suggested that a fatty acid pattern consisting of a large proportion of iso-branched C₁₅ and C₁₇ acids and a substantial amount of an n-16:1 acid is characteristic of myxobacteria.     

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.     

Heat-shock-induced proteins from Myxococcus xanthus

Optimal conditions for two-dimensional gel electrophoresis of total cellular proteins from Myxococcus xanthus were established. Using these conditions, we analyzed protein patterns of heat-shocked M. xanthus cells. Eighteen major spots and 15 minor spots were found to be induced by heat shock. From N-terminal sequences of 15 major spots, DnaK, GroEL, GroES, alkyl hydroperoxide reductase, aldehyde dehydrogenase, succinyl coenzyme A (CoA) synthetase, 30S ribosomal protein S6, and ATP synthase alpha subunit were identified. Three of the 18 major spots had an identical N-terminal sequence, indicating that they may be different forms of the same protein. Although a DnaK homologue, SglK, has been identified in M. xanthus (R. M. Weimer, C. Creghton, A. Stassinopoulos, P. Youderian, and P. L. Hartzell, J. Bacteriol. 180:5357-5368, 1998; Z. Yang, Y. Geng, and W. Shi, J. Bacteriol. 180:218-224, 1998), SglK was not induced by heat shock. In addition, there were seven substitutions within the N-terminal 30-residue sequence of the newly identified DnaK. This is the first report to demonstrate that succinyl CoA synthetase, 30S ribosomal protein S6, and ATP synthase alpha subunit are heat shock inducible.     

Microcyst Germination in Myxococcus xanthus

Germination of glycerol-prepared microcysts of Myxococcus xanthus was studied. The sequence of morphological events during germination resembled that of germinating fruiting body-microcysts. The turbidity drop of a culture of germinating microcysts could be described by McCormick's formula derived for germinating Bacillus spores. The rate of uptake of labeled glycine and acetate did not change during germination. Temperature, aeration, and pH optima for germination were the same as for vegetative cell growth. Germination was induced by protein hydrolysates and the individual amino acids glycine, alanine, valine, aspartic acid, and glutamic acid. A number of organic compounds, including sugars, alcohols, aldehydes, ketones, organic acids, and chelating agents, did not induce germination. The inorganic ions HPO(4) (2-), Mg(++), Ca(++), and NH(4) (+) induced germination, although ionic strength was not a factor. Microcysts incubated in distilled water at concentrations greater than about 10(9) cells/ml germinated; supernatant fluid from such suspensions (germination factor) induced germination of less concentrated suspensions. The activity of germination factor was resistant to boiling, but was lost on charring and dialysis. Germination of microcysts and growth of vegetative cells was equally sensitive to a variety of metabolic inhibitors, including penicillin and chloramphenicol. Germination was more resistant than vegetative growth to inhibition by antibiotics of the streptomycin family and by actinomycin D.