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.

Retroelements in bacteria.

A peculiar type of satellite DNA, called msDNA, has been discovered in myxobacteria and some natural isolates of E. coli. These molecules are characterized by the presence of single-stranded DNA branching out from an internal guanosine residue of an RNA molecule by a unique 2',5'-phosphodiester linkage. Reverse transcriptase is required for the synthesis of msDNA. The discovery of retroelements in bacterial populations raises many intriguing questions concerning the evolutionary origin of reverse transcriptase, the function and the biosynthesis of msDNA, and the nature of the mechanisms generating the extensive diversity found in msDNA and reverse transcriptase genes among different bacterial strains.

Purification and characterization of the Myxococcus xanthus FrzE protein shows that it has autophosphorylation activity.

Myxococcus xanthus exhibits multicellular interactions during vegetative growth and fruiting body formation. Gliding motility is needed for these interactions. The frizzy (frz) genes are required to control directed motility. FrzE is homologous to both CheA and CheY from Salmonella typhimurium. We used polyclonal antiserum raised against a fusion protein to detect FrzE in M. xanthus extracts by Western immunoblot analysis. FrzE was clearly present during vegetative growth and at much lower levels during development. A recombinant FrzE protein was overproduced in Escherichia coli, purified from inclusion bodies, and renatured. FrzE was autophosphorylated when it was incubated in the presence of [gamma-32P]ATP and MnCl2. Chemical analyses of the phosphorylated FrzE protein indicated that it contained an acylphosphate; probably phosphoaspartate. FrzE was phosphorylated in an intramolecular reaction. Based on these observations, we propose a model of the mechanism of FrzE phosphorylation in which autophosphorylation initially occurs at a conserved histidine residue within the "CheA" domain and then, via an intramolecular transphosphorylation, is transferred to a conserved aspartate residue within the "CheY" domain.

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.

Determination of bacterial ammonia pools using Myxococcus virescens as an example.

Samples (150 microliters) from liquid cultures of known cell density of Myxococcus virescens (Myxobacterales) were used for the determination of the intracellular NH3/NH+4 concentrations (= total ammonia). The cells were separated from the culture broth within 30 s by centrifugation through a silicone layer and were lysed immediately with 20 microliters of a disintegration liquid at the bottom of the centrifugation tube. The ammonia concentrations of the lysates were determined with a Dohrmann nitrogen analyzer. The intracellular ammonia concentrations were calculated after corrections for trapped supernatant had been made by adding radioactive glucose, which cannot be taken up by the organism. Control experiments with permeabilized cells and radioactive methylamine corroborated the reliability of the method.

A macrocyclic antibiotic M-230B produced by Myxococcus xanthus. Isolation and characterization.

Myxococcus xanthus strain M516E produced at least three related antibiotics against Gram-positive and Gram-negative bacteria. From physico-chemical properties, a main component was identical to myxovirescin A and a second component, designated M-230B was found to be an antibiotic which is closely related to myxovirescin A. The structure of M-230B was determined from its physico-chemical properties, especially from 13C NMR spectrum as compared with that of myxovirescin A. The addition of alcohol, such as isobutyl alcohol, to the culture medium markedly stimulated production of the antibiotics.

Occurrence of dialkyl ether phospholipids in Stigmatella aurantiaca DW4.

We investigated the lipid composition of vegetative cells of Stigmatella aurantiaca. Four phospholipids were isolated and identified: phosphatidylethanolamine as the main component, phosphatidylglycerol, lysophosphatidylethanolamine in an exceptionally large amount (17%), and phosphatidylinositol (18 to 25%), rare in procaryotic cells. This composition did not change significantly during growth. The fatty acids of total lipids were found to be rather similar to those of other strains of myxobacteria; the main fatty acids found were unsaturated and branched. We noted a different fatty acid pattern for each phospholipid. The presence of unusual alkyl ether linkages, established by chemical hydrolysis and infrared spectroscopy, was unexpected in these bacteria. Diacyl ester, dialkyl ether, and monoacyl-monoalkyl structures were shown in phosphatidylethanolamine and phosphatidylglycerol. Lysophosphatidylethanolamine was essentially a monoacyl form, whereas phosphatidylinositol was a unique dialkyl ether phospholipid.

The myxovalargins, new peptide antibiotics from Myxococcus fulvus (Myxobacterales). I. Cultivation, isolation, and some chemical and biological properties.

Antibiotic activity was isolated from the culture supernatant of the myxobacterium Myxococcus fulvus strain Mx f65. It was active against Gram-positive bacteria (MIC 0.3 approximately 5 micrograms/ml), at higher concentrations also against Gram-negative ones (MIC 6 approximately 100 micrograms/ml), and not at all against yeasts and molds. The activity could be resolved into 4 closely related peptides, the myxovalargins. One of them, myxovalargin A, was by far the most plentiful. The compounds appear to be new antibiotics and seem to interfere with protein synthesis.

The myxovirescins, a family of antibiotics from Myxococcus virescens (Myxobacterales).

The myxobacterium, Myxococcus virescens strain Mx v48 produced a family of at least 12 closely related antibiotics, the myxovirescins. At a concentration of 1 to 5 micrograms/ml, the main component, myxovirescin A, was bactericidal for many Gram-negative bacteria, in particular enterobacteria, and at 20 to 50 micrograms/ml it also inhibited some pseudomonads and Gram-positive bacteria. The antibiotics seem to interfere with cell wall synthesis. The molecular formula of myxovirescin A was C35H61NO8. It is a new antibiotic.

Development-specific protein S of Myxococcus xanthus: purification and characterization.

Protein S, a development-specific protein of Myxococcus xanthus, was purified from the cells of a late stage of development and crystallized. Its circular dichroism spectra indicated that protein S had a high content of beta-structure in both the presence and absence of calcium ion, which is required for self-assembly of protein S on the myxospore surface. Its amino and carboxyl terminal sequences were determined to be alanine-aspartic acid-isoleucine-glycine-valine-alanine-methionine-asparagine-asparagine-aspartic acid-threonine-serine-serine and isoleucine-arginine (isoleucine, serine), respectively. When protein S (molecular weight, 23,000) was digested with trypsin, a trypsin-resistant core of 10,000 molecular weight was obtained. The core peptide was purified, and its amino acid composition was compared with that of protein S. The core peptide was capable of self-assembly on the spore surface in the presence of calcium ion and competed with protein S for binding on the spore surface. The ratio of affinity to the spore surface for protein S to that for the core peptide was 1.55.

Reexamination of the genome size of myxobacteria, including the use of a new method for genome size analysis.

The genome sizes of two myxobacteria, Myxococcus xanthus and Stigmatella aurantiaca, were measured by renaturation analysis and also by a new method involving the quantitation of individual restriction fragments. In contrast to several previous reports, which indicate that M. xanthus has a genome size which is three to four times that of Escherichia coli, the present measurements indicated that the M. xanthus genome is only about 24 to 53% larger than that of E. coli. S. aurantiaca had a genome size nearly identical to that of M. xanthus. Of possible significance is the fact that the renaturation curves of M. xanthus and S. aurantiaca deoxyribonucleic acid both exhibited significant fractions which renatured with rapid, unimolecular kinetics. However, we were unable to establish that these fractions represented inverted repeats of repetitive sequences.

The primary structure of fulvocin C from Myxococcus fulvus.

The primary structure of fulvocin C, a bacteriocin produced by Myxococcus fulvus strain Mx f16, has been determined. This new bactericidal protein is composed of 45 amino acid residues and has a molecular weight of 4672. It contains no lipids or carbohydrates, indicating that only the protein molecule is responsible for its biological activity.

Preliminary crystallographic data for protein S, a development-specific protein of Myxococcus xanthus.

Protein S, which is produced only during the developmental cycle of Myxococcus xanthus, has been crystallized using 2-methyl-2,4-pentanediol as a precipitating agent. The crystals were very stable in the x-ray beam for up to 150 h and diffracted to a resolution of 2.2 A. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions a = 52.99 A, b = 60.10 A, and c = 102.16 A. Each asymmetric unit consists of two monomers of Protein S, each having a molecular weight of 23,000.

Separation and properties of the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus.

We have developed methods for separating the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus. The total membrane fraction from ethylenediaminetetraacetic acid-lysozyme-treated cells was resolved into three major fractions by isopycnic density centrifugation. Between 85 and 90% of the succinate dehydrogenase and cyanide-sensitive reduced nicotinamide adenine dinucleotide oxidase activity was found in the first (I) fraction (rho = 1.221 g/ml) and 80% of the membrane-associated 2-keto-3-deoxyoctonate was found in the third (III) fraction (rho = 1.166 g/ml). The middle (II) fraction (rho = 1.185 g/ml) appeared to be a hybrid membrane fraction and contained roughly 10 to 20% of the activity of the enzyme markers and 2-keto-3-deoxyoctonate. No significant amounts of deoxyribonucleic acid or ribonucleic acid were present in the three isolated fractions, although 26% of the total cellular deoxyribonucleic acid and 3% of the total ribonucleic acid were recovered with the total membrane fraction. Phosphatidylethanolamine made up the bulk (60 to 70%) of the phospholipids in the membrane fractions. However, virtually all of the phosphatidylserine and cardiolipin were found in fraction I. Fraction III appeared to contain elevated amounts of lysophospholipids and contained almost three times the amount of total phospholipid as compared with fraction I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved approximately 40 polypeptides in the total membrane fraction. Two-thirds of these polypeptides were enriched in fraction I, and the remainder was enriched in fraction III. Fraction II contained a banding pattern similar to the total membrane fraction. Electron microscopy revealed that vegetative cells of M. xanthus possessed an envelope similar to that of other gram-negative bacteria; however, the vesicular appearance of the isolated membranes was somewhat different from those reported for Escherichia coli and Salmonella typhimurium. The atypically low bouyant density of the outer membrane of M. xanthus is discussed with regard to the high phospholipid content of the outer membrane.

Iso-branched 2- and 3-hydroxy fatty acids as characteristic lipid constituents of some gliding bacteria.

The fatty acids present in the total hydrolysates of several gliding bacteria (Myxococcus fulvus, Stigmatella aurantiaca, Cytophaga johnsonae, Cytophaga sp. strain samoa and Flexibacter elegans) were analyzed by combined gas-liquid chromatography and mass spectrometry. In addition to 13-methyl-tetradecanoic acid, 15-methyl-hexadecanoic acid, hexadecanoic acid, and hexadecenoic acid, 2- and 3-hydroxy fatty acids comprised up to 50% of the total fatty acids. The majority was odd-numbered and iso-branched. Small amounts of even-numbered and unbranched fatty acids were also present. Whereas 2-hydroxy-15-methyl hexadecanoic acid was characteristic for myxobacteria, 2-hydroxy-13-methyl-tetradecanoic acid, 3-hydroxy-13-methyl-tetradecanoic acid, and 3-hydroxy-15-methyl-hexadecanoic acid were dominant in the Cytophaga-Flexibacter group.

Myxobacterial slime and proteolytic activity.

An extracellular protein-polysaccharide-lipide (PPL) complex from exponentially growing cultures of Myxococcus virescens was purified by phosphate precipitation and gel chromatography. The high molecular weight slime polymer appeared homogenous upon isoelectric focusing. The PPL complex exhibited proteolytic activity against gelatin and the activity was only partly reduced by heat treatment. The function of the slime polymer as protein denatured was studied. The complex formed micelles similar to anionic detergents and it inhibited the precipitation and coagulation of proteins by trichloroacetic acid. Lysozyme was totally inactivated when treated with the PPL complex. By gel chromatography binding studies, the PPL complex was found to bind lysozyme in the ratio of 1 to 5.8 (w/w). After separation of added protein from the complex the anticoagulation effect on the protein remained. The biological function of the PPL complex was demonstrated with hemoglobin. When all susceptible peptide bonds in PPL-treated hemoglobin were hydrolyzed by trypsin only 20% in the urea-denatured protein were attacked. The combined role of slime and proteolytic activity is discussed.