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Bacteriocin-like activities were found in several Myxococcus fulvus strains. One strain, Mx f16, exerted strong inhibitory effects on several myxobacterial strains. Synthesis of its bacteriocinic activity could not be induced by mitomycin. Electrophoresis and molecular sieve chromatography revealed at least three different bacteriocinic substances of low molecular weight.
1. Bacteriophage MX-1 is a virulent DNA phage whose hosts include strains of Myxococcus xanthus, M. fulvus and M. virescens. DNA was extracted from purified phage preparations. The molecular weight of phage DNA was measured by sedimentation-velocity and by rate-zonal ultracentrifugation. The apparent molecular weight was found to vary for reasons discussed in the text. From rate-zonal ultracentrifugation, using calibrated sucrose gradients, the molecular weight was calculated to be 149 (+/-22) X 10(6) daltons. The base composition of the DNA was estimated by different methods and was found to be 50-52% (G + C). The DNA demonstrated an anomalous thermal denaturation profile in dilute buffer. Denatured DNA was fractionated by ion-exchange chromatography and by buoyant-density centrifugation. No significant strand separation was obained and it was concluded that overall base compositions of the two strands are very similar. 2. DNA from bacteriophage MX-1 was hydrolysed with restriction endonucleases R. EcoRI, R.EcoRII and R. HindIII. The restriction fragments were catelogued and their apparent molecular weights calculated from electrophoresis gels calibrated with fragments from the DNA of coliphage lambda. From the total fragments obtained with nuclease R.EcoRI, the minimum apparent molecular weight of MX-1 DNA was found to be 130 X 10(6) daltons.
1. The plating efficiency of bacteriophage MX-1 on Myxococcus xanthus strains A and B and M. virescens V2 were compared. Comparison of strains V2 and A suggest that V2 is restrictive and A is not (restriction coefficient was approximately 8). A derivative of M. virescens V2 (strain V2-9) was obtained by repeated exposure of strain V2 to ultraviolet radiation. Strain V2-9 was also unrestrictive. Strain B is apparently unrestrictive too but analysis of phenotypic changes in phage derived from hosts V2, B and A suggested that some of the host-cell processes differ from orthodox restriction and modification. 2. Cell-free extracts from M. virescens V2 were fractionated by ion-exchange chromatography and two restriction endonucleases, R. MviV2I and R. MviV2II were identified. Nuclease I was found to hydrolyse coliphage lambdaDNA at apparently one site only and MX-1 DNA at approximatley 10 sites; nuclease II was found to hydrolyse MX-1 DNA at a very large number of sites and its restriction sequence was of comparable frequency with that of R. EcoRII. "Modified MX-1 DNA", obtained from phage whose last host was M. virescens V2 was hydrolysed by nuclease II but not by nuclease I. The significance of these findings for restriction in myxococci is discussed.
Mutants of Myxococcus xanthus FBt unable to form myxospores in response to 0.5 M glycerol arise spontaneously with a frequency of 1--3 X 10(-5). These mutants are designated glc. Ultraviolet mutagenesis increases the frequency to a maximum of 7% of the survivors. The reversion frequency following ultraviolet irradiation of spontaneous glc mutants is less than 10(-3). Of four glc mutants examined, none form myxospores in response to the alternative inducers, ethylene glycol and dimethyl sulphoxide. One glc mutant is induced by 1.5 M glycerol; strain FBt responds to this glycerol concentration with low efficiency myxospore formation. Strain FBt and glc mutants all produce myxospores with low efficiency in response to phenyl ethanol. Of 117 glc mutants tested, 109 form fruiting bodies containing mature myxospores; thus, mutations to the glc phenotype do not normally block myxospore formation within the fruiting cycle of the organism.
Acid and alkaline phosphatase of Myxococcus coralloides were examined during vegetative growth in a liquid medium. Two extracellular phosphatases and two cell-bound phosphatases, acid and alkaline in both cases, were produced. The phosphatase production was unaltered by the presence of high concentrations of inorganic phosphate. Both enzymes were produced constitutively. These two hydrolases were released into the growth medium during the exponential growth phase (approximately 10% of total activity). The production of these enzymes was modified by the presence of organic acids and metal ions in the medium.
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.
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.
The developmental events of sporulation and fruiting body formation in the prokaryote Myxococcus xanthus are preceded by a stage of massive cell death. Two phenotypically complementable strains of M. xanthus defective in developmental lysis were identified from a group of conditional sporulation mutants. Mixture of the two lysis groups resulted in full complementation of lysis, sporulation, and fruiting body formation; efficient sporulation was observed only in strain mixtures where lysis was complemented. We have identified a cell-free extract from developing cells that phenotypically complemented lysis, sporulation, and fruiting body formation in one group of mutants; the active component of this extract appeared to be tightly cell associated. The effect of the cell-free extract could be replaced by exogenously supplied glucosamine or mannosamine.
Certain developmental mutants of Myxococcus xanthus can be complemented (extracellularly) by wild-type cells. Insertions of Tn5 lac (a transposon which couples beta-galactosidase expression to exogenous promoters) into developmentally regulated genes were used to investigate extracellular complementation of the A group mutations. A- mutations reduced developmental beta-galactosidase expression from 18 of 21 Tn5 lac insertions tested and that expression was restored to A- Tn5 lac cells by adding wild-type cells. The earliest A-dependent Tn5 lac normally expresses beta-galactosidase at 1.5 hr of development indicating a developmental block at 1-2 hr in A- mutants. A substance which can rescue the expression of this early Tn5 lac is released by wild-type (A+) but not by A- cells. This substance appears in a cell-free wash of wild-type cells or in starvation buffer conditioned by wild-type cells 1-2 hr after development is initiated. The conditioned starvation buffer also restores normal morphological development to an A- mutant.
Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10(3) base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of beta-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.
In the chromosome of Myxococcus xanthus, two homologous genes for protein S, a development-specific protein, are tandemly repeated with a 1.4 X 10(3) base-pair sequence between the two genes. Two synthetic oligodeoxyribonucleotides were used as specific probes for individual transcripts from the upstream gene 1 and the downstream gene 2, respectively. The gene 2 transcript was detected only during developmental growth, while the gene 1 transcript was not detected during developmental or vegetative growth. The major initiation site for the gene 2 transcription was determined to be 51 bases upstream of the initiation codon for gene 2. The development-specific promoter of gene 2 was identified; it shows some homologies to the Escherichia coli promoter structures.
A physical map of 330 x 10(3) base-pairs near the replication origin of Myxococcus xanthus chromosome has been established already. Using DNA fragments from this region, Northern blot hybridization analysis was carried out in order to identify the genes expressed during vegetative growth. One of the genes, tentatively designated as vegA, was cloned and its entire DNA sequence was determined. The amino acid sequence of the gene product deduced from the DNA sequence reveals that the VegA protein is a very basic protein with a molecular weight of 18,700. The gene was expressed in Escherichia coli using an expression vector, and its gene product was identified using SDS/polyacrylamide gel electrophoresis. From the results of S1 nuclease mapping, the vegA promoter was found to contain the sequence TAGACA at the -35 region and the sequence AAGGGT at the -10 region. These two regions are separated by 18 nucleotides. Genetic analysis suggests that the vegA gene may be essential for the growth of M. xanthus. From a computer-aided search for homologies to know protein structures, it was found that the VegA protein has homologies to histone H4 of Tetrahymena thermophila and histone H2B of sea urchin.
Nucleoside diphosphate (NDP) kinase catalyzes the transfer of the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. Human and rodent forms of this enzyme have been shown to be suppressors of metastasis. Crystals that diffract X-rays to high resolution have been obtained for the recombinant Myxococcus xanthus NDP kinase expressed in and purified from Escherichia coli. Two crystal forms have been obtained. Both forms are orthorhombic, space group I222 (or I2(1)2(1)2(1)) with a = 267.1 A, b = 74.0 A and c = 75.1 A for form I and a = 53.5 A, b = 74.0 A and c = 75.1 A for form II. Form I appears to have five molecules in the asymmetric unit approximately related to each other by a translation of 0.2 along the a axis. Diffraction data have been recorded to 1.9 A for form I and to 2.2 A for form II.
A gram-negative bacterium, Myxococcus xanthus, was found to contain 500 to 700 copies per chromosome of a short single-stranded linear DNA fragment. When this DNA (multicopy single-stranded DNA; msDNA) labeled at the 5' end with kinase was used as a probe against total chromosomal blots, it hybridized to unique high molecular weight bands, which were cloned and sequenced. Labeling of msDNA was also possible using the Klenow fragment of DNA polymerase I as well as terminal deoxynucleotidyl transferase, permitting direct sequencing. The 5' end of msDNA was found to be primed by a short RNA segment. The DNA portion of msDNA consisted of 163 bases. Exact correspondence was seen between the msDNA sequence and the sequence of a chromosomal clone. An elaborate secondary structure is postulated for the msDNA sequence. A similar satellite DNA was also found in another myxobacterium, Stigmatella aurantiaca.
The site-specific recombination mechanism through which the plasmid RP4 has been previously shown to integrate into the chromosome of Myxococcus xanthus has been investigated further. Once integrated in one of the numerous chromosomal sites from two different strains, through a precise site on the plasmid, the latter can be excised either precisely or after a definite 14.5-kb deletion. In some cases, the integration is followed by different DNA rearrangements that yield a higher rate of excision and integration. A model for the site-specific integration and excision of the plasmid is proposed.
The inducibility of two promoter systems, one heterologous and one homologous, has been assessed in the Gram-negative bacterium Myxococcus xanthus. The heterologous system involved the hybrid tac promoter and the presence of lacIq, the lac repressor from Escherichia coli. This system is inducible in its natural host with isopropyl-beta-D-thiogalactopyranoside (IPTG). The homologous promoter system involves the light-inducible carQRS promoter, which is normally involved in the expression of the regulators of the light-inducible light-protective carotenoid synthesis regulon in M. xanthus. In each case, promoter activity and strength was assayed using the E. coli gene lacZ. In our constructs, which were present in a single copy in the M. xanthus chromosome, the carQRS promoter yielded at least a 47-fold increase in beta-galactosidase production upon light induction, whilst IPTG increased by 8-fold the amount of enzyme produced under the control of the ptac-lacIq system. Regulation by the latter was significantly higher than that obtained with the unmodified lacZ promoter.
Insertions of transposon Tn5 were used to examine the genetics of motility mutants in Myxococcus. Fifteen independent insertions of Tn5 were isolated that were linked to seven different loci that govern motility. Among the motility mutants that can be stimulated to move transiently by contact with other cells, a one-to-one correspondence was confirmed between specificity of stimulation and genetic locus. There are six different specificities and six corresponding loci, as if each locus governs a different protein required for gliding motility.
Myxococcus fulvus NK 35 has been shown to produce a soluble hemolysin which lysed rabbit, human, horse, and sheep erythrocytes. A medium (Varghese's medium) was devised in which a maximum of hemolysin was produced in 6 days at 28 degrees C under static conditions. The lysin was precipitated by complete saturation of the culture filtrate with ammonium sulphate, followed by dialysis against saline. Other enzyme systems were destroyed by heating at 100 degrees C. Further purification was achieved by passing through a Sephadex G-25 column, giving a single peak with 0.01 M of phosphate buffer, pH 6.6. Like the hemolysin of Pseudomonas aeruginosa, this hemolysin is non-proteinic and withstands 100 degrees C for 30 minutes.