Mutation and a high-throughput screening method for improving the production of Epothilones of Sorangium.

The epothilones are highly promising prospective anticancer agents that are produced by the myxobacterium Sorangium cellulosum. We mutated the epothilone producing S. cellulosum strain So0157-2 to improve the production of epothilones. For evaluation in high-throughput of a large number of mutants, we developed a simple microtiter method for primary screening. Using the classical UV-mutation method plus selection pressures, the production capacity was increased about 0.5 approximately 2.5 times the starting strain. The mutants with higher production and different phenotypes were further subjected to recursive protoplast fusions and the fusants products were screened under multi-selection pressure. Furthermore, the production was greatly increased by the genome shuffling. For epothilone B, the production of one fusant was increased about 130 times compared to the starting strain, increasing from 0.8 mg l(-1) to 104 mg l(-1).

Plasmid-mediated UV-protection in Myxococcus xanthus.

Plasmid R46 was successfully transferred from Escherichia coli K=12 into myxococcus xanthus strain MD-1 but not into M. xanthus strain XK. Plasmid R68.45 was transferred from E. coli K-12 into both strains of M. xanthus. The effects of these plasmids on survival of M. xanthus after ultraviolet (UV)-244 nm irradiation, the ability of M. xanthus to reactivate irradiated myxophages, and weigle reactivation of UV-irradiated effect on UV survival of M. xanthus, but increased the host's ability to reactivation irradiated myxophages. Plasmid R68.45 protected M. xanthus strains MD-1 and XK against the lethal effects of UV irradiation and also increased the host's ability to reactivate irradiated myxophages.

Misrepair mutagenesis in Myxococcus xanthus: induction of rifampicin-resistant mutants by N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet-irradiation.

In the ultraviolet (UV)-mutable bacterium, Myxococcus xanthus, dose response curves for the induction of rifampicin-resistant (Rifr) mutants were compared with dose response curves for Weigle(W)-reactivation of the UV-irradiated phage Mx4 at a phage survival of 5 X 10(-6). In most strains examined, including a uvr mutant, these curves are largely similar. Unexpectedly the UV-sensitive strain M. xanthus Bt, which is unable to perform W-reactivation, is nevertheless UV-mutable. This result may indicate that the repair pathway involved in phage reactivation is only partly responsible for UV-mutagenesis or alternatively is not able to act on phage DNA in M. xanthus Bt cells. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment of M. xanthus cells also results in marked W-reactivation of the UV-irradiated phage Mx4 at the same survival of 5 X 10(-6). The MNNG-stimulated phage reactivation is of the same order of magnitude as the UV-stimulated phage reactivation. Also the dose response curves for the induction of Rifr mutants by MNNG and the MNNG-stimulated phage reactivation are quite similar. This coincidence may indicate that misrepair mutagenesis is involved in both UV and MNNG-mutagenesis. It is suggested that M. xanthus is a useful organism with which to study misrepair mutagenesis in bacteria.

Photocontrol of development by Stigmatella aurantiaca.

Aggregation and fruiting body formation by Stigmatella aurantiaca were stimulated most effectively by low irradiances of blue light between 400 and 500 nm. At higher irradiances, other wavelengths of light, including those in the far-red region of the spectrum, were also effective.

Light-stimulated morphogenesis in the fruiting myxobacterium Stigmatella aurantiaca.

When the fruiting myxobacterium Stigmatella aurantiaca, a gliding prokaryote, is starved on an agar surface, the cells form multicellular aggregates resulting from morphogenetic movements. In the presence of incandescent light, each aggregate develops into a structurally complex fruiting body, possessing a stalk and several sporangia. In contrast, this pattern of development is not seen when cultures are incubated in the dark. The cells form irregular interconnecting aggregates, which rarely develop into fruits. However, aggregates formed in the light will develop into fruits even if placed in the dark, suggesting that the light produced a relatively stable alteration in the phenotype of the cells.

Evidence for repair of ultraviolet-induced damage in Cystobacter species (Myxobacterales).

Cystobacter species strain CK 1 does not grow with more than 0.2 microgram/ml acriflavine. Spontaneous two-step mutants growing with 2 microgram acriflavine per ml have been selected. One mutant (strain CK3) was used to investigate the effect of repair inhibitors. Both strains exhibit pronounced shoulders in their UV dose curves of inactivation. Acriflavine (AF), coumarin (CU), and caffeine (CA) when incorporated in the post-irradiation plating medium decreased survival of irradiated cells. Post-treatment with 2 microgram acriflavine/ml abolished the shoulder of the curve. Caffeine (1600 microgram/ml) and coumarin (350 microgram/ml) reduced it only to about 40%. It is concluded that probably two repair mechanisms are present. Pre-treatment of the cells with 2 microgram acriflavine/ml for two hours before UV-irradiation resulted in a constant dose enhancement factor of 1.9. The protective effect is increased with the time of treatment with acriflavine. This may indicate that pyrimidine dimers are responsible for UV-inactivation.

Pigmentation phenotype instability in Myxococcus xanthus.

Cells of Myxococcus xanthus FB2 produce tan or yellow colonies. Subcultures of tan colonies yielded tan and yellow colonies and subcultures of most yellow colonies yielded only yellow colonies. Strain FB2 variants in which the color type is more stable were obtained. Yellow cells were distinguishable from tan by the presence of pigment(s) with an absorption maximum at 379 nm. Fluctuation Test experiments and the presence of this pigment(s) in liquid cultures of FB2 indicated that tan phenotype cells spontaneously became or segregated yellow cells in liquid culture. The frequency of appearance of yellow cells was increased in low density cultures (less than 10(6)/ml). The increase cannot be explained by differences in growth rates of the two phenotypes. No evidence that cell-cell contact or culture medium constituents affect the appearance of the yellow phenotype was found. Ultraviolet irradiation of FB2 resulted in an increased proportion of cells producing yellow colonies among the survivors. Greater UV resistance of yellow cells and UV-induced conversion of tan to yellow accounts for this increase. Low level photoreactivation of viability and of the tan phenotype occurred. Incubation of FB2 in medium containing mitomycin C, nalidixic acid, phenethyl alcohol, or at 36.5 degrees C also resulted in conversion of tan to yellow cells.

Mutants of Myxococcus xanthus insensitive to glycerol-induced myxospore formation.

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.