DNA-methyltransferase activities in Streptomyces antibioticus

Abstract To quantitate ammonia production by the intestinal flora, ammonia levels in arterial blood and the venous effluent of the small and large bowel of conventional, selectively decontaminated, germ-free and gnotobiotic rats were measured.
When the anaerobic flora was removed by decontamination a significant decrease in ammonia levels was observed in the effluent of both the small and large intestine. Decontamination of aerobic flora did not result in depression of ammonia production. Gnotobiotic rats colonised with an anaerobic flora or with a mixed aerobic and anaerobic flora, showed a slight increase in ammonia levels. No increase in ammonia production was observed when rats were colonised with aerobic flora. These results indicate that the Enterobacteriaceae were not responsible for ammonia generation.
The increase in ammonia levels after colonisation with anaerobic or mixed anaerobic/aerobic flora did not completely restore ammonia levels, despite reaching bacterial counts which were comparable to those in conventional rats. This may be explained by the limited number of species with which the rats were colonized. The finding that aerobic flora does not significantly contribute to ammonia production suggests that neomycin, known to be exclusively effective against aerobic flora, must have other effects to explain improvement of hepatic encephalopathy.     

Fusion of protoplasts in Streptomyces antibioticus

The analysis of Streptomyces antibioticus recombinants isolated under non-selective conditions for stability of inheritance of parental properties demonstrated the existence of non-stable diploids and stable haploid recombinants. Also, genetic analysis of heteroclones isolated on selective medium after plating spores of regenerants was conducted. The results of analysis of haploid recombinant and heteroclones pointed to linkage of genetic loci and the possibility to use the technique of protoplast fusion for establishment of genetic map of S. antibioticus.     

Induction of mutations in Streptomyces antibioticus

Mutagenic action of UV-light, nitrosoguanidine and nitrosomethylbiuret was studied in Streptomyces antibioticus VNIIA 1607. Nitrosomethylbiuret appeared to be most effective inducer of auxotrophic mutations (mutation frequency reached 15%). By means of hybridological analysis, it was shown that heterokaryons predominated in the progeny of mixed cultures of multiply marked strains. The test for functional allelism using heterokaryons permitted us to divide 93 independently obtained mutations into 28 complementation groups.     

Restriction-modification systems determined by Pseudomonas plasmids

Four additional Pseudomonas R plasmids determining the PaeR7 restriction-modification system have been detected. All are transfer deficient and appear to belong to the same incompatibility group. The Pseudomonas fertility plasmid FP110 determines a different restriction-modification system and also inhibits the propagation of phage B39 by a separate mechanism. Pseudomonas R plasmid pMG73 has a third distinct restriction-modification specificity. PaeFP110 and PaeR73 are proposed as designations for these new plasmid-determined systems for restriction and modification.     

Respiratory Chain of Antimycin A-producing Streptomyces antibioticus

An active respiratory chain system was demonstrated in sonically treated mycelium of Streptomyces antibioticus, the producer of antimycin A. The respiratory electron transfer from substrate to oxygen proceeded successively through flavoprotein(s), b-, c-, and a-type cytochromes, and terminated with the cyanide-sensitive cytochrome oxidase. The cytochrome composition of the culture was not affected by the age of the mycelium, the intensity of antimycin A production, or differences in the media. Slater factor, coenzyme Q, and vitamin K were not interposed as hydrogen carriers in the respiratory chain between flavoproteins and cytochromes. The oxidation of reduced nicotinamide adenine dinucleotide and succinate was unaffected by antimycin A. Evidence is presented in support of the absence of the antimycin A-sensitive site from the electron transport system of S. antibioticus.     

relA Is Required for Actinomycin Production in Streptomyces antibioticus

The relA gene from Streptomyces antibioticus has been cloned and sequenced. The gene encodes a protein with an Mr of 93,653, which is 91% identical to the corresponding protein from Streptomyces coelicolor. Disruption of S. antibioticus relA produces a strain which grows significantly more slowly on actinomycin production medium than the wild type or a disruptant to which the intact relA gene was restored. Moreover, the disruptant was unable to accumulate ppGpp to the levels observed during the normal course of growth and actinomycin production in the wild type. The strain containing the disrupted relA gene did not produce actinomycin and contained significantly lower levels of the enzyme phenoxazinone synthase than the wild-type strain. Actinomycin synthetase I, a key enzyme in the actinomycin biosynthetic pathway, was undetectable in the relA disruptant. Growth of the disruptant on low-phosphate medium did not restore actinomycin production.     

Restriction-modification systems of type II in Shigella strains

Two restriction-modification systems specified by two plasmids are discovered in the clinical species of Shigella. The plasmids are designated pKMR114 and pKMR115. Both are of 60.800 bp and belong to the IncN incompatibility group. The EcoRI, EcoRV, HindIII restriction patterns of both plasmid DNAs are identical. As shown by efficiency of plating of bacteriophage lambda vir on the strains harbouring plasmids encoding EcoRI, EcoRII, EcoRIII, EcoRIV, EcoRV systems and plasmids studied, the discovered plasmids control synthesis of EcoRII specificity enzymes. The main distinctive feature of the pKMR114 is the ability to decrease efficiency of plating of bacteriophage T4 having glycolised DNA.     

Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans

In two separate studies a BclI-generated DNA fragment coding for the enzyme tyrosinase, responsible for melanin synthesis, was cloned from Streptomyces antibioticus DNA into two SLP1.2-based plasmid vectors (pIJ37 and pIJ41) to generate the hybrid plasmids, designated pIJ700 and pIJ701, using S. lividans 66 as the host. The fragment (1.55 kb) was subcloned into the multicopy plasmid pIJ350 (which carries thiostrepton resistance and has two non-essential BclI sites) to generate four new plasmids (pIJ702-pIJ705) with the tyrosinase insert located in either orientation at each site. All six plasmids conferred melanin production (the Mel+ phenotype) on their host. As in the S. antibioticus parent, strains of S. lividans carrying the gene specifying tyrosinase synthesis possessed an enzyme activity which was inducible. Most of the tyrosinase activity was secreted during growth of S. antibioticus; in contrast, the majority remained intracellular in the S. lividans clones. The specific activity of the induced tyrosinase activity (intracellular) was higher (up to 36-fold) when the gene was present on the multicopy vector in comparison with its location on the low copy plasmids, pIJ700 or pIJ701, or in S. antibioticus. Restriction mapping of the tyrosinase fragment in pIJ702 revealed endonuclease cleavage sites for several enzymes, including single sites for BglII, SphI and SstI that are absent from the parent vector (pIJ350). Insertion of DNA fragments at any one of these sites abolished the Mel+ phenotype. The results indicate that pIJ702 is a useful cloning vector with insertional inactivation of the Mel+ character as the basis of clone recognition.     

Glutamate-induced uptake of proline by Streptomyces antibioticus.

Streptomyces antibioticus possesses an energy-dependent, carrier mediated transport system for the uptake of L-glutamate and L-proline. Amino acid transport was found to have a temperature optimum of 35 degrees C and a pH optimum from 7.0 to 8.0 for glutamate and 6.5 to 7.5 for proline uptake. Uptake did not depend upon Mg2+, Ca2+, Zn2+, Na+, or Fe2+ ions. Reversible p-hydroxymercuribenzoate inhibition of uptake indicated the involvement of an active sulfhydryl group. L-Glutamate uptake was mediated by a glutamate-inducible, nonspecific transport system, which was extremely stable and was not subject to substrate inhibition by L-proline. On the other hand, L-proline transport was mediated by at least two systems. The L-glutamate-inducible nonspecific system can account for uptake of proline by the mycelium grown in glutamate. In addition, a proline-specific, constitutive transport system was found to be present in the mycelium grown in organic and inorganic nitrogen sources other than L-glutamate. Shift experiments revealed that proline transport is not as stable as glutamate transport when the glutamate-inducible nonspecific system is utilized.     

Structural changes induced by glycine on Streptomyces antibioticus

Abstract Germination and vegetative growth of Streptomyces antibioticus in liquid medium with different concentrations of glycine was examined. Both processes proved to be sensitive to the amino acid, being inhibited by 5 and 2.5% glycine, respectively. At concentrations of 5% or more, lysis of the vegetative mycelium occurred. Subinhibitory concentrations of glycine induced structural changes on germinating spores. These included an increase in the number of germ tubes produced by spore, in relation to the control. Moreover, soon after outgrowth the tubes bifurcate, giving rise to germinated spores with a characteristic aspect, and anomalous formation of cross-walls that appear both within the spores and in the newly formed germinative tubes, at or close to the region of outgrowth. The branching effect of glycine was also observed during vegetative growth of S. antibioticus .     

An exocytoplasmic endonuclease with restriction function in Streptomyces antibioticus.

Streptomyces antibioticus produces a strong endo-DNase which is located between the cytoplasmic membrane and the cell wall. All DNA substrates assayed, including the chromosomal DNA of this species and several bacteriophage DNAs, were completely degraded in vitro by the enzyme. The rate of synthesis of the nuclease depended on the growth medium. In NBG medium, in which the enzyme is not produced, the size of lytic plaques of several actinophages was larger than that in GYM or GAE medium, in which synthesis of the nuclease takes place late in growth. In addition, one of the phages assayed, phi A6, showed a diminution of its efficiency of plating in GYM medium with respect to that in NBG medium; another phage, phi A9, grew in NBG medium but not in the other two media. It is postulated that the presence of the host nuclease, together with the capability of the particular phage to absorb on S. antibioticus of different growth phases, determines the efficiency of growth and the plaque size of the phages on productive media. This hypothesis was confirmed when the growth of phi A6 and phi A9 in a mutant of S. antibioticus lacking the endonuclease activity was analyzed. It is concluded that the enzyme can assume, under some circumstances, a role in in vivo restriction.     

Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus

Streptomyces antibioticus accumulated glycogen and trehalose in a characteristic way during growth on solid medium. Glycogen storage in the substrate mycelium took place during development of the aerial mycelium. The concentration of nitrogen source in the culture medium influenced the time at which accumulation started as well as the maximum levels of polysaccharide stored. Degradation of these glycogen reserves was observed near the beginning of sporulation. The onset of sporogenesis was always accompanied by a new accumulation of glycogen in sporulating hyphae. During spore maturation the accumulated polysaccharide was degraded. No glycogen was observed in aerial non-sporulating hyphae or in mature spores. Trehalose was detected during all phases of colony development. A preferential accumulation was found in aerial hyphae and spores, where it reached levels up to 12% of the cell dry weight. The possible roles of both carbohydrates in the developmental cycle of Streptomyces are discussed.     

Resistance to oleandomycin in Streptomyces antibioticus, the producer organism

Resistance to oleandomycin in Streptomyces antibioticus, the producer organism, was studied. The organism was highly resistant in vivo to the antibiotic but sensitive to other macrolides and lincosamides. Protein synthesis in vivo by mycelium of S. antibioticus was more resistant to oleandomycin than that by mycelium of Streptomyces albus G, an oleandomycin-sensitive strain, and this resistance was dependent on the age of the culture, older mycelium of S. antibioticus being more resistant to oleandomycin than young mycelium. [3H]Oleandomycin was capable of binding to the same extent to the 50S subunits of the ribosomes of both organisms. Oleandomycin also inhibited in vitro protein synthesis by ribosomes obtained from an oleandomycin-production medium at the time when maximum levels of oleandomycin were being produced. A clear difference between the ability of the two organisms to incorporate exogenous oleandomycin was observed. Thus, while S. albus G took up oleandomycin, S. antibioticus showed a decreased permeability to the antibiotic, suggesting a role for cell permeability in self-resistance.     

Sporulation of Streptomyces antibioticus ETHZ 7451 in submerged culture.

Streptomyces antibioticus ETHZ 7451 formed spores in cultures grown in a liquid medium from either a spore or a mycelium inoculum. The spores formed were similar to those formed on surface-grown cultures, except for reduced heat resistance. Both types of spores were sensitive to lysozyme, which is unusual for Streptomyces spores. Glucose and other carbon sources, which promoted different growth rates, did not affect sporulation efficiency. Nitrogen sources, such as casamino acids, that allowed high growth rates suppressed the sporulation. A remarkable repression was also observed in media with some nitrogen sources that promoted noticeably lower growth rates. In permissive media, with nitrogen sources that permitted relatively high growth rates, sporulation was conditioned to the consumption of ammonium in the medium, but not to that of other nitrogen sources, such as asparagine. Phosphate did not show a repressive effect on sporulation in the assayed conditions.     

RNase III Is Required for Actinomycin Production in Streptomyces antibioticus

Using insertional mutagenesis, we have disrupted the RNase III gene, rnc, of the actinomycin-producing streptomycete, Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of the rnc disruption with the wild-type rnc gene from S. antibioticus restored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case in Streptomyces coelicolor, RNase III is required for antibiotic production in S. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of the S. antibioticus rnc mutant and its parental strain.