Genetic recombination in Streptomyces griseus.

Low-frequency (10(-6)) genetic recombination was observed in a cephamycin-producing strain of Streptomyces griseus. The recombinants were predominantly heteroclones. Heteroclone analysis was performed involving four heteroclones of one cross. In 100 mutants correlation was found between the type of auxotrophy and the level of antibiotic activity. A cross of this strain with a streptomycin-producing strain of S. griesus is described.     

Identification of bldA mutants of Streptomyces griseus

The bldA gene (encoding tRNA_UUA^Leu) from Streptomyces griseus (Sg) was cloned by hybridization with bldA from Streptomyces coelicolor (Sc). Introduction of Sg bldA into Sc bldA mutants restored sporulation and actinorhodin production. Sporulation of a subset of Sg bald mutants, which produce no aerial mycelium or spores, was restored in the presence of bldA from Sc or Sg. The nucleotide sequences of the bldA alleles from two such bald mutants revealed point mutations in the anticodon stem and the TΨC stem.     

Leukaemomycin-blocked mutants of Streptomyces griseus and their pigments

In a continued search for leukaemomycin-blocked mutants of three leukaemomycin-producing strains IMET JA 3933, IMET JA 5142 and IMET JA 5570 of Streptomyces griseus, 32 mutants producing aerial mycelium and spores were detected. Furthermore, in all mutants cosynthetic capability has been observed. This report describes characterization of leukaemomycin-blocked mutants obtained by mutagenic treatment experiments using NTG and combined UV-/X-rays. According to the biosynthetic capability for anthracyclinones or other pigments the mutants could be divided into six classes. The first class contains 14 leukaemomycin-blocked mutants unable to synthesize anthracyclinones. Besides two classes of mutants (12)synthesizing well-known anthracyclinones as epsilon-rhodomycinone, 7-deoxy-epsilon-rhodomycinone, 11-deoxy-derivatives of daunomycinone, three new classes of mutants (6) synthesizing reddish-brown, brown and blue-violet pigments on solid media with structures not elucidated as yet, will be described.     

Chromosomal circularization in Streptomyces griseus by nonhomologous recombination of deletion ends

Streptomyces linear chromosomes frequently cause deletions at both ends spontaneously or by various mutagenic treatments, and concomitantly display dynamic structural changes such as circularization and arm replacement. We have cloned and sequenced the fusion junctions of circularized chromosomes in two deletion mutants of Streptomyces griseus. No homology and a 1-bp overlap were found between the deletion ends of the mutant chromosomes. Taking this together with previous results, we concluded that chromosomal circularization in Streptomyces occurs by nonhomologous recombination between deletion ends.     

Streptomycin biosynthesis in Streptomyces griseus I. Characterization of streptomycin-idiotrophic mutants

Abstract A total of 16 idiotrophic mutants unable to produce the aminoglycoside antibiotic streptomycin ( smi ) were isolated from Streptomyces griseus N2-3-11. Cosynthesis of streptomycin, its formation from various precursors and analysis of accumulated intermediates allowed grouping of the mutants in 3 classes, blocked: (I) in the first transamination step of the streptidine pathway; (II) in later steps of the streptidine pathway; or (III) outside streptidine biosynthesis.     

Circularized chromosome with a large palindromic structure in Streptomyces griseus mutants

Streptomyces linear chromosomes display various types of rearrangements after telomere deletion, including circularization, arm replacement, and amplification. We analyzed the new chromosomal deletion mutants Streptomyces griseus 301-22-L and 301-22-M. In these mutants, chromosomal arm replacement resulted in long terminal inverted repeats (TIRs) at both ends; different sizes were deleted again and recombined inside the TIRs, resulting in a circular chromosome with an extremely large palindrome. Short palindromic sequences were found in parent strain 2247, and these sequences might have played a role in the formation of this unique structure. Dynamic structural changes of Streptomyces linear chromosomes shown by this and previous studies revealed extraordinary strategies of members of this genus to keep a functional chromosome, even if it is linear or circular.     

Genetic recombination in the cephamycin producer,Streptomyces clavuligerus

Summary In order to study the mechanism of cephamycin production by streptomycetes and to use genetic recombination in strain development, we undertook genetic studies inStreptomyces lipmanii andS. clavuligerus. S. lipmanii crosses gave 0.005–1.3% prototroph-like colonies, but all segregated back to parental genotypes. Crosses ofS. clavuligerus resulted in lower frequencies of prototroph-like colonies, i.e., 0.00002–0.9%. In ade x ura and ade x his crosses, the recombinant progeny did not segregate back. In arg x ade and arg x his crosses, segregation occurred in about 50% of the progeny. These data demonstrate that true haploid recombinants occur in crosses ofS. clavuligerus. S. lipmanii yielded only heterokaryons and, therefore, is less suitable thanS. clavuligerus for further genetic study.     

Pleiotropic effects of a butyrolactone-type autoregulator on mutants of Streptomyces griseus blocked in cytodifferentiation

Mutants of Streptomyces griseus blocked in cytodifferentiation regained their capacity to form differentiated mycelia and/or anthracycline pigments in the presence of butyrolactone-type autoregulatory effectors such as trans-2-(6'-methylheptanol-1'-yl)-3-hydroxymethyl-4-butanolide+ ++. In the pertinent indicator strains, the effect has been correlated with the increase of lipid synthesis, with changes in the composition of lipid fraction and with the restoration of the production of neutral proteinases. The results suggest that autoregulatory butyrolactones from streptomycetes stimulate cytodifferentiation of their producers at an early stage of development.     

Genetic analysis of A-factor synthesis in Streptomyces coelicolor A3(2) and Streptomyces griseus

A-factor is a potent pleiotropic effector produced by Streptomyces griseus and is essential for streptomycin production and spore formation in this organism. Its production is widely distributed among various actinomycetes including Streptomyces coelicolor A3(2). Genetic analysis of A-factor production was carried out with S. coelicolor A3(2), and two closely linked loci for A-factor mutations (afsA and B) were identified between cysD and leuB on the chromosomal linkage map. In contrast, genetic crosses of A-factor-negative mutants of S. griseus, using a protoplast fusion technique, failed to give a fixed locus for A-factor gene(s) and suggested involvement of an extrachromosomal or transposable genetic element in A-factor synthesis in this organism.     

Isolation and characterization of Streptomyces lividans mutants deficient in intraplasmid recombination

Summary Plasmid pIF132 containing two direct repeats of the mel (melanin) sequence was used to monitor intraplasmid recombination. Five mutants of Streptomyces lividans TK64 deficient in intraplasmid recombination were isolated. Four contained additional defects in aerial mycelium formation, pigmentation, and nutrient requirements; among these two showed extensive amplification of chromosomal sequences. Mutant JT46 had no pleiotropic defects but had the most severe blockage in recombination. Only one of the mutants was slightly more sensitive to UV and two were slightly more sensitive to mitomycin C. Plasmid pWCL1 (containing pIJ702, pUC12, and HBVsAg sequences; Lee et al. 1986) could not stably replicate in TK64 without spontaneous deletions. In contrast the mutant JT46 maintained the integrity of pWCL1 much more stably.     

Chromogenesis mirabilis in Streptomyces griseus

A number of chromogenic Streptomyces, producing diffusible melanoid pigment on complex organic media, fail to form melanin pigment on conventionally used synthetic tyrosine agar. By means of our new melanin formation test, almost all the chromogenic streptomyces can now be detected in chemically defined medium. In contrast to ordinary chromogenic streptomyces, two streptomyces species of the International Streptomyces Project, S. griseus ISP 5236 and S. ornatus ISP 5307, produce melanin pigment only on synthetic tyrosine agar, without showing chromogenicity on complex organic media. From the results obtained with S. griseus ISP 5236 and S. phaeochromogenes ISP 5073, it was revealed that melanin formation by Streptomyces, in general, is inhibited by L-cysteine present in organic nitrogen sources incorporated into natural media. Most chromogenic species of streptomyces produce a higher level of tyrosinase and rapidly utilize L-cysteine in the culture media which result in the manifestation of good chromogenicity on natural media. Peculiarity of chromogenicity of S. griseus and S. ornatus might be due to the lower ability to produce tyrosinase and to utilize L-cysteine in the culture medium.     

Biosynthesis of the antibiotic, grizin, by prototrophic revertants of Streptomyces griseus biochemical mutants

Spontaneous and nitrosomethylbiuret-induced prototrophic revertants of various biochemical mutants of Str. griseus producing grisin, a streptothricin antibiotic, were isolated. The antibiotic production level of the revertants was studied. It was found that most of the prototrophic revertants synthesized much higher amounts of grisin than the initial biochemical mutants. It was also shown that a number of the prototrophic revertants of the methionine- and arginine-dependent mutants synthesized 20-23% higher amounts of grisin as compared to the control.     

Hybridization of A-factor deficient mutants of Streptomyces griseus by means of fusion of protoplasts

Characteristics of 6 A-factor deficient mutants of S. griseus are presented. The common feature of the mutants was impairment of sporulation, formation of aerial mycelium and streptomycin synthesis. Pair-by-pair hybridization of the mutants was performed with protoplast fusion followed by regeneration. 9 pair couplings of the mutants were performed. In 3 of them sporulating recombinants were detected. The antibiotic production level in 70 hybrids was different and ranged from 0 to 1700 micrograms/ml. The morphological features of the colonies and the number of the spores formed were also different. The common feature of all the 70 sporulating hybrid strains was recovery of synthesis of A-factor, an endogenic regulator of S. griseus development. Therefore, in the A-factor deficient mutants impairment of A-factor synthesis was induced not by the plasmid elimination, as was suggested, but by mutation of separate genes.     

Genetic recombination between alpha-toxin mutants of Staphylococcus aureus

McClatchy, J. K. (The University of Texas Southwestern Medical School, Dallas), and E. D. Rosenblum. Genetic recombination between alpha-toxin mutants of Staphylococcus aureus. J. Bacteriol. 92:580-583. 1966.-A demonstration of genetic recombination between Staphylococcus aureus nonhemolytic mutants was attempted by means of transduction. The results of two-point reciprocal transductions placed the mutants into two genetic groups. Recombination within each group was not detectable within the limits of the method, but hemolytic recombinants were obtained in transductional crosses when donor and recipient were from different groups. At least two genetic loci are therefore involved in alpha-toxin production. The 11 mutants of group II were fibrinolysin-negative. The recombinants were always found to be restored to fibrinolysin production as well as to alpha-toxin production. These data suggest the existence of a pleiotropic gene simultaneously affecting the synthesis of both alpha toxin and fibrinolysin. The nine mutants of group I were fibrinolysin-positive. Group I members are postulated to be alpha-toxin structural mutants. Three mutants were also negative for bound coagulase, but no linkage was observed between the locus controlling bound coagulase and the loci for either fibrinolysin or alpha-toxin production.     

Properties of Streptomyces griseus aminopeptidase

The paper deals with studying the properties of aminopeptidase isolated from Str. griseus culture fluid. The preparation is characterized by a high specific activity and heat stability, it has no admixtures of carboxypeptidases and proteinases. The enzyme is easily inhibited by EDTA, but the addition of Ca2+ evokes its complete reactivation. A partial recovery of the activity may be also reached under the influence of some other bivalent metals. In hydrolysis of di- and tripeptides it is shown that the enzyme has a preferential effect on the substrates with N-terminal leucine. Peptides with N-terminal alanine, valine and glycine are almost not hydrolyzed. The use of the native insulin and decapeptide with the known amino acidic sequence as substrates shows that aminopeptidase can hydrolyze proteins and peptides with the successive release of some amino acids: phenylalanine, serine triptophane, valine, asparagine, etc. Glycine is difficult for removal and may inhibit the further hydrolysis of the polypeptide chain.