No abstract

Five non-streptomycin-producing non-aerial-mycelium-forming mutants (Str-Amy-) of Streptomyces griseus obtained either by spontaneous degenerat,ion or during continuous cultivation of the high-producing aerial-mycelium-forming parent strain HP (Str+Amy+) were checked with regard to t'he composit,ion of mycelial lipid material. All the Str-Amy-derivatives differed from their ancestor strain HP by an increased ratio of 13-methyltetradecanoic acid (aCl5:O) to isopalmitic acid (iC16 :0) during growth on a chemically defined medium lacking branched-chain amino acids. This finding attests alterations in the availability of precursors for t,he biosynthesis of methyl-branched fat,tJy acids. The qualitat'ive composition of phospholipids and other polar lipids in one mutant' group was found to be similar t o the progenitor strain but, addit'ionally, both it yellow pigment and a neutral lipid component were produced in excess. A second type of mutant differed by its incapability to form ornithinolipids even under phosphate limitation. Changes of phospholipid composition were demonstrated in the course of fermentation. Formation of ornithinolipid was suppressed by a n excess of inorga,nic phosphate in the medium, while the portions of phosphatidy1et)hanolamine and cardiolipin increased strongly. Furthermore, the formation of ornithinolipids was influenced by nitrogen sources. These results suggest t)hat the composition of membrane of 8. griseus varies in dependence upon the composition of the medium and the age of the mycelium. Yreeedingly, the non-aerial-m;yceliuin-forming (Amp-) and non-streptomycin-producing (Str-) derivatives LM 1, LM 2, LM 5, LM G and LM 7 of t'he industrial streptomycin-producing st'rain HP of Streptomyces qriseiis (Str+Amy+) were reported to differ froin their genetic ancest,or by very low intra-and extracelliilar levels of the NAD(1')-glycohydrolase (GRAFE et al. 1981). This work was undert'aken in order t'o hiocheniically characterize the Str-Amy-mutants obtained b y spontaneous degeneration (LM 1, JAM 2 ) or during continuous cultivation in a glycerol-limited charnostat (KOTH et al. in preparation). These strains appeared as particularly useful niodcls for studies on t.he biochemical background of the spontaneoiis variability of streptoniycetes whose true causes still await detailed elucidation (CHATER 1979, OKANISHI 1979, POGELL 1979. HOPWOOD 1978. Therefore. the lack of KAD(P)-glycohydrolase production in Str-Amy-strains (GRAFE et ul. 1981) prompted us to investigate the composition of the niycelial lipid fraction since the production of extracelliilar enzymes of Gram-positive bact,t:ria has been shown to depend on the coinposition of the cytoplasniic membrane (RAMALEY 1979). I t was the aim of this work to obtain more inforinat'ion about the differences between the Str-Amyderivatives and their ancestor, because we suppose that the genetic alterations during degeneration of strain HP were relatively small.

Eingegangen arn 27. 5. 1983) Arsenate-resistant (AsR) clones were obtained with high frequency from colony populations of streptothricin-producing strains of Streptomyces noursei by the paper strip method. Whereas in the AsR-strains obtained from both wild type and mutant NG 13 the antibiotic production was reduced to approx. 60% of the parental level, the AsR clones isolated from colony populations of mutant UV 12 displayed increased productivity (5150%).However, their improved capacity to produce streptothricins was lost rapidly after repeated cell propagation in submerged cultures, suggesting that unstable genetic elements were involved in enabling S. noursei to grow in the presence of toxic concentrations of arsenate.Many reports on antibiotic fermentations attest that excessive supply with inorganic phosphate suppresses the production of secondary metabolites of streptomycetes and PeniciZZium species (DEMAIN 1982). Therefore, most fermentations of antibiotics require the addition of inorganic phosphate in growth-limiting amounts, if high yields are to be obtained (MARTIN 1978, INOUE et al. 1982, OMURA et al. 1981).The molecular mechanism of the so-called 'phosphate effect' appears to be complex and may also depend on the particular microorganism (MARTIN 1977). Among the possible mechanisms are the inhibition and repression of the antibiotic-specific pathway as well as interference with the energy metabolism and the system of precursor generation (MARTLN and DEMAIN 1976).In order to release antibiotic fermentations from the regulation by inorganic phosphate, efforts have been mainly directed to the control of the phosphate pool in the medium. Recent results show that mutants deregulated in their response to inorganic phosphate can be obtained from the candicidine-producing Streptomyces griseus by either the conventional screening techniques or selection of arsenateresistant (AsR) strains (MARTIN etal. 1979, NAHARRO et aZ. 1981). Because corresponding data for other antibiotic-producing streptomycetes are missing, the general value of the latter methods for the isolation of superior producers of antibiotics cannot he assessed.I n the present work, we report on the isolation and some properties of arsenateresistant strains of Streptomyces noursei, a microorganism producing streptothricintype antibiotics (BRADLER and THRUM 1963). Materials and methodsStrains: Streptomyces ltoursei J A 3890b (BRADLER and THRUM 1963) was received from the culture collection of the Central Institute of Microbiology and Experimental Therapy, Jena.Mutant strains UV 12 and NG 13 were obtained by random selection for high antibiotic activity after using UV light and nitrosomethyl guanidine, respectively, as mutagens. The latter strains showed a 2-3 fold higher capacity to produce streptothricins (nourseothricin) than the parental strain.

Five non-streptomycin-producing non-aerial-mycelium-forming mutants (Str-Amy-) of Streptomyces griseus obtained either by spontaneous degeneration or during continuous cultivation of the high-producing aerial-mycelium-forming parent strain HP (Str+Amy+) were checked with regard to the composition of mycelial lipid material. All the Str-Amy- derivatives differed from their ancestor strain HP by an increased ration of 12-methyltetradecanoic acid (aC15:0) to isopalmitic acid (iC16:0) during growth on a chemically defined medium lacking branched-chain amino acids. This finding attests alterations in the availability of precursors for the biosynthesis of methyl-branched fatty acids. The qualitative composition of phospholipids and other polar lipids in one mutant group was found to be similar to the progenitor strain but, additionally, both a yellow pigment and a neutral lipid component were produced in excess. A second type of mutant differed by its incapability to form ornithinolipids even under phosphate limitation. Changes of phospholipid composition were demonstrated in the course of fermentation. Formation of ornithinolipid was suppressed by an excess of inorganic phosphate in the medium, while the portions of phosphatidylethanolamine and cardiolipin increased strongly. Furthermore, the formation of ornithinolipids was influenced by nitrogen sources. these results suggest that the composition of membrane of S. griseus varies in dependence upon the composition of the medium and the age of the mycelium.

Phosphate-dependent changes of the mycelial lipid composition were studied in the streptothricin-producing parental strain Streptomyces noursei JA 3890 b/2 and its mutant RG 2. In contrast to its ancestor, the mutant was capable of producing the antibiotic nourseothricin even when large quantities of inorganic phosphate were present in the medium. The apparent insensitivity of the secondary metabolism to phosphate inhibition corresponds to a decreased level of phospholipids in the presence of excessive inorganic phosphate and, during phosphate limitation, to a much higher production of the alkaline phosphatases. A model is discussed which proposed the control by a common genetic element of both the phospholipid and antibiotic production.