Pleiotropic effects of a relC mutation in Streptomyces antibioticus

Abstract

Ochi (Agric. Biol. Chem. 51:829-835, 1987) has isolated a relaxed mutant of Streptomyces antibioticus, designated relC49. relC49 accumulates significantly lower levels of ppGpp than the parent stain, IMRU3720. At its maximum, the ppGpp level in relC49 was only one-fourth that observed in strain IMRU3720. Interestingly, a burst of ppGpp synthesis between 18 and 22 h of growth in IMRU3720 coincided with the onset of actinomycin production in that strain. As shown previously, the activity in protein synthesis of ribosomes from strain IMRU3720 decreases with the age of the culture. The decrease in activity was less pronounced in cultures of relC49. relC49 mycelium contains reduced levels of phenoxazinone synthase, a key enzyme involved in actinomycin biosynthesis. The rel mutation prevents the normal increase in the activity of one of the other enzymes required for production of the antibiotic, 3-hydroxyanthanilate-4-methyltransferase, and a third enzyme, actinomycin synthetase I, appears to be completely absent from relC49 mycelium. Levels of phenoxazinone synthase mRNA were examined by RNA dot blotting with the cloned phenoxazinone synthase gene as a probe. mRNA levels for phenoxazinone synthase were dramatically reduced in relC49 compared with strain IMRU3720. These results are discussed in terms of the possible regulation of the onset of actinomycin production by ppGpp.The stringent response to amino acid starvation in Escherichia coli results in the shutdown of stable RNA synthesis. The guanine derivative, ppGpp, has been shown to be an effector of the stringent response (reviewed in reference 2). Relaxed (rel) mutants of E. coli accumulate lower levels of ppGpp than wild-type cells and continue to synthesize stable RNAs under conditions of amino acid starvation. Ochi has isolated a relaxed mutant of Streptomyces antibioticus (16). That mutant displays a relaxed response under conditions of amino acid starvation and, most interestingly, is unable to make the antibiotic (actinomycin) normally produced by wild-type cultures. One goal of the study of mechanisms of antibiotic biosynthesis is to identify the biochemical effectors responsible for the onset of antibiotic production. The results from Ochi's laboratory suggest the interesting possibility that ppGpp may be one of the effector molecules which regulates the production of actinomycin. To further examine the characteristics of the relaxed mutant, we have assayed (i) the levels of ppGpp and other nucleotides in wild-type and mutant cultures grown in production medium, (ii) the activity in cell-free protein synthesis of ribosomes from the mutant and parent cultures, (iii) the levels of several enzymes involved in actinomycin biosynthesis in wild-type S. antibioticus and in the rel mutant, and (iv) the levels of mRNA for phenoxazinone synthase (PHS), an enzyme involved in actinomycin biosynthesis in S. antibioticus. MATERIALS AND METHODSGrowth of organisms. S. antibioticus IMRU3720 and relC49 were cultured from spores in NZ-amine and galactose-glutamic acid me...

Guanosine pentaphosphate synthetase from Streptomyces antibioticus is also a polynucleotide phosphorylase

“…In addition, the relaxed mutant contained decreased levels of two enzymes involved in the biosynthesis of actinomycin, phenoxazinone synthase and actinomycin synthetase I (22,28). In the parental strain of S. antibioticus, there was a significant increase in ppGpp levels just prior to the onset of actinomycin production (22). Thus, studies of wild-type S. antibioticus as well as relaxed mutants support a relationship between ppGpp production and actinomycin biosynthesis.…”

“…Ochi isolated a relaxed (relC) mutant of S. antibioticus IMRU 3720 some years ago and demonstrated that this mutant contained reduced levels of ppGpp compared with the parental strain and was unable to synthesize actinomycin (28,29). In addition, the relaxed mutant contained decreased levels of two enzymes involved in the biosynthesis of actinomycin, phenoxazinone synthase and actinomycin synthetase I (22,28). In the parental strain of S. antibioticus, there was a significant increase in ppGpp levels just prior to the onset of actinomycin production (22).…”

Purification and properties of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) from Streptomyces antibioticus

“…To further examine this possibility, it is desirable to obtain mutants in which the production of ppGpp is reduced to negligible levels or is completed eliminated. Such is not the case in the relC49 mutant (15,20). An additional drawback of relC mutants is that the effect of the mutation on ppGpp production in those constructs is indirect.…”

“…The relC49 mutant was also found to lack detectable levels of actinomycin synthetase I, another enzyme involved in actinomycin biosynthesis (15). In the parental strain of S. antibioticus, there was a significant increase in ppGpp levels just prior to the onset of actinomycin production (15).…”

“…In more recent experiments, evidence was presented for an effect of the reiC mutation at the level of transcription of the structural gene for phenoxazinone synthase. Thus, the relC mutant contained decreased levels of phs mRNA (15). The relC49 mutant was also found to lack detectable levels of actinomycin synthetase I, another enzyme involved in actinomycin biosynthesis (15).…”