A 6.5 kb DNA fragment containing a chloramphenicol-resistance gene of Streptomyces venezuelae ISP5230 was cloned in Streptomyces fitlidans M252 using the high-copy-number plasmid vector pIJ702. The gene was located within a 2.4 kb KpnI-SstI fragment of the cloned DNA and encoded an enzyme (chloramphenicol hydrolase) that catalysed removal of the dichloroacetyl moiety from the antibiotic. The deacylated product, p-nitrophenylserinol, was metabolized to p-nitrobenzyl alcohol and other compounds by enzymes present in S. f i v i h s M252. Examination of the genomic DNA from several sources using the cloned 6.5 kb SstI fragment from S. venezuefue ISP5230 as a probe showed a hybridizing region in the DNA from S. venezuelae 13s but none in the DNA from another chloramphenicol producer, Streptomycesphaeochromogenes NRRLB 3559. The resistance phenotype was not expressed when the 6.5 kb SstI fragment or a subfragment was subcloned behind the lac-promoter of plasmid pTZ18R in Escherichia cofi.

Two chloramphenicol-producing strains of Streptomyces venezuelae accumulated small amounts of polyhydroxybutyrate during exponential growth; the compound disappeared from the mycelium as the cultures entered stationary phase. Depletion of polyhydroxybutyrate coincided with chloramphenicol production but the amount of polymer stored in the mycelium was insufficient to supply the precursor requirement for biosynthesis of the antibiotic. Accumulation of polyhydroxybutyrate in the S. venezuelae strains was appreciably lower than in two other streptomycetes examined. Glycogen and lipids accumulated in the mycelium of S. venezuelae 13s during the stationary phase, after nitrogen depletion; under the culture conditions used, they were the principal storage compounds in S. venezuelae. Trehalose was absent from the mycelium in vegetative cultures grown under nonsporulating conditions but it was abundant in spores obtained from submerged and surface cultures. Glycogen and polyhydroxybutyrate were absent from spores.

(1990).The ' H nuclear magnetic resonance spectrum of mollisin from cultures of the fungus Mollisia caesia supplemented with [2-2~3]acetate was consistent with biosynthesis of the metabolite via a single-chain polyketide. The presumption that chain scission accompanies dichlorination of a reactive methylene group in such a polyketide was supported by the detection of chloroperoxidase activity with monochlorodimedone as a substrate, and failure to detect chlorinating activity when the substrate was acetoacetyl coenzyme A. However, a decisive choice between single-chain and two-chain polyketide intermediates cannot be made with information provided by the techniques used so far. Les spectres en resonance magnktique nucleaire du 2~ de la mollisine provenant de milieux de cultures du champignon Mollisia caesia auxquels on a ajoutC du [2-2~3]acetate sont en accord avec ceux auxquels on pourrait s'attendre sur la base d'un mecanisme de biosynthese du mktabolite impliquant une chaine unique de polycktide. L'hypothkse d'aprks laquelle une scission de la chaine accompagne la dichloration d'un groupement methylkne rkactif d'un polycktide est en accord avec la dktection d'une activit6. de la choroperoxydase sur la monochlorodirnkdone comme substrat et le fait que l'on ne peut dktecter de chloration lorsque le substrat est 1'acCtoacetyl coenzyme A. Toutefois, sur la base des rksultats obtenus avec les techniques utilistes jusqu'i maintenant, on ne peut choisir entre les mecanismes impliquant des intermCdiaires polycktides i chaines uniques ou doubles.Mots ClCs : biosynthese, mollisine, marquage au deuterium et RMN; marquage au deuterium et RMN, biosynthkse de la mollisine; marquage de la mollisine par du deuterium, analyse par RMN; biosynthkse de la mollisine, marquage au deuterium et RMN; RMN, deuterium, biosynthese de la mollisine.[Traduit par la revue]