Transductional analysis of chloramphenicol biosynthesis genes in Streptomyces venezuelae.

Auxotrophs isolated from two chloramphenicol-nonproducing mutants of Streptomyces venezuelae included three requiring pyridoxal (Pxl-), VS248 (cml-11 pdx-2), VS253 (cml-11 pdx-3), and VS258 (cml-12 pdx-4), and one requiring thiosulfate, VS263 (cml-12 cys-28). Results of SV1-mediated transductions were consistent with the relative marker order cys-28-cml-12-cml-11-pdx-2,3,4,5, all of which were cotransducible and must therefore span less than 45 kilobases of DNA, the approximate length of DNA packaged by SV1. cys-28 was also cotransducible with arg-4 and arg-6, but arg and pdx were not cotransducible. Results of crosses with donors carrying any one of 11 cml mutations were consistent with the location of all cml mutations between cys-28 and pdx markers. Also, a new Pxl- auxotroph (pdx-6) and two new Cml- mutants were recovered after localized hydroxylamine mutagenesis of a cys-28 cml+ strain derived from VS263 by transduction.     

Isolation and characterization of mutants blocked in T-2 toxin biosynthesis

Mutants of Fusarium sporotrichioides NRRL 3299 that were blocked or altered in the biosynthesis of the trichothecene T-2 toxin were generated by UV treatment and identified by a rapid screen in which monoclonal antibodies to T-2 were used. Three stable mutants were isolated and chemically characterized. Two mutants accumulated diacetoxyscirpenol, which suggests that they were defective in the step required for the addition of a hydroxyl group to the C-8 position in the trichothecene core structure. The third mutant appeared to be partially blocked at an early step or regulatory point in the pathway. This represents the first isolation of mutants in a trichothecene biosynthetic pathway.     

Isolation and characterization of mutants blocked in T-2 toxin biosynthesis.

Mutants of Fusarium sporotrichioides NRRL 3299 that were blocked or altered in the biosynthesis of the trichothecene T-2 toxin were generated by UV treatment and identified by a rapid screen in which monoclonal antibodies to T-2 were used. Three stable mutants were isolated and chemically characterized. Two mutants accumulated diacetoxyscirpenol, which suggests that they were defective in the step required for the addition of a hydroxyl group to the C-8 position in the trichothecene core structure. The third mutant appeared to be partially blocked at an early step or regulatory point in the pathway. This represents the first isolation of mutants in a trichothecene biosynthetic pathway.     

Isolation of mutants blocked in calicheamicin biosynthesis.

Blocked mutants of Micromonospora echinospora defective in the production of the potent antitumor agent calicheamicin are described. Analysis of intermediates produced by blocked mutants indicates a branched biosynthetic pathway. Mutants that produced some, but not all, calicheamicin forms are also described.     

Chloramphenicol resistance in Streptomyces: cloning and characterization of a chloramphenicol hydrolase gene from Streptomyces venezuelae

A 6.5 kb DNA fragment containing a chloramphenicol-resistance gene of Streptomyces venezuelae ISP5230 was cloned in Streptomyces lividans 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. lividans M252. Examination of the genomic DNA from several sources using the cloned 6.5 kb SstI fragment from S. venezuelae ISP5230 as a probe showed a hybridizing region in the DNA from S. venezuelae 13s but none in the DNA from another chloramphenicol producer, Streptomyces phaeochromogenes 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 coli.     

Structural basis for chloramphenicol tolerance in Streptomyces venezuelae by chloramphenicol phosphotransferase activity

Streptomyces venezuelae synthesizes chloramphenicol (Cm), an inhibitor of ribosomal peptidyl transferase activity, thereby inhibiting bacterial growth. The producer escapes autoinhibition by its own secondary metabolite through phosphorylation of Cm by chloramphenicol phosphotransferase (CPT). In addition to active site binding, CPT binds its product 3-phosphoryl-Cm, in an alternate product binding site. To address the mechanisms of Cm tolerance of the producer, the crystal structures of CPT were determined in complex with either the nonchlorinated Cm (2-N-Ac-Cm) at 3.1 A resolution or the antibiotic's immediate precursor, the p-amino analog p-NH(2)-Cm, at 2.9 A resolution. Surprisingly, p-NH(2)-Cm binds CPT in a novel fashion. Additionally, neither 2-N-Ac-Cm nor p-NH(2)-Cm binds to the secondary product binding site.     

Conjugational fertility and location of chloramphenicol biosynthesis genes on the chromosomal linkage map of Streptomyces venezuelae

In Streptomyces venezuelae fertility, defined as chromosomal gene recombination, was enhanced over 1000-fold when one parent in a biparental conjugational cross lacked the physically-undetected plasmid SVP1, as compared with crosses in which both parents carried SVP1. The existence of SVP1 and at least two other fertility plasmids, SVP2 and SVP3, was detected in S. venezuelae by 'lethal zygosis' elicited by a plasmid-plus mycelium in contact with a plasmid-minus mycelium. Conjugational crosses were used to construct a linkage map of S. venezuelae which was highly consistent with the map of analogous loci in S. coelicolor A3(2). A cluster of genes governing chloramphenicol biosynthesis was located near arg, cys and pdxB genes at a position roughly equivalent to the 1-2 o'clock region of the S. coelicolor A3(2) map.     

Growth and ultrastructure of Streptomyces venezuelae during chloramphenicol production.

Streptomyces venezuelae (3022a) was grown in flask cultures and fermentors, using three media having differential effects on chloramphenicol production. Micromorphology, ultrastructure and chloramphenicol concentrations were studied during the growth cycle in each medium. Chloramphenicol production was greatest in the glycerol-serine-lactate (GSL) medium, less in the glycerol-nutrient broth-yeast extract (GNY) medium and very low in glucose-mineral salts (GA) medium. In GSL and GA, much growth was in the form of microcolonies, especially in flask cultures, while short hyphal fragments predominated in GNY. The major ultrastructural features were the high frequency of mesosomes in fragmenting hyphae in GNY, and electron-transparent zones which appeared during chloramphenicol synthesis in GSL. None of the structural abnormalities induced by chloramphenicol in sensitive organisms were observed in S. venezuelae despite high levels of the antibiotic in GSL medium.     

Biosynthesis and combinatorial biosynthesis of pikromycin-related macrolides in Streptomyces venezuelae

Pikromycin-related macrolides have recently attracted significant research interest because they are structurally related to the semisynthetic ketolide antibiotics that have demonstrated promising potential in combating multi-drug-resistant respiratory pathogens. Cloning and in-depth studies of the pikromycin biosynthetic gene cluster from Streptomyces venezuelae have led to new avenues in modular polyketide synthases, deoxysugar biosynthesis, cytochrome P450 hydroxylase, secondary metabolite gene regulation, and antibiotic resistance. Moreover, the knowledge and tools used for these studies are proving to be valuable in the development of advanced technologies for combinatorial biosynthesis of new macrolide antibiotics. This review summarizes these new developments and introduces S. venezuelae as a powerful new system for secondary metabolite pathway engineering from bench-top genetic manipulation to product fermentation.     

Isolation and characterization of yeast mutants blocked in mevalonic acid formation

Yeast mutants defective in beta-hydroxy-beta-methylglutaryl-CoA synthase and acetoacetyl-CoA thiolase have been isolated. Mutants impaired in acetoacetyl-CoA thiolase range into two linked complementation units, erg 10 A and erg 10 B. Mutants deficient in beta-hydroxy-beta-methylglutaryl-CoA synthase belong to two unlinked complementation groups, erg 11 and erg 13. In strictly anaerobic growth conditions, mutants impaired in beta-hydroxy-beta-methylglutaryl-CoA synthase require mevalonic acid in addition to sterol and oleic acid, pointing out the role of mevalonic acid in other physiological function than ergosterol precursor. Growth of mutants impaired in acetoacetyl-CoA thiolase cannot be recovered by mevalonic acid supplementation, suggesting a role of acetoacetyl-CoA or thiolase not linked to sterol pathway.     

Isolation and biochemical characterization of Streptomyces clavuligerus mutants in the biosynthesis of clavulanic acid and cephamycin C

Summary Seven mutants of Streptomyces clavuligerus blocked in the biosynthesis of clavulanic acid, cephamycin C, or both antibiotics, have been isolated and characterized. Mutants nca1 and nca2 were unable to synthesize clavulanic acid but produced cephamycin C. Mutants nce1 and nce2 were completely blocked in cephamycin C production but formed clavulanic acid. A third group (mutants ncc1, ncc4 and ncc5) failed to produce both antibiotics. Arginase activity (forming ornithine) was very low in mutants ncc1 and ncc5. All the mutants blocked in clavulanic acid biosynthesis showed a normal ornithine--aminotransferase activity. Mutant ncc1, blocked in cephamycin biosynthesis, lacked completely lysine--aminotransferase (forming -aminoadipic acid) and isopenicillin N synthase. Two other mutants (nce2 and nce5) lacked isopenicillin N synthase. There was a good correlation between the isopenicillin N synthase and the lysine--aminotransferase activities of the nca mutants and the ability of those strains to produce cephamycin C. The condensing enzyme involved in the formation of the clavulanic acid nucleus appears to be different from the isopenicillin N synthase.Dedicated to Professor H.-J. Rehm on the occasion of his 60th birthday     

Isolation and characterization of temperature-sensitive mutants of Streptomyces coelicolor A3(2) blocked in macromolecular synthesis.

A collection of temperature-sensitive mutants of Streptomyces coelicolor A3(2) was isolated. The majority of the mutants showed an osmotically remedial phenotype. Mutants defective in macromolecular synthesis were identified and characterized further. Four mutants were found in which DNA replication was defective, but which continued to synthesize RNA and protein at the restrictive temperature (39 degrees C). The kinetics of cessation of DNA synthesis allowed a tentative identification of slow (initiation) and fast (elongation) stop dna mutants. The inhibition of DNA replication in the four mutants was found to be reversible on returning to the permissive temperature (30 degrees C), but only after a delay of about 2 h. Three other mutants were identified which showed not only cessation of DNA replication at the restrictive temperature, but also defects in other macromolecular synthesis events.     

Isolation and characterization of nitrogen-deregulated mutants of Streptomyces clavuligerus

Two screening methods for isolation of mutants of Streptomyces clavuligerus with altered control of nitrogen metabolism enzymes are described. Thirty-eight prototrophic mutants with simultaneous deregulation of urease and glutamine synthetase were isolated. Nine mutants were examined in more detail and they also showed deregulated formation of arginase and ornithine aminotransferase. Different patterns of altered control of all four enzymes were observed. Inactivation of glutamine synthetase after ammonium shock took place to different extents in these nine strains, and seven of them had a thermosensitive glutamine synthetase activity. It is concluded that a system of nitrogen control, in which glutamine synthetase has a key role, is present in S. clavuligerus. Cephalosporin production was depressed by ammonium in all the mutants, irrespective of the alterations in nitrogen control of primary metabolism.     

Effects of addition of chloramphenicol on the growth and ultrastructure of Streptomyces venezuelae

The effects of adding chloramphenicol before inoculation and during exponential growth of Streptomyces venezuelae (3022a) in fermentors were studied. The responses of the organism during synthesis of chloramphenicol (in a glycerol-serine-lactate medium) were compared with those in media supporting less (glycerol-nutrient broth-yeast extract) or no synthesis (glucosemineral salts). In systems where little or no synthesis of the chloramphenicol occurred, addition of the antibiotic induced micromorphological and ultrastructural abnormalities similar to those reported for sensitive bacteria. There was also an increase in the frequency of mesosomes and electron-light areas. It was suggested that the former may be associated with activity of chloramphenicol hydrolase and the latter with storage and/or excretion of the breakdown product; N-acetyl p-nitro-phenylserinol. When chloramphenicol synthesis occurred, addition of the antibiotic had less effect on the micromorphology or ultrastructure of S. venezuelae as permeability barriers to external chloramphenicol had been established. Electron-light areas were frequent, possibly being associated with storage and excretion of precursors of chloramphenicol.     

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.     

Effect of ammonium on chloramphenicol production by Streptomyces venezuelae in batch and continuous cultures

Cultures of Streptomyces venezuelae presented with a mixture of ammonium and an amino acid as nitrogen sources used both compounds together. Absence of ammonium repression of alternative nitrogen assimilation pathways was also observed when ammonium was added to cultures already growing on proline. The presence of ammonium in the medium ab initio depressed the yield of chloramphenicol. However, its addition to a culture growing on proline caused only a temporary inhibition of antibiotic synthesis, even when sufficient ammonium was added to create an excess. Continuous cultures supplied with ammonium as the growth-limiting nutrient showed no significant change in specific antibiotic production at different specific growth rates. The overall results indicate that in S. venezuelae neither nitrogen utilization pathways nor chloramphenicol biosynthesis is controlled by nitrogen repression.     

Isolation and characterization ofSerratia marcescens mutants defective in prodigiosin biosynthesis

Mutants deficient in the biosynthesis of prodigiosin have been obtained by treatingSerratia marcescens with high doses of ultraviolet radiation. Mutants were selected on the basis of the color characteristics of their colonies when grown on peptone glycerol medium. New types of mutants, with unusual blocks in the biosynthetic pathway of prodigiosin, were obtained. All the mutants were classified under a new scheme on the basis of the syntrophic pigmentation characteristic and infrared spectroscopic analysis of their pigment. By these criteria mutants could be distinguished into eight distinct classes. Classes I to III include mutants of the three classes (M1, B3, and B1) reported previously [Morrison, DA (1966) J Bacteriol 91:1599–1604] and several new ones. Mutants blocked in the methylamylpyrrole (MAP) arm of the bifurcated pathway were assigned to class I. A class II mutant was distinguished by its inability to synthesize methoxybipyrrolecarboxyaldehyde (MBC), but was able to produce norprodigiosin. Class III mutants were deficient in the synthesis of hydroxybipyrrolecarboxaldehyde (HBC). Double mutants were obtained with defects in the expression of both MBC and MAP and were assigned to class IV. Mutants of class V were unable to synthesize HBC and MAP, but could form MBC when furnished with exogenous HBC. Class VI and VII mutants were defective in the synthesis of all three precursors, but differed in their ability to perform the coupling step. Finally, a mutant of class VIII was found to produce the three intermediates, but was deficient in prodigiosin or norprodigiosin biosynthesis, indicative of a defect in the enzymatic condensation of MAP with the bipyrroles MBC and HBC. The anomalous pattern of syntrophism among certain interclass mutants suggests that the physiology of pigment formation inS. marcescens is quite complex.     

Isolation and characterization of Francisella novicida mutants defective in lipopolysaccharide biosynthesis

In order to identify genes involved in LPS biosynthesis we isolated random mutants generated by transposon insertion in Francisella novicida. The resulting mutant bank yielded mutants with three distinct LPS phenotypes, and three representative mutants were chosen for further study. One mutant that had short O-antigen chains was sensitive to serum; this mutant and one other were more sensitive to killing by deoxycholate than control strains. The third mutant was resistant to deoxycholate killing but slightly sensitive to serum. The three mutants varied in their ability to grow in macrophages. The DNA sequences interrupted by the transposon in two of the three mutants showed similarity to known LPS biosynthetic genes at the deduced amino acid level.