The bialaphos biosynthetic genes of Streptomyces hygroscopicus: cloning and analysis of the genes involved in the alanylation step.

Hara, Osamu; Anzai, Hiroyuki; Imai, Shoichi; Kumada, Yoichi; Murakami, Takeshi; Itoh, Reiko; Takano, Eriko; Satoh, Atsuyuki; Nagaoka, Kozo

doi:10.7164/antibiotics.41.538

Cited by 30 publications

(21 citation statements)

References 23 publications

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“…The bialaphosproducing strain S. hygroscopicus ATCC 21705 (29) and S. hygroscopicus HP5-29, a bialaphos-over-producing derivative (2), were obtained from the Meiji Seika Culture Collection. Seed cultures were inoculated by placing a single colony of S. hygroscopicus grown on NE agar (41) into S1 medium (43) (10 ml in a 30-ml culture tube equipped with a stainless-steel spring to break up mycelial clumps) and l 111 [20]) were mapped by Murakami et al (41) and Hara et al (18). bar, bialaphos resistance gene which catalyzes step 10 in the pathway (31,58); ala, genes involved in peptide condensation; brpA, bialaphos regulatory protein.…”

Section: Methodsmentioning

confidence: 99%

“…Primary metabolic substrates are converted to the demethylated form of PT, which is then condensed with two alanine residues. Although the mechanism of this condensation has not been studied biochemically, mutants blocked in this step have been isolated (18). Genes which restore productivity to mutants having defects in either demethylated PT synthesis or peptide bond formation (bialaphos antibiotic production [bap]) have been cloned and localized to a 35-kb segment of the genome ( Fig.…”

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confidence: 99%

“…Genes which restore productivity to mutants having defects in either demethylated PT synthesis or peptide bond formation (bialaphos antibiotic production [bap]) have been cloned and localized to a 35-kb segment of the genome ( Fig. 1) (18,41). This region includes a gene which confers bialaphos resistance (bar) encoding a PT or demethylated PT acetyltransferase (58).…”

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Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus

et al. 1991

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Nucleotide sequence analysis of a 5,000-bp region of the bialaphos antibiotic production (bap) gene cluster defined five open reading frames (ORFs) which predicted structural genes in the order bah, ORF1, ORF2, and ORF3 followed by the regulatory gene, brpA (H. Anzai, T. Murakami, S. Imai, A. Satoh, K. Nagaoka, and C. J. Thompson, J. Bacteriol. 169:3482-3488, 1987). The four structural genes were translationally coupled and apparently cotranscribed from an undefined promoter(s) under the positive control of the brpA gene product. S1 mapping experiments indicated that brpA was transcribed by two promoters (brpAp1 and brpAp2) which initiate transcription 150 and 157 bp upstream of brpA within an intergenic region and at least one promoter further upstream within the bap gene cluster (brpAp3). All three transcripts were present at low levels during exponential growth and increased just before the stationary phase. The levels of the brpAp3 band continued to increase at the onset of stationary phase, whereas brpApl-and brpAp2-protected fragments showed no further change. BrpA contained a possible helix-turn-helix motif at its C terminus which was similar to the C-terminal regulatory motif found in the receiver component of a family of two-component transcriptional activator proteins. This motif was not associated with the N-terminal domain conserved in other members of the family. The structural gene cluster sequenced began with bah, encoding a bialaphos acetylhydrolase which removes the N-acetyl group from bialaphos as one of the final steps in the biosynthetic pathway. The observation that Bah was similar to a rat and to a bacterial (Acinetobacter calcoaceticus) lipase probably reflects the fact that the ester bonds of triglycerides and the amide bond linking acetate to phosphinothricin are similar and hydrolysis is catalyzed by structurally related enzymes. This was followed by two regions encoding ORF1 and ORF2 which were similar to each other (48% nucleotide identity, 31% amino acid identity), as well as to GrsT, a protein encoded by a gene located adjacent to gramicidin S synthetase in Bacillus brevis, and to vertebrate (mallard duck and rat) thioesterases. The amino acid sequence and hydrophobicity proffle of ORF3 indicated that it was related to a family of membrane transport proteins. It was strikingly similar to the citrate uptake protein encoded by the transposon Tn3411.Bialaphos, a linear tripeptide produced as a secondary metabolite by Streptomyces hygroscopicus, is composed of two L-alanine residues and the glutamate analog phosphinothricin (PT) (4, 29). After cleavage of bialaphos by intracellular nonspecific peptidases, PT acts as an inhibitor of glutamine synthetase (4, 29). For this reason, bialaphos has herbicide and antibiotic activities. The biosynthetic pathway has been well characterized by analyzing intermediates accumulated in cultures of blocked mutants (19). Primary metabolic substrates are converted to the demethylated form of PT, which is then condensed with two alanine residues. Althoug...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus

et al. 1991

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“…Simplification (28,29) and extension (1,11,20,39) of this method can help in the analysis of this and other biosynthetic gene clusters. The experimental approaches described in this work can be used to determine whether biosynthetic genes are associated with the other two resistance genes (srmA and srmC), to examine the physiological role of the resistance genes, and to study the regulation of spiramycin biosynthetic genes.…”

Section: Discussionmentioning

confidence: 99%

Cloning of spiramycin biosynthetic genes and their use in constructing Streptomyces ambofaciens mutants defective in spiramycin biosynthesis

et al. 1990

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Several cosmid clones from Streptomyces ambofaciens containing the spiramycin resistance gene srmB were introduced into S.fradiae PM73, a mutant defective in tylosin synthesis, resulting in tylosin synthesis. The DNA responsible for this complementation was localized to a 10.5-kilobase EcoRI fragment. A 32-kilobase DNA segment which included the srmB spiramycin resistance gene and DNA which complemented the defect in strain PM73 were mutagenie in vivo with TnlO carrying the gene for Nmr (which is expressed in Streptomyces spp.) or M vitro by insertional mutagenesis with a dnug resistance gene (Nmr) cassette. When these mutaenized DNA segments were crossed into the S. ambofaciens chromosome, three mutant classes blocked in spiramycin synthesis were obtained. One mutant accumulated two precursors of spiramycin, platenolide I and platenolide II. Two mutants, when cofermented with the platenolide-accumulating mutant, produced spiramycin. Tylactone supplementation of these two mutants resulted in the synthesis of a group of compounds exhibiting antibiotic activity. Two other mutants failed to coferment with any of the other mutants or to respond to tylactone supplementation.Spiramycin, a macrocyclic lactone antibiotic (macrolide) synthesized by Streptomyces ambofaciens (44), is used in both veterinary medicine (58) and human medicine (40). Spiramycin has a 16-member lactone ring (platenolide) to which three sugars are attached-mycaminose, mycarose, and forosamine (43). Structural similarities between spiramycin and tylosin (see Fig. 1) are mirrored by similarities in the two biosynthetic pathways, as evidenced by the ability of a cerulenin (polyketide synthase inhibitor)-inhibited culture of S. ambofaciens to synthesize chimeramycin, a tylosin-spiramycin hybrid, when supplemented with the tylosin lactone tylactone (42). We are interested in isolating spiramycin biosynthetic genes to study their regulation and to use them for the synthesis of novel antibiotics (3,24,26,41,42).Like many antibiotic-producing streptomycetes (14), S. ambofaciens is resistant to the antibiotic it produces, i.e., spiramycin. We have isolated three different spiramycin resistaince genes from S. ambofaciens in a shuttle cosmid vector (48). Since there is precedence for clustering of antibiotic biosynthetic genes in streptomycetes and for the association of resistance genes with these clusters (36, 52), we examined the possibility that DNA segments adjacent to the spiramycin resistance genes code for spiramycin biosynthetic enzymes. In this report, we present evidence for the existence of three spiramycin biosynthetic genes. These are located on both sides of the srmB spiramycin resistance gene. Culture conditions and genetic manipulations. Escherichia coli and Streptomyces strains were grown as described earlier (45, 48). E. coli transformations were done as described by Maniatis et al. (35). Streptomyces transformations were done as previously described (45). Selection for antibiotic resistance was on TY, TS, or modified R2 medium (45) sup...

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“…From a physicochemical point of view, direct formation of PPA by condensation between PF and PEP seemed unlikely, and the existence of an unidentified intermediate was implied. To dissect this potentially complex conversion, we attempted to obtain mutations in alleles distinct from that represented by NP213, using a gene replacement technique developed by Anzai et al (1,4). As a result, we isolated a mutant (NP71) whose cell extract could restore BA production to NP213.…”

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Carboxyphosphonoenolpyruvate phosphonomutase, a novel enzyme catalyzing C-P bond formation

et al. 1990

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An enzyme catalyzing the formation of an unusual C-P bond that is involved in the biosynthesis of the antibiotic bialaphos (BA) was isolated from the cell extract of a mutant (NP71) of Streptomyces hygroscopicus SF1293. This enzyme, carboxyphosphonoenolpyruvate (CPEP) phosphonomutase, was first identified as a protein lacking in a mutant (NP213) defective in one of the steps in the pathway to BA. The first 30 residues of the amino terminus of this protein were identical to those predicted by the nucleotide sequence of the gene that restored BA production to NP213. The substrate of the enzyme, a P-carboxylated derivative of phosphoenolpyruvate named CPEP, was also isolated from the broth filtrate of NP213 as a new biosynthetic intermediate of BA. CPEP phosphonomutase catalyzes the rearrangement of the carboxyphosphono group of CPEP to form the C-P bond of phosphinopyruvate.

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The bialaphos biosynthetic genes of Streptomyces hygroscopicus: cloning and analysis of the genes involved in the alanylation step.

Cited by 30 publications

References 23 publications

Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus

Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialaphos biosynthetic gene cluster of Streptomyces hygroscopicus

Cloning of spiramycin biosynthetic genes and their use in constructing Streptomyces ambofaciens mutants defective in spiramycin biosynthesis

Carboxyphosphonoenolpyruvate phosphonomutase, a novel enzyme catalyzing C-P bond formation

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