Kozo Nagaoka

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

SummaryWe have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.

The bialaphos biosynthetic genes of Streptomyces viridochromogenes: cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus

The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. lividans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in trans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. viridochromogenes bap genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. viridochromogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.

Biochemical Mechanism of C–P Bond Formation of Bialaphos: Use of Gene Manipulation for the Analysis of the C–P Bond Formation Step

One of the three C-P bond formation steps, defined as step 5 in the bialaphos (BA) biosynthetic pathway, was analyzed using a new BA non-producing mutant NP71. The mutant was derived from a BA producer by gene replacement of an unidentified region next to the gene responsible for the step 5 deficiency of the mutant NP213, obtained by conventional mutation procedures. Biochemical analysis of these two mutants indicated that NP71 was defective in the formation of carboxyphosphonoenolpyruvate (CPEP), while NP213 lacked the enzyme CPEP phosphonomutase, which catalyzed the intramolecular rearrangement of CPEP.

Purification and Properties of a Prokaryote Type Glutamine Synthetase from the Bialaphos Producer Streptomyces hygroscopicus SF1293

A prokaryote type glutamine synthetase (GS) was purified from a bialaphos (BA)-producing organism, Streptomyces hygroscopicus SF1293 (SF1293). The GS (GS I) consisted of a 55,000 dalton subunit, and its N-terminal amino acid sequence was similar to that of S. coelicolor GS. GS I was highly sensitive to GS inhibitor phosphinothricin (PPT). An increase of GS activity was observed accompanied by BA accumulation.

Isolation and Characterization of NewVirulent Actinophage φr5

A new virulent phage, φr5, was isolated from soil on Streptomyces ribosidificus SF733. Electron microscopic observation showed that this phage belongs to group B of Bradleys morphological classification. The molecular weight of φr5 DNAwas about 29×106. φr5 DNA showed resistance to many restriction enzymes. φr5 required Ca+ + for plaque formation and grew at 28°C on S. ribosidificus SF733, S. chartreusis SF1623, S. flavus, S. flavovirens, S. sulfonofaciens SF2103, S. sulfonofaciens SF21 38 and S. sulfonofaciens SF2 1 44. The restriction-modification of φr5 by the strains susceptible to this phage was examined.

Isolation and characterization of new virulent actinophage .PHI.r5.

A new virulent phage, Φr5, was isolated from soil on Streptomyces ribosidificus SF733. Electron microscopic observation showed that this phage belongs to group B of Bradley′s morphological classification. The molecular weight of Φr5 DNA was about 29 × 106. Φr5 DNA showed resistance to many restriction enzymes. Φr5 required Ca+ + for plaque formation and grew at 28°C on S. ribosidificus SF733, S. chartreusis SF1623, S.flavus, S. flavovirens, S. sulfonofaciens SF2103, S. sulfonofaciens SF2138 and S. sulfonofaciens SF2144. The restriction-modification of Φr5 by the strains susceptible to this phage was examined.