A DNA sequence (brpA) which regulates the expression of the genes of the bialaphos biosynthesis pathway (bap) in Streptomyces hygroscopicus was identfied and characterized. A newly isolated nonproducing mutant (NP57) had a pleiotropic defect involving at least 6 of the 13 known bap genes; only the step 6 conversion could be detected. NP57 was more sensitive to bialaphos than its parent and had depressed levels of the demethylphosphinothricin acetyltransferase activity (step 10 in the pathway) which confers bialaphos resistance. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of extracts of this mutant showed that it lacked proteins corresponding to steps 5 and 10. NP57 lacked mRNAs for steps 5, 10, and 13. Bialaphos productivity of NP57 was restored by transformation with a plasmid containing a 5.9-kilobase DNA fragment which was adjacent to the structural gene cluster. Subconing experiments showed that a 1.3-kilobase fragment from this primary dlone restored all the defects of NP5. We conclude that brpA can activate the transcription of the bialaphos resistance gene as well as at least six other bap structural genes.Under certain nutritional conditions, usually associated with the stationary phase of growth, streptomycetes can activate alternative (secondary) metabolic pathways which divert the intermediates of primary metabolism into antibiotic production pathways. The hlature of the nutritional imbalance and the intracellular regulatory factor(s) which leads to this activation or derepression of antibiotic biosynthesis genes are not well understood. In the case of streptomycin biosynthesis, it has been shown that the pathway enzymes are coordinately regulated (32). The availability of genetic engineering techniques and cloning vectors for Streptomyces species makes it easier to study the genes which control antibiotic production.Molecular cloning experiments have shown that structural genes which code for enzymes in a given antibiotic biosynthesis pathway are clustered (2-5, 15, 20, 23, 30). Studies of polar insertion mutations have demonstrated that the methylenomycin (3) and actinorhodin (16) biosynthesis genes are expressed as polycistronic transcriptional units. In the case of methylenomycin (3), streptomycin (23), and actinorhodin (16), the activator gene(s) is located near the structural genes under its control. These genes, which regulate specific antibiotic pathways, are presumably regulated by systems which turn on secondary metabolism and morphological development.We studied the biosynthesis of bialaphos, a secondary metabolite of Streptomyces hygroscopicus and a commercially important herbicide. Bialaphos is a tripeptide which consists of two L-alanine residues and the L-glutarnic acid analog phosphinothricin (13). In S. hygroscopicus, bialaphos is degraded to phosphinothricin, which is a potent inhibitor of glutamine synthetase (1; unpublished data). The bar gene encodes demethylphosphinothricin acetyltransferase * Corresponding author.
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.
A 32-year-old female presented with a history of intermittent, severe lower back pain, exacerbated by pregnancy and persisting for several years. Magnetic resonance (MR) imaging showed a sacral mass as a low signal intensity area on the T1-weighted image and a high signal intensity area on the T2-weighted image, consistent with a cystic structure containing cerebrospinal fluid. A tethered cord attached to the thecal sac was also confirmed. Sacral laminectomy revealed thinned sacral vertebral laminae, but no neuronal elements existed within the cyst. The cyst was attached to the distal thecal sac by a small fistula. The thecal sac was explored, revealing a thick filum terminale, which was transected. The severe pain resolved postoperatively. This type of meningeal cyst, often called "occult intrasacral meningocele," is best diagnosed by MR imaging. Surgery to obliterate the fistulous tract is the optimum treatment. Total removal of the cyst is unnecessary, but evaluation of other associated congenial abnormalities is important.
The purpose of this study was to scrutinize morphological characteristics of thin-section CT of the histopathological subtypes of adenocarcinoma of the lung. The subjects consisted of 83 patients with 87 adenocarcinomas measuring 3 cm or less in the largest. The tumors were divided into three groups (group I: Noguchi's histological subtypes type A and B tumors, group II: type C tumors, and group III: type D, E, and F tumors). In each group, tumor size, shape (round versus polygonal), presence of air bronchogram, bubble-like areas, coarse spiculation, pleural tag, and ratio of ground glass attenuation (GGA) were evaluated. Most of the group II lesions showed polygonal shape, whereas tumors in other groups were round in shape (P<0.01). Air bronchogram and bubble-like areas of low attenuation was seen more frequently in group II compared with those in group III (P<0.01). GGA areas were largest in group I and smallest in group III (P<0.01). We believe thin-section CT findings reflect the histopathological subtypes of adenocarcinoma of the lung. The presence of air bronchogram and bubble-like areas of low attenuation areas in particular is useful to differentiate replacement growth tumors from non-replacement growth tumors.
Cosynthesis in mixed culture and protoplast fusion of non-producing mutants of Streptomyces hygroscopicus which may produce biosynthetic intermediates of bialaphos (AMPBA) were studied. Non-producing mutants were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment, and six stable non-producing mutants were used for cosynthesis and protoplast fusion studies. The biosynthetic block sites of the non-producers were consistent with studies of the biosynthetic pathway of AMPBA.Bialaphos (AMPBA) is a metabolite of Streptomyces hygroscopicus ATCC 21705 having strong herbicide activity). The structure of this substance was determined to be 2-amino-4-(hydroxy)(methyl)-phosphinoylbutyrylalanylalanine2), and is the first natural product to have the unique C-P-C bond of the phosphinothricyl moiety). In previous papers4,5), we have described non-producing mutants of AMPBA producer obtained by mutagenesis with NTG treatment; these mutants produced derivatives or intermediates of AMPBA. We have further investigated the biosynthetic pathway of AMPBA by the technique of mixed culture cosynthesis and protoplast fusion using six additional AMPBA nonproducing mutants of S. hygroscopicus. The present paper deals with the determination of blocked sites in non-producers and the results of a complementation study by cosynthesis and protoplast fusion. Materials and MethodsOrganisms Streptomyces hygroscopicus ATCC 21705 was used parental strain. Proteus sp. MB-838 was used for the assay of AMPBA. Media Seed culture medium (S-1) was prepared with 2.0 % soluble starch, 1.0 % peptone, 0.3 % meat extract, 0.05 % KZHPO4, pH 7.0. Agar medium (A-4: 0.4% glucose, 0.35% wheat germ, 0.23% soluble vegetative protein, 0.03 % KH2PO4 0.0001 % CoCl2.6H2O, 2.0 % agar, pH 7.0) was used for cosynthesis experiments and selection of non-producing mutants by the use of agar piece method.') Cosynthesis on an Agar Plate Cosynthesis on agar plates was carried out by modifying the procedure derived by DELIC et al.') and FURUMAI et a1.8•0). The mycelial suspension of two non-producing mutants was harvested from S-1 medium (0.1 ml each) and spread about 1 -2 mm apart on opposite halves of an A-4 medium plate. The plate was incubated for 7 days at 28°C. An agar strip, 5 x 60 mm was cut from the plate at right angles to the line of separation between the two mutants. This strip was plated on the surface of an agar plate seeded with Proteus sp. MB-838, and incubated at 32°C overnight. The appearance of an inhibition zone along the agar strip was considered a sign of the production of AMPBA.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.