A DNA filter-binding technique was used to study the interaction of the biotin repressor and operator site. From a biotin saturation curve, the concentration for halfmaximal binding (Ko.5) was calculated to be 1 MM. However, in a similar study with the in vitro coupled transcription-translation system in which biotin served as the corepressor, the K0.5 for repression was 7.1 nM. This marked difference of over 2 orders of magnitude was attributed to the activation of biotin by the partially purified repressor preparation in the in vitro system. The activated product formed from biotin, ATP, and repressor preparation was identified as biotinyl 5'-adenylate by paper chromatography and hydroxamic acid formation. Synthetic biotinyl 5'-adenylate was as effective as biotin in the in vitro system (Ko.s, 10 nM) and much more effective than biotin in the DNA-binding assay (Ko.5 1.1 nM versus 1 M&M). These studies indicate that biotinyl 5'-adenylate has a more direct role in the regulation of the biotin genes than does biotin per se.A cluster of five genes coding for the enzymes of the biotin biosynthetic pathway is located at min 17 on the chromosomal map of Escherichia coli (1). These genes are transcribed divergently (2) and their expression is coordinately regulated by biotin through repression (3). The repressive action of biotin is mediated through the bioR gene situated at min 89 close to the bfe locus (4, 5).We have previously described an in vitro coupled transcription-translation system for synthesis of two of the enzymes of the biotin gene cluster; 7,8-diaminopelargonic acid aminotransferase and dethiobiotin synthetase, encoded by A and D genes on the I and r strands, respectively (6). Biotin was effective in repressing the synthesis of both enzymes only in the presence of a partially purified repressor preparation. The specificity for biotin was demonstrated by the inability of various biotin analogs, both natural and synthetic, to function as corepressors. In a preliminary study of the direct interaction of the biotin-repressor complex with the operator site on the DNA by using the DNA filter-binding technique (7, Preparation of DNA. 32P-Labeled X bNo DNA was prepared from a heat-inducible phage, Xc1857bioABFCDs7, kindly supplied by Max Gottesman. A biotin deletion mutant of E. coli K-12, T5-2, was grown to a density of 4 X 108 cells per ml in a low-phosphate medium (pH 7.3) containing per liter: 10 g of neopeptone, 2.5 g of yeast extract, 5 g of NaCl, 2 g of glucose, 5 ,ug of biotin, 5 mg of thiamine, and 1.2 g of MgSO4. The phage lysate was then added to give a multiplicity of infection of 3 and the incubation was continued until a cell density of 1.2 X 109 per ml was attained. After the addition of 10 mCi of [32P]orthophosphoric acid, the temperature of the culture was shifted to 42°C for 15 min for phage induction and then returned to 34°C for an additional 3-hr incubation.*The cells were collected by centrifugation, resuspended in 40 ml of phage buffer (10 mM Tris-HCl, pH 7.3/2 mM MgSO4/68 mM NaCl...
Some 60 biotin auxotrophs of Escherichia coli K-12 were isolated and classified into four groups according to their cross-feeding patterns, excretion products, and their ability to show a growth response to various biotin vitamers. Since all the mutants could be transduced with Xdbio phages known to carry the entire bioA locus, it was concluded that all of the mutation sites were located in this locus. It was also possible to derive a gene order for the different mutant groups on the basis of transduction studies with various Xdbio phages that carry portions of the bioA locus. A possible biochemical pathway for the biosynthesis of biotin in E. coli K-12 is discussed.
The enzymatic synthesis of 7-oxo-8-aminopelargonic acid (7-KAP) from pimelylcoenzyme A and L-alanine was demonstrated in cell-free extracts of a biotin mutant of Escherichia coli K-12 which excretes only 7-KAP into the growth medium. This biotin vitamer was identified by its chromatographic and electrophoretic properties. The enzyme (7-KAP synthetase) was repressed when the organism was grown in biotin concentrations greater than 0.2 ng/ml. The parent strain and members of other mutant groups that excrete 7-KAP, in addition to other vitamers, also exhibited synthetase activity. A mutant group that failed to excrete 7-KAP was further subdivided into three groups, one of which lacked synthetase activity. These results are discussed in relation to a previously proposed scheme for biotin biosynthesis in which the formation of 7-KAP is considered the point of entry for pimelic acid into the biotin pathway. Pimelic acid was established as a direct precursor of biotin by the isotopic studies of Eisenberg (3) and H
1. An unknown biotin vitamer was obtained in high yields in culture filtrates of Penicillium chrysogenum. 2. Production of this vitamer and desthiobiotin is controlled by the biotin concentration in the medium. 3. The unknown vitamer becomes labelled when the organism is grown in the presence of radioactive pimelic acid. 4. Chromatographic procedures were developed for the purification of the radioactive vitamer. 5. The vitamer is extremely stable in concentrated acid but gives rise to new vitamers under certain conditions. 6. The intermediate role of this vitamer in the synthesis of biotin is discussed.
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