Use of Tn5lac to study expression of genes required for production of the antibiotic TA.

AUTOR(ES)

Tolchinsky, S

RESUMO

The beta-galactosidase activities arising from Tn5lac insertions in several genes required for antibiotic TA production were measured under different growth conditions. In all of the non-TA-producing mutants, the beta-galactosidase specific activity was higher when the cells were grown in nutrient-limited 0.5CTS medium (0.5% Casitone plus alanine, serine, and glucose) than in rich 2CT medium (2% Casitone). One of the mutants, 420, had low beta-galactosidase specific activity in both media. The other seven mutants containing inserts in genes essential for TA production had specific activities of 139 to 367 U/mg of protein in 0.5CTS medium and 11 to 48 U/mg of protein in 2CT medium. The beta-galactosidase specific activities of two strains, 1030 and 420, increased during exponential growth in 0.5CTS medium. The beta-galactosidase specific activities of both strains increased greatly when the cells were grown in the presence of magnesium phosphate, which traps ammonium ions. The Tn5lac insertions in 1030 and 420 were used to screen for mutants with increased levels of transcription. An N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in 1030 that mapped 17 kb from the omega 1010 insert increased the specific activity of beta-galactosidase 21 times in 2CT medium. The regulatory mutation appears to release the repression caused by 2CT medium. A UV-induced mutation in 420 increased the beta-galactosidase specific activity 1.4 to 2.4 times. Medium conditions that affect the transcription of TA genes are discussed in terms of enhanced antibiotic TA production.

Documentos Relacionados

• Mode of action of Myxococcus xanthus antibiotic TA.
• Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis vir-regulated genes.
• Mutation and mapping of genes involved in production of the antibiotic TA in Myxococcus xanthus.
• Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus.
• Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus.