ABSTRACT
Evidence against the Bm1P1 protein as a positive transcription factor for barbiturate-mediated induction of cytochrome P450(BM-1) in Bacillus megaterium has been provided in our previous report. In the present study, we show that the basal-level expression of the P450(BM-1) promoter-xylE transcriptional fusion is significantly reduced when the bm1P1 gene is present on the same plasmid. Moreover, disruption of the chromosomal bm1P1 gene results in enhanced expression of the P450(BM-1) promoter-xylE fusion on a multicopy plasmid. By using a binary vector system, we found that expression of the P450(BM-1) promoter-xylE fusion from a high-copy plasmid is substantially repressed by expression of bm1P1 from a low-copy plasmid. Taken together, these results suggest that the basal-level expression of P450(BM-1) is negatively affected by bm1P1. Possible mechanisms for this control are discussed.
Subject(s)
Bacillus megaterium/genetics , Cytochrome P-450 Enzyme System/genetics , DNA-Binding Proteins/genetics , Dioxygenases , Gene Expression Regulation, Bacterial , Repressor Proteins , Transcription Factors/genetics , Animals , Bacillus megaterium/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catechol 2,3-Dioxygenase , Cytochrome P-450 Enzyme System/metabolism , DNA-Binding Proteins/metabolism , Mutation , Oxygenases/genetics , Oxygenases/metabolism , Plasmids/genetics , Promoter Regions, Genetic , Recombinant Fusion Proteins , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
A gene encoding beta-galactosidase, designated mbgA, was isolated from Bacillus megaterium ATCC 14581. Chromosomal beta-galactosidase production could be dramatically induced by lactose but not by isopropyl-beta-D-thiogalactopyranoside (IPTG) and was subject to catabolite repression by glucose. Disruption of mbgA in the B. megaterium chromosome resulted in loss of lactose-inducible beta-galactosidase production. A 27-bp inverted repeat was found to overlap the mbgA promoter sequence. Two partially overlapping catabolite-responsive elements (CREs) were identified within the inverted repeat. Base substitutions within CRE-I and/or CRE-II caused partial relief from catabolite repression. The results suggest that the 27-bp inverted repeat may serve as a target for a catabolite repressor(s).
Subject(s)
Bacillus megaterium/genetics , beta-Galactosidase/genetics , Bacillus megaterium/enzymology , Base Sequence , Cloning, Molecular , DNA Primers , Glucose/metabolism , Isopropyl Thiogalactoside/metabolism , Lactose/metabolism , Mutation , Promoter Regions, Genetic , Recombinant Fusion Proteins/genetics , Transcription, GeneticABSTRACT
The effects of iron and salicylate on the expression of cytochrome P450s in Bacillus megaterium were investigated in this report. Immunoblot analysis showed that the addition of 4 mM ferric iron or 10 mM salicylate to the culture medium resulted in a significant increase in the P450BM-1 level, while the same condition had little effect on P450BM-3 expression. Substantial induction of chloramphenicol acetyltransferase (CAT) activity by iron and salicylate in B. megaterium cells bearing a P450BM-1 promoter-cat transcriptional fusion vector suggests that the induction of P450BM-1 by iron and salicylate occurs at the transcriptional level. Unexpectedly, in contrast to the bm1P1-dependent induction of P450BM-1 by pentobarbital, disruption of bm1P1 gene did not affect induction of P450BM-1 by iron and salicylate. This result suggests that the induction of P450BM-1 by iron and salicylate occurs by a bm1P1-independent mechanism. To our knowledge, this is the first example of an iron-regulated cytochrome P450 gene in prokaryotes.
Subject(s)
Bacillus megaterium/enzymology , Cytochrome P-450 Enzyme System/biosynthesis , Iron/pharmacology , Salicylates/pharmacology , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , ImmunoblottingABSTRACT
To study the role of the cis-acting element(s) in controlling the expression of the cytochrome P450(BM-1) gene and its upstream regulatory gene, bm1P1, in Bacillus megaterium, various deletion derivatives were constructed. A 53-bp inverted repeat located midway between the P450(BM-1) gene and bm1P1 gene was found in vivo to negatively regulate the expression of both genes, the regulation of which may occur at the transcriptional level. The promoter of the P450(BM-1), gene was also identified and found to be similar to those recognized by the sigmaA RNA polymerase of Bacillus subtilis. Possible mechanisms by which the 53-bp inverted repeat regulates the gene expression are discussed.
Subject(s)
Bacillus megaterium/genetics , Bacterial Proteins , Cytochrome P-450 Enzyme System/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Bacterial/genetics , Repetitive Sequences, Nucleic Acid/genetics , Transcription Factors/genetics , Bacillus subtilis/enzymology , Base Sequence , DNA-Directed RNA Polymerases , Genes, Bacterial/genetics , Genes, Regulator/genetics , Molecular Sequence Data , Promoter Regions, Genetic/genetics , RNA, Bacterial/genetics , RNA, Messenger/genetics , Recombinant Fusion Proteins , Sigma Factor , Transcription, Genetic/geneticsABSTRACT
Target intermittence in tracking has been studied as frequency of target presentation at various time intervals. Task efficiency increased as a function of increased frequency of target display in open-loop tracking tasks, where the steady state of presentation resulted in the best performance. The present study examined effects of feedback intermittency in compensatory tracking as a major source of disruption of the motor-sensory feedback process in the closed-loop tracking system. Feedback intermittency is defined as the feedback of momentary sampling of the difference between target movements and the operator's control motion for specified time lengths before being displayed to him in a continuous tracking task. With a random wave pattern of 9.76 cpm, 7 magnitudes of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.5 sec. were used to represent various levels of feedback intermittency. Task efficiency decreased as a function of increased magnitudes of intermittency. Results are discussed relative to the difference between target intermittence and feedback intermittency and their effects on different tracking tasks. The findings also establish the concept of feedback intermittency as a disturbing factor in compensatory tracking in degrading the operator's performance.
