ABSTRACT
Expression of the Bacillus subtilis gluconate (gnt) operon is negatively regulated by the gnt repressor which is antagonized by D-gluconate or D-glucono-delta-lactone. The repressor specifically binds to a gnt operator near the gnt promoter. From the results of gel retardation assaying of the gnt repressor and various restriction enzyme digests of a fragment carrying the gnt promoter and the gntR gene, the gnt operator was found to be located within a 141-base pair region containing the gnt promoter. Deoxyribonuclease I protection experiments revealed that the gnt repressor protected a gnt promoter region, between -10 and +15, which contains a region showing dyad symmetry with a sequence showing half-symmetry, ATACTTGTA. A 35-base pair synthetic duplex DNA containing this region showing dyad symmetry specifically bound to the gnt repressor. Through cleavage at two AccI sites in the protected region an 8-base pair deletion was introduced into the region showing dyad symmetry, which made expression of the gnt promoter constitutive. A DNA fragment carrying this deletion did not bind to the repressor. These results clearly indicated that the gnt operator sequence is a sequence containing the region showing dyad symmetry located at the transcription initiation site of the gnt operon. It was also suggested that the gnt operon contains only one operator. The similarity in the recognition between the repressor-operator interaction of the gnt operon and those of Escherichia coli TrpR and its operators was discussed.
Subject(s)
Bacillus subtilis/genetics , Gluconates/metabolism , Operator Regions, Genetic , Repressor Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Base Sequence , DNA-Binding Proteins/metabolism , Gluconates/genetics , Molecular Sequence Data , Restriction MappingABSTRACT
The nucleotide sequence (742 bp) of the promoter region of the Bacillus subtilis gluconate (gnt) operon is presented. Nuclease Sl mapping revealed the start point of the transcription and suggested that the expression of this operon is probably regulated at the transcriptional level. The sequences of the -35 and -10 regions suggested that RNA polymerase possessing sigma-43 may recognize this structure. The 223 bp fragment containing 100 bp upstream from the transcription start site actually exhibited a promoter activity when cloned in a promoter probe vector of pPL603B. This promoter activity was highly derepressed and although still under catabolite repression. The fragment on a high copy plasmid could titrate a regulator of the gnt operon so that the expression of the operon on the host chromosome also became derepressed.
Subject(s)
Bacillus subtilis/genetics , Genes, Bacterial , Gluconates/genetics , Promoter Regions, Genetic , Amino Acid Sequence , Base Sequence , Chromosome Mapping , DNA Restriction Enzymes , DNA, Bacterial/genetics , Genes, Regulator , RNA, Bacterial/genetics , Repetitive Sequences, Nucleic Acid , Repressor Proteins/genetics , Transcription, GeneticABSTRACT
The enzymes involved in gluconate utilization in Bacillus subtilis seemed to be gluconate permease and gluconate kinase. Several mutants unable to grow on gluconate were isolated. The mutations they harboured (gnt) were clustered between iol-6 and fdp-74 on the B. subtilis chromosome (a tentative map order of gnt-10, gnt-4, gnt-26, gnt-23 and gnt-9 was obtained). The gnt-10 mutation seemed to be located within the structural gene of the kinase, and the gnt-23 and gnt-26 mutations seemed to be within that of the permease. An EcoRI fragment (4.5 MDal) containing an intact gluconate (gnt) operon consisting of these two structural genes was cloned in phage phi 105 by prophage transformation and was mapped physically. The physical location of the mutations coincided with their order on the genetic map. The HindIII-A fragment (2.4 MDal), which corrects all the gnt mutations, was subcloned in plasmid pC194. The fragment contained the structural genes for the gluconate permease and kinase, but not the regulatory region of the gluconate operon.
Subject(s)
Bacillus subtilis/genetics , Cloning, Molecular , Gluconates/genetics , Operon , Phosphotransferases (Alcohol Group Acceptor) , Bacillus subtilis/enzymology , Bacteriophages/genetics , Chromosome Mapping , DNA Restriction Enzymes , Electrophoresis, Agar Gel , Enzyme Induction , Membrane Transport Proteins/biosynthesis , Mutation , Phosphotransferases/biosynthesis , Transformation, BacterialABSTRACT
Spontaneous and Tn9-induced mutants of Erwinia chrysanthemi were isolated which affect the degradative pathway of galacturonate and ketodeoxygluconate. The mutations were characterized both biochemically and functionally by complementation analysis and localized in the E. chrysanthemi chromosome. The kdgK gene mapped very close to ile, the kdgA gene was between trp and his, and the exuT-uxaC-uxaB-uxaA cluster was linked to thy. The different types of mutants obtained were consistent with an organization of the exu-uxa cluster into two transcription units, one containing the exuT gene, and the other containing the three uxa genes, with the transcription going from uxaC to uxaA.
Subject(s)
Erwinia/genetics , Hexuronic Acids/metabolism , Mutation , Uronic Acids/metabolism , Biological Transport , Chromosome Mapping , Erwinia/metabolism , Gluconates/genetics , PlasmidsABSTRACT
The kdgT gene of Escherichia coli, which encodes for the 3-deoxy-2-oxo-D-gluconate transport system, was isolated as a ColE1-kdgT hybrid plasmid from the Clarke and Carbon bank. A restriction and genetic map of the min 88 region of the chromosome was established; by subcloning the restriction fragments into the plasmid vector pBR322, the kdgT gene was localized on a 1.4-megadalton PstI DNA fragment, and the direction of transcription of the gene was determined by making use of an in vitro gene fusion between kdgT and lacZ genes. Amplification of the gene product of kdgT was up to 14-fold the level found in a haploid strain. When plasmids bearing kdgT were expressed in an in vivo maxicell system, a specific polypeptide of 28,000 daltons appeared that was found to be associated with the membrane fraction.
Subject(s)
Carrier Proteins/genetics , Escherichia coli/genetics , Gluconates/metabolism , Hybridization, Genetic , Plasmids , Crosses, Genetic , DNA Restriction Enzymes , Gene Amplification , Genetic Vectors , Genotype , Gluconates/genetics , Species Specificity , Transcription, GeneticABSTRACT
A sample of the Isle of Man population was tested for the following red cell antigens, serum proteins and red cell enzymes: ABH; MNSs He; Cc CwD Du Ee Ce; K k Kpa Kpb; Lua; P1; Fya Fyb; haptoglobin; transferrin; Ag; acid phosphatase; phosphoglucomutase; adenylate kinase; esterase D; adenosine deaminase and 6-phosphogluconate dehydrogenase. The study comprised 219 blood donors, 338 secondary school children and 116 females attending the only antenatal clinic. The results were studied for intra-island variation and for their their relationship with other Irish Sea Basin populations. The total sample results were compared with data for England, Cumbria, Eire, Northern Ireland, S.W. Scotland and Wales using a genetic distance measure.