ABSTRACT
We isolated a mutant of plasmid pUB110 that has the following properties in Bacillus subtilis: (i) it is toxic for recA and add cells, particularly at elevated temperature; (ii) it has a copy number threefold higher than that of the parental plasmid, and the extra copies are present as multimers; and (iii) it can efficiently complement replication of a cmp- satellite plasmid, despite being cmp+. All these properties are due to a single change in the plasmid replication protein, i.e., Gly at position 148 to Glu. These properties of the mutant Rep protein reflect a diminished ability to terminate rolling circle replication. We propose that the Rep protein may have a diminished affinity for the plasmid origin; alternatively, it may be impaired for recognition of the plasmid conformations which distinguish initiation and termination.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , DNA Helicases , DNA Replication , DNA-Binding Proteins , Mutation , Plasmids/genetics , Trans-Activators , Amino Acid Sequence , Bacillus subtilis/growth & development , Bacterial Proteins/metabolism , Base Sequence , DNA, Bacterial , Gene Deletion , Genetic Complementation Test , Molecular Sequence DataABSTRACT
Bacillus subtilis GTP-cyclohydrolase gene and its deletion derivatives were subcloned in Escherichia coli cells. The position of the gene within the riboflavine operon was defined. The deletion of the 14 kDa fragment from the N-end of GTP-cyclohydrolase gene did not affect the enzyme activity.
Subject(s)
Bacillus subtilis/enzymology , GTP Cyclohydrolase/genetics , Cloning, Molecular , DNA, Bacterial/genetics , Electrophoresis , Magnesium/metabolism , Manganese/metabolism , Operon , Plasmids , Protein Biosynthesis , Restriction Mapping , Riboflavin/geneticsABSTRACT
Bacillus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells. Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells. The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein. The gene coding for the bacterial multidrug transporter, like the P-glycoprotein gene in mammalian MDR cells, was amplified in the resistant bacteria. On the other hand, the bacterial multidrug transporter showed no sequence similarity to P-glycoprotein but exhibited an obvious homology to tetracycline efflux pumps and carbohydrate-ion symporters. These results show that the transport of structurally unrelated molecules can be mediated by members of different families of membrane transporters.
Subject(s)
Antineoplastic Agents/pharmacology , Bacillus subtilis/genetics , Carrier Proteins/genetics , Drug Resistance, Microbial/genetics , Drug Resistance/genetics , Membrane Glycoproteins/genetics , Rhodamines/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Amino Acid Sequence , Animals , Bacillus subtilis/drug effects , Bacillus subtilis/physiology , Base Sequence , Chromosomes, Bacterial , Cloning, Molecular , DNA Transposable Elements , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Escherichia coli/genetics , Ethidium/metabolism , Mammals , Molecular Sequence Data , Open Reading Frames , Protein Conformation , R Factors , Restriction Mapping , Sequence Homology, Nucleic Acid , Tetracycline Resistance/geneticsABSTRACT
The plasmid pNB48 carring a pair of genetically distinct inverted repeats is constructed on the basis of plasmid pSM19035 replicon. The single Campbell-type recombination between inverted repeats of two circular monomers leads to the formation of the so called "inverted" dimers in Bacillus subtilis cells. The gene conversion is observed in the course of this recombination. The mechanism of structural rearrangements of plasmids with inverted repeats that was postulated earlier is confirmed now. Some replicon features may influence the recombination of plasmids in Bacillus subtilis cells. The plasmid pNB48 may be a suitable model for studying the genetic recombination in Bacillus subtilis.
Subject(s)
Bacillus subtilis/genetics , Chromosome Inversion , Plasmids , Recombination, Genetic , Repetitive Sequences, Nucleic AcidABSTRACT
A plasmid containing inverted repeats is constructed in Bacillus subtilis. Insertion of DNA fragments into the plasmid inverted repeats results either in the precise excision of the insert or in its duplication in the opposite inverted repeat. These rearrangements are due to the presence of inverted repeats only. Two recombination events are possibly responsible for these phenomena. During the first step of the recombination two plasmid monomers form a dimer molecule. During the second step the intramolecular recombination between the direct repeats in the dimer structure leads to the formation of two rearranged plasmid monomers devoid of insertion or containing two DNA inserts. Proposed dimeric intermediate is unstable in B. subtilis. However, it is isolated from Escherichia coli recA, cells. Plasmids containing the inverted repeats can serve as a model to study plasmid recombination in B. subtilis cells.