Manabu Inuzuka

Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K

A non-self-replicating segment (1370 base pairs) of plasmid R6K was cloned in E. coli and shown to trans-complement temperature-sensitive replication mutants of this plasmid. This segment contains the gene which codes for a protein required for initiation of replication of the plasmid, and was used as a helper in a functional assay for an origin of replication in R6K derivatives. A 420 bp fragment, derived from R6K DNA, was shown to carry a functional origin since it was capable of replicating as a plasmid in E. coli cells carrying the helper segment either on the host chromosome or on a plasmid Col E1 derivative. The copy number of the origin fragment in cells carrying the helper segment on the chromosome is essentially the same as the copy number of R6K. A model for the positive regulation of plasmid R6K replication is presented.

Specific Action of 4-Nitropyridine 1-Oxide on Escherichia coli K-12 Pro+ Strains Leading to the Isolation of Proline-Requiring Mutants: Isolation and Characterization of Pro− Mutants

A specific action of 4-nitropyridine 1-oxide on Escherichia coli K-12 Pro+ strains leading to highly efficient, selective isolation of Pro− mutants is described. Incubation of Pro+ cells with a sublethal concentration of 4-nitropyridine 1-oxide in Penassay broth gave Pro− mutants, which lacked either the biosynthetic pathway of proline from glutamic acid to glutamyl γ-phosphate (proB−) or the pathway from glutamyl γ-phosphate to glutamic γ-semialdehyde (proA−) or both. Pro− mutants, which have the metabolic block between Δ1 pyrroline-5-carboxylate (the cyclized dehydration product of glutamic γ-semialdehyde) and proline (proC−) were not found among survivors. Treatment of Pro+ cells with N-methyl-N′-nitro-N-nitrosoguanidine led to isolation of all three types of Pro− mutants, suggesting that the action of 4-nitropyridine 1-oxide on Pro+ cells is apparently distinct from the action of N-methyl-N′-nitro-N-nitrosoguanidine. F-duction and interrupted mating experiments led to determination of the correlation between proline loci and the biosynthetic pathway of proline from glutamic acid.

Specific Action of 4-Nitropyridine 1-Oxide on Escherichia coli K-12 Pro+ Strains Leading to the Isolation of Proline-Requiring Mutants: Mechanism of Action of 4-Nitropyridine 1-Oxide

Possible mechanisms involved in the action of 4-nitropyridine 1-oxide (4NPO) on Escherichia coli K-12 pro+ cells in Penassay broth leading to the selective isolation of proA− and/or proB− mutants but not proC− mutant were studied. Reconstruction experiments between pro+ and pro− cells, together with experiments on the bactericidal action of 4NPO on pro+ and pro− cells, indicated that 4NPO is more toxic for pro+ and proC− cells than for proA− and proB− cells. These results, coupled with data indicating little mutagenicity of 4NPO on E. coli cells, led us to conclude that the selection of proA− and/or proB− cells that arose spontaneously in the pro+ culture is a possible mechanism for the action of 4NPO. Examination of 4NPO sensitivity of pro+ transductants derived from proA− and proB− cells with P1 vir phage and pro+ cells as donor and of pro+ spontaneous revertants derived from those pro− cells suggested that 4NPO-sensitive gene(s) should be on, or very close to, the proA and proB loci and that both products of proA and proB genes may be involved in the sensitivity of bacteria to 4NPO. The fact that the 4NPO-sensitive allele is dominant over the 4NPO-resistant allele further indicated the possible correlation between gene products of proA and proB and the 4NPO sensitivity of bacteria. Experiments on metabolic conversion of 4NPO with bacterial cells proved that the major metabolic pathway of the agent is reduction to (possibly via 4-nitroso-) 4-hydroxylamino- and 4-amino-pyridine 1-oxides, and then to 4-aminopyridine. Investigation of the effect of structural modification of 4NPO on the elective selection of Pro− mutants in Pro+ culture further suggested that the structural feature indispensable for the action of the agent is the hydroxyl-amino or its more oxidized state at the 4 position and the N-oxide moiety at the 1 position on the pyridine skeleton. Action of 4NPO in minimal medium was found to be bacteriostatic on pro+ cells but not on pro− cells, leading to the formation of long nonseptate multinucleate filament cells on pro+ cells. Possible biochemical mechanisms of the selective toxicity of 4NPO for pro+ and pro− cells are discussed.

STRUCTURE OF THE REPLICATION ORIGINS OF PLASMIDS RK2 AND R6K AND THE REGULATION OF PLASMID DNA REPLICATION

ABSTRACT Regions of the plasmid genome essential for plasmid replication and maintenance in Escherichia coli have been identified for two antibiotic resistance Plasmids, RK2 and R6K. In both cases a small segment of the plasmid, containing an origin of replication, has been maintained as an extrachromosomal element when another essential region(s) of the plasmid that specifies a trans-acting product is present in the host cell. Nucleotide sequence analysis of the origin regions of plasmids RK2 and R6K revealed seven 22 base pair (bp) direct repeats in tandem for the R6K origin region and eight 17 bp direct repeats in clusters of three and five for RK2. A model is presented involving the interacting of the π protein, the trans-acting product of the R6K genome that is required for the initiation of R6K replication, with the 22 bp direct repeats as a necessary step in the initiation of plasmid R6K replication.