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Biomedical subjects

M Inuzuka

Publications and source records attributed to M Inuzuka.

At least 55 records · Page 3Linked to original sources
PubMed1985

A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K.

A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated. The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication. They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K. Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region. Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division.

Amino Acid Sequence↗
PubMed1980

Activity in vitro of three replication origins of the antibiotic resistance plasmid RSF1040.

Replicating molecules of plasmid RSF1040, a deletion mutant of R6K, were synthesized in vitro and analyzed by electron microscopy. Initiation of replication occurs at three unique sites, ori alpha, ori beta, and ori gamma, within a 3900-base pair segment of the R6K genome. These sites are indistinguishable from the origins that are active in vivo. Frequencies of initiation at these three origins, however, are different from those observed in vivo. Replication proceeds unidirectionally in either direction from ori beta and ori gamma and in one direction from ori alpha. The replication terminus of the R6K genome is inactive in the in vitro system.

Anti-Bacterial Agents↗
PubMed1978

Replication of antibiotic resistance plasmid R6K DNA in vitro.

A soluble extract prepared from cells of an Escherichia coli strain carrying the antibiotic resistance plasmid R6K is capable of carrying out the complete process of R6K DNA replication. DNA synthesis in vitro is dependent on the four deoxyribo- and ribonucleotide triphosphates and is sensitive to rifampin and streptolydigin, inhibitors of DNA-dependent RNA polymerase. The incorporation of deoxyribonucleotides into R6K DNA also is sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP, and N-ethylmaleimide. Kinetics of synthesis are linear for 60 to 120 min. Replication proceeds semiconservatively and supercoiled closed-circular DNA molecules are synthesized. Analysis by alkaline sucrose gradient centrifugation indicated that the early R6K DNA products contain DNA fragments of approximately 18 S in size, corresponding to the length between the R6K alpha origin of replication and the terminus of replication observed in vivo. Addition of exogenous supercoiled R6K DNA is inhibitory to the in vitro system, whereas the addition of R6K DNA in the form of relaxation complex stimulates R6K DNA synthesis to a small extent.

Anti-Bacterial Agents↗
PubMed1978

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.

Chromosome Mapping↗
PubMed1978

Requirement of a plasmid-encoded protein for replication in vitro of plasmid R6K.

Conditions are described for the replication of exogeneous R6K DNA in an in vitro system prepared from Escherichia coli cells. Replication of plasmid DNA in this system is semiconservative and sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP,N-ethylmaleimide, and inhibitors of DNA-dependent RNA polymerase. An ammonium sulfate fraction prepared from cells carrying the R6K plasmid is required for replication. A direct role in replication for a plasmid-encoded protein, designated pi, in this fraction is indicated by the inactivity of this fraction when prepared from cells carrying a temperature-sensitive mutant plasmid and the thermolability of this fraction when prepared from cells carrying a partial revertant of the mutant plasmid. This plasmid-encoded protein is necessary for the initiation of R6K DNA replication and functions before or during the formation of nascent RNA in the initiation process. The results of titration assays of this protein using various template DNAs suggest that the protein interacts with the plasmid DNA at the region essential for DNA replication.

Anti-Bacterial Agents↗
PubMed1976

Eliminatory action of glycine on drug resistance of Escherichia coli K12 harboring an R factor.

Glycine, known to inhibit the synthesis of a peptidoglycan component of the bacterial cell wall, was effective in eliminating drug resistance of Escherichia coli K12 JE2100 strain harboring theR100-1 factor although in lower frequencies than that of sodium dodecyl sulfate (SDS). The action of glycine was found to be less effective on the same R factor in JE177 strain, and not effective on the F factor in W6. Infection of R factors from R+ cells to R- cells was found to take place in the glycine broth as efficiently as in broth without glycine. This might result in lowering the apparent efficiency of the action of glycine on those plasmids. The segregation patterns of drug-susceptible clones obtained by the glycine treatment were different from those obtained after the SDS treatment. These results coupled with other evidences suggest that the mode of action of glycine on R+ cells maybe different from those of other curing agents and may involve mechanisms other than selection of R- or drug susceptible segregantsthat are present in R+ culture.

Culture Media↗
PubMed1976

Specific action of 4-nitropyridine 1-oxide on Excherichia 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 gamma-phosphate (proB(-)) or the pathway from glutamyl gamma-phosphate to glutamic gamma-semialdehyde (proA(-)) or both. Pro(-) mutants, which have the metabolic block between Delta(1) pyrroline-5-carboxylate (the cyclized dehydration product of glutamic gamma-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.

Culture Media↗
PubMed1976

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.

Escherichia coli↗
PubMed1974

Curing action of sodium dodecyl sulfate on a Proteus mirabilis R+ strain.

Growth of Proteus mirabilis harboring R100-1 (fi(+)drd str(r)cml(r)tet(r)sul(r)) factors in Penassay broth containing sodium dodecyl sulfate (SDS) leads to the loss of all or part of the genetic elements in high frequencies. In media containing SDS at concentrations as low as 0.03%, both lysis of R(+) cells and elimination of the R factors occur at high frequencies. Appearance of drug-susceptible cells in R(+) cultures occurs during the exponential phase of growth; however, the frequencies of susceptible cells increase substantially after the culture reaches the stationary phase. Reconstruction experiments, coupled with other observations, suggest that the major factor in altering the frequency of drug-susceptible variants is the greater resistance of the variants to the lytic action of SDS. This resistance correlates in most cases with the loss of the transfer functions in the resistance transfer factor.

Acridines↗
PubMed1972

Specific role of sex pili in the effective eliminatory action of sodium dodecyl sulfate on sex and drug resistance factors in Escherichia coli.

Evidence is presented for the specific role of sex pili in the eliminatory action of sodium dodecyl sulfate (SDS) on sex (F) and drug resistance (R) factors in Escherichia coli K-12 strains leading to their loss. SDS at 0.03% concentration lysed JE3100 F(8) (+) (F-gal)/gal(-)fla(-)pil(-) in Penassay broth after they had grown exponentially and reached maximum growth to the extent that the agent at concentrations higher than 1% did. However, the agent was only effective in eliminating sex factors from JE3100 in high frequencies at concentrations higher than 1%. Increase of osmotic pressure of the culture with SDS at concentrations as low as 0.03 to 0.1% by addition of sucrose led to the substantial increase of elimination efficiency. Reconstruction experiments between F(8) (+) and F(-) cells in the SDS culture revealed the selective growth of F(-) cells as well as a delay of maximum growth of F(-) variants derived from F(8) (+) cells, compared with those of F(8) (+) cells, as well as F(-) cells originally added to the culture. The agent was not very effective in eliminating sex factors from JE3427 F(8)m(+)5/fla(-)pil(-) cells which lack the function of production of F pili. F(8)m(+)5 cells showed a sensitivity toward SDS intermediate between those of F(8) (+) and F(-) cells. SDS was further effective in eliminating R factors from KE132 R(100-1) (+)/fla(-)pil(-) cells in high efficiency; however, the action was not efficient with KE133 F(100) (+) cells possibly with fewer sex pili than R(100-1) (+). Action of acridine orange on these F(+) or R(+) strains was found to be different in some aspects from that of SDS.

Acridines↗
PubMed1972

Studies on sex pili: mutants of the sex factor F in Escherichia coli defective in bacteriophage-adsorbing function of F pili.

Ultraviolet irradiation or nitrosoguanidine treatment of Escherichia coli K-12 strain JE3100 (F'(8)/fla pil) led to the isolation of six mutants defective in F pili function. The defects were shown to be caused by mutations in the F factor. The mutants retained conjugal fertility, although they were less efficient than parental F'(8) strain, and continued to synthesize F pili. Three of the mutants (strains KE196, 198, and 200) had lost sensitivity to male-specific MS2 phage, and the other three (strains KE161, 163, and 164) were insensitive to Qbeta and f1 as well as MS2 phages. F pili on strains KE196, 198, and 200 cells continued to adsorb MS2 phage, whereas those of strains KE161, 163, and 164 did not adsorb MS2 phage. The correlation of the mutant phenotypes with those of other F mutants reported in the literature is discussed.

Acridines↗
PubMed1969

Specific action of sodium dodecyl sulfate on the sex factor of Escherichia coli K-12 Hfr strains.

A specific action of sodium dodecyl sulfate (SDS) on the sex (F) factor in the integrated state of Escherichia coli K-12 Hfr H strain is reported. Growth of Hfr cells in Penassay Broth containing SDS results in the elimination of part or all of the F factor, yielding low and nonfertile variants of defective Hfr type and F(+) cells and also F(-) derivatives. Appearance of such variants was generally observed after the culture reached stationary phase. The frequencies of F(-) cells then increased. F(-) cells were usually isolated as the major population among survivors. Some defective variants of Hfr cells with an intermediate fertility between standard Hfr and F(+) cells had lost sensitivity toward the male-specific ribonucleic acid phage M12. Other defective Hfr variants with as much or less fertility than standard F(+) cells had also all lost sensitivity to phage M12. On single-colony isolation, they segregated nonfertile female H cells which, when infected with F, could restore high fertility with oriented transfer of the chromosome the same as that of the original Hfr H. Also, sensitivity to phage M12 was regained. Female H cells were characterized as those lacking fertility but still retaining a small segment of F or sfa locus at the original part of the chromosome, where newly infected F could attach. Similar results were obtained with two other Hfr strains. A possible mechanism of the specific action of SDS is discussed.

Coliphages↗
PubMed1968

Effective elimination of drug resistance and sex factors in Escherichia coli by sodium dodecyl sulfate.

A method for effective elimination of drug resistance (R) and sex (F) factors in Escherichia coli K-12 strains by treatment with sodium dodecyl sulfate (SDS) is presented. Growth of E. coli harboring R or F factors in Penassay Broth containing SDS led to the loss of all or part of these genetic elements. Appearance of drug-susceptible or F(-) cells among survivors was observed after the culture reached the stationary phase. Drug-susceptible cells which had lost all of their resistance markers by SDS treatment could be efficiently infected with R or F factors. Among isolated segregants which came from resistant cells, tetracycline-susceptible cells were the major segregant class. Drug-susceptible cells gave no revertants to drug resistance. By treatment of F(+) cells with SDS, unusual F(+) cells which retained mating ability but showed resistance to M12 phage were also isolated, together with mutants of another type which lost mating ability but retained sensitivity to M12 phage. Since SDS is more toxic to R(+) cells than R(-) cells, the isolation of drug-susceptible or F(-) cells under these conditions may be partly attributable to selective growth of drug-susceptible or F(-) cells in SDS-Penassay Broth.

Coliphages↗