ATP concentration in Escherichia coli during oxygen toxicity

Escherichia coli, strain E-26, grown in defined salts medium with glucose as the sole carbon and energy source, contained 1.50 ± 0.16 · 10⁶ molecules of ATP/cell. ATP was extracted with HClO₄ and assayed with a Dupont Luminescence Biometer using the luciferin-luciferase assay. Exposure during exponential growth at 37°C to 4.2 atm of oxygen resulted in complete growth cessation within 5 min, and to cyclic changes in cellular ATP concentration over a 2 h period. However, significant decrease in cellular ATP concentration occurred after growth inhibition in hyperbaric oxygen; hence, lack of ATP was not the cause of growth inhibition from oxygen toxicity.     

A permeabilized-cell assay for transaminase B activity in Escherichia coli K-12

The assay for transaminase B (EC 2.6.1.6) activity, developed by D. E. Duggan and J. A. Wechsler (1973, Anal. Biochem.51, 67–79) has been modified to allow for the measurement of activity in Escherichia coli cells made permeable by cetyltrimethylammonium bromide (CETAB). A concentration of 10 mg% CETAB was found to be most effective in treating the cells without having a significant effect on transaminase B activity. Extraction of the dinitrophenylhydrazone of 2-oxoisovalerate by toluene was not affected by the CETAB treatment. We further report that the Na₂CO₃ extraction step is not required to measure color formed by the dinitrophenylhydrazone of 2-oxoisovalerate. This CETAB-treated cell assay is accurate to study transaminase B activity through most of the logarithmic phase of growth of Escherichia coli.     

Isolation of a tetracycline-resistance plasmid excised from a chromosomal DNA sequence in Bacillus subtilis

When Bacillus subtilis GSY908 (recE4-) (H. C. Spatz and T. A. Trautner, 1971, Mol. Gen. Genet. 113, 174-190) protoplasts were infected with Staphylococcus aureus plasmid pNS1 specifying tetracycline resistance (Tcr) (N. Noguchi et al., 1983, Gene 21, 105-112), which was modified such that it either could not replicate or did not carry a functional Tcr gene, a plasmid with a molecular weight of 3.1 X 10(6) (4.9 kb) was generated in Tcr phenotypes. This plasmid, named Tcr pNS1981, exhibited completely different restriction endonuclease cleavage patterns to pNS1 and showed only negligible sequence homology in hybridization experiments. Southern hybridization experiments revealed that pNS1981 arises by excision of a B. subtilis chromosomal DNA sequence. No sequence corresponding to pNS1 was detectable on the chromosome of pNS1981-maintaining B. subtilis. The production of pNS1981 was also observed in B. subtilis RM125 (r-Mm-Mrec+) (T. Uozumi et al., 1977, Mol. Gen. Genet. 152, 65-69.) with almost the same frequency as B. subtilis GSY908. Since the recipient B. subtilis Marburg 168 derivatives stated above are sensitive to Tc, the results indicate that information essential for Tcr is under negative regulatory control in the integrated state on the chromosome. Restriction endonuclease analysis suggested that pNS1981 is essentially the same as pBC16, formerly found in B. cereus (K. Bernhard, H. Schrempf, and W. Goebel, 1978, J. Bacteriol. 133, 897-903).     

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants

Transposon-insertion mutants with vir^? Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir^? nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir^? octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir^? octopine Ti plasmid mutations and vir^? nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO^? mutants produced rooty tumors on Kalanchoë tubiflora.     

A mathematical model for lambda dv plasmid replication: analysis of copy number mutants

A mathematical model based on the molecular control mechanisms for lambda dv plasmid replication in a single Escherichia coli cell has been applied to simulate replication of mutant lambda dv plasmids. Model simulations of changes in repressor level and copy number resulting from mutations in the promoter-operator PROR region are consistent with experimental data. Calculated effects on lambda dv plasmid copy number of oligomer formation and of alternations in termination efficiency at tR1 also agree with experiment. The model has been employed to simulate the influence of cro mutants and of cro and tR1 double mutants on copy number and stable maintenance of lambda dv plasmid copy number. The genetic structure included in formulation of the replicon model provides a framework for relating changes in specific genetic loci on the plasmid with resulting alterations in host-plasmid system function.     

Saccharomyces cerevisiae ARS on a plasmid from Staphylococcus aureus

Staphylococcus aureus plasmid pC194 carries three sequences closely related to a consensus sequence defined previously by analysis of different genetic elements which replicate autonomously in yeast Saccharomyces cerevisiae. Two of these enable the plasmid to replicate in yeast, the third does not. A new consensus sequence A/T T T T A T R T T T, 1 bp shorter than the previous one, can be deduced from our results. Replacement of the T with G at the position 9 of the sequence abolishes its activity. The presence of the two active sequences on pC194 genome can be explained by the A + T-rich base composition of the plasmid.     

Inhibition of Escherichia coli DNA polymerase I by rose bengal

The fluorescein dye, rose bengal, inhibits Escherichia coli DNA polymerase I reversibly in the dark and irreversibly in the light. The reversible inhibition, which occurs in the micromolar concentration range, is competitive with respect to the poly(dA-T) template/ primer and noncompetitive with respect to the complementary deoxynucleoside triphosphates. The Hill coefficient for the inhibition by rose bengal is 3.0. Equilibrium dialysis experiments using ¹³¹I-labeled rose bengal have demonstrated direct binding of the inhibitor to the enzyme. No dye binds to poly(dA-T) at concentrations where the inhibition is observed. There are 22 ± 3 rose bengal binding sites per polymerase which can be subdivided into a class of high affinity sites and one of low affinity sites. The high affinity sites (3 μm) bind rose bengal with a Hill coefficient of 1.7 and are responsible for the observed inhibition. The low affinity sites (7μm) are more numerous (about 16) and bind rose bengal in a noncooperative manner. The displacement of rose bengal from the enzyme by poly(dA-T) at equilibrium confirms the competition between poly(dA-T) and rose bengal inferred from the kinetic data for the polymerization reaction. The inhibition of the 3′,5′ exonuclease activity and the template-directed dATP ? P-P exchange reaction by rose bengal is fully consistent with the interaction of rose bengal at the polynucleotide binding site. The enzyme induces an extrinsic Cotton effect in the visible absorption of rose bengal. The abolition of this Cotton effect by poly(dA-T) further supports the proposed site of binding of the dye.     

Identification of a protein from escherichia coli whose synthesis appears to be triggered by the initiation of DNA replication

Using a DNA temperature sensitive initiation mutant to synchronize the replication and cell division cycle, we have compared proteins which are synthesized during a period of DNA arrest with those synthesized after return to permissive temperature. This work has led to the identification of a DNA-binding protein of 60–65,000 molecular weight (SDS-gel electrophoresis) whose synthesis appears to be triggered by the initiation event.     

Characterization of the basic replicons of the chimeric R/Ent plasmid pCG86 and the related Ent plasmid P307

Restriction-enzyme fragments that can replicate autonomously after circularization were isolated from the chimeric R/Ent plasmid pCG86 and the Ent plasmid P307. Two such regions containing a basic replicon were located in each plasmid. One of the basic replicons of P307, RepFIB, is almost identical with one of the basic replicons of pCG86. The other basic replicon in P307, RepFIC, is partly homologous with the second basic replicon in pCG86, RepFIIA/RepFIC. The latter is a hybrid basic replicon and is in addition partly homologous with RepFIIA, a basic replicon present in IncFII R plasmids. By restriction-enzyme mapping and nucleotide-sequence analysis we have determined a site in the hybrid replicon where it ceases to be homologous with the RepFIIA basic replicon contained in the IncFII miniplasmid pSM1. The 2410-bp region of homology with pSM1 corresponds with a segment containing the origin of replication and all the genes responsible for replication control of pSM1.     

Construction and characterization of plasmid vectors for cloning in Staphylococcus aureus and Staphylococcus carnosus

Several plasmid vectors for cloning in Staphylococcus aureus and S. carnosus have been constructed and characterized. The chimeric plasmids are composed of parts of the following parental plasmids: The chloramphenicol-resistance plasmid, pC194, the tetracycline-resistance plasmid, pMK148, and the erythromycin-resistance plasmid, pE12. All the chimeric plasmids confer two selectable antibiotic-resistance markers on host cells. Insertional inactivation of the various antibiotic-resistance markers occurred at the BclI site of pE12, and the Sau96- or AvaII-site of pMK148; only a slight inactivation of the chloramphenicol-resistance marker occurred at the HaeIII-site of pC194. The chimeric plasmids pCT20 and pCE10 are both stable in S. aureus and S. carnosus. In addition, the hybrid plasmids of pCT20 and pCE10, containing lambda-DNA fragments in various restriction sites between 0.4 and 1.2 kb, are stably maintained. The inserted lambda-DNA fragments appear unchanged.     

Chemical composition and ultrastructure of the epicuticular wax in four mutants of Pisum sativum (L)

The action of mutations affecting the epicuticular wax of Pisum sativum has been investigated at the chemical and ultrastructural level. Upper and lower surfaces of the leaves were found to differ markedly in both ultrastructure and chemistry. Mutations affected primarily either the lower (wa, wb and wsp) or the upper surface (wlo), but some effects of all 4 genes could be seen on both surfaces. Specific biochemical lesions could be implied for wsp and wa but the chemical effects of wb and wlo were more diffuse. Generally a close relation between chemical composition and crystallite form of the wax was evident throughout the work.     

Restriction endonuclease mapping of a Rhizobium leguminosarum Sym plasmid

A circular map of the Sym plasmid pRle1001a corresponding to a genome size of 150 × 10⁶ D was established using the restriction endonucleases HpaI, SmaI, and KpnI. The map was derived from the results obtained by hybridizing individual HpaI fragments to blotted SmaI and KpnI digests of the plasmid. A complete map of the HpaI fragments was constructed in this way. Regions of homology with the structural nif-genes D and H of Klebsiella pneumoniae, with the Sym plasmid of Rhizobium trifolii 5, and with an octopine and nopaline Ti plasmid of Agrobacterium tumefaciens were mapped. A large area of plasmid pRle1001a, which carries nif structural genes, is highly conserved in the Sym plasmid of R. trifolii 5. It is assumed that this could be an area carrying genes involved in various functions that play a role in the process of symbiotic nitrogen fixation.