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Volume 80,
Issue 1,
1974
Volume 80, Issue 1, 1974
- Genetics And Molecular Biology
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Properties of Proteus and Providence Strains Harbouring Recombinant Plasmids between P-lac Rl drd 19 or R447b
More LessSUMMARYRecombinant plasmids between R1drd19 and P-lac and between R447b and P-lac were produced by phage PL25 transductions of the respective R factors to Providence p29 strains harbouring P-lac. The
(the superior line indicating the transduced R factor) recombinant plasmid transfers lac + and all determinants of the R factor to Proteus mirabilis strains and Escherichia coli at high frequencies which approach inter-Proteus transfer of the parental plasmids. The
recombinant transfers lac + and only marker K of the parent R factor, at frequencies of P-lac inter-Proteus transfer. Both recombinants possess the compatibility property and possibly most other features of P-lac. Transductions of the
recombinant with phages PL25 and 34 differ. Those with the former vector to p29 yield transductants which have markers ACKSu but not lac + or S and do not transmit markers conjugally. Conjugal transferability is restored with transductions of the recombinant to p29 carrying a resident P-lac; these transductants then transmit lac +ACKSu as a unit. Transductions of the recombinant
with phage 34 to strains of P. mirabilis produce transductants which possess all the resistance determinants and which transfer these conjugally, but in which lac + does not appear. A spontaneous lac segregant of the
recombinant retained the high transfer rate and other properties of the parent. Transduction of this segregant to strains of p29 and P. mirabilis resulted in transductants which carried markers ACKSu and ACKSSu respectively but could not transfer them by conjugation. Transductions of the segregant to strains harbouring P-lac again produced recombinant plasmids at low rates which transmitted the above groups of resistance determinants and lac + at frequencies similar to those of the original recombinant. Transductions of the
recombinant with either phage yielded transductants carrying K (and able to transmit it conjugally) but never any carrying lac +.
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Induction of R Bodies by Ultraviolet Light in Killer Paramecia
More LessSUMMARYKappa, a bacterial endosymbiont present in killer stocks of Paramecium aurelia, exists in two forms - brights, which contain a refractile (R) body, and non-brights which lack the R body. Non-brights are self-reproducing and give rise to brights, which cannot reproduce and are toxic to sensitive paramecia which ingest them. Associated with the R body are structures resembling bacteriophages in form and chemical composition. Animals of stock 51, syngen 4 of P. aurelia were given low doses of ultraviolet light. The percentage of brights increased markedly, beginning 6 h after irradiation. In three experiments, absolute numbers of kappas were determined 13 to 14 h after exposure to u.v.: the number of brights increased and non-brights decreased significantly, while the total number of kappas remained the same. At 24 h, after irradiation the numbers of kappa particles had decreased considerably; after 3 days they were completely gone. The results are consistent with the hypothesis that the phage-like structures exist as u.v.-inducible prophages in non-brights, and that when a non-bright becomes a bright, the prophage is spontaneously induced forming, at the same time, mature phage-like elements and the R body. These events constitute the lytic cycle which ends with the destruction of the bright.
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Chromosomal Integration of Klebsiella Nitrogen Fixation Genes in Escherichia coli
More LessSUMMARYEscherichia coli, c-m7, a His+Nif+ hybrid obtained by intergeneric mating with a Klebsiella pneumoniae donor strain, also inherited the unselected markers gnd and rfb. The R factor, R144drd3, which had been used to confer fertility on the donor, was present, detectable as covalently closed circular DNA of molecular weight 69 × 106 daltons. No other species of supercoiled DNA were isolated and the elimination of R144drd3 did not result in the loss of Klebsiella genes. Segregation analysis of donor markers indicated that the Klebsiella DNA was integrated at the his region of the E. coli chromosome in the probable order his-gnd-nif-rfb. Strain c-m7 produced a nitrogenase physiologically identical to that of K. pneumoniae, but synthesized a heteromeric species of gluconate-6-phosphate dehydrogenase.
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Plasmids Formed in Nitrogen-fixing Escherichia coli-Klebsiella pneumoniae Hybrids
More LessSUMMARYSegments of the Klebsiella pneumoniae chromosome, which included the nitrogen fixation (nif) genes, were maintained as covalently closed circular molecules of DNA in Escherichia coli hybrids, C-m9 and c-l4. The R factor, R 144drd3, which had been used to confer fertility on the donor, was also detected as covalently closed circular DNA of molecular weight (65 ± 4) × 106 daltons according to sedimentation studies and electron microscopy.
The nif genes in hybrid C-m9 were linked to his on a plasmid which had a molecular weight of 9·5 × 106 daltons. A third plasmid in this hybrid had a molecular weight of ii8 × 106 daltons. The Nif plasmid in C-m9 was transferred efficiently to other Escherichia coli strains only when R 1drd19 was present in the donor in addition to R 144drd3.
Presence of three electrophoretically distinguishable gluconate-6-phosphate dehydrogenases was evidence for duplication of gnd determinants in C-m9. Resistance of both hybrids to the rough-specific phage øX 174 suggested that they inherited Klebsiella his-linked rfb genes.
Four plasmids were isolated from hybrid c-14, one of which was R 144drd3. Two plasmids, one carrying his and nif and another met G, were mobilized by R 144drd3. The molecular weights of the c-l4 plasmids, excluding R 144drd3, were (105 ± 1) × 106, 46 × 106 and 12 × 106 daltons.
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Rifampicin-resistant Mutants of Streptomyces coelicolor A3(2)
More LessSUMMARY: Studies with whole cells and crude cell-free extracts of Streptomyces coelicolor a3(2) indicate that its RNA polymerase, though sensitive to rifampicin, is less so than most bacterial RNA polymerases. Of two other streptomycetes examined, one resembled S. coelicolor a3(2) and the other was more like other prokaryotes in respect of rifampicin sensitivity of its RNA polymerase. Three genetic map locations for rifampicin-resistance (rif) mutations of S. coelicolor a3(2) were determined, one (rifA) at 6 o’ clock on the conventional linkage map, very close to strA, one (rifB) at 11 o’ clock, between leuA and thiC, and a third (rifC) at 7 o’ clock, between strA and pheA. In representative rifB and C mutants, RNA synthesis was resistant to rifampicin in whole cells, but not in extracts, whereas in several rif A mutants RNA synthesis was equally resistant both in whole cells and in extracts, suggesting that the rifA gene may specify part of the RNA polymerase. None of more than 100 rifA mutants isolated possessed morphological abnormalities attributable to the rifA mutation.
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- Physiology And Growth
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The Inhibition of Growth of Escherichia coli Spheroplasts by Antibacterial Agents
More LessSUMMARY: Escherichia coli nctc86 organisms with impaired walls were prepared by three methods and the effect of antibacterial agents on their growth studied. The growth of penicillin spheroplasts was masked by the overgrowth of unaltered cells in the culture; the EDTA-lysozyme spheroplasts themselves were non-viable. The growth of penicillin spheroplasts was not affected by cell-wall inhibitors and ampicillin suppressed the overgrowth of unaltered cells. The sensitivity of penicillin spheroplasts and parent cells to inhibition by a range of agents was similar. EDTA treatment enhanced the susceptibility of E. coli nctc86 and other strains of Gramnegative species to several antibiotics, particularly erythromycin. Polyacetic acid chelating agents related to EDTA and some new amides derived from glycine, alanine, phenylalanine or methionine also potentiated erythromycin in vitro. Erythromycin showed some activity in protecting mice against infection by EDTA- treated E. coli nctc86. The antibiotic did not protect against infection by the untreated bacteria and its activity was not greatly enhanced by simultaneous administration of EDTA or the amide derivatives.
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Effect of Glucose on Thermal Injury of Yeast that may Define the Maximum Temperature of Growth
More LessSUMMARY: Anaerobically grown yeast, suspended in glucose solution, leaked cell contents non-selectively. The rate of leakage increased with temperature but reached a maximum rate close to the maximum temperature of growth (T max) of the yeast. The total leakage induced by glucose was much greater above T max than below it, because above T max the yeast lost the ability to take up released material. The rate of uptake of 14C-labelled amino acids was also substantially slowed above T max-Yeast heat-stressed in the presence of glucose lost the ability to establish and maintain a concentration gradient of sorbose, and simultaneously ATPase activity could be measured, all characteristics of yeast with a ruptured cytoplasmic membrane. These effects were uniquely caused by utilizable sugars, were essentially independent of sugar concentration and could be partially inhibited by Ca2+ or inhibitors that prevent sugar utilization. Yeast heated above T max in water suspension was essentially undamaged as determined by the tests used, but if glucose was subsequently added below T max an effect of heat damage could be demonstrated. We conclude that one factor that determines the T max of a yeast is the temperature sensitivity of the cytoplasmic membrane in the presence of the utilizable sugar of the growth medium.
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Formation of Ethylene by a Soil Fungus
More LessSUMMARY: Methionine is a substrate for ethylene formation in Mucor hiemalis, but glucose is also required for maximal ethylene production. The formation of ethylene from these substrates in a defined mineral salts medium was studied both with sealed shaken flasks and with a chemostat. Oxygen promoted growth and ethylene production per unit weight of organism. That anaerobic conditions appear to be necessary to observe ethylene accumulation in the soil is probably because soil anaerobiosis mobilizes the substrates required for ethylene biosynthesis.
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- Short Communication
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l-Lysine Decarboxylase as a Specific Inhibitor of the Growth of a Species of Pichia*
More LessIn conjunction with our interest in single-cell protein (Coty & Leavitt, 1971), a search was made for a screen which would supply a selection pressure for the isolation of a strain of yeast capable of excreting lysine during growth on hydrocarbons. Initially, we hoped to isolate over-producers by selecting for resistance to analogue of l-lysine, but failed because none of the analogues examined, either alone or in conjunction with compounds which were used to affect yeast-wall permeability, had any effect on the growth of our yeast. The use of L-lysine decarboxylase was then examined and found to be a successful means for inhibiting growth. The results of this study and its possible implications are discussed below.
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A Search for the Bacterial Mucopeptide Component, Muramic Acid, in Chlamydia
More LessSUMMARY: Chlamydiae are obligate intracellular parasites. They multiply by binary fission in a complex developmental cycle that involves a series of morphological forms. The mature infectious form, the elementary body, has a rigid wall similar in ultrastructure and amino acid content to the walls of Gram-negative bacteria (Manire & Tamura, 1967; Murray, 1968; Tamura, Matsumoto, Manire & Higashi, 1971). The intermediate form in multiplication, the reticulate body, lacks a rigid wall and is non-infectious.
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