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The isolation, properties, and genetic analysis of a strain of Escherichia coli K-12 with an amber recA mutation are described. The experiments demonstrate that the recA product is a protein that is probably not essential for growth.
To isolate mutants of Escherichia coli K-12 lacking endonuclease I activity (end), a method has been developed which detects, by differential methyl green staining, undegraded deoxyribonucleic acid (DNA) in colonies previously incubated in toluene. This procedure allows isolation of mutant strains in which DNA degradation is reduced. For half of these strains, this defect has been correlated with deficiencies of endonuclease I, ribonuclease I (rns), or ribonuclease II (rne) activities. The enzymatic deficiencies of the other strains remain unknown. An rne mutation is cotransducible with serA (which is located at 56 min on the genetic map). Most end mutations, called endA, are also cotransducible with serA and are located between serA and strA. One end mutation, called endB, is located between purE and trp (i.e., between 13 and 25 min on the genetic map).
Both fi(+) and fi(-) type R factors function as intact replicative units when segregated into Escherichia coli minicells. Hybridization studies demonstrate that at least 95% of the deoxyribonucleic acid (DNA) in R(+) minicells is episomal in origin. About half of the DNA can be extracted in a closed circular form and about 75% of the DNA is membrane associated. DNA, ribonucleic acid, and protein synthesis proceeds in R(+) minicells in contrast to R(-) minicell controls. The system offers a unique opportunity to study a relatively small replicative unit in a native cell environment and a simple means of isolating large quantities of episomal DNA.
A mutant of the repressed R factor R1a and two mutants of the derepressed R factor R1drd-19 showing a two- to fourfold increase in resistance to all of the antibiotics to which the wild-type R factors mediate resistance were studied. The increased resistance was due to a two- to fourfold increase in the number of R-factor copies per chromosome. The production of drug-metabolizing enzymes was linearly correlated to the gene dosage. There was also a linear correlation between resistance to the drugs and the production of the corresponding enzymes. The mutations were also expressed in Proteus mirabilis PM1. In Proteus, R factors are split into two plasmids, resistance transfer factor and the resistance part. The mutation in one of the mutant R factors seems to be located in the resistance part. A second fi(+) R factor (R100) was introduced into strains already carrying R1drd-19 or the mutant R factor R1drd-19B2. In the first case, R100 and R1drd-19 segregated with equal probability when the bacteria were grown on antibiotic-free medium, whereas, in the second case, R100 was rapidly and preferentially excluded.
A mutant deficient for detergent-resistant (DR) phospholipase A was isolated from Escherichia coli K-12. Because the enzyme is membrane-bound and the substrate is a lipid, a special procedure was developed for isolating mutants deficient for the enzyme from agar plates. A sodium dodecyl sulfate (SDS)-sensitive mutant was used as a parental strain for the isolation of DR phospholipase A-deficient mutant. Soft agar containing an unsaturated fatty acid auxotroph and SDS was poured over colonies of the parental strain. The cells were easily solubilized with SDS, and phospholipids were efficiently digested by DR phospholipase A from the colonies on an agar plate. Fatty acids released supported the growth of the indicator bacteria. After the cells of the parent were mutagenized with nitrosoguanidine, colonies which could not support the growth of an unsaturated fatty acid auxotroph in the presence of SDS were selected. Four mutants were isolated after in vitro scre[UNK]ning of DR phospholipase A activity of 30 halo-less clones. Since an extract of the parent strain mixed with that of a mutant strain was still active, it was concluded that the inability to hydrolyze phospholipids was not due to the accumulation of inhibitory substance; the activity of DR phospholipase A in the mutant was less than 1% of the parental activity. Physiological studies indicated that DR phospholipase A is not essential for the growth of E. coli.
R factor 1818 is shown to be eliminated from a thymineless strain of Escherichia coli J6-2 (R-1818) during thymine starvation. Readdition of thymine to the starved cultures produces a partial recovery in viable count but does not affect the proportion of R(-) cells. The R factor is not cured from exponential- or stationary-phase cultures which are starved of required amino acids as well as thymine, nor from cells which are deprived of thymine in the presence of chloramphenicol. However, in both of these cases, the extent of thymineless death is reduced. It is suggested that protein synthesis is a requirement for R-1818 elimination, and the possible nature of this protein is discussed.
A series of sheared donor-recipient complex samples were prepared from a competent Bacillus subtilis culture which had been transformed by using (3)H, (2)H-labeled deoxyribonucleic acid. Measurement of the molecular weights and buoyant densities of these samples led to the following conclusions. (i) The average size of the single-stranded donor segment which is integrated is 2.8 x 10(6). (ii) These donor segments are not randomly distributed in the recipient genome, but are clustered in groups, each group containing several discrete donor segments.
Cryptopleurine-resistant mutants of Saccharomyces cerevisiae were isolated. A single, recessive nuclear gene, very closely linked to the mating locus (2.1 centimorgans), is responsible for resistance. Ribosomes from the mutants were found to be resistant to cryptopleurine when analyzed by poly(U)-directed polyphenylalanine synthesis. Analysis of the distribution of ribosomes between monosomes and polysomes in sensitive cells exposed to cryptopleurine suggests that some step is inhibited during the elongation phase of protein synthesis.
The presence of a 138-kilobase plasmid (pD188) correlated with increased resistance to cadmium in Rhodococcus fascians D188. This plasmid could be transferred by a conjugation-like system in matings between R. fascians strains. Transconjugants expressed the cadmium resistance and could be used as donors in subsequent matings. Four other R. fascians strains (NCPPB 1488, NCPPB 1675, NCPPB 2551, and ATCC 12974) could also be used as donors for cadmium resistance in matings. Strain NCPPB 1675 showed a 100% cotransfer of cadmium and chloramphenicol resistance markers.
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Diluent composition, time consumed in experimental manipulations, and the presence of pyocine-like lethal agents affected the viability of sexual fertility factor-positive and fertility factor-negative auxotrophic stocks of Pseudomonas aeruginosa. In the same cultures, recovery of prototrophic revertants increased as the number of viable cells dispensed per plate was reduced. As a result, the number of revertants recovered was indirectly determined by the combined activities of the three conditions affecting viability. Possible modifications by these conditions affecting viability on the expression and interpretation of the fertility factor-positive sex factor-mediated system of genetic recombination are presented.
Bacteria carrying temperature-sensitive mutant R factors for chloramphenicol resistance were isolated. In the presence of chloramphenicol, these bacteria grew at 34 C but not at 43 C. The mutations in the chloramphenicol resistance gene of the R factors affected neither the resistance of the bacteria to dihydrostreptomycin and tetracycline nor the stability of the R factors at 43 C. The chloramphenicol acetyltransferase obtained from Escherichia coli K-12 carrying the mutant R factors was heat-labile as compared with that from a strain carrying the wild-type R factor. We could not find chloramphenicol acetyltransferase activity in 17 chloramphenicol-sensitive and 5 -resistant strains (selected in vitro) of E. coli examined. The results strongly suggest that the chloramphenicol resistance gene of the R factors is the structural gene of the chloramphenicol acetyltransferase rather than the genome controlling the expression of a chromosomal determinant for the enzyme. Furthermore, the studies confirm that the existence of the chloramphenicol acetyltransferase is the primary cause of chloramphenicol resistance of bacteria carrying the R factor. Both the enzyme activity producing the monoacetyl derivative from chloramphenicol and the subsequent formation of the diacetate from the monoacetyl product were heat-labile to the same degree. The results suggest that only one enzyme participates in two steps of chloramphenicol acetylation.
A variant of Neisseria meningitidis was found to carry a mutator factor which endowed the bacteria with generalized genetic instability. The reversion frequencies of several biochemical mutants were increased up to 1,000-fold when the factor was introduced. The factor is not unidirectional in preference, since the mutator induced mutants generally reverted with increased frequency in its presence. There could be found no indication of insufficient synthesis of nucleic acid precursors. Attempts to demonstrate an unusual, mutagenic base incorporated in deoxyribonucleic acid (DNA) were negative. Strains carrying the mutator factor had significantly increased sensitivity to ultraviolet light. A mutation to a more ultraviolet-resistant type coincided with a disappearance of the mutator property. The presence of the mutator factor in a competent strain resulted in a reduction of the transformation frequency to between 0.5 and 5% of that in the parental strain. A mutation to the more ultraviolet-resistant type resulted in simultaneous loss of the mutator property and reestablishment of a normal transformation efficiency. It has been suggested that this mutator factor may represent a defect in the DNA repair mechanism, which is also of importance for genetic recombination. The mutator factor showed cotransformation with the locus for streptomycin resistance, but a true linkage could not be proved.
The isolation and characterization of high-frequency recombining strains from different Escherichia coli host cells containing either the F factor or the Col V factor are described. The strains (with one exception) formed from three of the V(+) parents showed the same origin and polarity of transfer (xyl-arg-pro-trp-his-mal). The Hfr strains formed from the one remaining V(+) and the F(+) host cells showed a greater variety in their points of origin. In addition, several Hfr strains isolated from V(+) parents lost the ability to produce colicin V. F(v) (+) segregants of these were isolated, and the F(v) factors appeared to retain their preferential site for Hfr formation, but they lacked other propertes controlled by the Col V factor. Chromosomal integration of episomes and its relation to the fertility of F(+) and V(+) strains are discussed. Production of colicin V appeared to be uninfluenced by the state of the Col V factor within the cell.
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Timex, a strain of Neurospora crassa which exhibits a circadian rhythm of conidia formation in growth-tube cultures, has been found to differ from wild-type strains by two genes. One gene, inv, is responsible for an invertase deficiency, whereas the second gene, bd, is of unknown function. Both genes map independently from other genes known to induce Neurospora rhythmicity. The inv gene is not essential for the timex phenotype because bd strains express that phenotype on certain media. Although inv strains do exhibit some rhythmicity of their own, the rhythmicity apparently is not a direct result of the invertase deficiency, since there is no correlation between invertase level and rhymicity in 29 strains tested. Of the 29 strains tested, 20 exhibited some rhythmicity in growth-tube cultures, suggesting that morphological manifestations of rhythmicity in Neurospora may result from the function or the loss of function of numerous genes, or both. There was no correlation in these strains between rhythmicity and (i) genetic background; (ii) geographical origin; or (iii) nutritional requirements.
Strains of Escherichia coli that carry the mutation uvrA6 show no measurable excision of pyrimidine dimers and are easily killed by ultraviolet (UV) light, whereas strains that carry recA13 are defective in genetic recombination and are also UV-sensitive. An Hfr strain carrying uvrA6 was crossed with an F(-) strain carrying recA13. Among the recombinants identified, one carrying uvrA recA proved to be of exceptional sensitivity to UV light. It is estimated from the UV dose (0.2 erg/mm(2) at 253.7 nm) required to reduce the number of colony-forming cells by one natural logarithm that about 1.3 pyrimidine dimers were formed in a genome of 5 x 10(6) base pairs for each lethal event. This double mutant is 40 times more UV-sensitive than the excision-defective strain carrying uvrA6. The replication of one pyrimidine dimer is generally a lethal event in strains carrying recA13. Spontaneous breakdown and UV-induced breakdown of the deoxyribonucleic acid (DNA) of cells of the various genotypes were estimated by growing the cells in medium containing (3)H-thymidine and measuring both acid-precipitable and acid-soluble radioactivity. The UV-induced degradation in strains with recA13 did not require the uvr(+) genes and hence appears to depend upon a mechanism other than dimer excision. The greater level of survival after irradiation in Rec(+) as compared to Rec(-) bacteria may be due to a recovery mechanism involving the reconstruction of the bacterial chromosome through genetic exchanges which occur between the newly replicated sister duplexes and which effectively circumvent the damaged bases remaining in the DNA.