Biochemical and genetic studies of recombination proficiency in Escherichia coli K12. IV. Analysis of recombinants formed by a recombination deficient (recB21 recC22) strain

Summary Residual genetic recombination is carried out by recB ^- recC ^- mutants of E. coli. Recombinants (for one gene) formed by a recB ^- recC ^- parent were shown to be as recombination deficient as their parent, when recombination of a second gene is measured. Therefore the resididual recombination cannot be attributed to a genetically recombination proficient fraction of the parent recB ^- recC ^- culture. I conclude that each recB ^- recC ^- parent cell is capable of carrying out genetic recombination. This conclusion is consistent with the existence of an alternate (and minor) recombination mechanism in E. coli K12, independent of the recB ⁺ recC ⁺ mediated steps.The previous paper in this series was Capaldo-Kimball and Barbour, Involvement of Recombination Genes in Growth and Viability, J. Bact. April (1971).     

Restoration by the rac locus of recombinant forming ability in recB? and recC? merozygotes of Escherichia coli K-12

Summary The recombinant forming ability of recB ^– or recC ^– strains of E. coli K12 is almost totally recovered in merozygotes which are heterozygous for a genetic locus denoted rac which is located five minutes clockwise from trp on the genetic map. This transient recovery phenomenon only occurs when the donor strain is rac ⁺ (wild type) and the recipient strain is rac ^-. The recombinants derived from such crosses all have the normal phenotype characteristic of recB ^– (or recC ^–) strains, and they are almost always rac ^-. The results imply that the rac ⁺ locus (or loci) is zygotically expressed and excised from the chromosome in a manner which is analogous to the zygotic induction of a prophage.     

Plasmid control of recombination in E. coli K 12

Summary The recombination proficiency of three recipient strains of Escherichia coli K 12 carrying different plasmids was investigated by conjugal mating with Hfr Cavalli. Some plasmids (e.g. R1drd 19, R6K) caused a marked reduction in the yield of recombinants formed in crosses with Hfr but did not reduce the ability of host strains to accept plasmid F104. The effect of plasmids on recombination was host-dependent. In Hfr crosses with AB1157 (R1-19) used as a recipient the linkage between selected and unselected proximal markers of the donor was sharply decreased. Plasmid R1-19 also decreased the yield of recombinants formed by recF, recL, and recB recC sbcA mutants, showed no effect on the recombination proficiency of recB recC sbcB mutant, and increased the recombination proficiency of recB, recB recC sbcB recF, and recB recC sbcB recL mutants. An ATP-dependent exonuclease activity was found in all tested recB recC mutants carrying plasmid R1-19, while this plasmid did not affect the activity of exonuclease I in strain AB1157 and its rec ^– derivatives. The same plasmid was also found to protect different rec ^– derivatives of the strain AB1157 against the lethal action of UV light. We suppose that a new ATP-dependent exonuclease determined by R1-19 plays a role in both repair and recombination of the host through the substitution of or competition with the exoV coded for by the genes recB and recC.     

Mutants of Escherichia coli with altered deoxyribonucleases. II. Isolation and characterization of mutants for exonuclease I

Mutants of Escherichia coli K12 deficient in exonuclease I (xon^?)³ were identified by enzymic assay of randomly selected, heavily mutagenized clones. From one of the six mutants of independent origin a thermolabile variant of exonuclease I was partially purified and identified, indicating that the mutation is probably in a structural gene for the enzyme. Transduction of this mutation into a recB^? recC^? strain did not result in the suppression of any of the phenotypic traits of the recipient. Although the five other mutants also appear to have temperature-sensitive exonuclease I activities in crude extracts, these enzymes were not sufficiently stable to permit purification. These latter mutations were of the xonA^? type; they produced a temperature-dependent suppression of the sensitivity to ultraviolet light and to mitomycin C manifested by a recB^? recC^? strain. None of the six mutations were of the sbcB^? type; that is, they did not suppress the recombination deficiency of a recB^? recC^? strain.In experiments with bacteriophage Plke, the six mutations were 41 to 62% cotransducible with the his region of E. coli. Heterozygous F′-merodiploids were constructed and studied for possible complementation of exonuclease I activity. All six mutations and an sbcB^? mutation were recessive to the wild-type alleles, and all were found to belong to a single complementation group. The results suggest that alterations of a structural gene for exonuclease I may result in the indirect suppression of the ultraviolet and mitomycin sensitivity manifested by recB^? recC^? strains.     

Properties of strains of Escherichia coli K12 carrying mutant lex and rec alleles

Summary Strains of Escherichia coli K12 have been constructed which carry the lex-3 mutation in combination with recA56, recB21, or recC22. The lex ^– recA ^–strain is equally as sensitive to ultraviolet light (UV) and ionizing radiation and as recombination-deficient as the corresponding lex ⁺ recA ^–strain, whereas the lex ^– recB ^–and lex ^– recC ^–strains are somewhat more sensitive to UV and ionizing radiation than the corresponding lex ^– rec ⁺strain and have approximately the same recombination deficiencies as the respective lex ⁺ recB ^–and Lex ⁺ recC ^–strains. When cultures of the lex ^– recB ^–and lex ^–recC^–strains are UV-irradiated, they degrade their DNA at the low rate characteristic of lex ⁺ recB ^–and lex ⁺ recC ^–single mutants, in contrast to the high rate of degradation seen with lex ^– rec ⁺single mutants.These results imply that the lex ⁺and recA ⁺products act in the same pathway of DNA repair and that both are needed to limit the DNA breakdown due to the recB ⁺/C⁺nuclease.     

Across Genus Plasmid Transformation Between <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and the Effect of <Emphasis Type="Italic">Escherichia coli</Emphasis> on the Transforming Ability of Free Plasmid DNA

The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.     

Induction of E. coli recA protein via recBC and alternate pathways: quantitation by enzyme-linked immunosorbent assay (ELISA)

Summary An enzyme-linked immunosorbent assay (ELISA) has been adapted to measure E. coli recA protein in the 1 to 10 ng range in whole-cell sonicates, membrane extracts, and osmotic shock fluid from 2x10⁸ cells. The specific activity of recA protein is maintained at a relatively constant basal level (800 to 1,200 molecules per cell for wild-type E. coli in L-broth, salt-depleted broth and minimal media) during early-log and mid-log phase growth, but it increases by two- to ten-fold as the culture approaches saturation density. Nalidixate-induced levels are 20- to 50-fold higher, and 100-fold higher in a constitutive tif ^- spr ^-mutant.Induction of recA protein synthesis by nalidixic acid, which normally requires functional recBC enzyme, also occurs in recB ^-and recC ^-cells by pathways activated by mutation in the sbcA and sbcB indirect suppressors. In recB ^- sbcA ^-mutants, exonuclease VIII, the recE gene product, is required for induction of recA protein. Abolition of exonuclease I activity by mutation in sbcB allows induction of recA protein by nalidixate in recB ^-and recC ^-cells. Mutation in recF does not affect induction by nalidixate in RecBC⁺ cells, but it enables induction to occur in RecBC^- cells, suggesting that recF gene product is involved in regulation of recA protein.     

Involvement of Recombination Genes in Growth and Viability of Escherichia coli K-12

We have studied the growth properties of 17 isogenic strains of Escherichia coli K-12 differing only in the recA, recB, recC, and sbcA alleles. We have observed the following. (i) All recombination deficient strains have decreased growth rates and decreased viabilities compared with recombination proficient strains. The large populations of nonviable cells in Rec⁻ cultures may arise by spontaneous lethal sectoring (9). (ii) A recA mutant strain which is entirely recombination deficient and which shows high ultraviolet sensitivity and “reckless” deoxyribonucleic acid (DNA) breakdown has approximately the same growth rate and twice the viability as recB and recC mutant strains which have residual recombination proficiency, moderate ultraviolet sensitivity, and “cautious” DNA breakdown. (iii) Indirectly suppressed (sbcA⁻) recombination proficient (Rec⁺) revertants of recB and recC mutant strains have approximately normal growth rates and are three times as viable as their Rec⁻ ancestors (but not as viable as rec⁺ cells). We suggest the following hypothesis to account for the low viability of Rec⁻E. coli. Single-strand breaks in the DNA duplex, necessary for normal bacterial growth, may be repaired in a Rec⁺ cell. Failure of Rec⁻ cells to repair this normal DNA damage may lead to the observed loss of viability.     

Molecular aspects of genetic instability of an artificial 68 bp perfect palindrome in Escherichia coli

Summary An artificial 68 bp perfect palindrome carried on a plasmid (pAS807) is genetically unstable. An increase in the population of cells harbouring palindrome-deleted pAS807 derivatives (pAS807-) is observed as the number of cell generations increases. The calculated frequency of palindrome excision events per cell generation and per plasmid replication round in Escherichia coli is 0.95x10^-4. Sequence analysis of eight independent isolates of palindrome-deleted molecules, reveals two symmetrical deletion types (three of type I and five of type II). The two types of pAS807- molecules retain 19 bp of the original sequence of the 68 bp palindrome but differ in the content of the central 3 bp. The generation of the two deletion types is best explained by formation of intermediate cruciform structures. Following the fate of the palindrome in various bacterial mutants, we find that the excision events depend on functional polA1, polA(ex1), lig, texA343(recC343) and texA344(recB344) gene products. However, recB21 recC22 mutations do not affect palindrome excision.     

Alteration of plasmid DNA-mediated transformation and mutation induced by covalent binding of benzo[a]pyrene- 7,8-dihydrodiol-9,10-oxide in Escherichia coli

Plasmid-mediated transformation and mutagenesis induced by (±)-trans- benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BP-DEI) in recipient Escherichia coli (E. coli) have been studied. Because plasmid DNA is used, the system is entirely free from direct toxic effects of BP-DEI on the recipient cells. Plasmid pK0482 DNA, which has two dominant genes, β-lactamase (amp-r) and galactokinase (galK) was modified with BP-DEI prior to its transformation of E. coli N99, AB1157, AB2463(recA^?) and AB1886(uvrA^?). Transformants were selected by ampicillin resistance and mutations were analyzed simultaneously by the altered expression of the galK gene. (1) Approx. 3 molecules of BP-DEI per molecule of pK0482 DNA decreased the transformation efficiency to 37% in AB1157 and the mutation frequency in this strain was proportional to the amount of BP-DEI covalently bound to pK0482 DNA. (2) In AB1886(uvrA^?) a 37% transformation efficiency was produced by only 1 molecule of BP-DEI per molecule of pK0482 DNA, and the mutation frequency in this strain was higher than in AB1157. (3) In AB2463(recA^?), the transformation efficiency was similar to that obtained with AB1157, but mutagenesis was clearly suppressed. (4) Polyacrylamide gel patterns of restriction digests of the pK0482 mutated at the galK gene were indistinguishable from those of the unmutated plasmid DNA.     

Specific Effects of a recB Mutation on the HfrH Strain of Escherichia coli

The recB268::Tn10 mutation was introduced into the HfrH strain of Escherichia coli. Compared with recB F⁻ and recB F⁺ cells, the viability of this mutant strain was much lower. Compared with wild-type HfrH, the recB derivative donated much shorter fragments of its chromosome to the recipient. It is suggested that the recB gene product (i.e., RecBCD enzyme) participates in Hfr transfer.     

Development of an electro-transformation system for Escherichia coli DH10B

Summary Escherichia coli can be transformed to high efficiencies by subjecting a mixture of cells and DNA to a brief but intense electrical field. Factors that affect the transformation efficiency of E.coli strain DH10B were analysed. Optimal conditions gave an efficiency of 10⁸ to 10⁹ transformants/g DNA with E.coli strains K803 and DH10B, and plasmids pB1221.23 and pBSK+. The use of ligated DNA resulted in 10⁶ transformants/g DNA. Detailed protocols for these systems are given.     

Interaction of the exrA mutation with rec mutants of Escherichia coli K12

Mutants of Escherichia coli K₁₂ were constructed which carry the exrA mutation addition to the various recombination deficient mutations recA recB and recC. The double mutant containing the exrA recA genotype is found to be slightly more sensitive to UV irradiation at very low doses of UV but essentially is very similar to the exrA + recA at the high UV doses. The recombination deficiency, λ induction and DNA degradation of the exrA recA shows a slight increase in the defectiveness of each of these functions. The double mutants of exrA recB and exrA recC show an increase in UV sensitivity and recombination deficiency and λ induction. The DNA degradation following UV-irradiation of these mutants is more characteristic of the recB and recC mutant alone.These results give further support to the theory that exrA and lex are probably mutants within the same cistron and also suggest that exrA, lex and recA are involved in a common DNA repair pathway and that the gene products of all three functions are required to regulate recB+ and recC+ endonuclease induced DNA degradation.     

The Evolution of RecD Outside of the RecBCD Complex

The common understanding of the function of RecD, as derived predominantly from studies in Escherichia coli, is that RecD is one of three enzymes in the RecBCD double-stranded break repair DNA recombination complex. However, comparative genomics has revealed that many organisms possess a recD gene even though the other members of the complex, recB and recC, are not present. Further, bioinformatic analyses have shown that there is substantial sequence dissimilarity between recD genes associated with recB and recC (recD1), and those that are not associated with recBC (recD2). Deinococcus radiodurans, known for its extraordinary DNA repair capability, is one such organism that does not possess either recB or recC, and yet does possess a recD gene. The recD of D. radiodurans was deleted and this mutant was shown to have a capacity to repair double-stranded DNA breaks equivalent to wild-type. The phylogenetic history of recD was studied using a dataset of 120 recD genes from 91 fully sequenced species. The analysis focused upon the role of gene duplication and functional genomic context in the evolution of recD2, which appears to have undergone numerous independent events resulting in duplicate recD2 genes. The role of RecD as part of the RecBCD complex appears to have a divergence from an earlier ancestral RecD function still preserved in many species including D. radiodurans.     

A correlation between sensitization to gamma-rays by purine starvation and the rec genes in Escherichia coli K-12

Summary After purine staration the recombination proficient (Rec⁺) strains of E. coli K-12 tested were sensitized to inactivation by gamma-rays (Figs. 1–4). No such change was noted in the Rec^– strains tested (Fig. 5). These results suggest that purine starvation may sensitize cells to ionizing radiation by inhibiting repair controlled by the recA, recB and recC genes.This paper was reported in part at the Fourteenth Annual meeting of the Biophysical Society in Baltimore, Md., 25–27 February 1970.     

Exchange of chromosomal markers by natural transformation between the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell

Both the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell, have been shown previously to be naturally transformable. This study reports the detection of genetic exchange by natural transformation between these two isolates. Transformation frequency was determined by filter transformation procedures. Three independent antibiotic resistance loci were used as chromosomal markers to monitor this exchange event: resistance to rifampicin, streptomycin, and nalidixic acid. The maximum frequencies of transformation were on the order of 3.1 to 3.8×10^-6 transformants per recipient; frequencies over an order of magnitude greater than those for spontaneous antibiotic resistance, although they are lower than those observed for soil: soil or marine: marine strain crosses. This exchange was inhibited by DNase I. Transformation was observed between soil and marine strains, both by filter transformation using purified DNA solutions and when transforming DNA was added in the form of viable donor cells. The results from this study support the close genetic relationship betweenP. stutzeri JM300 andP. stutzeri strain ZoBell. These results also further validate the utility ofP. stutzeri as a benchmark organism for modeling gene transfer by natural transformation in both soil and marine habitats.     

Restriction of plasmid-mediated transformation in Bacillus subtilis 168.

Summary When plasmids carrying leucine genes of Bacillus subtilis 168 were isolated from a restriction and modification deficient (r^-m^-) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec ⁺ strain (transformation by integration of the donor DNA into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r⁺ or r^- phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r^-m^- strains should be used for construction of recombinant DNA molecules in B. subtilis 168.     

Genetic analysis of the recF pathway to genetic recombination in Escherichia coli k12: Isolation and characterization of mutants

A recombination proficient strain ofEscherichia coli which is recB^? recC^? sbcB^? has been subjected to mutagenesis by nitrosoguanidine. Among the recombination deficient mutants isolated one was sbcB⁺, three were recA^～ and 11 were mutants in at least four newrec genes: recF, recJ, recK and recL. recF143 and recL152 are cotransducible with ilv but they lie on opposite sides of the ilv operons as determined by F$\text{\$}\text{?}$studies. recF, recL and recK are not involved in the RecBC pathway of recombination since a recB⁺recC⁺sbcB^? strain carrying a mutation in one of these genes is recombination proficient. Hence the hypothesis that a RecF pathway of recombination can operate as a partially independent substitute for the RecBC pathway of recombination is supported. recF^?recB⁺ and recF⁺recB^? single mutants are sensitive to u.v. irradiation while the recF^?recB^? double mutant is more sensitive than either single mutant. The sensitivity of the recB^?recC^?sbcB^?recF^? strain approaches the sensitivity of a recA^? single mutant. This is interpreted to mean that there are partially independent RecF and RecBC pathways for the repair of u.v. damage. recJ and mutations were not mapped precisely; hence the mutant properties they confer can not be stated conclusively.