Recombination deficient mutant of Caulobacter crescentus

Summary A recombination-deficient (Rec^-) strain of Caulobacter crescentus has been isolated from a collection of mutants sensitive to ultraviolet irradiation. The Rec^- mutant fails to give recombinants following Cr30-mediated generalized transduction or following RP4-mediated conjugation. The recombination frequency in the Rec^- strain is at least 5000-fold lower than in the wild type strains. The Rec^- mutant is indistinguishable from wild type in terms of morphology, growth rate, viability, and phage sensitivities, differing only in properties known to be associated with recA-type mutations in other organisms: recombination frequency, ultraviolet sensitivity, and Weigle reactivation. The map location of the rec-526 allele has not been identified, but rec-526 can be cotransferred with the fla-169 mutation by RP4-mediated conjugation at low frequency. This apparent linkage has been used to move the rec mutation to other strains. The Rec^- mutant resembles recA strains of other organisms and provides a healthy strain severely deficient in recombination for use in complementation and cloning studies involving C. crescentus.     

Mini-muduction: A new mode of gene transfer mediated by mini-Mu

Summary We compared the transducing properties of Mucts62 and Mucts62/mini-Mu lysates, using Mu immune and non immune Rec⁺ and recA recipient strains. The Mu/mini-Mu lysates transduced all bacterial markers tested 10 times more efficiently than the Mucts62 lysates in Rec⁺ recipients. Most of the transductants obtained after infection with the Mu/mini-Mu lysates result from the substitution of the mutated gene of the recipient by the wild type allele from the donor, most probably carried on the gigantic variable end linked to the mini-Mu genome.Moreover the Mu/mini-Mu lysates gave a new type of Rec-independent transduction that we called mini-muduction. Mini-muduction requires the activity of Mu gene A and provides transductants which carry the transduced marker surrounded by two mini-Mu genomes similarly oriented, and inserted at random location in the recipient chromosome. The mini-Mu/transduced DNA/mini-Mu structures are able to transpose spontaneously, for instance into a transmissible plasmid, in the presence of Mu gene A product.     

The segregation of the SbcA and Rac phenotypes in an Escherichia coli recB mutant

Summary In certain HfrxF^– recB ^– crosses recombinant progeny were examined for their SbcA and Rac phenotypes. Recombinants which inherited either his ⁺ or trp ⁺ from the donor in an Hfr recB21 sbcA8xF^– recB21 Rac^–SbcA⁺ cross acquired the RecB⁺ phenotype in most instances (presumably by inheriting the sbcA8 allele). Several independent Rec⁺ (sbcA8) recombinants from this cross were converted back to the Rec^– (sbcA ⁺) phenotype by mating with a Rac⁺ SbcA⁺ Hfr. Ten out of 14 of these Rec^– recombinants retained the Rac^– phenotype of the original parent. It was concluded that these results were inconsistent with the hypothesis that sbcA is a gene carried by a Rac prophage.     

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.     

A molecular model for conjugational recombination in Escherichia coli K12

Summary Conjugational recombination in Escherichia coli was investigated by measuring lacZ ⁺ product, -galactosidase, in crosses between lacZ mutants. Enzyme production in both Hfr and F-prime crosses was detected very soon after transfer of the donor lacZ allele. The level of enzyme activity was reduced by no more than two-fold when the recipient carried a recB mutation. With an F-prime donor, recombination appeared to be restricted largely to a short period immediately after transfer, with little evidence of recombination during subsequent exponential growth of the transconjugant cells. These observations are interpreted to suggest that recA dependent recombination is able to initiate with high efficiency at gaps present in the donor DNA before synthesis of a complementary strand is completed, and independently of recB function. A molecular model for conjugational recombination based on this idea is presented in terms of the known activities of recA and recBC products. Some of the predictions of the model are tested by analysing the recombinant genotypes produced in Hfr crosses with multiply marked strains.     

Inactivation of a newly transferred gene in recombination-deficient recipients of Escherichia coli

Summary The expression of a newly transferred lacZ ⁺ gene in lacZ recipients carrying various mutations in the recA and recB genes was studied by measuring the rates of induced synthesis of -galactosidase in zygotes formed after mating with either F or Hfr donors. The ability to synthesize -galactosidase decreases with time in both recA and recB zygotes when the lacZ ⁺ gene is transferred from an Hfr donor, but not when the lacZ gene is transferred from an F donor. There is no such inactivation of the newly transferred lacZ ⁺ gene in Rec⁺ zygotes. We conclude that the functioning of the transferred DNA is progressively inactivated in rec recipients unless the DNA is contained in an episome such as F.     

Initiation of recombination during transformation of Bacillus subtilis requires no extensive homologous sequences

Summary Lysates obtained shortly after entry of transforming DNA to Bacillus subtilis contain donor-recipient DNA complexes, in which the donor moiety is associated with the recipient DNA in an unstable way. The complexes could be artificially stabilized by crosslinking with 4,5,8-trimethylpsoralen. The unstable complexes dissociated upon helix-destabilizing treatments, such as heating at 70°C, and CsCl gradient centrifugation at pH 11.2, but remained stable during CsCl gradient centrifugation at pH 10. Donor-recipient DNA complexes were not formed after entry of heterologous pUB110 DNA. These observations suggest that base-pairing is involved in the unstable association. The donor moiety of the unstable complexes was completely, or almost completely, digestible by nuclease S₁, indicating that the donor and recipient base-sequences are only paired over very short distances.The unstable donor-recipient DNA complexes are true recombination intermediates because (i) strain 7G224 (recE4) was impaired in the formation of the unstable complexes, and (ii) the unstable complexes were rapidly converted to stable complexes in recombination proficient strains, whereas their conversion was delayed in the recombination deficient strain 7G84.Unstable complexes were also formed with Escherichia coli donor DNA, but to a lesser extent. Apparently a limited degree of base-sequence homology is sufficient to initiate recombination.     

In vivo loss of supercoiled DNA carrying a palindromic sequence

Summary Interest in the fate of long palindromic DNA sequences in E. coli has been kindled by the observation that their inviability is overcome in recBC sbcB strains and that these hosts permit the construction of DNA libraries containing long palindromic sequences present in the human genome. In this paper we show that a reduction in the level of intracellular supercoiled DNA occurs as the result of the presence of a 530 bp palindrome in bacteriophage lambda. This reduction occurs in Rec⁺ and recA strains but not in strains lacking exonucleases V and I (recBC sbcB). However, the DNA must be active (not repressed) for this reduction to be observed, since it is not seen in a Rec⁺ host lysogenic for phage lambda. These results argue against two hypotheses: firstly, that the palindrome causes inviability solely by interfering with packaging, and secondly, that it dose so solely by interfering with recombination. Conversely, these results suggest that a feature of active monomeric DNA (probably its replication) is involved in inviability.     

Does Tn10 transpose via the cointegrate molecule?

Summary It has been well established that Tn3 and its relatives transpose from one replicon to another by two successive reactions: formation of the cointegrate molecule and resolution from it. Whether or not the 9300 base pair tetracycline resistance transposon Tn10 transposes in the same manner as Tn3 was investigated by two methods.In the first method, 55, a lambda phage carrying Tn10 was lysogenized in an Escherichia coli strain carrying a Tn10 insertion; the phage has a deletion in attP, hence it was lysogenized in a Tn10 sequence in the E. coli chromosome by reciprocal recombination. The chromosomal structure in these lysogens is equivalent to the Tn10-mediated cointegrate molecule of lambda and the E. coli chromosomal DNA. The stability of the cointegrate molecule was examined by measuring the rate of excision of lambda from the host chromosome, and was found to be stable, especially in a Rec^- strain. Because of this stability, the cointegrate molecule should be accumulated if Tn10 transposes via the cointegrate molecule. Then, we examined the configuration of products made by transposition of Tn10 from 55 to the E. coli chromosome. The cointegrate molecule was found in products of Tn10 transposition in a Rec⁺ strain at a frequency of 5% per Tn10 transposition, but this molecule could not be found in a Rec^- strain. Since transposition of Tn10 was recA-independent, absence of the cointegrate molecule formed in a RecA^- strain strongly suggested that the cointegrate molecule is not an obligatory intermediate of transposition of Tn10.In the second method, mobilization of pACYC177 by R388 and by R388:: Tn10 was examined. The pACYC177 plasmid was mobilized by R388::Tn10 at a frequency of 10^-4 per donor but not by R388. It occurred, in most cases, by inverse transposition of R388::Tn10 to pACYC177 forming plasmids such as pACYC177::IS10-R388-IS10. Mobilization of pACYC177 by a Tn10-mediated cointegrate in the form of pACYC177::Tn10-R388-Tn10 was not observed in crosses using a Rec^- donor. These observations also suggested that transposition of Tn10 in Rec^- cells does not occur via the cointegrate molecule.     

Vegetative recombination of bacteriophage Mu-1 in Escherichia coli

Summary Twenty-two amber mutants of the thermoinducible mutator phage Mu-c4ts were isolated. These mutants fall into 11 complementation groups. The data obtained by crossing these amber mutants suggest that bacteriophage Mu-1 has a linear vegetative linkage map. In a recombination deficient host of the RecA type the recombination frequencies are extremely low, indicating that Mu-1, in contrast to many other E. coli phages, is dependent on the recombination system of its host. With as a helper phage, recombination between Mu phages in a RecA host is restored to about 1/3 of the frequency in a Rec⁺ host. Although Mu-1 is able to integrate efficiently into the chromosome of a RecA strain, it seems that its integration system does not contribute to vegetative recombination.The survival of UV-irradiated Mu-1 was measured on different radiation sensitive mutants of E. coli. The survival on a UvrB strain was very low as compared to the wild-type; the survival on a RecA strain was almost the same as on the wild-type.Research Fellow from the Laboratory of Genetics, State University, Leiden, The Netherlands.     

Studies on radiation-sensitive mutants of E. coli. 3. Participation of the rec system in induction of mutation by ultraviolet irradiation

Summary The bacterial recA gene participates in the induction by UV irradiation of the clear mutation of phage and the Lac^- mutation of bacteria. The necessary function is induced by irradiation of Rec⁺ bacteria and acts upon DNA irradiated with UV light.     

Ultraviolet reactivation of the single stranded DNA phage phiX 174.

Summary When UV-irradiated X174 was grown in pre-irradiated host cells of various strains, ultraviolet reactivation (UVR) was observed only in recombination proficient strains such as E. coli C (uvrA ⁺ recA ⁺) and HF4704 (uvrA ^- recA ⁺), but not in the recombination deficient strain HF4712 (uvrA ⁺ recA ^-). By increasing the multiplicity of infection, no rise in the amount of such reactivation was observed. From the study of the neutral and alkaline sucrose gradient sedimentation patterns of DNA samples extracted from unirradiated cells infected with unirradiated phage, it appears that after the conversion of the viral single stranded (SS) DNA to the double stranded form (DS), nicks or scissions were produced on it within all three strains, which were ultimately sealed up in the recA ⁺ but persisted within the recA ^- host cells. When UV-irradiated phage infected unirradiated host cells, such nicking of the DS DNA appeared to be much more extensive in uvrA ⁺ recA ⁺, but slightly reduced in uvrA ⁺ recA ^- and severely suppressed in uvrA ^- recA ⁺ strains. When the host cells were also UV-irradiated, the conversion of the infecting viral SS DNA to DS DNA as well as its subsequent nicking were reduced in all the three strains to a much greater extent. Although nicking of the DS DNA molecule is an essential step even in the normal intracellular replication of X DNA, the production and the sealing up of such nicks appear not to have any positive correlation with UVR of these phages. A drastic reduction in nicking due te pre-irradiation of the host cells might, however, mean slowing down of the replication of the damaged parental RF molecules which would facilitate their repair perhaps through recombination with the homologous parts of the host genome.     

Recombination in Escherichia coli. VI. Characterization of a recombination-deficient mutation with unusual properties

A detailed study of the rec-34 mutation shows that this mutation is located near pheA on the E. coli chromosome, like the recA and recH genes. The rec-34 recipients, which are radiation-sensitive and “reckless”, yield about 0.1–1% of the number of recombinants obtained with a Rec⁺ recipient in conjunction experiments. The recombinants obtained in conjunction experiments, in which rec-4⁺ allele transfer is avoided, are still recombination deficient.rec-34 definitely is not a mutation in the recH gene. Although definite proof is lacking we suggest that rec-34 is an unusual recA mutation.     

Effect of mismatched base pairs on the fate of donor DNA in transformation of Streptococcus pneumoniae

Summary Investigation of the mechanism that discriminates against mismatched base pairs in transformation of Streptococcus pneumoniae of genotype hex ⁺ was based on the use of a radioactively labeled cloned fragment of pneumococcal DNA as donor in transformation. The fate of the donor label was followed by lysis of the transformed cells and separation by agarose gel electrophoresis of DNA fragments generated by restriction endonucleases. As a result of Hex action, most of the donor DNA fragment, which was a few kilobases in length, was lost when a mismatched base pair occurred between donor and recipient DNA. This was not observed in hex ^- recipient cells. Kinetic studies of mismatch-induced donor DNA loss showed that the process is faster in strain 800, an R6 derivative, than in DP 1601, a strain of different origin. In the latter strain, the amount of donor label that becomes double stranded rises substantially, indicating extensive formation of donorrecipient heteroduplex structures, before falling to the expected level. At 30°C the process is essentially completed 15 min after entry.     

Carbohydrate metabolism and transport in Bacillus subtilis. A study of ctr mutations

Summary Indirect ultraviolet induction of prophage occurs when lysogenic E. coli K12 cells are mated with ultraviolet-irradiated donor strains carrying a transmissible episome such as F lac ⁺. Indirect induction occurs in wild type, uvrA, or recB recipient lysogens, but not in recA lysogens. When nonpermissive lysogens carrying prophages susO or susP are similarly mated, the defective prophages are induced and indirect curing takes place.Although indirect induction is independent of the capacity of the lysogen for repair by pyrimidine dimer excision, indirect curing (and hence indirect induction) is subject to photoreactivation when the recipient lysogen is exposed to visible light after mating. This confirms that the structure initiating indirect ultraviolet induction is a damaged transferred episome consisting of one DNA strand containing ultraviolet photoproducts and a newly synthesized discontinuous DNA strand such that pyrimidine dimers remain in single-stranded regions.F^- lac ⁺ recombinants are formed in either nonlysogenic or lysogenic Lac^- cells receiving damaged F lac ⁺ episomes from ultraviolet irradiated F lac ⁺ donors. prophage induction occurs more frequently in zygotes that form Lac⁺ recombinants than in zygotes that remain Lac^-. In contrast, cells receiving intact (undamaged) episomes are converted to F lac ⁺ secondary donors, but are rarely induced or cured.     

Isolation and characterisation of Hfr strains from a recombination-deficient strain of Escherichia coli

Summary A T6^s RecA^- strain carrying a lac proB deletion and F_ts lac ⁺ was challenged with phage T6 and survivors which were both T6^r and Lac⁺ at 42° were tested for fertility. Among these were a number of Hfr strains which had their points of origin at or near the tsx locus and which still carried the recA allele. These arose in comparable frequencies in the RecA^- strain and in a Rec⁺ analogue. We conclude that such integration does not require the RecA function. The rate of chromosome transfer was similar in one such RecA^- Hfr and its Rec⁺ derivative; the yield of recombinants from the RecA^- strain was slightly lower than from its Rec⁺ derivative.     

Lambda red-mediated synthesis of plasmid linear multimers in Escherichia coli K12

Summary Expression of the red ⁺ and gam ⁺ genes of bacteriophage in plasmids cloned in Escherichia coli wild-type cells leads to plasmid linear multimer (PLM) formation. In mutants that lack exonuclease I (sbcB sbcC), either of these functions mediates PLM formation. In order to determine whether PLM formation in sbcB sbcC mutants occurs by conservative (break-join) recombination of circular plasmids or by de novo DNA synthesis, thyA sbcB sbcC mutants were transferred from thymine- to 5-bromo-2-deoxyuridine (BUDR)-supplemented medium, concurrently with induction of red ⁺ or gam ⁺ expression, and the density distribution of plasmid molecular species was analyzed. After a period of less than one generation in the BUDR-supplemented medium, most PLM were of heavy/heavy density. Circular plasmids, as well as chromosomal DNA, were of light/light or light/heavy density. These results indicate that Red or Gam activities mediate de novo synthesis of PLM in sbcB sbcC mutants. Examination of plasmid DNA preparations from sbcB sbcC mutants expressing gam ⁺ or red ⁺ reveals the presence of two molecular species that may represent intermediates in the PLM biosynthesis pathway: single-branched circles (-structures) and PLM with single-stranded DNA tails. While Gam-mediated PLM synthesis in sbcB mutants depends on the activity of the RecF pathway genes, Red-mediated PLM synthesis, like Red-mediated recombination, is independent of recA and recF activities. One of the red ⁺ products, protein, suppresses RecA deficiency in plasmid recombination and PLM synthesis in RecBCD^– Exol^– cells. The dependence of PLM synthesis on the RecE, RecF or Red recombination pathways and the dependence of plasmid recombination by these pathways on activities that are required for plasmid replication support the proposal that PLM synthesis and recombination by these pathways are mutually dependent. We propose the hypothesis that DNA double-stranded ends, which are produced in the process of PLM synthesis, are involved in plasmid recombination by the RecE, RecF and Red pathways. Conversely, recombination-dependent priming of DNA synthesis at 3 singles-tranded DNA ends is hypothesized to initiate PLM synthesis on circular plasmid DNA templates.Abbreviations PLM plasmid linear multimers - BUDR 5-bromo-2-deoxyuridine - bp base pair     

Formation of concatemers of lambda phage DNA in a recombination-deficient system

Summary Concatenand molecules of lambda DNA were formed even in a recombination dificient system (Int^-Red^-Rec^-) in the late stage of phage growth. No significant difference was observed in the formation of concatemers between recombination deficient and proficient systems. These results suggest that concatemer formation is an intrinsic nature of replication in the late stage. The possibility of concatemer formation by molecular exchanges which do not contribute genetic recombination is not excluded.This paper is the sixth of a series Replication of bacteriophage DNA the fourth and fifth of which are J. Tomizawa, and Y. Sakakibara and J. Tomizawa in Bacteriophage lambda ed. A. D. Hershey, Cold Spring Harbor, N. Y. in press.