Conjugational recombination in resolvase-deficient ruvC mutants of Escherichia coli K-12 depends on recG.

ruvC mutants of Escherichia coli appear to lack an activity that resolves Holliday intermediates into recombinant products. Yet, these strains produce close to normal numbers of recombinants in genetic crosses. This recombination proficiency was found to be a function of recG. A "mini-kan" insertion in recG was introduced into ruvA, ruvB, and ruvC strains. Conjugational recombination was reduced by more than 100-fold in recG ruvA::Tn10, recG ruvB, and recG ruvC strains and by about 30-fold in a recG ruvA strain carrying a ruvA mutation that is not polar on ruvB. The double mutants also proved very deficient in P1 transduction and are much more sensitive to UV light than ruv single mutants. Since mutation of recG alone has very modest effects on recombination and sensitivity to UV, it is concluded that there is a functional overlap between the RecG and Ruv proteins. However, this overlap does not extend to circular plasmid recombination. The possibility that RecG provides a second resolvase that can substitute for Ruv is discussed in light of these findings.     

Role of the ruvB gene in homologous and homeologous recombination in Rhizobium etli

The Rhizobium etli ruvA and ruvB genes were cloned through a PCR-based approach, using degenerate primers matching conserved sectors in the amino acid sequences of RuvB from eight bacterial species. Comparative analysis of the predicted polypeptides for RuvA and RuvB of R. etli showed highly conserved blocks with the corresponding homologs in other bacteria; RuvB depicts characteristic motifs for DNA helicases (ATP-binding and DEXH-box motifs). An R. etli ruvB::loxP Sp mutant was constructed by interposon mutagenesis. This mutant was highly sensitive to DNA-damaging agents, such as methyl methanesulfonate and nitrofurantoin, implying a deficiency in DNA repair. Homologous and homeologous conjugational recombination was reduced almost tenfold in the ruvB::loxP Sp mutant; a recombination defect was also observed in assays employing recombination between small plasmids, albeit at a smaller magnitude. Although the ruvA and ruvB genes are contiguous in R. etli, complementation studies suggest that they are expressed independently.     

UV-inducible DNA repair in the cyanobacteria Anabaena spp.

Strains of the filamentous cyanobacteria Anabaena spp. were capable of very efficient photoreactivation of UV irradiation-induced damage to DNA. Cells were resistant to several hundred joules of UV irradiation per square meter under conditions that allowed photoreactivation, and they also photoreactivated UV-damaged cyanophage efficiently. Reactivation of UV-irradiated cyanophage (Weigle reactivation) also occurred; UV irradiation of host cells greatly enhanced the plaque-forming ability of irradiated phage under nonphotoreactivating conditions. Postirradiation incubation of the host cells under conditions that allowed photoreactivation abolished the ability of the cells to perform Weigle reactivation of cyanophage N-1. Mitomycin C also induced Weigle reactivation of cyanophage N-1, but nalidixic acid did not. The inducible repair system (defined as the ability to perform Weigle reactivation of cyanophages) was relatively slow and inefficient compared with photoreactivation.     

Identification and characterization of Thermus thermophilus HB8 RuvA protein, the subunit of the RuvAB protein complex that promotes branch migration of Holliday junctions

The Escherichia coli ruvA and ruvB genes constitute an SOS-regulated operon. The products of these genes form a protein complex that promotes branch migration of the Holliday junction, an intermediate of homologous recombination. RuvA protein binds specifically to the Holliday junction and recruits RuvB protein to the junction. RuvB is an ATP-driven motor protein involved in branch migration. We previously cloned the ruvB gene of the thermophilic bacterium Thermus thermophilus HB8 (Tth) and found that, in contrast to the operon structure in most mesothermic bacteria, the ruvA gene is absent from the vicinity of ruvB. In this work, we cloned the ruvA gene from T. thermophilus HB8 and analyzed its nucleotide sequence. Tth RuvA is a protein of 20,414 Da consisting of 191 amino acid residues, and is 37% identical in amino acid sequence to E. coli RuvA. Tth ruvA complemented the DNA repair defect of E. coli deltaruvA mutants. The purified Tth RuvA protein stimulated Tth RuvB activities, such as hydrolysis of ATP and promotion of branch migration of the Holliday junction, in a manner similar to the RuvA-RuvB interactions observed in E. coli. In addition, Tth RuvA stimulated the E. coli RuvB activities in vitro, which was well consistent with the results of in vivo hetero-complementation experiments.     

Recombination is essential for viability of an Escherichia coli dam (DNA adenine methyltransferase) mutant

Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, or ruvC could not be constructed, whereas dam derivatives with recD, recF, recJ, and recR were viable. The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates. A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a dam lexA (Ind(-)) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recA and ruvAB need to be derepressed to allow for dam mutant survival. The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind(-)) derivatives. The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents.     

Ultraviolet- and X-Ray-Induced Responses of a Deoxyribonucleic Acid Polymerase-Deficient Mutant of Escherichia coli

Escherichia coli K-12, polAl(-) is a mutant strain whose extracts are deficient in Kornberg deoxyribonucleic acid (DNA) polymerase activity. We have compared the mutant and parental strains on the basis of a number of responses to ultraviolet (UV) and X-irradiation. For both types of radiation, the mutant is more sensitive by approximately the same factor as measured by reduction in colony formation, depression of DNA synthesis, and enhancement of DNA degradation. The rate of repair of X-ray-induced single-strand breaks in the mutant is also slower, as is the repair of breaks after excision repair of UV damage. On the other hand, the mutant has a significant capability to reactivate UV-irradiated lambda phage, although it is almost totally deficient in the ability to carry out UV reactivation. The data indicate that the polAl mutation leaves the cells with some ability to perform excision and strand-rejoining repair but that an exonuclease, whose identity remains obscure, is the agent responsible for the extensive breakdown of the DNA in polAl(-) cells after irradiation.     

The effect of cell irradiation on mutation in ultraviolet-irradiated and intact simian virus 40

The induction of phenotypic wild-type revertants in the progeny of an unirradiated or UV-irradiated temperature-sensitive late mutant of simian virus 40 was studied after low multiplicity passages in normal or UV-irradiated confluent monkey kidney cells. The production of wild-type revertants in the progeny of undamaged tsBC245 was followed by infecting the cells at distinct times after irradiation of the cells. Mutation frequencies reached a maximum when infection was delayed for 3--4 days after irradiation of the host cells, and declined gradually thereafter. Virus grown in unirradiated cells did not show such an alteration in mutation frequency. The temporarily higher mutation frequency of virus in UV-pretreated cells is due to a transient mutator activity operating in these cells rather than to an increased number of replications performed in UV-irradiated cells. A similar time course was found for the reactivation of UV-damaged SV40. This might suggest that reactivation and mutagenesis are manifestations of the same process. The yield of mutants due to irradiation of the virus alone was enhanced when infection was delayed for some days after the cells reached confluency; UV pretreatment of the host cells did not enhance the level of mutation obtained by UV irradiation of the virus.     

Ultraviolet enhanced reactivation of a human virus: effect of delayed infection

The ability of UV-irradiated herpes simplex virus to form plaques was examined in monolayers of CV-1 monkey kidney cells preexposed to UV radiation at different intervals before virus assay. From analysis of UV reactivation (Weigle reactivation) curves it was found that as the interval between cell UV irradiation (0-20 J/m2) and initiation of the virus assay was increased over a period of five days, (1) the capacity of the cells to support unirradiated virus plaque formation, which was decreased immediately following UV exposure to the monolayers, increased and returned to approximately normal levels within five days, and (2) at five days an exponential increase was observed in the relative plaque formation of irradiated virus as a function of UV fluence to the monolayers. For high UV fluence (20 J/m2) to the cells, the relative plaque formation by the UV-irradiated virus at five days was about 10-fold higher than that obtained from assay on unirradiated cells. This enhancement in plaque formation is interpreted as a delayed expression of Weigle reactivation. The amount of enhancement resulting from this delayed reactivation was several fold greater than that produced by the Weigle reactivation which occurred when irradiated herpes virus was assayed immediately following cell irradiation.     

Lethal and mutation frequency responses of Spiroplasma citri cells to UV irradiation

The effect of UV irradiation on viability and mutant colony frequency in the Mollicute Spiroplasma citri was investigated at 3 phases of growth. The first UV-induced mutants obtained in Mollicutes were selected: xylitol-resistant (XylR) and arsenic acid-resistant mutants (ArsR). Lethal and mutation frequency responses of S. citri cells increase with the age of the cell cultures. In all UV-irradiated populations, light exposure slightly increases the number of survivors and decreases the induced mutation frequency; liquid holding conditions increase the number of both survivors and mutant colonies. This suggests that, in UV-irradiated S. citri cells maintained under liquid holding conditions, there is no dark reactivation but induction of an error-prone repair system of the SOS type. In S. citri, the error-free light and dark repair systems are inefficient. Results allow the development of a method to select UV-induced mutations usable as markers in genetic studies of Spiroplasma cells.     

Kinetics of induction and decay of error-prone DNA repair activity in Escherichia coli after treatment with nalidixic acid

Inducible error-prone DNA repair activity was detected by infecting nalidixic acid-pretreated E. coli cells with UV-irradiated phage phi X174. Induction and decay kinetics of reactivation very much resembled that of mutagenesis of the UV-damaged phage. Repair as well as mutagenic activity increased for about 30 min. The maximal error-prone repair capacity, which was induced in the cell during the 30 min nalidixic acid treatment, rapidly died out during subsequent cell growth in absence of nalidixic acid. Induction of this repair mode was not observed in a recA- mutant. In the presence of nalidixic acid plus rifampicin both repair and mutagenic effects were abolished.     

Excision Repair Characteristics of recB−res− and uvrC− Strains of Escherichia coli

An Escherichia coli strain carrying the recB21 and res-1 mutations showed an abnormally low level of colony-forming ability although it grew essentially normally in liquid medium. The recB21 res-1 strain showed little, if any, of the ultraviolet (UV)-induced deoxyribonucleic acid (DNA) breakdown characteristic of the res-1 mutant. Nevertheless, the double mutant was far more sensitive to UV than either the res-1 or the recB21 strain. When compared with a wild-type strain, the rate of release of dimers from UV-irradiated DNA was very slow in the recB21 res-1, but normal in the res-1 recB(+) or recB21 res(+) mutants. However, the ratio of dimer-to-thymine released into the acid-soluble fraction was three times higher than the wild type in recB21 res(+) and recB21 res-1 and only one-tenth as high as the wild type in res-1 rec(+). Alkaline sucrose gradient centrifugation revealed occurrence of single-strand incision of UV-irradiated DNA and the restitution of nicked DNA at a similar rate in the recB21 res-1 and recB21 res(+) strains. Mutants uvrC(-) showed increased amounts of nicks in their DNA with increasing incubation time after UV irradiation, although no detectable amounts of dimers were excised from UV-irradiated DNA. From these results, it is concluded that the increased sensitivity of the res-1 strain to UV light is due to a reduced ability to excise dimers from UV-irradiated DNA and that the high rate of UV-induced breakdown of DNA is not the primary cause. A possible role of uvrC gene in the excision repair is discussed.     

UV-induced mutation in bacteriophage T4.

Two late gene am mutants of bacteriophage T4 that can be induced to revert by UV were crossed to a temperature-sensitive ligase mutant. In the double mutants, UV-induced reversion was eliminated at a semirestrictive temperature. When the single am mutants were irradiated and then allowed a single passage in a permissive host, the UV-induced reversion frequency was increased by 15- to 25-fold. This increased mutagenesis was also abolished by the presence of the ligase allele. When the UV-irradiated single am mutants multiply infected a permissive host, allowing multiplicity reactivation to occur, the induced reversion frequency was reduced similarly to the reduction in lethality. The mutagenesis that remained was again abolished by the presence of the ligase allele. It is concluded that UV induces mutations in phage T4 through the action of a pathway that includes polynucleotide ligase. The increase in mutation frequency after growth in a permissive host implies that mutagenesis can occur at more than one stage of the infection rather than only in an early stage before expression of the mutant genome. The process of multiplicity reactivation appears to be error-free since it overcomes lethal lesions without inducing new mutations.     

Host cell and ultraviolet reactivation of ultraviolet-irradiated Mycoplasmaviruses.

The mycoplasma Acholeplasma laidlawii was shown to have mechanisms for both host cell and ultraviolet (UV) reactivation of UV-irradiated mycoplasmaviruses. Host cell reactivation was examined by comparing the survival abilities of UV-irradiated double-stranded deoxyribonucleic acid mycoplasmavirus plated on both untreated and on acriflavine-treated cells. Acriflavine treatment inhibited cell exision repair. Decreased survival on the acriflavine-treated cells demonstrated host cell reactivation. UV reactivation was studied by comparing the survival of UV-irradiated virus plated on untreated cells with its survival on cells that received a small UV dose before plating. The UV-irradiated cells gave increased virus survival, showing UV reactivation. Similar experiments with a single-stranded deoxyribonucleic acid mycoplasmavirus showed that this virus could be UV reactivated, but not host cell reactivated.     

One-Step Microbial Conversion of a Racemic Mixture of Pantoyl Lactone to Optically Active d-(-)-Pantoyl Lactone

Washed cells of Rhodococcus erythropolis IFO 12540 were found to convert only the l-(+)-isomer of pantoyl lactone to the d-(-)-isomer in a racemic mixture of pantoyl lactone. Under suitable reaction conditions, the amount of d-(-)-pantoyl lactone synthesized was 18.2 mg/ml (94.4% enantiomer excess; molar yield, 90.5%). This conversion was suggested to proceed through the following successive reactions: first, the enzymatic oxidation of l-(+)-pantoyl lactone to ketopantoyl lactone; second, the rapid and spontaneous hydrolysis of the ketopantoyl lactone to ketopantoic acid; and then, the enzymatic reduction of the ketopantoic acid to d-(-)-pantoic acid. After the reaction d-(-)-pantoic acid could be lactonized by means of acid treatment. During the conversion, the d-(-)-isomer, which was initially present in the reaction mixture, did not undergo any modification.     

Defective Excision Repair of Pyrimidine Dimers in the Ultraviolet-Sensitive Escherichia coli ras− Mutant

The ras(-) mutant of Escherichia coli K-12 is sensitive to ultraviolet (UV) light but only slightly sensitive to X-irradiation (1.5-fold increase). Other phenotypic properties include normal recombination ability and normal host cell reactivation ability but an abnormally high frequency of UV-induced mutation. The response of the ras(-) mutant to UV has been studied biochemically. After low doses of UV, the ras(-) mutant degraded excessive amounts of deoxyribonucleic acid, and long delays in resumption of deoxyribonucleic acid synthesis occurred. Pyrimidine dimers were excised at the normal rate. Although the mutant had the capability of initiating repair replication, the process was not completed after the high UV dose required to allow detection of repair replication. The ras(-) mutant, after low UV doses, left three to four times as many single-strand breaks not rejoined as did the wild-type strain.     

Relationship between the functional regions of the RecA protein and ATP hydrolysis in UV-irradiated Escherichia coli cells.

The time course of the intracellular ATP concentration in several UV-irradiated RecA protease constitutive (Cptc) mutants of E. coli has been studied. All Cptc mutants harboring a mutation in region 3 of the RecA protein (including amino acid residues 298-301) increased ATP after UV damage but without any subsequent decrease. Nevertheless, these mutants induced the SOS response after UV irradiation. Likewise, truncated RecA proteins lacking region 3 are also unable to carry out massive ATP hydrolysis in UV-irradiated cells. On the other hand, mutants in region 1 (including amino acids 25-39) or 2 (amino acids 157-184) of the RecA protein showed an increase in ATP concentration during the first 20 min following UV irradiation, which dropped afterwards to the basal level. All these data indicate that region 3 of the RecA protein must be involved in the ATP hydrolysis process. Furthermore, a relationship between the quantity of the UV-mediated ATP produced and the strength of the different RecA Cptc mutants has also been found. Accordingly, both lexA71::Tn5 and null lexA mutants of E. coli only show a cellular ATP increase after UV irradiation when containing a multicopy plasmid carrying either a wild-type lexA or a lexA (Ind-) gene.     

Growth and Division of Filamentous Forms of Escherichia coli.

Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), and Alice A. Hardigree. Growth and division of filamentous forms of Escherichia coli. J. Bacteriol. 90:223-226. 1965.-Cells of certain mutant strains of Escherichia coli grow into long multinucleate filaments after exposure to radiation. Deoxyribonucleic acid, ribonucleic acid, and protein synthesis proceed, but cytokinesis does not occur. Cytokinesis (cross-septation) can be initiated by exposure of the filaments to pantoyl lactone or a temperature of 42 C. If growing filaments are treated with mitomycin C, nuclear division does not occur, and nuclear material is confined to the central region of the filament. Cytokinesis cannot be induced in mitomycin C-treated filaments by pantoyl lactone or treatment at 42 C.     

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage lambda. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut(+) strains. UV irradiation induced mutations in a mutU4 strain, and phage lambda was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4.