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Genetics, Department of Biology and Environmental Sciences, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany
Correspondence
Wilfried Wackernagel
wilfried.wackernagel{at}uni-oldenburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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recJ mutant of A. baylyi was only slightly altered in transformability and was not affected in UV survival. In contrast, a recBCD mutant was UV-sensitive, and had a low viability and altered transformation. Compared to wild-type, transformation with large chromosomal DNA fragments was decreased about 5-fold, while transformation with 1.5 kbp DNA fragments was increased 3.3- to 7-fold. A recD mutation did not affect transformation, viability or UV resistance. However, double mutants recJ recBCD and recJ recD were non-viable, suggesting that the RecJ DNase or the RecBCD DNase (presumably absent in recD) becomes essential for the recombinational repair of spontaneously inactivated replication forks if the other DNase is absent. A model of recombination during genetic transformation is discussed in which the two ends of the single-stranded donor DNA present in the cytoplasm frequently integrate separately and often with a time difference. If replication runs through that genomic region before both ends of the donor DNA are ligated to recipient DNA, a double-strand break (DSB) is formed. In these cases, transformation becomes dependent on DSB repair.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and has orthologues in bacteria, archaea and eukaryotes (Aravind & Koonin, 1998; Sutera et al., 1999; Rajman & Lovett, 2000). In E. coli, RecJ is essential in the RecF pathway of recombination (Lovett & Clark, 1984). The enzyme is proposed to degrade the 5' strand from a duplex DNA end during unwinding by a helicase such as RecQ, resulting in a 3' overhang. With the help of the RecFOR proteins, RecA then binds to the single strand to give the nucleoprotein filament, promoting strand transfer to a homologous DNA (Lovett & Kolodner, 1989; Clark & Low, 1988; Kowalczykowski et al., 1994; Lloyd & Low, 1996; Kuzminov, 1999). RecJ is also required in the recovery of blocked replication forks in UV-irradiated cells by degrading the newly synthesized 5' strand in regressed forks (Courcelle & Hanawalt, 1999, 2003). Further, RecJ is thought to trim single-stranded 5' tails from duplex DNA ends to make ends available for the RecBCD enzyme or helicases, and from branched DNA after synapsis to stabilize joint molecules and to prepare ligatable structures. In these ways RecJ is also important for the RecBCD pathway, so that a recJ mutation decreases recombination, particularly when another single-stranded exonuclease, the 3'-specific exonuclease I, is inactivated by mutation (Miesel & Roth, 1996; Razavy et al., 1996; Friedman-Ohana & Cohen, 1998; Viswanathan & Lovett, 1998).
The central enzyme of the main pathway of recombination in E. coli, the RecBCD pathway, is the RecBCD enzyme, consisting of the three proteins encoded by the recB, recC and recD genes (Kowalczykowski et al., 1994). The enzyme is the strongest duplex DNA exonuclease in E. coli, degrading both strands while unwinding the duplex as a helicase. The main role of the enzyme in recombination is seen in the generation of a RecA protein-covered 3' tail on a duplex DNA end, giving the recombinogenic nucleoprotein filament (Kowalczykowski et al., 1994; Anderson & Kowalczykowski, 1997). This occurs when the RecBCD enzyme, degrading DNA from an end, reaches a properly oriented Chi site (5'-GCTGGTGG-3'; present in the genome about every 4500 bp), where the enzyme switches to degrade only the 5' strand and to actively load RecA onto the 3' strand. Recombination initiated in this way can repair double-strand breaks (DSBs) and restore inactivated replication forks. Broken or inactivated forks are produced e.g. when the replication complex runs into a single-strand break in DNA or is blocked by DNA structure, bound proteins or DNA damage. Then the fork is reversed, followed by subsequent steps which may include cleavage by Holliday junction resolvase (Kuzminov, 2001; Michel et al., 2001, 2004). A broken replication fork results in a contiguous double strand and partially replicated genome with a duplex DNA end. The end can be repaired by a recombination process termed DSB end repair (Kuzminov, 1999), reconstituting a fork. As a consequence of defective DSB repair the recB and recC mutants have low viability, resulting from lethal sectoring, and are very sensitive to DNA-damaging agents including UV irradiation (Kuzminov, 1999). The recombination deficiency of these mutants also drastically reduces the integration of DNA transferred by horizontal gene transfer, including conjugation and transduction (Clark & Low, 1988). In contrast to recB or recC strains, recD mutants show approximately normal viability and UV resistance, and somewhat increased recombination proficiency (Amundsen et al., 1986; Biek & Cohen, 1986). In recD mutants, recombination events are no longer localized to Chi sites but occur close to the DNA end (Thaler et al., 1989). Deficiency of recD leads to a RecBC(D–) enzyme devoid of the strong duplex DNA exonuclease activity of RecBCD, while the helicase and RecA-loading functions are maintained (Amundsen et al., 1986; Churchill et al., 1999). The presence of a recJ mutation, which in otherwise wild-type E. coli cells also has no strong effect on UV sensitivity and recombination, confers high UV sensitivity to recD mutants and also decreases their recombination proficiency (Lloyd et al., 1988; Lovett et al., 1988; Dermic, 2006).
We asked whether recJ and recBCD are involved in the recombination process necessary to integrate DNA taken up during the natural transformation of Acinetobacter baylyi. A. baylyi (formerly termed A. calcoaceticus BD413 or Acinetobacter sp. ADP1) belongs to a genus whose members are widely present in the environment, including aquatic habitats, soil, sediments and living organisms (Barbe et al., 2004). These organisms are nutritionally versatile chemoheterotrophs (Young et al., 2005). In addition, A. baylyi, with its high competence for natural transformation, has been extensively studied with respect to horizontal gene transfer by extracellular DNA in vitro and in environmental microcosms (Young et al., 2005). Horizontal gene transfer is a major route in prokaryotes to increase genetic diversity (Arber, 2000), and the acquisition of foreign non-homologous DNA by natural transformation has been specifically studied in A. baylyi (de Vries & Wackernagel, 2002; de Vries et al., 2004). During natural transformation the cells take up and integrate free DNA present in their environment (Goodgal, 1982; Lorenz & Wackernagel, 1994; Palmen & Hellingwerf, 1997; Dubnau, 1999; Chen & Dubnau, 2004). In the process, studied mainly in Haemophilus influenzae, Neisseria gonorrhoeae, Bacillus subtilis and Streptococcus pneumoniae, double-stranded DNA binds to the cell, a DSB is introduced into the DNA, and one of the molecule fragments is transported into the cell, becoming resistant to experimentally added DNase. One strand of the duplex transits into the cytoplasm while the other strand is degraded. The single strand in the cytoplasm can recombine via heteroduplex formation with the resident DNA in a RecA-dependent process, which is the final step in transformation. In A. baylyi also, the donor DNA is present as single-strand (Palmen et al., 1993). The influence of recJ on natural transformation has not yet been studied while in some transformable organisms, e.g. N. gonorrhoeae, H. influenzae and B. subtilis, the deficiency of RecBCD or its orthologues has been found to affect transformability (Kooistra & Venema, 1976; Mehr & Seifert, 1998; Haijema et al., 1996). We found that the elimination of recBCD in A. baylyi affected transformation differently depending on the length of the transforming DNA molecules, whereas deficiency of recJ did not have a strong effect. We discovered that double DNase mutants like recJ recBCD and recJ recD were non-viable. This phenomenon had not been observed in E. coli or Salmonella enterica sv. Typhimurium. We propose a model of the recombinational integration of transforming single-stranded DNA which frequently leads to the formation of a DSB and, therefore, due to the requirement of DSB end repair, makes transformation largely dependent on RecBCD.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The A. baylyi strains all contain the auxotrophic marker trpE27. The E. coli strain BT544 was derived from WA735, which is AB1157 with the recD1011 and the recJ284 : : Tn10 alleles (Rinken et al., 1992), by selection of an imprecise Tn10 eductant as described by Maloy & Nunn, (1981) and bears a defective recJ gene (identified by the high UV sensitivity of the strain). The plasmid pRK415iq (GenBank accession no. EF435043) was derived from pRK415 (Keen et al., 1988) by insertion of a 1.2 kbp PCR product covering the lacIq gene. The plasmid pQLICE consists of a derivative of RSF1010 (Scholz et al. 1989) containing the 1.9 kbp BamHI–AgeI fragment covering the lacIq gene and the ptac promoter (de Boer et al., 1983) of plasmid pYanni1 (Graupner & Wackernagel, 2000), and also has the strAB genes (streptomycin resistance). The GenBank accession number of that plasmid is EF189157. The A. baylyi and E. coli strains were grown in LB medium (Sambrook et al., 1989) at 30 °C. The minimal medium was M9 (Sambrook et al., 1989) with 20 mM succinate (pH 7.5) as carbon source. If necessary, liquid or solid media contained antibiotics: kanamycin (Km; 10 µg ml–1), chloramphenicol (Cm; 10 µg ml–1), ampicillin (Ap; 100 µg ml–1), tetracycline (Tc; 10 µg ml–1) or streptomycin (Sm; 20 µg ml–1). Plasmids were transferred into E. coli strains by electroporation as described by de Vries & Wackernagel, (1998). To identify clones with sacB (phenotype: sucrose-sensitive, SucS), the plate medium contained 50 g sucrose l–1.
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) and cloned into plasmid vector pGT41 (derived from the ColE plasmid cloning vector pPCR-Script Cam; Stratagene) upstream and downstream of a gene cassette containing a selectable (nptII, KmR) and a counter-selectable (sacB, SucS; Dedonder, 1966) marker combination. The resulting plasmid was termed pEK2. A. baylyi was naturally transformed with pEK2 DNA linearized by cleavage with SalI, leading to KmR and SucS transformants that have the recJ gene substituted by the nptII sacB cassette. One transformant (verified by PCR) was termed EK3. For the second step, the nptII sacB cassette was deleted from pEK2 by cleavage with XbaI and religation (leading to the fusion of both cloned flanks, generating a recJ allele). The XbaI cleavage sites had been introduced to the sides of the nptII sacB cassette by using primers with XbaI recognition sequences in the 5' tails of primers used during amplification of the flanks of recJ (Table 2). The resulting plasmid (pEK3) was utilized to naturally transform strain EK3, leading to SucR (and KmS) transformants having the nptII sacB gene cassette replaced by the recJ allele (strain EK4; verified by PCR). The recBCD and recD substitution and deletion strains were constructed correspondingly, using MluI restriction sites to remove the nptII sacB cassette. The primers for amplification of flanking DNA segments for recJ, recBCD and recD and the lengths of these segments are listed in Table 2.
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) into the EcoICRI site of pRK415iq, putting recJ under the control of the lac promoter. pEKX2 was constructed by insertion of a PCR-amplified DNA segment covering recJ, including its ribosome-binding sequence (the primers were recJ-ORF-f2 and recJ-ORF-r2; Table 2), which was cleaved with Cfr9I and cloned into the Cfr9I site of pQLICE, putting recJ under the control of the tac promoter.
Verification of transformants and PCR conditions.
Transformants were verified by PCR using primers suitable to indicate insertions or deletions as required, using primers listed in Table 2. Amplification of DNA segments over 2000 bp and DNA segments used for cloning was performed with Phusion DNA polymerase according to the protocol provided by the supplier (Finnzymes) with the addition of DMSO (10 %) in the reaction mix. For amplification of other DNA segments, MolTaq DNA polymerase was used under reaction conditions as recommended by the supplier (Molzym). When cells were used for PCR, cell material from a fresh plate was resuspended in sterile water, the cell concentration determined under the microscope and a sample (0.2–1 µl) containing 105 cells included in the PCR mix.
UV irradiation.
Cells from an exponential-phase culture in LB were sedimented, resuspended at about 2x108 ml–1 in phosphate buffer and irradiated with UV under stirring as described by Thoms & Wackernagel (1982). The dose rate was 1.8 J m–2 s–1. Cells were plated after appropriate dilution on LB medium and colonies counted after about 24 h incubation at 30 °C.
Natural transformation.
Concentrated competent cell suspensions were prepared from various strains as described by de Vries & Wackernagel, (1998) and stored at –80 °C. For maintenance of plasmids the culture media contained the respective antibiotic if required. Samples were thawed at room temperature and diluted 40-fold in LB (giving a cell titre of 2.5x108 ml–1), and DNA was added to a final concentration of 100 ng ml–1. After 90 min of aeration at 30 °C (in a roller incubator) samples were plated on selective (transformants) and unselective (total c.f.u) medium. Colonies were counted after about 20 h incubation on LB plates and about 48 h on M9 plates. Transformation frequencies are given as the transformant titre divided by the titre of c.f.u. For transformation with the 1.5 kbp fragment covering trpE, the DNA was amplified from a trp+ strain by primers trp-ORF-f and trp-ORF-r (Table 2).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In order to test the functional equivalence of the two proteins, we examined complementation of an E. coli recJ mutation by the recJ+ gene from A. baylyi. The recJ ORF of A. baylyi was amplified by PCR and cloned into the broad-host-range expression vector pRK415iq (Table 1) downstream of the lac promoter controlled by LacI, giving plasmid pEKX1. This plasmid was transferred by electroporation into E. coli strain BT544, which is recJ recD and therefore highly UV-sensitive (Lovett et al., 1988). As shown in Fig. 1, the presence of pEKX1 in BT544 increased the survival of cells measurably at UV doses of 54 and 72 J m–2. When the cells were incubated for 1 h in the presence of 1 mM IPTG for derepression of the recJ gene before irradiation, the survival was significantly higher at low and high UV doses compared with the parental strain BT544 (Fig. 1). The presence of the vector plasmid did not influence the UV sensitivity of the strain, with IPTG in the growth medium before irradiation or without (data not shown). These observations indicate that the recJ+ gene of A. baylyi can at least partially complement a recJ null mutation of E. coli, suggesting that the RecJ protein of A. baylyi has a function in recombinational DNA repair similar to that of the RecJ DNase of E. coli. The only partial nature of complementation could be due to a suboptimal chemical environment in the cytoplasm of E. coli for the activity of the enzyme from A. baylyi, or be due to limited cooperation of the RecJ of A. baylyi with other components of the E. coli recombinative DNA repair machinery as a result of divergent amino acid sequences of the proteins. A partial interspecific complementation of an E. coli recJ mutant by a recJ gene from Methanococcus jannaschii has also been reported (Rajman & Lovett, 2000).
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recJ mutant is transformation-proficient and not UV-sensitive), suggesting that the RecJ protein was not involved in the recombinational repair of UV damage or that its function was taken over by another enzyme with a redundant activity. Also, the generation time of EK4 cells during exponential-phase growth in broth medium (48 min) was indistinguishable from that of the wild-type cells (46 min) and the viability of EK4 cells was as high as that of the parental strain (Table 3). The transformation frequency of competent EK4 cells with chromosomal trp+ DNA was unimpaired (Table 3). We also used a 1.5 kbp PCR-amplified DNA segment of the A. baylyi trp+ chromosome for transformation which covered the trpE27 mutation present in our strains. With the shorter DNA fragments any intracellular exonucleolytic degradation of transforming DNA could perhaps become apparent as a marker loss. That transformation by PCR fragments does not depend on the intracellular hybridization of two fragments of different polarity for recombinational integration (two-hit mechanism) was shown by the direct proportionality (one-hit mechanism) between DNA concentration (from 0.1 to 300 ng ml–1) and the number of transformants formed in wild-type and mutant strains (data not shown). With the 1.5 kbp fragment the transformation frequency of the recJ mutant was 50 % higher than that of the wild-type (Table 3). Although observed repeatedly, the increase was not significant by a Student's t-test. A strain overexpressing recJ+ from plasmid pEKX2 was not changed in its transformation with chromosomal DNA and with the 1.5 kbp PCR fragments (not shown).
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recJ, to give the recBCD deletion mutant KOM18. This strain grew slowly (generation time 71 min) and had a low viability in overnight cultures (Table 3). This phenomenon is in accord with the inability of E. coli recBCD mutants to effectively rescue spontaneous broken replication forks by recombination repair, resulting in slow growth and low viability (Kuzminov, 1999; Miranda & Kuzminov, 2003). The strain KOM18 was UV-sensitive (Fig. 2), giving a survival of about 10–4 at a UV dose of 72 J m–2, which corresponds to the sensitivity of an E. coli recBCD mutant (Ivan
i
-Bae et al., 2005).
With chromosomal trp+ DNA, the transformability of the recBCD strain was fivefold lower than that of the wild-type (Table 3). The significant reduction of transformation did not result from low viability because we report transformation frequencies relative to total c.f.u. rather than absolute numbers. A very similar result was obtained with another set of strains as recipients. In these strains, a cassette (2.3 kbp) consisting of an nptII gene with an internal 10 bp deletion (nptIIi; KmS) and the eukaryotic transcription terminator tg4 were present in the alkM (alkane monooxygenase) gene, preventing growth on minimal medium with hexadecane as the carbon source. A recBCD derivative of this strain was transformed with wild-type DNA to alkM+ (selection on M9 medium with hexadecane as carbon source) at a frequency of 1.1 (±0.6)x10–4, which was 0.21 of that obtained with a recBCD+ derivative [5.4 (±2.3)x10–4]. These data show that the recBCD deletion decreased transformation by chromosomal DNA of recipients having a point or an insertion mutation.
With the 1.5 kbp DNA fragment covering trpE+, the transformation frequency of strain KOM18 was 3.3-fold higher than that of the wild-type (Table 3). Similarly, the transformation of the strain with the cassette insertion in alkM with a 1.5 kbp PCR fragment covering part of the cassette with an nptII+ gene (selection: KmR) was 7-fold higher in the recBCD derivative [4.0 (±2.7)x10–3] than in the recBCD+ strain [5.7 (±0.9)x10–4]. The higher transformation frequency in the recBCD mutant indicates that during transformation with short DNA molecules, the RecBCD enzyme has an anti-transformation activity, eliminating 70 % or more of the transformation events.
A recD mutant is transformation-proficient
recD mutants of E. coli are very similar to wild-type cells in their recombination proficiency, UV resistance and viability (Amundsen et al., 1986; Biek & Cohen, 1986). We constructed a recD mutant of A. baylyi (strain EK6) in which the recB and recC genes are expressed as in the wild-type, because recD is the last gene of the recBCD operon (Barbe et al., 2004). The viability of the recD mutant was at the same level as that of the wild-type (Table 3). The survival of the strain after UV irradiation was slightly higher than that of wild-type cells at UV doses of 54 and 72 J m–2 (Fig. 2). The transformation of the recD mutant with both the chromosomal DNA and the 1.5 kbp trp+ PCR fragment was not different from that of the wild-type (Table 3). These results were confirmed by transformation of a recD derivative of the strain with the nptIIi gene cassette in the alkM gene (see previous section) by chromosomal DNA [2.4 (±1.2)x10–4 in wild-type and 2.3 (±0.6)x10–3 in recD] and 1.5 kbp PCR fragments covering nptII+ [0.6 (±0.1)x10–3 in wild-type and 1.0 (±0.3)x10–3 in recD]. It is concluded that the RecBC(D–) enzyme supports recombination required for transformation with long and short DNA fragments to the same level as the RecBCD enzyme does. Moreover, the anti-transformation effect produced by the RecBCD enzyme with the short transforming DNA fragments is also produced by the RecBC(D–) enzyme. The recBCD deletion abolished this effect.
recBCD recJ and recD recJ double mutants are non-viable
We wanted to see if a recJ mutation would further decrease the transformability of a recBCD strain. In order to construct a recJ recBCD : : nptII sacB double mutant by transformation, competent cells of strain EK4, which are recombination-proficient, were treated with the linearized DNA of plasmid pKH83 and selected for kanamycin resistance. Only rare and small colonies of variable size were obtained on the Km medium, which mostly did not grow on the same medium upon restreaking. Those colonies that eventually grew were only weakly resistant to Km. Among these colonies (50 tested) SucS clones were not found, suggesting that the colonies were formed by spontaneous partial KmR mutants. Thus, recJ strains with the integrated recBCD : : nptII sacB allele were not obtained. We then tried to add the recJ : : nptII sacB allele to a recBCD mutant by transforming strain KOM18 with linearized pEK2 DNA. Again, after several attempts, stable KmR clones, which were also SucS, were not obtained. From both approaches it appeared that null mutations of recJ and recBCD are not combinable in the same genome.
We tried to combine the recJ and recD mutations, but these attempts were also not successful. When strain EK4 was transformed with linearized pEK5 [recD : : (nptII sacB)] DNA or strain EK6 was transformed with linearized pEK2 DNA, KmR and SucS clones were not obtained (50 and 35 colonies tested, respectively). In these experiments, small colonies of variable size and transient or permanent partial resistance against kanamycin appeared on the plates as described above. The results suggested that recJ and recD null mutations were also incompatible.
If recJ recD strains were not obtained because they are non-viable, it should be possible to construct them when one of the deleted genes was provided in trans. This was the case. When strain EK4 with the recJ+-expressing plasmid pEKX1 was transformed with linearized plasmid DNA containing the recD : : nptII sacB allele, the majority of the KmR transformants had the recD+ gene replaced by the recD substitution allele (13 out of 15 transformants tested). These strains displayed a stable and strong KmR phenotype. A corresponding result was obtained when the recD strain EK6 carrying pEKX1 was transformed with linearized pEK2 DNA. Then the majority of KmR transformants had recJ+ replaced by the recJ : : nptII sacB allele (11 out of 13 colonies tested). As confirmed by PCR controls, in both attempts true genomic recD recJ double mutants were obtained. In these strains the plasmid pEKX1 was extremely stable. After 45 generations without selection for the plasmid by tetracycline in the LB medium, plasmid-free segregants were not detectable (less than 0.1 %). In M9-succinate medium with tryptophan the cells grew only to about 6x108 cells ml–1. Then the cells were mostly filaments and only about 10 % were able to form colonies on LB. All colonies (500 tested) consisted of TcR cells. The results indicate that in minimal medium, lethal sectoring occurs and survivors always maintain the plasmid. In a control experiment, the recJ single mutant with pEXK1 produced plasmid-free segregants amounting to 50 % of cells after 12 generations grown without tetracycline selection. These observations support the view that the double mutant cells became non-viable as soon as the plasmid with recJ+ was lost. Similarly to the recD : : nptII sacB allele, the recBCD : : nptII sacB allele could also be integrated by transformation in strain EK4 carrying pEKX1 (data not shown).
Deletion mutants of several putative helicase genes were viable even in the presence of recJ or recBCD
In E. coli, RecJ is thought to cooperate with a DNA helicase such as RecQ to initiate recombination, presumably by degrading the 5' strand during helicase action, leading to a recombinogenic 3' single-strand tail (Mendonca et al., 1993; Kuzminov, 1999). We hypothesized that the absence of the RecBCD helicase plus DNase activities in a recBCD strain was compensated by the RecJ DNase activity together with a helicase. A gene encoding a protein homologous to RecQ from E. coli or from other bacteria could not be identified in the A. baylyi genome by BLAST searches (Altschul et al., 1990). Also, no genes encoding putative homologues of UvrD (helicase II) or HelD (helicase IV) were found. However, several other putative helicase genes were identified which could encode candidate enzymes for cooperation with RecJ. Two essential replication-associated helicases, DnaB (ACIAD2433; 50 % identity and 71 % similarity) and Rep (ACIAD0900; 49 % identity and 70 % similarity) were not considered, and insertional inactivation of the latter was not successful (data not shown), supporting the assumed essential function. Among the remaining putative helicase genes, four appeared likely to encode enzymes functioning together with RecJ because these enzymes had similarity with experimentally confirmed helicases, and corresponding enzymes were present in other prokaryotes as detected by TBLASTN searches. These genes were: (i) ACIAD0379 (overall 29 % identity and 48 % similarity to the Homo sapiens F-box helicase 18 and overall 59 % identity and 74 % similarity to a putative helicase of Psychrobacter arcticus); (ii) ACIAD0495 (overall 28 % identity and 56 % similarity to the PcrA helicase of B. subtilis); (iii) ACIAD2014 (38 % identity and 56 % similarity to 379 aligned amino acids of the Homo sapiens PIF1 DNA helicase isoform beta and overall 47 % identity and 64 % similarity to a putative helicase of P. arcticus); (iv) ACIAD2185 (overall 29 % identity and 47 % similarity to DnaB helicase of E. coli). We cloned PCR-amplified portions of each of the four genes into plasmid vectors, inserted a resistance marker (ACIAD0379 : : tetA; ACIAD0495 : : tetA; ACIAD2014 : : nptII sacB; ACIAD2185 : : nptII) and used the constructs to inactivate the genomic genes of A. baylyi by allelic exchange through transformation. We found that the resulting four mutants (confirmed by PCR) were viable, suggesting that none of the genes was essential in otherwise wild-type cells. We further found that each of the four mutants was also viable when we additionally introduced the recBCD, recJ or recD allele, respectively. These results suggest that none of the putative helicases solely complements along with recJ+ the recBCD mutation. It remains unknown if any of the putative helicases operates in recombinational pathway(s) or if several of them have redundant functions during recombination. Our data do not exclude the possibility that the Rep helicase is involved in transformation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Vijayakumar & Morrison, 1986; Campbell et al., 1998). On the other hand, the unimpaired transformation of the recJ mutant also indicates that RecJ is not essential for the homologous recombination to integrate the transforming DNA, including the trimming of single strands to produce ligatable structures of DNA. However, the trimming of a single-stranded non-homologous part of otherwise homologous DNA is performed by RecJ as recently observed (Harms et al., 2007), but in the experiments presented here the transforming DNA was fully homologous. In contrast to the findings with recJ, the absence of RecBCD significantly influenced transformation, although the enzyme is known to interact in vivo with duplex DNA ends. The low transformability of the recBCD strain with large chromosomal DNA fragments indicates that, in wild-type cells, the RecBCD enzyme is required for about 80 % of transformation events. At the same time, the enzyme prevents most of potential transformation events elicited by the 1.5 kbp DNA fragments covering the trpE27 marker (or the nptIIi marker). Further, since the recD mutant is not different from recBCD+ cells in the transformation with chromosomal or 1.5 kbp DNA fragments, it is concluded that (i) in the recD mutant the presumably remaining RecBC(D–) helicase, along with an unknown DNase (possibly RecJ), functions as efficiently as the RecBCD enzyme and that (ii) the phenotype of the recBCD mutant results from the elimination of both the helicase and the DNase of RecBCD.
How can the role of the recombination-initiating duplex DNA exonuclease be reconciled with the genomic integration of the single-stranded donor DNA present in the cytoplasm after DNA uptake? One possibility could be that the single-strand is converted into a duplex, which would be the recombination substrate for RecBCD. Initiation of replication on a single strand in the absence of a replication fork or a D-loop is normally not possible. However, in the B. subtilis dnaB75 mutant, it was observed that the single-stranded replication intermediate of plasmid pC194 was converted into duplex DNA independently of the presence of a primosome assembly site and PriA protein, suggesting that complementary strand synthesis could initiate at any place on single-stranded DNA (Bruand et al., 2001). Partial conversion of the linear plasmid DNA taken up into duplex DNA was also one of the explanations of plasmid monomer transformation in B. subtilis (Michel et al., 1982). Conversion of a single strand into duplex DNA is probably a minor process in transformation with genomic DNA, because the huge mass of single-stranded DNA internalized by competent B. subtilis remains single-stranded until it is degraded if recombination is prevented by absence of RecA activity or nucleotide sequence homology (Dubnau et al., 1973; te Riele & Venema, 1982). Therefore, we propose a model in which single-strand integration occurs during transformation and frequently leads to a DSB requiring DSB end repair, performed mainly by the RecBCD pathway and, to a smaller extent, by alternative route(s). Our first assumption is that the single-strand 3' and 5' ends are often integrated into the recipient DNA by independent events (Fig. 3a–c). If the two integrations occurred simultaneously (leading to the covalent joining of 3' and 5' ends to resident DNA at about the same time), a round of replication would segregate the donor and recipient strands, resulting in one transformant and one parental genome (Fig. 3d). Our second assumption is that the integrations of the 3' and 5' ends mostly occur with a time difference. If during this period the replication fork passes through the region of the chromosome with a one-side-integrated donor DNA (constituting a strand interruption), a DSB would be formed (Fig. 3e, g), along with disassembly of the replisome (Kuzminov, 2001). The DSB would be formed irrespective of the replication fork approaching from the side with the fused donor DNA end (Fig. 3b, i) or the not-yet-fused end (Fig. 3b, ii). Reconstitution of a replication fork will require DSB end repair and new assembly of the replisome (Kuzminov, 1999). Depending on the location of the genetic marker on the transforming DNA and the relative direction of the replication fork during DSB formation, the marker will be either on a duplex DNA end (Fig. 3e) or on a single-stranded DNA end (Fig. 3g). When the marker is located on the duplex end it can be integrated during DSB end repair (Fig. 3f). If the marker is present on the single-strand tail it will be lost (Fig. 3h) or may be integrated by a second attempt of strand fusion of the DNA end after replication fork restoration, if the marker-containing single strand is preserved long enough. Even when present on the duplex DNA end, the marker may be lost if located close to the DNA end, which will be resected by RecBCD until the enzyme reaches a Chi-sequence, where its duplex DNA degradation is attenuated and the single-strand transfer is initiated. These considerations would explain why transformation with chromosomal DNA is decreased in a recBCD strain which is impaired in DSB repair. The approximately 20 % remaining transformation events in the recBCD deletion strain of A. baylyi may result from a DSB end repair system not dependent on RecBCD and/or from strand integrations occurring without a DSB (Fig. 3a–d). The model described in Fig. 3 is compatible with the observations that donor DNA is recovered up to 30 min after uptake into the recipient cell as single-stranded DNA, and only after hybridization and covalent fusion with resident DNA increasingly as duplex DNA (Vovis, 1973; Kooistra & Venema, 1976). Remarkably, in the recD strain transformation was not affected, suggesting that any DSBs produced through transformation or spontaneous replication fork breakdown are repaired efficiently. In this repair the RecBC(D–) helicase could act together with RecJ, which would degrade the unwound 5' strand to generate a recombinogenic 3' single-strand overhang. The requirement for RecJ in the recD strain for transformation could not be tested because of the non-viability of the recJ recD double mutant. Since transformation in a recD mutant is as high as in the wild-type (in which RecBCD-DNA resection occurs, eliminating the marker in a certain proportion of events), this suggests that the presumed resection of the duplex DNA end by the RecJ DNase (or other exonucleases) is roughly similar to that occurring in wild-type by RecBCD.
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). Compared to transformation with chromosomal DNA, the transformation with PCR fragments was threefold lower. Two things must be considered here. First, the marker on a small DNA fragment might be directly inactivated by the double-strand cleavage event during DNA uptake or one homologous flank might be shortened, leading to lower recombinational integration. Second, in the PCR-amplified DNA, a much higher number of DNA molecules carry the genetic marker (6x1010 per 100 ng) than in chromosomal DNA (2.7x107 per 100 ng). These two influences could obscure an effect of RecBCD and RecBC(D–) on transformation with the 1.5 kbp fragments. However, there was the clear finding that, in the recBCD mutant, the transformation with the 1.5 kbp PCR fragments was increased 3.3-fold (trp+; Table 3) and 7-fold (nptII+), respectively, compared to the recBCD+ and recD strains. These data indicate that the alternate DSB end repair system active in the recBCD mutant does not resect as much of the duplex DNA end as RecBCD does or RecBC(D–) plus the unknown exonuclease (Table 3). It is not yet known which enzymes are necessary for the RecBCD-independent transformation process. RecJ is a candidate, but may also merely be required for viability.
What evidence do we have for proposing that both the 3' and 5' ends of transforming DNA integrate by independent events in A. baylyi and not that integration starts at one end and, by branch migration, runs through the entire molecule as is the case in S. pneumoniae (Claverys & Lacks, 1986)? First, as shown by transformation with heteroduplex DNA in A. baylyi, the donor DNA end which is ligated to the resident DNA within a homologous region can be the 3' or the 5' end (de Vries & Wackernagel, 2002), indicating that both ends can perform a homology search and undergo covalent ligation to resident DNA. Further, in a mismatch repair (MMR)-deficient mutant (mutS) of A. baylyi having the trpE27 single base exchange mutation, the transformation with chromosomal trp+ DNA was at best 1.5-fold increased compared to mutS+, indicating rare formation of a mismatch (K. Harms & W. Wackernagel, unpublished). Absence of mismatch formation would be expected if most transformation events occurred either by 3' plus 5' integrations followed by replicative segregation (Fig. 3a–d) or by the proposed DSB end repair model (Fig. 3e, f). A strong effect of MMR would be expected in the branch migration model of S. pneumoniae, in which absence of MMR increases transformation, depending on the marker, 10-fold or higher (Claverys & Lacks, 1986). In this context, it might be recalled that MMR in transformable bacteria is directed by gaps or strand interruption towards elimination of the donor strand (Claverys & Lacks, 1986). In Pseudomonas stutzeri, the transformation with genomic DNA of strains with single nucleotide exchange mutations was only 1.9-fold increased by mutS deficiency, but on average was more than 10-fold increased when a 1.5 kbp fragment was used (Meier & Wackernagel, 2005), indicating that mismatches were formed relatively rarely during transformation with large DNA molecules and more frequently with short fragments. Similarly, in B. subtilis, the transformation with large DNA molecules having a single nucleotide exchange was also only about 1.3-fold increased in an MMR mutant (Majewski & Cohan, 1998). Finally, transforming homologous single strands integrated in B. subtilis by independent invasion of both the 3' and 5' ends at sites with homology, and did not integrate by branch migration (Majewski & Cohan, 1999).
At present, we do not have any direct evidence for a time difference between the integration of the two ends of a transforming DNA molecule. In vitro studies have shown that 3' and 5' ends are about equally effective in E. coli RecA-promoted homology search and D-loop formation (McIlwraith & West, 2001). Although in A. baylyi both 3' and 5' ends were observed to integrate in vivo during transformation of wild-type cells (de Vries & Wackernagel, 2002), recent studies showed that in the recJ mutant junctions of donor with resident DNA occur more frequently and always at the 3' end within homologous regions (in these experiments the 5' end was integrated by an illegitimate recombination event; Harms et al., 2007). Possibly, the two ends of homologous DNA integrate with different efficiency producing a time difference, with the 3' end apparently being faster in homology finding, strand invasion and/or final ligation to recipient DNA.
RecBCD enzymes or their functional equivalents have a role in natural transformation of other bacteria. In N. gonorrhoeae, deletion of recB, recC or recD decreased transformation about 40-fold (Mehr & Seifert, 1998). In this organism RecD is probably required to constitute a functional RecBCD complex, because deletion of only the recD gene gave the same phenotype as the recB or recC deletions (Mehr & Seifert, 1998). Considering that RecBCD normally interacts with duplex DNA ends, which are not expected during single-strand assimilation, Mehr & Seifert (1998) proposed that RecBCD might act as a helicase on resident DNA, removing one strand to allow hybridization of the donor DNA strand. In add mutants of H. influenzae (deficient for the ATP-dependent DNase which is the RecBCD counterpart), the natural transformation efficiency was decreased about 10-fold (Kooistra & Venema, 1976) and 4- to 5-fold (Wilcox & Smith, 1975). From studies with labelled DNA, it was concluded that the Add enzyme acted after physical integration of donor DNA into the host genome, e.g. by removing a remaining tail or performing a repair step necessary for viability of the transformant (Vovis, 1973; Wilcox & Smith, 1975). The add mutations in B. subtilis decreased transformation up to 15-fold (Haijema et al., 1996) or no more than 3-fold (Fernández et al., 2000). The combination of addAB and recF mutations abolished transformation (Alonso et al., 1993; Haijema et al., 1996). Several add point mutants of S. pneumoniae displayed 6- to 7-fold reduced transformation efficiency compared to wild-type (Vovis & Buttin, 1970; Vovis, 1973), while in deletion mutants, impaired transformation was not observed (Halpern et al., 2004). Altogether, the role of RecBCD type enzymes in the repair of transformation-induced DSB proposed here could be a rather general contribution of RecBCD to transformation and would account for 66 to 98 % of transformation events.
The non-viability of the recJ recBCD and the recJ recD double mutants of A. baylyi was unexpected. In E. coli such double mutants are viable. The recD recJ mutants of E. coli are as highly UV-sensitive as recB or recC mutants and show reduced recombination (Lloyd et al., 1988; Lovett et al., 1988). Strongly reduced recombination was also found in a recD recJ mutant of S. enterica sv. Typhimurium (Miesel & Roth, 1996). The recB recJ double mutants of E. coli are extremely UV-sensitive and have lost the residual recombination observed in recB or recBC mutants (Lovett & Clark, 1984; Lloyd et al., 1987). A recB recJ mutant of S. enterica sv. Typhimurium not only displayed extreme UV sensitivity and recombination deficiency but also strongly reduced viability, attributed to severe deficiency in repair of spontaneous DNA damage and inactivated replication forks (Garzón et al., 1996). The non-viability of the A. baylyi double mutants therefore could indicate their inability to repair spontaneous DNA damage and arrested replication forks, both of which require recombination enzymes (Kowalczykowski, 2000; Michel et al., 2001, 2004). An inference from the non-viability of the recJ recBCD mutant could be that RecJ and RecBCD are essential components of separate repair pathways, of which one must be functional. The A. baylyi pathway being dependent on RecJ could be equivalent to the RecF pathway of E. coli. Genes homologous to other essential RecF pathway genes of E. coli besides recJ, including recF, recO and recR, were identified in the A. baylyi genome sequence (Barbe et al., 2004). However, the A. baylyi double mutants recBCD recO and recD recO were found to be viable (K. Harms & W. Wackernagel, unpublished), indicating that mutational inactivation of the hypothetical RecBCD and RecF pathways did not impair genome maintenance. Further, the helicase active in the RecF pathway of E. coli is mainly RecQ and, to lower extents, also UvrD and HelD (Mendonca et al., 1995), but genes homologous to recQ, uvrD and helD are lacking in A. baylyi. The elimination of four different putative helicase genes of A. baylyi did not reveal that one of them has a major function in a presumed alternate pathway, but they may act in a redundant fashion. The RecBC(D–) enzyme, presumably having no DNase activity, makes the DSB repair or/and replisome restart in a recD mutant dependent on RecJ. Therefore, the RecJ may not represent a separate pathway but merely functions as a redundant DNase in the RecBCD pathway (Lovett et al., 1988), and A. baylyi apparently has only this one pathway. In contrast, E. coli cells lacking essential functions of both the RecBCD and RecF pathway are viable, because they can apparently cope with spontaneous replication blocks due to a further back-up system (Michel et al., 2004). This notion is in accord with the recent finding of lethality of E. coli recD recJ mutants (Dermic, 2006), which have additional mutations in xonA (exonuclease I) and xseA (exonuclease VII), two genes that have no homologues in A. baylyi.
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ACKNOWLEDGEMENTS |
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Edited by: L. Jannière
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), BT125 (recD; 
), BT544 (recD recJ; 
), BT544 with pEKX1 (
) and BT544 with pEKX1 grown in the presence of IPTG (
). Data are means from three independent determinations; error bars represent standard deviation.
