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. 1998 Jun;180(12):3227–3232. doi: 10.1128/jb.180.12.3227-3232.1998

Cloning, Sequencing, and Characterization of the recA Gene from Rhodopseudomonas viridis and Construction of a recA Strain

I-Peng Chen 1, Hartmut Michel 1,*
PMCID: PMC107827  PMID: 9620976

Abstract

A recombination-deficient strain of the phototrophic bacterium Rhodopseudomonas viridis was constructed for the homologous expression of modified photosynthetic reaction center genes. The R. viridis recA gene was cloned and subsequently deleted from the R. viridis genome. The cloned R. viridis recA gene shows high identity to known recA genes and was able to complement the Rec− phenotype of a Rhizobium meliloti recA strain. The constructed R. viridis recA strain showed the general Rec− phenotype, i.e., increased sensitivity to DNA damage and severely impaired recombination ability. The latter property of this strain will be of advantage in particular for expression of modified, nonfunctional photosynthetic reaction centers which are not as yet available.

Rhodopseudomonas viridis is a gram-negative, photosynthetic, purple nonsulfur bacterium (10). Its photosynthetic reaction center (RC) mediates the conversion of light energy to chemical energy and is the key protein complex of photosynthesis. The R. viridis RC was the first membrane protein complex for which the structure has been determined at a near-atomic level (6, 13). Due to the structural data of the RC, our understanding of the molecular mechanism of light energy conversion has been greatly improved. However, for the acquisition of further information regarding structure-function relationships, studies on modified reaction centers are essential.

Normally, R. viridis grows under photoheterotrophic conditions; however, under microaerophilic conditions the bacterium can also grow chemoheterotrophically, albeit with a greatly reduced growth rate (14). Under phototrophic growth conditions, the RC protein is expressed at extremely high levels, whereas under microaerophilic conditions, expression of the RC is dramatically reduced, and thus isolation of the RCs is impossible. Studies on modified R. viridis RCs have therefore been severely limited due both to the difficulty in obtaining RCs from this bacterium under nonphotosynthetic conditions and to the fact that no suitable expression system has been available for production of nonfunctional RCs. However, such RCs are of great importance in studying the role of essential amino acids in more detail. Coexpression of mutated, together with wild-type, RC genes is therefore considered to be promising for obtaining nonfunctional RCs. A prerequisite for this kind of expression is a recombination-deficient strain. However, up to now very few studies on molecular genetic properties have been performed with R. viridis, and homologous recombination in this bacterium has not been investigated.

In many bacteria the recA gene is involved in homologous recombination and DNA repair. The first recA gene was isolated and sequenced from Escherichia coli K-12 (16). The gene contains 1,059 bp encoding a 38-kDa protein (22). In E. coli the RecA protein promotes homologous pairing and strand exchange between homologous DNA molecules. Furthermore, the RecA protein accelerates cleavage of the LexA repressor, which serves as the direct repressor of SOS genes. After proteolytic cleavage of the LexA repressor, various SOS genes which work coordinately in DNA repair in response to DNA damage are expressed at elevated levels (25). In addition to its indirect role in DNA repair, the RecA protein directly participates in DNA repair by promoting single-strand DNA exchange after replication of the damaged DNA (5, 17, 20).

Mutant strains constructed by inactivation of the recA gene in general show increased sensitivity to UV irradiation, as well as to chemical DNA-damaging agents such as methyl methanesulfonate (MMS). Furthermore, the ability to perform homologous recombination is severely reduced. Due to the extreme decrease in homologous recombination, recA strains are ideal hosts for homologous gene expression. Since no recombination-deficient strain has yet been reported for R. viridis, it was thus necessary to find out if, similar to many other organisms, a recA gene is present in R. viridis and, if so, to delete it. Here, we report the cloning of the R. viridis recA gene as well as construction of a R. viridis recombination-deficient strain which will serve as an appropriate host for homologous expression of modified RCs by stably maintaining modified host genes in trans.

Bacterial strains, plasmids, growth conditions, and general methods.

The bacterial strains and plasmids used in this work are listed in Table 1. E. coli strains were grown aerobically at 37°C in Luria-Bertani medium (10 g of Bacto Tryptone per liter, 5 g of yeast extract per liter, 10 g of NaCl per liter [pH 7.2]). Rhizobium meliloti 2011-2 (recA) and L33 (Rec+ [wild-type recA]) were grown under aerobic conditions at 30°C in RGMC medium (10 g of Bacto Tryptone per liter, 1 g of yeast extract per liter, 8 g of NaCl per liter, 0.3 g of CaCl2 per liter, 1 g of MgCl2 per liter, 1 g of glucose per liter [pH 7.4]). R. viridis DSM133 and the respective recA strain were grown phototrophically and anaerobically at 28°C in sodium succinate medium 27 (4) under cold-light bulbs (120 W for liquid cultures; 60 W for plates). An R. viridis pufC strain was grown microaerophilically. Appropriate antibiotics (ampicillin [100 μg/ml], chloramphenicol [30 μg/ml], gentamycin [60 μg/ml], kanamycin [20 μg/ml], streptomycin [500 μg/ml], and/or tetracycline [12.5 μg/ml]) at the indicated concentrations supplemented the growth media. Methods for DNA manipulation and transformation were carried out as described by Sambrook et al. (21). For colony and Southern hybridizations, 32P-labeled transcripts synthesized in vitro by T7 RNA polymerase were used as probes. DNA sequencing was performed by using the T7 sequencing kit from Pharmacia Biotech. The Genetics Computer Group software package (7) was used for analysis of DNA and protein sequences.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid Relevant genotype or characteristics Source or reference
Strains
 E. coli
  XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)] 2
 R. viridis
  DSM133 (ATCC 19567) Wild type 9
  pufC strain pufC derivative of DSM133; Kmr D. Oesterhelt
  recA strain recA derivative of DSM133; Kmr This study
 R. meliloti
  2011-2 recA derivative of strain 2011; Gmr 23
  L33 bioluminescent derivative of strain 2011; Smr Rec+ 24
Plasmids
 pBluescriptIIKS+ (pBSIIKS+) Cloning vector; Apr Stratagene
 pBSIIKS+RvrecA R. viridis recA coding sequence This study
 pBSIIKS+puhH-Tetr R. viridis puhH with an insertion of tetracycline cartridge This study
 pBS+ Cloning vector; Apr Stratagene
 pBS+RvrecAint 0.5-kb R. viridis recA internal fragment This study
 pBS+RvrecA 2.6-kb PstI fragment containing R. viridis recA This study
 pKV1 Apr Kmr 15
 pRK404 Broad-host-range vector; Tetr 8
 pRKRvrecA 2.6-kb PstI fragment containing R. viridis recA This study
 pSVB20 Cloning vector; Apr 1
 pSVB20RvrecA 2.6-kb PstI fragment containing R. viridis recA This study
 pSVB20RvrecA−Kmr See Fig. 3a This study
 pUC18 Cloning vector; Apr 18
 pUC18RvrecAint 0.5-kb R. viridis recA internal fragment This study
 pUC18RvrecA 2.6-kb PstI fragment containing R. viridis recA This study
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Cloning and sequencing of the R. viridis recA gene.

Amino acid sequence comparison of the RecA proteins from a multitude of organisms revealed extremely high sequence similarities (12, 20). Of the 350 amino acids of different bacterial RecA proteins, about 100 residues were found to be absolutely conserved in all known sequences. Therefore, for the amplification of an internal part of the R. viridis recA gene, PCR primers corresponding to two of the most conserved regions were chosen. The distance between these two PCR primers corresponded to 150 amino acid residues. In order to reduce the degeneracy of these oligonucleotides, preferential codon usage was applied for the 5′ ends, whereas at the 3′ ends all possible codons were used for generating the PCR primers. Both 29-mer primers revealed a 384-fold degeneracy. The coding-strand primer was based on the sequence of amino acids 32 to 38 (amino acids are numbered according to the alignment in Fig. 1b) with the sequence 5′-AAT CTA GAT TCG G(C,T)A A(G,A)G G(A,C,T)T C(G,A,T,C)(G,A) T(G,A,T,C)A TG-3′. The complementary-strand primer was based on the sequence of amino acids 189 to 183 and had the sequence 5′-AAT CTA GAC TT(G,A) CG(T,C) A(G,A,T)(T,C) GCC TG(G,A,T,C) (G,C)(A,T)C AT-3′. For cloning purposes, recA gene-unrelated sequences (underlined) were introduced into both primers to create XbaI recognition sites in the PCR fragment. Using these primers for PCR, a 500-bp fragment was amplified from the R. viridis genome, and this PCR fragment was cloned and sequenced. As expected, the fragment contained a portion of a recA gene which showed 60 and 70% identities to the E. coli and R. meliloti recA genes, respectively.

FIG. 1.

FIG. 1

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The R. viridis recA gene. (a) Restriction map and ORFs identified in the R. viridis recA gene region. The orientation of each ORF is indicated by an arrow. The probe recAint (PCR fragment) for Southern analysis is shown above the schematic diagram. Abbreviations: P, PstI; H, HindIII; S, SalI; Sm, SmaI; Sp, SphI; X, XbaI. The XbaI sites were introduced by PCR primers. (b) Multiple sequence alignment of RecA proteins. The deduced primary sequences were aligned by the multiple sequence alignment program Pileup (Genetics Computer group software, version 7). The numbering of residues begins with a methionine residue, the N-terminal residue in the deduced Rhodobacter capsulatus RecA sequence. Dots represent deletions in the sequences. From top to bottom, the bacterial strains are as follows: R. sphaeroides, R. capsulatus, R. viridis, R. meliloti, and E. coli. The derived consensus sequence (Con) is indicated in the bottom row. Lowercase letters indicate partially conserved residues; uppercase letters indicate invariant residues. Invariant residues are indicated by boldface type. DNA sequences were obtained from GenBank under the following accession numbers: RsrecA, X72705; RcrecA, X82183; RmrecA, X59957; EcrecA, J01672.

In order to clone the recA gene from R. viridis, the genomic DNA of R. viridis was digested with BamHI, EcoRI, and PstI and subjected to Southern analysis. An in vitro-synthesized transcript of the PCR fragment served as a probe (probe recAint [Fig. 1a]). Hybridization signals of approximately 10 kb were detected when the R. viridis chromosomal DNA was digested either with BamHI or EcoRI. When the DNA was digested with PstI, a 2.6-kb fragment hybridized to the probe (data not shown). For cloning of the complete gene, a size-selected R. viridis PstI genomic library was constructed by cloning the PstI fragments in the size range of 1 to 5 kb into pUC18. Subsequently, colony hybridization of this size-selected genomic library was performed. From more than 2,500 PstI fragment recombinant clones, only one clone scored positive after hybridization with the PCR probe. The plasmid (pUC18RvrecA) from this clone was isolated and analyzed. The restriction map of the 2.6-kb fragment present in this plasmid is shown in Fig. 1a.

Sequence analysis of the complete PstI fragment revealed three complete open reading frames (ORFs) and one partial ORF (Fig. 1a). The third complete ORF (nucleotides 1399 to 2445) is located in the 3′ part of the PstI fragment, consists of 1,047 bp, and showed high sequence identity to known recA genes. Similar to some bacteria, such as E. coli and R. meliloti, an SOS box (25) with the sequence CTG-N10-CAG, known to be the binding site for the LexA repressor, is located 100 bp upstream of the start codon of this ORF. The amino acid sequence deduced from this ORF revealed high identity to bacterial RecA proteins (Fig. 1b). The highest identity was found with the R. meliloti RecA protein (79% identity; 93% similarity). The partial ORF (bases 1 to 282) coding for 93 amino acids was detected at the 5′ end of the fragment. An amino acid sequence search program (FASTA [19]) revealed that this ORF showed similarity to the family of chemotactic response regulatory proteins, CheY. In general, CheY proteins are 120 to 130 amino acids long and are involved in the transmission of extracellular stimuli to the motor of the flagella, which results in directional changes of movement. The sequence similarity of the putative R. viridis CheY to its counterparts from R. meliloti, Rhodobacter sphaeroides, and E. coli ranged from 52 to 59%. However, chemotaxis in R. viridis has not yet been studied. Cloning of the complete R. viridis cheY gene would be helpful for the investigation of chemotactic response in this organism. Sequence analysis of the other two complete ORFs downstream of that encoding CheY, ORF149 (nucleotides 527 to 976) and ORF79 (nucleotides 1043 to 1282), did not reveal any sequence similarity to known genes.

Interspecific complementation studies with the R. viridis recA gene.

As the sequence comparison showed that the R. meliloti recA gene has the highest identity with R. viridis recA, both on the DNA and protein levels, we used the R. meliloti recA strain (strain 2011-2) for complementation studies. For such studies with R. meliloti, the 2.6-kb PstI R. viridis DNA fragment was cloned into vector pRK404, a broad-host-range vector which can be propagated in R. meliloti. The resulting plasmid, pRKRvrecA, was introduced into R. meliloti 2011-2 by electroporation (3).

To test whether the R. viridis recA gene can restore the ability of DNA repair to R. meliloti, R. meliloti recA cells at exponential phase were treated with different doses of MMS or UV light, as described previously (3). In comparison to the R. meliloti recA cells transformed with vector pRK404 alone, R. meliloti cells containing plasmid pRKRvrecA were more resistant to both MMS and UV irradiation (Fig. 2a and c). Quantitative analysis of the restoration of the Rec+ phenotype in the R. meliloti recA strain is presented in Fig. 2b and d. In the case of UV irradiation, R. meliloti 2011-2(pRKRvrecA) cells were at least 10-fold more resistant than cells without pRKRvrecA at all UV doses used. At 10 and 20 s of UV exposure, the recovery of DNA repair ability was even greater (up to 100-fold [Fig. 2d]). Extremely elevated resistance was obtained after treatment of recombinant R. meliloti clones with MMS. At 0.5 mM MMS, the survival rate of the recombinant clones was 102-fold higher than that of R. meliloti 2011-2(pRK404) cells (Fig. 2b). The difference was even more pronounced at 1.5 mM MMS. At this MMS concentration, 7 × 105 surviving colonies were observed for the R. meliloti 2011-2(pRKRvrecA) clone; however, for the R. meliloti 2011-2(pRK404) clone, no survivors were detected. It is interesting to note that whereas the R. viridis recA gene only partially restored DNA repair ability after UV irradiation, this gene conferred to the R. meliloti recA cells MMS resistance comparable to the wild-type (recA+) level (Fig. 2b, d). Only at a very high concentration of MMS (5 mM) was the growth of R. meliloti 2011-2(pRKRvrecA) cells slightly inhibited, with smaller colonies formed (about five times smaller in diameter than R. meliloti L33 cells [Rec+; wild-type recA]) (not shown). The less efficient complementation after UV treatment in comparison to MMS treatment by the R. viridis recA gene might indicate that R. viridis recA-mediated recombination repair in R. meliloti cells is not comparable with that by the host recA gene. This assumption has still to be proved. Whereas the R. viridis recA gene was functionally complemented in R. meliloti, it was unable to complement the Rec− phenotype in E. coli XL1-Blue. Distinct differences in codon usage have been observed between R. viridis recA and E. coli recA. Some codons which are rarely used in E. coli (11), such as CTC (for Leu), TCG (for Ser), CGG (for Arg), and GGG (for Gly), are used in R. viridis recA at moderate to high levels. This difference in codon usage may cause problems in heterologous expression systems (26). However, whether this difference in codon usage between E. coli and R. viridis can explain the failure of complementation of the E. coli recA strain by the R. viridis recA gene is still unclear. In addition, low-level expression or instability of the R. viridis RecA protein in E. coli should be taken into account as a possible reason. Substantial differences in RecA functions between these two organisms are considered to be unlikely as the main reason for the failed complementation, since recA genes from both organisms have as high as 65% identity and 82% similarity on the protein level.

FIG. 2.

FIG. 2

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Complementation studies of the R. viridis recA gene in R. meliloti. (a) Restoration of MMS resistance to the R. meliloti recA strain (strain 2011-2) by R. viridis recA. R. meliloti exponential-phase cells (optical density at 580 nm, 0.5 to 0.6) were spread onto RGMC plates containing the appropriate concentration of MMS and were incubated at 30°C for 60 h. Approximately the same number of R. meliloti cells were spread onto plates containing 0, 0.5, or 1 mM MMS. (1) R. meliloti recA(pRK404) (vector only). Number of cells, ≈800. (2) One clone of R. meliloti recA(pRKRvrecA) with R. viridis recA. Number of cells, ≈1,200. (b) Quantitative analysis of complementation studies after MMS treatment. Cells were properly diluted and spread onto RGMC plates containing MMS and incubated, and CFU were counted. RmrecA+, R. meliloti L33; RmrecA−, R. meliloti 2011-2. At 1.5 mM and beyond this concentration, no viable cells were detected when 106 R. meliloti recA(pRK404) cells were spread. (c) Restoration of UV resistance to the R. meliloti recA strain by R. viridis recA. R. meliloti cell suspensions (from exponential-phase cells) were irradiated with UV light, and approximately the same number of cells was dotted onto RGMC plates and incubated. (1) R. meliloti recA(pRK404). Number of cells, ≈4 × 106. (2) One clone of R. meliloti recA(pRKRvrecA). Number of cells, ≈6 × 106. (d) Quantitative analysis of complementation studies after UV treatment. The cell suspensions were irradiated with different doses of UV light. After proper dilutions, cell suspensions were spread onto RGMC plates and incubated. Survivors are expressed as the ratio of CFU obtained from DNA damaging-agent-treated cells to untreated cells. All data are mean values of at least two independent experiments.

Construction and characterization of the R. viridis recA strain.

In order to obtain an R. viridis recA strain by gene disruption, a suicide plasmid was constructed. The cloned PstI fragment containing the R. viridis recA gene was subcloned into pSVB20, resulting in pSVB20RvrecA. To disrupt the R. viridis recA gene, a 550-bp SphI fragment which encompasses 100 bp of the upstream region and 450 bp at the 5′ end of the recA gene was deleted from plasmid pSVB20RvrecA by digestion with SphI endonuclease. Subsequently, a HindIII/BamHI DNA fragment bearing the kanamycin resistance gene isolated from plasmid pKV1 (15) was blunt-end ligated into the digested pSVB20RvrecA, resulting in pSVB20RvrecA−Kmr (Fig. 3a). This plasmid contains no origin of replication for R. viridis and therefore cannot be propagated in this host. By a double-crossover event between the disrupted and the wild-type recA gene copy, the chromosomal recA gene is expected to be inactivated by the kanamycin cartridge under appropriate selection pressure.

FIG. 3.

FIG. 3

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Generation of an R. viridis recA strain. (a) Construction of the suicide plasmid pSVB20RvrecA−Kmr. In this plasmid, the recA gene is interrupted by replacing a 550-bp SphI fragment with a kanamycin cartridge from Tn5. The vector part is not shown. Probe C, for Southern analysis, is indicated above the diagram. B, BamHI; all other abbreviations are the same as in the legend to Fig. 1. (b) Southern analysis of the R. viridis recA gene in wild-type and recA strains. R. viridis chromosomal DNA (1 μg) from the wild-type (lane 1) and recA (lane 2) strains was digested completely with the restriction enzyme PstI and separated on a 1% agarose gel. The DNA fragments were then transferred to a nylon membrane (Biodyne A; pore size, 1.2 μm) and hybridized with 32P-labeled transcripts (probe C). The approximate size of each DNA fragment is given on the right.

The suicide plasmid was electroporated (3) into wild-type R. viridis cells, and electroporants showing kanamycin resistance were considered to have the kanamycin cartridge integrated into the genome via homologous exchange and to be recA. Resistant colonies can arise due to incorporation of the kanamycin cartridge into the chromosome by either a double-crossover or a single-crossover event. Only a double-crossover event will result in inactivation of the chromosomal recA gene. Southern analysis with 32P-labeled probe C (Fig. 3a) was performed on chromosomal DNA prepared from positive clones. A double-crossover event between the wild-type recA located on the chromosome and the disrupted one on the plasmid was detected in these positive clones by Southern hybridization. When total DNA was digested with PstI, genomic DNA isolated from wild-type cells showed a signal at 2.6 kb corresponding to the fragment encoding the intact recA gene, whereas genomic DNA isolated from the positive clones (recA clones) revealed a signal at 1.4 kb, which indicated disruption of the recA gene (Fig. 3b). These results of the Southern analysis proved the presence of a single copy of the R. viridis recA gene in the wild-type genome and the successful disruption of it in the recA strain.

One of the clones possessing the recA genotype was further characterized. First, the R. viridis recA strain was examined for its DNA repair ability. The recA strain revealed extremely high sensitivity to MMS in comparison to the wild-type strain. At a concentration of 0.5 mM MMS the survival rate of this strain was 104 times lower than that of the wild type. At this MMS concentration, the survival rate of the wild type remained unchanged (Fig. 4a). It is worth mentioning that R. viridis is more sensitive to MMS than many other bacteria, such as E. coli and R. meliloti (Fig. 2). Also, as reported for several other bacterial recA strains, the R. viridis recA strain showed a slightly reduced growth rate compared to the wild-type strain (Fig. 4b).

FIG. 4.

FIG. 4

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Characterization of the R. viridis recA strain. (a) Viability of R. viridis recA+ (wild type [WT]) and recA (recA−) strains in the presence of MMS. Bacteria from liquid cultures at exponential phase were grown phototrophically in liquid cultures containing MMS at 28°C for 10 days. The survival rates were determined as described in the legend to Fig. 2. (b) Phototrophic growth of the wild-type R. viridis strain and the recA strain in liquid cultures. The cell density was determined by measuring the turbidity (Klett units) of each culture at the indicated time after anaerobic incubation at 28°C.

Inability to perform homologous recombination is another characteristic feature of bacterial recA strains. To test this impaired feature of the R. viridis recA strain, recombination between homologous sequences located in the chromosome and in the suicide plasmid pBSIIKSpuhH-Tetr was studied. Plasmid pBSIIKSpuhH-Tetr contains the R. viridis puhH gene with a tetracycline cartridge insertion. After electroporation (3) of R. viridis cells (109 cells) with the suicide plasmid (0.1 μg), in the wild-type Rec+ background (R. viridis pufC) 200 tetracycline-resistant colonies were obtained, whereas in the Rec− background (R. viridis recA) no tetracycline-resistant colonies were detected. These results clearly demonstrate the severely impaired recombination ability in the recA strain due to the inactivation of the recA gene. This recombination-deficient R. viridis recA strain will enable us to obtain nonfunctional or functionally impaired RCs labeled by tags for isolation and wild-type RCs in the same cells, with the wild-type RCs producing the energy for growth. Therefore, a study of structure-function relationships in R. viridis RCs now appears to be feasible without the previously mentioned limitations.

Nucleotide sequence accession number.

The nucleotide sequence of the R. viridis recA gene region has been deposited in the Genbank database under accession no. AF022175.

Acknowledgments

We thank Werner Klipp, Alfred Pühler, and Werner Selbitschka for providing R. meliloti strains and plasmids and Dieter Oesterhelt for the R. viridis pufC strain. We are also grateful to Laura Baciou, Alastair Gardiner, Carola Hunte, Jules Jacobsen, Helmut Reiländer, and Daniel Ungàr for constructive suggestions.

This work was supported by the Max-Planck-Gesellschaft and the Fonds der Chemischen Industrie.

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