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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.

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.

View larger version (42K): [in this window] [in a new window]   FIG. 1.   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.

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).

View larger version (57K): [in this window] [in a new window]   FIG. 2.   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 pSVB20RvrecAKm^r (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.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Generation of an R. viridis recA strain. (a) Construction of the suicide plasmid pSVB20RvrecAKmr. 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.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   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.

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.