INTRODUCTION

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Mycobacterium bovis BCG is a live vaccine against tuberculosis that has been administered to more than one billion people worldwide (6). In addition, BCG is used as a nonspecific immunotherapeutic agent in cancer treatment (38, 41). Although BCG shows a great deal of geographic variability in its ability to protect against lung tuberculosis (17), in most trials, BCG revealed significant protection against early childhood tuberculosis and disseminated manifestations of the disease (6). M. bovis BCG has been suggested as an ideal delivery system for expression of foreign antigens due to the long persistence of BCG in the immunized host (1, 21, 23, 33, 48, 58).

Despite its widespread use BCG is known to cause severe infections in immunocompromised individuals (24, 45, 46, 52), indicating that this organism is endowed with residual virulence properties which may be manifested in the absence of an effective immune response. It is thought that the ability of BCG to survive for prolonged periods without causing progressive infection in immunocompetent individuals is an important component of its protective properties (2).

BCG is known to have undergone significant genetic rearrangements, and recent evidence suggests that major recombinational events resulting in duplication of large segments of the chromosome have occurred and are still occurring (3, 16). Thus, if BCG is to be retained as a vaccine in its own right, developed as a carrier for generating novel vaccines and still used as an immunotherapeutic agent, it is essential that a more genetically stable strain be developed.

Phagocytic cells are able to generate superoxide, hydrogen peroxide, and other reactive oxygen metabolites which are capable of damaging microbial DNA, proteins, and membranes (22). Microorganisms possess multiple defenses against oxidative stress including enzymes such as catalase and DNA repair systems, which repair damage resulting from oxidative stress (47). The enzyme catalase catalyzes the decomposition of toxic hydrogen peroxide to water and oxygen; a correlation between catalase activity and virulence has been observed for the Mycobacterium tuberculosis complex (7, 28).

RecA is the regulator of the error-prone DNA repair mechanism (SOS response) and a key element of homologous recombination (53). The RecA of the M. tuberculosis complex has an unusual structure, in that it contains a protein-splicing element, termed intein (10). Difficulties in achieving homologous recombination in the M. tuberculosis complex have, at least partially, been attributed to this unusual structure (34). However, recent data suggest that the M. tuberculosis RecA intein does not interfere with RecA function (18, 39). DNA repair mechanisms in general and RecA function in particular have been shown to be essential for the survival of intracellular pathogens by repairing DNA damage resulting from oxidative stress, e.g., in Salmonella enterica serovar Typhimurium (5).

In general, live vaccine strains should possess a RecA phenotype. (i) Mutant recA strains are genetically more stable than their recA⁺ counterparts, reducing concerns about major genetic changes resulting in an altered phenotype (25). (ii) RecA acts as an inducible positive regulator of interspecies gene exchange in bacteria (31, 32); a mutant recA allele averts reversion of virulence attenuation by interspecies gene transfer (35). (iii) Mutant recA strains are more sensitive to UV irradiation and other DNA-damaging agents and thus show reduced persistence in the environment (35). (iv) Expression of foreign antigens is more stable in a mutant recA background (43). However, if the lack of a functional RecA results in a reduced ability to survive in the host, the immunogenicity of, for example, BCG could be compromised.

To generate M. bovis BCG recA knockout mutants, we wanted to adapt a technique previously established successfully for Mycobacterium smegmatis (44). This technique is based on the dominant-negative selectable marker rpsL⁺, which confers streptomycin sensitivity to a streptomycin-resistant host, allowing the isolation of allelic replacement mutants from single-crossover recombinants. The successful adoption of this strategy would complement other techniques to generate targeted mutants in the M. tuberculosis complex which make use of a combined counterselection provided by sacB and a thermosensitive origin of plasmid replication (42). Here we describe the generation of an M. bovis BCG recA knockout mutant by use of rpsL as a counterselectable marker and report on the characterization of the mutant with respect to DNA repair, in vitro induced dormancy, and survival in vivo.

	MATERIALS AND METHODS

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DNA manipulations and isolation of plasmids. Standard techniques were used for DNA manipulation. All initial cloning procedures were performed with E. coli XL1-Blue MRF. Plasmids were prepared with a Qiagen plasmid preparation kit according to the manufacturer's recommendations. Plasmid DNA was dissolved in Tris-EDTA buffer in concentrations of 500 to 1,000 ng/µl.

Generation of suicide vectors. For the generation of suicide vector precA::aph-rpsL, the following cloning steps were performed. A 5.2-kb ApaI fragment from plasmid pEJ126 (10) containing M. tuberculosis recA was subcloned into the PstI site of plasmid pBluescript KSII() (Stratagene), resulting in plasmid pBluescript-recA. From this vector, a 1.3-kbp internal PstI fragment was replaced by a 1.3-kbp aph cassette isolated as a PstI fragment from plasmid pUC4K (Pharmacia), resulting in plasmid precA::aph. A fragment comprising the inactivated recA was removed by digestion with EcoRV and SpeI and cloned into ptrpA-1-rpsL previously digested with SacI, blunt ended, and subsequently digested with SpeI (44), resulting in suicide vector precA::aph-rpsL. The cloning procedures were confirmed by DNA sequencing.

Cultivation of mycobacteria. When cultivated on solid medium, M. bovis BCG was grown on Middlebrook 7H10 agar supplemented with oleic acid-albumin-dextrose (OADC) (Difco) for 3 weeks at 37°C. Tween 80 was added to liquid broth 7H9-OADC to avoid clumping; incubation was performed in a roller bottle for 10 to 20 days. Antibiotics were added to the following concentrations: kanamycin, 25 µg/ml; streptomycin, 25 µg/ml.

Southern blot analyses. For Southern blot analyses, 200 ng of genomic DNA was digested with an appropriate restriction enzyme, separated on an agarose gel, and treated according to standard protocols. DNA was transferred to a Hybond-N membrane (Amersham) and cross-linked by UV irradiation. DNA was hybridized to a recA probe (a 1.6-kbp ApaI-PstI fragment from pBluescript-recA) labeled with digoxigenin according to the manufacturer's instructions (Boehringer GmbH, Mannheim, Germany), washed under stringent conditions, and developed with an antibody directed against digoxigenin coupled with horseradish peroxidase.

Western blot analyses. M. bovis BCG strains were grown in 100 ml of Dubos broth and induced with ofloxacin (1 µg/ml) for 24 h, and cell extracts were prepared as described previously (39). Cell extracts corresponding to 30 µg of protein were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis through a 10% polyacrylamide gel, and the proteins were electroblotted onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore). The membrane was blocked with 10% nonfat milk in TTBS (20 mM Tris [pH 7.5)], 0.5 M NaCl buffer containing 0.1% Tween 20) and incubated with a 1:1,000 dilution of a mouse antiserum raised against the purified M. tuberculosis RecA protein. A mouse antibody conjugated to horseradish peroxidase (Dako) was used as secondary antibody. After being washed with TTBS, the blot was developed with diaminobenzidine reagent solution as described previously (11).

Generation of M. bovis BCG SMR1. To generate streptomycin-resistant mutants, M. bovis BCG (strain Pasteur) was spread on 7H10-OADC agar containing streptomycin (20 µg/ml). After 4 weeks of incubation, single colonies were picked. The genotypes of the streptomycin-resistant strains were determined by PCR-mediated amplification of rrs and rpsL. The sequencing of rrs and rpsL PCR products revealed a single A  G transition in rpsL codon 88 conferring an amino acid change from lysine to arginine.

Transformation of M. bovis BCG SMR1. M. bovis BCG SMR1 was grown in a 2-liter roller bottle containing 400 ml of 7H9-OADC-Tween until an optical density (OD) of 0.6 was achieved. One day before harvesting the cells, glycine was added to a final concentration of 1.5% (vol/vol), and cells were incubated for an additional 24 h. All the following steps were performed at room temperature. Cells were harvested by centrifugation, washed several times with 10% glycerol, and finally resuspended in a volume of 5 ml. For electroporation, 400 µl of competent cells was mixed with 1 µg of supercoiled plasmid DNA and electroporated (Gene pulser II; Bio-Rad) with the following settings: 2.5 kV, 1,000 ohms, 25 µF. After electroporation cells were resuspended in 4 ml of 7H9-OADC-Tween and incubated for 20 h with vigorous shaking at 37°C. Following incubation, appropriate dilutions were plated on selective agar. Single colonies were picked, restreaked, and grown in liquid broth.

EMS and MMS assay. 7H9-OADC medium containing ethylmethane sulfonic ethyl ester (EMS) or ethylmethane sulfonic methyl ester (MMS) was inoculated with a 1/50 volume of a freshly grown culture (OD, 0.5). After 6 days of incubation, the OD was determined. 7H9-OADC medium without alkylating chemicals served as a control.

UV irradiation assay. For the UV irradiation assay, 100-µl aliquots of a freshly grown culture (OD at 600 nm [OD₆₀₀] = 0.1) were placed in an inverted lid of a 24-well culture plate and put under a standard germicidal UV lamp (distance, 20 cm). Cells were irradiated for different time periods, and samples were removed and plated. Mean values and standard deviations from three independent experiments are shown. Appropriate dilutions of each culture were plated out in duplicate.

In vitro induced dormancy. Experiments were performed as described previously using an in vitro dormancy model (29). Briefly, screw-cap test tubes (20 by 125 mm) with a total fluid capacity of 25.5 ml were used. An early-log-phase culture was diluted to an OD₆₀₀ of 0.005 in a total volume of 17 ml of Dubos broth (Difco). Solid caps with latex liners were tightly screwed down (limited oxygen supply), and the cultures were gently stirred at 170 rpm for 20 days. Self-generated oxygen depletion was monitored via the decolorization of the oxygen indicator dye methylene blue. The growth of cultures was monitored by determining OD₆₀₀; viable counts were determined by plating appropriate dilutions on Dubos oleic acid-albumin-agar (Difco). In some cases, metronidazole was added at a concentration of 10 µg/ml. Mean values and standard deviations were determined from three independent experiments. Each experiment was carried out with duplicate cultures. Appropriate dilutions of each culture were plated out in triplicate.

Infections. BALB/c and nude mice (6 to 8 weeks old) were obtained from the breeding facility at the National Institute for Medical Research (Mill Hill, United Kingdom). M. bovis BCG strains were grown in Dubos broth. Logarithmically growing cultures were diluted in saline to an OD of 0.8; 0.2 ml (approximately 10⁶ CFU) was injected into the tail vein. Mice were sacrificed according to ethical guidelines at various times (three mice per BCG strain for each time point), and the spleens and lungs were removed, weighed, and homogenized. The suspensions were serially diluted in saline and then plated on 7H10 agar supplemented with OADC. The plates were incubated for 3 weeks. The results were calculated and expressed as CFU per organ.

	RESULTS

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Generation of M. bovis BCG recA Strain M. bovis BCG SMR1 (for a list of strains and plasmids, see Table 1) is a streptomycin-resistant derivative of M. bovis BCG; this strain has a mutation in rpsL codon 88 (Lys  Arg), a mutation known to confer a streptomycin-resistant phenotype.

Genetic analysis of transformants and counterselection. Nine of 11 kanamycin-resistant (KanR) clones investigated contained the aph cassette, indicating that these colonies arose from transformation with the suicide vector rather than representing spontaneous KanR mutants (data not shown). Genomic DNA was isolated and investigated by Southern blot analyses using a recA fragment as the probe (Fig. 1). Two of the nine transformants revealed a pattern indicative of a 5' single crossover at the recA locus (a 2.6-kbp fragment and a 4.9-kbp fragment; the wild type shows a 2-kbp fragment) and were subjected to counterselection on medium containing kanamycin plus streptomycin. Transformants resistant to kanamycin plus streptomycin (frequency, 10⁴ to 10⁵) were screened by PCR for the absence of the deleted recA intein coding sequence (7 of 40 transformants were investigated; data not shown). recA deletion was confirmed by Southern blot analyses. Following digestion with SmaI the kanamycin- and streptomycin-resistant transformants revealed a single 2.6-kbp fragment (Fig. 1), indicating a second crossover event, resulting in loss of the functional recA copy. For further investigations, one of the recA mutants was chosen.

View larger version (40K): [in this window] [in a new window]   FIG. 1.   (A) Southern blot analysis of the recA locus. Lane 1, parental M. bovis BCG; lane 2, single-crossover transformant obtained by transformation with plasmid precA::aph-rpsL; lanes 3 to 5, recA (recA::aph) knockout mutants obtained after counterselection of a single-crossover transformant on medium containing kanamycin plus streptomycin. Approximately 200 ng of genomic DNA was digested with SmaI and hybridized to a recA probe. (B) Schematic drawing of the BCG recA locus: the wild-type locus is shown along with the vector used for inactivation, a 5' single-crossover transformant, and a knockout mutant. S, SmaI recognition site.

Western blot analyses. Western blot analyses using an antiserum raised against M. tuberculosis RecA was performed with the parental strain, a single-crossover transformant, and the corresponding knockout mutant (Fig. 2). A single band with a molecular mass of approximately 40 kDa corresponding to the mature, spliced form of M. tuberculosis RecA was observed in extracts of both the parental strain and the single-crossover transformant but not in the recA mutant, indicating the absence of the RecA protein and thus confirming the data obtained by Southern blot analysis. A faint band with a mass of approximately 20 kDa possibly corresponding to a truncated N-terminal RecA fragment was detected in recA single-crossover transformants and in the recA knockout mutant.

View larger version (60K): [in this window] [in a new window]   FIG. 2.   Western blot analysis of RecA. Lane 1, parental M. bovis BCG; lane 2, single-crossover transformant; lane 3, recA knockout mutant. Approximately 30 µg of protein was separated on a polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and probed with an antibody raised against M. tuberculosis RecA.

In vitro characterization of M. bovis BCG recA. (i) M. bovis BCG recA mutant strains are sensitive to alkylating agents. One of the most noticeable phenotypes of recA mutant strains is their increased sensitivity to DNA-damaging agents (26). To investigate the physiological effects of recA inactivation, several tests with different DNA-damaging agents were performed. The ability of recA⁺ and recA mutant strains to grow in the presence of EMS or MMS was determined in liquid broth (Table 2).

(ii) M. bovis BCG recA mutants are sensitive to UV irradiation. Irradiation with UV light is frequently used to compare the levels of effectiveness of DNA repair mechanisms. Inactivation of recA rendered BCG sensitive to UV irradiation: the recA strain exhibited a 10,000-fold-decreased viability after 30 s of irradiation compared with a 25-fold decrease for the recA⁺ strain (Fig. 3). The survival rate decreased dramatically at higher irradiation dosages; very few mutant survivors were detected after 60 s of irradiation (<0.0001%), compared to about a 0.01% survival rate for the parental strain. These results support previous observations that, despite the presence of an intein, M. bovis BCG RecA is functionally expressed and promotes DNA repair mechanisms in mycobacteria (18, 40).

View larger version (11K): [in this window] [in a new window]   FIG. 3.   Survival after UV irradiation. Parental M. bovis BCG (), a recA single-crossover transformant (), and a recA knockout mutant () were irradiated with UV light for the indicated times. Following irradiation, the numbers of viable cells were determined by plating.

(iii) In vitro induced dormancy and metronidazole resistance. The obligate aerobe M. tuberculosis complex can enter an anaerobic dormant state in which it survives for extended periods of time (54). To determine whether recA is involved in the dormancy response, we investigated recA⁺ and recA mutant strains with respect to their abilities to survive in an in vitro dormancy model (57). Mycobacteria were grown in sealed and stirred tubes to achieve self-generated oxygen depletion, a signal which triggers entry into the dormant state. Growth of the cultures was monitored by measuring the OD; the number of cells surviving oxygen depletion was determined by plating. Self-generated oxygen depletion was judged by fading or decolorization of the indicator methylene blue.

View larger version (12K): [in this window] [in a new window]   FIG. 4.   Growth under dormancy conditions. Parental M. bovis BCG () and the recA knockout mutant () were grown under dormancy culture conditions. Growth was determined by measuring the OD. F and D, fading and complete decolorization of the methylene blue indicator, respectively.

View larger version (13K): [in this window] [in a new window]   FIG. 5.   Survival under dormancy conditions. Survival of parental M. bovis BCG and the recA knockout mutant under dormancy culture conditions was investigated by determining the numbers of CFU after 20 days of incubation in the presence or absence of metronidazole (MTZ; 10 µg/ml).

Survival of the M. bovis BCG recA mutant in mice. DNA repair mechanisms in general and RecA in particular are important virulence factors for the survival of intracellular pathogens, such as Salmonella (5). Although wild-type M. bovis BCG does not cause a progressive infection in mice, it does persist in tissue for a significant period of time. We thus investigated the effect of recA inactivation on the survival of M. bovis BCG in a high-dose animal infection model. BALB/c mice were infected by intravenous injection with M. bovis BCG recA⁺, the single-crossover transformant, and the recA mutant strain. After days 1, 28, and 84, organs (spleen and lung) were removed and homogenized and appropriate dilutions were plated on 7H10 agar. The plates were incubated for 3 weeks, and the numbers of CFU per organ were calculated. As shown in Fig. 6, the knockout mutant showed no difference in the course of infection compared to the wild-type strain, either in the spleens or in the lungs of infected BALB/c mice. These data indicate that RecA is not essential for survival in the high-dose mouse infection model.

View larger version (13K): [in this window] [in a new window]   FIG. 6.   Course of infection in BALB/c mice. Parental M. bovis BCG (), a recA single-crossover transformant (), and a recA knockout mutant () were injected into the tail vein (approximately 106 CFU/animal). The numbers of bacteria in spleens (A) and lungs (B) were determined at different time points.

View larger version (13K): [in this window] [in a new window]   FIG. 7.   Course of infection in nude mice. Parental M. bovis BCG (), a recA single-crossover transformant (), and a recA knockout mutant () were injected into the tail vein (approximately 106 CFU/animal). The numbers of bacteria in spleens (A) and lungs (B) were determined at different time points.

	DISCUSSION

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Infections with M. tuberculosis are a major cause of human morbidity and mortality. Vaccines are the most cost-effective intervention to prevent disease, and M. bovis BCG is a widely used vaccine; it can be given as a single dose at birth and confers long-lasting immunity. M. bovis BCG not only is used as vaccine against tuberculosis but also offers great potential for innovative approaches for development of polyvalent vaccines (1, 33, 48, 58). Genetic stability and in vivo persistence are of special importance for the use of live vaccines; a mutant BCG which is rapidly eliminated is unlikely to be an effective vaccine (2).

RecA is a multifunctional and ubiquitous protein involved both in general recombination and in DNA repair. Thus, RecA-mediated DNA repair mechanisms have been shown to be essential for intracellular survival and persistence (5), while simultaneously RecA-dependent recombination mediates genetic rearrangements resulting in increased genetic instability (25). As an inducer of the SOS response RecA regulates at least 20 genes, most of which are usually suppressed by LexA (36); mycobacteria possess the key elements of a functional SOS system (13, 37).

Investigations of M. tuberculosis RecA so far have been performed in vitro, with Escherichia coli and M. smegmatis (10, 11, 12, 18, 27, 40, 51). These investigations demonstrated that the mature RecA is able to promote DNA repair mechanisms and homologous recombination (18, 40). However, as no isogenic M. tuberculosis complex mutants were available, the possibility that a homologous gene, e.g., radA (Rv3585) (8), could compensate for RecA function could not be excluded. The results presented here show that BCG recA mutants are sensitive to DNA-damaging agents (DNA alkylation and UV irradiation) and thus have an in vitro phenotype similar to those of recA mutants of other species (36). These investigations suggest that M. bovis BCG has a nonredundant recA gene, which is essential to promote DNA repair mechanisms.

M. bovis BCG and M. tuberculosis are able to enter a dormant state (29). This response is triggered by slow self-generated depletion of oxygen (54). Entry into the dormant state is an adaptive process, as sudden oxygen depletion results in cell death (55). Experiments in an in vitro dormancy model were performed to investigate whether RecA is involved in dormancy survival. As the numbers of viable bacterial cells after oxygen depletion were essentially identical for the recA⁺ and recA mutant strains, RecA does not appear to play an essential role in entry, survival, or exit from the dormant state.

Originally developed as an antiparasitic agent, metronidazole has been recognized as an effective drug for treatment of infections with anaerobic bacteria. Metronidazole is also active against M. tuberculosis and M. bovis BCG when grown under anaerobic conditions (29, 56). Our investigations in the in vitro dormancy model demonstrated that M. bovis BCG recA knockout mutants show increased susceptibility to metronidazole. These results support previous findings that metronidazole acts by damaging DNA after reduction to form a toxic metabolite (15).

Numerous reports have demonstrated that recA represents an important virulence factor: RecA is involved in stress survival (14), mediates aerotolerance in microaerophilic bacteria (9), induces production of colicins, pyocins (36), and extracellular degradative enzymes (30), and mediates amplification of toxin genes (19). Most notably, Salmonella recA mutant strains are highly attenuated, both in cultured macrophage cells (4) and in a mouse infection model (5). This effect has been attributed to the DNA-damaging effect of the oxidative burst and the reduced ability of the mutants to perform DNA repair (47). However, a recA mutation does not necessarily affect bacterial virulence, as demonstrated, e.g., for Campylobacter jejuni (20), Corynebacterium pseudotuberculosis (43), Brucella abortus (50), and some Vibrio cholerae strains (49). It was thus of interest to investigate the contribution of RecA to the survival of M. bovis BCG in mice. The results indicate that RecA does not contribute to the establishment and maintenance of infection. This is an important finding since persistence of BCG following vaccination is thought to be a significant contributory factor to its immunogenicity; a mutant BCG which is rapidly eliminated is unlikely to be an effective vaccine.

In analogy to M. tuberculosis (3, 16) differences between strains of M. bovis BCG can be attributed to RecA-dependent genetic rearrangements. The BCG recA mutant constructed in this study is deficient in the major recombination pathway but is not affected in its in vivo survival. Due to the increased genetic stability of recA mutants, this strain is of interest as a tuberculosis vaccine and for further development of M. bovis BCG as an antigen delivery system for expression of foreign antigens.

The BCG mutant was generated by adapting an rpsL-based strategy which has previously been used to generate allelic exchange mutants in M. smegmatis (44). As transformation efficiencies are a critical issue, the original strategy has been modified to a two-step allelic exchange procedure incorporating successive steps of positive and negative selection. The procedure described is a valuable alternative to strategies which use sacB and thermosensitive vectors for the generation of allelic exchange mutants in mycobacteria (42).