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J Bacteriol. Jan 2002; 184(2): 592–595.
PMCID: PMC139588

Role of the RecBCD Recombination Pathway in Salmonella Virulence

Abstract

Mutants of Salmonella enterica lacking the RecBC function are avirulent in mice and unable to grow inside macrophages (N. A. Buchmeier, C. J. Lipps, M. Y. H. So, and F. Heffron, Mol. Microbiol. 7:933–936, 1993). The virulence-related defects of RecBC mutants are not suppressed by sbcB and sbcCD mutations, indicating that activation of the RecF recombination pathway cannot replace the virulence-related function(s) of RecBCD. Functions of the RecF pathway such as RecJ and RecF are not required for virulence. Since the RecBCD pathway, but not the RecF pathway, is known to participate in the repair of double-strand breaks produced during DNA replication, we propose that systemic infection by S. enterica may require RecBCD-mediated recombinational repair to prime DNA replication inside phagocytes. Mutants lacking both RecD and RecJ are also attenuated in mice and are unable to proliferate in macrophages, suggesting that exonucleases V and IX provide alternative functions for RecBCD-mediated recombinational repair during Salmonella infection.

Salmonella enterica is a gram-negative bacterial pathogen that causes gastrointestinal disorders and systemic infections in humans and livestock animals (29). A hallmark of Salmonella pathogenesis is the capacity of the bacterium to survive and proliferate within phagocytic cells (29). In vitro and in vivo studies have provided evidence that bacterial growth inside macrophages and neutrophils is required for systemic infection (3, 8, 18, 25, 31). Phagocytic cells synthesize DNA-damaging agents such as nitric oxide and oxygen radicals (37) as mammalian defense mechanisms against pathogens; hence, Salmonella must face the attack of compounds that challenge genome integrity (21, 33, 37). Active functions and mechanisms that protect Salmonella from oxidative stress inside phagocytes have been described recently (2, 7, 38). In addition, almost a decade ago Buchmeier et al. showed that mutants of S. enterica lacking RecA or RecBC functions are avirulent in the murine typhoid model and highly sensitive to oxidative compounds synthesized by macrophages, suggesting that recombination is required for the repair of oxidative DNA damage (4). This view is supported by the recent report that Escherichia coli uses recombinational repair for survival of exposure to nitric oxide (35).

Like E. coli and other bacterial species, S. enterica can perform recombination between homologous DNA molecules by means of two main processes or pathways, RecBCD and RecF (20). Each pathway has distinct DNA substrate preferences and requires a specific set of recombination functions (20). In this study, we have examined the ability of S. enterica recombination mutants to cause systemic infection in mice and to proliferate inside macrophages. The strains used lack recombination functions of the RecBCD pathway and/or the RecF pathway. Our data indicate that the RecBCD enzyme is the key function required for both systemic infection and growth inside macrophages. Suppressor mutations that restore recombination proficiency in RecBC mutants by activating the RecF pathway do not restore virulence, indicating that a process different from standard recombination is affected. Because the RecF pathway cannot functionally replace RecBCD-mediated recombination for the repair of double-strand DNA breaks (15, 36), the inability of RecBC SbcB and RecBC SbcB SbcCD mutants of S. enterica to grow inside macrophages may result from arrested chromosome replication upon DNA damage. We also show that the functions of the RecF pathway are not required for virulence; however, mutants lacking both RecD and RecJ are avirulent, suggesting that exonuclease IX (RecJ) can substitute for the exonuclease activity of RecBCD.

Tests for virulence of S. enterica serovar Typhimurium recombination mutants in BALB/c mice.

To identify recombination functions required for virulence in the murine typhoid model, BALB/c mice were challenged with recombination-deficient strains of S. enterica serovar Typhimurium (Table (Table1).1). In parallel, infections were performed with the virulent strain SV1445, a His+ derivative of the mouse virulent strain SL1344 (12). The insertion zeb-6312::Tn10dTc carried by strains SV4189 and SV4190 is a Tn10dTc insertion linked to the sbcB locus and has no effect on virulence (data not shown). The 50% lethal doses (LD50) shown in Table Table22 support the following conclusions. (i) Among the single mutants, the most attenuated is the RecBC mutant, which has an oral LD50 more than 104-fold higher than that of the parental strain. Actually, the RecBC mutant was unable to cause the death of any animal, even at the higher doses used (7.2 × 109 CFU). We also observed that strain SV4191 (recA1) was able to kill mice at doses 10 to 100 times lower than those of SV4188 (recB497::MudJ) (data not shown). The difference observed between the RecA and RecBC mutants may reflect the pleiotropy of recBC mutations; i.e., they impair growth in standard culture media and cause various kinds of DNA repair defects (1, 16, 19, 22). RecF, RecJ, and RecF RecJ strains were all virulent, suggesting that, aside from the RecA function, functions of the RecF pathway of recombination are not required for systemic infection. LD50 trials by intraperitoneal inoculation confirmed that RecA and RecBC strains are the only avirulent strains among the single mutants tested (data not shown).

TABLE 1.
Strain lista
TABLE 2.
Virulence of S. enterica recombination mutants in BALB/c mice

(ii) RecA RecF and RecA RecJ mutants were attenuated at levels similar to that of a RecA strain, confirming that RecF and RecJ functions are not required for virulence, regardless of the presence of RecA.

(iii) A RecBC RecF mutant showed attenuation at a level similar to that of a RecBC strain, thereby suggesting that the only recombination pathway required for systemic infection is that of RecBCD. A corollary is that the RecF pathway cannot provide the recombination functions required for virulence, not even when the RecBCD pathway is absent. LD50 trials with RecBC RecJ mutants were not feasible because of their low viability (11).

(iv) Unexpectedly, a RecD RecJ mutant turned out to be avirulent. This result suggests that either exonuclease V or IX is required for systemic infection and hence that RecJ can participate in RecBCD-mediated recombinational repair in the absence of the RecD subunit. Previous observations have suggested that RecD and RecJ provide alternative functions for recombinational repair of DNA damage in both Salmonella (11, 23, 27) and E. coli (39). Function redundancy may thus explain why RecD and RecJ single mutants are virulent (Table (Table2;2; see also reference 40).

Effect of suppressors of recB and recC mutations on the virulence of S. enterica.

Mutations that suppress the recombination defect of RecBC mutants without restoring the activities of the RecB and RecBC enzymes have been characterized for both E. coli and Salmonella (1, 16, 19). In E. coli, sbc mutations are known to activate the RecF and RecE pathways of recombination (19, 20). The prophage-associated recE gene (13) does not exist in Salmonella. Mutations in the sbcB locus of S. enterica suppress the recombination deficiency and the UV sensitivity of RecBC mutants by activating the RecF pathway (1). If combined with mutations in another locus, sbcCD, suppression of mitomycin C sensitivity is also observed (1). RecBC SbcB SbcCD mutants also regain the ability to grow normally in standard media (1). With these facts in mind, we examined the ability of sbc mutations to suppress the virulence defect of RecBC mutants of S. enterica. Both RecBC SbcB and RecBC SbcB SbcCD strains remained avirulent by the oral and intraperitonal routes (Table (Table3).3). The avirulence of the RecBC SbcB SbcCD mutant is specially significant, because these strains are recombination proficient in transductional tests, are resistant to both UV and mitomycin C, and do not exhibit growth defects (data not shown; see also reference 1). The inability of sbc suppressors to restore virulence in RecBC mutants confirms the idea that the role of RecBC in Salmonella virulence cannot be performed by functions of the RecF pathway. The hypothetical nature of this role is discussed below.

TABLE 3.
Effect of sbc suppressor mutations on Salmonella virulence in BALB/c mice

Ability of recombination mutants of S. enterica to proliferate in macrophages.

Growth of Salmonella inside phagocytes is a strict requirement for systemic infection (8). Furthermore, a correlation between the avirulence of S. enterica RecA and RecBC mutants and their impaired ability to repair DNA was established in the seminal study of Buchmeier et al.: RecA and RecBC mutants can grow within macrophages that do not synthesize reactive oxygen compounds (4). As expected, we also found a straight correlation between the virulence of S. enterica recombination mutants and their ability to proliferate in macrophages. Unlike the virulent recombination mutants, those with virulence defects when tested in mice (RecA, RecBC, RecD RecJ, RecBC SbcB, and RecBC SbcB SbcCD) also showed defects for intracellular proliferation within J774.A1 mouse macrophages (Fig. (Fig.1).1). A noteworthy observation is, however, that sbc suppressor mutations were unable to restore intracellular proliferation within macrophages. Since sbc mutations restore recombination proficiency by activating pathways other than RecBCD, our observations indicate that S. enterica requires recombinational repair via the RecBCD pathway to grow inside phagocytes. Because of the unique ability of the RecBCD pathway to repair double-strand DNA breaks (15, 17, 36), it seems logical to conclude that S. enterica relies on the RecBCD recombination pathway to repair this kind of DNA lesion inside macrophages. In fact, a recent study has shown the occurrence of double-strand breaks in bacteria growing inside J774.A1 macrophages (33).

FIG. 1.
Ability of recombination mutants of S. enterica to proliferate in J774.A1 macrophages. Growth, infection, and lysis of J774.A1 mouse macrophages were carried out as described elsewhere (9, 10). The bacterial strains used for infection were SV1445, SV1488, ...

What is the role of the RecBCD pathway in Salmonella virulence?

A tight association between DNA replication and recombination in phage T4 has long been known (28). In the last 5 years, the idea that DNA replication requires recombinational repair has been extended to both bacteria (14, 32) and Saccharomyces cerevisiae (24). Whenever a double-strand DNA break is produced, recombinational repair via the RecBCD pathway is required to prime DNA synthesis and hence for the resumption of chromosome replication (17, 36). Our observation that the RecBC enzyme is required for growth inside macrophages correlates with the extreme sensitivity of RecBCD mutants to NO (35) and with the occurrence of double-strand breaks among the DNA lesions found in E. coli cells grown inside macrophages (33). Since the RecF pathway cannot functionally replace RecBCD-mediated recombination for the repair of double-strand DNA breaks, sbcB and sbcCD suppressor mutations do not restore recombinational repair (17). Hence, the inability of RecBC SbcB and RecBC SbcB SbcCD mutants of S. enterica to grow inside macrophages may result from arrested chromosome replication upon DNA damage.

Our finding that RecD RecJ mutants are avirulent and unable to grow inside macrophages seems to indicate that, in the absence of the RecD exonuclease, recombinational repair via the RecBCD pathway can be carried out with the participation of RecJ. This situation is similar to that of Rep mutants of E. coli, which require either RecD or RecJ for double-strand break repair (36). Furthermore, there is evidence that RecJ is involved in the recovery of replication forks upon DNA damage (6) and in other RecBC-dependent recombination processes (27, 39).

In summary, we propose that S. enterica uses the RecBCD recombination pathway to repair DNA double-strand breaks produced during growth inside macrophages and that RecD and RecJ provide alternative exonuclease activities for the repair process. The absence of RecBC or both RecD and RecJ renders Salmonella avirulent. A tentative explanation, supported by abundant literature (reviewed in reference 15), may be that Salmonella needs RecBC-mediated DNA repair to prime DNA replication inside phagocytes.

Acknowledgments

This work was supported by grants from the Dirección General de Enseñanza Superior of the Government of Spain (PM97-0148-CO2), the European Union (QLK2-1999-00310), and the Comunidad de Madrid (08.2/0045.1/2000). M.G.P. is supported by a postdoctoral fellowship from the Comunidad de Madrid.

We thank Nello Bossi and John Roth for providing strains, Mike Mahan for discussions on recombination, and Andrés Aguilera for critical reading of the manuscript.

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