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The RecA protein is ubiquitous among the bacteria studied and possesses one of the most conserved primary amino acid sequences. The function of the RecA protein as a mediator of homologous recombination and as a regulator of the inducible SOS response in both Escherichia coli and Bacillus subtilis has been elucidated in detail (33, 36). The primary role of RecA in E. coli, and perhaps in all bacteria, appears to be its participation in the housekeeping function of repairing and restarting stalled replication forks (5).

The importance of this housekeeping function is illustrated by the fact that E. coli PriA, which is disposable for initiation of DNA replication at oriC, but essential for restarting stalled replication forks, cannot be functionally inactivated without suffering a severe penalty: in growing cultures about 90% of priA mutant cells are dead (16, 25). Similarly, the absence of recombinational repair in E. coli recA mutants results in a 50% loss of viability (4). In recA mutants, the primary cause of death appears to be chromosomal degradation, presumably at sites of stalled DNA replication forks. About 10% of E. coli recA cells are anucleate, and an additional portion show signs of chromosomal degradation (29, 38).

During our analysis of site-specific recombination and chromosome dimerization in B. subtilis, we noted that mutations in recA did not completely suppress the nucleoid segregation defects associated with the inability to resolve dimeric chromosomes in ripX mutants (26). However, the recA mutation by itself had little effect on nucleoid morphology. Subsequently, we noted that about 12% of recA cells were viable during exponential growth and stationary phase ("viable" in this report refers to colony-forming ability). In addition, we have observed that a B. subtilis strain with a recA mutation developed a subpopulation of cells that displayed gross division defects and a loss of nucleoid integrity during the transition from exponential growth to stationary phase. These phenotypes were not seen in E. coli recA mutants. To our knowledge, there have been no previous reports of aberrant nucleoids in recombination-deficient strains of B. subtilis.

The parent strain in this study was BR151 (trpC2 lys-3 metB10). The SP phage strain used (YB886) and the SP addB72 strain (SL7576) were obtained from the Bacillus Genetic Stock Center and are derivatives of BR151 (37). SL7131, SL7360, and SL7370 have been described previously (26). The B. subtilis recF strain (SL7609) was made by transformation of BR151 with pSAS28, a pBluescript plasmid containing recF::spc (spc between the StyI sites within recF). The SP addB72 recF strain (SL7611) was made by transformation of SL7576 with pSAS28. W3110 is an E. coli recA::Tn9 strain (34). The recA-lacZ strain (SL7937) was made by transformation of BR151 with YB3001 DNA (28). comG-lacZ (SL7952) and comK-lacZ (SL7954) strains were made by transformation of BR151 with BD1960 and BD1991 DNA, respectively. Bacterial cultures were grown in Luria-Bertani (LB) medium or modified Schaeffer's sporulation medium (MSSM) (21) at 37°C with shaking at 150 rpm. Media were used fresh or were stored for short periods in the dark before use; glass vessels were used throughout. Medium volume was maintained between 6 and 9% of total flask volume. recA, recF, and addB mutants were grown in glass flasks wrapped in aluminum foil to reduce the effects of ambient light. Mitomycin C (MMC) was used at a concentration of 20 ng ml¹. Nucleoids were stained with 4', 6-diamidino-2-phenylindole (DAPI) and were observed and photographed as described previously (26). Cell and nucleoid measurements were performed as described earlier (26). -Galactosidase and sporulation-frequency assays were performed essentially as described previously (19). "T₀ " in these studies refers to the end of exponential growth. "T₁, T₂, T_3, " etc., are 1, 2, and 3 h, etc., after the end of exponential growth. Plating efficiencies were derived from dilutions of exponential- or stationary-phase cultures. Dilutions were made in Spizizen's minimal salts plus 55 mM glucose, 0.02% casein hydrolysate, 0.1% yeast extract, 20 µg of tryptophan per ml, and 50 µg (each) of methionine and lysine per ml. Plating was performed in duplicate on freshly prepared LB agar, and incubation was at 37°C in the dark. Only plates with between 30 and 400 colonies were used to determine viable counts. In general, Difco components were used in the media. Preliminary experiments indicated that growth rate, cell and nucleoid morphologies, and plating efficiencies of recA mutants were very similar when Oxoid components were substituted for Difco components.

Nucleoid and cell morphology phenotypes of B. subtilis rec mutants during exponential-phase growth. In Table 1, we present an assessment of nucleoid and cell morphologies for various rec mutant strains and their derivatives during exponential growth. A small proportion of the recA mutant (SL7360) cells showed evidence of partitioning failures and cell wall irregularities. The cell wall defects observed were typically bulges in the lateral wall (Fig. 1E and F). recA cells with cell wall defects almost always contained complex nucleoid structures (Fig. 1E and F). Because the nucleoids in cells with abnormal wall morphologies had a highly variable appearance, we did not attempt to quantify them in our scoring. Anucleate cells in the recA strain were rare (Table 1). In other experiments in which a larger number of cells were observed, the anucleate frequency of two different recA alleles was 0.16% (4 anucleate cells out of 2,500 cells scored). This is notable because a hallmark of E. coli recA strains is their high frequency of anucleate cells (~10%) (38). Anucleate cells in E. coli recA strains are thought to result from "rec-less" degradation of chromosomal DNA mediated by RecD (29).

View larger version (114K): [in this window] [in a new window]   FIG. 1.   Micrographs of B. subtilis strains treated with DAPI to visualize nucleoids. (A) Parent strain BR151 grown to stationary phase (T1.5) in LB medium. (B) recA mutant SL7360 grown to stationary phase (T1.5) in LB medium. (C) recA mutant SL7360 grown to stationary phase (T1.5) in sporulation medium MSSM. (D) Parent strain BR151 grown in LB medium with MMC (20 ng/ml) added at mid-exponential phase; photo was taken 1.5 h after MMC addition. (E) Example of cell wall defect in recA mutant SL7360 during the exponential phase. (F) Example of cell wall defect in recA ripX mutant SL7370 during the exponential phase. Arrows point to elongated nucleoids. The scale bar in panel A is 10 µm and applies to all panels.

Viability of B. subtilis rec mutants during exponential-phase growth. The generation time of the recA strain in LB medium was significantly longer than that of the parent strain (recA, 31 min; parent, 22 min). The plating efficiency of an exponentially growing B. subtilis recA strain was about 12% of that seen in the parent strain (Table 1). This score is in agreement with previous results for a recA mutant grown at 30°C (14) and is particularly striking given the mild nucleoid phenotype we have observed. These data are consistent with the concept that recA cells suffer from an inability to repair stalled DNA replication forks. The B. subtilis recA viability as indicated by plating efficiency is less than the 50% viability score reported for E. coli recA mutants (4). The viabilities of the addB and recF mutants were about 50 and 60%, respectively (Table 1). The addB recF double mutant was approximately 30% viable, reinforcing the concept that both recombination paths are utilized during exponential growth. The coparticipation of the B. subtilis AddAB and RecF recombination pathways has previously been shown in transformation and transduction assays (1). In addition, the double-mutant data suggest that a small amount of recombination repair can be mediated independent of AddB and RecF. We note that the presence of a ripX mutation (which impairs chromosome dimer resolution) in a recA strain lowered the plating efficiency twofold (from 12% to 5.6%). This result supports our observation that ripX mutations are not silent in a recA derivative of strain BR151 (26).

Cell division and nucleoid phenotypes of stationary-phase recA cells. After the end of exponential growth, recA mutants developed a subpopulation of filamented cells with elongated nucleoids (Fig. 1). These unusual cells have been measured as a separate class apart from normal cells to highlight the differences between the two cell types in the recA stationary-phase population. In Table 2, we show that the peak representation of the aberrant subpopulation is about 17% (a similar frequency was observed for a recA4 point mutant strain [data not shown]). The average nucleoid length in the aberrant subpopulation class was found to be two- to fourfold greater than that in the normal-appearing recA cells and in the parent strain. Also, the average cell length of the aberrant subpopulation was two- to threefold larger than that of unaffected recA cells and that of the parent strain (Table 2 and Fig. 1). We observed no signs of septum formation in the areas occupied by these distended recA nucleoids, either by phase-contrast microscopy or by fluorescent microscopy employing the vital membrane stain FM4-64 (22) (Fig. 1) (data not shown).

recA phenotypes during sporulation. As was the case in LB medium, only about 10% of sporulating recA cells were viable (Table 3). The same morphological phenotypes found in recA cells making the transition from the exponential phase into the stationary phase under nonsporulation conditions were also seen in postexponential recA cells in the sporulation-inducing medium MSSM (Fig. 1C). The elongated nucleoid structures seen in postexponential recA cells were distinguishable from sporulation-dependent axial filaments that form during stage I of sporulation in B. subtilis (3) as follows. (i) Axial filaments were not formed by the parent strain in LB medium. (ii) The cell and nucleoid lengths were both noticeably larger in affected recA cells than those of the parent strain in sporulation medium at stage I of development. (iii) Unlike stage I sporulation axial filaments, whose appearance is temporary, the phenotypes associated with recA cells that became apparent at the end of exponential growth were maintained for at least 7 h (data not shown).

Induction of recA during the stationary phase. Using a recA-lacZ transcriptional fusion, we have shown that at approximately the same time recA cells begin developing overt cell division and nucleoid irregularities, transcription of the recA gene in BR151 was induced at the end of exponential gowth in LB medium (Fig. 2). (In other experiments, a low, basal level of transcription was observed during exponential growth.) This postexponential induction is in agreement with previous observations of recA transcription in B. subtilis in LB medium (23) and in competence medium (15, 23). RecA is capable of positively regulating gene expression (33, 36). Therefore, we reasoned that the elongated nucleoids in a subpopulation of cells in the stationary phase (Fig. 1B) might be the result of gene silencing in the absence of the normally induced RecA. For example, B. subtilis mecA mutants release comK transcription and the transcription of other ComK-regulated genes, such as comC, -D, -E, and -G, from medium dependence and have been reported to have cell and nucleoid abnormalities similar to those we have seen in stationary-phase recA cells (12). Based on these similarities, we speculated that RecA might be involved in the positive regulation of mecA during the stationary phase in the non-competence-inducing medium used here. Therefore, we evaluated the expression of comK and comG in our parent and recA strains during their transitions into the stationary phase. Neither the comK-lacZ nor the comG-lacZ reporter activity was enhanced in the absence of RecA (data not shown). These data indicate that RecA does not exert a positive regulatory effect on mecA or other genes responsible for regulating comK during the stationary phase in LB medium.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   recA-lacZ activity during the transition into the stationary phase. Solid diamonds, SL7937 (recA-lacZ). The endogenous activity of the parent strain BR151 has been subtracted from each time point. Results are the average of two experiments.