Growth Phase Variation in Cell and Nucleoid Morphology in a Bacillus subtilis recAMutant

Nucleoid and cell morphology phenotypes of B. subtilis rec mutants during exponential-phase growth.In Table1, 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 recAstrain were rare (Table 1). In other experiments in which a larger number of cells were observed, the anucleate frequency of two differentrecA 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).

• Open in new tab
• Download powerpoint

Fig. 1.

Micrographs of B. subtilis strains treated with DAPI to visualize nucleoids. (A) Parent strain BR151 grown to stationary phase (T_1.5) in LB medium. (B)recA mutant SL7360 grown to stationary phase (T_1.5) in LB medium. (C) recAmutant SL7360 grown to stationary phase (T_1.5) 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 inrecA 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.

Two major homologous recombination pathways (RecA dependent) are responsible for processing of stalled replication forks in E. coli: the recBCD pathway and the recFpathway (5). The RecD component of RecBCD has been identified as the nuclease responsible for degradation of chromosomes in recA cells of E. coli (20). The counterparts for E. coli recBCD and recF inB. subtilis are addAB and recF(9). Like the recA strain, both theaddB and recF strains showed a low frequency of nucleoid partitioning defects. Neither strain, however, showed substantial signs of the cell wall irregularities noted inrecA and recA combination mutants (Table 1). Consistent with previous experiments, ripX and ripX recA strains displayed strong evidence of nucleoid partitioning defects. We also note that the frequency of cell wall defects observed with the ripX recA strain was more than triple that seen with the recA strain, suggesting that there is an additive penalty for carrying both mutations (Table 1).

Viability of B. subtilis rec mutants during exponential-phase growth.The generation time of therecA 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 arecA 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 thatrecA 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 recFdouble 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 aripX 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 thatripX mutations are not silent in a recAderivative of strain BR151 (26).

Although the frequency of anucleate cells in B. subtilis was substantially lower than that seen in E. coli, the 12% viability of the B. subtilis recA mutant suggests that most of the nucleated recA population is nonviable. Also, the sum of the nucleoid defects in B. subtilis recA cells, recorded here as 6%, presumably underreports the extent of nucleoid defects in a population. Based on these considerations, we speculate that lethal nucleoid damage indeed occurs in B. subtilis, but degradation is not as rapid or complete as it is in E. coli.

Cell division and nucleoid phenotypes of stationary-phaserecA 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 recAstationary-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 recA4point 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 unaffectedrecA 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).

Based on the role of RecA in restoration of stalled replication forks, it is plausible that some or all of the cells in stationary-phase cultures of the B. subtilis recA strains that contained elongated nucleoids had accumulated DNA damage that prevented replication fork progression. This supposition is strengthened by our observation that treatment of B. subtilisRecA⁺ cells with MMC resulted in the development of filamented cells containing elongated nucleoids, a phenotype similar to that seen in recA stationary-phase cells (compare Fig. 1B and D). Additionally, recF and addB mutants showed the same distinctively elongated cells and nucleoids during stationary phase, although not as frequently (50 and 10% relative to the recA mutant [data not shown]). We discuss the growth-phase-specific nature of the aberrant subpopulation in B. subtilis recA cells later.

The reduced frequency of aberrant nucleoids in addB cells was unexpected. A possible explanation is that in the absence of RecA or RecF, unprocessed DNA substrates subsequently become targets for the exonuclease activity of AddAB. This combination of DNA degradation without repair might lead to the observed stationary-phase nucleoid phenotype in recA and recF cells. If this proposed scheme is correct, addB mutants would not be expected to present similar frequencies of the aberrant nucleoid phenotype to those seen in recA and recF mutants, because chromosomal DNA would not be efficiently degraded in the absence of a fully functional AddAB complex.

In an E. coli recA mutant (W3110), we observed no comparable signs of nucleoid elongation during the transition from exponential growth into the stationary phase (data not shown). Furthermore, the amount of cell filamentation in E. coli recA cells actually decreased during this time (exponential, ∼13%; stationary, ∼4%). It is also worth noting in this context that even when treated with MMC, E. coli recA cells continued growing and dividing at stages where plating yielded no CFU (13). Therefore, a second distinction can now be made between E. coli recA andB. subtilis recA cells. Whereas chromosome degradation is extensive and cell division occurs frequently in E. coli recA cells, the more stable nucleoids appear to persist as barriers to septation during the transition into the stationary phase in B. subtilis recA cells (31, 35). A similar absence of septation in cells harboring aberrant nucleoids has been seen in B. subtilis ripX mutants (26).

Why should E. coli and B. subtilis nucleoids appear so different upon entry into stationary phase? E. coli H-NS and integration host factor (IHF) are DNA-binding proteins involved in maintaining nucleoid structure and have been shown to be transcriptional regulators of gene expression. The IHF protein is up-regulated upon entry into the stationary phase (7). While several groups have shown substantial H-NS up-regulation at the stationary phase (6, 30, 32), another study showed little growth phase variation in H-NS levels (10). The B. subtilis DNA-binding proteins with the strongest similarity to these E. coli proteins are HBsu (IHF) and Smc (H-NS) (2, 8, 17, 18). Interestingly, B. subtilistranscription of hbs (encoding HBsu) and the Smc level are both reduced in stationary phase (8, 11), although the HBsu/DNA ratio in spores is similar to that in vegetative cells (24). It is possible, therefore, that E. colihas evolved a more comprehensive system of stationary-phase DNA compaction and protection than B. subtilis (with the exception of the highly protective stationary-phase sporulation program).

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 postexponentialrecA cells in the sporulation-inducing medium MSSM (Fig.1C). The elongated nucleoid structures seen in postexponentialrecA 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 recAcells that became apparent at the end of exponential growth were maintained for at least 7 h (data not shown).

In addition to the cell division and nucleoid irregularities seen early in the sporulation program, we have observed two separate and unfavorable consequences for sporulating recA mutants. First, the timing of prespore nucleoid condensation (27) is delayed by ∼2 h and occurs at a lower frequency than that of the parent strain (Table 4). Second,recA strains sporulate less frequently than their parent strain (Table 3) (26); the reduced sporulation frequency is distinct from the altered resistance properties of recAspores (28). The impairment in prespore condensation and diminished frequency of sporulation are likely related to the aberrant morphologies discussed previously. Regardless of the cause, it should be remembered that the reduced sporulation frequency in recAmutants is superimposed on a severely reduced plating efficiency.

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 ofrecA 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 releasecomK 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 andrecA strains during their transitions into the stationary phase. Neither the comK-lacZ nor the comG-lacZreporter 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 regulatingcomK during the stationary phase in LB medium.

• Open in new tab
• Download powerpoint

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.