The RecA-Dependent SOS Response Is Active and Required for Processing of DNA Damage during Bacillus subtilis Sporulation.

The expression of and role played by RecA in protecting sporulating cells of Bacillus subtilis from DNA damage has been determined. Results showed that the DNA-alkylating agent Mitomycin-C (M-C) activated expression of a PrecA-gfpmut3a fusion in both sporulating cells' mother cell and forespore compartments. The expression levels of a recA-lacZ fusion were significantly lower in sporulating than in growing cells. However, M-C induced levels of ß-galactosidase from a recA-lacZ fusion ~6- and 3-fold in the mother cell and forespore compartments of B. subtilis sporangia, respectively. Disruption of recA slowed sporulation and sensitized sporulating cells to M-C and UV-C radiation, and the M-C and UV-C sensitivity of sporangia lacking the transcriptional repair-coupling factor Mfd was significantly increased by loss of RecA. We postulate that when DNA damage is encountered during sporulation, RecA activates the SOS response thus providing sporangia with the repair machinery to process DNA lesions that may compromise the spatio-temporal expression of genes that are essential for efficient spore formation

A two-step transport pathway allows the mother cell to nurture the developing spore in <i>Bacillus subtilis</i>

<div><p>One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA) in the developing spore. This small molecule comprises 5–15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes) that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ) is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria.</p></div

<i>B</i>. <i>subtilis</i> strains and plasmids used in this study.

<p><i>B</i>. <i>subtilis</i> strains and plasmids used in this study.</p

Levels of ß-galactosidase from <i>recA-lacZ</i> with and without DNA damage during sporulation in SOS-proficient (a and b) and –deficient (c and d) <i>B</i>. <i>subtilis</i> strains.

<p><i>B</i>. <i>subtilis</i> strains LAS600 (parental) and LAS523 (SOS-deficient) containing a <i>recA-lacZ</i> fusion were grown and induced to sporulate in DSM. The optical densities of cultures were measured without (○) or after (●) DNA damaging treatment. 4 h after the onset of sporulation (T₀), the culture was divided into two subcultures; one subculture was challenged with M-C (500 ng/mL) and the other one was untreated. Cells samples from untreated (open symbols) or treated (filled symbols) were collected at the indicated times and ß-galactosidase specific activity in the mother cell (a and c, triangles) and forespore (b and d, squares) fractions was determined, all as described in Materials and Methods.</p

Resistance of Δ<i>recA</i> and SOS-deficient strains to M-C (a, c) and UV-C radiation (b, d) during sporulation.

<p>Sporulating cells of strains 168 (wild-type), PERM1030 (<b>Δ</b><i>recA</i>), LAS600 (parental) and LAS523 (<i>lexA</i>[ind^-]) were treated (open symbols) or not (filled symbols) with increasing doses of M-C (a and c, circles) or UV-C light (b and d, triangles) at 4.5 h (wild-type, LAS600 and LAS523) or 6.5 h (Δ<i>recA</i>) after the onset of sporulation, and cell survival was determined as described in Materials and Methods. Data are expressed as the average ± SD of at least three independent experiments.</p

Levels of ß-galactosidase from <i>recA-lacZ</i> in growth and sporulation and with and without DNA damage.

<p><i>B</i>. <i>subtilis</i> strain YB3001 containing a <i>recA-lacZ</i> fusion was grown and induced to sporulate in DSM (a-c) The optical densities of cultures were measured without (○) or after (●) DNA damaging treatment. Samples were also collected at different times during growth and sporulation and were processed and assayed for ß-galactosidase specific activity. In <b>(a)</b> ß-galactosidase from <i>recA-lacZ</i> was assayed throughout growth and sporulation (◆). In <b>(b,c)</b> 4 h after the onset of sporulation (T₀), the culture was divided into two subcultures; one subculture was challenged with M-C (500 ng/mL) and the other one was untreated. Cells samples from untreated (open symbols) or treated (filled symbols) were collected at the indicated times and ß-galactosidase specific activity in the mother cell (b, triangles) and forespore (c, squares) fractions was determined, all as described in Materials and Methods. In (d), <i>B</i>. <i>subtilis</i> YB3001 was propagated in PAB medium, when the culture reached an OD_600nm = 0.5 (Exponential) or 4 h after T₀ (Stationary), vegetative cells were treated (black bars) or not (gray bars) with M-C (500 ng/mL) for 1.5 h and then the cultures were processed for determination of ß-galactosidase as described above. Results are the average of values from three independent experiments ± standard deviations (SD) of ß-galactosidase specific activity.</p

Expression of <i>spoVFA-lacZ</i> during sporulation of wild-type and Δ<i>recA B</i>. <i>subtilis</i> strains.

<p><i>B</i>. <i>subtilis</i> strains PERM1233 (wild-type) (open symbols) and PERM1280 (<b>Δ</b><i>recA</i>) (closed symbols) containing the <i>spoVFA-lacZ</i> fusion were grown and induced to sporulate in DSM and the OD_600nm (circles) was measured. Cells were collected during sporulation at the indicated times, treated with lysozyme and the extracts were assayed for ß–galactosidase in the mother cell fractions only (triangles). Results are the averages of values from three independent experiments ± SD of ß-galactosidase specific activity.</p

SpoVV (YlbJ) is required for DPA accumulation in dormant spores.

<p>A. Representative phase-contrast images of the indicated strains sporulated by nutrient exhaustion for 30 h at 37°C. Examples of phase-dark prematurely germinated spores are highlighted (yellow carets). Sporulation efficiencies, as determined by colony forming units after heat treatment (80°C, 20 min) are indicated below each image. Strains lacking the B subunit of the GerA receptor (GerAB) are designated ∆<i>gerA</i> for clarity. B. Representative images of purified spores used to measure DPA content. Indicated strains were sporulated by resuspension in defined minimal medium for 30 h at 37°C. Spores were purified as described in Methods. Scale bars indicate 2 μm. C. Bar graph showing DPA content in purified spores from the indicated strains. Purified spores were concentrated to an OD₆₀₀ of 5, and incubated at 100°C for 30 min to release DPA. DPA content in the supernatant was then quantified using a colorimetric assay. Commercial DPA was used to generate a standard curve. Results shown are the average ± standard deviation of three independent biological replicates.</p

The DPA transport pathway during spore morphogenesis.

<p>Schematic model of the spatio-temporal regulation of the DPA transport pathway and forespore accumulation of DPA. During the morphological process of engulfment, SpoVV is produced in the mother cell under SigE control where it specifically localizes in the outer forespore membrane in a manner that depends on the SpoIIQ-SpoIIIAH complex (not shown) (left panel). Prior to the completion of engulfment, SpoIIID represses transcription of the <i>spoVV</i> gene. Accordingly, upon completion of engulfment SpoVV is no longer produced and cannot accumulate in the cytoplasmic membranes of the mother cell. After completion of engulfment, the proteins of the <i>spoVA</i> operon are produced in the forespore under the control of SigG (middle panel). Finally, SpoVFAB proteins are produced in the mother cell under the control of SigK leading to the synthesis of DPA (dark circles). DPA is then transported across the two membranes that separate the mother cell and forespore.</p

SpoVV resembles concentrative nucleoside transporters.

<p>A. Structure of the <i>V</i>. <i>cholerae</i> concentrative nucleoside transporter (vcCNT) in cross-section bound to uridine in the inward open orientation. Close-up of the substrate-binding pocket of vcCNT bound to uridine and the putative substrate-binding pocket of SpoVV highlighting amino acid residues (yellow) predicted to interact with DPA. B. Sporulation efficiencies of the indicated strains harboring amino acid substitutions in the putative substrate-binding pocket of SpoVV. The mutations were generated in the context of the <i>spoVV-gfp</i> fusion to enable the assessment of protein levels. Cells were sporulated in liquid medium at 37°C and sporulation efficiency (assessed by heat-resistance to 80°C for 20 min) was determined. C. Bar graph showing DPA content in dormant spores of the indicated strains. Purified spores were generated by resuspension and DPA was assayed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007015#pgen.1007015.g001" target="_blank">Fig 1</a>. All <i>spoVV</i> mutant strains lacked the B subunit of the GerA receptor (designated ∆<i>gerA</i> for clarity) to prevent premature germination. Results shown are the average ± standard deviation of two independent biological replicates. D. Immunoblot showing the levels of the SpoVV-GFP mutants (in the ∆<i>spoVV</i> ∆<i>gerAB</i> background) compared to wild-type. Whole cell lysates from sporulating cells collected 3 h after the onset of sporulation were analyzed using anti-GFP antibodies. SpoIIP levels were used to control for loading. We note that as reported previously [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007015#pgen.1007015.ref075" target="_blank">75</a>], small differences in DPA levels appear to have dramatic effects on heat resistance (compare, for example, SpoVV(N97A) and SpoVV(Q310A)). The defects in sporulation efficiencies reported here and likely those reported previously reflect both the loss in heat resistance and the percentage of sporulating cells in which the GerA receptor was prematurely activated [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007015#pgen.1007015.ref024" target="_blank">24</a>]. Analysis of sporulation efficiency of the SpoVV mutants in a strain lacking GerAB followed similar trends.</p