Bacillus subtilis serine/threonine protein kinase YabT is involved in spore development via phosphorylation of a bacterial recombinase

Abstract

We characterized YabT, a serine/threonine kinase of the Hanks family, from Bacillus subtilis. YabT is a putative transmembrane kinase that lacks the canonical extracellular signal receptor domain. We demonstrate that YabT possesses a DNA-binding motif essential for its activation. In vivo YabT is expressed during sporulation and localizes to the asymmetric septum. Cells devoid of YabT sporulate more slowly and exhibit reduced resistance to DNA damage during sporulation. We established that YabT phosphorylates DNA-recombinase RecA at the residue serine 2. A non-phosphorylatable mutant of RecA exhibits the same phenotype as the ΔyabT mutant, and a phosphomimetic mutant of RecA complements ΔyabT, suggesting that YabT acts via RecA phosphorylation in vivo. During spore development, phosphorylation facilitates the formation of transient and mobile RecA foci that exhibit a scanning-like movement associated to the nucleoid in the mother cell. In some cells these foci persist at the end of spore development. We show that persistent RecA foci, which presumably coincide with irreparable lesions, are mutually exclusive with the completion of spore morphogenesis. Our results highlight similarities between the bacterial serine/threonine kinase YabT and eukaryal kinases C-Abl and Mec1, which are also activated by DNA, and phosphorylate proteins involved in DNA damage repair.

Fig. 1

YabT is activated by DNA binding. A. Schematized structure of YabT aligned to reference Hanks kinase PrkC, showing the kinase domain (blue shading) and the transmembrane helix (dark grey). Truncated versions YabTΔ1 and YabTΔ2 are shown, with the putative DNA-binding region (residues 230–315) highlighted in orange. Catalytic mutant YabT K55A has the mutated residue K55 highlighted in red. B. Binding of YabT to random sequence ssDNA (140 bases fragment, 0.4 μM) and dsDNA (210 base pairs fragment, 0.25 μM), in a gel-shift assay. B. subtilis kinases PrkD and PrkC were included (4.5 μM each) as controls in lanes 1 and 2, YabT concentrations are 0, 2.0, 3.0, 4.0, 5.0 μM (lanes 3–7) and 0, 1.5, 3.0, 4.5, 6.0 μM (lanes 8–12). C. Autoradiography of SDS-polyacrylamide gels showing the influence of DNA binding on YabT autophosphorylation. 0.25 μM YabT was incubated for 2 h with 0, 3, 6, 12 nM of random-sequence ssDNA (140 bases fragment) in lanes 1 to 4, and 0, 3, 6, 12 nM of dsDNA (210 base-pairs fragment) in lanes 4 to 8, in the in vitro phosphorylation assay. Quantification was performed on results from three independent experiments and the representative gels are shown. Columns 1 to 8 correspond to lanes 1 to 8 in the gels respectively. Signals were normalized with respect to autophosphorylation of YabT incubated without DNA (lanes 1 and 5) that was taken as 100%.

Fig. 2

YabT DNA binding site and trans-autophosphorylation. A. Binding of YabTΔ1 and YabTΔ2 to 0.4 μM 140 bases ssDNA fragment, in a gel-shift assay. No YabT was added in lane 1 as control, and 4 μM of each protein were in lanes 2 to 4. B. In vitro autophosphorylation assay of YabT, YabTΔ1 and YabTΔ2. 0.25 μM YabT, YabTΔ1 and YabTΔ2 were incubated in the presence of 12 nM ssDNA fragment (lanes 2, 4 and 6), and in the absence of ssDNA fragment (lanes 1, 3 and 5). Quantification was performed from three independent experiments and the representative gel is shown. All data were normalized with respect to the autophosphorylation signal of YabT incubated without DNA (lane 1) that was defined as 100%. Reactions were incubated for 2 h before separation on SDS-PAGE. C. ClustalW alignment showing the conservation of the catalytic YabT residue K55. Other bacterial Hanks kinases shown are PrkC and PrkD from B. subtilis and PknD and PknF from Mycobacterium tuberculosis. D. In vitro phosphorylation assay showing trans-autophosphorylation reaction between YabT wild type and YabT K55D. The presence or absence of key reactants (12 nM 140 nt ssDNA, 0.08 μM YabT WT and 0.16 μM YabT K55D) is indicated as +/− above each lane. Reactions were incubated for 2 h before separation on SDS-PAGE.

Fig. 3

YabT enriched at the septum during sporulation. A–C. Fluorescence microscopy of B. subtilis cells expressing a xylose-inducible GFP-YabT fusion. For the images at the top of each panel, red fluorescence indicates cell membranes stained with FM4-64 (left), green fluorescence indicates localization of GFP-YabT (middle), and they are also shown in overlay (right). Quantification of fluorescence along the cell axis is shown for a typical cell in the bottom left part of each panel. Based on this quantification for all examined cells, an enrichment profile at the septum has been calculated for FM4-64 and GFP-YabT fluorescence, bottom right of each panel. The enrichment is calculated as the ratio of fluorescence at the septum divided by the fluorescence at the opposite polar peak for sporulating cells, and average value of both polar peaks for exponential cells. A. Localization of GFP-YabT during sporulation, images taken at T3. B. Localization of GFP-YabTΔTM during sporulation, images taken at T3. C. Localization of GFP-YabT expressed during exponential growth.

Fig. 4

Phenotypes of ΔyabT and recA point-mutants during sporulation. A. Kinetics of spore formation of wild type B. subtilis and strains recA S2A, recA S2D, ΔyabT and recA S2D ΔyabT in the sporulation medium from stage T0 to T7. Spore counts are expressed as % of total number of viable cells. Error bars represent standard deviation from 5 biological replicates. B. Spore survival after mitomycin treatment (20 ng ml⁻¹) applied at time-point T1 to the sporulating cultures of B. subtilis strains recA S2A, recA S2D, ΔyabT, recA S2D ΔyabT and wild type. Spore counts are expressed as number of spores in treated culture/number of spores in untreated culture for each strain, normalized with respect to the wild type. Error bars represent standard deviation from 5 biological replicates.

Fig. 5

Phosphorylation of RecA by YabT. A. Autoradiography of SDS-polyacrylamide gels showing in vitro phosphorylation assays with YabT and RecA fused with an N-terminal His₆-tag (6xHis-RecA) or C-terminal His₆-tag (RecA-6xHis). Experiment was performed with 0.25 μM YabT and 1 μM RecA (either N- or C-terminally tagged). Presence of key proteins in the assays is indicated with +/− above each lane. Reactions were incubated for 2 h prior to separation by SDS-PAGE. Bands corresponding to autophosphorylated YabT and phosphorylated RecA are indicated by arrows. B. In lanes 1–3, phosphorylation of RecA by three versions of YabT was examined. 1 μM RecA was incubated with 0.25 μM YabT, YabTΔ1 and YabTΔ2. Reactions were incubated for 2 h before separation on SDS-PAGE. C. Autoradiography of SDS-polyacrylamide gels showing in vitro YabT-dependent phosphorylation of RecA wild type and RecA S2A. Time dependence of the reaction was followed and the incubation times are given above each lane. Bands corresponding to autophosphorylated YabT and phosphorylated RecA are indicated. D. Yeast two-hybrid phenotypic interaction assay with YabT and RecA (wild type, truncated and mutant derivatives). Yeast haploid cells expressing the YabT protein, the RecA full sized, or N and C-terminal domains (NTD and CTD respectively), as well as the S2A and S2D mutant derivatives were fused with the binding domain of Gal4 (BD) and mated against compatible haploid cells expressing the matching RecA derivatives fused with the activating domain (AD). The interacting phenotypes were monitored by the ability of mating products to grow on the selective medium -LUH. E. RecA levels in different strains were probed by Western blot using anti-RecA antibodies on crude extracts obtained at T3. All protein extracts were normalized to the same total protein concentration using a Bradford assay, and equal amount of total protein was loaded in each lane. Strains are indicated above each lane, and the band corresponding to RecA is indicated by an arrow.

Fig. 6

RecA foci observed during spore development. A. GFP-RecA foci visualized by fluorescent microscopy at T3. The overlay pictures of the same cell represent membrane-staining (FM4-64) in red, nucleoid (DAPI) in violet and GFP-RecA is green. The GFP-RecA fusion was induced at T0. The proportion of cells containing GFP-RecA foci at T3 is shown in wild type and different mutant stains: ΔyabT, recA S2A, recA S2D and recA S2D ΔyabT. Detection of foci for counting was done with the help of the fluorescent spot detector software (FluorSpotRecognition). Error bars represent standard deviation from 3 independent experiments. B. Externally induced DNA damage transforms RecA foci into filaments/threads. DNA damage was induced at T1 with 40 ng ml⁻¹ of mitomycin. Cells were observed with FITC/DAPI-staining overlap at T3. Strain names are indicated in each panel.

Fig. 7

RecA foci persisting at T6 are incompatible with sporulation in wild type cells. A. A sample of sporulating wild type cells observed at T6 with FITC/brightfield overlap. Green spots in FITC/brightfield overlap represent RecA foci and black spots are mature spores. Strains are indicated in each panel. White arrows highlight the cells containing RecA foci. B. Coexistence of RecA foci and spores at T6 in different strains. Over 500 cells were counted for each strain. Total bar height represents the fraction of cells containing a RecA focus. Light-grey bar represents the absence of a spore and dark grey bare the presence of a spore in cells containing RecA foci. C. Over 500 wild type cells were examined to count the ratio of cells with spores and cells with RecA foci/threads at T6. To the left is the ratio in the untreated wild type cells, and to the right the ratio in wild type cells treated with mitomycin.