Effects of the SpoVT Regulatory Protein on the Germination and Germination Protein Levels of Spores of Bacillus subtilis

Abstract

bBacillus subtilis isolates lacking the SpoVT protein, which regulates gene expression in developing forespores, gave spores that released their dipicolinic acid (DPA) via germinant receptor (GR)-dependent germination more rapidly than wild-type spores. Non-GR-dependent germination via dodecylamine was more rapid with spoVT spores, but germination via Ca-DPA was slower. The effects of a spoVT mutation on spore germination were seen with spores made in rich and poor media, and levels of SpoVTLacZ were elevated 2-fold in poor-medium spores; however, elevated SpoVT levels were not the only cause of the slower GR-dependent germination of poor-medium spores. The spoVT spores had >5-fold higher GerA GR levels, ϳ2-fold elevated GerB GR levels, wild-type levels of a GerK GR subunit and the GerD protein required for normal GR-dependent germination, ϳ2.5-fold lower levels of the SpoVAD protein involved in DPA release in spore germination, and 30% lower levels of DNA protective ␣/␤-type small, acid-soluble spore proteins. With one exception, the effects on protein levels in spoVT spores are consistent with the effects of SpoVT on forespore transcription. The spoVT spores were also more sensitive to UV radiation and outgrew slowly. While spoVT spores' elevated GR levels were consistent with their more rapid GR-dependent germination, detailed analysis of the results suggested that there is another gene product crucial for GR-dependent spore germination that is upregulated in the absence of SpoVT. Overall, these results indicate that SpoVT levels during spore formation have a major impact on the germination and the resistance of the resultant spores.

Numbers of Individual Nutrient Germinant Receptors and Other Germination Proteins in Spores of Bacillus subtilis

“…In individual experiments, aliquots from the same amounts of various spore fractions (termed 1ϫ) were run on SDSpolyacrylamide (11 to 15%) gel electrophoresis (PAGE) gels and subjected to Western blot analysis with antisera specific for various germination proteins, as described previously (6,7,18,19). In some cases, after the use of one antiserum, the polyvinylidene difluoride (PVDF) Western blot membranes were stripped with Restore Western blot stripping buffer (Thermo Scientific, Rockford, IL) for 15 to 20 min at 37°C and then probed with antiserum against another germination protein.…”

“…Dormant B. subtilis spores (ϳ15 mg [dry weight]) of various strains were routinely chemically decoated to remove the outer membrane and much spore coat protein and disrupted by bursts of sonication after lysozyme treatment, and the disrupted spores were fractionated as previously described (5,7,18,19,23). Briefly, after low-speed centrifugation of the disrupted spores for 5 min at 16,000 ϫ g, the supernatant fluid was saved and termed the low-speed supernatant fluid (LS) fraction.…”

“…Quantitation by Western blot analysis was done with M or LS fractions from wild-type spores and the lysate (L) from wild-type spores that were analyzed on the same Western blot with different amounts of the purified (Ͼ95%) B. subtilis proteins used to raise various antisera. These proteins were obtained and purified as previously described (6,7,18,19,24) and were (i) amino acids (aa) 2 to 239 of GerAA (27.1 kDa), (ii) GerAC without its signal sequence (40.5 kDa), (iii) GerBC without its signal sequence (40.4 kDa), (iv) aa 39 to 276 of GerKA (26.7 kDa), (v) GerD without its signal sequence (18.4 kDa), and (vi) full-length SpoVAD (36.0 kDa). The concentrations of these proteins were determined by their absorbance at 280 nm and their molecular extinction coefficient at this wavelength.…”

“…Since GR subunits have been localized to the IM (5,7,12,18,19), the IM pellet (M fraction) was isolated from wild-type spores to compare levels of GR subunits in this fraction to known amounts of purified GerAA protein. As expected (6, 7), the M sample gave a GerAA band at ϳ54 kDa in Western blot analysis with the GerAA-specific antiserum (Fig.…”

“…For example, L-valine germination is significantly increased when GerA is overexpressed, and these elevated GerA levels inhibit the GerB/GerK pathway's response to AGFK, although levels of GerBC and GerKA are unchanged (7). In addition, (i) decreases in GR subunit levels by sporulation in nutrientpoor medium are accompanied by lowered spore germination rates with nutrient germinants, (ii) spores that are termed superdormant and germinate extremely slowly with nutrient germinants have very low GR levels, and (iii) spores lacking the regulatory protein SpoVT have higher GR subunit levels and higher rates of nutrient germination (6,18,19). These data strongly suggest that GR levels are a major factor determining rates of spore germination.…”