Germination Protein Levels and Rates of Germination of Spores of Bacillus subtilis with Overexpressed or Deleted Genes Encoding Germination Proteins

Abstract

Deletion of Bacillus subtilis spores' GerA germinant receptor (GR) had no effect on spore germination via the GerB plus GerK GRs, and loss of GerB plus GerK did not affect germination via GerA. Loss of one or two GRs also did not affect levels of GRs that were not deleted. Overexpression of GRs 5- to 18-fold increased rates of germination via the overexpressed GR and slowed germination by other GRs up to 15-fold. However, overexpression of one or two GRs had no effect on levels of GRs that were not overexpressed. These results suggest that either interaction between different GRs reduces the activity of GRs in triggering spore germination or all GRs compete for interaction with a limiting amount of a downstream signaling molecule in the germination pathway. Overexpression or deletion of GRs also had no effect on spores' levels of the GerD protein needed for normal GR-dependent germination or of the SpoVAD protein likely involved in dipicolinic acid release early in germination. Loss of GerD also had no effect on levels of GRs or SpoVAD. Spores of a strain lacking the only B. subtilis prelipoprotein diacylglycerol transferase, GerF, also had no detectable GerD or the GerA's C subunit, both of which are most likely lipoproteins; GerA's A subunit was also absent. However, levels of GerB's C subunit, also almost certainly a lipoprotein, and GerK's A subunit were normal in gerF spores. These results with g erF spores were consistent with effects of loss of GerF on spore germination by different GRs.

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.…”

“…Analyses of a number of engineered strains of B. subtilis have shown that rates of spore germination with various nutrient germinants can be significantly altered when the cognate GR(s) is overexpressed (7,17). 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).…”

“…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).…”

“…One such layer is the inner membrane (IM), a lipid bilayer with extremely low permeability in which lipid probes and presumably proteins are immobile prior to germination (3). The IM also contains a number of proteins crucial for spore germination, including the nutrient germinant receptors (GRs) that trigger spore germination in response to specific nutrient germinants (4)(5)(6)(7)(8)(9).…”