Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores

Abstract

Dormant Bacillus subtilis spores germinate in response to specific nutrients called germinants, which are recognized by multisubunit receptor complexes encoded by members of the gerA family of operons, of which the gerB operon is a member. The germinant receptors are expected to be membrane associated, but there is some debate about whether they are located in the inner or outer spore membrane. In this study we have used Western blot analysis to determine the precise location of GerBA, a gerB-encoded receptor protein, in various spore fractions. GerBA was not extracted from spores by a decoating treatment that removes the coat and outer membrane but was present in lysates from decoated spores and in the insoluble fraction (termed P100) from such lysates that contained inner-membrane vesicles. GerBA was also solubilized from the P100 fraction with detergent but not with high salt. These findings suggest that GerBA is an integral membrane protein located in the spore's inner membrane. Consistent with this idea, GerBA was present in the cell membrane of the outgrowing spore, a membrane that is derived from the dormant spore's inner membrane. Based on these observations we propose that GerBA and probably the entire GerB germinant receptor are located in the inner membrane of the dormant spore. We also estimated that there are only 24 to 40 molecules of GerBA per spore, a number that is consistent with the previously reported low level of gerB operon expression and with the putative receptor function of the proteins encoded by the gerB operon.

FIG. 1

SDS-PAGE analysis of purified His10-GerBA protein. Synthesis of the His10-GerBA protein was induced in E. coli cells of strain BL21 λ(DE3) carrying plasmid pFE254, the soluble proteins were isolated, and GerBA was purified as described in Materials and Methods. An aliquot (8 μg) of the purified protein preparation (lane P) was run on an SDS-10% PAGE gel and visualized by Coomassie blue staining. The arrowhead points to the major species that was purified, and asterisks mark the smaller species, which are degradation products of the major protein (see text). Arrows indicate the migration positions of the molecular mass markers in kilodaltons (lane M).

FIG. 2

GerBA in extracts from untreated and decoated spores. Extracts from identical amounts (∼3 OD₆₀₀ units) of disrupted spores without (lanes A and C) or with (lanes B and D) prior decoating treatment as described in Materials and Methods were run on an SDS-PAGE gel and transferred to an Immobilon membrane, and GerBA was detected as described in Materials and Methods. The spores were from strains FB58 (P_sspB::gerB; lanes A and B) and FB60 (ΔgerB; lanes C and D). Bands marked with an asterisk are nonspecific background bands, whereas those in regions marked I and II represent GerBA-specific signals; bands in region II lie close to the predicted GerBA molecular mass (53 kDa), whereas those in region I probably contain highly modified or complexed forms of GerBA. Migration positions of molecular mass markers in kilodaltons are indicated with arrows.

FIG. 3

GerBA in spore lysates. Lysates from equal amounts (15 OD₆₀₀ units) of spores of strains PS832 (wild type; lane A), FB60 (ΔgerB; lane B), FB86 (ΔgerA ΔgerK; lane C), and FB72 (ΔgerA ΔgerB ΔgerK; lane D) were run on an SDS-PAGE gel and transferred to an Immobilon membrane, and GerBA was detected as described in Materials and Methods. The arrowhead points to GerBA bands in region II as described in the legend to Fig. 2. Background bands and molecular mass markers are labeled as in Fig. 2. Note that the samples run in Fig. 2 were from spores that overexpressed GerBA while the samples run in this experiment were from spores that did not overexpress GerBA. That difference and the low exposure level of the Western blot shown here account for the relative lack of higher molecular mass GerBA observed in this figure compared to Fig. 2 and 6.

FIG. 4

GerBA in fractionated spore lysates. Lysates from equal amounts (15 OD₆₀₀ units) of decoated spores of strains PS832 (wild type; lanes A, C, and E) and FB60 (ΔgerB; lanes B, D, and F) were subjected to centrifugation at 100,000 × g for 1 h. The initial lysate (lanes A and B), S100 supernatant fractions (lanes C and D), and P100 pellet fractions (lanes E and F) were run on an SDS-PAGE gel, proteins were transferred to an Immobilon membrane, and GerBA was detected as described in Materials and Methods. The arrowheads denoting GerBA bands, the background bands, and molecular mass markers are labeled as in Fig. 2.

FIG. 5

Effects of detergent and salt treatments on GerBA fractionation. Lysates from decoated spores (15 OD₆₀₀ units) of strain FB60 (ΔgerB; lanes A to C and A′ to C′) or strain PS832 (wild type; lanes D to F and D′ to F′) were incubated with no additions (lanes A, D, A′, and D′), with 1% Triton X-100 (lanes B, E, B′, and E′), or with 0.5 M NaCl (lanes C, F, C′, and F′) for 30 min on ice, followed by centrifugation at 100,000 × g to obtain the P100 pellet (lanes A to F) and S100 supernatant (lanes A′ to F′) fractions. The P100 fractions from 10 OD₆₀₀ units of spores and S100 fractions from 7 to 8 OD₆₀₀ units of spores were run on an SDS-PAGE gel and transferred to an Immobilon membrane, and GerBA was detected as described in Materials and Methods. The arrowheads denoting GerBA bands, the background bands, and molecular mass markers are labeled as in Fig. 2. Note that detergent and salt treatments also affect fractionation of some of the background bands.

FIG. 6

GerBA signal in spores with different levels of expression of gerB. The P100 pellet fractions of spores (15 OD₆₀₀ units) from strains FB60 (ΔgerB; lane A), PS832 (wild type; lane B), FB49 (PS832 amyE::gerB; lane C), or FB58 (P_sspB::gerB; lane D) were run on an SDS-PAGE gel, transferred to an Immobilon membrane, and probed for GerBA as described in Materials and Methods. The arrowheads labeled I and II denote the GerBA-specific signals in the 100-kDa and 40- to 50-kDa ranges, respectively. Background bands and molecular mass markers are labeled as in Fig. 2.

FIG. 7

GerBA in outgrowing spores. P100 fractions from outgrowing (10 OD₆₀₀ units) spores of strains FB60 (ΔgerB; lane A) and PS832 (wild type; lane B) prepared as described in Materials and Methods were run on an SDS-PAGE gel, proteins were transferred to an Immobilon membrane, and GerBA was detected as described in Materials and Methods. Arrowheads labeled I and II denote GerBA bands in regions I and II, respectively, and background bands and molecular mass markers are labeled as in Fig. 2.