Localization of a Germinant Receptor Protein (GerBA) to the Inner Membrane of Bacillus subtilis Spores

Abstract

DormantBacillus 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.Dormant spores of Bacillus subtilis are metabolically inert structures that are formed when growing cells are starved for particular nutrients. Despite their dormancy, these spores can detect nutrients in the environment and respond to such cues by triggering a number of reactions that result in the resumption of metabolism and growth. The initial steps in this process, called spore germination, thus represent a novel paradigm for studying the communication between a cell and its surroundings.A variety of nutrients can induce spore germination, and in B. subtilis two such germinants have been extensively studied: L-alanine (L-Ala) and a combination of L-asparagine, D-fructose, D-glucose, and potassium ions (AFGK) (19). Previous work suggested that germinants act by binding to and activating spore receptors (9,13,19). This idea has been substantiated by several types of experiments which suggest that a family of homologous operons (the gerA family, which includes gerA, gerB, and gerK) encode the predicted receptors. First, mutations in gerA, gerB, and gerK block germination in a germinant-specific manner (17-19). Second, missense mutations in one family member, gerB, allow spores to recognize novel germinants (23). Finally, spores lacking all of the gerA family members show a severe defect in nutrient-induced germination (24). At the DNA sequence level, each gerA family member is a tricistronic operon that encodes three predicted proteins named A, B, and C, and there is evidence t...

Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile

“…F, dormant wild-type spores; s, germinated wild-type spores; OE, dormant cwlD spores; , germinated cwlD spores; », dormant cotE spores. (3,59,60). The activities of some enzymes present in the spore's inner membrane have also been shown to change markedly in the first minute of spore germination in the absence of protein synthesis (48).…”

Cooperativity Between Different Nutrient Receptors in Germination of Spores of Bacillus subtilis and Reduction of This Cooperativity by Alterations in the GerB Receptor

“…Model A needs no additional (and at present hypothetical) components to explain signal processing in germination, as it requires only the formation of receptor complexes. However, there must be high affinity between receptors for significant complex formation between different nutrient receptors, since levels of nutrient receptors in the spore's inner membrane are low (22). It also seems unlikely that stimulation by a germinant or cogerminants would drive receptor complex formation.…”

“…An early event triggered by the binding of germinants to nutrient receptors is DPA release, and there is significant evidence that the proteins encoded by the spoVA operon are involved in this DPA release (8,26,28). At least one SpoVA protein, SpoVAD, is located in the spore's inner membrane (29), the location of the nutrient receptors (10,22). Consequently, it is reasonable to assume that all SpoVA proteins are in the inner membrane, perhaps as a protein complex.…”

Functional Consequences of Amino Acid Substitutions to GerVB, a Component of the Bacillus megaterium Spore Germinant Receptor

“…Biochemical and structural analyses of the germinant receptor have been hindered by an inability to express key receptor proteins at preparative levels, which, consistent with their status as receptors for environmental stimuli, have been identified as integral or membrane-associated proteins (12,22). Thus, virtually all information regarding functionality of the germinant receptor has been accumulated from geneticsbased analyses.…”