Factors influencing germination of Bacillus subtilis spores via activation of nutrient receptors by high pressure

Different nutrient receptors varied in triggering germination of Bacillus subtilis spores with a pressure of 150 MPa, the GerA receptor being more responsive than the GerB receptor and even more responsive than the GerK receptor. This hierarchy in receptor responsiveness to pressure was the same as receptor responsiveness to a mixture of nutrients. The levels of nutrient receptors influenced rates of pressure germination, since the GerA receptor is more abundant than the GerB receptor and elevated levels of individual receptors increased spore germination by 150 MPa of pressure. However, GerB receptor variants with relaxed specificity for nutrient germinants responded as well as the GerA receptor to this pressure. Spores lacking dipicolinic acid did not germinate with this pressure, and pressure activation of the GerA receptor required covalent addition of diacylglycerol. However, pressure activation of the GerB and GerK receptors displayed only a partial (GerB) or no (GerK) diacylglycerylation requirement. These effects of receptor diacylglycerylation on pressure germination are similar to those on nutrient germination. Wild-type spores prepared at higher temperatures germinated more rapidly with a pressure of 150 MPa than spores prepared at lower temperatures; this was also true for spores with only one receptor, but receptor levels did not increase in spores made at higher temperatures. Changes in inner membrane unsaturated fatty acid levels, lethal treatment with oxidizing agents, or exposure to chemicals that inhibit nutrient germination had no major effect on spore germination by 150 MPa of pressure, except for strong inhibition by HgCl2.     

Cooperativity between different nutrient receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by alterations in the GerB receptor

The GerA nutrient receptor alone triggers germination of Bacillus subtilis spores with L-alanine or L-valine, and these germinations were stimulated by glucose and K+ plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K+ (GFK) (termed cogerminants) plus GerK for triggering of germination with a number of L-amino acids. GerB and GerA also triggered spore germination cooperatively with l-asparagine, fructose, and K+ and either L-alanine or L-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to D-alanine do not respond to D-alanine but respond to the same L-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these L-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to l-alanine germination via GerA, spore germination via L-alanine and GerB or GerB* was not inhibited by D-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K+ somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to L-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to L-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to L-amino acids, and GerA interacts only with L-valine, L-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to L-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore germination.     

Effects of a gerF (lgt) mutation on the germination of spores of Bacillus subtilis

One of the proteins of the membrane-bound receptors that recognize individual nutrients that trigger germination of spores of Bacillus subtilis contains the recognition sequence for diacylglycerol addition to a cysteine residue near the protein's N terminus. B. subtilis spores lacking the gerF (lgt) gene that codes for prelipoprotein diacylglycerol transferase exhibited significantly slowed germination in response to nutrient germinants as found previously, but germination of gerF spores with a mixture of Ca2+ and dipicolinic acid or with dodecylamine was normal, as was the spontaneous germination of gerF spores lacking all nutrient germinant receptors. The deleterious effects of the gerF mutation on nutrient germination were highest on germination triggered by the GerA nutrient receptor and were less so (but still significant) on germination triggered by the GerB nutrient receptor. However, there was little, if any, effect on GerK nutrient receptor-mediated spore germination. As predicted from the latter results, replacement by alanine of the cysteine residue to which diacylglycerol is thought to be added to these nutrient receptors had a large effect on GerA receptor function, less effect on GerB receptor function, and little, if any, effect on GerK receptor function.     

Structure-based functional studies of the effects of amino acid substitutions in GerBC, the C subunit of the Bacillus subtilis GerB spore germinant receptor

Highly conserved amino acid residues in the C subunits of the germinant receptors (GRs) of spores of Bacillus and Clostridium species have been identified by amino acid sequence comparisons, as well as structural predictions based on the high-resolution structure recently determined for the C subunit of the Bacillus subtilis GerB GR (GerBC). Single and multiple alanine substitutions were made in these conserved residues in three regions of GerBC, and the effects of these changes on B. subtilis spore germination via the GerB GR alone or in concert with the GerK GR, as well as on germination via the GerA GR, were determined. In addition, levels of the GerBC variants in the spore inner membrane were measured, and a number of the GerBC proteins were expressed and purified and their solubility and aggregation status were assessed. This work has done the following: (i) identified a number of conserved amino acids that are crucial for GerBC function in spore germination via the GerB GR and that do not alter spores' levels of these GerBC variants; (ii) identified other conserved GerBC amino acid essential for the proper folding of the protein and/or for assembly of GerBC in the spore inner membrane; (iii) shown that some alanine substitutions in GerBC significantly decrease the GerA GR's responsiveness to its germinant l-valine, consistent with there being some type of interaction between GerA and GerB GR subunits in spores; and (iv) found no alanine substitutions that specifically affect interaction between the GerB and GerK GRs.     

Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis

The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K(+) (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.     

The Effects of Heat Activation on Bacillus Spore Germination,with Nutrients or under High Pressure,with or without Various Germination Proteins

Nutrient germination of spores of Bacillus species occurs through germinant receptors (GRs) in spores'' inner membrane (IM) in a process stimulated by sublethal heat activation. Bacillus subtilis spores maximum germination rates via different GRs required different 75°C heat activation times: 15 min for l-valine germination via the GerA GR and 4 h for germination with the l-asparagine–glucose–fructose–K⁺ mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores'' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores'' IM and only indirectly affecting GRs. The 150- and 550-MPa HP germinations of Bacillus amyloliquefaciens spores, a potential surrogate for Clostridium botulinum spores in HP treatments of foods, were also stimulated by heat activation.     

Germination protein levels and rates of germination of spores of Bacillus subtilis with overexpressed or deleted genes encoding germination proteins

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 gerF spores were consistent with effects of loss of GerF on spore germination by different GRs.     

The GerW Protein Is Not Involved in the Germination of Spores of Bacillus Species

Germination of dormant spores of Bacillus species is initiated when nutrient germinants bind to germinant receptors in spores’ inner membrane and this interaction triggers the release of dipicolinic acid and cations from the spore core and their replacement by water. Bacillus subtilis spores contain three functional germinant receptors encoded by the gerA, gerB, and gerK operons. The GerA germinant receptor alone triggers germination with L-valine or L-alanine, and the GerB and GerK germinant receptors together trigger germination with a mixture of L-asparagine, D-glucose, D-fructose and KCl (AGFK). Recently, it was reported that the B. subtilis gerW gene is expressed only during sporulation in developing spores, and that GerW is essential for L-alanine germination of B. subtilis spores but not for germination with AGFK. However, we now find that loss of the B. subtilis gerW gene had no significant effects on: i) rates of spore germination with L-alanine; ii) spores’ levels of germination proteins including GerA germinant receptor subunits; iii) AGFK germination; iv) spore germination by germinant receptor-independent pathways; and v) outgrowth of germinated spores. Studies in Bacillus megaterium did find that gerW was expressed in the developing spore during sporulation, and in a temperature-dependent manner. However, disruption of gerW again had no effect on the germination of B. megaterium spores, whether germination was triggered via germinant receptor-dependent or germinant receptor-independent pathways.     

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

Germination of dormant Bacillus subtilis spores with specific nutrient germinants is dependent on a number of inner membrane (IM) proteins, including (i) the GerA, GerB, and GerK germinant receptors (GRs) that respond to nutrient germinants; (ii) the GerD protein, essential for optimal GR function; and (iii) SpoVA proteins, essential for the release of the spore-specific molecule dipicolinic acid (DPA) during spore germination. Levels of GR A and C subunit proteins, GerD, and SpoVAD in wild-type spores were determined by Western blot analysis of spore fractions or total disrupted spores by comparison with known amounts of purified proteins. Surprisingly, after disruption of decoated B. subtilis spores with lysozyme and fractionation, ∼90% of IM fatty acids and GR subunits remained with the spores'' insoluble integument fraction, indicating that yields of purified IM are low. The total lysate from disrupted wild-type spores contained ∼2,500 total GRs/spore: GerAA and GerAC subunits each at ∼1,100 molecules/spore and GerBC and GerKA subunits each at ∼700 molecules/spore. Levels of the GerBA subunit determined previously were also predicted to be ∼700 molecules/spore. These results indicate that the A/C subunit stoichiometry in GRs is most likely 1:1, with GerA being the most abundant GR. GerD and SpoVAD levels were ∼3,500 and ∼6,500 molecules/spore, respectively. These values will be helpful in formulating mathematic models of spore germination kinetics as well as setting lower limits on the size of the GR-GerD complex in the spores'' IM, termed the germinosome.     

Interaction between individual protein components of the GerA and GerB nutrient receptors that trigger germination of Bacillus subtilis spores

Germination of Bacillus subtilis spores via the GerA nutrient receptor was suppressed by GerAC lacking the diacylglycerylated cysteine essential for receptor function. Overexpression of the C protein of the GerB nutrient receptor also suppressed the function of both the GerA receptor and a variant GerB receptor, GerB*. These findings suggest that GerAC and GerBC interact with their respective A and B proteins in GerA or GerB receptors and that GerBC potentially interacts with GerAA-GerAB. However, GerAC did not appear to interact with GerBA-GerBB.     

Comparative study of pressure- and nutrient-induced germination of Bacillus subtilis spores

Germination experiments with specific germination mutants of Bacillus subtilis, including a newly isolated mutant affected in pressure-induced germination, suggest that a pressure of 100 MPa triggers the germination cascades that are induced by the nutrient germinant alanine (Ala) and by a mixture of asparagine, glucose, fructose, and potassium ions (AGFK), by activating the receptors for alanine and asparagine, GerA and GerB, respectively. As opposed to germination at 100 MPa, germination at 600 MPa apparently short-cuts at least part of the Ala- and AGFK-induced germination pathways. Inhibitors of nutrient-induced germination (HgCl(2) and Nalpha-P-tosyl-L-arginine methyl ester) also inhibit pressure-induced germination at 600 MPa, suggesting that germination at 600 MPa involves activation of a true physiological germination pathway and is therefore not merely a physico-chemical process in which water is forced into the spore protoplast.     

Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis

Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of β-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.     

Expression of genes coding for GerA and GerK spore germination receptors is dependent on the protein phosphatase PrpE

The ability of Bacillus subtilis to form spores is a strategy for survival under unfavorable environmental conditions. It is equally crucial to break spore dormancy and return to vegetative growth at the appropriate time. Here we present data showing that the PrpE phosphatase is involved in the control of expression of genes coding for GerA receptors, which are necessary for L-alanine-induced spore germination. Moreover, PrpE is also involved in aspartic acid, glucose, fructose, and potassium (AGFK)-induced spore germination by controlling expression of genes coding for GerK receptors. In the absence of PrpE, the production of spores was essentially normal. However, L-alanine-induced spore germination and, to a lesser extent, the AGFK-induced pathway were abolished. In contrast, the germination pathway dependent on Ca2+-dipicolinate or dodecylamine remained intact. A protein phosphatase PrpE-green fluorescent protein fusion was localized to the prespore and to the dormant spore, consistent with a role in controlling expression of genes coding for GerA receptors. We propose that PrpE is an important element in a signal transduction pathway in Bacillus subtilis that controls the expression of genes coding for germination receptors.     

Role of GerD in germination of Bacillus subtilis spores

Spores of a Bacillus subtilis strain with a gerD deletion mutation (Delta gerD) responded much slower than wild-type spores to nutrient germinants, although they did ultimately germinate, outgrow, and form colonies. Spores lacking GerD and nutrient germinant receptors also germinated slowly with nutrients, as did Delta gerD spores in which nutrient receptors were overexpressed. The germination defect of Delta gerD spores was not suppressed by many changes in the sporulation or germination conditions. Germination of Delta gerD spores was also slower than that of wild-type spores with a pressure of 150 MPa, which triggers spore germination through nutrient receptors. Ectopic expression of gerD suppressed the slow germination of Delta gerD spores with nutrients, but overexpression of GerD did not increase rates of spore germination. Loss of GerD had no effect on spore germination induced by agents that do not act through nutrient receptors, including a 1:1 chelate of Ca2+ and dipicolinic acid, dodecylamine, lysozyme in hypertonic medium, a pressure of 500 MPa, and spontaneous germination of spores that lack all nutrient receptors. Deletion of GerD's putative signal peptide or change of its likely diacylglycerylated cysteine residue to alanine reduced GerD function. The latter findings suggest that GerD is located in a spore membrane, most likely the inner membrane, where the nutrient receptors are located. All these data suggest that, while GerD is not essential for nutrient germination, this protein has an important role in spores' rapid response to nutrient germinants, by either direct interaction with nutrient receptors or some signal transduction essential for germination.     

Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore.

The GerAA, -AB, and -AC proteins of the Bacillus subtilis spore are required for the germination response to L-alanine as the sole germinant. They are likely to encode the components of the germination apparatus that respond directly to this germinant, mediating the spore's response; multiple homologues of the gerA genes are found in every spore former so far examined. The gerA operon is expressed in the forespore, and the level of expression of the operon appears to be low. The GerA proteins are predicted to be membrane associated. In an attempt to localize GerA proteins, spores of B. subtilis were broken and fractionated to give integument, membrane, and soluble fractions. Using antibodies that detect Ger proteins specifically, as confirmed by the analysis of strains lacking GerA and the related GerB proteins, the GerAA protein and the GerAC+GerBC protein homologues were localized to the membrane fraction of fragmented spores. The spore-specific penicillin-binding protein PBP5*, a marker for the outer forespore membrane, was absent from this fraction. Extraction of spores to remove coat layers did not release the GerAC or AA protein from the spores. Both experimental approaches suggest that GerAA and GerAC proteins are located in the inner spore membrane, which forms a boundary around the cellular compartment of the spore. The results provide support for a model of germination in which, in order to initiate germination, germinant has to permeate the coat and cortex of the spore and bind to a germination receptor located in the inner membrane.     

The Bacillus subtilis germinant receptor GerA triggers premature germination in response to morphological defects during sporulation

During sporulation in Bacillus subtilis, germinant receptors assemble in the inner membrane of the developing spore. In response to specific nutrients, these receptors trigger germination and outgrowth. In a transposon‐sequencing screen, we serendipitously discovered that loss of function mutations in the gerA receptor partially suppress the phenotypes of > 25 sporulation mutants. Most of these mutants have modest defects in the assembly of the spore protective layers that are exacerbated in the presence of a functional GerA receptor. Several lines of evidence indicate that these mutants inappropriately trigger the activation of GerA during sporulation resulting in premature germination. These findings led us to discover that up to 8% of wild‐type sporulating cells trigger premature germination during differentiation in a GerA‐dependent manner. This phenomenon was observed in domesticated and undomesticated wild‐type strains sporulating in liquid and on solid media. Our data indicate that the GerA receptor is poised on a knife's edge during spore development. We propose that this sensitized state ensures a rapid response to nutrient availability and also elicits premature germination of spores with improperly assembled protective layers resulting in the elimination of even mildly defective individuals from the population.     

Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure

AIMS: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination. METHODS AND RESULTS: Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50 degrees C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60 degrees C, in contrast to an optimum temperature of 40 degrees C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23 degrees C to 44 degrees C, and decreased twofold when 1 mol l(-1) salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg(2+), but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment. CONCLUSIONS: VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear. SIGNIFICANCE AND IMPACT OF THE STUDY: There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.     

Effects of moderately high pressure plus heat on the germination and inactivation of Bacillus cereus spores lacking proteins involved in germination

Aims:  To determine the germination and inactivation of Bacillus cereus spores lacking various germination proteins using moderately high pressure (MHP) and heat.
Methods:  The inactivation and germination of wild-type B. cereus spores in buffer by MHP (150 MPa) at various temperatures, as well as the MHP inactivation and germination of B. cereus spores lacking individual germinant receptors and monovalent cation antiporters, was determined.
Results:  Loss of individual germinant receptors had no large effects on spore inactivation or germination, although germination of receptor-deficient spores was generally slightly decreased. Loss of the GerN in particular the GerN and GerT antiporters also decreased spore germination by MHP, especially at 40 and 50°C.
Conclusions:  Both inactivation and germination of B. cereus spores by MHP increased with rise of temperature; however, mutant strains lacking individual germinant receptor had similar levels of germination as compared to wild-type spores. To evaluate the role of germinant receptors in MHP, a strain lacking a large number of germinant receptors is needed.
Significance and Impact of the Study:  The results of this work may lead to a better understanding of how MHP causes germination of spores of B. cereus .     

Role of ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis

Dormant Bacillus subtilis spores germinate in the presence of particular nutrients called germinants. The spores are thought to recognize germinants through receptor proteins encoded by the gerA family of operons, which includes gerA, gerB, and gerK. We sought to substantiate this putative function of the GerA family proteins by characterizing spore germination in a mutant strain that contained deletions at all known gerA-like loci. As expected, the mutant spores germinated very poorly in a variety of rich media. In contrast, they germinated like wild-type spores in a chemical germinant, a 1-1 chelate of Ca(2+) and dipicolinic acid (DPA). These observations showed that proteins encoded by gerA family members are required for nutrient-induced germination but not for chemical-triggered germination, supporting the hypothesis that the GerA family encodes receptors for nutrient germinants. Further characterization of Ca(2+)-DPA-induced germination showed that the effect of Ca(2+)-DPA on spore germination was saturated at 60 mM and had a K(m) of 30 mM. We also found that decoating spores abolished their ability to germinate in Ca(2+)-DPA but not in nutrient germinants, indicating that Ca(2+)-DPA and nutrient germinants probably act through parallel arms of the germination pathway.