Germinant Receptors Research Articles

Isolation and characterization of Bacillus subtilis spores that are superdormant for germination with dodecylamine or Ca2+ -dipicolinic acid

To isolate and characterize spores superdormant (SD) for germination with either Ca(2+)-dipicolinic acid (CaDPA) or dodecylamine. Bacillus subtilis spores were germinated three times with either CaDPA or dodecylamine and germinated spores removed after each germination treatment, yielding 0.9% (CaDPA-SD spores) or 0.4% (dodecylamine-SD spores) of initial dormant spores. Compared to dormant spores, CaDPA-SD spores germinated poorly with CaDPA and better with dodecylamine and nutrient germinants, although release of DPA from individual CaDPA-SD spores was slow during nutrient germination, and this germination was strongly inhibited by TbCl3. The CaDPA-SD spores were sensitive to hypochlorite and had elevated levels of nutrient germinant receptors (GRs) relative to levels in dormant spores. Dodecylamine-SD spores' germination with dodecylamine and nutrients was similar to that of dormant spores, their germination with Ca-DPA was slower than that of dormant spores, and these SD spores' GR levels were lower than in dormant spores. However, dodecylamine-SD spores were not sensitive to hypochlorite, and the nutrient germination of these SD spores was only partially inhibited by TbCl3 . CaDPA-SD spores appear to have a coat defect and accompanying low levels of the cortex-lytic enzyme CwlJ. The defect in dodecylamine-SD spores, however, is not clear. The results suggest that triggering germination by non-GR-dependent germinants is a potential strategy for efficient spore inactivation.

Topology and Accessibility of Germination Proteins in the Bacillus subtilis Spore Inner Membrane

Access to a membrane-impermeant biotinylation reagent as well as protease sensitivity was used to determine germination proteins' topology in the inner membrane (IM) of decoated dormant spores and intact germinated Bacillus subtilis spores. The proteins examined were four nutrient germinant receptor (GR) subunits, the GerD protein, essential for normal GR-dependent spore germination, the SpoVAD protein, essential for dipicolinic acid movement across the IM, the SleB cortex-lytic enzyme, and the YpeB protein, essential for SleB assembly in spores, as well as green fluorescent protein (GFP) in the spore core. GerD and SpoVAD as well as GFP in the spore were not biotinylated in decoated dormant spores. However, GR subunits, SleB, and YpeB were biotinylated 4 to 36% in decoated dormant spores, although these levels were not increased by higher biotinylation reagent concentrations or longer reaction times. In contrast, the germination proteins were largely biotinylated in germinated spores, although GFP was not. All of the germination proteins in the germinated spore's IM, but not spore core GFP, were largely sensitive to an exogenous protease. These results, coupled with predicted or experimentally determined structural data, indicate that (i) these germination proteins are at least partially and in some cases completely on the outer surface of the spore's IM and (ii) there is significant reorganization of these germination proteins' structure or environment in the IM during spore germination.

Analysis of the effects of a gerP mutation on the germination of spores of Bacillus subtilis.

As previously reported, gerP Bacillus subtilis spores were defective in nutrient germination triggered via various germinant receptors (GRs), and the defect was eliminated by severe spore coat defects. The gerP spores' GR-dependent germination had a longer lag time between addition of germinants and initiation of rapid release of spores' dipicolinic acid (DPA), but times for release of >90% of DPA from individual spores were identical for wild-type and gerP spores. The gerP spores were also defective in GR-independent germination by DPA with its associated Ca(2+) divalent cation (CaDPA) but germinated better than wild-type spores with the GR-independent germinant dodecylamine. The gerP spores exhibited no increased sensitivity to hypochlorite, suggesting that these spores have no significant coat defect. Overexpression of GRs in gerP spores did lead to faster germination via the overexpressed GR, but this was still slower than germination of comparable gerP(+) spores. Unlike wild-type spores, for which maximal nutrient germinant concentrations were between 500 μM and 2 mM for l-alanine and ≤10 mM for l-valine, rates of gerP spore germination increased up to between 200 mM and 1 M l-alanine and 100 mM l-valine, and at 1 M l-alanine, the rates of germination of wild-type and gerP spores with or without all alanine racemases were almost identical. A high pressure of 150 MPa that triggers spore germination by activating GRs also triggered germination of wild-type and gerP spores identically. All these results support the suggestion that GerP proteins facilitate access of nutrient germinants to their cognate GRs in spores' inner membrane.

The genomic basis for the evolution of a novel form of cellular reproduction in the bacterium Epulopiscium

BackgroundEpulopiscium sp. type B, a large intestinal bacterial symbiont of the surgeonfish Naso tonganus, does not reproduce by binary fission. Instead, it forms multiple intracellular offspring using a process with morphological features similar to the survival strategy of endospore formation in other Firmicutes. We hypothesize that intracellular offspring formation in Epulopiscium evolved from endospore formation and these two developmental programs share molecular mechanisms that are responsible for the observed morphological similarities.ResultsTo test this, we sequenced the genome of Epulopiscium sp. type B to draft quality. Comparative analysis with the complete genome of its close, endospore-forming relative, Cellulosilyticum lentocellum, identified homologs of well-known sporulation genes characterized in Bacillus subtilis. Of the 147 highly conserved B. subtilis sporulation genes used in this analysis, we found 57 homologs in the Epulopiscium genome and 87 homologs in the C. lentocellum genome.ConclusionsGenes coding for components of the central regulatory network which govern the expression of forespore and mother-cell-specific sporulation genes and the machinery used for engulfment appear best conserved. Low conservation of genes expressed late in endospore formation, particularly those that confer resistance properties and encode germinant receptors, suggest that Epulopiscium has lost the ability to form a mature spore. Our findings provide a framework for understanding the evolution of a novel form of cellular reproduction.

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

Bacillus 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 SpoVT-LacZ 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, ∼2-fold elevated GerB GR levels, wild-type levels of a GerK GR subunit and the GerD protein required for normal GR-dependent germination, ∼2.5-fold lower levels of the SpoVAD protein involved in DPA release in spore germination, and 30% lower levels of DNA protective α/β-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.

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.

Role of the gerA operon in L-alanine germination of Bacillus licheniformisspores

BackgroundThe genome of Bacillus licheniformis DSM 13 harbours three neighbouring open reading frames showing protein sequence similarities to the proteins encoded from the Bacillus subtilis subsp. subtilis 168 gerA operon, GerAA, GerAB and GerAC. In B. subtilis, these proteins are assumed to form a germinant receptor involved in spore germination induced by the amino acid L-alanine.ResultsIn this study we show that disruption of the gerAA gene in B. licheniformis MW3 hamper L-alanine and casein hydrolysate-triggered spore germination, measured by absorbance at 600 nm and confirmed by phase contrast microscopy. This ability was restored by complementation with a plasmid-borne copy of the gerA locus. Addition of D-alanine in the casein hydrolysate germination assay abolished germination of both B. licheniformis MW3 and the complementation mutant. Germination of both B. licheniformis MW3 and the gerA disruption mutant was induced by the non-nutrient germinant Ca2+-Dipicolinic acid.ConclusionsThese results demonstrate that the B. licheniformis MW3 gerA locus is involved in germination induced by L-alanine and potentially other components present in casein hydrolysate.

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.

Effects of Sporulation Conditions on the Germination and Germination Protein Levels of Bacillus subtilis Spores

Bacillus subtilis spores prepared in rich medium germinated faster with nutrient germinants than poor-medium spores as populations in liquid and multiple individual spores on a microscope slide. Poor-medium spores had longer average lag times between mixing of spores with nutrient germinants and initiation of Ca-dipicolinic acid (CaDPA) release. Rich-medium spores made at 37°C germinated slightly faster with nutrient germinants than 23°C spores in liquid, but not when spores germinated on a slide. The difference in germination characteristics of these spore populations in liquid was paralleled by changes in expression levels of a transcriptional lacZ fusion to the gerA operon, encoding a germinant receptor (GR). Levels of GR subunits were 3- to 8-fold lower in poor-medium spores than rich-medium spores and 1.6- to 2-fold lower in 23°C spores than 37°C spores, and levels of the auxiliary germination protein GerD were 3.5- to 4-fold lower in poor medium and 23°C spores. In contrast, levels of another likely germination protein, SpoVAD, were similar in all these spores. These different spores germinated similarly with CaDPA, and poor-medium and 23°C spores germinated faster than rich-medium and 37°C spores, respectively, with dodecylamine. Since spore germination with CaDPA and dodecylamine does not require GerD or GRs, these results indicate that determinants of rates of nutrient germination of spores prepared differently are primarily the levels of the GRs that bind nutrient germinants and trigger germination and secondarily the levels of GerD.

Analysis of the slow germination of multiple individual superdormant Bacillus subtilis spores using multifocus Raman microspectroscopy and differential interference contrast microscopy

To analyse the dynamic germination of hundreds of individual superdormant (SD) Bacillus subtilis spores. Germination of hundreds of individual SD B. subtilis spores with various germinants and under different conditions was followed by multifocus Raman microspectroscopy and differential interference contrast microscopy for 12h and with temporal resolutions of ≤30s. SD spores germinated poorly with the nutrient germinant used to isolate them and with alternate germinants targeting the germinant receptor (GR) used originally. The mean times following mixing of spores and nutrient germinants to initiate and complete fast release of Ca-dipicolinic acid (CaDPA) (T(lag) and T(release) times, respectively) of SD spores were much longer than those of dormant spores. However, the ΔT(release) times (T(release) -T(lag) ) of SD spores were essentially identical to those of dormant spores. SD spores germinated almost as well as dormant spores with nutrient germinants targeting GRs different from the one used to isolate the SD spores and with CaDPA that does not trigger spore germination via GRs. Since (i) ΔT(release) times were essentially identical in GR-dependent germination of SD and dormant spores; (ii) rates of GR-independent germination of SD and dormant spores were identical; (iii) large increases in T(lag) times were the major difference in the GR-dependent germination of SD as compared with spores; and (iv) higher GR levels are correlated with shorter T(lag) times, these results are consistent with the hypothesis that low levels of a GR are the major reason that some spores in a population are SD with germinants targeting this same GR. This study provides information on the dynamic germination of individual SD spores and improves the understanding of spore superdormancy.

Coatings Capable of Germinating and Neutralizing Bacillus anthracis Endospores

Endospores are formed by various bacterial families, including Bacillus and Clostridium, in response to environmental stresses as a means to survive conditions inhospitable to vegetative growth. Although metabolically inert, the endospore must interact with its environment to determine an optimal time to return to a vegetative state, a process known as germination. Germination has been shown to occur in response to a variety of chemical stimuli from specific nutrient germinants including amino acids, sugars and nucleosides. This process is known to be mediated primarily by the GerA family of spore-specific receptor proteins which initiates a signal transduction cascade that results in a return of oxidative metabolism in response to germinant receptor interactions. Herein, we report the development of a novel coating system capable of germinating B. anthracis endospores, followed by rapid killing of the vegetative bacteria by a novel incorporated amphiphilic biocide. The most effective formulation tested exhibited an ability to germinate and kill B. anthracis endospores and vegetative bacteria, respectively. The formulation reported resulted in a 90% reduction in as little as 5 min, and a 6 log reduction by 45 min.

Membrane Topology of the Bacillus anthracis GerH Germinant Receptor Proteins

Bacillus anthracis spores are the etiologic agent of anthrax. Nutrient germinant receptors (nGRs) packaged within the inner membrane of the spore sense the presence of specific stimuli in the environment and trigger the process of germination, quickly returning the bacterium to the metabolically active, vegetative bacillus. This ability to sense the host environment and initiate germination is a required step in the infectious cycle. The nGRs are comprised of three subunits: the A-, B-, and C-type proteins. To date there are limited structural data for the A- and B-type nGR subunits. Here the transmembrane topologies of the B. anthracis GerH(A), GerH(B), and GerH(C) proteins are presented. C-terminal green fluorescent protein (GFP) fusions to various lengths of the GerH proteins were overexpressed in vegetative bacteria, and the subcellular locations of these GFP fusion sites were analyzed by flow cytometry and protease sensitivity. GFP fusion to full-length GerH(C) confirmed that the C terminus of this protein is extracellular, as predicted. GerH(A) and GerH(B) were both predicted to be integral membrane proteins by topology modeling. Analysis of C-terminal GFP fusions to full-length GerH(B) and nine truncated GerH(B) proteins supports either an 8- or 10-transmembrane-domain topology. For GerH(A), C-terminal GFP fusions to full-length GerH(A) and six truncated GerH(A) proteins were consistent with a four-transmembrane-domain topology. Understanding the membrane topology of these proteins is an important step in determining potential ligand binding and protein-protein interaction domains, as well as providing new information for interpreting previous genetic work.

Effects of Cortex Peptidoglycan Structure and Cortex Hydrolysis on the Kinetics of Ca 2+ -Dipicolinic Acid Release during Bacillus subtilis Spore Germination

The kinetic parameters of the release of Ca(2+)-dipicolinic acid (CaDPA) during germination of spore populations and multiple individual spores of Bacillus subtilis strains with major alterations in the structure of the spore peptidoglycan (PG) cortex or lacking one or both of the two redundant enzymes involved in cortex hydrolysis (cortex-lytic enzymes [CLEs]) were determined. The lack of the CLE CwlJ greatly slowed CaDPA release with a germinant receptor (GR)-dependent germinant, l-valine, or a non-GR-dependent germinant, dodecylamine. The absence of the cortex-specific PG modification muramic acid-δ-lactam also increased the time needed for full CaDPA release during germination with both types of germinants. In contrast, increased cortex PG cross-linking was associated with faster times for initiation of CaDPA release with both l-valine and dodecylamine but not with faster CaDPA release once this release had been initiated. These data suggest that the precise structure of the spore cortex plays a significant role in determining the timing and the rate of CaDPA release during B. subtilis spore germination and, further, that this effect is independent of effects of GRs.

Synergism between Different Germinant Receptors in the Germination of Bacillus subtilis Spores

Synergism between Different Germinant Receptors in the Germination of Bacillus subtilis Spores

The different pathways of spore germination and inactivation in dependence of pressure and temperature

Bacillus subtilis spores (PS832) and isogenic strains, which lack the germinant receptors (FB114 and FB115), were treated up to 24 h between 100 and 700 MPa and 30–80 °C to improve the understanding of spore inactivation during the high pressure thermal sterilization. Flow cytometry was used to analyze the physiological state of the spores after pressure-treatment. A two step kinetic model was capable to derive a global model with an acceptable fit for all strains. The kinetic data demonstrated that spore germination without germination receptors is possible even at 300 MPa and no inactivation occurred up to 700 MPa and 50 °C. Contrary for PS832, a spore inactivation of more than 3 log 10 was detected at 300 MPa, whereas no inactivation was observed at 550 MPa and moderate temperature. Possibly, spores with nutrient receptors could proceed through stage II of germination at low pressure levels, whereas this step is retarded at higher pressures. The high pressure thermal sterilization (HPTS) is an emerging technology to reduce the thermal load of a sterilized food product, which consequently increases its sensorial and nutritional quality. However, an industrial installation of this promising technology has not been implemented, yet. An improved understanding of the underlying inactivation mechanisms of bacterial spores may contribute to an optimization of this process. The kinetic data of the current manuscript proves that a significant spore inactivation is possible at moderate treatment pressures and temperatures for long pressure dwell times. However, for a full spore inactivation in an economical treatment time, high pressures and elevated temperatures are needed. For B. subtilis a threshold pressure of 600 MPa was identified, whereas above this pressure the process temperature dominates the inactivation rate. Furthermore, the used two step kinetic model, applied for different kinetic data sets showed an excellent fit in the shared pressure–temperature range, which confirms that inactivation data derived under isothermal isobaric conditions could be transferred to other HP units. ► Spore germination is possible without nutrient receptors even at 200 MPa. ► Spores treated at pressure ≤ 400 MPa possibly proceed through germination stage II. ► To germinate the entire spore population high pressures and temperatures are needed. ► A two step kinetic model was capable to derive a global model for all spore strains. ► Kinetics from isothermal isobaric conditions could be transferred to other HP units.

Analysis of the germination of individual Clostridium perfringens spores and its heterogeneity

To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens. Germination of individual wild-type Cl. perfringens spores was followed by monitoring Ca-dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (T(lag)) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔT(release)) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex-lytic enzyme (CLE) (sleC spores) exhibited the same features during GR-dependent germination, but with longer average T(lag) values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The T(lag) of wild-type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild-type and sleC spores had identical average T(lag) and ΔT(release) values in dodecylamine germination that does not utilize GRs. Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE-deficient Bacillus spores, indicating that germination of these species' spores is not completely identical. This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.

Host serum factor triggers germination of Clostridium perfringens spores lacking the cortex hydrolysis machinery

Clostridium perfringens type A is the causative agent of a variety of histotoxic and enteric diseases. The ability of C. perfringens spores to germinate in vivo might be due to the presence of nutrient germinants in the host tissue and blood. In the current study, we investigated the ability of spores of C. perfringens wild-type and mutation strains to germinate in blood. Results indicate that spores of all three surveyed C. perfringens wild-type isolates germinated better in blood than in brain heart infusion (BHI) broth. However, as expected, spores lacking germinant receptor (GR) protein GerAA or GerKB germinated like wild-type spores in BHI broth and blood. Strikingly, while spores lacking GR proteins GerKA and GerKC showed significantly decreased germination in BHI broth, these spores germinated well in blood, suggesting that blood factor(s) can trigger spore germination through a GR-independent pathway. Using C. perfringens spores lacking cortex lytic enzymes (ΔcspB or ΔsleC ΔsleM), we were able to identify a host serum germination factor with peptidoglycan hydrolysing activity that (i) restored the colony-forming efficiencies of ΔcspB and ΔsleC ΔsleM spores up to ~5-20% of that of total colony-forming spores; (ii) increased the number of c.f.u. of decoated ΔcspB and ΔsleC ΔsleM spores to ~99% of that of colony-forming spores; (iii) and finally lost enzymic activity after heat inactivation, consistent with serum germination factor being an enzyme. Further characterization demonstrated that serum germination factor is very likely lysozyme, which can form a stable high molecular mass complex of ~120 kDa in serum. In conclusion, the current study indicates that a host serum germination factor with peptidoglycan hydrolysing activity is capable of triggering germination of C. perfringens spores by directly degrading the spore peptidoglycan cortex. Collectively, this study contributes to our understanding of the mechanism of in vivo germination of spores of C. perfringens.

Germination proteins in the inner membrane of dormant Bacillus subtilis spores colocalize in a discrete cluster

Dormant bacterial spores are extraordinarily resistant to environmental insults and are vectors of various illnesses. However, spores cannot cause disease unless they germinate and become vegetative cells. The molecular details of initiation of germination are not understood, but proteins essential in early stages of germination, such as nutrient germinant receptors (GRs) and GerD, are located in the spore inner membrane. In this study, we examine how these germination proteins are organized in dormant Bacillus subtilis spores by expressing fluorescent protein fusions that were at least partially functional and observing spores by fluorescence microscopy. We show that GRs and GerD colocalize primarily to a single cluster in dormant spores, reminiscent of the organization of chemoreceptor signalling complexes in Escherichia coli. GRs require all their subunits as well as GerD for clustering, and also require diacylglycerol addition to GerD and GRs' C protein subunits. However, different GRs cluster independently of each other, and GerD forms clusters in the absence of all the GRs. We predict that the clusters represent a functional germination unit or 'germinosome' in the spore inner membrane that is necessary for rapid and cooperative response to nutrients, as conditions known to block nutrient germination also disrupt the protein clusters.

Synergism between Different Germinant Receptors in the Germination of Bacillus subtilis Spores

Rates of commitment to germinate and germination of Bacillus subtilis spores with mixtures of low concentrations of germinants acting on different germinant receptors (GRs) were much higher than the sums of the rates of commitment and germination with individual germinants alone. This synergism with mixtures of nutrient germinants was not seen with spores lacking GRs responsible for recognizing one or several components of the germinant mixtures and was not eliminated by either a gerD mutation or overexpression of one of the GRs involved in this synergism. This synergism was also not seen between the germinant L-valine, which acts via a GR, and the germinant dodecylamine, which does not act via any GR. These results indicate that spores not only integrate but can also amplify signals from multiple germinants and multiple GRs in determining rates of commitment and overall spore germination. This amplification can be explained by a simple mechanism in which a single signal integrator triggers germination above an accumulation threshold. Direct cooperative action between GRs may further add to the synergism seen in germination triggered by multiple GRs. Further experiments and modeling are required to determine the relative contributions of these different mechanisms.

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.