Characterization of Bacillus anthracis germinant receptors in vitro

Bacillus anthracis begins its infectious cycle as a metabolically dormant cell type, the endospore. Upon entry into a host, endospores rapidly differentiate into vegetative bacilli through the process of germination, thus initiating anthrax. Elucidation of the signals that trigger germination and the receptors that recognize them is critical to understanding the pathogenesis of B. anthracis. Individual mutants deficient in each of the seven putative germinant receptor-encoding loci were constructed via temperature-dependent, plasmid insertion mutagenesis and used to correlate these receptors with known germinant molecules. These analyses showed that the GerK and GerL receptors are jointly required for the alanine germination pathway and also are individually required for recognition of either proline and methionine (GerK) or serine and valine (GerL) as cogerminants in combination with inosine. The germinant specificity of GerS was refined from a previous study in a nonisogenic background since it was required only for germination in response to aromatic amino acid cogerminants. The gerA and gerY loci were found to be dispensable for recognition of all known germinant molecules. In addition, we show that the promoter of each putative germinant receptor operon, except that of the gerA locus, is active during sporulation. A current model of B. anthracis endospore germination is presented.     

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.     

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.     

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.     

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.     

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.     

Isolation and characterization of mutations in Bacillus subtilis that allow spore germination in the novel germinant D-alanine.

Bacillus subtilis spores break their metabolic dormancy through a process called germination. Spore germination is triggered by specific molecules called germinants, which are thought to act by binding to and stimulating spore receptors. Three homologous operons, gerA, gerB, and gerK, were previously proposed to encode germinant receptors because inactivating mutations in those genes confer a germinant-specific defect in germination. To more definitely identify genes that encode germinant receptors, we isolated mutants whose spores germinated in the novel germinant D-alanine, because such mutants would likely contain gain-of-function mutations in genes that encoded preexisting germinant receptors. Three independent mutants were isolated, and in each case the mutant phenotype was shown to result from a single dominant mutation in the gerB operon. Two of the mutations altered the gerBA gene, whereas the third affected the gerBB gene. These results suggest that gerBA and gerBB encode components of the germinant receptor. Furthermore, genetic interactions between the wild-type gerB and the mutant gerBA and gerBB alleles suggested that the germinant receptor might be a complex containing GerBA, GerBB, and probably other proteins. Thus, we propose that the gerB operon encodes at least two components of a multicomponent germinant receptor.     

Role of the gerP Operon in Germination and Outgrowth of Bacillus anthracis Spores

Germination of Bacillus anthracis spores occurs when nutrients such as amino acids or purine nucleosides stimulate specific germinant receptors located in the spore inner membrane. The gerP_ABCDEF operon has been suggested to play a role in facilitating the interaction between germinants and their receptors in spores of Bacillus subtilis and Bacillus cereus. B. anthracis mutants containing deletions in each of the six genes belonging to the orthologue of the gerP_ABCDEF operon, or deletion of the entire operon, were tested for their ability to germinate. Deletion of the entire gerP operon resulted in a significant delay in germination in response to nutrient germinants. These spores eventually germinated to levels equivalent to wild-type, suggesting that an additional entry point for nutrient germinants may exist. Deletions of each individual gene resulted in a similar phenotype, with the exception of ΔgerPF, which showed no obvious defect. The removal of two additional gerPF-like orthologues was necessary to achieve the germination defect observed for the other mutants. Upon physical removal of the spore coat, the mutant lacking the full gerP operon no longer exhibited a germination defect, suggesting that the GerP proteins play a role in spore coat permeability. Additionally, each of the gerP mutants exhibited a severe defect in calcium-dipicolinic acid (Ca-DPA)–dependent germination, suggesting a role for the GerP proteins in this process. Collectively, these data implicate all GerP proteins in the early stages of spore germination.     

Cooperativity and interference of germination pathways in Bacillus anthracis spores

Spore germination is the first step to Bacillus anthracis pathogenicity. Previous work has shown that B. anthracis spores use germination (Ger) receptors to recognize amino acids and nucleosides as germinants. Genetic analysis has putatively paired each individual Ger receptor with a specific germinant. However, Ger receptors seem to be able to partially compensate for each other and recognize alternative germinants. Using kinetic analysis of B. anthracis spores germinated with inosine and L-alanine, we previously determined kinetic parameters for this germination process and showed binding synergy between the cogerminants. In this work, we expanded our kinetic analysis to determine kinetic parameters and binding order for every B. anthracis spore germinant pair. Our results show that germinant binding can exhibit positive, neutral, or negative cooperativity. Furthermore, different germinants can bind spores by either a random or an ordered mechanism. Finally, simultaneous triggering of multiple germination pathways shows that germinants can either cooperate or interfere with each other during the spore germination process. We postulate that the complexity of germination responses may allow B. anthracis spores to respond to different environments by activating different germination pathways.     

Histidine acts as a co‐germinant with glycine and taurocholate for Clostridium difficile spores

Aims: It is well established that the bile salt sodium taurocholate acts as a germinant for Clostridium difficile spores and the amino acid glycine acts as a co‐germinant. The aim of this study was to determine whether any other amino acids act as co‐germinants. Methods and Results: Clostridium difficile spore suspensions were exposed to different germinant solutions comprising taurocholate, glycine and an additional amino acid for 1 h before heating shocking (to kill germinating cells) or chilling on ice. Samples were then re‐germinated and cultured to recover remaining viable cells. Only five amino acids out of the 19 common amino acids tested (valine, aspartic acid, arginine, histidine and serine) demonstrated co‐germination activity with taurocholate and glycine. Of these, only histidine produced high levels of germination (97·9–99·9%) consistently in four strains of Cl. difficile spores. Some variation in the level of germination produced was observed between different PCR ribotypes, and the optimum concentration of amino acids with taurocholate for the germination of Cl. difficile NCTC 11204 spores was 10–100 mmol l^?1. Conclusions: Histidine was found to be a co‐germinant for Cl. difficile spores when combined with glycine and taurocholate. Significance and Impact of the Study: The findings of this study enhance current knowledge regarding agents required for germination of Cl. difficile spores which may be utilized in the development of novel applications to prevent the spread of Cl. difficile infection.     

Diversity of spore germination in response to inosine and L-alanine and its interaction with NaCl and pH in the Bacillus cereus group

Aims: Our aim was to assess the diversity of the nutrient germination response of Bacillus cereus spores. Methods and Results: B. cereus spore germination was monitored by decrease in optical density using a Bioscreen C analyser in response to the major germinant substances inosine and l -alanine. Spores of a set of 12 strains taken to illustrate the diversity of the B. cereus group showed ranging germination capacities. Two strains never germinated in the presence of l -alanine, at any of the germinant concentrations tested. Both the extent and rate of spore germination were affected by low pH and high NaCl concentration, but differently according to the strain. Conclusions: A broad diversity was observed in nutrient-triggered spore germination among the members of the B. cereus group. Spore germination of some strains occurred at low concentrations of inosine or l -alanine, suggesting high receptor sensitivity to germinants. The activity of these receptors was also affected by pH or high NaCl concentration. Significance and Impact of the Study: The greater ability of some strains to germinate in response to l -alanine and inosine is one criterion among others for B. cereus strain selection in food processing or storage studies, before confirmation in complex food or laboratory media. The diversity in response to germinants found among the B. cereus strains suggests a differential expression and (or) absence of some germination genes involved in the response, mainly to l -alanine.     

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.     

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.     

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

Aim: To analyse the dynamic germination of hundreds of individual superdormant (SD) Bacillus subtilis spores. Methods and Results: 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 12 h and with temporal resolutions of ≤30 s. 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. Conclusions: 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. Significance and Impact of the Study: This study provides information on the dynamic germination of individual SD spores and improves the understanding of spore superdormancy.     

Analysis of interactions between nutrient germinant receptors and SpoVA proteins of Bacillus subtilis spores

Yeast two-hybrid and Far Western analyses were used to detect interactions between Bacillus subtilis spores' nutrient germinant receptor proteins and proteins encoded by the spoVA operon, all of which are involved in spore germination and located in the spores' inner membrane. These analyses indicated that two subunits of the GerA nutrient germinant receptor interact, consistent with previous genetic data, and that some GerA proteins interact with SpoVAD and some with SpoVAE. SpoVA proteins appear to be involved in the release of the spore's dipicolinic acid during spore germination, an event triggered by the binding of nutrient germinants to their receptors. Consequently, these new findings suggest that nutrient germinant receptors physically contact SpoVA proteins, and presumably this is a route for signal transduction during spore germination.     

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.     

Monitoring of Commitment,Blocking, and Continuation of Nutrient Germination of Individual Bacillus subtilis Spores

Short exposures of Bacillus spores to nutrient germinants can commit spores to germinate when germinants are removed or their binding to the spores'' nutrient germinant receptors (GRs) is inhibited. Bacillus subtilis spores were exposed to germinants for various periods, followed by germinant removal to prevent further commitment. Release of spore dipicolinic acid (DPA) was then measured by differential interference contrast microscopy to monitor germination of multiple individual spores, and spores did not release DPA after 1 to 2 min of germinant exposure until ∼7 min after germinant removal. With longer germinant exposures, percentages of committed spores with times for completion of DPA release (T_release) greater than the time of germinant removal (T_b) increased, while the time T_lag − T_b, where T_lag represents the time when rapid DPA release began, was decreased but rapid DPA release times (ΔT_release = T_release − T_lag) were increased; Factors affecting average T_release values and the percentages of committed spores were germinant exposure time, germinant concentration, sporulation conditions, and spore heat activation, as previously shown for commitment of spore populations. Surprisingly, germination of spores given a 2nd short germinant exposure 30 to 45 min after a 1st exposure of the same duration was significantly higher than after the 1st exposure, but the number of spores that germinated in the 2nd germinant exposure decreased as the interval between germinant exposures increased up to 12 h. The latter results indicate that spores have some memory, albeit transient, of their previous exposure to nutrient germinants.     

Summer meeting 2013 – when the sleepers wake: the germination of spores of Bacillus species

Spores of Bacillus species can remain dormant and resistant for years, but can rapidly ‘come back to life’ in germination triggered by agents, such as specific nutrients, and non‐nutrients, such as CaDPA, dodecylamine and hydrostatic pressure. Major events in germination include release of spore core monovalent cations and CaDPA, hydrolysis of the spore cortex peptidoglycan (PG) and expansion of the spore core. This leads to a well‐hydrated spore protoplast in which metabolism and macromolecular synthesis begin. Proteins essential for germination include the GerP proteins that facilitate germinant access to spores' inner layers, germinant receptors (GRs) that recognize and respond to nutrient germinants, GerD important in rapid GR‐dependent germination, SpoVA proteins important in CaDPA release and cortex‐lytic enzymes that degrade cortex PG. Rates of germination of individuals in spore populations are heterogeneous, and methods have been developed recently to simultaneously analyse the germination of multiple individual spores. Spore germination heterogeneity is due primarily to large variations in GR levels among individual spores, with spores that germinate extremely slowly and termed superdormant having very low GR levels. These and other aspects of spore germination will be discussed in this review, and major unanswered questions will also be discussed.     

Clostridium perfringens spore germination: characterization of germinants and their receptors

Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of L-asparagine and KCl was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in C-cpe isolates only; and (ii) L-alanine or L-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with L-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca2+ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.     

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.