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.     

Mechanisms of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination.

The mechanism by which potassium sorbate inhibits Bacillus cereus T and Clostridium botulinum 62A spore germination was investigated. Spores of B. cereus T were germinated at 35 degrees C in 0.08 M sodium-potassium phosphate buffers (pH 5.7 and 6.7) containing various germinants (L-alanine, L-alpha-NH2-n-butyric acid, and inosine) and potassium sorbate. Spores of C. botulinum 62A were germinated in the same buffers but with 10 mM L-lactic acid, 20 mM sodium bicarbonate, L-alanine or L-cysteine, and potassium sorbate. Spore germination was monitored by optical density measurements at 600 nm and phase-contrast microscopy. Inhibition of B. cereus T spore germination was observed when 3,900 micrograms of potassium sorbate per ml was added at various time intervals during the first 2 min of spore exposure to the pH 5.7 germination medium. C. botulinum 62A spore germination was inhibited when 5,200 micrograms of potassium sorbate per ml was added during the first 30 min of spore exposure to the pH 5.7 medium. Potassium sorbate inhibition of germination was reversible for both B. cereus T and C. botulinum 62A spores. Potassium sorbate inhibition of B. cereus T spore germination induced by L-alanine and L-alpha-NH2-n-butyric acid was shown to be competitive in nature. Potassium sorbate was also a competitive inhibitor of L-alanine- and L-cysteine-induced germination of C. botulinum 62A spores.     

Factors affecting the germination of spores of Bacillus anthracis

Spores of Bacillus anthracis germinated poorly at high cell densities unless the alanine racemase inhibitor O-carbamyl-D-serine was added to the germination medium. Spores derived from a variety of strains of B. anthracis germinated optimally at 22 degrees C. No correlation was found between rate of spore germination and virulence or between susceptibility of animal species to anthrax and spore germination rate using sera from those animals as the germination medium.     

Factors affecting the germination of spores of Bacillus anthracis

Spores of Bacillus anthracis germinated poorly at high cell densities unless the alanine racemase inhibitor O-carbamyl-D-serine was added to the germination medium. Spores derived from a variety of strains of B. anthracis germinated optimally at 22°C. No correlation was found between rate of spore germination and virulence or between susceptibility of animal species to anthrax and spore germination rate using sera from those animals as the germination medium.     

Involvement of the spore coat in germination of Bacillus cereus T spores

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.     

Involvement of the spore coat in germination of Bacillus cereus T spores.

Bacillus cereus T spores were prepared on fortified nutrient agar, and the spore coat and outer membrane were extracted by 0.5% sodium dodecyl sulfate-100 mM dithiothreitol in 0.1 M sodium chloride (SDS-DTT) at pH 10.5 (coat-defective spores). Coat-defective spores in L-alanine plus adenosine germinated slowly and to a lesser extent than spores not treated with SDS-DTT, as determined by decrease in absorbance and release of dipicolinic acid and Ca2+. Spores germinated in calcium dipicolinate only after treatment with SDS-DTT. Biphasic and triphasic germination kinetics were observed with normal and coat-defective spores, respectively, in an environment with temperature increasing from 20 to 65 degrees C at a rate of 1 degree C/min. Therefore, the physical and biochemical processes involved in germination are modified by coat removal. The data suggest that a portion of the germination apparatus located interior to the coat may be protected by the coat and outer membrane or that the coat and outer membrane otherwise enhance germination in L-alanine plus adenosine. When coat-defective spores were heat activated with the dialyzed (12,000-Mr cutoff) components extracted from the spores, germination of the SDS-DTT-treated spores was enhanced; thus, one or more components located in the spore coat or outer membrane with a molecular weight greater than 12,000 were essential for fast germination.     

Identification of an in vivo inhibitor of Bacillus anthracis spore germination

Germination of Bacillus anthracis spores into the vegetative form is an essential step in anthrax pathogenicity. This process can be triggered in vitro by the common germinants inosine and alanine. Kinetic analysis of B. anthracis spore germination revealed synergy and a sequential mechanism between inosine and alanine binding to their cognate receptors. Because inosine is a critical germinant in vitro, we screened inosine analogs for the ability to block in vitro germination of B. anthracis spores. Seven analogs efficiently blocked this process in vitro. This led to the identification of 6-thioguanosine, which also efficiently blocked spore germination in macrophages and prevented killing of these cells mediated by B. anthracis spores. 6-Thioguanosine shows potential as an anti-anthrax therapeutic agent.     

GerN, an antiporter homologue important in germination of Bacillus cereus endospores

A homologue of the grmA spore germination gene of Bacillus megaterium and of a NaH-antiporter gene (napA) of Enterococcus hirae has been identified in Bacillus cereus 569 (ATCC 10876). The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively. Insertional inactivation of this B. cereus gene, named gerN, did not affect vegetative growth or sporulation. The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to L-alanine (10 mM). The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed. At a suboptimal germinant concentration (50 microM), inosine germination was completely blocked in the mutant, while the rate of germination in 50 microM L-alanine was reduced to one-third of that of the wild type. The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN. Since germination in suboptimal concentrations of L-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations.     

Identification of Bacillus anthracis proteins associated with germination and early outgrowth by proteomic profiling of anthrax spores

The use of anthrax spores as a bioweapon has spurred efforts aimed at identifying key proteins expressed in Bacillus anthracis. Because spore germination and outgrowth occur prior to and are required for disease manifestations, blocking germination and early outgrowth with novel vaccines or inhibitors targeting critical B. anthracis germination and outgrowth-associated factors is a promising strategy in mitigating bioterror. By screening 587 paired protein spots that were isolated from dormant and germinating anthrax spores, respectively, we identified 10 spore proteins with statistically significant germination-associated increases and decreases. It is likely that proteins whose levels change during germination may play key roles in the germination and outgrowth processes, and they should be listed as priority targets for development of prophylactic and therapeutic agents against anthrax. The 31 new proteins identified in this study also complement an emerging proteomic database of B. anthracis.     

Stimulation of germination of unactivated Bacillus cereus spores by ammonia

Inclusion of ammonia in germinant mixtures containing L-alanine and inosine stimulated germination of unactivated Bacillus cereus spores at rates equal to those obtained using heat-activated spores without ammonia. D-Alanine had little effect on germination of heat-activated spores, but severely inhibited germination of unactivated spores in the presence of ammonia. Ammonia did not replace the requirement for either L-alanine or inosine: all three compounds were required for rapid germination. Kinetic analysis suggested that the functions of ammonia and L-alanine were more closely related than the functions of ammonia and inosine. With rate-saturating concentrations of L-alanine and inosine, germination rates showed saturation kinetics for ammonia with a Km for NH4Cl of 5 mM. Comparisons of the effects of salts, amines and pH on germination rates suggested that NH4OH rather than NH+4 was the rate-limiting form of ammonia. In comparisons of various strains of B. cereus, stimulation of germination by ammonia occurred in all cases, although spores of most soil isolates germinated more rapidly than B. cereus T spores in the absence of ammonia.     

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.     

Monoclonal antibodies against spore antigens of Bacillus anthracis

A murine monoclonal antibody produced against heat inactivated spores of Bacillus anthracis Ames, reacted with live or inactivated spores of several anthrax strains in indirect immunofluorescence (IF) tests. The reactive anthrax strain gave only a moderate degree of reaction. No staining of anthrax vegetative cells was observed. The monoclonal did not react with spores of non-anthrax Bacillus strains that gave cross reactions with mouse hyperimmune antiserum raised against Ames spores. The staining of individual spores in B. anthracis preparations was more heterogeneous with the monoclonal antibody than with the hyperimmune serum. Evidence is produced that the epitope for this monoclonal is not stable during long-term storage of inactivated spore preparations, and is not fully available for reaction with antibody until late in spore maturation. The monoclonal did not react by immunoblotting (Western blotting) of spore extracts. A monoclonal antibody produced against Ames spore extracts reacted with about 1% of Ames spores in IF tests, but not reproducible reactions with other anthrax strains were recorded. This monoclonal interacted with three bands in Western blots of anthrax spore extracts.     

Localization of the germination protein GerD to the inner membrane in Bacillus subtilis spores

GerD of Bacillus subtilis is a protein essential for normal spore germination with either L-alanine or a mixture of L-asparagine, D-glucose, D-fructose, and potassium ions. GerD's amino acid sequence suggests that it may be a lipoprotein, indicating a likely location in a membrane. Location in the spore's outer membrane seems unlikely, since removal of this membrane does not result in a gerD spore germination phenotype, suggesting that GerD is likely in the spore's inner membrane. In order to localize GerD within spores, FLAG-tagged GerD constructs were made, found to be functional in spore germination, and detected in immunoblots of spore extracts as not only monomers but also dimers and trimers. Upon fractionation of spore extracts, GerD-FLAG was found in the inner membrane fraction from dormant spores and was present at approximately 2,000 molecules/spore. GerD-FLAG in the inner membrane fraction was solubilized by Triton X-100, suggesting that GerD is a lipoprotein, and the protein was also solubilized by 0.5 M NaCl. GerD-FLAG was not processed proteolytically in a B. subtilis strain lacking gerF (lgt), which encodes prelipoprotein diacylglycerol transferase (Lgt), indicating that when GerD does not have a diacylglycerol moiety, signal sequence processing does not occur. However, unprocessed GerD-FLAG still gave bands corresponding to monomers and dimers of slightly higher molecular weight than that of GerD-FLAG from a strain with Lgt, further suggesting that GerD is a lipoprotein. Upon spore germination, much GerD became soluble and then appeared to be degraded as the germinated spores outgrew and initiated vegetative growth. All of these results suggest that GerD is a lipoprotein associated with the dormant spore's inner membrane that may be released in some fashion from this membrane upon spore germination.     

Heat resistance, spore germination, and enterotoxigenicity of Clostridium perfringens

Heat resistance at 95 C, heat activation at 75 C, and germination response were determined for spores of 10 serotype strains of Clostridium perfringens type A, including five heat-resistant and five heat-sensitive strains. The D95-values ranged from 17.6 to 63.0 and from 1.3 to 2.8 for the heat-resistant and the heat-sensitive strains, respectively. The heat-activation values, the ratios between the heated and unheated viable counts of spore suspensions, ranged from 0.0035 to 0.65 and from 6.5 to 60.0 for the heat-sensitive and the heat-resistant strains, respectively. Spores of these strains were divided into two distinct germination types on the basis of their germination response; spores of the heat-resistant strains germinated in KC1 medium after heat activation (K-type), and spores of the heat-sensitive strains germinated in a mixture of L-alanine, inosine, and CaCl2 in the presence of CO2 without heat activation (A-type). The strains were tested for enterotoxigenicity by a reversed passive latex-agglutination (RPLA) test. All the heat-resistant strains were RPLA-positive, whereas the heat-sensitive strains were all RPLA-negative. A total of 37 strains of the organism isolated from food-poisoning outbreaks were tested for spore germination and enterotoxin formation. All of the 20 heat-resistant strains showed K-type spore germination and, except for three strains, were RPLA-positive, whereas all of the 17 heat-sensitive strains showed A-type spore germination and, except for only one strain, were RPLA-negative.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

Spore pool glutamic acid as a metabolite in germination

Spore glutamic acid pools were examined in dormant and germinating spores using colorimetric and (14)C analytical procedures. Germination of spores of Bacillus megaterium (parent strain), initiated by d-glucose, was accompanied by a rapid drop in the level of spore pool glutamate, from 12.0 mug/mg of dry spores to 7.7 mug/mg of dry spores after 30 sec of germination. Similar decreases in extractable spore pool glutamate were observed with l-alanine-initiated germination of B. licheniformis spores. On the other hand, glutamate pools of mutant spores of B. megaterium, with a requirement of gamma-aminobutyric acid for spore germination, remained unchanged for 9 min of germination, at which time more than 50% of the spore population had germinated. Evidence for conversion of spore pool glutamate to gamma-aminobutyric acid during germination of spores of B. megaterium (parent strain) was obtained.     

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.     

Mechanism of Ca2+ and dipicolinic acid requirement for L-alanine induced germination of Bacillus cereus BIS-59 spores

Spores prepared from different sporulating media containing varying amounts of Ca and dipicolinic acid (DPA), exhibited differential responses to germination in L-alanine (0.25 M). Ca-spores with moderately high Ca and DPA contents could be triggered to germination by L-alanine, whereas P-spores with low contents of Ca and DPA could not be germinated by L-alanine unless Ca2+ or DPA was exogenously added. The initiation of L-alanine induced germination by P-spores in the presence of 45CaCl2 was associated with a marked uptake of 45Ca2+. Experiments involving stepwise extraction of 45Ca from prelabelled spores indicated that a part of the spore calcium may be involved in L-alanine induced germination. Both Ca2+ and DPA seemed to have a stimulatory effect on the incorporation of 14C-L-alanine.     

炭疽灭活和裂解芽胞抗原免疫家兔后血清中的IgG抗体应答

炭疽杆菌芽胞在炭疽免疫中发挥基本作用。实验中以炭疽活芽胞疫苗为原形,建立了制备灭活和裂解炭疽芽胞的方法,研究了各种灭活和裂解炭疽芽胞疫苗不同浓度、不同剂次免疫家兔的抗芽胞和毒素IgG应答,总结分析了各种灭活和裂解炭疽芽胞疫苗用于新疫苗成分之一的可能性。甲醛灭活炭疽芽胞疫苗设芽胞浓度2.5×108剂量组、5×108剂量组、1×109剂量组,于0、4、8周时3次免疫。在3剂免疫后血清抗炭疽芽胞IgG水平持续升高,首次免疫后4、8、12周时家兔血清中抗芽胞IgG几何平均滴度可达到600～16000。裂解炭疽芽胞疫苗的制备和动物免疫中,只采取了2.5×108芽胞浓度,两剂免疫,免疫时间为0、4周。在首次免疫后4、8、12周时家兔血清中抗芽胞IgG几何平均滴度分别为362、776和388。各时间点采集的家兔血清未能测出或只测出极微量的抗炭疽毒素IgG。通过上述研究认为,以裂解炭疽芽胞抗原作为炭疽疫苗成分之一,其抗原性和免疫原性是适宜的;免疫剂量可以设定为2.5×108芽胞浓度上下;免疫次数可定为2剂间隔1个月。     

Monoclonal antibodies against spore antigens of Bacillus anthracis

Abstract A murine monoclonal antibody produced against heat inactivated spores of Bacillus anthracis Ames, reacted with live or inactivated spores of several anthrax strains in indirect immunofluorescence (IF) tests. The reactive anthrax strain gave only a moderate degree of reaction. No staining of anthrax vegetative cells was observed. The monoclonal did not react with spores of non-anthrax Bacillus strains that gave cross reactions with mouse hyperimmune antiserum raised against Ames spores. The staining of individual spores in B. anthracis preparations was more heterogeneous with the monoclonal antibody than with the hyperimmune serum. Evidence is produced that the epitope for this monoclonal is not stable during long-term storage of inactivated spore preparations, and is not fully available for reaction with antibody until late in spore maturation. The monoclonal did not react by immunoblotting (Western blotting) of spore extracts.