Germination Response Of Spores Research Articles

Nutrient L-Alanine-Induced Germination of Bacillus Improves Proliferation of Spores and Exerts Probiotic Effects in vitro and in vivo

As alternatives to antibiotics in feed, probiotic Bacillus carries multiple advantages in animal production. Spores undergo strain-related germination in the gastrointestinal tract, but it is still unknown whether the probiotic function of the Bacillus depends on the germination of spores in vivo. In this study, based on 14 potential probiotic Bacillus strains from fermented food and feed, we detected the germination response of these Bacillus spores in relation to different germinating agents. The results showed the germination response was strain-specific and germinant-related, and nutrient germinant L-alanine significantly promoted the growth of strains with germination potential. Two strains of Bacillus subtilis, S-2 and 312, with or without a high spore germination response to L-alanine, were selected to study their morphological and genic differences induced by L-alanine through transmission electron microscopy and comparative transcriptomics analysis. Consequently, after L-alanine treatment, the gray phase was largely increased under microscopy, and the expression of the germination response genes was significantly up-regulated in the B. subtilis S-2 spores compared to the B. subtilis 312 spores (p < 0.05). The protective effect of L-alanine-induced spore germination of the two strains was comparatively investigated both in the IPEC-J2 cell model and a Sprague–Dawley (SD) rat model challenged by enterotoxigenic Escherichia coli K99. The result indicated that L-alanine helped B. subtilis S-2 spores, but not 312 spores, to decrease inflammatory factors (IL-6, IL-8, IL-1 β, TNF-α; p < 0.05) and promote the expression of occludin in IPEC-J2 cells. Besides, supplement with L-alanine-treated B. subtilis S-2 spores significantly improved the growth of the SD rats, alleviated histopathological GIT lesions, and improved the ratio of jejunal villus length to crypt depth in comparison to the B. subtilis S-2 spores alone (p < 0.05). Improved species diversity and abundance of fecal microbiota were only observed in the group with L-alanine-treated S-2 spores (p < 0.05). The study demonstrates L-alanine works well as a probiotic Bacillus adjuvant in improving intestinal health, and it also provides a solution for the practical and accurate regulation of their use as antibiotic alternatives in animal production.

Flow Cytometry Combined With Single Cell Sorting to Study Heterogeneous Germination of Bacillus Spores Under High Pressure.

Isostatic high pressure (HP) of 150 MPa can trigger the germination of bacterial spores, making them lose their extreme resistance to stress factors, and increasing their susceptibility to milder inactivation strategies. However, germination response of spores within a population is very heterogeneous, and tools are needed to study this heterogeneity. Here, classical methods were combined with more recent and powerful techniques such as flow cytometry (FCM) and fluorescence activated cell sorting (FACS) to investigate spore germination behavior under HP. Bacillus subtilis spores were treated with HP at 150 MPa and 37°C, stained with SYTO16 and PI, and analyzed via FCM. Four sub-populations were detected. These sub-populations were for the first time isolated on single cell level using FACS and characterized in terms of their heat resistance (80°C, 10 min) and cultivability in a nutrient-rich environment. The four isolated sub-populations were found to include (1) heat-resistant and mostly cultivable superdormant spores, i.e., spores that remained dormant after this specific HP treatment, (2) heat-sensitive and cultivable germinated spores, (3) heat-sensitive and partially-cultivable germinated spores, and (4) membrane-compromised cells with barely detectable cultivability. Of particular interest was the physiological state of the third sub-population, which was previously referred to as “unknown”. Moreover, the kinetic transitions between different physiological states were characterized. After less than 10 min of HP treatment, the majority of spores germinated and ended up in a sublethally damaged stage. HP treatment at 150 MPa and 37°C did not cause inactivation of all geminated spores, suggesting that subsequent inactivation strategies such as mild heat inactivation or other inactivation techniques are necessary to control spores in food. This study validated FCM as a powerful technique to investigate the heterogeneous behavior of spores under HP, and provided a pipeline using FACS for isolation of different sub-populations and subsequent characterization to understand their physiological states.

The CspC pseudoprotease regulates germination of Clostridioides difficile spores in response to multiple environmental signals.

The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile is thought to use the soluble pseudoprotease, CspC, to detect bile acid germinants. To gain insight into CspC’s unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile acid germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile acid-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since mutations in specific residues altered CspC’s responsiveness to these different signals, CspC plays a critical role in regulating C. difficile spore germination in response to multiple environmental signals. Taken together, these studies implicate CspC as being intimately involved in the detection of distinct classes of co-germinants in addition to bile acids and thus raises the possibility that CspC functions as a signaling node rather than a ligand-binding receptor.

Fern spore germination in response to environmental factors

Fern spore germination gives rise to the rhizoid and protonemal cell through asymmetric cell division, and then develops into a gametophyte. Spore germination is also a representative single-cell model for the investigation of nuclear polar movement, asymmetrical cell division, polarity establishment and rhizoid tip-growth. These processes are affected by various environmental factors, such as light, gravity, phytohormones, metal ions, and temperature. Here, we present a catalog of spore germination in response to different environmental factors. They are as follows: (1) Representative modes of light affecting spore germination from different fern species include red light-stimulated and far red light-inhibited spore germination, far red light-uninhibited spore germination, blue light-inhibited spore germination, and spore germination in the dark. The optimal light intensity and illumination time for spore germination are different among various fern species. Light response upon spore germination is initiated from the cell mitosis that regulated by phytochromes (PHYs) and cryptochromes (CRYs). AcPHY2, AcCRY3 and/or AcCRY4 are hypothesized to be involved in spore germination; (2) Gravity and calcium are crucial to early nuclear movement and polarity establishment of spores; (3) Gibberellin and antheridiogen can initiate and promote spore germination in many species, but abscisic acid, jasmonic acid, and ethylene pose only minor effects; (4) Spores can obtain the maximal germination rate in their favorable growth medium. Moreover, metal ions, pH, and spore density in the culture medium also affect spore germination; (5) Most fern spores germinate at 25°C, and an optimal CO2 concentration is necessary for spore germination of certain fern plants. These provide valuable information for understanding fern spore germination in response to environmental factors.

D-cycloserine or similar physiochemical compounds may be uniquely suited for use inBacillus anthracisspore decontamination strategies

As observed in the aftermath of the anthrax attacks of 2001, decontamination and remediation of a site contaminated by the accidental or intentional release of Bacillus anthracis spores is difficult, costly and potentially damaging to the environment. The identification of novel strategies that neutralize the threat of spores while minimizing environmental damage remains a high priority. We investigated the efficacy of d-cycloserine (DCS), an antibiotic and inhibitor of the spore-associated enzyme (alanine racemase) responsible for converting l-alanine to d-alanine, as a spore germination enhancer and antimicrobial agent. We characterized the impact of DCS exposure on both germinating spores and vegetative cells of fully virulent B.anthracis by evaluating spore germination kinetics, determining the minimum inhibitory concentrations (MICs) required to affect growth of the bacteria and performing macrophage viability assays. DCS enhanced germination induced by l-alanine and also efficiently killed the newly germinated spores. Furthermore, DCS proved nontoxic to macrophages at concentrations that provided protection from the killing effects of spores. Similar tests were conducted with Bacillus thuringiensis (subspecies kurstaki and Al Hakam) to determine its potential as a possible surrogate for B.anthracis field trials. Bacillus thuringiensis spores responded in a similar manner to B.anthracis spores when exposed to DCS. These results further support that DCS augments the germination response of spores in the presence of l-alanine but also reveal that DCS is bactericidal towards germinating spores. DCS (or similar compounds) may be uniquely suited for use as part of decontamination strategies by augmenting the induction of spore germination and then rendering the germinated spores nonviable.

Spores of Clostridium difficile Clinical Isolates Display a Diverse Germination Response to Bile Salts

Clostridium difficile spores play a pivotal role in the transmission of infectious diarrhoea, but in order to cause disease spores must complete germination and return to vegetative cell growth. While the mechanisms of spore germination are well understood in Bacillus, knowledge of C. difficile germination remains limited. Previous studies have shown that bile salts and amino acids play an important role in regulating the germination response of C. difficile spores. Taurocholate, in combination with glycine, can stimulate germination, whereas chenodeoxycholate has been shown to inhibit spore germination in a C. difficile clinical isolate. Our recent studies of C. difficile sporulation characteristics have since pointed to substantial diversity among different clinical isolates. Consequently, in this study we investigated how the germination characteristics of different C. difficile isolates vary in response to bile salts. By analysing 29 isolates, including 16 belonging to the BI/NAP1/027 type, we show that considerable diversity exists in both the rate and extent of C. difficile germination in response to rich medium containing both taurocholate and glycine. Strikingly, we also show that although a potent inhibitor of germination for some isolates, chenodeoxycholate does not inhibit the germination, or outgrowth, of all C. difficile strains. Finally, we provide evidence that components of rich media may induce the germination of C. difficile spores, even in the absence of taurocholate. Taken together, these data suggest that the mechanisms of C. difficile spore germination in response to bile salts are complex and require further study. Furthermore, we stress the importance of studying multiple isolates in the future when analysing the nutrients or chemicals that either stimulate or inhibit C. difficile spore germination.

Germination response of spores of the pathogenic bacterium Clostridium perfringens and Clostridium difficile to cultured human epithelial cells

Spores of pathogenic Clostridium perfringens and Clostridium difficile must germinate in the food vehicle and/or host’s intestinal tract to cause disease. In this work, we examined the germination response of spores of C. perfringens and C. difficile upon incubation with cultured human epithelial cell lines (Caco-2, HeLa and HT-29). C. perfringens spores of various sources were able to germinate to different extents; while spores of a non-food-borne isolate germinated very well, spores of food-borne and animal isolates germinated poorly in human epithelial cells. In contrast, no detectable spore germination (i.e., loss of spore heat resistance) was observed upon incubation of C. difficile spores with epithelial cells; instead, there was a significant ( p < 0.01) increase in heat-resistant spore titers. In C. perfringens, the highest spore germination response observed with the HT-29 cell line, might be in part, due to the expression of germination factor with peptidoglycan cortex hydrolysis activity by HT-29 cells. Collectively, these findings might well have implications in understanding the mechanism of clostridial spore germination in vivo.

Identification of a Receptor Subunit and Putative Ligand-Binding Residues Involved in the Bacillus megaterium QM B1551 Spore Germination Response to Glucose

The molecular basis for the recognition of glucose as a germinant molecule by spores of Bacillus megaterium QM B1551 has been examined. A chromosome-located locus (BMQ_1820, renamed gerWB) is shown to encode a receptor B-protein subunit that interacts with the GerUA and GerUC proteins to form a receptor that is cognate for both glucose and leucine. GerWB represents the third receptor B protein that binds to glucose in this strain. Site-directed mutagenesis (SDM) experiments conducted on charged proline and aromatic residues predicted to reside in the transmembrane domains of a previously characterized receptor B protein, GerVB, reveal the importance to receptor function of a cluster of residues predicted to reside in the middle of the transmembrane 6 (TM6) domain. Reductions in the region of 70- to 165-fold in the apparent affinity of receptors for glucose in which Glu196, Tyr191, and Phe192 are individually replaced by SDM indicate that some or all of these residues may be directly involved in the binding of glucose and perhaps other germinants to the germinant receptor.

Diversity of spore germination in response to inosine and L-alanine and its interaction with NaCl and pH in theBacillus cereusgroup

Our aim was to assess the diversity of the nutrient germination response of Bacillus cereus spores. 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. 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. 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.

Arbuscular mycorrhizal fungi in chronically petroleum‐contaminated soils in Mexico and the effects of petroleum hydrocarbons on spore germination

Arbuscular mycorrhizal fungi (AMF) have been hypothesized to enhance plant adaptation and growth in petroleum-contaminated soils. Nevertheless, neither AMF-biodiversity under chronically petroleum-contaminated soils nor spore germination response to petroleum hydrocarbons has been well studied. Chronically petroleum-contaminated rhizosphere soil and roots from Echinochloa polystachya, Citrus aurantifolia and C. aurantium were collected from Activo Cinco Presidentes, Tabasco, Mexico. Root colonization and spore abundance were evaluated. Additionally, rhizosphere soil samples were propagated using Sorghum vulgare L. as a plant trap under greenhouse conditions; subsequently, AMF-spores were identified. AMF-colonization ranged from 63 to 77% while spore number ranged from 715 to 912 in 100 g soil, suggesting that AMF tolerate the presence of petroleum hydrocarbons in the rhizosphere. From grass species, four AMF-morphospecies were identified: Glomus ambisporum, G. sinuosum (previously described as Sclerocystis sinuosum), Acaulospora laevis, and Ambispora gerdermanni. From citrus trees, four AMF-species were also identified: Scutellospora heterogama, G. ambisporum, Acaulospora scrobiculata, and G. citricola. In a second study, it was observed that spore germination and hyphal length of G. mosseae, G. ambisporum, and S. heterogama were significantly reduced by either volatile compounds of crude oil or increased concentrations of benzo[a ]pyrene or phenanthrene in water-agar.

Identification and characterization of a germination operon from Bacillus thuringiensis

Spore cortex-lytic enzymes are essential for germination in Bacillus. A homologue of the cwlJ gene involved in spore germination was isolated from Bacillus thuringiensis. The deduced product of this gene exhibits striking sequence similarity to CwlJ of Bacillus subtilis. Another open reading frame (ORF) was found 27 bp downstream of cwlJ and its deduced product shows high similarity to YwdL of B. subtilis. Reverse transcription polymerase chain reaction analysis indicated that cwlJ and ywdL formed a bicistronic operon in B. thuringiensis. Disruption of this operon did not affect sporulation and the spores of corresponding mutant showed the same refractility as the wild-type strain. In contrast, the fall of optical density at 600 nm in the mutant culture was slower than that of the wild-type strain during the spore germination response to different germinants such as L: -alanine, inosine or CaDPA. The spore germination of the cwlJ mutant was restored by introducing this operon into the disruption mutant. These results suggest this operon is essential for normal spore germination in B. thuringiensis. The expression of cwlJ was observed in the sporulating cells through Western blot experiments.

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.

Phenotypic and genotypic characterisation of Bacillus cereus isolates from Bangladeshi rice

We report the first phenotypic and genotypic characterisation of Bacillus cereus from Bangladeshi rice. Seven strains of B. cereus were isolated from two high-yielding rice varieties (BR5 and BRRI Dhan28) which are cultivated during different rice-growing seasons in Bangladesh. The strains were identified as B. cereus based on colonial and cellular (light microscopic) morphology and were confirmed using the API 50 CHB test system and 16 S rDNA analysis. They could also be distinguished from each other based on their substrate–utilisation patterns using the API system. No psychrotrophic (cold-tolerant) isolates were obtained and all were mesophilic with a minimum growth temperature of 10 °C, but with an ability to grow at a higher rate than normal temperature (45 °C) for B. cereus. All strains had temperature optima between 35 and 45 °C, but two groups could be distinguished based on having either a narrow or a broad optimum range. The different strains could not be distinguished based on their phospholipid compositions including major fatty acyl chains which were typical for B. cereus. However, capillary–GC/MS analysis of trace fatty acyl components of the lipids has been used for the first time to group the isolates. Genotypic analysis of the rDNA region has also been used to distinguish the strains. Restriction fragment length polymorphism (RFLP) analysis of the spacer region between the 16 S and 23 S rRNA genes yielded two groups based on different base sequences, whereas sequence analysis of variable regions in the 16 S rRNA gene gave four different groups by base differences at two positions. Despite their association with rice, none of the strains produced emetic toxin. The spore germination response of heat-activated spores of the Bangladeshi B. cereus strains was typical of the bacterium, but three different groups could be identified based on differences in germination rates. The use of this polyphasic approach to taxonomy has enabled all the B. cereus strains to be distinguished from each other.

Variability in spore germination response by strains of proteolytic Clostridium botulinum types A, B and F.

The objective of the study was to evaluate the variability of germination response of 10 strains of proteolytic Clostridium botulinum. An automated turbidometric method was used to follow the fall in optical density. Spores of proteolytic Cl. botulinum germinated in response to l-alanine alone, with rate and extent of germination increased by addition of l-lactate or bicarbonate ions. Other hydrophobic amino acids also triggered germination of spores of proteolytic Cl. botulinum but not AGFK and inosine, germinants for Bacillus subtilis or B. cereus. Unlike spores of nonproteolytic Cl. botulinum, all proteolytic Cl. botulinum germinate in hydrophobic l-amino acids without l-lactate. However, a great variability of response to germinant is evidenced between the species. The selection of a model strain to study germination of Cl. botulinum spores should consider the variability in sensitivity to germinants shown in this work. In particular, the sequenced strain ATCC 3502 may not be the most appropriate model for germination studies.

Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores.

The gerP1 transposon insertion mutation of Bacillus cereus is responsible for a defect in the germination response of spores to both L-alanine and inosine. The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold. The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant. Permeabilization of gerP mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete. The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced. Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence. A homologous operon (yisH-yisC) can be found in the Bacillus subtilis genome sequence; null mutations in yisD and yisF, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in B. cereus, though somewhat less extreme and equally repairable by spore permeabilization. Normal rates of germination, as estimated by loss of heat resistance, are also restored to a gerP mutant by the introduction of a cotE mutation, which renders the spore coats permeable to lysozyme. The B. subtilis operon is expressed solely during sporulation, and is sigma K-inducible. We hypothesize that the GerP proteins are important as morphogenetic or structural components of the Bacillus spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore.

Nucleotide sequence and gene organization of the gerK spore germination locus of Bacillus subtilis 168.

The nucleotide sequence of the whole gerK spore germination locus of Bacillus subtilis has been determined by sequencing the remaining first and third genes of the locus. The gerK locus contained three open reading frames capable of encoding polypeptides of 546, 407 and 373 amino acids. Each predicted gene product had homologues in the gerA and gerB operon, but the gene order was different from that of gerA and gerB. Sequences regarded as -10 and -35 promoter sequences for the sigma-G type RNA polymerase was found upstream of the first gene suggesting that the gerK also is an operon expressed specifically in the forespore during sporulation. All three genes were suggested to be essential for spore germination in response to glucose.

Relationship between germination of spores and two characteristics(enterotoxin production and Hobb's serotypability) of Clostridium perfringens from natural environments.

The germination response of spores of C. perfringens from natural environments was examined, and relationship between germination type of spores and both enterotoxin production and Hobbs' serotypability of the isolates was studied.A large number of isolates from the samples heated at 75°C for 20min showed alanine-type germination (A-type germination), but almost all isolates from the samples heated at 100°C for 60min showed potassium-type germination (K-type germination).Most of enterotoxigenic strains and a large number of Hobbs' serotype strains showed K-type germination. Also, all of the enterotoxigenic Hobbs' serotype strains showed K-type germination. However, both K-type and A-type germination were detected in enterotoxigenic Hobbs' untypable strains and non-enterotoxigenic Hobbs' serotype strains.It was considered that germination type of spores of isolates was not always closely related to two characteristics of them.

Substrate Restriction in Entomophilous Splachnaceae: II. Effects of Hydrogen Ion Concentration on Establishment of Gametophytes

The authors tested the hypothesis that establishment of Splachnum gametophytes is limited to neutral substrates by examining the effect of hydrogen ion concentration on spore germination, protonemal growth, and gametophore production in Splachnum ampullaceum, S. luteum, and S. sphaericum. Decreased germination was observed in S. sphaericum spores grown at pH 4 as compared to pH 7. Splachnum ampullaceum and S. luteum, however, exhibited no spore germination response to pH. Protonemal growth and gametophore production were inhibited at pH 4 compared to pH 7 in all taxa. Numbers of cells and protonemal branches per filament, chlorophyll a content, and total protein content were significantly reduced at pH 4. In addition, dark oxygen uptake was higher and light-dependent oxygen evolution was lower in protonema of S. ampullaceum grown at pH 4. In vivo ({sup 35}S) sulfate-labelled protonema of S. ampullaceum grown at pH 4 and pH 7 produced generally similar polypeptide patterns on one-dimensional polyacrylamide gels, but four major differences were detected. Total ({sup 35}S) sulfate uptake and incorporation into protein were reduced in protonema grown at pH 4. These observations are consistent with the neutrophilic hypothesis of substrate restriction in Splachnum.

The use of inhibitors to identify early events during Bacillus megaterium KM spore germination.

The germination response of spores of Bacillus megaterium KM, as measured by loss of A600, is more than 95% inhibited by 1 mM-HgCl2. Two Hg2+-sensitive sites (referred to as 'sites I and II') have been identified during germination. Site I represents a pre-commitment event and can be protected from HgCl2 by 50 mM-D-alanine, whereas site II represents a post-commitment event and is not D-alanine-protectable. At 1 mM-HgCl2, 25% of the spore population becomes committed to germinate, but an A600 loss of less than 5% occurs. In this system, loss of heat resistance was associated with commitment, whereas selective cortex hydrolysis, release of pyridine-2,6-dicarboxylic acid, Zn2+ and soluble peptidoglycan, as well as loss of refractility, were identified as post-commitment events. The commitment event was reversibly inhibited by several proteinase inhibitors and a membrane bulking agent. A model of spore germination based on these results is presented.

Identification of three complementation units in the gerA spore germination locus of Bacillus subtilis.

The gerA locus, mutations in which affect the germination response of spores to L-alanine and related amino acids, is contained within a 6-kilobase region of DNA cloned in phage and plasmid vectors. Fragments from this region, subcloned in the shuttle vector pHV33, were introduced into Bacillus subtilis, and their ability to complement chromosomal gerA mutations in a recE4 background was examined. Although the plasmids were somewhat unstable, it was possible to score complementation within spore-containing colonies on nutrient agar by their ability to reduce 2,3,5-triphenyltetrazolium chloride in an overlay. These studies have assigned the 10 gerA mutations tested to three complementation groups. An analysis of Tn1000 insertions into the cloned DNA of two relatively stable plasmids that together encompass the entire gerA region has identified more precisely the location and extent of the complementation units; recombination studies and in vitro mutagenesis were used to further delineate the extents of two of the units. The evidence suggests that the three complementation units are adjacent and that they are probably capable of separate transcription.