A coating of lipoproteins provides a stabilizing environment on the inner membrane of Bacillus subtilis spores.

A new study by M. J. Flores, K. Duricy, S. Choudhary, M. Laue, and D. L. Popham (J Bacteriol 205:e00142-23, 2023, https://doi.org/10.1128/jb.00142-23) demonstrates a role for the YlaJ/YhcN family of lipoproteins in the immobilization of the spore's inner membrane. In the absence of these lipoproteins, membrane fluidity increases and membrane-associated proteins like the GerA receptor complexes are more exposed to inimical conditions. The role of these proteins in stabilizing the Bacillus spore inner membrane is now being explored.

Spore germination receptors - a new paradigm.

David Rudner and his team (Gao et al.) predict a pentameric structure for the GerA alanine-responsive germination receptor of Bacillus subtilis and demonstrate that it behaves as a nutrient-gated ion channel, finally establishing a function for this novel family of receptors and focussing research on early ion movements in germination.

Genetic Suppression Meets Structure Prediction: Probing a Spore Germination Receptor Complex.

Despite the thousands of spore germinant receptor operons identified in genomes of bacilli and clostridia, understanding how the three essential receptor components act as a signal transduction machine in germination remains limited. The paper by Amon et al. in this issue uses the classical genetic approach of suppression to define a region of likely interaction between the GerAA and GerAB proteins: it provides a first glimpse into potential events within the receptor complex (J. D. Amon, L. Artzi, and D. Z. Rudner, J Bacteriol 204:e00470-21, 2022, https://doi.org/10.1128/JB.00470-21).

Architecture and Self-Assembly of Clostridium sporogenes and Clostridium botulinum Spore Surfaces Illustrate a General Protective Strategy across Spore Formers.

Spores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even distant relatives can have similar spore architectures although some display unique features; they all incorporate protective proteinaceous envelopes. We previously found that Bacillus spores can achieve these protective properties through extensive disulfide cross-linking of self-assembled arrays of cysteine-rich proteins. We predicted that this could be a mechanism employed by spore formers in general, even those from other genera. Here, we tested this by revealing in nanometer detail how the outer envelope (exosporium) in Clostridium sporogenes (surrogate for C. botulinum group I), and in other clostridial relatives, forms a hexagonally symmetric semipermeable array. A cysteine-rich protein, CsxA, when expressed in Escherichia coli, self-assembles into a highly thermally stable structure identical to that of the native exosporium. Like the exosporium, CsxA arrays require harsh "reducing" conditions for disassembly. We conclude that in vivo, CsxA self-organizes into a highly resilient, disulfide cross-linked array decorated with additional protein appendages enveloping the forespore. This pattern is remarkably similar to that in Bacillus spores, despite a lack of protein homology. In both cases, intracellular disulfide formation is favored by the high lattice symmetry. We have identified cysteine-rich proteins in many distantly related spore formers and propose that they may adopt a similar strategy for intracellular assembly of robust protective structures.IMPORTANCE Bacteria such as those causing botulism and anthrax survive harsh conditions and spread disease as spores. Distantly related species have similar spore architectures with protective proteinaceous layers aiding adhesion and targeting. The structures that confer these common properties are largely unstudied, and the proteins involved can be very dissimilar in sequence. We identify CsxA as a cysteine-rich protein that self-assembles in a two-dimensional lattice enveloping the spores of several Clostridium species. We show that apparently unrelated cysteine-rich proteins from very different species can self-assemble to form remarkably similar and robust structures. We propose that diverse cysteine-rich proteins identified in the genomes of a broad range of spore formers may adopt a similar strategy for assembly.

Proteins YlaJ and YhcN contribute to the efficiency of spore germination in Bacillus subtilis.

The YlaJ and YhcN spore lipoproteins of Bacillus subtilis contain a common domain, and are of unknown function. Homologues of YlaJ or YhcN are widespread in Bacilli and are also encoded in those Clostridia that use cortex lytic enzymes SleB and CwlJ for cortex hydrolysis during germination. In B. subtilis, we report that single and double mutants lacking YlaJ and/or YhcN show a reduced rate of spore germination in L-alanine, with a delay in loss of heat resistance, release of dipicolinic acid and OD fall. If B. subtilis spores lack the cortex lytic enzyme CwlJ, spore cortex degradation and subsequent outgrowth to form colonies is strictly dependent on the other cortex lytic enzyme SleB, allowing a test of SleB function; in a cwlJ mutant background, the combined loss of both ylaJ and yhcN genes resulted in a spore population in which only 20% of spores germinated and outgrew to form colonies, suggesting that SleB activity is compromised. YlaJ and YhcN have a role in germination that is not yet well defined, but these proteins are likely to contribute, directly or indirectly, to early events in germination, including effective SleB function.

Molecular tiling on the surface of a bacterial spore - the exosporium of the Bacillus anthracis/cereus/thuringiensis group.

Bacteria of the genera Bacillus and Clostridium form highly resistant spores, which in the case of some pathogens act as the infectious agents. An exosporium forms the outermost layer of some spores; it plays roles in protection, adhesion, dissemination, host targeting in pathogens and germination control. The exosporium of the Bacillus cereus group, including the anthrax pathogen, contains a 2D-crystalline basal layer, overlaid by a hairy nap. BclA and related proteins form the hairy nap, and require ExsFA (BxpB) for their localization on the basal layer. Until now, the identity of the main structural protein components of the basal layer was unknown. We demonstrate here that ExsY forms one of the essential components. Through heterologous expression in Escherichia coli, we also demonstrate that ExsY can self-assemble into ordered 2D arrays that mimic the structure of the exosporium basal layer. Self-assembly is likely to play an important role in the construction of the exosporium. The ExsY array is stable to heat and chemical denaturants, forming a robust layer that would contribute to overall spore resistance. Our structural analysis also provides novel insight into the location of other molecular components anchored onto the exosporium, such as BclA and ExsFA.

Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains.

Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75-100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores.

Spore Germination.

Despite being resistant to a variety of environmental insults, the bacterial endospore can sense the presence of small molecules and respond by germinating, losing the specialized structures of the dormant spore, and resuming active metabolism, before outgrowing into vegetative cells. Our current level of understanding of the spore germination process in bacilli and clostridia is reviewed, with particular emphasis on the germinant receptors characterized in Bacillus subtilis, Bacillus cereus, and Bacillus anthracis. The recent evidence for a local clustering of receptors in a "germinosome" would begin to explain how signals from different receptors could be integrated. The SpoVA proteins, involved in the uptake of Ca2+-dipicolinic acid into the forespore during sporulation, are also responsible for its release during germination. Lytic enzymes SleB and CwlJ, found in bacilli and some clostridia, hydrolyze the spore cortex: other clostridia use SleC for this purpose. With genome sequencing has come the appreciation that there is considerable diversity in the setting for the germination machinery between bacilli and clostridia.

YwdL in Bacillus cereus: its role in germination and exosporium structure.

In members of the Bacillus cereus group the outermost layer of the spore is the exosporium, which interacts with hosts and the environment. Efforts have been made to identify proteins of the exosporium but only a few have so far been characterised and their role in determining spore architecture and spore function is still poorly understood. We have characterised the exosporium protein, YwdL. ΔywdL spores have a more fragile exosporium, subject to damage on repeated freeze-thawing, although there is no evidence of altered resistance properties, and coats appear intact. Immunogold labelling and Western blotting with anti-YwdL antibodies identified YwdL to be located exclusively on the inner surface of the exosporium of B. cereus and B. thuringiensis. We conclude that YwdL is important for formation of a robust exosporium but is not required to maintain the crystalline assembly within the basal layer or for attachment of the hairy nap structure. ΔywdL spores are unable to germinate in response to CaDPA, and have altered germination properties, a phenotype that confirms the expected defect in localization of the cortex lytic enzyme CwlJ in the coat.

Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale.

Bacteria of the Bacillus cereus family form highly resistant spores, which in the case of the pathogen B. anthracis act as the agents of infection. The outermost layer, the exosporium, enveloping spores of the B. cereus family as well as a number of Clostridia, plays roles in spore adhesion, dissemination, targeting, and germination control. We have analyzed two naturally crystalline layers associated with the exosporium, one representing the "basal" layer to which the outermost spore layer ("hairy nap") is attached, and the other likely representing a subsurface ("parasporal") layer. We have used electron cryomicroscopy at a resolution of 0.8-0.6 nm and circular dichroism spectroscopic measurements to reveal a highly α-helical structure for both layers. The helices are assembled into 2D arrays of "cups" or "crowns." High-resolution atomic force microscopy of the outermost layer showed that the open ends of these cups face the external environment and the highly immunogenic collagen-like fibrils of the hairy nap (BclA) are attached to this surface. Based on our findings, we present a molecular model for the spore surface and propose how this surface can act as a semipermeable barrier and a matrix for binding of molecules involved in defense, germination control, and other interactions of the spore with the environment.

Amino acid residues in the GerAB protein important in the function and assembly of the alanine spore germination receptor of Bacillus subtilis 168.

The paradigm gerA operon is required for endospore germination in response to c-alanine as the sole germinant, and the three protein products, GerAA, GerAB, and GerAC are predicted to form a receptor complex in the spore inner membrane. GerAB shows homology to the amino acid-polyamine-organocation (APC) family of single-component transporters and is predicted to be an integral membrane protein with 10 membrane-spanning helices. Site-directed mutations were introduced into the gerAB gene at its natural location on the chromosome. Alterations to some charged or potential helix-breaking residues within membrane spans affected receptor function dramatically. In some cases, this is likely to reflect the complete loss of the GerA receptor complex, as judged by the absence of the germinant receptor protein GerAC, which suggests that the altered GerAB protein itself may be unstable or that the altered structure destabilizes the complex. Mutants that have a null phenotype for Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real, 1, 24006 León, Spain-alanine germination but retain GerAC protein at near-normal levels are more likely to define amino acid residues of functional, rather than structural, importance. Single-amino-acid substitutions in each of the GerAB and GerAA proteins can prevent incorporation of GerAC protein into the spore; this provides strong evidence that the proteins within a specific receptor interact and that these interactions are required for receptor assembly. The lipoprotein nature of the GerAC receptor subunit is also important; an amino acid change in the prelipoprotein signal sequence in the gerAC1 mutant results in the absence of GerAC protein from the spore.

Effect of amino acid substitutions in the GerAA protein on the function of the alanine-responsive germinant receptor of Bacillus subtilis spores.

Spores of Bacillus subtilis require the GerAA, GerAB, and GerAC receptor proteins for L-alanine-induced germination. Mutations in gerAA, both random and site directed, result in phenotypes that identify amino acid residues important for receptor function in broad terms. They highlight the functional importance of two regions in the central, integral membrane domain of GerAA. A P324S substitution in the first residue of a conserved PFPP motif results in a 10-fold increase in a spore's sensitivity to alanine; a P326S change results in the release of phase-dark spores, in which the receptor may be in an "activated" or "quasigerminated" state. Substitutions in residues 398 to 400, in a short loop between the last two likely membrane-spanning helices of this central domain, all affect the germination response, with the G398S substitution causing a temperature-sensitive defect. In others, there are wider effects on the receptor: if alanine is substituted for conserved residue N146, H304, or E330, a severe defect in L-alanine germination results. This correlates with the absence of GerAC, suggesting that the assembly or stability of the entire receptor complex has been compromised by the defect in GerAA. In contrast, severely germination-defective mutants such as E129K, L373F, S400F, and M409N mutants retain GerAC at normal levels, suggesting more local and specific effects on the function of GerAA itself. Further interpretation will depend on progress in structural analysis of the receptor proteins.

Localization of the GerD spore germination protein in the Bacillus subtilis spore.

The GerD protein of Bacillus subtilis is required for efficient spore germination in l-alanine, and for germination in the alternative germinant combination of amino acids plus sugars. Only germination via nutrient receptors is affected in the mutant. The GerD protein is predicted to be a lipoprotein that is produced in the forespore compartment of the sporulating cell. Using antibody raised against the GerD protein, Western blots of proteins from spore fractions revealed that, as might be expected, the protein was detected in the inner membrane of spores, but it was also present at a high level in spore integuments (comprising coat, cortex and germ cell wall layers), and to some extent in the soluble fraction. It is likely that the GerD protein in the outer layers of dormant spores is located in the germ cell wall, as it was detected in coat-defective spores, and in the cell wall fraction of cells that were outgrowing from spores. Which of the multiple locations of GerD is important for its function is not known, but the inner membrane association would be appropriate for any interaction with germinant receptor proteins or SleB cortex lytic enzyme. Substitution of alanine for cysteine in the conserved cleavage site of the predicted prelipoprotein signal sequence of GerD resulted in mutant spores that lacked the GerD protein entirely.

The Bacillus cereus GerN and GerT protein homologs have distinct roles in spore germination and outgrowth, respectively.

The GerT protein of Bacillus cereus shares 74% amino acid identity with its homolog GerN. The latter is a Na(+)/H(+)-K(+) antiporter that is required for normal spore germination in inosine. The germination properties of single and double mutants of B. cereus ATCC 10876 reveal that unlike GerN, which is required for all germination responses that involve the GerI germinant receptor, the GerT protein does not have a significant role in germination, although it is required for the residual GerI-mediated inosine germination response of a gerN mutant. In contrast, GerT has a significant role in outgrowth; gerT mutant spores do not outgrow efficiently under alkaline conditions and outgrow more slowly than the wild type in the presence of high NaCl concentrations. The GerT protein in B. cereus therefore contributes to the success of spore outgrowth from the germinated state during alkaline or Na(+) stress.

Structure of the exosporium and sublayers of spores of the Bacillus cereus family revealed by electron crystallography.

We report on the first step in mapping out the spatial location of structural proteins within the exosporium, namely a description of its three-dimensional architecture. Using electron microscopy and image analysis, we have characterized crystalline fragments from the exosporium of Bacillus cereus, B. thuringiensis and B. anthracis strains and identified up to three distinct crystal types. Type I and type II crystals were examined in three dimensions and shown to form arrays of interlinked crown-like structures each enclosing a cavity approximately 26-34 A deep with threefold symmetry. The arrays appear to be permeated by tunnels allowing access from one surface to the other, possibly indicating that the exosporium forms a semi-permeable barrier. The pore size of approximately 23-34 A would allow passage of the endospore germinants, alanine or inosine but not degradative enzymes or antibodies. Thus the structures appear compatible with a protective role for the exosporium. Furthermore the outermost crystalline layer must act as a scaffold for binding the BclA protein that contributes to the 'hairy nap' layer. The array of crowns may also act as a matrix for the binding or adsorption of other proteins that have been identified in the exosporium such as GroEL, immune inhibitor A and arginase.

SigM-responsive genes of Bacillus subtilis and their promoters.

Promoters of nine Bacillus subtilis genes (bcrC, yacK, ydaH, yfnI, yjbD, ypbG, ypuA, yraA, and ysxA), all responsive to artificially induced increases in the stress-responsive extracytoplasmic function sigma factor, SigM, were mapped by rapid amplification of cDNA ends-PCR. The resulting promoter consensus suggests that overlapping control by SigX or SigW is common.

ExsY and CotY are required for the correct assembly of the exosporium and spore coat of Bacillus cereus.

The exosporium-defective phenotype of a transposon insertion mutant of Bacillus cereus implicated ExsY, a homologue of B. subtilis cysteine-rich spore coat proteins CotY and CotZ, in assembly of an intact exosporium. Single and double mutants of B. cereus lacking ExsY and its paralogue, CotY, were constructed. The exsY mutant spores are not surrounded by an intact exosporium, though they often carry attached exosporium fragments. In contrast, the cotY mutant spores have an intact exosporium, although its overall shape is altered. The single mutants show altered, but different, spore coat properties. The exsY mutant spore coat is permeable to lysozyme, whereas the cotY mutant spores are less resistant to several organic solvents than is the case for the wild type. The exsY cotY double-mutant spores lack exosporium and have very thin coats that are permeable to lysozyme and are sensitive to chloroform, toluene, and phenol. These spore coat as well as exosporium defects suggest that ExsY and CotY are important to correct formation of both the exosporium and the spore coat in B. cereus. Both ExsY and CotY proteins were detected in Western blots of purified wild-type exosporium, in complexes of high molecular weight, and as monomers. Both exsY and cotY genes are expressed at late stages of sporulation.

The ExsA protein of Bacillus cereus is required for assembly of coat and exosporium onto the spore surface.

The outermost layer of spores of the Bacillus cereus family is a loose structure known as the exosporium. Spores of a library of Tn917-LTV1 transposon insertion mutants of B. cereus ATCC 10876 were partitioned into hexadecane; a less hydrophobic mutant that was isolated contained an insertion in the exsA promoter region. ExsA is the equivalent of SafA (YrbA) of Bacillus subtilis, which is also implicated in spore coat assembly; the gene organizations around both are identical, and both proteins contain a very conserved N-terminal cortex-binding domain of ca. 50 residues, although the rest of the sequence is much less conserved. In particular, unlike SafA, the ExsA protein contains multiple tandem oligopeptide repeats and is therefore likely to have an extended structure. The exsA gene is expressed in the mother cell during sporulation. Spores of an exsA mutant are extremely permeable to lysozyme and are blocked in late stages of germination, which require coat-associated functions. Two mutants expressing differently truncated versions of ExsA were constructed, and they showed the same gross defects in the attachment of exosporium and spore coat layers. The protein profile of the residual exosporium harvested from spores of the three mutants--two expressing truncated proteins and the mutant with the original transposon insertion in the promoter region--showed some differences from the wild type and from each other, but the major exosporium glycoproteins were retained. The exsA gene is extremely important for the normal assembly and anchoring of both the spore coat and exosporium layers in spores of B. cereus.

Identification of proteins in the exosporium of Bacillus anthracis.

Spores of Bacillus anthracis, the causative agent of anthrax, possess an exosporium. As the outer surface layer of these mature spores, the exosporium represents the primary contact surface between the spore and environment/host and is a site of spore antigens. The exosporium was isolated from the endospores of the B. anthracis wild-type Ames strain, from a derivative of the Ames strain cured of plasmid pXO2(-), and from a previously isolated pXO1(-), pXO2(-) doubly cured strain, B. anthracis UM23Cl2. The protein profiles of SDS-PAGE-separated exosporium extracts were similar for all three. This suggests that avirulent variants lacking either or both plasmids are realistic models for studying the exosporium from spores of B. anthracis. A number of loosely adsorbed proteins were identified from amino acid sequences determined by either nanospray-MS/MS or N-terminal sequencing. Salt and detergent washing of the exosporium fragments removed these and revealed proteins that are likely to represent structural/integral exosporium proteins. Seven proteins were identified in washed exosporium: alanine racemase, inosine hydrolase, ExsF, CotY, ExsY, CotB and a novel protein, named ExsK. CotY, ExsY and CotB are homologues of Bacillus subtilis outer spore coat proteins, but ExsF and ExsK are specific to B. anthracis and other members of the Bacillus cereus group.

Bacterial spore germination and protein mobility.

Fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) has been used to report on protein mobility in single spores. Proteins found in dormant Bacillus spores are not mobile; however, mobility is restored when germination occurs and the core rehydrates. Spores of a cwlD mutant, in which the cortex is resistant to hydrolysis, are able to complete the earliest stages of germination in response to a specific germinant stimulus; in these circumstances, the protein in the spore remains immobile. Therefore, the earliest stages of spore germination, including loss of resistance to extreme heat and the complete release of the spore component dipicolinic acid, are achieved without the restoration of protein mobility.