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Sporulation is a finely regulated morphogenetic program important in the ecology and epidemiology of Clostridium botulinum. Exogenous elements disrupting sporulation-associated genes contribute to sporulation regulation and introduce diversity in the generally conserved sporulation programs of endospore formers. We identified a novel prophage-like DNA segment, termed the yin element, inserted within yabG, encoding a sporulation-specific cysteine protease, in an environmental isolate of C. botulinum. Bioinformatic analysis revealed that the genetic structure of the yin element resembles previously reported mobile intervening elements associated with sporulation genes. Within a pure C. botulinum culture, we observed two subpopulations of cells with the yin element either integrated into the yabG locus or excised as a circular DNA molecule. The dynamics between the two observed conformations of the yin element was growth-phase dependent and likely mediated by recombination events. The yin element was not required for sporulation by C. botulinum but triggered an earlier entry into sporulation than in a related isolate lacking this element. So far, the yin element has not been found in any other C. botulinum strains or other endospore-forming species. It remains to be demonstrated what kind of competitive edge it provides for C. botulinum survival and persistence. [ABSTRACT FROM AUTHOR]

A genomic signature for endosporulation includes a gene coding for a protease, YabG, which in the model organism Bacillus subtilis is involved in assembly of the spore coat. We show that in the human pathogen Clostridioidesm difficile, YabG is critical for the assembly of the coat and exosporium layers of spores. YabG is produced during sporulation under the control of the mother cell-specific regulators σE and σK and associates with the spore surface layers. YabG shows an N-terminal SH3-like domain and a C-terminal domain that resembles single domain response regulators, such as CheY, yet is atypical in that the conserved phosphoryl-acceptor residue is absent. Instead, the CheY-like domain carries residues required for activity, including Cys207 and His161, the homologues of which form a catalytic diad in the B. subtilis protein, and also Asp162. The substitution of any of these residues by Ala, eliminates an auto-proteolytic activity as well as interdomain processing of CspBA, a reaction that releases the CspB protease, required for proper spore germination. An in-frame deletion of yabG or an allele coding for an inactive protein, yabGC207A, both cause misassemby of the coat and exosporium and the formation of spores that are more permeable to lysozyme and impaired in germination and host colonization. Furthermore, we show that YabG is required for the expression of at least two σK-dependent genes, cotA, coding for a coat protein, and cdeM, coding for a key determinant of exosporium assembly. Thus, YabG also impinges upon the genetic program of the mother cell possibly by eliminating a transcriptional repressor. Although this activity has not been described for the B. subtilis protein and most of the YabG substrates vary among sporeformers, the general role of the protease in the assembly of the spore surface is likely to be conserved across evolutionary distance. Author summary: Clostridioides difficile, an anaerobic spore-forming bacterium, colonizes the gastro-intestinal tract when, as during antibiotic treatment, the protective effect of the microbiota is disrupted. A leading agent of nosocomial infections, causing a range of symptoms from mild diarrhea to life-threatening conditions, the organism is recognized as a global and urgent threat. Infection begins with the ingestion of spores, which will germinate in response to bile salts. Two proteinaceous spore surface layers, the coat and the exosporium, play a crucial role in infection and colonization, as they contribute to spore resistance, binding to host cells and the interaction with and the response to germinants. The yabG gene, part of a genomic signature for sporulation, codes for a cysteine protease, with residues required for catalysis embedded in a CheY-like response regulator receiver domain. YabG is required for proper morphogenesis of the spore surface layers, germination and host colonization. YabG also regulates the mother cell line of gene expression by allowing the expression of genes required for assembly of the coat and exosporium. While this latter function has not been described for other organisms, the general role of yabG in the assembly of the spore surface layers is likely to be conserved among spore-formers. [ABSTRACT FROM AUTHOR]

Clostridium difficile, a toxin-producing, spore-forming bacillus, is a main cause of nosocomial antimicrobial drug-associated diarrhea in industrialized countries (1). C. difficile infection (CDI) usually develops in previously hospitalized persons with [...]

Activation of the late prespore-specific RNA polymerase sigma factor [[sigma].sup.G] during Bacillus subtilis sporulation coincides with completion of the engulfment process, when the prespore becomes a protoplast fully surrounded by the mother cell cytoplasm and separated from it by a double membrane system. Activation of [[sigma].sup.G] also requires expression of spoIIIJ, coding for a membrane protein translocase of the YidC/Oxa1p/Alb3 family, and of the mother cell-specific spoIIIA operon. Here we present genetic and biochemical evidence indicating that SpoIIIAE, the product of one of the spoIIIA cistrons, and SpoIIIJ interact in the membrane, thereby linking the function of the spoIIIJ and spoIIIA loci in the activation of [[sigma].sup.G]. We also show that SpoIIIAE has a functional Sec-type signal peptide, which is cleaved during sporulation. Furthermore, mutations that reduce or eliminate processing of the SpoIIIAE signal peptide arrest sporulation following engulfment completion and prevent activation of [[sigma].sup.G]. SpoIIIJ-type proteins can function in cooperation with or independently of the Sec system. In one model, SpoIIIJ interacts with SpoIIIAE in the context of the Sec translocon to promote its correct localization and/or topology in the membrane, so that it can signal the activation of [[sigma].sup.G] following engulfment completion.

We report evidence that CotC and CotU, two previously identified components of the Bacillus subtilis spore coat, are produced concurrently in the mother cell chamber of the sporulating cell under the control of [[sigma].sup.l] and GerE and immediately assembled around the forming spore. In the coat, the two proteins interact to forma coat component of 23 kDa. The CotU-CotC interaction was not detected in two heterologous hosts, suggesting that it occurs only in B. subtilis. Monomeric forms of both CotU and CotC failed to be assembled at the surface of the developing spore and accumulated in the mother cell compartment of cells mutant for cotE. In contrast, neither CotU nor CotC accumulated in the mother cell compartment of cells mutant for cotH. These results suggest that CotH is required to protect both CotU and CotC in the mother cell compartment of the sporangium and that CotE is needed to allow their assembly and subsequent interaction at the spore surface.

The Bacillus subtilis SpoVE integral membrane protein is essential for the heat resistance of spores, probably because of its involvement in spore peptidoglycan synthesis. We found that an SpoVE-yellow fluorescent protein (YFP) fusion protein becomes localized to the forespore during the earliest stages of engulfment, and this pattern is maintained throughout sporulation. SpoVE belongs to a well-conserved family of proteins that includes the FtsW and RodA proteins of B. subtilis. These proteins are involved in bacterial shape determination, although their function is not known. FtsW is necessary for the formation of the asymmetric septum in sporulation, and we found that an FtsW-YFP fusion localized to this structure prior to the initiation of engulfment in a nonoverlapping pattern with SpoVE-cyan fluorescent protein. Since FtsW and RodA are essential for normal growth, it has not been possible to identify loss-of-function mutations that would greatly facilitate analysis of their function. We took advantage of the fact that SpoVE is not required for growth to obtain point mutations in SpoVE that block the development of spore heat resistance but that allow normal protein expression and targeting to the forespore. These mutant proteins will be invaluable tools for future experiments aimed at elucidating the function of members of the SEDS ('shape, elongation, division, and sporulation') family of proteins.

The synthesis of structural components and morphogenetic factors required for the assembly of the Bacillus subtilis spore coat is governed by a mother cell-specific transcriptional cascade. The first two temporal classes of gene expression, which involve RNA polymerase sigma [[sigma.sup.E] factor and the ancillary regulators GerR and SpoIIID, are deployed prior to engulfment of the prespore by the mother cell. The two last classes rely on [[sigma].sup.K], whose activation follows engulfment completion, and GerE. The cotE gene codes for a morphogenetic protein essential for the assembly of the outer coat layer and spore resistance to lysozyme, cotE is expressed first from a [[sigma].sup.E]-dependent promoter and, in a second stage, from a promoter that additionally requires SpoIIID and that remains active under [[sigma.sup.k] control. Cote localizes prior to engulfment completion close to the surface of the developing spore, hut formation of the outer coat is a late, [[sigma].sup.K]-controlled event. We have transplanted cotE to progressively later classes of mother cell gene expression. This created an early class of mutants in which cotE is expressed prior to engulfment completion and a late class in which expression of cotE follows the complete engulfment of the prespore. Mutants of the early class assemble a nearly normal outer coat structure, whereas mutants of the late class do not. Hence, the early expression of Cote is essential for outer coat assembly. Surprisingly, however, all mutants were fully resistant to lysozyme. The results suggest that Cote has genetically separable functions in spore resistance to lysozyme and spore outer coat assembly.

The article presents structure, assembly and functions of the coat and exosporium layers of the spore through studies in the Bacillus subtilis. This shows the role between morphogenetic factors and structural components for accurate assembly and in ecological context.

Morphogenetic proteins such as SpoVID and SafA govern assembly of the Bacillus subtilis endospore coat by guiding the various protein structural components to the surface of the developing spore. Previously, a screen for peptides able to interact with SpoVID led to the identification of a PYYH motif present in the C-terminal half of the SafA protein and to the subsequent demonstration that SpoVID and SafA directly interact, spoVID and safA spores show deficiencies in coat assembly and are lysozyme susceptible. Both proteins, orthologs of which are found in all Bacillus species, have LysM domains for peptidoglycan binding and localize to the cortex-coat interface. Here, we show that the interaction between SafA and SpoVID involves the PYYH motif (region B) but also a 13-amino-acid region (region A) just downstream of the N-terminal LysM domain of SafA. We show that deletion of region B does not block the interaction of SafA with SpoVID, nor does it bring about spore susceptibility to lysozyme. Nevertheless, it appears to reduce the interaction and affects the complex. In contrast, lesions in region A impaired the interaction of SafA with SpoVID in vitro and, while not affecting the accumulation of SafA in vivo, interfered with the localization of SafA around the developing spore, causing aberrant assembly of the coat and lysozyme sensitivity. A peptide corresponding to region A interacts with SpoVID, suggesting that residues within this region directly contact SpoVID. Since region A is highly conserved among SafA orthologs, this motif may be an important determinant of coat assembly in the group of Bacillus spore formers.

Bacillus subtilis is considered a soil organism for which endospore formation provides a means to ensure long-term survival in the environment. We have addressed here the question of what happens to a spore when ingested. Spores displaying on their surface a heterologous antigen, tetanus toxin fragment C (TTFC), were shown to generate anti-TTFC responses not to the antigen contained in the primary oral inoculum but to those displayed on spores that had germinated and then resporulated. We then used reverse transcription-PCR to determine expression of vegetative genes and sporulation-specific genes in the mouse gut following oral dosing with spores. Significant levels of germination and sporulation were documented. Using natural isolates of B. subtilis that could form biofilms, we showed that these strains could persist in the mouse gut for significantly longer than the laboratory strain. Moreover, these isolates could grow and sporulate anaerobically and exhibited a novel phenomenon of being able to form spores in almost half the time required for the laboratory isolate. This suggests that spores are not transient passengers of the gastrointestinal tract but have adapted to carry out their entire lifecycle within this environment. This is the first report showing an intestinal life cycle of B. subtilis and suggests that other Bacillus species could also be members of the gut microflora.

The Bacillus subtilis murB gene, encoding UDP-N-acetylenolpyruvoylglucosamine reductase, a key enzyme in the peptidoglycan (PG) biosynthetic pathway, is embedded in the dcw (for 'division and cell wall') cluster immediately upstream of divIB. Previous attempts to inactivate murB were unsuccessful, suggesting its essentiality. Here we show that the cell morphology, growth rate, and resistance to cell wall-active antibiotics of murB conditional mutants is a function of the expression level of murB. In one mutant, in which murB was insertionally inactivated in a merodiploid bearing a second xylose-inducible PxylA-murB allele, DivIB levels were reduced and a normal growth rate was achieved only if MurB levels were threefold that of the wild-type strain. However, expression of an extra copy of divIB restored normal growth at wild-type levels of MurB. In contrast, DivlB levels were normal in a second mutant containing an in-frame deletion of marB ([DELTA]murB) in the presence of the PxylA-murB gene. Furthermore, this strain grew normally with wild-type levels of MurB. During sporulation, the levels of MurB were highest at the time of synthesis of the spore cortex PG. Interestingly, the [DELTA]murB PxylA-murB mutant did not sporulate efficiently even at high concentrations of inducer. Since high levels of inducer did not interfere with sporulation of a mur[B.sup.+]PxylA-murB strain, it appears that ectopic expression of murB fails to support efficient sporulation. These data suggest that coordinate expression of divIB and murB is important for growth and sporulation. The genetic context of the murB gene within the dcw cluster is unique to the Bacillus group and, taken together with our data, suggests that in these species it contributes to the optimal expression of cell division and PG biosynthetic functions during both vegetative growth and spore development.

The assembly of a multiprotein coat around the Bacillus subtilis spore confers resistance to lytic enzymes and noxious chemicals and ensures normal germination. Part of the coat is cross-linked and resistant to solubilization. The coat contains [epsilon]-([gamma]-glutamyl)lysyl cross-links, and the expression of the gene (tgl) for a spore-associated transglutaminase was shown before to be required for the cross-linking of coat protein GerQ. Here, we have investigated the assembly and function of Tgl. We found that Tgl associates, albeit at somewhat reduced levels, with the coats of mutants that are unable to assemble the outer coat (cotE), that are missing the inner coat and with a greatly altered outer coat (gerE), or that are lacking discernible inner and outer coat structures (cotE gerE double mutant). This suggests that Tgl is present at various levels within the coat lattice. The assembly of Tgl occurs independently of its own activity, as a single amino acid substitution of a cysteine to an alanine (C116A) at the active site of Tgl does not affect its accumulation or assembly. However, like a tgl insertional mutation, the tglC116A allele causes increased extractability of polypeptides of about 40, 28, and 16 kDa in addition to GerQ (20 kDa) and affects the structural integrity of the coat. We show that most Tgl is assembled onto the spore surface soon after its synthesis in the mother cell under [[sigma].sup.K] control but that the complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later. We also show that a multicopy allele of tgl causes increased assembly of Tgl and affects the assembly, structure, and functional properties of the coat.

We report here studies of expression and functional analysis of a Bacillus subtilis gene, ywcE, which codes for a product with features of a holin. Primer extension analysis of ywcE transcription revealed that a single transcript accumulated from the onset of sporulation onwards, produced from a [[sigma].sup.A]-type promoter bearing the TG dinucleotide motif of 'extended' -10 promoters. No primer extension product was detected in vivo during growth. However, specific runoff products were produced in vitro from the ywcE promoter by purified [[sigma].sup.A]-containing RNA polymerase (E[[sigma].sup.A]), and the in vivo and in vitro transcription start sites were identical. These results suggested that utilization of the ywcE promoter by E[[sigma].sup.A] during growth was subjected to repression. Studies with a lacZ fusion revealed that the transition-state regulator AbrB repressed the transcription of ywcE during growth. This repression was reversed at the onset of sporulation in a Spo0A-dependent manner, but Spo0A did not appear to contribute otherwise to ywcE transcription. We found ywcE to be required for proper spore morphogenesis. Spores of the ywcE mutant showed a reduced outer coat which lacked the characteristic striated pattern, and the outer coat failed to attach to the underlying inner coat. The mutant spores also accumulated reduced levels of dipicolinic acid. ywcE was also found to be important for spore germination.

Temporal and compartment-specific control of gene expression during sporulation in Bacillus subtilis is governed by a cascade of four RNA polymerase subunits. [[sigma].sup.F] in the prespore and [[sigma].sup.E] in the mother cell control early stages of development, and are replaced at later stages by [[sigma].sup.G] and [[sigma].sup.K], respectively. Ultimately, a comprehensive description of the molecular mechanisms underlying spore morphogenesis requires the knowledge of all the intervening genes and their assignment to specific regulons. Here, in an extension of earlier work, DNA macroarrays have been used, and members of the four compartment-specific sporulation regulons have been identified. Genes were identified and grouped based on: i) their temporal expression profile and ii) the use of mutants for each of the four sigma factors and a bofA allele, which allows [[sigma].sup.K] activation in the absence of [[sigma].sup.G]. As a further test, artificial production of active alleles of the sigma factors in non-sporulating cells was employed. A total of 439 genes were found, including previously characterized genes whose transcription is induced during sporulation: 55 in the [[sigma].sup.F] regulon, 154 [[sigma].sup.E]-governed genes, 113 [[sigma].sup.G]-dependent genes, and 132 genes under [[sigma].sup.K] control. The results strengthen the view that the activities of [[sigma].sup.F], [[sigma].sup.E], [[sigma].sup.G] and [[sigma].sup.K] are largely compartmentalized, both temporally as well as spatially, and that the major vegetative sigma factor ([[sigma].sup.R]) is active throughout sporulation. The results provide a dynamic picture of the changes in the overall pattern of gene expression in the two compartments of the sporulating cell, and offer insight into the roles of the prespore and the mother cell at different times of spore morphogenesis.

RNA polymerase sigma factor [[sigma].sup.F] initiates the prespore-specific program of gene expression during Bacillus subtilis sporulation. [[sigma].sup.F] governs transcription of spoIIIG, encoding the late prespore-specific regulator [[sigma].sup.G]. However, transcription of spoIIIG is delayed relative to other genes under the control of [[sigma].sup.F], and after synthesis, [[sigma].sup.G] is initially kept in an inactive form. Activation of [[sigma].sup.G] requires the complete engulfment of the prespore by the mother cell and expression of the spoIIIA and spoIIIJ loci. We screened for random mutations in spoIIIG that bypassed the requirement for spoIIIA for the activation of [[sigma].sup.G]. We found a mutation (spoIIIGE156K) that resulted in an amino acid substitution at position 156, which is adjacent to the position of a mutation (E155K) previously shown to prevent interaction of SpoIIAB with [[sigma].sup.G]. Comparative modelling techniques and in vivo studies suggested that the spoIIIGE156K mutation interferes with the interaction of SpoIIAB with [[sigma].sup.G]. The [[sigma].sup.GE156K] isoform restored [[sigma].sup.G]-directed gene expression to spoIIIA mutant cells. However, expression of sspE-lacZ in the spoIIIA spoIIIGE156K double mutant was delayed relative to completion of the engulfment process and was not confined to the prespore. Rather, [beta]-galactosidase accumulated throughout the entire cell at late times in development. This suggests that the activity of [[sigma].sup.GE156K] is still regulated in the prespore of a spoIIIA mutant, but not by SpoIIAB. In agreement with this suggestion, we also found that expression of spoIIIGE156K from the promoter for the early prespore-specific gene spoIIQ still resulted in sspE-lacZ induction at the normal time during sporulation, coincidently with completion of the engulfment process. In contrast, transcription of spoIIIGE156K, but not of the wild-type spoIIIG gene, from the mother cell-specific spoIID promoter permitted the rapid induction of sspE-lacZ expression. Together, the results suggest that SpoIIAB is either redundant or has no role in the regulation of [[sigma].sup.G] in the prespore.

Five commercially available Bacillus probiotic strains consisting of Bactisubtil, Enterogermina, Biosubtyl Nha Trang, Biosubtyl Da Lat and Subtyl, were characterized for determining their potential attributes that would account for their claimed probiotic properties and are available for human use. Evidence was provided for the colonization, immunostimulation and antimicrobial activity, which facilitate the hypothesis that the organisms have a potential probiotic effect.

Over 30 polypeptides are synthesized at various times during sporulation in Bacillus subtilis, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat. The coat consists of three main layers, an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-dense striated outer layer. The product of the B. subtilis oxdD gene was previously shown to have oxalate decarboxylase activity when it was produced in Escherichia coli and to be a spore constituent. In this study, we found that OxdD specifically associates with the spore coat structure, and in this paper we describe regulation of its synthesis and assembly. We found that transcription of oxdD is induced during sporulation as a monocistronic unit under the control of [[sigma].sup.K] and is negatively regulated by GerE. We also found that localization of a functional OxdD-green fluorescent protein (GFP) at the surface of the developing spore depends on the SafA morphogenetic protein, which localizes at the interface between the spore cortex and coat layers. OxdD-GFP localizes around the developing spore in a cotE mutant, which does not assemble the spore outer coat layer, but it does not persist in spores produced by the mutant. Together, the data suggest that OxdD-GFP is targeted to the interior layers of the coat. Additionally, we found that expression of a multicopy allele of oxdD resulted in production of spores with increased levels of OxdD that were able to degrade oxalate but were sensitive to lysozyme.

Spores formed by wild-type Bacillus subtilis are encased in a multilayered protein structure (called the coat) formed by the ordered assembly of over 30 polypeptides. One polypeptide (CotB) is a surface-exposed coat component that has been used as a vehicle for the display of heterologous antigens at the spore surface. The cotB gene was initially identified by reverse genetics as encoding an abundant coat component, cotB is predicted to code for a 43-kDa polypeptide, but the form that prevails in the spore coat has a molecular mass of about 66 kDa (herein designated CotB-66). Here we show that in good agreement with its predicted size, expression of cotB in Escherichia coli results in the accumulation of a 46-kDa protein (CotB-46). Expression of cotB in sporulating cells of B. subtilis also results in a 46-kDa polypeptide which appears to be rapidly converted into CotB-66. These results suggest that soon after synthesis, CotB undergoes a posttranslational modification. Assembly of CotB-66 has been shown to depend on expression of both the cotH and cotG loci. We found that CotB-46 is the predominant form found in extracts prepared from sporulating cells or in spore coat preparations of cotH or cotG mutants. Therefore, both cotH and cotG are required for the efficient conversion of CotB46 into CotB-66 but are dispensable for the association of CotB-46 with the spore coat. We also show that CotG does not accumulate in sporulating cells of a cotH mutant, suggesting that CotH (or a CotH-controlled factor) stabilizes the otherwise unstable CotG. Thus, the need for CotH for formation of CotB-66 results in part from its role in the stabilization of CotG. We also found that CotB-46 is present in complexes with CotG at the time when formation of CotB-66 is detected. Moreover, using a yeast two-hybrid system, we found evidence that CotB directly interacts with CotG and that both CotB and CotG self-interact. We suggest that an interaction between CotG and CotB is required for the formation of CotB-66, which may represent a multimeric form of CotB.

We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms. Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediately after their synthesis, since no accumulation of either of them was detected in the mother cell compartment, where their synthesis occurs. The other two components, 12.5 and 30 kDa, were generated 2 h later and were probably the products of posttranslational modifications of the two early forms occurring directly on the coat surface during spore maturation. None of the CotC forms was found either on the spore coat or in the mother cell compartment of a cotH mutant. This indicates that CotH serves a dual role of stabilizing the early forms of CotC and promoting the assembly of both early and late forms on the spore surface.

Streptococcus pneumoniae is a human pathogen responsible for high morbidity and mortality worldwide. Disease is incidental and is preceded by asymptomatic nasopharyngeal colonization in the form of biofilms. Simultaneous colonization by multiple pneumococcal strains is frequent but remains poorly characterized. Previous studies, using mostly laboratory strains, showed that pneumococcal strains can reciprocally affect each other's colonization ability. Here, we aimed at developing a strategy to investigate pneumococcal intra-species interactions occurring in biofilms. A 72h abiotic biofilm model mimicking long-term colonization was applied to study eight pneumococcal strains encompassing 6 capsular types and 7 multilocus sequence types. Strains were labeled with GFP or RFP, generating two fluorescent variants for each. Intra-species interactions were evaluated in dual-strain biofilms (1:1 ratio) using flow cytometry. Confocal microscopy was used to image representative biofilms. Twenty-eight dual-strain combinations were tested. Interactions of commensalism, competition, amensalism and neutralism were identified. The outcome of an interaction was independent of the capsular and sequence type of the strains involved. Confocal imaging of biofilms confirmed the positive, negative and neutral effects that pneumococci can exert on each other. In conclusion, we developed an experimental approach that successfully discriminates pneumococcal strains growing in mixed biofilms, which enables the identification of intra-species interactions. Several types of interactions occur among pneumococci. These observations are a starting point to study the mechanisms underlying those interactions. [ABSTRACT FROM AUTHOR]

Bacteria of the Firmicutes phylum are able to enter a developmental pathway that culminates with the formation of highly resistant, dormant endospores. Endospores allow environmental persistence, dissemination and for pathogens, are also infection vehicles. In both the model Bacillus subtilis , an aerobic organism, and in the intestinal pathogen Clostridioides difficile , an obligate anaerobe, sporulation mobilizes hundreds of genes. Their expression is coordinated between the forespore and the mother cell, the two cells that participate in the process, and is kept in close register with the course of morphogenesis. The evolutionary mechanisms by which sporulation emerged and evolved in these two species, and more broadly across Firmicutes, remain largely unknown. Here, we trace the origin and evolution of sporulation using the genes known to be involved in the process in B. subtilis and C. difficile , and estimating their gain-loss dynamics in a comprehensive bacterial macroevolutionary framework. We show that sporulation evolution was driven by two major gene gain events, the first at the base of the Firmicutes and the second at the base of the B. subtilis group and within the Peptostreptococcaceae family, which includes C. difficile. We also show that early and late sporulation regulons have been coevolving and that sporulation genes entail greater innovation in B. subtilis with many Bacilli lineage-restricted genes. In contrast, C. difficile more often recruits new sporulation genes by horizontal gene transfer, which reflects both its highly mobile genome, the complexity of the gut microbiota, and an adjustment of sporulation to the gut ecosystem. [ABSTRACT FROM AUTHOR]

The studies have shown that the inner-coat oxalate decarboxylate (OxdD) has exposed an endogenous phytase, a commonly used feed for monogastric animals, in an active form at the spore surface. The potential of OxdD as a carrier protein is documented through the spore display of a bioactive heterologous passenger, the tetrameric [beta]-glucuronidase enzyme from Escherichia coli.

A study is conducted where the isolation of 237 presumptive gut-associated Bacillus spp. isolates that were obtained by heat and ethanol treatment of fecal material from organically reared broilers followed by aerobic plating. The result suggests that some of the sporeformers isolated have the potential to persist in or transiently associate with the complex gut ecosystem.

In many cases protein assemblies are stabilized by covalent bonds, one example of which is the formation of intra- or intermolecular ε-(γ-glutamyl)lysil cross-links catalyzed by transglutaminases (TGases). Because of the potential for unwanted cross-linking reactions, the activities of many TGases have been shown to be tightly controlled. Bacterial endospores are highly resilient cells in part because they are surrounded by a complex protein coat. Proteins in the coat that surrounds Bacillus subtilis endospores are crosslinked by a TGase (Tgl). Unlike other TGases, however, Tgl is produced in an active form, and efficiently catalyzes amine incorporation and protein cross-linking in vitro with no known additional requirements. The absence of regulatory factors raises questions as to how the activity of Tgl is controlled during spore coat assembly. Here, we show that substrates assembled onto the spore coat prior to Tgl production govern the localization of Tgl to the surface of the developing spore. We also show that Tgl residues important for substrate recognition are crucial for its localization. We identified the glutamyl (Q) and lysil (K) substrate docking sites and we show that residues on the Q side of Tgl are more important for the assembly of Tgl than those on the K side. Thus, the first step in the reaction cycle, the interaction with Q-substrates and formation of an acyl-enzyme intermediate, is also the determinant step in the localization of Tgl. Consistent with the idea that Tg exerts a “spotwelding” activity, cross-linking pre-formed assemblies, we show that C30 is an oblong hexamer in solution that is cross-linked in vitro into high molecular weight forms. Moreover, during the reaction, Tgl becomes part of the cross-linked products. We suggest that the dependency of Tgl on its substrates is used to accurately control the time, location and extent of the enzyme´s activity, directed at the covalent fortification of pre-assembled complexes at the surface of the developing spore. [ABSTRACT FROM AUTHOR]

Prokaryote genomes are the result of a dynamic flux of genes, with increases achieved via horizontal gene transfer and reductions occurring through gene loss. The ecological and selective forces that drive this genomic flexibility vary across species. Bacillus subtilis is anaturally competent bacterium that occupies various environments, including plant-associated, soil, and marineniches, and the gut of both invertebrates and vertebrates. Here, we quantify the genomic diversity of B. subtilis and infer the genome dynamics that explain the high genetic and phenotypic diversity observed. Phylogenomic and comparative genomic analyses of 42 B. subtilis genomes uncover a remarkable genome diversity that translates into a core genome of 1,659 genes and an asymptotic pangenome growth rate of 57 new genes per new genome added. This diversity is due to a large proportion of low-frequency genes that are acquired from closely related species. We find no gene-loss bias among wild isolates, which explains why the cloud genome, 43%of the species pangenome, represents only a small proportion of each genome. We show that B. subtilis can acquire xenologous copies of core genes that propagate laterally among strains within a niche. While not excluding the contributions of other mechanisms, our results strongly suggest a process of gene acquisition that is largely driven by competence, where the long-term maintenance of acquired genes depends on local and global fitness effects. This competence-driven genomic diversity provides B. subtilis with its generalist character, enabling it to occupy a wide range of ecological niches and cycle through them. [ABSTRACT FROM AUTHOR]

The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages of sporulation require the mother cell regulatory protein σK. In Bacillus subtilis, the onset of σK activity requires both excision of a prophage-like element (skinBs) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skinBs recombinase is produced specifically in this cell. In C. difficile, σK lacks a pro-sequence but a skinCd element is present. The product of the skinCd gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skinCd excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skinCd excision is under the control of mother cell regulatory proteins σE and SpoIIID. We then demonstrate that σE and SpoIIID control the expression of the skinCd gene CD1234, and that this gene is required for sporulation and skinCd excision. CD1231 and CD1234 appear to interact and both proteins are required for skinCd excision while only CD1231 is necessary for skinCd integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skinCd excision to the terminal mother cell. Finally, while the skinCd element is not essential for sporulation, deletion of skinCd results in premature activity of σK and in spores with altered surface layers. Thus, skinCd excision is a key element controlling the onset of σK activity and the fidelity of spore development. [ABSTRACT FROM AUTHOR]

Engulfment of the forespore by the mother cell is a universal feature of endosporulation. In Bacillus subtilis, the forespore protein SpoIIQ and the mother cell protein SpoIIIAH form a channel, essential for endosporulation, through which the developing spore is nurtured. The two proteins also form a backup system for engulfment. Unlike in B. subtilis, SpoIIQ of Clostridium difficile has intact LytM zinc-binding motifs. We show that spoIIQ or spoIIIAH deletion mutants of C. difficile result in anomalous engulfment, and that disruption of the SpoIIQ LytM domain via a single amino acid substitution (H120S) impairs engulfment differently. SpoIIQ and SpoIIQH120S interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQH120S, binds Zn2+, and metal absence alters the SpoIIQ-SpoIIIAH complex in vitro. Possibly, SpoIIQH120S supports normal engulfment in some cells but not a second function of the complex, required following engulfment completion. We show that cells of the spoIIQ or spoIIIAH mutants that complete engulfment are impaired in post-engulfment, forespore and mother cell-specific gene expression, suggesting a channel-like function. Both engulfment and a channel-like function may be ancestral functions of SpoIIQ-SpoIIIAH while the requirement for engulfment was alleviated through the emergence of redundant mechanisms in B. subtilis and related organisms. [ABSTRACT FROM AUTHOR]