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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.

At an early stage during Bacillus subtilis endospore development the bacterium divides asymmetrically to produce two daughter cells. The smaller cell (forespore) differentiates into the endospore, while the larger cell (mother cell) becomes a terminally differentiated cell that nurtures the developing forespore. During development the mother cell engulfs the forespore to produce a protoplast, surrounded by two bilayer membranes, which separate it from the cytoplasm of the mother cell. The activation of [sigma]G, which drives late gene expression in the forespore, follows forespore engulfment and requires expression of the spoIIIA locus in the mother cell. One of the spoIIIA-encoded proteins SpoIIIAH is targeted specifically to the membrane surrounding the forespore, through an interaction of its C-terminal extracellular domain with the C-terminal extracellular domain of the forespore membrane protein SpollQ. We identified a homologous relationship between the C-terminal domain of SpoIIIAH and the YscJ/FliF protein family, members of which form multimeric rings involved in type III secretion systems and flagella. If SpoIIIAH forms a similar ring structure, it may also form a channel between the mother cell and forespore membranes. To test this hypothesis we developed a compartmentalized biotinylation assay, which we used to show that the C-terminal extracellular domain of SpoIIIAH is accessible to enzymatic modification from the forespore cytoplasm. These and other results lead us to suggest that SpoIIIAH forms part of a channel between the forespore and mother cell that is required for the activation of [sigmam]G. Bacillus subtilis|flagellar protein export apparatus | sporulation | type III secretion system

Spo0A, a classical two-component-type response regulator in Bacillus subtilis, binds to a specific DNA sequence found in many promoters to repress or activate the transcription of over 100 genes. On the spoIIG promoter, one of the Spo0A binding sites, centered at position -40, overlaps a consensus -35 element that may also interact with region 4 of the sigma A ([[sigma].sup.A]) subunit of RNA polymerase. Molecular modeling corroborated by genetic evidence led us to propose that the binding of Spo0A to this site repositions [[sigma].sup.A] region 4 on the promoter. Therefore, we used a chemical nuclease, p-bromoacetamidobenzyl-EDTA-Fe, that was covalently tethered to a single cysteine in region 4 of [[sigma].sup.A] to map the position of [[sigma].sup.A] on the promoter. The results indicated that in the absence of Spo0A, [[sigma].sup.A] region 4 of the RNA polymerase was located near the -35 element sequence centered at position -40. However, in the presence of SpoOA, [[sigma].sup.A] region 4 was displaced downstream from the -35 element by 4 bp. These and other results support the model in which the binding of Spo0A to the spoIIG promoter stimulates promoter utilization by repositioning prebound RNA polymerase and stabilizing the repositioned RNA polymerase-promoter complex at a new position that aligns [[sigma].sup.A] region 2 with the -10 region sequences of the promoter, thus facilitating open complex formation.

The Bacillus subtilis spoIIIA locus encodes eight proteins, SpoIIIAA to SpoIIIAH, which are expressed in the mother cell during endospore formation and which are essential for the activation of [[sigma].sup.G]; in the forespore. Complementation studies indicated that this locus may be transcribed from two promoters, one promoter upstream from the first gene and possibly a second unidentified promoter within the locus. Fragments of the spoIIIA locus were expressed at an ectopic site to complement the sporulation-defective phenotype of a spoIIIAH deletion, and we determined that complementation required a fragment of DNA that extended into spoIIIAF. To confirm that there was a promoter located in spoIIIAF, we constructed transcriptional fusions to lacZ and found strong sporulation-induced promoter activity. Primer extension assays were used to determine the transcription start site, and point mutations introduced into the -10 and -35 regions of the promoter reduced its activity. This promoter is transcribed by [[sigma].sup.E]-RNA polymerase and is repressed by SpoIIID. Therefore, we concluded that the spoIIIA locus is transcribed from two promoters, one at the start of the locus ([P1.sub.IIIA]) and the other within the locus ([P2.sub.spoIIIA]). Based on Campbell integrations and reverse transcription-PCR analysis of the [P2.sub.spoIIIA] region, we determined that [P2.sub.spoIIIA] is sufficient for transcription of [P2.sub.spoIIIAG] and [P2.sub.spoIIIAH]. Inactivation of [P2.sub.spoIIIA] blocked spore formation, indicating that [P2.sub.spoIIIA] is essential for expression of [P2.sub.spoIIIAG] and [P2.sub.spoIIIAH]. The [P2.sub.spoIIIA] activity is twice the [P2.sub.spoIIIA] activity; therefore, larger amounts of SpoIIIAG and SpoIIIAH than of proteins encoded at the upstream end of the locus may be required.

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.

At the onset of endospore formation in Bacillus subtilis the DNA-binding protein Spo0A directly activates transcription from promoters of about 40 genes. One of these promoters, Pskf, controls expression of an operon encoding a killing factor that acts on sibling cells. AbrB-mediated repression of Pskf provides one level of security ensuring that this promoter is not activated prematurely. However, Spo0A also appears to activate thepromoter directly, since Spo0A is required for Pskf activity in a [DELTA]abrB strain. Here we investigate the mechanism of Pskf activation. DNase I footprinting was used to determine the locations at which Spo0A bound to the promoter, and mutations in these sites were found to significantly reduce promoter activity. The sequence near the -10 region of the promoter was found to be similar to those of extended -10 region promoters, which contain a TRTGn motif. Mutational analysis showed that this extended -10 region, as well as other base pairs in the -10 region, is required for Spo0A-dependent activation of the promoter. We found that a substitution of the consensus base pair for the nonconsensus base pair at position -9 of Pskf produced a promoter that was active constitutively in both [DELTA]abrB and [DELTA]spo0A [DELTA]abrB strains. Therefore, the base pair at position -9 of Pskf makes its activity dependent on Spo0A binding, and the extended -10 region motif of the promoter contributes to its high level of activity.

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.

An interaction between two membrane proteins expressed in different cells of the Bacillus subtilis sporangium, the mother cell protein SpoIIIAH and the forespore protein SpoIIQ is described. The SpoIIQ-SpoIIIAH complex reaches from the mother cell cytoplasm to the forespore cytoplasm and is ideally positioned to govern the activity of engulfment-dependent transcription factors.

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.

At the onset of endospore formation in Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two types of promoters. The first type includes the spoIIG and spoIIE promoters, which are used by [[sigma].sup.A]-RNA polymerase, whereas the second type includes the spoIIA promoter, which is used by RNA polymerase containing the secondary sigma factor [[sigma].sup.H]. Previous genetic analyses have identified specific amino acids in [alpha]-helix E of Spo0A that are important for activation of Spo0A-dependent, [[sigma].sup.A]-dependent promoters. However, these amino acids are not required for activation of the [[sigma].sup.H]-dependent spoIIA promoter. We now report the effects of additional single-amino-acid substitutions and the effects of deletions in [alpha]-helix E. The elects of alanine substitutions revealed one new position (239) in Spo0A that appears to be specifically required for activation of the [[sigma].sup.A]-dependent promoters. Based on the effects of a deletion mutation, we suggest that [alpha]-helix E in Spo0A is not directly involved in interaction with [[sigma].sup.H]-RNA polymerase.

In Bacillus subtilis, the DNA binding protein SpoOA activates transcription from two classes of promoters, those used by RNA polymerase containing the primary sigma factor, [[sigma].sup.A] (e.g., spoIIG), and those used by RNA polymerase containing the secondary sigma factor, [[sigma].sup.H] (e.g., spoIIA). Several single amino acid substitutions in region 4 of [[sigma].sup.A] define positions in [[sigma].sup.A] that are specifically required for Spo0A-dependent promoter activation. Similarly, several single amino acid substitutions in Spo0A define positions in Spo0A that are required for [[sigma].sup.A]-dependent promoter activation but not for other functions of Spo0A. It is unknown whether these amino acids in Spo0A interact directly with those in region 4 of [[sigma].sup.A] or whether they interact with another subunit of RNA polymerase to effect promoter activation. Here we report the identification of a new amino acid in region 4 of [[sigma].sup.A], arginine at position 355 (R355), that is involved in Spo0A-dependent promoter activation. To further investigate the role of R355, we used the coordinates of Spo0A and sigma region 4, each in complex with DNA, to build a model for the interaction of [[sigma].sup.A] and Spo0A at the spoIIG promoter. We tested the model by examining the effects of amino acid substitutions in the putative interacting surfaces of these molecules. As predicted by the model, we found genetic evidence for interaction of R355 of [[sigma].sup.A] with glutamine at position 221 of SpoOA. These results appear to define the surfaces of Spo0A and [[sigma].sup.A] that directly interact during activation of the spoIIG promoter.

Secondary RNA polymerase sigma factors in many bacteria are responsible for regulating a vast range of processes including virulence. A protein ([[sigma].sup.x]) in the gram-positive human pathogen Streptococcus pyogenes (the group A Streptococcus or GAS) was recently shown to function in vitro as a secondary sigma factor. We report here the isolation of a mutant in which both sigX genes are inactivated, show that [[sigma].sup.x] functions in GAS cells, and show that the amount of [[sigma].sup.x] is controlled at two levels. Primer extension analysis indicates that sigX transcription is low in GAS cells grown in Todd-Hewitt yeast broth, and immunoblot assays with a [[sigma].sup.x]-specific polyclonal antibody demonstrate that the protein does not accumulate in these cells. To increase the level of sigX transcription in GAS, we constructed a strain that constitutively expresses the sigX gene from a heterologous promoter. Expression of sigX from this promoter led to transcription of the [[sigma].sup.x]-dependent cinA promoter in GAS cells. We found that expression of the sigX gene in a clpP mutant strain resulted in greater accumulation of [[sigma].sup.x] protein, which resulted in higher levels of transcription from the [[sigma].sup.x]-dependent promoters cinA, smf, and cglA. In addition, a clpP mutant containing sigX only at its wild-type loci on the chromosome generated more transcription from the [[sigma].sup.x]-dependent cinA promoter than did the wild-type parental strain. Therefore, [[sigma].sup.x] activity in GAS is limited by low-level transcription of the sigX structural genes and by clpP, which appears to negatively regulate [[sigma].sup.x] accumulation.

Amino acid substitution studies reveal that substitution of alanine for the phenylalanine at position 6 and to the leucine at position 67 in the GerE transcription factor give rise to region 1 and region 2 promoter activation sites in Bacillus subtilis.

Results demonstrate that amino acyl residues in or near the putative helix-turn-helix motif of the DNA-binding protein GerE determine the specificity of DNA binding in Bacillus subtilis. Nucleotides at positions +2, +3, and +4 with reference to transcription start site on the sigk promoter are involved in the binding site.

Results reveal that during spore coat assembly in Bacillus subtilis, morphogenetic proteins SpoVID and SafA interact and SafA interacts with itself. Immunofluorescence analysis indicate that SafA is targeted to the forespore but its localization to the forespore requires SpoIVD.

Research shows that in Bacillus subtilis, LonB protease is encoded by lonB gene whose transcription occurs during sporulation, which in turn is under the control of sigma(sup)F factor. Data indicate that LonB is restricted to prespore compartment of sporulating cells.

Research reveals a translation mechanism for the production of structural components of the endospore coat of Bacillus subtilis. Data indicate that expression of the shorter form 30-kilodalton SafA protein arises from translation initiation at codon 164 and overproduction of this protein compromises sporulation.

Research discusses the genetic aspects of spore-coat formation in bacillus subtilis. Data point out that the yrbA gene product is associated with SpoVID protein during the early stage of coat assembly.

The DNA binding protein GerE stimulates transcription from several promoters that are employed by RNA polymerase containing sigmaK during endospore formation in Bacillus subtilis. The model that GerE interacts with sigmaK at the cotX promoter was tested by looking for amino acid substitutions in sigmaK that interfered with GerE-dependent activation of the cotX promoter but which did not affect utilization of the sigmaK-dependent, GerE-independent promoter gerE. Findings showed that amino acid residues at positions 216 and 225 are needed for GerE-dependent cotX promoter activity, that the histidine at position 225 of sigmaK may interact with GerE at the cotX promoter and that this interaction may facilitate the initial binding of sigmaK RNA polymerase to the cotX promoter.

A study was conducted to examine the role of single alanine substitution at the Bacillus subtilis transcription factor SpoOA on the activity of spoIIG promoters. The amino acid substitution in SpoOA which suppressed the phenotype of the H359R substitution in sigmaA subunit of the RNA polymerase showed that H359R substitution on sigmaA prevented its interaction with SpoOA. Results suggested the relevance of the region of SpoOA near position 229 in sigmaA-dependent promoter activation.

The sodA locus encodes a manganese-dependent superoxide dismutase (SOD) in Bacillus subtilis. Disruption of this locus leads to a higher extraction rate for the spore coat protein CotG, an incomplete inner coat in the spores and the absence of the characteristic striated appearance of the spore coat. This indicates that SOD plays an important role in the covalent cross-linking of CotG to the insoluble matrix of the spore coat.

The significance of the interaction of Spo0A and the sigma subunit of RNA polymerase in the activation of the Bacillus subtilis spoIIG promoter was analyzed. Results of the study revealed that B. subtilis Spo0A activates form both sigmaA-and-sigmaH dependent promoters. Alanine substitutions at these positions were discovered to be significantly reduced from the sigmaA-dependent, Spo0A-dependent promoters, spoIIG and spoIIE in vivo.

The Bacillus subtilis genomic library was screened for promoters that are dependent on sigmaE or sigmaK to characterize the genes responsible for the assembly of the spore coat in the gram-positive bacterium. Analysis of the Bacillus subtilis genomic library indicated the presence of the cotM gene which was negatively regulated by GerE and induced during development by sigmK. The Bacillus subtilis cotM gene also encoded a product that was related to the alpha-crystallin family of stress proteins.

Transcriptional units that are activated during sporulation such as cse15, cse60 and csk22 were analyzed to characterize the mother-cell-specific promoters of sporulation in Bacillus subtilis. Cloning and nucleotides sequence analysis of the transcriptional units indicated the role of the Escherichia coli RNA polymerase sigmaE factor in regulating the expression of the promoters during sporulation. Furthermore, the activity of the SpoIIID DNA-binding protein was necessary for the expression of cse15 and cse60.

An operon consisting of at least five open reading frames (ORFs) and dependent on a mother-cell-specific sigma factor has been isolated while studying sporulation. The operon is subjected to catabolite repression. The gene products of the first three ORFs are similar to the enzymes related with beta-oxidation of fatty acids while the fourth ORF encodes a third citrate synthase other than CitA and CitZ. Although the environmental condition, under which expression of this operon is essential for sporulation, is unknown, such expression probably affects catabolite repression.

The specific amino acid residues in sigma(supra E) from Bacillus subtilis that identify what particular nucleotides at the -10 region of its cognate promoters to which it will react with, are determined. Findings indicate that amino acid residues at positions 119 and 120 are partly responsible for specifying the type of interactions between sigma-E and the nucleotides in the -10 region of its cognate promoters.

The effects of amino acid substitutions in sigmaE from Bacillus subtilis were investigated to determine the role of the most highly conserved region of bacterial RNA polymerase sigma factors in the formation of the enzyme. Two single amino acid substitutions, one at position 68 and the other at position 94, were detected. Genetic and molecular techniques suggested that the substitution at position 68 do not contribute to the overall structure of the sigma factor.

Molecular cloning of an operon, cotJ, one of the 13 coat proteins which when inactivated induces sporulation with an altered pattern of coat polypeptides in Bacillus subtilis, revealed a tricistronic operon preceded by a strong sigmaE-like promoter. DNA mapping showed that expression of cotJ is completely dependent on spoIIGB, a DNA binding protein that encodes sigmaE, and that its precedes expression of cotA, but not cotE. This suggests that cotJ is involved in the early stage of coat assembly in B. subtilis.

The lon-1 mutation that leads to the improper transcription of genes under sporulation inhibition conditions governed by the Bacillus subtilis RNA polymerase sigma factor (sigma G) resides at 245 degree Celsius on the genetic map inside a lon protease homolog-encoding open reading frame. SigmaG is generated under non-sporulating conditions of the cells by the positive autoregulation of its structural gene spoIIIG, indicating the independent actions of spoIIIAB and Lon protease in the inhibition of the improper synthesis of sigmaG and improper transcription of its genes.

The spoVR gene from Bacillus subtilis is associated with spore cortex development. spoVR mutants contain cortexless and phase-dark spores. The mutants reduce dipicolinate annihilation and the number of chloroform- and heat-resistant spores. spoVR gene downstream of B. Subtilis is directed away from phoAIV gene. Sigma E-dependent promoter influences spoVR gene expression. The gene encodes 468 residues.

An analysis of the function of regulatory protein SpoOA during phosphorylation in spoIIG promoter activation revealed that it enhanced the affinity to specific sites. SpoOA, which binds to sites 1 and 2 in the promoter, is essential for the activation of spoIIG at the onset of sporulation in Bacillus subtilis. Phosphorylation of SpoOA is crucial to the stimulation of sporulation.

A gene essential for the normal assembly of the spore coat of Bacillus subtilis has been isolated and characterized. The gene, spoVID, is located downstream to the hemAXCDBL operon and is activated in the mother cell during the second hour of endospore formation. The gene product is a 575-residue acidic protein. Mutations in the spoVID locus produce cells with aberrant assembly of coat proteins.

The effects of single- and double-amino-acid substitutions in the -10 binding region of sigma factor E from Bacillus subtilis was examined. Sporulation was used to assay sigma factor E function, since it was required for endospore formation. The results showed that substitutions at some positions, including highly conservedamino acids, had little effect on sigma factor E function. However, single-amino-acid substitutions at only a few positions produced severely defective sigma factors.

The early stages of sporulation in Bacillus subtilis is partially regulated by the spoIIE operon. Upstream from spoIIE is its promoter sequence which is utilized by Esigma(A), amajor vegetative form of polymerase. Research shows that the spoIIE promoter sequence functions as a binding site for the Spo0A protein, a transription factor which initiates sporulation. Further analyses showed that the Spo0A protein also recognizes the promoter of another sporulation operon, spoIIG. These results suggest that spoIIE and spoIIG encode early-intermediate sporulation factors whose transcription by Esigma(A) is activated by the Spo0A protein.

An additional role for the -10 region recognition sequenceof sigma factor E was demonstrated. A C117R mutation was introduced in the -10 recognition sequence of sigma factor E, and the mutant protein was tested for the ability to recognize and transcribe genes. The results showed that the mutant sigma factor could recognize and bind to correct promoter sequences. However, it could not initiate transcription. The results suggest that the -10 recognition region of sigma factors is also involved in transcription steps following promoter binding and involving transcription initiation.

The activation of the spoIIG promoter was investigated to determine if the binding of Spo0A protein is responsible for the activity of the promoter region used by RNA polymerase during transcription. DNase I protection assays were also conducted to verify the findings. Results indicate that binding of Spo0A protein on the spoIIG promoter region activates the sequence. Further, the assays showed that mutations that decrease Spo0A affinity to the promoter sequence reduces the promoter-activating ability of Spo0A.

The coat assembly of bacterial endospores is made possible through the expression of the tasA gene, a protein by-product of gerE mutant strains. This protein, which may be synthesized through NaOH treatment, enables the predivisional cell of the bacteria to create a highly complex coat that acts as shield against noxious chemicals. TasA has also been found to induce protein secretion during coat assembly, thereby influencing the bacteria's germination response.

Sporulation in Bacillus subtilis involves two cells that follow separate but coordinately regulated developmental programs. Late in sporulation, the developing spore (the forespore) resides within a mother cell. The regulation of the forespore transcription factor σG that acts at this stage has remained enigmatic. σG activity requires eight mother-cell proteins encoded in the spoIIIA operon and the forespore protein SpoIIQ. Several of the SpoIIIA proteins share similarity with components of specialized secretion systems. One of them resembles a secretion ATPase and we demonstrate that the ATPase motifs are required for σG activity. We further show that the SpoIIIA proteins and SpoIIQ reside in a multimeric complex that spans the two membranes surrounding the forespore. Finally, we have discovered that these proteins are all required to maintain forespore integrity. In their absence, the forespore develops large invaginations and collapses. Importantly, maintenance of forespore integrity does not require σG. These results support a model in which the SpoIIIA-SpoIIQ proteins form a novel secretion apparatus that allows the mother cell to nurture the forespore, thereby maintaining forespore physiology and σG activity during spore maturation.

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., Competing Interests: The authors have declared that no competing interests exist.

At a late stage in spore development in Bacillus subtilis , the mother cell directs synthesis of a layer of peptidoglycan known as the cortex between the two forespore membranes, as well as the assembly of a protective protein coat at the surface of the forespore outer membrane. SafA, the key determinant of inner coat assembly, is first recruited to the surface of the developing spore and then encases the spore under the control of the morphogenetic protein SpoVID. SafA has a LysM peptidoglycan-binding domain, SafA LysM , and localizes to the cortex-coat interface in mature spores. SafA LysM is followed by a region, A, required for an interaction with SpoVID and encasement. We now show that residues D10 and N30 in SafA LysM , while involved in the interaction with peptidoglycan, are also required for the interaction with SpoVID and encasement. We further show that single alanine substitutions on residues S11, L12, and I39 of SafA LysM that strongly impair binding to purified cortex peptidoglycan affect a later stage in the localization of SafA that is also dependent on the activity of SpoVE, a transglycosylase required for cortex formation. The assembly of SafA thus involves sequential protein-protein and protein-peptidoglycan interactions, mediated by the LysM domain, which are required first for encasement then for the final localization of the protein in mature spores. IMPORTANCE Bacillus subtilis spores are encased in a multiprotein coat that surrounds an underlying peptidoglycan layer, the cortex. How the connection between the two layers is enforced is not well established. Here, we elucidate the role of the peptidoglycan-binding LysM domain, present in two proteins, SafA and SpoVID, that govern the localization of additional proteins to the coat. We found that SafA LysM is a protein-protein interaction module during the early stages of coat assembly and a cortex-binding module at late stages in morphogenesis, with the cortex-binding function promoting a tight connection between the cortex and the coat. In contrast, SpoVID LysM functions only as a protein-protein interaction domain that targets SpoVID to the spore surface at the onset of coat assembly., (Copyright © 2019 American Society for Microbiology.)

During sporulation in Bacillus subtilis, a group of mother cell-specific proteins guides the assembly of the coat, a multiprotein structure that protects the spore and influences many of its environmental interactions. SafA and CotE behave as party hubs, governing assembly of the inner and outer coat layers. Targeting of coat proteins to the developing spore is followed by encasement. Encasement by SafA and CotE requires E, a region of 11 amino acids in the encasement protein SpoVID, with which CotE interacts directly. Here, we identified two single alanine substitutions in E that prevent binding of SafA, but not of CotE, to SpoVID, and block encasement. The substitutions result in the accumulation of SafA, CotE and their dependent proteins at the mother cell proximal spore pole, phenocopying a spoVID null mutant and suggesting that mislocalized SafA acts as an attractor for the rest of the coat. The requirement for E in SafA binding is bypassed by a peptide with the sequence of E provided in trans. We suggest that E allows binding of SafA to a second region in SpoVID, enabling CotE to interact with E and SpoVID to function as a non-competitive hub during spore encasement., (© 2018 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

The coat of Bacillus subtilis spores is a multiprotein protective structure that also arbitrates many of the environmental interactions of the spore. The coat assembles around the cortex peptidoglycan layer and is differentiated into an inner and an outer layer and a crust. SafA governs assembly of the inner coat, whereas CotE drives outer coat assembly. SafA localizes to the cortex-coat interface. Both SafA and its short form C30 are substrates for Tgl, a coat-associated transglutaminase that cross-links proteins through ε-(γ-glutamyl)lysyl isopeptide bonds. We show that SafA and C30 are distributed between the coat and cortex layers. The deletion of tgl increases the extractability of SafA, mainly from the cortex. Tgl itself is mostly located in the inner coat and cortex. The localization of Tgl-cyan fluorescent protein (Tgl-CFP) is strongly, but not exclusively, dependent on safA However, the association of Tgl with the cortex requires safA Together, our results suggest an assembly pathway in which Tgl is first recruited to the forming spore in a manner that is only partially dependent on SafA and then is drafted to the cortex by SafA. Tgl, in turn, promotes the conversion of coat- and cortex-associated SafA into forms that resist extraction, possibly by catalyzing the cross-linking of SafA to other coat proteins, to the cortex, and/or to cortex-associated proteins. Therefore, the final assembly state of SafA relies on an autoregulatory pathway that requires the subcellular localization of a protein cross-linking enzyme. Tgl most likely exerts a "spotwelding" activity, cross-linking preformed complexes in the cortex and inner coat layers of spores. IMPORTANCE In this work, we show how two proteins work together to determine their subcellular location within the coat of bacterial endospores. Bacillus subtilis endospores are surrounded by a multilayer protein coat composed of over 80 proteins, which surrounds an underlying peptidoglycan layer (the spore cortex) protecting it from lytic enzymes. How specific coat proteins are targeted to specific layers of the coat is not well understood. We found that the protein SafA recruits a protein-cross-linking enzyme (a transglutaminase) to the cortex and inner layers of the coat, where both are cemented, by cross-linking, into macromolecular complexes., (Copyright © 2018 American Society for Microbiology.)

Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.

During sporulation in Bacillus subtilis, the onset of activity of the late forespore-specific sigma factor σG coincides with completion of forespore engulfment by the mother cell. At this stage, the forespore becomes a free protoplast, surrounded by the mother cell cytoplasm and separated from it by two membranes that derive from the asymmetric division septum. Continued gene expression in the forespore, isolated from the surrounding medium, relies on the SpoIIIA-SpoIIQ secretion system assembled from proteins synthesised both in the mother cell and in the forespore. The membrane protein insertase SpoIIIJ, of the YidC/Oxa1/Alb3 family, is involved in the assembly of the SpoIIIA-SpoIIQ complex. Here we show that SpoIIIJ exists as a mixture of monomers and dimers stabilised by a disulphide bond. We show that residue Cys134 within transmembrane segment 2 (TM2) of SpoIIIJ is important to stabilise the protein in the dimeric form. Labelling of Cys134 with a Cys-reactive reagent could only be achieved under stringent conditions, suggesting a tight association at least in part through TM2, between monomers in the membrane. Substitution of Cys134 by an Ala results in accumulation of the monomer, and reduces SpoIIIJ function in vivo. Therefore, SpoIIIJ activity in vivo appears to require dimer formation.

Proteins SpoIIQ and SpoIIIAH interact through two membranes to connect the forespore and the mother cell during endospore development in the bacterium Bacillus subtilis. SpoIIIAH consists of a transmembrane segment and an extracellular domain with similarity to YscJ proteins. YscJ proteins form large multimeric rings that are the structural scaffolds for the assembly of type III secretion systems in gram-negative bacteria. The predicted ring-forming motif of SpoIIIAH and other evidence led to the model that SpoIIQ and SpoIIIAH form the core components of a channel or transporter through which the mother cell nurtures forespore development. Therefore, to understand the roles of SpoIIIAH and SpoIIQ in channel formation, it is critical to determine whether SpoIIIAH adopts a ring-forming structural motif, and whether interaction of SpoIIIAH with SpoIIQ would preclude ring formation. We report a 2.8-Å resolution structure of a complex of SpoIIQ and SpoIIIAH. SpoIIIAH folds into the ring-building structural motif, and modeling shows that the structure of the SpoIIQ-SpoIIIAH complex is compatible with forming a symmetrical oligomer that is similar to those in type III systems. The inner diameters of the two most likely ring models are large enough to accommodate several copies of other integral membrane proteins. SpoIIQ contains a LytM domain, which is found in metalloendopeptidases, but lacks residues important for metalloprotease activity. Other LytM domains appear to be involved in protein-protein interactions. We found that the LytM domain of SpoIIQ contains an accessory region that interacts with SpoIIIAH.

Two highly similar RNA polymerase sigma subunits, σ(F) and σ(G), govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σ(F) drives synthesis of σ(G) but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of σ(G) while discriminating between σ(F) and σ(G) in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of σ(G) (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σ(G) in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σ(G). CsfB is normally produced in the forespore, under σ(F) control, but sigGN45E mutant cells also expressed csfB and did so in a σ(G)-dependent manner, autonomously from σ(F). Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σ(G) activity. N45 is invariant in the homologous position of σ(G) orthologues, whereas its functional equivalent in σ(F) proteins, E39, is highly conserved. While CsfB does not bind to wild-type σ(F), a E39N substitution in σ(F) resulted in efficient binding of CsfB to σ(F). Moreover, under certain conditions, the E39N alteration strongly restrains the activity of σ(F) in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two forespore-specific sigma factors in B. subtilis., Competing Interests: The authors have declared that no competing interests exist.