Electron Microscope Study of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis

A comprehensive ultrastructural analysis of sporulation and parasporal crystal development is described for Bacillus thuringiensis. The insecticidal crystal of B. thuringiensis is initiated at the start of engulfment and is nearly complete by the time the exosporium forms. The crystal and a heretofore unobserved ovoid inclusion develop without any clear association with the forespore septum, exosporium, or mesosomes. These observations contradict previous hypotheses that the crystal is synthesized on the forespore membrane, exosporium, or mesosomes. Formation of forespore septa involves densely staining, double-membrane-bound, vesicular mesosomes that have a bridged appearance. Forespore engulfment is subpolar and also involves mesosomes. Upon completion of engulfment and the following cytoplasmic changes occur: decrease in electron density of the incipient forespore membrane; loss of bridged appearance of incipient forespore membrane; change in stainability of incipient forespore, forespore, and mother cell cytoplasms; and alteration in staining quality of plasma membrane. These changes are involved in the conversion of the incipient forespore into a forespore and reflect "commitment" to sporulation.     

Ultrastructural Analysis of Spores and Parasporal Crystals Formed by Bacillus sphaericus 2297

Bacillus sphaericus 2297, growing from a boiled, relatively nontoxic spore inoculum, increased about 30-fold in toxicity for mosquito larvae during early exponential growth but showed an approximately 1,000-fold toxicity increase during the late-exponential phase, as spores began to appear in the culture. The development of spores in the bacterial cells was accompanied by the formation of parasporal crystals. These parasporal crystals appeared during stage III as the forespore septum engulfed the incipient forespore. The paraspores were separated from the forespores by a branch of the exosporium across the cell. Measurements of the parasporal substructure revealed a 6.3-nm distance between the striations. When spores and paraspores were fed to mosquito larvae and the larvae were fixed 15 min after feeding, it was found that the spores remained relatively unchanged but that the matrix of the paraspores was dissolved. After dissolution of the paraspore matrix, a meshlike envelope remained which retained the paraspore shape and which was often in contact with the cross-cell portion of the exosporium. The parasporal crystals may be a source of the mosquito larval toxin in this strain of B. sphaericus, but proof will require their isolation from other cellular components.     

A forespore checkpoint for mother cell gene expression during development in B. subtilis

Gene expression in the mother cell compartment of sporulating cells of B. subtilis is partly governed by the mother cell RNA polymerase sigma factor sigma K. Paradoxically, sigma K-directed gene expression also depends on sigma G, the product of the forespore compartment regulatory gene spoIIIG, and on other forespore regulatory proteins. We now identify mutations in the genes bofA and bofB that relieve the dependence of mother cell gene expression on forespore regulatory proteins but not on sigma K. We establish that the dependence of mother cell gene expression on the forespore regulatory proteins is mediated at the level of the conversion of pro-sigma K to its mature, active form. We propose that the bofA and/or bofB proteins govern this conversion in response to a signal generated by the forespore. Activation of pro-sigma K could be a checkpoint for coordinating gene expression between the mother cell and forespore compartments of the developing sporangium.     

Sporeless mutants of Bacillus thuringiensis. III. The process of crystal formation

In order to investigate the formation of the parasporal crystal of B. thuringiensis with special reference to the spore, sequential ultrastructural analysis of sporulation was performed using a sporeless mutant strain (sp^?) as well as its parent wild strain (sp⁺). From the logarithmic growth to the end of forespore formation, the same sequential process of sporulation proceeded in both strains and a forespore with double membranes appeared. Thereafter, subsequent sporulation in the sp strain was either partly or completely arrested and finally spore (mainly the forespore) became deformed. On the other hand, crystal formation took place throughout by the same processes both in sp⁺ and sp^? strains. During the forespore formation, a primordial crystal and an ovoid inclusion appeared and after this stage, the crystal displayed a characteristic diamond-shaped body with lattice fringes increasing its size. No regularity was found in the position of the crystal with respect to the spore. As far as the present ultrastructural observations were concerned, the crystal developed without any special association with the membranes of the spore. However, without the formation of the forespore (including the incipient forespore), no crystal formation was observed.     

spoIIQ, a forespore-expressed gene required for engulfment in Bacillus subtilis

A crucial step in converting an actively growing Bacillus subtilis cell into a dormant spore is the formation of a cell within a cell. This unusual structure is created by a phagocytosis-like process in which the larger mother cell progressively engulfs the adjacent smaller forespore. Only mutations blocking engulfment at an early stage and affecting genes expressed in the mother cell have been identified. Here we describe a new locus, spoIIQ , which is transcribed in the forespore and which encodes a membrane-bound protein required at a late stage of engulfment. Immunofluorescence microscopy analysis have shown that SpoIIQ is initially targeted to the septum at the boundary between the two cells and then spreads around the entire membrane of the forespore. Septum targeting requires only the first 52 residues of SpoIIQ as well as unidentified forespore-specific components. Electron-microscopy studies of cells engineered to activate the mother-cell program of gene expression independently of the forespore indicate that other as yet uncharacterized genes are involved in engulfment and that this morphological process is driven from both sides of the forespore envelope.     

Dynamic patterns of subcellular protein localization during spore coat morphogenesis in Bacillus subtilis

Endospores of Bacillus subtilis are encased in a thick, proteinaceous shell known as the coat, which is composed of a large number of different proteins. Here we report the identification of three previously uncharacterized coat-associated proteins, YabP, YheD, and YutH, and their patterns of subcellular localization during the process of sporulation, obtained by using fusions of the proteins to the green fluorescent protein (GFP). YabP-GFP was found to form both a shell and a ring around the center of the forespore across the short axis of the sporangium. YheD-GFP, in contrast, formed two rings around the forespore that were offset from its midpoint, before it eventually redistributed to form a shell around the developing spore. Finally, YutH-GFP initially localized to a focus at one end of the forespore, which then underwent transformation into a ring that was located adjacent to the forespore. Next, the ring became a cap at the mother cell pole of the forespore that eventually spread around the entire developing spore. Thus, each protein exhibited its own distinct pattern of subcellular localization during the course of coat morphogenesis. We concluded that spore coat assembly is a dynamic process involving diverse patterns of protein assembly and localization.     

A 54-kilodalton protein encoded by pBtoxis is required for parasporal body structural integrity in Bacillus thuringiensis subsp. israelensis

Strains of Bacillus thuringiensis such as B. thuringiensis subsp. israelensis (ONR-60A) and B. thuringiensis subsp. morrisoni (PG-14) pathogenic for mosquito larvae produce a complex parasporal body consisting of several protein endotoxins synthesized during sporulation that form an aggregate of crystalline inclusions bound together by a multilamellar fibrous matrix. Most studies of these strains focus on the molecular biology of the endotoxins, and although it is known that parasporal body structural integrity is important to achieving high toxicity, virtually nothing is known about the matrix that binds the toxin inclusions together. In the present study, we undertook a proteomic analysis of this matrix to identify proteins that potentially mediate assembly and stability of the parasporal body. In addition to fragments of their known major toxins, namely, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, we identified peptides with 100% identity to regions of Bt152, a protein coded for by pBtoxis of B. thuringiensis subsp. israelensis, the plasmid that encodes all endotoxins of this subspecies. As it is known that the Bt152 gene is expressed in B. thuringiensis subsp. israelensis, we disrupted its function and showed that inactivation destabilized the parasporal body matrix and, concomitantly, inclusion aggregation. Using fluorescence microscopy, we further demonstrate that Bt152 localizes to the parasporal body in both strains, is absent in other structural or soluble components of the cell, including the endospore and cytoplasm, and in ligand blots binds to purified multilamellar fibrous matrix. Together, the data show that Bt152 is essential for stability of the parasporal body of these strains.     

The structure of bypass of forespore C, an intercompartmental signaling factor during sporulation in Bacillus

Sporulation in Bacillus subtilis begins with an asymmetric cell division giving rise to smaller forespore and larger mother cell compartments. Different programs of gene expression are subsequently directed by compartment-specific RNA polymerase sigma-factors. In the final stages, spore coat proteins are synthesized in the mother cell under the control of RNA polymerase containing sigma(K), (Esigma(K)). sigma(K) is synthesized as an inactive zymogen, pro-sigma(K), which is activated by proteolytic cleavage. Processing of pro-sigma(K) is performed by SpoIVFB, a metalloprotease that resides in a complex with SpoIVFA and bypass of forespore (Bof)A in the outer forespore membrane. Ensuring coordination of events taking place in the two compartments, pro-sigma(K) processing in the mother cell is delayed until appropriate signals are received from the forespore. Cell-cell signaling is mediated by SpoIVB and BofC, which are expressed in the forespore and secreted to the intercompartmental space where they regulate pro-sigma(K) processing by mechanisms that are not yet fully understood. Here we present the three-dimensional structure of BofC determined by solution state NMR. BofC is a monomer made up of two domains. The N-terminal domain, containing a four-stranded beta-sheet onto one face of which an alpha-helix is packed, closely resembles the third immunoglobulin-binding domain of protein G from Streptococcus. The C-terminal domain contains a three-stranded beta-sheet and three alpha-helices in a novel domain topology. The sequence connecting the domains contains a conserved DISP motif to which mutations that affect BofC activity map. Possible roles for BofC in the sigma(K) checkpoint are discussed in the light of sequence and structure comparisons.     

Engulfment during sporulation in Bacillus subtilis is governed by a multi-protein complex containing tandemly acting autolysins

The conversion of a growing cell into an endospore in Bacillus subtilis involves a phagocytic-like process in which the developing spore (the forespore) is wholly engulfed by the adjacent mother cell. A prerequisite for engulfment is the removal of peptidoglycan from the septum that separates the forespore from the mother cell, a process that depends on the autolysin SpoIID and two proteins of unknown function, SpoIIM and SpoIIP. Here we present evidence that SpoIIP is also an autolysin, that it acts in tandem with SpoIID, and that all three proteins are in a complex with each other. We further show that the members of the complex exhibit a hierarchical relationship in which SpoIIM is responsible for localization to the septal membrane, SpoIIP localizes to the septal membrane by interacting with SpoIIM, and SpoIID, in turn, localizes by interacting with SpoIIP. Finally, we show that localization of SpoIIM depends on a fourth protein SpoIIB, raising the possibility that the complex contains an additional component and creating an overall hierarchy of the form: SpoIIB-->SpoIIM-->SpoIIP-->SpoIID.     

Levels of Small Molecules and Enzymes in the Mother Cell Compartment and the Forespore of Sporulating Bacillus megaterium

We have determined the amounts of a number of small molecules and enzymes in the mother cell compartment and the developing forespore during sporulation of Bacillus megaterium. Significant amounts of adenosine 5'-triphosphate and reduced nicotinamide adenine dinucleotide were present in the forespore compartment before accumulation of dipicolinic acid (DPA), but these compounds disappeared as DPA was accumulated. 3-Phosphoglyceric acid (3-PGA) accumulated only within the developing forespore, beginning 1 to 2 h before DPA accumulation. Throughout its development the forespore contained constant levels of enzymes of both 3-PGA synthesis (phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) and 3-PGA utilization (phosphoglycerate mutase, enolase, and pyruvate kinase) at levels similar to those in the mother cell and the dormant spore. Despite the presence of enzymes for 3-PGA utilization, this compound was stable within isolated forespores. Two acid-soluble proteins (A and B proteins) also accumulated only in the forespore, beginning 1 to 2 h before DPA accumulation. At this time the specific protease involved in degradation of the A and B proteins during germination also appeared, but only in the forespore compartment. Nevertheless, the A and B proteins were stable within isolated forespores. Arginine and glutamic acid accumulated within the forespore in parallel with DPA accumulation. The forespore also contained the enzyme arginase at a level similar to that in the mother cell and a level of glutamic acid decarboxylase 2- to 25-fold higher than that in the mother cell, depending on when in sporulation the forespores were isolated. The specific activities of several other enzymes (protease active on hemoglobin, ornithine transcarbamylase, malate dehydrogenase, aconitase, and isocitrate dehydrogenase) in forespores were about 10% or less of the values in the mother cell. Aminopeptidase was present at similar levels in both compartments; threonine deaminase was not found in either compartment.     

GerM is required to assemble the basal platform of the SpoIIIA–SpoIIQ transenvelope complex during sporulation in Bacillus subtilis

Sporulating Bacillus subtilis cells assemble a multimeric membrane complex connecting the mother cell and developing spore that is required to maintain forespore differentiation. An early step in the assembly of this transenvelope complex (called the A–Q complex) is an interaction between the extracellular domains of the forespore membrane protein SpoIIQ and the mother cell membrane protein SpoIIIAH. This interaction provides a platform onto which the remaining components of the complex assemble and also functions as an anchor for cell–cell signalling and morphogenetic proteins involved in spore development. SpoIIQ is required to recruit SpoIIIAH to the sporulation septum on the mother cell side; however, the mechanism by which SpoIIQ specifically localizes to the septal membranes on the forespore side has remained enigmatic. Here, we identify GerM, a lipoprotein previously implicated in spore germination, as the missing factor required for SpoIIQ localization. Our data indicate that GerM and SpoIIIAH, derived from the mother cell, and SpoIIQ, from the forespore, have reciprocal localization dependencies suggesting they constitute a tripartite platform for the assembly of the A–Q complex and a hub for the localization of mother cell and forespore proteins.     

Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium

Ellar, D. J. (Syracuse University, Syracuse, N.Y.), and D. G. Lundgren. Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium. J. Bacteriol. 92:1748-1764. 1966.-A study was made of the fine structure of sporulating cells of Bacillus cereus grown in a chemically defined medium. The developmental stages of sporulation occurred in a fairly synchronous manner and were complete by 14 hr. This time period was shortened when spore wall peptide components were added to the medium, but the addition had no effect upon fine structure except to thicken the cell wall. Sporulation could be separated into six morphological stages which generally agreed with those published for other sporulating bacteria. The initiation of the spore (forespore) septum takes the form of an inward folding of the cytoplasmic membrane toward the pole of the cell. The inward folding forms a characteristic Y-shaped membrane structure enclosing an area within which vesicles are found. These vesicles comprise the perisporal mesosome of the cell. The membranes on opposite sides of the cell progress toward the cell center where they fuse to form the double unit membrane of the spore septum. As the proliferation of the spore septum continues, the vesicular areas move towards the pole. The end result is a double forespore membrane which completely encloses a part of the vegetative cell's chromatin. Sporal mesosomes, as well as membrane vesicles, are involved in the proliferation of the forespore. Vesicles are generally bounded by a single unit membrane, whereas in the sporal mesosomes several unit membranes are arranged concentrically. The latter become associated with the segregation of a portion of the nuclear material into the forespore region of the cell.     

Parasiticidal 2-alkoxy- and 2-aryloxyiminoalkyl trifluoromethanesulfonanilides

A series of novel 2-alkoxy- and 2-aryloxyiminoalkyl trifluoromethanesulfonanilide derivatives have shown significant in vitro parasiticidal activity against the ectoparasites Ctenocephalides felis and Rhipicephalus sanguineus. A number of these compounds also displayed significant in vitro endoparasite activity against the nematode Haemonchus contortus.     

Condensation of the forespore nucleoid early in sporulation of Bacillus species.

Fluorescence microscopic examination coupled with digital videoimage analysis of 4',6-diamidino-2-phenylindole-stained sporulating cells of Bacillus megaterium or Bacillus subtilis revealed a striking condensation of the forespore nucleoid. While both mother cell and forespore compartments had equal amounts of DNA, the forespore nucleoid became greater than 2-fold more condensed than the mother cell nucleoid. The condensation of the forespore nucleoid began after only the first hour of sporulation, 2 to 3 h before expression of most forespore-specific genes including those for small, acid-soluble spore proteins, and was abolished in spo0 mutants but not in spoII or spoIII mutants. It is possible that this striking condensation of forespore DNA plays some role in modulating gene expression during sporulation.     

Forespore signaling is necessary for pro-sigmaK processing during Bacillus subtilis sporulation despite the loss of SpoIVFA upon translational arrest

The sigmaK checkpoint coordinates gene expression in the mother cell with signaling from the forespore during Bacillus subtilis sporulation. The signaling pathway involves SpoIVB, a serine peptidase produced in the forespore, which is believed to cross the innermost membrane surrounding the forespore and activate a complex of proteins, including BofA, SpoIVFA, and SpoIVFB, located in the outermost membrane surrounding the forespore. Activation of the complex allows proteolytic processing of pro-sigmaK, and the resulting sigmaK RNA polymerase transcribes genes in the mother cell. To investigate activation of the pro-sigmaK processing complex, the level of SpoIVFA in extracts of sporulating cells was examined by Western blot analysis. The SpoIVFA level decreased when pro-sigmaK processing began during sporulation. In extracts of a spoIVB mutant defective in forespore signaling, the SpoIVFA level failed to decrease normally and no processing of pro-sigmaK was observed. Although these results are consistent with a model in which SpoIVFA inhibits processing until the SpoIVB-mediated signal is received from the forespore, we discovered that loss of SpoIVFA was insufficient to allow processing under certain conditions, including static incubation of the culture and continued shaking after the addition of inhibitors of oxidative phosphorylation or translation. Under these conditions, loss of SpoIVFA was independent of spoIVB. The inability to process pro-sigmaK under these conditions was not due to loss of SpoIVFB, the putative processing enzyme, or to a requirement for ongoing synthesis of pro-sigmaK. Rather, it was found that the requirements for shaking of the culture, for oxidative phosphorylation, and for translation could be bypassed by mutations that uncouple processing from dependence on forespore signaling. This suggests that ongoing translation is normally required for efficient pro-sigmaK processing because synthesis of the SpoIVB signal protein is needed to activate the processing complex. When translation is blocked, synthesis of SpoIVB ceases, and the processing complex remains inactive despite the loss of SpoIVFA. Taken together, the results suggest that SpoIVB signaling activates the processing complex by performing another function in addition to causing loss of SpoIVFA or by causing loss of SpoIVFA in a different way than when translation is blocked. The results also demonstrate that the processing machinery can function in the absence of translation or an electrochemical gradient across membranes.