Forespore Compartment Research Articles

Membrane fission during bacterial spore development requires cellular inflation driven by DNA translocation

Bacteria require membrane fission for both cell division and endospore formation. In Bacillus subtilis, sporulation initiates with an asymmetric division that generates a large mother cell and a smaller forespore that contains only a quarter of its genome. As the mother cell membranes engulf the forespore, a DNA translocase pumps the rest of the chromosome into the small forespore compartment, inflating it due to increased turgor. When the engulfing membrane undergoes fission, the forespore is released into the mother cell cytoplasm. The B.subtilis protein FisB catalyzes membrane fission during sporulation, but the molecular basis is unclear. Here, we show that forespore inflation and FisB accumulation are both required for an efficient membrane fission. Forespore inflation leads to higher membrane tension in the engulfment membrane than in the mother cell membrane, causing the membrane to flow through the neck connecting the two membrane compartments. Thus, the mother cell supplies some of the membrane required for the growth of the membranes surrounding the forespore. The oligomerization of FisB at the membrane neck slows the equilibration of membrane tension by impeding the membrane flow. This leads to a further increase in the tension of the engulfment membrane, promoting its fission through lysis. Collectively, our data indicate that DNA translocation has a previously unappreciated second function in energizing the FisB-mediated membrane fission under energy-limited conditions.

Genetically Intact Bioengineered Spores of Bacillussubtilis.

Developments in genome editing offer potential solutions to challenges in agriculture, industry, medicine, and the environment. However, many technologies remain unexploited due to limitations in the use of genetically altered organisms. In this study, we use B. subtilis spores to explore the possibility of bioengineering organisms while leaving their genome intact. Taking advantage of the differential expression between the mother cell and the fore-spore compartments during sporulation, we created plasmids programmed to modify the spore phenotype from the mother cell compartment, but to "self-digest" in the fore-spore. At the end of sporulation, the mother cell undergoes lysis and releases the phenotypically engineered, genetically unaltered spores. Using this approach, we demonstrated the potential to express foreign proteins in B. subtilis spores without genome alterations by producing spores expressing GFP in their protective coats, where approximately 90% of the spore population had no detectable plasmid or chromosome alterations. In a separate demonstration, we programmed KinA overexpression during vegetative growth to artificially induce sporulation, and also obtained spores with nearly 90% of them free of detectable plasmid. Artificial induction of sporulation could potentially simplify the bioprocess for industrial spore production, as it reduces the number of steps involved. Overall, these findings demonstrate the potential to create genetically intact bioengineered organisms.

Channels modestly impact compartment-specific ATP levels during Bacillus subtilis sporulation and a rise in the mother cell ATP level is not necessary for Pro-σK cleavage.

Starvation of Bacillus subtilis initiates endosporulation involving formation of mother cell (MC) and forespore (FS) compartments. During engulfment, the MC membrane migrates around the FS and protein channels connect the two compartments. The channels are necessary for postengulfment FS gene expression, which relieves inhibition of SpoIVFB, an intramembrane protease that cleaves Pro-σK , releasing σK into the MC. SpoIVFB has an ATP-binding domain exposed to the MC cytoplasm, but the role of ATP in regulating Pro-σK cleavage has been unclear, as has the impact of the channels on MC and FS ATP levels. Using luciferase produced separately in each compartment to measure relative ATP concentrations during sporulation, we found that the MC ATP concentration rises about twofold coincident with increasing cleavage of Pro-σK , and the FS ATP concentration does not decline. Mutants lacking a channel protein or defective in channel protein turnover exhibited modest and varied effects on ATP levels, which suggested that low ATP concentration does not explain the lack of postengulfment FS gene expression in channel mutants. Furthermore, a rise in the MC ATP level was not necessary for Pro-σK cleavage by SpoIVFB, based on analysis of mutants that bypass the need for relief of SpoIVFB inhibition.

A model of cell-wall dynamics during sporulation in Bacillus subtilis.

To survive starvation, Bacillus subtilis forms durable spores. After asymmetric cell division, the septum grows around the forespore in a process called engulfment, but the mechanism of force generation is unknown. Here, we derived a novel biophysical model for the dynamics of cell-wall remodeling during engulfment based on a balancing of dissipative, active, and mechanical forces. By plotting phase diagrams, we predict that sporulation is promoted by a line tension from the attachment of the septum to the outer cell wall, as well as by an imbalance in turgor pressures in the mother-cell and forespore compartments. We also predict that significant mother-cell growth hinders engulfment. Hence, relatively simple physical principles may guide this complex biological process.

Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation.

In this study, evidence is presented revealing that aag, encoding a DNA glycosylase implicated in processing of hypoxanthine and alkylated DNA bases, exhibits a forespore-specific pattern of gene expression during B. subtilis sporulation. Consistent with this spatiotemporal mode of expression, Aag was found to protect the sporulating cells of this microorganism from the noxious and mutagenic effects of base deamination and alkylation.

The RecA-Dependent SOS Response Is Active and Required for Processing of DNA Damage during Bacillus subtilis Sporulation.

The expression of and role played by RecA in protecting sporulating cells of Bacillus subtilis from DNA damage has been determined. Results showed that the DNA-alkylating agent Mitomycin-C (M-C) activated expression of a PrecA-gfpmut3a fusion in both sporulating cells’ mother cell and forespore compartments. The expression levels of a recA-lacZ fusion were significantly lower in sporulating than in growing cells. However, M-C induced levels of ß-galactosidase from a recA-lacZ fusion ~6- and 3-fold in the mother cell and forespore compartments of B. subtilis sporangia, respectively. Disruption of recA slowed sporulation and sensitized sporulating cells to M-C and UV-C radiation, and the M-C and UV-C sensitivity of sporangia lacking the transcriptional repair-coupling factor Mfd was significantly increased by loss of RecA. We postulate that when DNA damage is encountered during sporulation, RecA activates the SOS response thus providing sporangia with the repair machinery to process DNA lesions that may compromise the spatio-temporal expression of genes that are essential for efficient spore formation.

A DNA-binding protein defines the precise region of chromosome capture during Bacillus sporulation.

During sporulation, Bacillus subtilis divides around the nucleoid near one cell pole, initially capturing approximately one quarter of one chromosome in the newly formed forespore compartment. While it is known that a specific region of the nucleoid is reproducibly captured in the forespore, the mechanism underlying the precision of capture is unknown. Here we describe a role for RefZ, a DNA-binding protein that regulates FtsZ, and its cognate binding motifs (RBMs) in defining the specific region of chromosome initially captured in the forespore. RefZ is conserved across the Bacillus genus and remains functional as an inhibitor of cell division in a species-swapping experiment. The RBMs are also conserved in their positioning relative to oriC across Bacillus, suggesting that the function of the RBMs is both important and position-dependent in the genus. In B. subtilis, the RBMs flank the region of the chromosome captured at the time of cell division, and we find that RefZ binds the five oriC-proximal RBMs with similar apparent affinity in units of two and four. refZ and RBM mutants capture chromosomal regions normally excluded from the forespore, suggesting that RefZ-RBM complexes play a role in regulating the position of cell division relative to the chromosome during sporulation.

Bistable forespore engulfment in Bacillus subtilis by a zipper mechanism in absence of the cell wall.

To survive starvation, the bacterium Bacillus subtilis forms durable spores. The initial step of sporulation is asymmetric cell division, leading to a large mother-cell and a small forespore compartment. After division is completed and the dividing septum is thinned, the mother cell engulfs the forespore in a slow process based on cell-wall degradation and synthesis. However, recently a new cell-wall independent mechanism was shown to significantly contribute, which can even lead to fast engulfment in 60 of the cases when the cell wall is completely removed. In this backup mechanism, strong ligand-receptor binding between mother-cell protein SpoIIIAH and forespore-protein SpoIIQ leads to zipper-like engulfment, but quantitative understanding is missing. In our work, we combined fluorescence image analysis and stochastic Langevin simulations of the fluctuating membrane to investigate the origin of fast bistable engulfment in absence of the cell wall. Our cell morphologies compare favorably with experimental time-lapse microscopy, with engulfment sensitive to the number of SpoIIQ-SpoIIIAH bonds in a threshold-like manner. By systematic exploration of model parameters, we predict regions of osmotic pressure and membrane-surface tension that produce successful engulfment. Indeed, decreasing the medium osmolarity in experiments prevents engulfment in line with our predictions. Forespore engulfment may thus not only be an ideal model system to study decision-making in single cells, but its biophysical principles are likely applicable to engulfment in other cell types, e.g. during phagocytosis in eukaryotes.

Interaction of Apurinic/Apyrimidinic Endonucleases Nfo and ExoA with the DNA Integrity Scanning Protein DisA in the Processing of Oxidative DNA Damage during Bacillus subtilis Spore Outgrowth

Oxidative stress-induced damage, including 8-oxo-guanine and apurinic/apyrimidinic (AP) DNA lesions, were detected in dormant and outgrowing Bacillus subtilis spores lacking the AP endonucleases Nfo and ExoA. Spores of the Δnfo exoA strain exhibited slightly slowed germination and greatly slowed outgrowth that drastically slowed the spores' return to vegetative growth. A null mutation in the disA gene, encoding a DNA integrity scanning protein (DisA), suppressed this phenotype, as spores lacking Nfo, ExoA, and DisA exhibited germination and outgrowth kinetics very similar to those of wild-type spores. Overexpression of DisA also restored the slow germination and outgrowth phenotype to nfo exoA disA spores. A disA-lacZ fusion was expressed during sporulation but not in the forespore compartment. However, disA-lacZ was expressed during spore germination/outgrowth, as was a DisA-green fluorescent protein (GFP) fusion protein. Fluorescence microscopy revealed that, as previously shown in sporulating cells, DisA-GFP formed discrete globular foci that colocalized with the nucleoid of germinating and outgrowing spores and remained located primarily in a single cell during early vegetative growth. Finally, the slow-outgrowth phenotype of nfo exoA spores was accompanied by a delay in DNA synthesis to repair AP and 8-oxo-guanine lesions, and these effects were suppressed following disA disruption. We postulate that a DisA-dependent checkpoint arrests DNA replication during B. subtilis spore outgrowth until the germinating spore's genome is free of damage.

A Small Protein Required for the Switch from σ F to σ G during Sporulation in Bacillus subtilis

A cascade of alternative sigma factors governs the program of developmental gene expression during sporulation in Bacillus subtilis. Little is known, however, about how the early-acting sigma factors are inactivated and replaced by the later-acting factors. Here we identify a small protein, Fin (formerly known as YabK), that is required for efficient switching from σ(F)- to σ(G)-directed gene expression in the forespore compartment of the developing sporangium. The fin gene, which is conserved among Bacillus species and species of related genera, is transcribed in the forespore under the control of both σ(F) and σ(G). Cells mutant for fin are unable to fully deactivate σ(F) and, conversely, are unable to fully activate σ(G). Consistent with their deficiency in σ(G)-directed gene expression, fin cells are arrested in large numbers following the engulfment stage of sporulation, ultimately forming 50-fold fewer heat-resistant spores than the wild type. Based in part on the similarity of Fin to the anti-σ(G) factor CsfB (also called Gin), we speculate that Fin is an anti-σ(F) factor which, by disabling σ(F), promotes the switch to late developmental gene expression in the forespore.

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

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

SpoIIIE strips proteins off the DNA during chromosome translocation

The FtsK/SpoIIIE family of DNA transporters are responsible for translocating missegregated chromosomes after the completion of cell division. An extreme example of this post-cytokinetic DNA segregation occurs during spore formation in the bacterium Bacillus subtilis, where SpoIIIE pumps three-quarters of the chromosome (>3 megabases) into one of the two daughter cells. Here, we investigate the fate of the proteins associated with the translocated DNA. Taking advantage of several unique features of Bacillus sporulation, we demonstrate that RNA polymerase, transcription factors, and chromosome remodeling proteins are stripped off the DNA during translocation of the chromosome into the forespore compartment. Furthermore, we show that in vitro the soluble ATPase domain of SpoIIIE can displace RNA polymerase bound to DNA, suggesting that SpoIIIE alone is capable of this wire-stripping activity. Our data suggest that the bulk of the forespore chromosome is translocated naked into the forespore compartment. We propose that the translocation-stripping activity of SpoIIIE plays a key role in reprogramming developmental gene expression in the forespore.

SpoIIQ Anchors Membrane Proteins on Both Sides of the Sporulation Septum in Bacillus subtilis

During the process of spore formation in Bacillus subtilis, many membrane proteins localize to the polar septum where they participate in morphogenesis and signal transduction. The forespore membrane protein SpoIIQ plays a central role in anchoring several mother-cell membrane proteins in the septal membrane. Here, we report that SpoIIQ is also responsible for anchoring a membrane protein on the forespore side of the sporulation septum. Co-immunoprecipitation experiments reveal that SpoIIQ resides in a complex with the polytopic membrane protein SpoIIE. During the early stages of sporulation, SpoIIE participates in the switch from medial to polar division and co-localizes with FtsZ at the polar septum. We show that after cytokinesis, SpoIIE is released from the septum and transiently localizes to all membranes in the forespore compartment. Upon the initiation of engulfment, it specifically re-localizes to the septal membrane on the forespore side. Importantly, the re-localization of SpoIIE to the engulfing septum requires SpoIIQ. These results indicate that SpoIIQ is required to anchor membrane proteins on both sides of the division septum. Moreover, our data suggest that forespore membrane proteins can localize to the septal membrane by diffusion-and-capture as has been described for membrane proteins in the mother cell. Finally, our results raise the intriguing possibility that SpoIIE has an uncharacterized function at a late stage of sporulation.

SpoIVB and CtpB Are Both Forespore Signals in the Activation of the Sporulation Transcription Factor σKinBacillus subtilis

The proteolytic activation of the mother cell transcription factor pro-sigma(K) is controlled by a signal transduction pathway during sporulation in the bacterium Bacillus subtilis. The pro-sigma(K) processing enzyme SpoIVFB, a membrane-embedded metalloprotease, is held inactive by two other integral membrane proteins, SpoIVFA and BofA, in the mother cell membrane that surrounds the forespore. Two signaling serine proteases, SpoIVB and CtpB, trigger pro-sigma(K) processing by cleaving the regulatory protein SpoIVFA. The SpoIVB signal is absolutely required to activate pro-sigma(K) processing and is derived from the forespore compartment. CtpB is necessary for the proper timing of sigma(K) activation and was thought to be a mother cell signal. Here, we show that the ctpB gene is expressed in both the mother cell and forespore compartments but that synthesis in the forespore under the control of sigma(G) is both necessary and sufficient for the proper timing of pro-sigma(K) processing. We further show that SpoIVB cleaves CtpB in vitro and in vivo but that this cleavage does not appear to be necessary for CtpB activation. Thus, both signaling proteins are made in the forespore and independently target the same regulatory protein.

A Novel Lipolytic Enzyme, YcsK (LipC), Located in the Spore Coat of Bacillus subtilis , Is Involved in Spore Germination

The predicted amino acid sequence of Bacillus subtilis ycsK exhibits similarity to the GDSL family of lipolytic enzymes. Northern blot analysis showed that ycsK mRNA was first detected from 4 h after the onset of sporulation and that transcription of ycsK was dependent on SigK and GerE. The fluorescence of the YcsK-green fluorescent protein fusion protein produced in sporulating cells was detectable in the mother cell but not in the forespore compartment under fluorescence microscopy, and the fusion protein was localized around the developing spores dependent on CotE, SafA, and SpoVID. Inactivation of the ycsK gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, lysozyme, or chloroform. The germination of ycsK spores in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride and LB medium was also the same as that of wild-type spores, but the mutant spores were defective in L-alanine-stimulated germination. In addition, zymogram analysis demonstrated that the YcsK protein heterologously expressed in Escherichia coli showed lipolytic activity. We therefore propose that ycsK should be renamed lipC. This is the first study of a bacterial spore germination-related lipase.

Targeting proteins to the cell wall of sporulating Bacillus anthracis

Dormant spores of Bacillus anthracis germinate during host infection and their vegetative growth and dissemination precipitate anthrax disease. Upon host death, bacilli engage a developmental programme to generate infectious spores within carcasses. Hallmark of sporulation in Bacillus spp. is the formation of an asymmetric division septum between mother cell and forespore compartments. We show here that sortase C (SrtC) cleaves the LPNTA sorting signal of BasH and BasI, thereby targeting both polypeptides to the cell wall of sporulating bacilli. Sortase substrates are initially produced in different cell compartments and at different developmental stages but penultimately decorate the envelope of the maturing spore. srtC mutants appear to display no defect during the initial stages of infection and precipitate lethal anthrax disease in guinea pigs at a similar rate as wild-type B. anthracis strain Ames. Unlike wild-type bacilli, srtC mutants do not readily form spores in guinea pig tissue or sheep blood unless their vegetative forms are exposed to air.

Signalling network with a bistable hysteretic switch controls developmental activation of the σF transcription factor in Bacillus subtilis

The sporulation process of the bacterium Bacillus subtilis unfolds by means of separate but co-ordinated programmes of gene expression within two unequal cell compartments, the mother cell and the smaller forespore. sigmaF is the first compartment-specific transcription factor activated during this process, and it is controlled at the post-translational level by a partner-switching mechanism that restricts sigmaF activity to the forespore. The crux of this mechanism lies in the ability of the anti-sigma factor SpoIIAB (AB) to form alternative complexes either with sigmaF, holding it in an inactive form, or with the anti-anti-sigma factor SpoIIAA (AA) and a nucleotide, either ATP or ADP. In the complex with AB and ATP, AA is phosphorylated on a serine residue and released, making AB available to capture sigmaF in an inactive complex. Subsequent activation of sigmaF requires the intervention of the SpoIIE serine phosphatase to dephosphorylate AA, which can then attack the AB-sigmaF complex to induce the release of sigmaF. By incorporating biochemical, biophysical and genetic data from the literature we have constructed an integrative mathematical model of this partner-switching network. The model predicts that the self-enhancing formation of a long-lived complex of AA, AB and ADP transforms the network into an essentially irreversible hysteretic switch, thereby explaining the sharp, robust and irreversible activation of sigmaF in the forespore compartment. The model also clarifies the contributions of the partly redundant mechanisms that ensure correct spatial and temporal activation of sigmaF, reproduces the behaviour of various mutants and makes strong, testable predictions.

Bacteriophages: How Bacterial Spores Capture and Protect Phage DNA

A recent study explains how bacterial spores capture and protect phage DNA, which remains free in the host cytoplasm but is unable to initiate the virulence pathway that leads to lysis of actively growing bacterial cells.

Role of the Nfo (YqfS) and ExoA Apurinic/Apyrimidinic Endonucleases in Protecting Bacillus subtilis Spores from DNA Damage

The Bacillus subtilis enzymes ExoA and Nfo (originally termed YqfS) are endonucleases that can repair apurinic/apyrimidinic (AP) sites and strand breaks in DNA. We have analyzed how the lack of ExoA and Nfo affects the resistance of growing cells and dormant spores of B. subtilis to a variety of treatments, some of which generate AP sites and DNA strand breaks. The lack of ExoA and Nfo sensitized spores (termed alpha-beta-) lacking the majority of their DNA-protective alpha/beta-type small, acid-soluble spore proteins (SASP) to wet heat. However, the lack of these enzymes had no effect on the wet-heat resistance of spores that retained alpha/beta-type SASP. The lack of either ExoA or Nfo sensitized wild-type spores to dry heat, but loss of both proteins was necessary to sensitize alpha-beta- spores to dry heat. The lack of ExoA and Nfo also sensitized alpha-beta-, but not wild-type, spores to desiccation. In contrast, loss of ExoA and Nfo did not sensitize growing cells or wild-type or alpha-beta- spores to hydrogen peroxide or t-butylhydroperoxide. Loss of ExoA and Nfo also did not increase the spontaneous mutation frequency of growing cells. exoA expression took place not only in growing cells, but also in the forespore compartment of the sporulating cell. These results, together with those from previous work, suggest that ExoA and Nfo are additional factors that protect B. subtilis spores from DNA damage accumulated during spore dormancy.

Characterization of a Polysaccharide Deacetylase Gene Homologue (pdaB) on Sporulation of Bacillus subtilis

The predicted amino acid sequence of Bacillus subtilis ybaN (renamed pdaB) exhibits high similarity to those of several polysaccharide deacetylases. Northern hybridization analysis with sporulation sigma mutants indicated that the pdaB gene is transcribed by EsigmaE RNA polymerase and negatively regulated by SpoIIID. The pdaB mutant was deficient in spore formation. Phase- and electron microscopic observation showed morphological changes of spores in late sporulation periods. The pdaB spores that had lost their viability were empty. Moreover, GFP driven by the promoter of the sspE gene was localized in the forespore compartment for the wild type, but was localized in both the mother cell and forespore compartments for phase-gray/dark forespores of the pdaB mutant. This indicates that GFP expressed in the forespores of the mutant leaks into the mother cells. Therefore, PdaB is necessary to maintain spores after the late stage of sporulation.