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Asymmetric division during sporulation by Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation. The program is governed by a hierarchical cascade consisting of the transcription factors: sigma(E), sigma(K), GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The sigma(E)...
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... profiling was carried out with three independent preparations of form- aldehyde-treated cells, twice with two of the preparations and once with the third, for a total of five analyses. An enrichment factor was calculated for each gene, representing the enrich- ment of that gene by immunoprecipitation relative to DNA that had not been subjected to immunoprecipitation, and the entire dataset is displayed in Table S3. ...
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... though pro-r K was expected to be synthesized somewhat prematurely in BDR1663, the appearance of mature r K remained subject to the pathway governing the proteolytic processing of pro-r K and hence would have occurred at the normal time (Cutting et al. 1991a). Indeed, cells harboring the amyE::P spoIVF -sigK construct sporulated as efficiently as the wild type and did so in a manner that did not depend on the presence of spoIVCB (Table 3). We conclude that bypassing the requirement for SpoIIID in r K synthesis does not measurably affect sporula- tion efficiency. ...
Context 3
... when the amyE::P spoIVF -sigK construct was intro- duced into cells harboring a spoIIID mutation (generating strain BDR1666), sporulation efficiency was still reduced by about a 100,000-fold compared to the wild type (Table 3). This result reinforces the findings of Lu and Kroos (1994), who showed that sporulation was impaired in spoIIID mutant cells even in the presence of a construct that allowed pro-r K to be produced in a SpoIIID-independent manner. ...
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... possible explanation for these results is that, in addition to its role in r K synthesis, SpoIIID is required for the synthesis of some other unidentified protein or proteins that are needed for sporulation. To investigate this possibility, we systematically inactivated all of the newly identified SpoIIID-activated transcription units (Table 3). With three exceptions, those of spoVK, asnO, and ycgM, the resulting mutants sporulated at levels comparable to that of the wild type. ...
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Citations
... SpoIVFB is embedded in the membrane but kept inactive in a complex with SpoIVFA and BofA (Olenic et al., 2022). Cleavage of SpoIVFA by SpoIVB in the intermembrane space causes a conformational change in SpoIVFB, which can then cleave proσ K , to release active σ K (De Hoon et al., 2010;Eichenberger et al., 2004;Ramírez-Guadiana et al., 2018). B. subtilis SpoIVB has another substrate, the forespore-specific SpoIIQ protein (Chiba et al., 2007;Jiang et al., 2005). ...
In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro‐protein, pro‐σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro‐sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in‐frame deletion mutant fails to produce heat‐resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.
... Taking into consideration the exceptional complexity of the issue and the imperfection of methodological approaches, most scientists have studied the processes of microorganism development more oft en at the level of populations, sometimes at the level of individual cells, as well as passing into subcellular and molecular biological studies. Th e most numerous peculiarities of the growth and diff er-Peculiarities of Bacilli Ontogenesis During Cycle From a Spore to a Vegetative Cell entiation of bacterial cells have been established in the study of aerobic spore-forming bacteria (Gould & Hurst, 1969;Gould, 1971;Smirnov et al., 1993;Galperin et al., 2022;Setlow, 2006;Khanna et al., 2020;Eichenberger et al., 2004;Meeske et al., 2016;Zheng et al., 2016). ...
In the review, on the example of aerobic spore-forming bacteria, the problems of the development and ontogenesis of the bacterial cell are considered along with the possibilities of influencing the course of these processes. The characteristics of the main concepts "growth", "differentiation", and "development" as independent processes with their dynamic interrelation are presented. Attention is focused on the analysis of literature data on the peculiarities of vegetative cell development, starting from a spore in a dormant state and finishing with vegetative form formation. In particular, the mechanisms that maintain the spore dormant state and subsequent processes of activation, initiation, outgrowth, and vegetative cell formation are described. There are emphasized certain problems with research on the ontogenesis of bacterial cells due to the deficiency of appropriate methods, as well as the lack of a single opinion regarding individual stages of the development and vegetative form formation. It was concluded that the study of individual stages of the development of prokaryotes, which differ in spore-forming and non-spore-forming microorganisms, is still relevant. Knowledge of these processes will help scientists to develop mechanisms of influence on the ontogenesis of microorganisms.
... Numerous studies show that environmental conditions affect the resistance and surface characteristics of endospores from Bacillus species (Bressuire-Isoard et al., 2018;Hamiot et al., 2023;Isticato et al., 2020). In Bacillus subtilis, several transcription factors positively and negatively fine-tune the expression of hundreds of genes during sporulation (Arrieta-Ortiz et al., 2015;Eichenberger et al., 2004;Kroos, 2007), ensuring that the resulting spores are endowed with resistance and surface properties tailored for their environment (McKenney et al., 2013;. ...
Upon starvation, rod‐shaped Myxococcus xanthus bacteria form mounds and then differentiate into round, stress‐resistant spores. Little is known about the regulation of late‐acting operons important for spore formation. C‐signaling has been proposed to activate FruA, which binds DNA cooperatively with MrpC to stimulate transcription of developmental genes. We report that this model can explain regulation of the fadIJ operon involved in spore metabolism, but not that of the spore coat biogenesis operons exoA‐I, exoL‐P, and nfsA‐H. Rather, a mutation in fruA increased the transcript levels from these operons early in development, suggesting negative regulation by FruA, and a mutation in mrpC affected transcript levels from each operon differently. FruA bound to all four promoter regions in vitro, but strikingly each promoter region was unique in terms of whether or not MrpC and/or the DNA‐binding domain of Nla6 bound, and in terms of cooperative binding. Furthermore, the DevI component of a CRISPR‐Cas system is a negative regulator of all four operons, based on transcript measurements. Our results demonstrate complex regulation of sporulation genes by three transcription factors and a CRISPR‐Cas component, which we propose produces spores suited to withstand starvation and environmental insults.
... Numerous studies show that environmental conditions affect the resistance and surface characteristics of endospores from Bacillus species (78)(79)(80). InBacillus subtilis , several transcription factors positively and negatively fine-tune the expression of hundreds of genes during sporulation (81)(82)(83), ensuring that the resulting spores are endowed with resistance and surface properties tailored for their environment (84)(85)(86). Understanding how ecology and evolution impact the developmental GRN of M. xanthus and related species is an ongoing and future challenge (6,87,88). ...
Upon starvation rod-shaped Myxococcus xanthus bacteria form mounds and then differentiate into round stress-resistant spores. Little is known about the regulation of late-acting operons important for spore formation. C-signaling has been proposed to activate FruA, which binds DNA cooperatively with MrpC to increase transcription of many genes. We report that this model can explain regulation of the fadIJ operon involved in spore metabolism, but not that of the spore coat biogenesis operons exoA-I , exoL-P , and nfsA-H . Rather, a mutation in fruA increased the transcript levels from these operons early in development, suggesting negative regulation by FruA initially, and a mutation in mrpC affected transcript levels from each operon differently. FruA bound to all four promoter regions in vitro , but strikingly each promoter region was unique in terms of whether or not MrpC and the DNA-binding domain of Nla6 bound, and in terms of cooperative binding. Furthermore, the DevI component of a CRISPR-Cas system is a negative regulator of all four operons, based on transcript measurements. Our results demonstrate complex regulation of sporulation genes by three transcription factors and a CRISPR-Cas component, which we propose thwarts viral intrusion while making spores suited to withstand starvation and environmental insults.
... (A) Schematic representation of the genetic organization of the cge, cot, and sps genes involved in crust synthesis. Broken arrows, arrows and, stem-loop, respectively, represent the σ K dependent promoters, direct or indirect activation of transcription by a transcriptional regulator and transcriptional terminators defined previously (Zhang et al., 1994;Roels and Losick, 1995;Eichenberger et al., 2004;Kuwana et al., 2005;Nicolas et al., 2012;Cangiano et al., 2014;Arrieta-Ortiz et al., 2015). Primers used for qRT-PCR are indicated by half arrows. ...
Spore-forming bacteria of the Bacillus subtilis group are responsible for recurrent contamination of processing lines in the food industry which can lead to food spoilage. The persistence of B. subtilis would be due to the high resistance of spores to extreme environmental condition and their propensity to contaminate surfaces. While it is well known that sporulation conditions modulate spore resistance properties, little is known about their effect on surface and adhesion properties. Here, we studied the impact of 13 sporulation conditions on the surface and adhesion properties of B. subtilis 168 spores. We showed that Ca ²⁺ or Mg ²⁺ depletion, lower oxygen availability, acidic pH as well as oxidative stresses during sporulation lead to the release of more hydrophobic and adherent spores. The consequences of these sporulation conditions on crust composition in carbohydrates and proteins were also evaluated. The crust glycans of spores produced in a sporulation medium depleted in Ca ²⁺ or Mg ²⁺ or oxygen-limited conditions were impaired and contained lower amounts of rhamnose and legionaminic acid. In addition, we showed that lower oxygen availability or addition of hydrogen peroxide during sporulation decreases the relative amount of two crust proteins (CgeA and CotY) and the changes observed in these conditions could be due to transcriptional repression of genes involved in crust synthesis in late stationary phase. The fact that sporulation conditions affect the ease with which spores can contaminate surfaces could explain the frequent and recurrent presence of B. subtilis spores in food processing lines.
... Furthermore, we confirmed SpoIIID binding to the spoIID promoter region by using EMSA. We found that the DNA fragment containing the SpoIIID box located between −35 and −10 elements of the spoIID promoter region migrated as a discrete shifted band in the presence of varying concentrations of SpoIIID ( Figure 2c, lanes 1-3), as reported previously (Eichenberger et al., 2004). By contrast, when the DNA fragment containing the mutated SpoIIID box was examined, little or no shifted bands were detected (Figure 2c, lanes 4-6). ...
... By contrast, when the DNA fragment containing the mutated SpoIIID box was examined, little or no shifted bands were detected (Figure 2c, lanes 4-6). These results confirmed that the previously suggested SpoIIID box was a genuine SpoIIID binding site, contributing to repressing spoIID expression (Eichenberger et al., 2004). We note that the shifted DNAs with Spo0A~P were detected as smeared bands under our tested conditions ( Figure 2a, lanes 3-6), while the shifted band with SpoIIID was discrete (Figure 2d, lanes 2 and 3). ...
... We further found that the reporter activities were higher in the IIID* mutant than in the wild-type strain. These results directly verified that SpoIIID acted as a negative regulator for spoIID expression (Figure 3a), as suggested previously (Eichenberger et al., 2004). The double mutants (0A1*IIID* and 0A2*IIID*) displayed similar activities to that in the IIID* single mutant, suggesting that the derepression effects of IIID* are more predominant than the positive effects of 0A1 and 0A2 under the tested conditions. ...
The Spo0A transcription factor is activated by phosphorylation in starving Bacillus subtilis cells. The activated Spo0A (Spo0A~P) regulates genes controlling entry into sporulation and appears to control mother-cell-specific gene expression after asymmetric division, but the latter remains elusive. Here, we found that Spo0A~P directly binds to three conserved DNA sequences (0A1-3) in the promoter region of the mother cell-specific lytic transglycosylase gene spoIID, which is transcribed by σE -RNA polymerase (RNAP) and negatively controlled by the SpoIIID transcription factor and required for forespore engulfment. Systematic mutagenesis of the 0A boxes revealed that the 0A1 and 0A2 boxes located upstream of the promoter positively control the transcription of spoIID. In contrast, the 0A3 box located downstream of the promoter negatively controls the transcription of spoIID. The mutated SpoIIID binding site located between the -35 and -10 promoter elements causes increased expression of spoIID and reduced sporulation. When the mutations of 0A1, 0A2, and IIID sites are combined, sporulation is restored. Collectively, our data suggest that the mother cell-specific spoIID expression is precisely controlled by the coordination of three factors, Spo0A~P, SpoIIID, and σE -RNAP, for proper sporulation. The conservation of this mechanism across spore-forming species was discussed.
... During this process,~1.5 million ATP molecules are hydrolysed, each pumping 2 bp across the septum [91]. SpoIIIE-driven translocation of DNA and associated counterions increases turgor pressure and inflates the forespore, allowing subsequent engulfment and maturation of the spore, which takes~8 h to complete [90,92]. Therefore, the physiochemical properties of the DNA pumped into the phage capsid or into the forespore contribute to the formation of infective virions or mature spores that provide a strategy for surviving multiple environmental challenges [93,94]. ...
As part of their survival strategy under harsh environmental conditions, endospore-forming bacteria can trigger a sporulation developmental program. Although the regulatory cascades that precisely control the transformation of vegetative bacteria into mother cells and resilient spores have been described in detail, less is known about how bacteriophages that prey on endospore-formers exploit sporulation. Herein, we argue that phages infecting these bacteria have evolved several specific molecular mechanisms, not yet known in other bacteria, that manifest from the phage-driven alliance to negative effects on the host. We anticipate that the relationships between phages and endospore-formers outlined here will inspire studies on phage ecology and evolution, and could facilitate important advances in the development of phage therapies against pathogenic spore-formers.
... The spsA and the spsB genes are inactive during vegetative growth and are activated by a sporulation-specific sigma factor, s K , in response to the deterioration of nutrients in a growing environment. 31,32 Thus, the spsAB segment is suitable for analysis of the effect of the non-denaturing sodium bisulfite treatment on cell activities other than transcription. ...
Bacterial condensin preferentially loads onto single-stranded DNA (ssDNA) in vitro and onto rDNA in vivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines (CT-conversion) at melted DNA of a genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the rDNA segment . In principle, the CT-conversion rate is an accurate guide to detect ssDNA segment. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA hindered the ssDNA formation at the 100–500 bp segment downstream the promoter. These data support the idea that the ssDNA segment plays a crucial role as the condensin-loading site and suggest the mechanism of condensin loading onto rDNA.
... B z and δ z represents same for Z. n is co-operativity which accounts for the multimer formation of proteins. Existence of this motif in yeast [2], C. elegence [22], B. subtilis [23,24], Sea urchin [23], E. coli [23,22], fruit fly [23], human [25] and in many more diverse organisms are already seen. Instead of having this diversity, all two gene input circuits are not a FFL motif. ...
One of the major reason behind failure of the experimental synthetic circuit implementation is ignoring the hidden couplings in gene regulatory dynamics and its consequences. Dependency of the components of a genetic circuit upon its host, due to requirement for resources like ribosome, ATP, transcription factors, tRNA etc., and related effects are of utmost importance. Several times these dependencies are capable of giving rise to unexpected emergent responses. In a resource-limited environment, two apparently unconnected genes can compete for resources for their respective expression and may exhibit indirect regulatory connection; an emergent response thus arises in the system completely because of resource competition. In this work, we have shown how the responses of Feed-Forward loop (FFL), a well-studied regulatory genetic motif, can get recreated considering the resource competition in a three-gene pathway. The chosen motifs show many of the characteristic features of the conventional FFL structure, like response delay and pulse generation. This study pinpoints towards a larger area of research and exploration in synthetic and cellular systems which will reveal novel controlling ideas and unique behavioral changes in the system for its context dependencies.
... B. subtilis laboratory strains have long been used as a model to study sporulation, and ;150 nonessential genes required for efficient sporulation and germination have been identified through a variety of screening strategies (24)(25)(26)(27)(28)(29)(30)(31). Some of these genes are expressed during sporulation under the control of a sporulation sigma factor (32)(33)(34)(35)(36), while others cause a reduction in sporulation efficiency when deleted. Many of these screens relied on transposon insertion screening (7, 37) or on gene knockout libraries (6) and hence focused on only nonessential genes; thus, the role of essential genes in sporulation remains unknown. ...
For many bacteria, life typically involves growth in dense, three-dimensional communities called biofilms that contain cells with differentiated roles held together by extracellular matrix. To examine how essential gene function varies between vegetative growth and the developmental states of biofilm formation and sporulation, we created and screened a comprehensive library of strains using CRISPRi to knockdown expression of each essential gene in the biofilm-capable Bacillus subtilis strain 3610.
