7.20A: Sporulation in Bacillus - Biology

7.20A:  Sporulation in Bacillus - Biology

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Sporulation is the last-ditch response to starvation; it is suppressed until alternative responses prove inadequate.

Learning Objectives

  • Explain sporulation in Bacillus

Key Points

  • B. subtilis can divide symmetrically to make two daughter cells (binary fission), or asymmetrically, producing a single endospore that is resistant to environmental factors such as heat, desiccation, radiation, and chemical insult which can persist in the environment for long periods of time.
  • The process of endospore formation has profound morphological and physiological consequences: radical post-replicative remodelling of two progeny cells, accompanied eventually by cessation of metabolic activity in one daughter cell (the spore ) and death by lysis of the other (the ‘mother cell’).
  • Sporulation in B. subtilis is induced by starvation; the sporulation developmental program is not initiated immediately when growth slows due to nutrient limitation.

Key Terms

  • endospore: A dormant, tough, and non-reproductive structure produced by certain bacteria from the Firmicute phylum.
  • sporulation: The process of a bacterium becoming a spore.

Bacillus subtilis is a rod-shaped, Gram-postive bacteria that is naturally found in soil and vegetation. It is known for its ability to form a small, tough, protective, and metabolically dormant endospore. B. subtilis can divide symmetrically to make two daughter cells (binary fission), or asymmetrically, producing a single endospore that is resistant to environmental factors such as heat, desiccation, radiation, and chemical insult which can persist in the environment for long periods of time. The endospore is formed at times of nutritional stress, allowing the organism to persist in the environment until conditions become favourable. The process of endospore formation has profound morphological and physiological consequences: radical post-replicative remodeling of two progeny cells, accompanied eventually by cessation of metabolic activity in one daughter cell (the spore) and death by lysis of the other (the ‘mother cell’).

Although sporulation inB. subtilis is induced by starvation, the sporulation developmental program is not initiated immediately when growth slows due to nutrient limitation. A variety of alternative responses can occur:

  • The activation of flagellar motility to seek new food sources by chemotaxis
  • The production of antibiotics to destroy competing soil microbes
  • The secretion of hydrolytic enzymes to scavenge extracellular proteins and polysaccharides, or the induction of ‘competence’ for uptake of exogenous DNA for consumption, with the occasional side-effect that new genetic information is stably integrated.

Sporulation is a last-ditch response to starvation, and it is suppressed until alternative responses prove inadequate. Even then, certain conditions must be met, such as chromosome integrity, the state of chromosomal replication, and the functioning of the Krebs cycle.

Sporulation requires a great deal of time and energy, and it is essentially irreversible, making it crucial for a cell to monitor its surroundings efficiently and ensure that sporulation is embarked upon at only the most appropriate times. The wrong decision can be catastrophic: a vegetative cell will die if the conditions are too harsh, while bacteria-forming spores in an environment which is conducive to vegetative growth will be outcompeted. In short, initiation of sporulation is a very tightly regulated network with numerous checkpoints for efficient control.

Two transcriptional regulators, σH and Spo0A, play key roles in initiation of sporulation. Several additional proteins participate, mainly by controlling the accumulated concentration of Spo0A~P. Spo0A lies at the end of a series of inter-protein phosphotransfer reactions, Kin–Spo0F–Spo0B–Spo0A, termed as a ‘phosphorelay’.

Stage II sporulation protein E

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May play the central regulatory role in sporulation. It may be an element of the effector pathway responsible for the activation of sporulation genes in response to nutritional stress. Spo0A may act in concert with Spo0H (a sigma factor) to control the expression of some genes that are critical to the sporulation process. Repressor of abrB, activator of the spoIIa operon. Binds the DNA sequence 5'-TGNCGAA-3' (0A box).

Sporulation of Bacteria (With Diagram)

Bacteria produce several types of spores called gonidia, sporangiospores, arthrospores (oidia), conidia, cysts and endospores.

They are highly thick-walled and resistant spores which are formed in response to adverse environment, presence of harmful waste products or ageing of bacterial colony.

A part of protoplast of the bacterial cell containing the nuclear body or nucleoid stores food undergoes dehydration and separates from the rest by means of mesosome and in growth of plasma membrane.

It is called endospore prunordium. The primordium secretes a wall around it. More wall materials are deposited over it by the surrounding cytoplasm to form the endospore. The residual cytoplasm and wall of parent bacterium undergo autolysis.

The liberated endospore is dispersed by air currents and on germination forms a new bacterium. Endospores can easily tolerate a temperature of ± 100°C. Toxic chemicals have no effect on them.

The resistant nature of endospores is due to their thick wall, low water content and the presence of an anticoagulant chemical known as dipicolinic acid. Fortunately, only two pathogenic bacteria Clostridium tetani and Bacillus anthracis produce endospores.

The Sporulation Module

The sporulation module, which acts as a timer, controls the cell progression toward sporulation (9). The master regulator of this module, Spo0A, upon phosphorylation activates the transcription of itself and the other response regulator Spo0F via σ H , in a positive feedback loop activated when Spo0A* > SAct (29). The module is build of two submodules, the Kin–Spo0F and the Spo0B–Spo0A two-component systems, which are coupled in series by Spo0B quickly transferring phosphate between Spo0F and Spo0A, as is analyzed in detail in SI Appendix.

Kin–Spo0F is a stress-sensing system, the structure of which is similar to that of the ComA–ComP two-component system. The schematic diagram shown in Fig. 1 represents the operation of at least five different histidine kinases (KinA–KinE), each autophosphorylating in response to a different stress signal. In the absence of or low stress levels, the histidine kinases KinA–E, can also dephosphorylate Spo0F*. Therefore, the rate of production of Spo0F* ω = d(Spo0F*)/dt has a sigmoid dependence on the stress level. This result, together with the dephosphorylation of Spo0F* by Rap, implies that the Kin–Spo0F sensing system acts as a gate that turns on the sporulation timer only above some minimum stress level. The clock rate ω is regulated by a competition between the positive effect of stress level and the negative effect of the Rap input [dephosphorylation of Spo0F* (34)], as detailed in the next section.

When a sufficiently high stress level is encountered, phosphate is transferred to the Spo0B histidine kinase, which rapidly transfers the phosphate to the response regulator Spo0A, beginning the accumulation of Spo0A*. The accumulation rate Ω = d(Spo0A*)/dt of Spo0A* (the clock rate of the sporulation timer) changes during the progression of sporulation. When Spo0A* accumulates above the threshold concentration SAct, Ω rapidly increases from a base level Ω0 to a higher-level Ωup.

Shaping an Endospore: Architectural Transformations During Bacillus subtilis Sporulation

Endospore formation in Bacillus subtilis provides an ideal model system for studying development in bacteria. Sporulation studies have contributed a wealth of information about the mechanisms of cell-specific gene expression, chromosome dynamics, protein localization, and membrane remodeling, while helping to dispel the early view that bacteria lack internal organization and interesting cell biological phenomena. In this review, we focus on the architectural transformations that lead to a profound reorganization of the cellular landscape during sporulation, from two cells that lie side by side to the endospore, the unique cell within a cell structure that is a hallmark of sporulation in B. subtilis and other spore-forming Firmicutes. We discuss new insights into the mechanisms that drive morphogenesis, with special emphasis on polar septation, chromosome translocation, and the phagocytosis-like process of engulfment, and also the key experimental advances that have proven valuable in revealing the inner workings of bacterial cells.

Treatments of CDI Based on Sporulation/Germination

Currently, the standard treatment of CDI is the use of vancomycin, metronidazole, or fidaxomicin, each of which has some level of recurring disease due to the continued insult to the colonic microbiome and the presence of spores within the colon/environment (Allen et al., 2013). To meet this challenge, non-antibiotic and immune-based therapies against CDI have been developed, such as anti-toxins, vaccines, fecal microbiota transplant (FMT), and anti-germination-based compounds (Gerding et al., 2008 Howerton et al., 2013 Kociolek and Gerding, 2016). Many anti-toxins and vaccines for CDI have been developed in the past two decades (Cox et al., 2013 Monteiro et al., 2013 Mathur et al., 2014 Zhao et al., 2014 Wang Y. K. et al., 2015 Yang et al., 2015 Qiu et al., 2016). Though these treatments can effectively decrease the morbidity and mortality of CDI, most of the anti-toxins and vaccines cannot suppress C. difficile colonization and kill C. difficile spores. Therefore, with these treatments, there are still risks of potential CDI relapse in the host.

Instead of merely neutralizing C. difficile toxins in host, strategies which can directly decrease C. difficile colonization, kill the vegetative cells, and suppress sporulation/germination are desirable treatments for CDI. FMT is an effective strategy to reconstruct the gut microbiota to suppress C. difficile colonization, especially for patients who have multiple bouts of recurring disease and who have failed conventional treatment methods (Borody and Khoruts, 2012 Weingarden et al., 2014 Khoruts and Sadowsky, 2016 Kim et al., 2016). Although FMT is deemed relatively safe and low-cost, the unappealing aesthetics of the procedure is often a concern of patients (Sampath et al., 2013 Varier et al., 2015). Because the C. difficile spore form is necessary for dissemination and persistence, sporulation/germination are critical steps for CDI. Thus, it is worth developing therapeutic strategies for disrupting C. difficile disease transmission and spread according to C. difficile spore biology. Basing on the progress of C. difficile spore germination, the PBA CDCA and secondary bile acids LCA, UDCA, and iLCA are potent inhibitors of C. difficile spore germination (Sorg and Sonenshein, 2010 Zhang and Klaassen, 2010 Heeg et al., 2012). Moreover, several mouse-derived bile acids, such as α-muricholic acid, β-muricholic acid, and ω-muricholic acid inhibit C. difficile spore germination and growth (Francis et al., 2013b). Excitingly, synthesized bile acid analogs, such as CAmSA, methylchenodeoxycholic acid diacetate, and compound 21b (derived from UDCA) have been identified to inhibit C. difficile spore germination (Sorg and Sonenshein, 2009 Howerton et al., 2013 Stoltz et al., 2017). Of these compounds, CAmSA showed promise in inhibiting/delaying C. difficile disease in a mouse model of CDI. These anti-germination-based strategies could work in a couple of different ways. (i) High risk patients who are to be treated with antibiotics could also take an anti-germinant to prevent the germination of spores within the host's gut. This patient continues to take the anti-germinant during and post-antibiotic treatment so that the normal, colonic, microbiome has a chance to repopulate and provide natural protection against CDI. (ii) Patients with CDI could take the recommended course of antibiotics plus the anti-germinants. This strategy would prevent recurring disease by allowing the microbiome to re-establish colonization resistance post-antibiotic treatment. Because both strategies block germination, and thus downstream events (vegetative growth, toxin production, and spore formation), anti-germination therapy would limit the presence of spores within the surrounding environment because C. difficile would not have a chance to expand in population and produce spores. In contrast germination-inducing strategies are a viable option for environmental cleanup inducing in vivo germination has the potential for toxin-production and, thus, exacerbation of symptoms. Due to the inherent nature of the dormant spore, harsh chemicals (e.g., bleach) are required to clean environmental surfaces. But by germinating the spores in the environment, the germinated spores become susceptible to a wider range of sanitizing agents (Nerandzic and Donskey, 2010, 2013, 2017 Nerandzic et al., 2016). More studies should be investigated for further application of germination inhibitors.


An endospore is structurally and chemically more complex than the vegetative cell. It contains more layers than vegetative cells. Resistance of Bacterial spores may be mediated by dipicolinic acid, a calcium ion chelator found only in spores. When the favorable condition prevails, (i.e. availability of water, appropriate nutrients) spores germination occurs which forms vegetative cells of pathogenic bacteria.

  • Calcium dipicolinate in core
  • Keratin spore coat
  • New enzymes (i.e., dipicolinic acid synthetase, heat-resistant catalase)
  • Increases or decreases in other enzymes.

A mature endospore contains a complete set of the genetic material (DNA) from the vegetative cell, ribosomes and specialized enzymes.

Mature endospores are released from the vegetative cell to become free endospores. When the free endospores are placed in an environment that supports growth, the endospores will revert back to a vegetative cell in a process called germination. It should be noted that unlike the process of binary fission observed with vegetative cells, endospore formation is not a reproductive process but a process of differentiation that provides the bacteria with a mechanism for survival.

Extracellular control of spore formation in Bacillus subtilis

Spore formation in the Gram-positive bacterium Bacillus subtilis has been classically viewed as an example of unicellular differentiation that occurs in response to nutritional starvation. We present evidence that B. subtilis produces an extracellular factor(s) that is required, in addition to starvation conditions, for efficient sporulation. This factor is secreted and accumulates in a cell density-dependent fashion such that cells at a low density sporulate poorly under conditions in which cells at a high density sporulate efficiently. Conditioned medium (sterile filtrate) from cells grown to a high density contains this extracellular differentiation factor (EDF-A) and stimulates spore formation of cells at low density under normal starvation conditions. EDF-A is heat-resistant, protease-sensitive, and dialyzable, indicating that it is at least in part an oligopeptide. Production of EDF-A is reduced or eliminated in spoOA and spoOB mutants, which are defective in many processes associated with the end of vegetative growth. Mutations in abrB, which suppress many of the pleiotropic phenotypes of spoOA mutants, restore production of EDF-A.

Gene expression in Bacillus subtilis surface biofilms with and without sporulation and the importance of yver for biofilm maintenance

Five independent DNA microarray experiments were used to study the gene expression profile of a 5-day Bacillus subtilis air–liquid interface biofilm relative to planktonic cells. Both wild-type B. subtilis and its sporulation mutant (ΔspoIIGB::erm) were investigated to discern the important biofilm genes (in the presence and absence of sporulation). The microarray results indicated that suspension cells were encountering anaerobic conditions, and the air–liquid interface biofilm was metabolically active. For the statistically significant differential expression (P < 0.05), there were 342 genes induced and 248 genes repressed in the wild-type biofilm, whereas 371 genes were induced and 128 genes were repressed in the sporulation mutant biofilm. The microarray results were confirmed with RNA dot blotting. A small portion of cells (1.5%) in the wild-type biofilm formed spores and sporulation genes were highly expressed. In the biofilm formed by the sporulation mutant, competence genes (comGA, srfAA, srfAB, srfAD, and comS) were induced which indicate a role for quorum sensing (bacterial gene expression controlled by sensing their population) in biofilms. There were 53 genes consistently induced in the biofilms of both the wild-type strain and its spoIIGB mutant—those genes have functions for transport, metabolism, antibiotic production—and 26 genes with unknown functions. Besides the large number of genes with known functions induced in the biofilm (121 genes in the wild-type biofilm and 185 genes in the sporulation mutant biofilm), some genes with unknown functions were also induced (221 genes in the wild-type biofilm and 186 genes in the sporulation mutant biofilm), such as the yve operon which appears to be involved in polysaccharide synthesis and the ybc operon which inhibits the growth of competitors for nutrients. A knockout mutant of yveR was constructed, and the mutant showed major defects in biofilm maintenance. Both the wild-type strain and its sporulation mutant formed normal biofilms, suggesting complete sporulation is not necessary for biofilm formation. The expression profiles of these two strains share more repressed genes than induced genes, suggesting that the biofilm cells repress similar pathways in response to starvation and high cell density. © 2004 Wiley Periodicals, Inc.