The Bacterial Spore: from Molecules to Systems

The taxonomy of spore-forming Gram-positive bacteria has a long and colorful history. In 1872, 35 years after Christian Ehrenberg provided the initial description of Vibrio subtilis (and also Vibrio bacillus), Ferdinand Cohn assigned it to the genus Bacillus and family Bacillaceae, specifically noting the existence of heat-sensitive vegetative cells and heat-resistant endospores (see reference 1 ). Soon after that, Robert Koch identified Bacillus anthracis as the causative agent of anthrax in cattle and the endospores as a means of the propagation of this organism among its hosts. In subsequent studies, the ability to form endospores, the specific purple staining by crystal violet-iodine (Gram-positive staining, reflecting the presence of a thick peptidoglycan layer and the absence of an outer membrane), and the relatively low (typically less than 50%) molar fraction of guanine and cytosine in the genomic DNA have been used as diagnostic characteristics of the phylum Firmicutes (low-G+C Gram-positive bacteria).

The family Bacillaceae (domain Bacteria; kingdom Bacteria; phylum Firmicutes; class Bacilli; order Bacillales) ( Fig. 1 ) is a globally dispersed and phenotypically heterogeneous group of bacteria ( 1 , 2 ). It therefore follows that the family possesses a considerable evolutionary history for scientists to unravel. In recent years, the issue of evolution in the Bacillaceae has been probed from two directions. Some researchers have taken an ecological approach to understand the organisms’ adaptive evolution to particular environmental niches; others have pursued a laboratory-based approach in which single laboratory strains are directed to evolve under particular conditions determined by the experimenter. Both approaches have yielded new insights. As in so many other facets of the biology of the Bacillaceae, most of our knowledge has been derived from the intense study of relatively few members of the family, most notably Bacillus subtilis ( 3 , 4 ). Much less information has been obtained concerning the vast majority of the Bacillaceae, prompting the present examination of diversity and evolution within this ubiquitous family. The present article builds upon previous reviews of the topic ( 5 – 10 ). Additional information on the genomic diversity of spore-forming Firmicutes and on the ecology of the Bacillaceae can be found in articles by Galperin ( 480 ) and Mandic-Mulec et al. ( 481 ).

The most distinguishing feature of most members of the family Bacillaceae (phylum Firmicutes) is their ability to form endospores that provide high resistance to heat, radiation, chemicals, and drought, allowing these bacteria to survive adverse conditions for a prolonged period of time. Bacillaceae are widely distributed in natural environments, and their habitats are as varied as the niches humans have thought to sample. Over the years of microbiological research, members of this family have been found in soil, sediment, and air, as well as in unconventional environments such as clean rooms in the Kennedy Space Center, a vaccine-producing company, and even human blood ( 1 – 3 ). Moreover, members of the Bacillaceae have been detected in freshwater and marine ecosystems, in activated sludge, in human and animal systems, and in various foods (including fermented foods), but recently also in extreme environments such as hot solid and liquid systems (compost and hot springs, respectively), salt lakes, and salterns ( 4 – 6 ). Thus, thermophilic genera of the family Bacillaceae dominate the high-temperature stages of composting and have also been found in hot springs and hydrothermal vents, while representatives of halophilic genera have mostly been isolated from aquatic habitats such as salt lakes and salterns, but less often from saline soils ( 7 , 8 ). The isolates that have been obtained, in particular, from the varied extreme habitats, produce a wide range of commercially valuable extracellular enzymes, including those that are thermostable ( 9 , 10 ).

Bacteria thrive in amazingly diverse ecosystems and often tolerate large fluctuations within a particular environment. One highly successful strategy that allows a cell or population to escape life-threatening conditions is the production of spores. Bacterial endospores, for example, have been described as the most durable cells in nature ( 1 ). These highly resistant, dormant cells can withstand a variety of stresses, including exposure to temperature extremes, DNA-damaging agents, and hydrolytic enzymes ( 2 ). The ability to form endospores appears restricted to the Firmicutes ( 3 ), one of the earliest branching bacterial phyla ( 4 ). Endospore formation is broadly distributed within the phylum. Spore-forming species are represented in most classes, including the Bacilli, the Clostridia, the Erysipelotrichi, and the Negativicutes (although compelling evidence to demote this class has been presented [ 5 ]). To the best of our knowledge endospores have not been observed in members of the Thermolithobacteria, a class that contains only a few species that have been isolated and studied. Thus, sporulation is likely an ancient trait, established early in evolution but later lost in many lineages within the Firmicutes ( 4 , 6 ).

Biologists often think of regulation as deterministic; causes have invariant consequences, and changes predictably beget further changes. However, with the advent of techniques for the study of individual cell phenotypes, it has become evident that random cell-to-cell variation in the amounts of mRNA and protein is prevalent and often entails significant consequences, particularly for developmental processes ( 1 , 2 ). This review focuses on the developmental pathways of Bacillus subtilis, with attention to the roles of this variation and of stochastic reactions in competence, motility, sporulation, and biofilm formation. We do not comprehensively review the regulatory mechanisms that control these pathways but present only what is needed for our purposes. Figure 1 identifies the major players that are discussed below and illustrates the competence, motility, cannibalism, biofilm, and sporulation modules that represent the major known developmental pathways of B. subtilis.

The principal B. subtilis laboratory strain, strain 168, is derived from a parent strain isolated in Marburg, Germany, following a mutagenesis procedure ( 1 ). The popularity of this strain arose after it was shown to be competent for genetic transformation ( 2 , 3 ), which paved the way for myriad molecular genetics analyses that led to a detailed understanding of the biology of B. subtilis and related Gram-positive bacteria. It is therefore not surprising that strain 168 was the first Gram-positive species to have its entire genome sequenced, at a time when sequencing was a laborious and expensive process. The project to sequence the genome was set up in 1987 by a consortium of over 30 laboratories and took about 10 years to complete. Each laboratory was assigned a different region of the chromosome and used their own cloning and sequencing strategies to manage their assigned portion of the genome ( 4 ). The final genome sequence contained 4,214,810 base pairs, and the original annotation included 4,100 protein-coding genes ( 5 ). Following the development of sequencing technologies that were considerably faster and more efficient, the genome of B. subtilis strain 168 was resequenced and cleared of sequencing errors in 2009 ( 6 ). The most recent update of the annotation brought the total of protein-coding genes to 4,458 ( 7 ).

The first published reports of sporulation were by Ferdinand Cohn in Bacillus subtilis and Robert Koch in Bacillus anthracis in 1877 in the Zeitschrift für Plänzenbiologie ( 1 , 2 ). In addition to being among the earliest published reports of bacteria, these articles provided the initial demonstration that bacteria have an internal cellular organization. Cohn and Koch described intracellular membrane-bound compartments of different sizes and positions within the cell. Despite these striking original observations, bacteria were considered for much of the 20th century to be “bags of enzymes” that lacked any discernible spatial organization ( 3 ). However, in the past two decades, numerous examples, including dedicated intracellular compartments such as magnetosomes, the presence of a defined orientation of the chromosome, and the polarity of the chemotactic apparatus have emphatically demonstrated that bacterial cells exhibit an intracellular organization that, while less complex than eukaryotic cells, is nonetheless a critical and significant part of their physiology.

The spore is simply a cell with some extremely novel properties and structural elements. The primary morphological elements shared with vegetative bacterial cells are the spore core (the cytoplasm), the inner spore membrane (the cytoplasmic membrane), and the peptidoglycan (PG) wall ( Fig. 1 ). A spore stripped of coat protein layers outside the PG retains its dormancy and many of its resistance properties ( 1 , 2 ). The primary factor contributing to spore dormancy and heat resistance, and a major factor in resistance to chemical and physical damaging agents, is the relative dehydration of the spore core ( 3 – 7 ). A predominant factor in maintaining this dehydration, and potentially a factor in attaining it, is the PG wall ( 2 ).

The coat varies considerably in width among species. In Bacillus subtilis, where the coat is relatively wide, it is just less than 200 nm in width, and its multilayered organization is unmistakable by transmission electron microscopy (TEM). Importantly, the number of coat layers and the presence or absence of appendages extending from the coat surface vary among species. This interspecies variation and differences in complexity drew attention as soon as spores were imaged at high resolution, and in the decades since ( 1 – 7 ). The coat is readily distinguished from the cortex (see reference 178 ) because of its higher electron density. In a large subset of species, the spore also possesses an additional layer surrounding the coat, called the exosporium ( Fig. 1 ; see also references 8 and 9 ).

The extreme resistance of spores of members of the Bacillales and Clostridiales orders is probably the property most closely associated with these spores. In the past, this extreme resistance contributed to claims for spontaneous generation and, in more recent years, has contributed to the applied importance of spores in a number of different areas including the following. (i) The food industry. Given that spores of a number of species are ubiquitous in the environment, they routinely contaminate foodstuffs. Since spores of many species are vectors for food spoilage and food-borne disease, the food industry commits significant resources to eliminating spores in order to make foods sterile, in particular to eliminate extremely dangerous spores such as those of Clostridium botulinum ( 1 , 2 ). Indeed, many of the requirements for food sterilization regimens in the United States are designed to completely inactivate C. botulinum spores. (ii) The medical products industry. Just as in the food industry, spores present similar concerns in the medical products industry, including the manufacture of medical devices and parenteral drugs, again because of the involvement of spores in a number of human diseases. (iii) The health care industry. There is an increasing prevalence of disease due to Clostridium difficile in hospital and long-term nursing care facilities, largely because of the resistance of C. difficile spores and thus their persistence in patient care environments unless stringent environmental decontamination regimens are followed. (iv) Vaccine development. There is increasing interest in spores as carriers of proteins important as vaccines ( 3 , 91 ), in large part because of spores’ extreme stability to normal and even extreme environmental conditions. This may allow the delivery of vaccines to areas where cold storage is difficult and is facilitated by utilizing the spore coat as a means to deliver immunogens. (v) Probiotics. Since spores are dormant, as such, they will not be probiotics. However, the administration of spores with their resistance to low pH conditions in the stomach is a route to effectively deliver potentially beneficial bacteria to the lower gastrointestinal tract ( 4 , 91 ). Notably, it is the C. difficile spore’s resistance to stomach acidity that is the reason that the oral route is the major mechanism for C. difficile infection. (vi) Biological warfare. While the disease-causing potential of Bacillus anthracis is one reason that this organism has come to the fore as a biological weapon, in particular of bioterror (S. L. Welkos, unpublished data), the major reason for this organism’s visibility in this area is that B. anthracis spores are so resistant. This makes their dispersal either in water or as an aerosol relatively simple and ensures that these spores will persist in contaminated environments and will thus require stringent decontamination methods for their elimination.

The specialized structure that maintains the dormancy and resistance properties of endospores provides an opportunity for wide dispersal of spores in the environment, and for survival over long periods under conditions unfavorable for growth. Although dormant and uniquely resistant to environmental insult ( 1 , 206 ), a spore remains sensitive to changes in its environment. Specific germinants are detected by receptors in the spore inner membrane; this signal is then transduced by mechanisms that are not understood in detail, but result in the activation of proteins that variously allow movement of small molecules across the membrane and deconstruct protective layers, restoring normal hydration and active metabolism ( 2 , 3 ).

The three main species of the Bacillus cereus sensu lato, B. cereus, B. thuringiensis, and B. anthracis, were recognized and established by the early 1900s because they each exhibited distinct phenotypic traits. B. thuringiensis isolates and their parasporal crystal proteins have long been established as a natural pesticide and insect pathogen ( 1 ). B. anthracis, the etiological agent for anthrax, was used by Robert Koch in the 19th century as a model to develop the germ theory of disease ( 2 ), and B. cereus, a common soil organism, is also an occasional opportunistic pathogen of humans ( 3 – 5 ). In addition to these three historical species designations, are three less-recognized and -understood species: B. mycoides, B. weihenstephanensis, and B. pseudomycoides. All of these “species” combined comprise the B. cereus sensu lato group. Despite these apparently clear phenotypic definitions, early molecular approaches to separate the first three by various DNA hybridization and 16S/23S ribosomal sequence analyses led to some “confusion” because there were limited differences to differentiate between these species ( 6 ). These and other results have led to frequent suggestions that a taxonomic change was warranted to reclassify this group to a single species ( 7 , 8 ). But the pathogenic properties of B. anthracis and the biopesticide applications of B. thuringiensis appear to “have outweighed pure taxonomic considerations” and the separate species categories are still being maintained ( 9 ). B. cereus sensu lato represents a classic example of a now common bacterial species taxonomic quandary where relatively new molecular data must somehow be incorporated into a traditional hierarchical classification system ( 10 ).

In some Bacillus species, including Bacillus subtilis, the coat and a glycoprotein layer referred to as the spore crust are the outermost layer of the spore ( Fig. 1 ). These spore structures are discussed in more detail in Driks and Eichenberger ( 108 ). In others, such as the Bacillus cereus family, there is an additional layer that envelops the coat, called the exosporium, which is distinct from the crust. In the case of Bacillus anthracis, one of the three pathogenic species of the B. cereus family, a series of fine hair-like projections, also referred to as a “hair-like” nap, extends from the exosporium basal layer ( 1 – 4 ) ( Fig. 1 ). Other exosporium-producing Bacillus species, such as Bacillus megaterium, lack this “hairy” nap ( Fig. 1 ). Separating the exosporium from the rest of the spore structure is an area referred to as the interspace ( 5 ).

Anthrax is primarily a zoonotic disease caused by the Gram-positive spore-forming bacterium Bacillus anthracis, which occurs in domesticated and wild animals, primarily herbivores. B. anthracis is found throughout the world and exists in the soil as the dormant, highly resistant spore form. Animals become infected when they ingest spores while grazing on contaminated land or ingesting spore-laden feed, although mechanical spreading by flies or vultures from one animal environmental locus to another could be possible ( 1 – 3 ). Humans are incidental and relatively rare hosts that, in natural settings, become infected by contact with infected animals or contaminated animal products ( 3 – 7 ). These infections occur most often in agricultural settings and lesser-developed countries. Before the development of effective vaccines and disinfection practices, industrial exposures were also common in European and North American countries. The latter were associated with the processing of animal materials (hides, hair, wool, and bones), as illustrated by the highly lethal illness known as woolsorter’s disease. This disease occurred after inhalation of spore-laden dust and aerosols in wool and textile mills in England and the industrialized regions of the northeastern United States ( 4 , 6 , 8 ).

Clostridia are anaerobic bacteria, although many species can tolerate oxygen to various extents. They are able to form endospores and are not capable of dissimilatory sulfate reduction. Most of them show a positive Gram reaction. These criteria have been used in the past for classification. However, phylogenetic analyses based on 16S rRNA sequences led to reattribution of many former clostridia to numerous other and also novel genera, such as Blautia, Butyrivibrio, Caloramator, Cellulosilyticum, Dendrosporobacter, Eubacterium, Filifactor, Flavonifractor, Moorella, Oxalophagus, Oxobacter, Paenibacillus, Thermoanaerobacter, Thermoanaerobacterium, Sedimentibacter, Sporohalobacter, Syntrophomonas, Syntrophospora, and Tissierella (J. P.Euzéby, List of prokaryotic names with standing in nomenclature – genus Clostridium, http://www.bacterio.cict.fr/c/clostridium.html). For the genus Clostridium, approximately 180 species have been validly described, rendering it one of the largest bacterial genera. Only a few of these species are pathogenic, however, involving microbes producing very dangerous toxins. On the other hand, a large number of species are used in biotechnological applications (enzyme, bulk chemicals, and biofuels production) and tested for use in cancer therapy. This is due to the enormous metabolic diversity within the clostridia, rendering them the avant-garde of biotechnologically exploited microorganisms. During past years, techniques have been developed that allowed establishment of genetic systems for many clostridia. Thus, the tools are at hand for further elucidation and exploitation. Due to the limited space of this article, many aspects cannot be presented in detail. Thus, the interested reader is referred to recent references for additional information ( 1 – 6 ).

The ability of the Gram-positive, anaerobic rod Clostridium perfringens to form resistant spores contributes to its survival in many environmental niches, including soil, waste water, feces, and foods ( 1 ). In addition, sporulation and germination play a significant role when this important pathogen causes disease ( 2 , 3 ). As introduced in the next section of this review, spores often facilitate the transmission of C. perfringens to hosts and then germinate in vivo to cause disease.

Display systems that present biologically active molecules on the surface of microorganisms have become increasingly used to address environmental and biomedical issues ( 1 – 3 ). Strategies using environmentally relevant proteins or peptides for display on the surface of phages or bacterial cells have been extensively reviewed by Wu et al. ( 4 ). Examples include proteins able to bind metal ions that can be used as bioadsorbents or biocatalysts, including cysteine-rich metallothioneins (MTs) or Cys-His rich synthetic peptides, known to bind Cd2+ and Hg2+ with a very high affinity. Eukaryotic MTs have been expressed on the surface of Escherichia coli cells through fusion to the porin LamB, with a 20-fold increased ability of Cd2+ accumulation of the recombinant cell with respect to its parental strain ( 5 , 6 ). In addition, metal-binding peptides have also been expressed on the surface of soil bacteria known to survive in contaminated environments. The mouse MT was displayed on the surface of Pseudomonas putida ( 7 ) and Ralstonia metallidurans CH34 ( 8 ), resulting in a 3-fold increase in binding and removal of Cd2+, sufficient to improve plant growth in a contaminated soil ( 8 ). Synthetic phytochelatins (ECn) with the repetitive metal-binding motif (Glu-Cys)nGly were displayed on the surface of Moraxella sp. cells causing a 10-fold improvement in Hg2+ intracellular accumulation ( 9 – 11 ). In addition to heavy metals, organic contaminants can be removed from the environment by the use of microbial cells displaying heterologous enzymes. Examples include organophosphorus hydrolases (OPHs). These bacterial enzymes are able to degrade organophosphates, which are toxic compounds widely used as pesticides. E. coli cells expressing OPH on their surface via the Lpp-OmpA fusion system were able to degrade parathion and paraoxon 7-fold faster than cells expressing OPH intracellularly ( 12 ). Surface display approaches have also been used to develop whole-cell diagnostic tools and vaccine delivery systems. Functional single-chain antibody fragments have been expressed on bacterial cells and used as diagnostic devices in immunological tests. In the first report of an antibody fragment expressed in an active form on a bacterial surface, the murine anti-human-IgE scFv antibody fragment was exposed on the surface of Staphylococcus xylosus and S. carnosus cells ( 13 ). More recently, the oral commensal bacterium Streptococcus gordonii was engineered to display a single-chain Fv (scFv) antibody fragment, derived from a monoclonal antibody raised against the major adhesin of the dental caries-producing bacterium Streptococcus mutans (streptococcal antigen I/II or SA I/II). Recombinant S. gordonii was found to specifically bind to immobilized SA I/II and represents the first step toward the development of a stable system for the delivery of recombinant antibodies ( 14 ).

Life and elemental cycles are intertwined through biogeochemistry. Organisms not only order atoms into dynamic molecules, they also help control the composition of their natural environments along with chemical, physical, and geological processes. Elements such as C, H, O, N, P, and S make up the backbone of life on earth. These, combined with a suite of trace nutrients including metals such as Fe, Cu, and Mn, compose all the structural, mechanical, and messaging components of the cell. They are fixed from the environment and cycled through metabolic transformations. Eukaryotic and prokaryotic microorganisms are abundant and perform many geochemical cycling processes including biotransformation, mineral dissolution, and biomineralization. This review focuses on the contribution of bacteria and, more specifically, bacterial spores to metal speciation in the environment. Many of these metal transformations are required for cellular metabolism and are facilitated by metals via electron transfer in metal-protein centers.