Review

[Link]

Milestones in Bacillus subtilis sporulation research

Eammon P. Riley1, Corinna Schwarz2, Alan I. Derman2 and Javier Lopez-Garrido2,*
1 Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA.
2 Max Planck Institute for Evolutionary Biology, Plön, Germany.
* Corresponding Author:
Javier Lopez-Garrido, August-Thienemann-Str. 2, 24306 Plön, Germany; Phone: +49 4522 763 216;
E-mail: lopezgarrido@[Link]

ABSTRACT Endospore formation has been a rich field of research for more doi: 10.15698/mic2021.01.739
than a century, and has benefited from the powerful genetic tools available in Received originally: 17.07.2020;
Bacillus subtilis. In this review, we highlight foundational discoveries that in revised form: 21.10.2020,
Accepted 03.11.2020,
shaped the sporulation field, from its origins to the present day, tracing a
Published 27.11.2020.
chronology that spans more than one hundred eighty years. We detail how
cell-specific gene expression has been harnessed to investigate the existence
and function of intercellular proteinaceous channels in sporulating cells, and Keywords: Bacillus subtilis, sporulation,
we illustrate the rapid progress in our understanding of the cell biology of genetics, SpoIIIE, SpoIIIA, SpoIIQ,
sporulation in recent years using the process of chromosome translocation as sporulation history.
a storyline. Finally, we sketch general aspects of sporulation that remain
largely unexplored, and that we envision will be fruitful areas of future re- Abbreviations:
search. SASP – small acid-soluble protein,
Spo– – asporogenous and
oligosporogenous mutants,
Spo+ - sporulating strains,
SRS – SpoIIIE Recognition Sequence,
TCA – tricarboxylic acid,
TZM – 2,3,5-triphenyltetrazolium
chloride.

INTRODUCTION A BRIEF HISTORY OF ENDOSPORE FORMATION

Since their discovery, bacterial endospores (a.k.a. spores) Arguably the most important milestone in endospore re-
have attracted the attention of the scientific community search was the actual discovery of endospores. The first
and, at times, even the general public. This should not reported observation of bacterial endospores dates back to
come as a surprise, as some properties of endospores 1838, when Christian Gottfried Ehrenberg noted refractile
could well be in the headlines of the popular press. Bacte- bodies inside bacterial cells [4]. It was not until nearly four
rial endospores are among the most resilient cell-types decades later that endospores started to be characterized
known, and can survive for very long periods of time with- in seminal studies by Ferdinand Cohn and Robert Koch.
out any nutrients. In fact, it has been claimed that viable Cohn initiated the study of the resistance properties of
endospores were isolated from the gut of extinct bees bur- spores with the observation that B. subtilis spores survive
ied in Dominican amber more than 25 million years ago [1], periods of boiling [5]. Koch, in collaboration with Cohn,
evoking the imagery of Jurassic Park [2]. Furthermore, the followed the sporulation-germination cycle in B. anthracis
use of Bacillus anthracis (anthrax) spores in biological war- [6], and realized that spores and vegetative cells are differ-
fare and, more recently, in bioterrorist attacks has brought ent cellular forms of the same bacterial species, and that
the bacterial spore to the attention of modern society [3]. cells can interconvert between these two forms, from veg-
The study of endospore formation, however, is full of fas- etative cells to spores via sporulation, and from spores to
cinating discoveries that, while subtle enough to escape vegetative cells via germination. Their hand-drawn sketch-
the attention of the mainstream media, have contributed es of these processes are shown in Fig. 1.
to shape our current perception of bacterial cells. This re- At the same time, John Tyndall independently postu-
view aims to discuss some of the key findings that have lated the existence of latent bacterial forms able to survive
shaped the sporulation field, through the lens of bacterial extreme temperatures. He described a sterilization tech-
genetics. nique (now designated “tyndallization”) that eradicates

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E.P. RIley et al. (2020) Milestones in B. subtilis sporulation

FIGURE 1: Early depictions of sporulation and

germination in Bacillus spp. In one of his most
well-known studies [6], Robert Koch investi-
gated the etiology of anthrax. (A) Koch fol-
lowed the sporulation process in B. anthracis.
He placed a slice of spleen containing the Bacil-
lus into cow sera or aqueous humor, and incu-
bated the specimens at 35-37°C in an incubator
that he himself had constructed. During the
incubation, he observed the cells growing into
string-like structures. After 10-15 h, these
strings contained light refracting bodies, which
he identified as spores. Koch depicted the
structures in this sketch and likened them to
fragile strings of pearls. The strings gradually
decomposed, the spores were released and sank to the bottom of the droplet where they accumulated and could be kept for weeks. (B)
Koch followed spore germination and outgrowth, and determined that spores can form viable cells. He fixed dried spores on a slide and
incubated the sample with aqueous humor. After 3-4 hours, he observed various stages of germination and outgrowth, which are depicted
in Cohn’s sketch. Koch described spores as egg-shaped structures, enclosed by a thin layer of protoplasm that he called bright matter. He
hypothesized that during outgrowth this bright matter stretched and became the vegetative cell, whereas the spore remained at one cell
pole, lightened, and shrank, before it dissociated and disappeared. He proposed that the spore core consists of an oily substance necessary
for the cell to resume vegetative growth.

spores from culture media through the application of in- In 1965, Antoinette Ryter defined six different stages of
termittent heating periods that kill vegetative cells, inter- sporulation [17], based on her own studies of B. subtilis
spersed with periods of no heat that permit the spores that and on those of Young and Fitz-James in B. cereus, and this
survive to germinate into vegetative cells, which are then classification was later expanded to eight stages [18, 19].
killed during subsequent heating periods [7]. An up-to-date description of the different stages in B. sub-
Throughout the next few decades, researchers at- tilis, including details derived from fluorescence microsco-
tempted to characterize the process of endospore for- py and genetic studies, is shown in Fig. 2. It is important to
mation in different bacterial species, but it was difficult for note that this classification represents discrete cytological
them to achieve a unified understanding due to the limita- steps of a process that is actually continuous. Thus, not
tions of the optical microscopy and staining methods avail- only do several steps occur concomitantly, but also details
able at that time [8, 9]. Some researchers, led by Koch, about the transition between different stages are often
thought that spores were formed through the growth of a neglected. For example, the transition between stages II
single refractile granule inside the cytoplasm of the vegeta- and III happens through a phagocytosis-like process called
tive cell [6]. A related model postulated that spores were engulfment, in which the membrane of the mother cell
not the result of the growth of a single granule, but of the migrates around the forespore, generating the cell within a
fusion of multiple sporogenic granules inside the vegeta- cell that is a hallmark of endospore formation (Fig. 2); the
tive cell [10]. These putative sporogenic granules were synthesis of the different spore layers is also gradual and
probably cytoplasmic inclusion bodies that are present in starts shortly after polar septation (Fig. 2) [20]. Neverthe-
various species of endospore forming bacteria, and are less, the classification proposed by Ryter represents a con-
unrelated to spore formation. A different model, which ceptual framework that facilitated the subsequent genetic
comports better with our current perception of endospore dissection of spore formation.
formation (see below), proposed that spores were formed
through the condensation of part of the vegetative cell B. SUBTILIS AND THE GENETIC ANALYSIS OF
protoplasm, which gained refractility over time [11]. A SPORULATION MUTANTS
shared feature of these models, however, was that spore During the first half of the 20th century, sporulation was
formation happened close to a single cell pole. studied simultaneously in different bacterial species. Two
The advent of electron microscopy allowed for a more major discoveries, however, positioned B. subtilis to be the
comprehensive cytological model for sporulation to be ideal model system for characterizing the process of sporu-
developed. In the late 1950’s and early 60’s, Young and lation during the second half of the century: in 1958 John
Fitz-James provided detailed cytological descriptions of Spizizen reported that B. subtilis can be transformed with
endospore formation in Bacillus cereus [12, 13]. Electron DNA [21]; shortly thereafter, Thorne and Takahashi inde-
microscopy studies on sporulation in other Bacillus and pendently isolated phages capable of mediating general-
Clostridium species were published shortly thereafter [14– ized transduction in the same organism [22–25]. These
16]. By the mid 60’s, the field had achieved a consensus powerful tools opened new avenues for genetic studies,
view of the cytological transformations leading to endo- and several laboratories turned their attention to B. subtilis
spore formation, which are highly conserved among differ- to perform thorough genetic analyses of sporulation.
ent species.

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E.P. RIley et al. (2020) Milestones in B. subtilis sporulation

FIGURE 2: Sporulation stages in B. subtilis.

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E.P. RIley et al. (2020) Milestones in B. subtilis sporulation

Mutants unable to form endospores (asporogenous), or ized in a classic review by Piggot and Coote in 1976 [19].
with significantly reduced ability to form spores (oligospo- The spo loci were defined as those, that when mutated,
rogenous) have been reported for different species of en- impaired the ability of the cell to form spores, without hav-
dospore-forming bacteria ever since the inception of ing a noticeable effect on vegetative growth. The loci were
sporulation research [26]. The defining characteristic of classified into different categories (spo0, spoII, spoIII, spoIV,
oligosporogenous mutants is that the spores produced by and so on), according to the stage at which sporulation was
them yield vegetative cells with equally low ability to pro- blocked in the mutants (Fig. 2). No distinction was made
duce spores. In contrast, asporogenous mutants, by defini- between stages 0 and I, and mutations that caused block-
tion, do not form viable spores, unless they accumulate age in either stage were globally referred to as spo0. A
secondary mutations that suppress the asporogenous phe- capital letter was used to distinguish different loci, muta-
notype. Such spores yield vegetative cells with significantly tions in which produced blockages at the same stage. Thus,
increased ability to form new spores compared to the for example, spoIIIA and spoIIIE refer to two distinct loci
asporogenous parental strain. For simplicity, we will refer that, when mutated, produce a blockage at stage III. In
to both asporogenous and oligosporogenous mutants as some cases, it was later discovered that a specific locus
Spo−, and strains able to sporulate efficiently will be re- was actually an operon containing several genes. In those
ferred to as Spo+. cases, the different genes of the operon were assigned a
Researchers quickly devised simple, forward genetic second capital letter (for example, the spoIIIA locus actual-
screens to isolate Spo− mutants in large numbers, and ly consists of an operon of at least eight genes, which are
mapped the mutations using classical genetic methods named in order from spoIIIAA to spoIIIAH).
such as transformation and transduction [27, 28]. Those In addition to the spo genes, three more developmen-
studies were facilitated by the development of simple tests tal gene categories were described: ger, cot and ssp (Fig. 3).
to distinguish Spo+ and Spo− strains. Spo− mutants form Many ger genes were identified in elegant genetic screens
white colonies in certain rich media, whereas strains able for mutations that rendered spores unable to germinate
to sporulate efficiently form brown colonies. The brown properly [35–40]. These screens relied on an agar plate
color is due to the accumulation of a pigment whose syn- assay in which colonies formed by mutagenized cells were
thesis requires a component of the spore coat, CotA [29, treated with heat or chloroform vapor to kill vegetative
30]. Secondly, the heat resistance of the spores provides a cells [35]. The remaining spores were then overlaid with
definitive test to distinguish Spo+ and Spo− strains [31]. agar containing two key components: (i) nutrients to in-
Determining the linkage between mutations conferring the duce germination, and (ii) 2,3,5-triphenyltetrazolium chlo-
Spo− phenotype and different markers in the chromosome ride (TZM), which turns red when reduced by metabolical-
led to the understanding that there are multiple sporula- ly-active cells. Spores are metabolically dormant, and are
tion (spo) loci, scattered around the chromosome [27, 28, unable to reduce TZM to produce the red pigment, but
32–34]. upon germination become metabolically active and can
The initial analysis of Spo− mutants indicated that spor- reduce TZM to give the colonies a pink coloration. Colonies
ulation was blocked at different stages in different Spo − containing germination-deficient spores fail to reduce TZM,
mutants [31], suggesting that each sporulation stage was and therefore, do not produce the red pigment. While
controlled by a dedicated set of genes. This was formally some of the ger genes identified in germination screens
confirmed in 1966, when Ryter and collaborators per- encode proteins involved exclusively in germination (for
formed a cytological characterization of B. subtilis strains example, the genes encoding germinant receptors, such as
carrying different spo mutations using electron microscopy, those in the gerA and gerB operons), others encode pro-
and classified them according to the stage at which sporu- teins that are actually required for proper spore develop-
lation was blocked [18]. This study led to the establishment ment, generating some overlap between ger and spo genes
of a specific nomenclature for the spo loci that was formal- (for instance, gerM is required for proper sporulation, but

FIGURE 3: Developmental loci in B. subtilis. Diagram of the

sporulation-germination cycle, with the different classes of
developmental loci noted (in parentheses). The spo loci are
required for spore formation, but not for vegetative growth.
The ger loci are required for proper spore germination.
After germination, metabolism is reactivated and the spore
transforms into a growing vegetative cell in a process called
outgrowth, which is thought to depend on the same path-
ways that control vegetative growth. The cot and ssp loci
were identified after the isolation and characterization of
coat and small acid-soluble proteins (SASPs), respectively,
from spores. In addition to the these categories, there are
hundreds of developmentally-regulated genes whose roles
in sporulation and germination are not yet understood.
Membranes, red; DNA, blue; cell wall, gray; spore cortex,
light gray; spore coat, black; SASPs, green circles.

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E.P. RIley et al. (2020) Milestones in B. subtilis sporulation

it was first identified in a screen for germination mutants lying this interpretation is as follows (Fig. 4): of the two
[41, 42]). cells required to form a spore, only the forespore retains
The two major remaining categories of developmental its chromosome in the mature spore, since the mother cell
genes, cot (for coat) and ssp (for spore-specific protein), lyses upon sporulation completion (Fig. 2). Thus, spo genes
were identified by reverse genetics, after the isolation and required in the forespore must be complemented in the
biochemical characterization of coat proteins and small forespore chromosome and the wild-type version of the
acid-soluble proteins (SASPs) [29, 43–47] from spores, re- gene will therefore always present in mature spores, and
spectively (Fig. 3). SASPs are abundant spore proteins that these will give rise to a homogenous population of Spo +
saturate and protect the spore chromosome [45]. Since transformant spores. But if a spo gene is required in the
individual SASPs and most coat proteins are dispensable mother cell and not in the forespore, then sporulating cells
for B. subtilis sporulation under standard laboratory condi- in which only the mother-cell chromosome is transformed
tions [29, 46, 48, 49], they were missed in the genetic with the wild-type allele will be able to form mature spores,
screens that led to the identification of spo genes. but the non-transformed forespore chromosome would
The discovery and characterization of developmental still contain the mutated version of the spo gene, and the
genes accelerated after the rise of DNA cloning and se- spores would remain Spo−.
quencing technology. Researchers were able to clone spo Results from these biochemical and genetic analyses
genes by selecting for chromosomal DNA fragments that underscored that different genetic programs were activat-
complemented the respective spo mutations. From these ed in the two cells, at different times during sporulation.
clones, lacZ fusions were generated, and these in turn al- From here, the focus moved on to deciphering the genetic
lowed for the study of the spo genes in vivo. New genetic regulation of sporulation.
tools, such as Tn917-based transposition mutagenesis [50– In 1969, it was discovered that bacterial RNA polymer-
52] and integrational plasmid vectors [52–55], further facil- ases contain a factor, called Sigma (σ), that is necessary for
itated the identification and manipulation of spo loci. Dur- promoter recognition and transcription initiation [61, 62].
ing the late 1970’s and 80’s, many developmental loci were This finding led researchers to hypothesize the existence of
cloned and sequenced, their expression characterized, and alternative σ factors, which could direct RNA polymerase
the role of the proteins they encode started to be deci- to specific subsets of promoters. The existence of such
phered. alternative σ factors was first demonstrated in B. subtilis
phage SP01 [63], which encodes σ factors that drive ex-
CELL-SPECIFIC GENE EXPRESSION pression from middle and late phage promoters. The first
Three main conclusions arose from the initial genetic anal- bacterial alternative σ factor to be discovered was σ37 [64],
ysis of sporulation: first, most developmental genes are not a general-stress response σ factor of B. subtilis. Initially, the
expressed in growing cells, and their expression is induced name of the different σ factors was based on their molecu-
at different times during sporulation; second, there is a lar weight, in kDa. Thus, σ37 was ~37 kDa, as determined by
dependency hierarchy in developmental gene expression, the apparent size of the purified protein in an acrylamide
such that the expression of genes that participate in later gel. However, the discovery of alternative σ factors with
events in the sporulation pathway tends to depend on the similar sizes or that migrated aberrantly in acrylamide gels
expression of genes that participate in earlier events; third, accelerated over the following years, which led researchers
the majority of the sporulation genes are expressed in only in the B. subtilis field to replace the molecular weight des-
one of the two cells required to form a spore, either the ignation with a letter reflecting order of discovery [65, 66].
mother cell or the forespore. This last conclusion was Thus, σ37 was renamed σB, the name by which it is com-
achieved by using fractionation techniques to separate monly known today, and the genes encoding the σ factors
forespore and mother-cell protoplasms [56–58], and by the were named sig followed by their corresponding letter.
implementation of genetic strategies to determine the cell Shortly after the discovery of σB, it was shown that the
in which different spo genes were required [59, 60]. In one sporulation regulatory program also relies on alternative σ
such genetic strategy, developed by Lencastre and Piggot factors that are activated in the mother cell or in the fore-
[59] and illustrated in Fig. 4, Spo− mutants were trans- spore at different developmental stages. The first sporula-
formed with genomic DNA from the wild-type (Spo+) strain tion-specific σ factor to be discovered was σE (originally
at the start of sporulation, thereby rendering the Spo − mu- referred to as σ29) [67]. σE was discovered using predomi-
tants capable of forming heat-resistant spores. The paren- nantly biochemical and molecular biology techniques [67].
tal Spo− mutants fell into two categories, depending on the The protein was isolated from RNA polymerase purified
ability of the resultant spores to produce additional spores from sporulating cultures, and was shown to change the
following germination: mutants in the first category pro- promoter specificity of RNA polymerase in vitro. Not sur-
duced spores that were homogeneously Spo+; mutants in prisingly, it was subsequently shown that σE was encoded
the second category produced a heterogeneous population by a previously identified spo locus, spoIIGB (later renamed
of Spo+ and Spo− spores. The spo genes mutated in the first as sigE) [68, 69]. After σE, three additional sporulation σ
category of parental strains were inferred to be required in factors were identified by means of both sequence similari-
the forespore, and the ones mutated in the second were ty with previously identified σ factors, and in vitro tran-
inferred to be required in the mother cell. The logic under- scription assays of sporulation genes: σF [70, 71], σG [72–

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E.P. RIley et al. (2020) Milestones in B. subtilis sporulation

FIGURE 4: Genetic strategy to identify the cell in which a spo gene is required. A Spo– strain with a mutation in a known spo gene (yellow
star) is depicted in Stage I (see Fig. 2) with its two chromosomes, one that will be packed into the forespore (upper chromosome) and one
that will remain in the mother cell and be destroyed when the mother cell lyses (lower chromosome). The strain is transformed with ge-
nomic DNA from a Spo+ strain at the onset of sporulation. Transformed chromosomes are drawn in purple, non-transformed chromosomes,
in blue. Either or both chromosomes are capable of being transformed to spo+. If the spo gene is required in the forespore (left panel),
transformation of the forespore chromosome to spo+ rescues the process and generates spores that can germinate and go on to sporulate
again (left). But transformation of the mother-cell chromosome to spo+ with no accompanying transformation of the forespore chromosome
leaves the forespore chromosome spo– so the cells cannot complete the process, and no spores are produced (center). Hence only Spo +
spores are generated if the spo gene is required in the forespore. If the spo gene is required in the mother cell (right panel), transformation
of the mother-cell chromosome to spo+ rescues the process (center and right), but only if the forespore chromosome is transformed as well
will the spores that are produced be able to germinate into cells that can go on to sporulate again (right). Hence, both Spo+ and Spo– spores
can be generated if the spo gene is required in the mother cell.

74] and σK [75]. In B. subtilis, the activation of the different After the discovery of the sporulation-specific σ factors,
sporulation σ factors follows a hierarchical order in which many of the developmental genes previously identified
different σ factors are sequentially activated in alternating were assigned to different regulons, depending on the σ
cellular compartments, and the activation of a later factor factor controlling their expression. Microarray technology
depends on the activation of the previous one (Fig. 5): first, allowed researchers to discover additional genes under the
σF becomes active in the forespore shortly after polar sep- control of sporulation σ factors [80, 81]. Together the cell-
tation, followed by σE in the mother cell. Roughly coinci- specific σ factors control the expression of more than 500
dent with engulfment completion, σG is activated in the different genes [82, 83], the majority of which are not ex-
forespore and, finally, σK is activated in the mother cell. pressed during vegetative growth. Over the course of the
The variety of mechanisms involved in the sequential acti- past few decades, the study of sporulation has focused on
vation of these σ factors includes phosphorelays, proteo- deciphering the function of genes that are under the con-
lytic processing, and even a gene splicing event [76–79]. trol of sporulation-specific σ factors. But the functions of
nearly 1/3 of these genes remain a mystery, and for the

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E.P. RIley et al. (2020) Milestones in B. subtilis sporulation

tion systems from Gram-negative bacteria [103, 106–108],

and one of them, SpoIIIAG, has been shown to form mul-
timeric rings in vitro [109, 110], leading to the proposal
that A-Q complexes constitute proteinaceous channels that
connect the mother cell and the forespore, through which
molecules required for σG activation might be transferred
between the two cells. Meisner and collaborators devel-
oped a compartmentalized biotinylation assay to test this
idea (Fig. 6A) [111]. The authors used mother cell- and
forespore-specific promoters to express the gene encoding
E. coli biotin ligase (birA) in either cell, and fused biotin
acceptor peptides to the extracellular domains of A and Q
FIGURE 5: Cascade of sporulation-specific σ factors in B. subtilis. proteins. They found that BirA produced in the forespore
Immediately after polar septation, σF is activated in the forespore. could biotinylate the extracellular domain of SpoIIIAH,
σF activation triggers an intercompartmental signaling cascade which supported the idea that A-Q complexes are channels
that leads to the activation of σE in the mother cell. Together σF that are open at least on the forespore side.
and σE control gene expression during engulfment. Roughly coin- In a separate study, Camp and Losick took advantage of
cident with the completion of engulfment, σF is replaced by σG in cell-specific gene expression to study whether A-Q chan-
the forespore, which leads to the subsequent activation of σ K in nels were specifically required for σG activation, or if they
the mother cell. σG and σK control gene expression in the forespore
also played a role in the expression of σG-independent
and the mother cell, respectively, after engulfment.
genes (Fig. 6B) [112]. They monitored the activity of the
heterologous RNA polymerase, T7 RNAP, when produced
majority of sporulation genes with assigned function, we in the forespore from a σF-dependent promoter by placing
have only a superficial knowledge of how specific gene a lacZ gene under the control of a T7 RNAP-dependent
products contribute to spore formation. We envision that promoter. In the presence of A-Q, β-galactosidase activity
the study of developmentally-regulated genes will continue could be detected at the onset of sporulation, and it in-
to be a fruitful area of research for years to come. creased gradually over time until it reached a plateau that
was maintained throughout spore formation. In mutants
CELL-SPECIFIC GENE EXPRESSION AS A GENETIC TOOL lacking functional A-Q complexes, β-galactosidase activity
Beyond its central importance to the developmental pro- was also detected at the onset of sporulation, but dropped
gram of sporulation, cell-specific gene expression has come off later in sporulation, at roughly the time when σG would
to provide a powerful genetic tool for the study of different be normally activated. These observations indicated that A-
aspects of spore formation. After the discovery of the Q plays a general role in regulating forespore gene expres-
sporulation-specific σ factors, many researchers carefully sion at late sporulation stages, rather than affecting σG
characterized promoters regulated by the different σ fac- activity specifically. The authors proposed that A-Q chan-
tors (i.e. [84–92]), enabling these promoters to be co- nels constitute a feeding tube through which the mother
opted for expression of heterologous genes in either the cell provides metabolic resources to the forespore in order
mother cell or the forespore, at different developmental to maintain forespore biosynthetic activities at late sporu-
stages. Cell-specific gene expression has been used to lation stages [112].
study many of the fundamental features of spore for- We have recently accumulated more evidence that the
mation such as the temporal compartmentalization of the mother cell nurtures the forespore, by implementing a cell-
different sporulation σ factors [93], the organization of the specific protein degradation system that also relies on cell-
chromosomes after sporulation initiation [94], and the specific gene expression (Fig. 6C and D) [113, 114]. The
transport of the forespore chromosome from the mother system uses modified ssrA* tags from E. coli fused to the C-
cell to the forespore [95–101], among other things. In or- terminus of target proteins [114], and the cognate E. coli
der to illustrate the power of this approach, we describe SspB adaptor protein produced under the control of moth-
three experiments that have helped to disentangle, at least er-cell or forespore-specific promoters [113] (note that E.
partially, the function of the sporulation loci spoIIIA and coli SspB is not related to the sporulation ssp genes de-
spoIIQ (Fig. 6): scribed previously in this manuscript; to avoid confusion, E.
Mother-cell proteins encoded in the spoIIIA operon coli SspB will be referred to as SspBEc). When SspBEc is pro-
(which contains eight genes, spoIIIAA to spoIIIAH) together duced, it binds to ssrA* and delivers the target protein to
with the forespore protein SpoIIQ form a trans-envelope the endogenous B. subtilis protease ClpXP for degradation
multimeric complex (henceforth A-Q complexes) that (Fig. 6C and D) [114]. We are using this platform to sys-
spans the mother-cell and forespore membranes (Fig. 6A) tematically assess the requirement of different metabolic
[102, 103]. Mutants lacking spoIIIA or spoIIQ are asporoge- pathways in the mother cell and in the forespore for sporu-
nous, as they fail to activate the late forespore σ factor, σ G lation to progress normally. Our results indicate that the
[104, 105]. Some of the SpoIIIA proteins show sequence tricarboxylic acid (TCA) cycle is required in the mother cell,
and structural homology to components of various secre- but is largely dispensable in the forespore (Fig. 6E) [115].
We have also observed that protein synthesis in the fore-

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E.P. RIley et al. (2020) Milestones in B. subtilis sporulation

spore requires TCA cycle activity in the mother cell, sug- precursors are transported to the forespore to support
gesting that the mother cell produces TCA cycle-derived protein synthesis, in line with the feeding tube model pro-
metabolic precursors, such as amino acids, for protein syn- posed by Camp and Losick [112].
thesis in both the mother cell and the forespore (Fig. 6E) Overall, cell-specific gene expression has been a fantas-
[115]. These findings indicate that mother-cell metabolic tic addition to the list of genetic tools available in B. subtilis.

FIGURE 6: Harnessing cell-specific gene expression to study the function of A-Q complexes. (A) The forespore protein SpoIIQ (Q, orange
circle) and the mother-cell proteins encoded in the spoIIIA operon (A, light blue circle), form trans-envelope complexes that bridge the fore-
spore and mother-cell membranes during engulfment. Zoomed in panel: Biotin ligase (BirA, green) produced in the forespore is able to bio-
tinylate a biotin acceptor peptide (pink) fused to the extracellular domain of the A protein, SpoIIIAH [111], indicating that A-Q complexes are
channels (see panel B), and that the channel pore is open on the forespore side and large enough for BirA to reach the extracellular domain
of SpoIIIAH. (B) The activity of a heterologous RNA polymerase (T7 RNAP, yellow) produced in the forespore under σF control is monitored
by the accumulation of β-galactosidase (β-gal, dark blue circles) produced through the expression of a lacZ gene under the control of a T7
RNAP-dependent promoter. In the presence of A-Q channels (A-Q+, left zoomed in panel), sustained β-galactosidase activity is detected
throughout sporulation. In the absence of A-Q channels (A-Q–, right zoomed in panel), β-galactosidase activity drops at later sporulation
stages. Camp and Losick [112] proposed that A-Q channels constitute feeding tubes through which the mother cell transfers metabolic re-
sources to the forespore to maintain biosynthetic activities at late sporulation stages. (C) The ssrA*/SspBEc inducible protein degradation
system [114]. SspBEc (purple) binds to ssrA* (red) fused to the C-terminus of target proteins (green), and delivers the target proteins to the
endogenous B. subtilis protease ClpXP (orange pacman) for degradation. (D) Cell-specific degradation of target proteins during sporulation is
achieved by expressing sspBEc from mother cell- or forespore-specific promoters [113]. Target proteins represented by green ovals tagged
with ssrA (red line); degradation represented by orange pacman. (E) Left cell: Mother-cell TCA cycle provides metabolic precursors, such as
amino acids (AAs, yellow circles), to support protein synthesis in both the mother cell and the forespore [115]. Mother-cell metabolic pre-
cursors could be transported to the forespore via A-Q channels, in keeping with the feeding tube model. Right cell: Degradation of TCA cycle
enzymes in the mother cell blocks protein synthesis in both the mother cell and the forespore [115].

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E.P. RIley et al. (2020) Milestones in B. subtilis sporulation

We are looking forward to seeing new creative approaches copy has made it possible to resolve two different foci
using those tools to explore the inner workings of sporulat- within the SpoIIIE complex (Fig. 7C), suggesting that the
ing bacteria. complex consists of two subcomplexes of opposite polarity,
one anchored to the mother cell and the other to the fore-
THE GENETIC BASIS OF CELLULAR DYNAMICS spore septal membrane. Each subcomplex contains enough
The study of sporulation has helped to overturn the tradi- SpoIIIE monomers to assemble at least two hexameric rings
tional dogma that bacterial cells lack a defined internal [101], which would allow the two arms of the chromosome
organization owing to the absence of membrane-bound to be translocated in parallel from the mother cell to the
organelles. Over the last 25 years, we have obtained pre- forespore (Fig. 7D) [98].
cise descriptions of how proteins are redistributed during Assembly of the SpoIIIE translocation complex requires
sporulation to mediate dynamic cellular processes. Ad- that the DNA be trapped at the septum [126], and the
vances in our understanding of sporulation during this pe- complex is in turn required to mediate septal membrane
riod have been driven by the development of a wealth of fission while the chromosome is translocated [124]. This
new imaging technologies, particularly in fluorescence mi- has led to the proposal that the mother-cell and forespore
croscopy, which have allowed the field of bacterial cell SpoIIIE subcomplexes pair to form a continuous channel
biology to emerge. We illustrate the rapid progress made spanning both septal membranes (Fig. 7D). In support of
in this field by focusing on a single protein, SpoIIIE. this idea, it has been shown that both forespore and
Since the mid 20th century, it was recognized that the mother-cell subcomplexes are required to keep the septal
two chromosomes resulting from a replication event prior membranes separated during chromosome translocation
to sporulation initiation are segregated during sporulation, [101]. However, the mother-cell subcomplex by itself is
such that one remains in the mother cell and the other is sufficient to mediate the transport of the chromosome into
packed into the small forespore [12]. In 1994, Wu and Err- the forespore [95, 101]. If the SpoIIIE mother-cell subcom-
ington [116] reported that mutations in spoIIIE, one of the plex is absent, the forespore subcomplex can transport the
spo loci that was identified during the genetic analysis of chromosome out of the forespore and into the mother cell
sporulation mutants, prevented complete segregation of [95, 101], indicating that the SpoIIIE translocation complex
the forespore chromosome, such that only ~30% of the can, in principle, function as a bidirectional motor. During
chromosome was present in the forespore with the rest sporulation, however, the SpoIIIE translocation complex
still in the mother cell (Fig. 7A). They inferred that the po- always transports the chromosome from the mother cell to
lar septum trapped the forespore chromosome, and that the forespore (Fig. 7D). The mechanism that determines
SpoIIIE mediated the translocation of the chromosome translocation directionality has been at least partially elu-
across the septum into the forespore. cidated by means of a combination of in vivo, in vitro and
Further studies showed that SpoIIIE is a membrane- in silico approaches. The motor domain of SpoIIIE is able to
anchored FtsK-like protein that has a large C-terminal cy- recognize specific, highly-skewed octameric sequences in
toplasmic motor domain [117] with DNA-dependent the DNA called SpoIIIE Recognition Sequences (SRSs),
ATPase activity, that is capable of tracking along DNA in the which are present in each arm of the chromosome but on
presence of ATP [118]. More recently, in vitro studies with opposite strands [97]. Since the B. subtilis chromosome is
purified SpoIIIE motor domains have shown that they as- circular and the two chromosome arms are trapped at the
semble into hexameric rings [119, 120], with an inner di- septum, the opposite orientation of SRSs on each arm
ameter large enough to accommodate a double-stranded might serve as a cue to determine translocation direction-
DNA molecule [119]. Single molecule experiments using ality. In fact, it has been shown that the interaction of
optical tweezers have allowed the characterization of the SpoIIIE with the SRS in the orientation preferentially en-
inter-subunit coordination [121] and of the mechanochem- countered by SpoIIIE as each chromosome arm is directed
istry [122] of the hexameric rings as they move along DNA into the forespore stimulates SpoIIIE ATPase activity [120,
molecules, providing exquisite details of the mechanism of 127, 128]. This might account for the direction of translo-
chromosome translocation at the molecular level. cation and the simultaneous transport of both chromo-
SpoIIIE assembles into a translocation complex at the some arms to the forespore.
septal midpoint during the early stages of sporulation (Fig. In addition to guaranteeing that the forespore receives
7B). This was shown first in fixed cells using immunofluo- a full complement of genetic material, SpoIIIE-mediated
rescence microscopy [117] and later in living cells with a chromosome translocation also contributes to the genera-
SpoIIIE-GFP fusion and fluorescent membrane dyes that tion and maintenance of forespore shape during engulf-
are compatible with live cell imaging [123] (Fig. 7B). More ment [100]. Packaging an entire chromosome into the
recently, studies of the assembly of the SpoIIIE transloca- small forespore compartment leads to a high turgor pres-
tion complex with super-resolution optical microscopy sure, which inflates the forespore like a balloon and dis-
techniques [101, 124, 125] have revealed that SpoIIIE oli- tends the forespore membranes (Fig. 7E).
gomers localize to the leading edge of the polar septum as While spoIIIE is among the best characterized spo loci,
it forms, and ultimately assemble a stable translocation the field has made significant progress in the understand-
complex at the septal midpoint. In mutants that produce ing of a myriad of dynamic processes associated with spor-
thicker than normal polar septa, super-resolution micros- ulation, such as the formation of the polar septum [129–
131], the migration of the mother-cell membrane around

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E.P. RIley et al. (2020) Milestones in B. subtilis sporulation

FIGURE 7: Progress in our understanding of SpoIIIE-mediated chromosome translocation. (A) Fluorescence microscopy images of a wild-
type (WT, left) and of a spoIIIE mutant (spoIIIE, right) strain of B. subtilis. The upper panels show the DNA stained with DAPI (blue), and the
lower panels the overlay of DAPI-stained DNA and the membranes stained with FM 4-64 (red). In the upper panel, forespores are indicated
by single arrowheads, and mother cells by double arrowheads. Wild-type forespores contain a complete chromosome. However, forespores
of spoIIIE mutant strains contain only ~30% of a chromosome, and the rest remains trapped in the mother cell. (B) Fluorescence microscopy
image of a sporulating cell producing a SpoIIIE-GFP fusion during chromosome translocation. The upper panel shows the DNA stained with
DAPI (blue) and the GFP signal, and the lower panel shows in addition the membranes stained with FM 4-64 (red). SpoIIIE forms a focus
(green) at septal midpoint, where the chromosome is trapped. (C) SpoIIIE visualized by super-resolution microscopy (PALM) in living cells
with thicker polar septa. SpoIIIE forms two foci (dual foci), which are separated by a distance equivalent to the septal thickness, indicating
that one cluster is present at one side of the septum and the other at the opposite side. Reproduced from [101]. (D) Model for the organiza-
tion and function of the SpoIIIE translocation complex at the septal midpoint. SpoIIIE forms two side-by-side channels spanning both septal
membranes (red lines), thereby allowing the simultaneous transport of both arms of the chromosome from the mother cell (MC) to the
forespore (FS). Translocation is powered by SpoIIIE motor domains at the mother-cell side of the septum (green circles), which are activated
to export the chromosome to the forespore. Forespore motor domains (red circles) remain inactive. (E) Cryo-electron microscopy of a wild-
type sporulating cell (top), and of a spoIIIE mutant (bottom). Membranes are annotated as follows: forespore membrane, pink; mother-cell
membrane, purple. Chromosome translocation is required to maintain the shape of the forespore. In the absence of chromosome transloca-
tion, the forespore appears deflated. Reproduced with permission from [100].

the forespore during engulfment [132–139], the synthesis folding, and translational regulation play a significant role
of the peptidoglycan cortex [140], and the assembly of the in assembling the spore, and if so, if they are required in a
proteinaceous coat [20, 141], to name a few. specific cell or during a specific stage of sporulation. Recent
high-throughput studies have identified vegetative pro-
HISTORICAL CONTINGENCIES teins that likely play a role in sporulation [142, 143]. Genet-
Sporulation in B. subtilis is one of the most well- ic tools to interfere with the function of specific proteins in
understood developmental processes, but there are still a precise, cell- and developmental stage-specific manner
gaps in our understanding, which are due in part to the during spore formation have also recently been developed
historical contingencies of sporulation research. Much of (Fig. 6C and D) [113], and these present a promising new
the sporulation research performed so far has focused on approach for understanding the role played by these and
understanding the role of different developmental genes in other vegetative proteins during endospore formation.
spore formation. The definition of spo genes—those that, Although many facets of sporulation remain unclear, new
when mutated, impair the ability to form spores without technologies and innovative approaches will continue to
affecting vegetative growth—has likely biased our under- advance our knowledge of this remarkable process.
standing of sporulation, as it necessarily excluded all the A more general aspect of endospore formation that
essential genes and non-essential genes that are also re- remains poorly studied is how the process functions in
quired for optimal vegetative growth. It has therefore re- different species. Endospore formation occurs in a broad
mained unclear whether essential housekeeping proteins range of bacterial species that belong to an ancient and
involved in central metabolism, redox reactions, protein exceptionally diverse bacterial phylum, the Firmicutes

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E.P. RIley et al. (2020) Milestones in B. subtilis sporulation

[144]. However, most mechanistic studies on sporulation of endospore formation may allow us to address key ques-
over the previous 50 years have been done in B. subtilis, as tions regarding the ecological roles and the evolutionary
this organism is particularly amenable to genetic studies. origin of this fascinating developmental process.
Placing the focus on a single species has provided us with
extraordinarily detailed descriptions of sporulation but, at ACKNOWLEDGMENTS
the same time, has prevented us from fully appreciating We thank Jan Böhning for assistance with translation of the
the diversity of endospore formation. In recent years, Clos- Koch and Cohn publications. This work was supported by ERC
tridioides difficile —formerly known as Clostridium dif- starting grant 853323.
ficile— has become a model for endospore formation in
anaerobic bacteria. Studies in this organism reveal that, CONFLICT OF INTEREST
although the general scheme of endospore formation is The authors declare no conflict of interest.
similar to that of B. subtilis, there are mechanistic differ-
ences that affect every sporulation stage, from initiation to COPYRIGHT
germination [145–151]. In addition, while B. subtilis and © 2020 Riley et al. This is an open-access article released
C. difficile produce one spore per sporangium, there are under the terms of the Creative Commons Attribution (CC
some species that can produce two [152, 153], and others BY) license, which allows the unrestricted use, distribution,
that can produce multiple spores [154–156], opening the and reproduction in any medium, provided the original
intriguing possibility that the sporulation pathway was co- author and source are acknowledged.
opted as a reproductive strategy in some species, or that
sporulation evolved from what was originally a reproduc-
tive strategy [157–160]. Clearly, this is a rich and interest- Please cite this article as: Eammon P. Riley, Corinna Schwarz, Alan
ing area of future research that will require the develop- I. Derman and Javier Lopez-Garrido (2020). Milestones in Bacillus
ment of new culturing techniques and genetic tools to ma- subtilis sporulation research. Microbial Cell 8(1): 1-16. doi:
10.15698/mic2021.01.739
nipulate non-model bacteria. Understanding the diversity

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