Updates on the sporulation process in Clostridium species
Talukdar, P. K., Olguín-Araneda, V., Alnoman, M., Paredes-Sabja, D., & Sarker, M. R. (2015). Updates on the sporulation process in Clostridium species. Research in Microbiology, 166(4), 225-235. doi:10.1016/j.resmic.2014.12.001
10.1016/j.resmic.2014.12.001 Elsevier
Accepted Manuscript http://cdss.library.oregonstate.edu/sa-termsofuse *Manuscript
1 Review article for publication in special issue: Genetics of toxigenic Clostridia
2
3 Updates on the sporulation process in Clostridium species
4
5 Prabhat K. Talukdar1, 2, Valeria Olguín-Araneda3, Maryam Alnoman1, 2, Daniel Paredes-Sabja1, 3,
6 Mahfuzur R. Sarker1, 2.
7
8 1Department of Biomedical Sciences, College of Veterinary Medicine and 2Department of
9 Microbiology, College of Science, Oregon State University, Corvallis, OR. U.S.A; 3Laboratorio
10 de Mecanismos de Patogénesis Bacteriana, Departamento de Ciencias Biológicas, Facultad de
11 Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile.
12
13
14 Running Title: Clostridium spore formation.
15
16
17 Key Words: Clostridium, spores, sporulation, Spo0A, sigma factors
18
19
20 Corresponding author: Dr. Mahfuzur Sarker, Department of Biomedical Sciences, College of
21 Veterinary Medicine, Oregon State University, 216 Dryden Hall, Corvallis, OR 97331. Tel: 541-
22 737-6918; Fax: 541-737-2730; e-mail: [email protected]
23
1 24
25 Abstract
26 Sporulation is an important strategy for certain bacterial species within the phylum Firmicutes to
27 survive longer periods of time in adverse conditions. All spore-forming bacteria have two phases
28 in their life; the vegetative form, where they can maintain all metabolic activities and replicate to
29 increase numbers, and the spore form, where no metabolic activities exist. Although many
30 essential components of sporulation are conserved among the spore-forming bacteria, there are
31 differences in the regulation and the pathways among different genera, even at the species level.
32 While we have gained much information from the most studied spore-forming bacterial genus,
33 Bacillus, we still lack an in-depth understanding of spore-formation in the genus Clostridium.
34 Clostridium and Bacillus share the master regulator of sporulation, Spo0A, and its downstream
35 pathways, but there are differences in the activation of the Spo0A pathway. While Bacillus
36 species use a multicomponent phosphorylation pathway for phosphorylation of Spo0A, termed
37 phosphorelay, such a phosphorelay system is absent in Clostridium. On the other hand, a number
38 of genes regulated by the different sporulation-specific transcription factors are conserved
39 between different Clostridium and Bacillus species. In this review, we discuss the recent findings
40 on Clostridium sporulation and compare the sporulation mechanism in Clostridium and Bacillus.
41
2 42 1. Introduction
43 Sporulation is an intriguing bacterial property of a certain low G+C group of Gram-
44 positive bacteria, which have existed from ancient time (2.5 billion years ago) [1]. It is a
45 complex developmental process, which leads to the generation of metabolically dormant spores
46 from vegetative cells [2]. While the exact reason is not known for the decision of bacterial cell to
47 form spores, it has been hypothesized that nutrient depletion or the presence of toxic compounds
48 triggers the sporulation process [3]. Spore formation is a helpful strategy for the spore-forming
49 bacteria leading to survival in unfavorable conditions in the environment or inside the hosts for
50 prolonged periods of time, and transmission to other hosts or environments. Spores can
51 withstand physical and chemical stresses, such as high temperatures, pressures, solvents,
52 oxidizing agents, lytic enzymes, irradiation, acceleration, and antimicrobials [4, 5], which could
53 rapidly destroy the vegetative form of the bacterium. In several instances, spores serve as an
54 infective particle in human and animal diseases [6]. Each of these fascinating characteristics led
55 microbiologists to engage their profound interest on dissecting spore structure and the
56 mechanism of spore formation.
57 Extensive studies have been conducted on the sporulation process of Bacillus species,
58 especially Bacillus subtilis for many years, and thus it is regarded as the model organism for
59 sporulation. Due to the availability of techniques for genetic manipulation, molecular
60 microbiologists showed the most interest in this species to illustrate the sporulation mechanism.
61 However, after gaining significant knowledge from B. subtilis, researchers have switched their
62 focus to other spore-forming bacteria, especially Clostridium species. Although, it has been
63 suggested that sporulation in Bacillus and Clostridium employ similar mechanisms based on
3 64 their morphological similarities, studies have shown that there are some differences in the early
65 stages of sporulation process in these two species [2, 7-9].
66 Clostridium species are Gram-positive, anaerobic, spore-forming prokaryotes including
67 strains of importance to human and animal health (C. tetani, C. perfringens, C. botulinum and C.
68 difficile), cellulose degradation (C. phytofermentans and C. thermocellum), solvent production
69 (C. acrtobutylicum and C. beijerinckii) and strains involved in bioremediation (C. cadavaris).
70 This heterogeneous group of Clostridium is divided into 19 clusters [10]. These strains are
71 widely distributed throughout the world in all sorts of environments, but most likely found in
72 soils and animal intestines in the form of vegetative cells or dormant spores.
73 It has been hypothesized that Bacilli and Clostridia were in the same class until about 2.5
74 billion years ago when the rise of atmospheric oxygen occurred, also known as the ‘great
75 oxidation’ event [1]. Bacilli diverged from the Clostridia as a separate class during that period.
76 After that separation, Bacilli remain as an aerobic spore-former whereas Clostridia persisted as
77 an anaerobic-spore former. Different environmental requirements for the growth of these two
78 classes of bacteria may explain why there are differences in the molecular mechanism of
79 sporulation, especially at the initial stages in sporulation. In contrast, signature sporulation genes
80 are still conserved between these two classes long time after their separation indicating that both
81 had originated from the same origin [11, 12].
82 Despite having importance in the field of human and animal health and physiology,
83 cellulose degradation, solvent production and bioremediation, the molecular events in
84 Clostridium sporulation are not well understood, primarily as a result of limited genetic
85 manipulation. Recent developments in molecular techniques such as, high-throughput genome
86 sequencing, genome-wide transcriptional profiling, and directed or random mutagenesis
4 87 techniques such as group II introns for insertional mutagenesis and transposons for random
88 mutagenesis, have enabled researcher to find out the hints of molecular mechanism leads to the
89 sporulation in Clostridium.
90 In this review, we discuss the recent advancements in the sporulation study on four major
91 Clostridium species including C. acetobutylicum, C. botulinum, C. difficile, and C. perfringens.
92 We discuss how the components of the sporulation process differ between Clostridium than
93 Bacillus species. Also, we compare the sporulation process among Clostridium species.
94
95 2. Stages of sporulation and spore structure
96 The morphological stages for spore formation are similar in all spore-forming bacteria. In
97 every sporulation event, there are two forms present in the cell; the mother cell and the forespore.
98 The total sporulation process can be divided into seven stages (stage I-VII) [13]. The first stage,
99 called stage 0 is actually the growth of vegetative cells before the beginning of sporulation. In
100 stage I and II, the cell DNA releases as an axial filament and the asymmetric cell division results
101 in forming of two compartments, one with smaller prespore compartment and the other is larger
102 mother cell compartment. Initially, one-third of the DNA material is deposited in the prespore,
103 although the rest of the DNA is rapidly pumped into the prespore compartment via the action of
104 the DNA translocase, SpoIIIE. During stage III, the prespore is engulfed by the mother cell and
105 called forespore, which has inner and outer membranes surrounding and floating as a protoplast.
106 In step IV, peptidoglycan (PG) layer synthesizes the primordial germ cell wall and the cortex in
107 the space between inner and outer membranes surrounding the forespore. The outcome of stage
108 V is the formation of the complex structure of proteins known as the spore coat, outside on the
109 surface of the forespore. Despite these changes in the morphological structure of spores, there is
5 110 one more stage to make the newly formed spore more durable. In stage VI, spore’s resistance to
111 UV radiation and heat is established. After going through all these changes, mature spores are
112 liberated from the mother cell into the environment during the stage VII of sporulation.
113 The basic structure of spores and morphological stages are conserved among all spore-
114 forming bacteria. The spore structures contribute to the dormant microorganism to sustain in a
115 variety of environmental stresses like high temperature, pressure, extreme pH, and radiation until
116 the spore finds itself in more favorable condition for vegetative growth. Usually, the bacterial
117 genome is deposited inside the central compartment of the spore surrounded by the lipid bilayer
118 covered with a layer of PG, which is known as the germ cell wall. This germ cell wall also serves
119 as the cell wall of vegetative forms after the completion of spores germination. The Germ cell
120 wall is wrapped in a thick layer of another layer of modified PG, termed the cortex. It is essential
121 for the acquisition and the maintenance of the heat resistance [14, 15]. Finally, this cortex layer
122 is encapsed by a multiprotein coat protecting it from the action of the PG-lytic enzymes. In
123 several species such as B. anthracis, B. cereus and C. difficile, this multiprotein coat is further
124 enclosed by another structure known as the exposporium [16-18]. If present, the exosporium or
125 otherwise the coat serves as the interacting structure of the spore to the environment. The spore’s
126 inner membrane contains the essential components for spore germination, including various
127 germinant receptors, which interacts with small molecules that trigger germination and a return
128 to the vegetative form [14, 19-21].
129
130 3. Initiation of sporulation
131 The full initiation pathway has been identified in the Bacillus species; however, the clear
132 picture in Clostridium has yet to be elucidated. In Bacillus, a multi-component signal
6 133 transduction system termed ‘phosphorelay’ is present [7]. Proper functioning of this system
134 leads to the activation of a master regulator, Spo0A [22] (Fig. 1). At present, no such
135 phosphorylation system has been found in Clostridium species. Another important component
136 for the initiation of sporulation are kinases, termed orphans, because they lack the cognate
137 response regulator [23]. These orphan kinases are involved in receiving different stimuli, both
138 from the extracellular or intracellular and initiate the process of sporulation. In Bacillus, at least
139 five orphan kinases (KinA - KinE) are present, but the KinA and KinB are the most efficient
140 ones. Each of the kinases is able to respond to different stimuli [24].
141 In Bacillus, all orphan kinases autophosphorylate upon receiving the respective signal(s)
142 and transfer the phosphoryl group from their phosphotransferase domain to an aspartate residue
143 in Spo0F, a single domain response regulator [24]. The phosphoryl group is then passed to a
144 histidine residue in a phosphotransferase domain within Spo0B. Finally, Spo0B phosphorylates
145 the key sporulation regulator, Spo0A, that eventually starts the second phase of the sporulation
146 process (Fig. 1). There is no evidence for the presence of Spo0F in any Clostridium species [7].
147 However, a homolog of Spo0B was found in C. tetani, but its function has yet to be known [25].
148 There are more components involved in Bacillus sporulation and those can regulate the
149 phosphorelay system either positively or negatively. An alternative sigma factor, SigH, is a
150 transcription factor and activates Spo0A, Spo0F, KinA and KinE [26]. While Spo0A is activated
151 directly by SigH, Spo0A increases the transcription of spo0H gene encoding SigH by repressing
152 AbrB, a global repressor. In contrast to Bacillus, Clostridium spo0H is constitutively expressed
153 at a low level, without any sign of increase at the onset of spore formation [9].
154 In total, thirty-nine histdine kinases have been found in B. subtilis, of which nine are
155 orphans [7, 24]. Among five sporulation specific histidine kinases, only three have been found to
7 156 contain transmembrane domains [27], which means those three might response to the
157 environmental signal. In an earlier review, Paredes et al [2] described the presence of putative
158 orphan histidine kinases in three Clostridium species; C. acetobutylicum, C. perfringens, and C.
159 tetani based on the completed genome sequences of Clostridium species at that time. These
160 authors used a BLAST- based approach to identify 35 kinases in C. acetobutylicum and six of
161 them are orphans (Table 1). Another study identified two different pathways for sporulation
162 initiation in C. acetobutylicum [28], and by insertional inactivation of kinase genes (single and
163 double mutants), they showed that CAC0903, CAC3319, and CAC0323 directly activate Spo0A.
164 Our group then extended the search for putative orphan histidine kinases in other
165 Clostridium species. We followed the strategy of Paredes et al [2], to find out the orphan
166 histidine kinases from the Clostridium genomes in NCBI database. In C. perfringens Type-A
167 food poisoning strain SM101, six putative orphan histidine kinases (ORFs CPR1953, CPR1493,
168 CPR1316, CPR0195, CPR1055, and CPR1728) were identified based on the BLASTP analyses
169 with kinases from B. subtilis. Knock-out mutations in two of these (CPR1055, and CPR1728)
170 showed sporulation and germination defects suggesting their putative role in activating the
171 Spo0A by phosphorylation (P. Udompijitkul and M. R. Sarker, unpublished).
172 The phosphorelay system might explain why Bacillus is more environmentally versatile
173 than Clostridium. In the process of evolution, Bacillus adopted themselves to the environmental
174 changes by adding different functional genes for reacting to multiple signals. The absence of a
175 phosphorelay system leaves remaining questions about how Clostridia initiate sporulation. One
176 hypothesis is that, Clostridium Spo0A is activated by direct phosphorylation from the orphan
177 histidine kinases. Another possibility is that there might be a different unknown phosphorelay
178 system present in Clostridium.
8 179
180 4. The master regulator, Spo0A
181 The most widely studied and functionally characterized component of the sporulation
182 machinery in spore-forming bacteria is the key regulator, Spo0A. It is a transcription factor that
183 controls the transition of the bacterium into the spore form [2, 26]. The structure and function of
184 Spo0A protein has been mostly studied in Bacillus species. In that system, Spo0A is activated by
185 Spo0B [29-31]. When the number of phosphorylated Spo0A reaches a threshold [32], the protein
186 binds directly to specific DNA sequences (TGNCGAA) upstream of several early sporulation
187 genes and thus activates the sporulation process [33-35]. This particular DNA sequence is known
188 as ‘0A box’ or the binding site for Spo0A. The structure of Spo0A is critical for its subsequent
189 changes from de-phosphorylation to phosphorylation state and vice-versa. Spo0A has two
190 functional domains, the N-terminal phosphorylation and dimerization domain (receiver), and the
191 C-terminal DNA binding (effector) domain. These two domains are separated by a hinge region
192 [36]. Phosphorylation leads to structural rearrangement that facilitates Spo0A dimerization [37,
193 38], resulting in the disruption of transcription-inhibitory contacts between the receiver and
194 effector domains. This disruption leads to the binding of DNA binding domains to the Spo0A
195 protein. The crystal structure of the DNA binding domain confirms specific and non-specific
196 contacts between Spo0A protein and the consensus sequence [38, 39]. Upon activation, Bacillus
197 Spo0A directly activates 121 genes, including genes required for polar septum formation [34].
198 Spo0A is conserved between both Bacillus and Clostridium [40, 41]. The inactivation of
199 spo0A in several Clostridium species resulted in the blocking of sporulation and synthesis of
200 sporulation-specific sigma factors [9, 42-44]. Transcriptomic and proteomic analyses identified
201 C. dificile Spo0A as a global regulator that regulates metabolic and virulence factors outside the
9 202 sporulation process [45]. Similarly as in B. subtilis [46], Spo0A has a role in biofilm formation in
203 C. difficile [47, 48] and C. perfringens [49].
204 One additional function of Spo0A is to regulate different Clostridial toxins [42, 50].
205 Recent works have suggested that it may be responsible for the regulation of toxin A and toxin B
206 production by C. difficile [35, 43, 51, 52]. Study demonstrated that Spo0A positively regulates
207 toxin A and toxin B expression. The spo0A mutant in an erythromycin mutant strain of C.
208 difficile, C. difficile strain 630 delta erm resulted in the productions of toxins [43]. However,
209 another study showed a contradictory result; inactivation of spo0A in 630 delta erm strain had no
210 affect on the toxin production [35]. Deakin et al [51] reported that a C. difficile R20291 spo0A
211 mutant caused more severe disease in a murine model than the wild type strain. Recently, one
212 study has shown the various affects of Spo0A on the toxin production in different C. difficile
213 strains [52]. Spo0A has been implicated in virulence in mice models [43, 51]. In C.
214 acetobutylicum, Spo0A as well as sporulation affect the solvent production [53].
215
216 5. Sigma (σ) factors
217 After the initial trigger of spore formation, the cell transitions through different
218 morphological stages to form mature spores, facilitated by the contribution of different σ factors.
219 These are the dissociable RNA polymerase subunits that alter the promoter specificity of the
220 RNA polymerase complex under different environmental and growth phase-dependent
221 conditions [26]. Four sporulation-specific σ factors were first identified in B. subtilis [13, 54-56].
222 These σ factors are compartment specific; σ factor F (σF) and G (σG) are forespore specific and
223 regulated by anti- σ factors and anti-anti- σ factors. On the other hand, σ factor E (σE) and K (σK)
224 are mother cell specific, synthesized as precursor protein, which needs to be cleaved for the
10 225 activation. Generally, σF is the first σ factor appeared in the sporulation process by controlling
226 the early stages of the forespore followed by σE in the mother cells. Later, σG and σK have their
227 actions in the formation of mature spores and vegetative cells, respectively (Fig. 1). The
228 sequence similarities to all four σ factors have been identified in C. acetobutylicum [57] and
229 confirmed by PCR based approach [41]. Similar results have also been observed in other
230 Clostridium species; homologs of all four σ factors have been found in C. perfringens [58, 59].
231 This has been confirmed by transcriptional and protein analyses [60, 61].
232
233 5.1 σF
234 In B. subtilis, the RNA polymerase σF is synthesized prior to the formation of polar
235 septum and held inactive until septation is completed [26]. A similar pattern has been found in
236 few Clostridium species (Fig. 1). In B. subtilis, σF encoding gene, spoIIAC, is transcribed by σH
237 associated RNA polymerase during the initiation of sporulation [62, 63]. σF remains inactive in
238 the pre-divisional cell by binding with anti- σ factor SpoIIB, until it is relieved by the anti-anti- σ
239 factor SpoIIA. The functionality of SpoIIA becomes inactive by the phosphorylation with
240 SpoIIAB, which is a kinase and anti- σ factor. In contrast, SpoIIA becomes active by the
241 dephosphorylation with a phosphatase, SpoIIE. The non-phosphorylated SpoIIA interacts with
242 the SpoIIB- σF complex and displace the σF. Released σF becomes active and directs gene
243 expression via the control of different genes. The role of σF in sporulation of different
244 Clostridium species has been demonstrated by gene-knock-out studies. Clostridium mutants
245 lacking σF completely blocked for sporulation and this defect could be restored to nearly wild-
246 type levels by complementation with wild-type sigF gene, indicating that σF is essential for spore
247 formation in these organisms [61, 64, 65].
11 248
249 5.2 σE
250 In both Bacillus and Clostridium species, σE is synthesized as an inactive precursor
251 protein (pro- σE) (Fig. 1), which is activated by the proteolysis event by the protease activity of
252 SpoIIGA [66, 67]. Pro- σE is transcribed from spoIIG operon, in which sigE (earlier named as
253 spoIIGB), is the second gene in the operon, initiated transcription before asymmetric septation
254 and continues after septum formation, only in the mother cell [68]. This indicates that the σE is
255 produced even after the septum formation. The expression of spoIIGA, the first gene of the
256 spoIIG operon, requires the σF -controlled SpoIIR protein. This indicates that the mother-cell-
257 specific σE is controlled by the forespore-specific σF -directed gene transcription and the presence
258 of an intercellular gene transcription pathway [26, 64]. spoIIGA expression is also controlled by
259 the Spo0A after asymmetric division [26, 69]. Like σF, σE mutant strains in C. perfringens and C.
260 difficile showed sporulation defects in sporulation-inducing conditions [60, 64]. C.
261 acetobutylicum σE mutant strain also blocks the sporulation before the asymmetric division [70].
262
263 5.3 σG
264 In B. subtilis, σG is synthesized in the pre-engulfment prespore, but is not activated until
265 the end of stage III (complete engulfment of prespore by mother cell). Transcription of sigG
266 (earlier named spoIIG) is dependent on σF [26, 71]. The products of spoIIIA and spoIIIJ are
267 needed for the release of σG from inhibition in the forespore compartment. spoIIIA is selectively
268 expressed in the mother cell under σE control, whereas spoIIIJ is expressed in the forespore [26,
269 71]. Mutations in sigG blocks the sporulation in C. perfringens and C. difficile [61, 64]. In C.
270 acetobutylicum, sigG mutant halted the sporulation during the maturation stage [70].
12 271
272 5.4 σK
273 σK is known as the late stage (stage IV) mother cell specific σ factor. It regulates spore
274 coat formation during the late-stage of sporulation [72, 73]. An intervening element of 42-kb
275 size, named skin (sigma K intervening) separates sigK into two parts in some Bacillus species
276 [74]. Both halves are required, as mutations in either halves results in the failure of spore
277 formation at late stage [75]. The absence of the skin element does not affect the growth or
278 sporulation suggesting that this element does not contain any genes essential for survival [74].
279 Inside the mother cell, the skin undergoes site-specific recombination to form sigK. Because it
280 has no visible role in survival, it has been assumed that skin is a rudimentary element left from
281 the ancestors [76]. Interestingly, this skin element has been found in C. difficile, and unlike B.
282 subtilis, it is important for sporulation [77]. Although a 47-kb skin element, named skinCt has
283 been identified in C. tetani [78], other known Clostridium genomes do not contain this element
284 and have an uninterrupted sigK.
285 The homolog of Bacillus σK has been found in the genomes of C. botulinum [79] and C.
286 perfringens [58] and shown to be essential for early stage of sporulation [60, 80] (Fig. 1). Krik et
287 al [80] constructed two sigK mutants in C. botulinum and demonstrated that σK also acts in the
288 early stage of sporulation and plays as a transcriptional activator of Spo0A. σF transcript is also
289 lower in σK muatnts. Also, σK was shown to have role in different stress tolerance such as cold
290 and osmotic stress in C. botulinum ATCC3502 strain [81].
291
292 5.5 Regulation of sigma factors in Clostridium
13 293 Among the Clostridium species, the most studied organism for σ factor expression during
294 sporulation is C. acetobutylicum [9, 41, 82]. In this species, the pattern of σ factor expression and
295 solventogenesis for SpoIIA, σE, σG, and σK matched that in B. subtilis [9], although it was spread
296 over a much longer time (35 h) than that seen in B. subtilis (8 h). Some σ factors have two
297 separate developmental roles during sporulation. For example, in C. acetobutylicum, σK acts both
298 early, even prior to Spo0A expression, and late stages of sporulation, past of σG activation [83].
299 The expression and regulation of all four σ factors in C. perfringens has been evaluated in
300 two separate studies. By introducing mutations in sigE and sigK genes of C. perfringens SM101,
301 Harry et al [60] discovered some differences in expressions and functions of these σ factors
302 during the sporulation of C. perfringens SM101 versus B. subtilis (Fig. 1). 1) Unlike B. subtilis,
303 where σK is the last σ factor expressed during sporulation, normal production of sigF and sigE
304 transcripts in sporulating SM101 cells is dependent on sigK. 2) sigF transcript production was
305 delayed in a SM101 sigE-mutant, but sigF is the first transcript in B. subtilis. 3) sigG transcripts
306 were detected in SM101-sigE and -sigK mutants while sigG transcription in B. subtilis requires
307 both σE and σF. 4) Transcripts of all four σ factor genes were detected much earlier in C.
308 perfringens SM101 than that reported for B. subtilis. Finally, unlike B. subtilis, where expression
309 of spoIIID (a key mother cell specific transcription factor) requires σE-associated RNA
310 polymerase, the transcription and translation of spoIIID in C. perfringens does not require either
311 σK or σE.
312 But these findings were questioned by another study conducted by Li and McClane [61].
313 To investigate the role of σF and σG in sporulation and CPE production in C. perfringens SM101
314 strain, they constructed isogenic sigF and sigG null mutants and corresponding complemented
315 strains. By the Western blot analyses, they showed that there were little or no production of σG
14 316 σE, and σK in sigF mutant, which were completely restored in complementing strain [61]. These
317 findings suggest that regulation of σ-factors in C. perfringens is similar to B. subtilis.
318 In 2013, three extensive studies [64, 84, 85] were conducted on expression and regulation
319 of σ factors in C. difficile. Saujet et al [85] identified 225 genes under the regulation of σ factors
320 by genome-wide transcriptional analyses and promoter mapping. In a separate study, Fimlaid et
321 al [84] has found almost the same number of genes (224) regulated by the sporulation specific σ
322 factors. Pereira et al showed the intra-regulation of σ factors by disrupting each of the σ genes
323 [64]. Collectively, all these studies revealed that mother-cell-specific σ factors, σE and σK are not
324 regulated by forespore-specific σF and σG, respectively [64, 84, 85]. Also, σG is not dependent on
325 σE [64, 84].
326 When comparing the normalized reference genes, Kirk et al [86] demonstrated the
327 expressions of four σ factors in C. botulinum ATCC3502 strain. Expressions of sigF, sigE, and
328 sigG were simultaneously high at the end of the exponential growth phase. Although very low
329 sigK expression was detected during the exponential phase, this expression was peaked after 18
330 h, means very late during the stationary phase.
331
332 5.6 Role of σ factors in toxin production
333 In addition to regulation of sporulation process, σ factors control the expression and
334 production of some clostridial toxins. First example of sporulation and/or σ factors regulated
335 toxin is CPE, an essential virulence factor for C. perfringens pathogenesis [87]. Sporulation
336 controls the expression of CPE encoding gene (cpe) at the transcriptional level, as both northern
337 blot and reporter construct studies detected cpe transcription in sporulating, but not in vegetative
338 cultures [88, 89]. The transcription of cpe is dependent on three promoters: promoter 1 (P1) was
15 339 proposed to be σK-dependent, while promoter 2 (P2) and promoter 3 (P3) σE-dependent based on
340 the consensus recognition sequences [89]. The presence of these strong promoters probably
341 explains why C. perfringens type A isolates produce such high levels of CPE during sporulation.
342 Since putative σK -and σE-dependent promoters had been identified upstream of the cpe [89],
343 Harry et al [60] constructed sigK and sigE mutants of C. perfringens SM101 and evaluated the
344 importance of σK and σE in cpe expression. Both mutants failed to produce β-glucuronidase when
345 transformed with a recombinant plasmid carrying the cpe promoter fused to Escherichia coli β-
346 glucuronidase gene gusA [60], indicating that cpe expression is dependent on σK and σE. In a
347 follow-up study, Li and McClane [61] demonstrated that cpe transcription and CPE production
348 are blocked in an SM101 sigF-mutant, however, normal levels of cpe transcription and CPE
349 production were observed in sporulating SM101 sigG-mutant cultures. Collectively, findings
350 from these two studies showed that only σE, σF, and σK are needed for CPE synthesis.
351 The second sporulation-regulated C. perfringens toxin is TpeL, which belongs to the
352 family of large clostridial toxins. No expression of tpeL-gusA fusion was observed in SM101
353 spo0A-or sigE-mutant, indicating that tpeL expression is dependent on the master regulator of
354 sporulation, Spo0A, and the sporulation-specific σ factor, σE [50].
355 A recent study has identified a repressor protein named VirX, which significantly
356 inhibited the sporulation and CPE production in C. perfringens SM101 strain [90]. The higher
357 levels of cpe transcription and CPE production were observed in SM101 virX-mutant compared
358 to wild-type. Also, the transcription level of sigE, sigF and sigK was strongly induced at 2.5 h of
359 culture of the virX mutant, suggesting that VirX negatively regulates the transcription of cpe and
360 production of CPE through the sporulation-specific σ factors [90].
361
16 362 6. Other factors in Clostridium sporulation
363 Sporulation is regulated by bacterial cell density and quorum sensing. The homologues of
364 AgrB and AgrD of the well-studied Staphylococcus aureus Agr quorum sensing system have
365 been found in a few Clostridium species [91-93]. Mutations in both putative agrB and agrD in C.
366 sporogenes and C. botulinum result in the reduction of sporulation as well as toxin production
367 [93]. Agr-dependent quorum sensing is also involved in the regulation of sporulation and
368 granulose formation in C. acetobutylicum [92]. Sporulation and CPE production in C.
369 perfringens also seems to be positively regulated by Agr-like quorum-sensing (QS) system [91].
370
371 7. New sporulation genes in Clostridium
372 Sporulation studies are mainly focused on two important spore-forming genera; Bacillus
373 (aerobic) and Clostridium (anaerobic). Moreover, other organisms resembling both aerobic and
374 anaerobic within the firmicutes have a sporulating nature, although the differences in
375 morphology and life style are widely spread. Despite the universal presence of master regulator,
376 Spo0A, and the four cell-type specific σ factors in all spore-forming bacteria, there are number of
377 other genes conserved in all spore-forming bacteria. Phylogenetic analysis with all spore-forming
378 bacteria reveals the core sporulation genes.
379 Both forward and reverse genetics have been applied to find out the genes directly under
380 the control of sporulation factors. In this aspect, again Bacillus is the model strain to find out the
381 sporulation associated genes. Approaches like identification of sporulation specific proteins such
382 as small acid-soluble proteins (SASPs), and transcriptional analyses revealed new sporulation
383 genes [94, 95]. Using phylogenetic analysis, studies found 111 core genes that are highly
384 conserved among the spore-forming bacteria [12]. Spo0A directly regulates the expression of
17 385 121 genes in Bacillus [34] and significantly enhances the expression of over 500 genes [96].
386 Tragg et al [97] analyzed the presence of all gene products in B. subtilis subsp. subtilis 168 with
387 a total of 626 bacterial genomes including 46 genomes of endospore-formers and found that
388 fifty-eight genes are highly enriched among spore-forming bacteria. Eight of the previously
389 unidentified putative sporulation genes in Bacillus species were inactivated and found to have
390 roles in sporulation in Bacillus [97]. The presence of these newly identified sporulation genes
391 were also found in C. perfringens SM101 strain and mutational analyses demonstrated that ylmC
392 and bkdR mutant showed sporulation defect under spore-forming conditions (P. K. Talukdar and
393 M. R. Sarker, unpublished data). In our study, we extended the search of putative sporulation
394 genes in more Clostridium species. We have selected seven proteins (BkdR, CwlD, DapG, YlxY,
395 YlyA, YlzA and Yqhq) from the pool of 127 proteins identified in B. subtilis [12, 97] because: 1)
396 these are conserved among spore formers (mostly Bacillus and Clostridium strains) and mostly
397 not found in other non-spore-forming bacteria, and 2) role of these proteins yet to be determined.
398 The distribution of these seven putative sporulation proteins in different Clostridium species are
399 shown in Table 2.
400
401 8. Spore components involved in spore resistance
402 Different components of spore structure can protect spores from different physical and
403 chemical stresses. For example, the spore coat and the relatively impermeable spore inner
404 membrane contribute in the spore resistance. Spore core’s low water content, and high levels of
405 dipicolinic acid and associated divalent cations are other important factors involved in spore
406 resistance [98, 99]. Spore’s DNA in the core is protected by sporulation-specific proteins named
407 as α/β-type small acid soluble proteins (SASPs). These proteins bind to the DNA and alter its
18 408 chemical and photochemical reactivity, which are important to protect DNA from heat, many
409 genotoxic chemicals, and UV radiation [5, 100-102]. These SASP proteins are highly conserved
410 in both Bacillus and Clostridium species and appear at the same time in sporulation in both genus
411 [103]. Three SASP genes (ssp1, ssp2, and ssp3) have been identified in different C. perfringens
412 food poisoning (FP) and non-food-borne strains [103, 104]. Gene knock-out studies
413 demonstrated that α/β-type SASPs play a major role in mediating resistance of C. perfringens
414 spores to UV radiation, moist heat and chemicals [103, 105], but not to dry heat [103]. C.
415 perfringens ssp2 was expressed in B. subtilis spores lacking one or both major α/β-type SASP
416 and restored the resistance of α-β- spores to UV and nitrous acid and of α- spores to dry heat,
417 indicate the interchangeability of α/β-type SASP in DNA protection in spores [106]. However,
418 similar levels of SASPs production by FP and NFB strains [104] could not explain the reason
419 why spores of NFB isolates exhibit lower heat resistance than spores of FP isolates [107]. To
420 this end, Li and McClane identified a new SASP protein, named Ssp4, in C. perfringens FP and
421 NFB strains [108] and showed that a single amino acid substitution at Ssp4 residue 36 is critical;
422 a glycine at 36 residue of Ssp4 in NFB isolate is responsible for mediating spores’ heat
423 sensitivity, while an asparagine at the same site in Ssp4 of FP isolate for spores’ heat resistance
424 [108].
425
426 9. Concluding remarks and future perspective
427 Sporulation is the unique survival strategy for bacterial cell besides other strategies like
428 quorum sensing or biofilm formation. To date, most of the knowledge on sporulation has been
429 gathered from the genus, Bacillus. In contrast, information about sporulation in Clostridium
430 species is still scarce and insufficient to allow a full understanding of the complete regulatory
19 431 circuit of sporulation. Although the early stage of Clostridium sporulation is vastly different from
432 that in Bacillus because of the absence of phosphorelay system, the major regulator for
433 sporulation, Spo0A and the downstream signaling systems are mostly conserved. Studies done
434 on four industrial and pathogenic Clostridium species revealed the intra generic differences in
435 the regulation of sporulation. For understanding the full mechanism of Clostridium sporulation,
436 we have to answer the following questions:
437 1) Is there any phosphorelay system present in Clostridium species?
438 2) If not, how many kinase genes are present and how do they regulate the activation of
439 Spo0A?
440 3) Are these kinases regulating each other or independently working on Spo0A?
441 4) What are the environmental signals triggering sporulation in Clostridium?
442 5) Do kinases environmental signal-specific?
443 Clostridium genetic manipulation is much harder than in Bacillus species. But with the invention
444 of newer molecular tools like TargeTron/ClosTron for genetic manipulation, a wealth of new
445 information on Clostridium sporulation has been gathered. This new information with the
446 comparison with other sporulation mechanism, one can reveal the intrinsic mechanism of
447 sporulation.
448
449 Acknowledgements
450 This work was supported by a grant from the Agricultural Research Foundation of
451 Oregon State University, and by a Department of Defense Multidisciplinary University Research
452 Initiative (MURI) award through the U.S. Army Research Laboratory and the U. S. Army
453 Research Office under contract number W911NF-09-1-0286 (all to M.R.S); and by grants from
20 454 Fondo Nacional de Ciencia y Tecnología de Chile (FONDECYT Grant 1110569), by a grant
455 from the Research Office of Universidad Andres Bello (DI-275-R/13), and by a grant from
456 Fondo de Fomento al Desarrollo Científico y Tecnológico (FONDEF) CA13I10077 to D.P-S.
457 MA was supported by the Ministry of Higher Education in Saudi Arabia. We thank Dr. Daniel
458 D. Rockey for his editorial help.
459
460 461 462 463 464 465 466 467
468
469
470
471
472
473
474
475
476
477
478
21 479 References
480 [1] Battistuzzi FU, Feijao A, Hedges SB, A genomic timescale of prokaryote evolution: insights
481 into the origin of methanogenesis, phototrophy, and the colonization of land. BMC Evol Biol
482 2004;4:44.
483 [2] Paredes CJ, Alsaker KV, Papoutsakis ET, A comparative genomic view of clostridial
484 sporulation and physiology. Nat Rev Microbiol 2005;3:969-978.
485 [3] Higgins D, Dworkin J, Recent progress in Bacillus subtilis sporulation. FEMS Microbiol Rev
486 2012;36:131-148.
487 [4] Nicholson WL, Munakata N, Horneck G, Melosh HJ, Setlow P, Resistance of Bacillus
488 endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev
489 2000;64:548-572.
490 [5] Setlow P, Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and
491 chemicals. J Appl Microbiol 2006;101:514-525.
492 [6] Viswanathan VK, Mallozzi MJ, Vedantam G, Clostridium difficile infection: An overview of
493 the disease and its pathogenesis, epidemiology and interventions. Gut microbes 2010;1:234-242.
494 [7] Stephenson K, Hoch JA, Evolution of signalling in the sporulation phosphorelay. Mol
495 Microbiol 2002;46:297-304.
496 [8] Durre P, Ancestral sporulation initiation. Mol Microbiol 2011;80:584-587.
497 [9] Durre P, Hollergschwandner C, Initiation of endospore formation in Clostridium
498 acetobutylicum. Anaerobe 2004;10:69-74.
499 [10] Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J, Garcia P, et al.,
500 The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species
501 combinations. Int J Syst Bacteriol 1994;44:812-826.
22 502 [11] Galperin MY, Mekhedov SL, Puigbo P, Smirnov S, Wolf YI, Rigden DJ, Genomic
503 determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-
504 specific genes. Environ Microbiol 2012;14:2870-2890.
505 [12] Abecasis AB, Serrano M, Alves R, Quintais L, Pereira-Leal JB, Henriques AO, A genomic
506 signature and the identification of new sporulation genes. J Bacteriol 2013;195:2101-2115.
507 [13] Hilbert DW, Piggot PJ, Compartmentalization of gene expression during Bacillus subtilis
508 spore formation. Microbiol Mol Biol Rev 2004;68:234-262.
509 [14] Henriques AO, Moran CP, Jr., Structure, assembly, and function of the spore surface layers.
510 Annu Rev Microbiol 2007;61:555-588.
511 [15] McKenney PT, Driks A, Eichenberger P, The Bacillus subtilis endospore: assembly and
512 functions of the multilayered coat. Nat Rev Microbiol 2013;11:33-44.
513 [16] Ball DA, Taylor R, Todd SJ, Redmond C, Couture-Tosi E, Sylvestre P, et al., Structure of
514 the exosporium and sublayers of spores of the Bacillus cereus family revealed by electron
515 crystallography. Mol Microbiol 2008;68:947-958.
516 [17] Kailas L, Terry C, Abbott N, Taylor R, Mullin N, Tzokov SB, et al., Surface architecture of
517 endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale. Proc
518 Natl Acad Sci U S A 2011;108:16014-16019.
519 [18] Permpoonpattana P, Tolls EH, Nadem R, Tan S, Brisson A, Cutting SM, Surface layers of
520 Clostridium difficile endospores. J Bacteriol 2011;193:6461-6470.
521 [19] Banawas S, Paredes-Sabja D, Korza G, Li Y, Hao B, Setlow P, et al., The Clostridium
522 perfringens germinant receptor protein GerKC is located in the spore inner membrane and is
523 crucial for spore germination. J Bacteriol 2013;195:5084-5091.
23 524 [20] Paredes-Sabja D, Setlow P, Sarker MR, Germination of spores of Bacillales and
525 Clostridiales species: mechanisms and proteins involved. Trends in microbiology 2011;19:85-94.
526 [21] Setlow P, Germination of spores of Bacillus species: what we know and do not know. J
527 Bacteriol 2014;196:1297-1305.
528 [22] Burbulys D, Trach KA, Hoch JA, Initiation of sporulation in B. subtilis is controlled by a
529 multicomponent phosphorelay. Cell 1991;64:545-552.
530 [23] Fabret C, Feher VA, Hoch JA, Two-component signal transduction in Bacillus subtilis: how
531 one organism sees its world. J Bacteriol 1999;181:1975-1983.
532 [24] Jiang M, Shao W, Perego M, Hoch JA, Multiple histidine kinases regulate entry into
533 stationary phase and sporulation in Bacillus subtilis. Mol Microbiol 2000;38:535-542.
534 [25] Stephenson K, Lewis RJ, Molecular insights into the initiation of sporulation in Gram-
535 positive bacteria: new technologies for an old phenomenon. FEMS Microbiol Rev 2005;29:281-
536 301.
537 [26] Errington J, Regulation of endospore formation in Bacillus subtilis. Nat Rev Microbiol
538 2003;1:117-126.
539 [27] Krogh A, Larsson B, von Heijne G, Sonnhammer EL, Predicting transmembrane protein
540 topology with a hidden Markov model: application to complete genomes. J Mol Biol
541 2001;305:567-580.
542 [28] Steiner E, Dago AE, Young DI, Heap JT, Minton NP, Hoch JA, et al., Multiple orphan
543 histidine kinases interact directly with Spo0A to control the initiation of endospore formation in
544 Clostridium acetobutylicum. Mol Microbiol 2011;80:641-654.
24 545 [29] Baldus JM, Green BD, Youngman P, Moran CP, Jr., Phosphorylation of Bacillus subtilis
546 transcription factor Spo0A stimulates transcription from the spoIIG promoter by enhancing
547 binding to weak 0A boxes. J Bacteriol 1994;176:296-306.
548 [30] Bird TH, Grimsley JK, Hoch JA, Spiegelman GB, Phosphorylation of Spo0A activates its
549 stimulation of in vitro transcription from the Bacillus subtilis spoIIG operon. Mol Microbiol
550 1993;9:741-749.
551 [31] Ferrari FA, Trach K, LeCoq D, Spence J, Ferrari E, Hoch JA, Characterization of the spo0A
552 locus and its deduced product. Proc Natl Acad Sci U S A 1985;82:2647-2651.
553 [32] Fujita M, Gonzalez-Pastor JE, Losick R, High- and low-threshold genes in the Spo0A
554 regulon of Bacillus subtilis. J Bacteriol 2005;187:1357-1368.
555 [33] Liu J, Tan K, Stormo GD, Computational identification of the Spo0A-phosphate regulon
556 that is essential for the cellular differentiation and development in Gram-positive spore-forming
557 bacteria. Nucleic Acids Res 2003;31:6891-6903.
558 [34] Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, Liu JS, et al., The
559 Spo0A regulon of Bacillus subtilis. Mol Microbiol 2003;50:1683-1701.
560 [35] Rosenbusch KE, Bakker D, Kuijper EJ, Smits WK, C. difficile 630Deltaerm Spo0A
561 regulates sporulation, but does not contribute to toxin production, by direct high-affinity binding
562 to target DNA. PLoS One 2012;7:e48608.
563 [36] Grimsley JK, Tjalkens RB, Strauch MA, Bird TH, Spiegelman GB, Hostomsky Z, et al.,
564 Subunit composition and domain structure of the Spo0A sporulation transcription factor of
565 Bacillus subtilis. J Biol Chem 1994;269:16977-16982.
25 566 [37] Lewis RJ, Scott DJ, Brannigan JA, Ladds JC, Cervin MA, Spiegelman GB, et al., Dimer
567 formation and transcription activation in the sporulation response regulator Spo0A. J Mol Biol
568 2002;316:235-245.
569 [38] Muchova K, Lewis RJ, Perecko D, Brannigan JA, Ladds JC, Leech A, et al., Dimer-induced
570 signal propagation in Spo0A. Mol Microbiol 2004;53:829-842.
571 [39] Zhao H, Msadek T, Zapf J, Madhusudan, Hoch JA, Varughese KI, DNA complexed
572 structure of the key transcription factor initiating development in sporulating bacteria. Structure
573 2002;10:1041-1050.
574 [40] Brown DP, Ganova-Raeva L, Green BD, Wilkinson SR, Young M, Youngman P,
575 Characterization of spo0A homologues in diverse Bacillus and Clostridium species identifies a
576 probable DNA-binding domain. Mol Microbiol 1994;14:411-426.
577 [41] Sauer U, Treuner A, Buchholz M, Santangelo JD, Durre P, Sporulation and primary sigma
578 factor homologous genes in Clostridium acetobutylicum. J Bacteriol 1994;176:6572-6582.
579 [42] Huang IH, Waters M, Grau RR, Sarker MR, Disruption of the gene (spo0A) encoding
580 sporulation transcription factor blocks endospore formation and enterotoxin production in
581 enterotoxigenic Clostridium perfringens type A. FEMS Microbiol Lett 2004;233:233-240.
582 [43] Underwood S, Guan S, Vijayasubhash V, Baines SD, Graham L, Lewis RJ, et al.,
583 Characterization of the sporulation initiation pathway of Clostridium difficile and its role in toxin
584 production. J Bacteriol 2009;191:7296-7305.
585 [44] Harris LM, Welker NE, Papoutsakis ET, Northern, morphological, and fermentation
586 analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824. J
587 Bacteriol 2002;184:3586-3597.
26 588 [45] Pettit LJ, Browne HP, Yu L, Smits WK, Fagan RP, Barquist L, et al., Functional genomics
589 reveals that Clostridium difficile Spo0A coordinates sporulation, virulence and metabolism.
590 BMC genomics 2014;15:160.
591 [46] Hamon MA, Lazazzera BA, The sporulation transcription factor Spo0A is required for
592 biofilm development in Bacillus subtilis. Mol Microbiol 2001;42:1199-1209.
593 [47] Dawson LF, Valiente E, Faulds-Pain A, Donahue EH, Wren BW, Characterisation of
594 Clostridium difficile biofilm formation, a role for Spo0A. PLoS One 2012;7:e50527.
595 [48] Ethapa T, Leuzzi R, Ng YK, Baban ST, Adamo R, Kuehne SA, et al., Multiple factors
596 modulate biofilm formation by the anaerobic pathogen Clostridium difficile. J Bacteriol
597 2013;195:545-555.
598 [49] Obana N, Nakamura K, Nomura N, A sporulation factor is involved in the morphological
599 change of Clostridium perfringens biofilms in response to temperature. J Bacteriol
600 2014;196:1540-1550.
601 [50] Paredes-Sabja D, Sarker N, Sarker MR, Clostridium perfringens tpeL is expressed during
602 sporulation. Microb Pathog 2011;51:384-388.
603 [51] Deakin LJ, Clare S, Fagan RP, Dawson LF, Pickard DJ, West MR, et al., The Clostridium
604 difficile spo0A gene is a persistence and transmission factor. Infect Immun 2012;80:2704-2711.
605 [52] Mackin KE, Carter GP, Howarth P, Rood JI, Lyras D, Spo0A Differentially Regulates
606 Toxin Production in Evolutionarily Diverse Strains of Clostridium difficile. PLoS One
607 2013;8:e79666.
608 [53] Ravagnani A, Jennert KC, Steiner E, Grunberg R, Jefferies JR, Wilkinson SR, et al., Spo0A
609 directly controls the switch from acid to solvent production in solvent-forming clostridia. Mol
610 Microbiol 2000;37:1172-1185.
27 611 [54] Haldenwang WG, The sigma factors of Bacillus subtilis. Microbiol Rev 1995;59:1-30.
612 [55] Kroos L, The Bacillus and Myxococcus developmental networks and their transcriptional
613 regulators. Annu Rev Genet 2007;41:13-39.
614 [56] Losick R, Stragier P, Crisscross regulation of cell-type-specific gene expression during
615 development in B. subtilis. Nature 1992;355:601-604.
616 [57] Wong J, Sass C, Bennett GN, Sequence and arrangement of genes encoding sigma factors in
617 Clostridium acetobutylicum ATCC 824. Gene 1995;153:89-92.
618 [58] Myers GS, Rasko DA, Cheung JK, Ravel J, Seshadri R, DeBoy RT, et al., Skewed genomic
619 variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens. Genome Res
620 2006;16:1031-1040.
621 [59] Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, Shiba T, et al., Complete
622 genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci U S
623 A 2002;99:996-1001.
624 [60] Harry KH, Zhou R, Kroos L, Melville SB, Sporulation and enterotoxin (CPE) synthesis are
625 controlled by the sporulation-specific sigma factors SigE and SigK in Clostridium perfringens. J
626 Bacteriol 2009;191:2728-2742.
627 [61] Li J, McClane BA, Evaluating the involvement of alternative sigma factors SigF and SigG
628 in Clostridium perfringens sporulation and enterotoxin synthesis. Infect Immun 2010;78:4286-
629 4293.
630 [62] Gholamhoseinian A, Piggot PJ, Timing of spoII gene expression relative to septum
631 formation during sporulation of Bacillus subtilis. J Bacteriol 1989;171:5747-5749.
632 [63] Wu JJ, Howard MG, Piggot PJ, Regulation of transcription of the Bacillus subtilis spoIIA
633 locus. J Bacteriol 1989;171:692-698.
28 634 [64] Pereira FC, Saujet L, Tome AR, Serrano M, Monot M, Couture-Tosi E, et al., The spore
635 differentiation pathway in the enteric pathogen Clostridium difficile. PLoS Genet
636 2013;9:e1003782.
637 [65] Jones SW, Tracy BP, Gaida SM, Papoutsakis ET, Inactivation of σF in Clostridium
638 acetobutylicum ATCC 824 blocks sporulation prior to asymmetric division and abolishes σE and
639 σG protein expression but does not block solvent formation. J Bacteriol 2011;193:2429-2440.
640 [66] Jonas RM, Weaver EA, Kenney TJ, Moran CP, Jr., Haldenwang WG, The Bacillus subtilis
641 spoIIG operon encodes both σE and a gene necessary for σE activation. J Bacteriol
642 1988;170:507-511.
643 [67] Kenney TJ, Moran CP, Jr., Organization and regulation of an operon that encodes a
644 sporulation-essential sigma factor in Bacillus subtilis. J Bacteriol 1987;169:3329-3339.
645 [68] Fujita M, Losick R, An investigation into the compartmentalization of the sporulation
646 transcription factor σE in Bacillus subtilis. Mol Microbiol 2002;43:27-38.
647 [69] Fujita M, Losick R, The master regulator for entry into sporulation in Bacillus subtilis
648 becomes a cell-specific transcription factor after asymmetric division. Genes Dev 2003;17:1166-
649 1174.
650 [70] Tracy BP, Jones SW, Papoutsakis ET, Inactivation of σE and σG in Clostridium
651 acetobutylicum illuminates their roles in clostridial-cell-form biogenesis, granulose synthesis,
652 solventogenesis, and spore morphogenesis. J Bacteriol 2011;193:1414-1426.
653 [71] Piggot PJ, Hilbert DW, Sporulation of Bacillus subtilis. Curr Opin Microbiol 2004;7:579-
654 586.
29 655 [72] Kodama T, Matsubayashi T, Yanagihara T, Komoto H, Ara K, Ozaki K, et al., A novel
656 small protein of Bacillus subtilis involved in spore germination and spore coat assembly. Biosci
657 Biotechnol Biochem 2011;75:1119-1128.
658 [73] Oke V, Losick R, Multilevel regulation of the sporulation transcription factor σK in Bacillus
659 subtilis. J Bacteriol 1993;175:7341-7347.
660 [74] Kunkel B, Losick R, Stragier P, The Bacillus subtilis gene for the development transcription
661 factor σK is generated by excision of a dispensable DNA element containing a sporulation
662 recombinase gene. Genes Dev 1990;4:525-535.
663 [75] Piggot PJ, Coote JG, Genetic aspects of bacterial endospore formation. Bacteriol Rev
664 1976;40:908-962.
665 [76] Takemaru K, Mizuno M, Sato T, Takeuchi M, Kobayashi Y, Complete nucleotide sequence
666 of a skin element excised by DNA rearrangement during sporulation in Bacillus subtilis.
667 Microbiology 1995;141 ( Pt 2):323-327.
668 [77] Haraldsen JD, Sonenshein AL, Efficient sporulation in Clostridium difficile requires
669 disruption of the σK gene. Mol Microbiol 2003;48:811-821.
670 [78] Bruggemann H, Baumer S, Fricke WF, Wiezer A, Liesegang H, Decker I, et al., The
671 genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc Natl Acad
672 Sci U S A 2003;100:1316-1321.
673 [79] Sebaihia M, Peck MW, Minton NP, Thomson NR, Holden MT, Mitchell WJ, et al., Genome
674 sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative
675 analysis of the clostridial genomes. Genome Res 2007;17:1082-1092.
30 676 [80] Kirk DG, Dahlsten E, Zhang Z, Korkeala H, Lindstrom M, Involvement of Clostridium
677 botulinum ATCC 3502 sigma factor K in early-stage sporulation. Appl Environ Microbiol
678 2012;78:4590-4596.
679 [81] Dahlsten E, Kirk D, Lindstrom M, Korkeala H, Alternative sigma factor SigK has a role in
680 stress tolerance of group I Clostridium botulinum strain ATCC 3502. Appl Environ Microbiol
681 2013;79:3867-3869.
682 [82] Jones SW, Paredes CJ, Tracy B, Cheng N, Sillers R, Senger RS, et al., The transcriptional
683 program underlying the physiology of clostridial sporulation. Genome Biol 2008;9:R114.
684 [83] Al-Hinai MA, Jones SW, Papoutsakis ET, σK of Clostridium acetobutylicum is the first
685 known sporulation-specific sigma factor with two developmentally separated roles, one early and
686 one late in sporulation. J Bacteriol 2014;196:287-299.
687 [84] Fimlaid KA, Bond JP, Schutz KC, Putnam EE, Leung JM, Lawley TD, et al., Global
688 analysis of the sporulation pathway of Clostridium difficile. PLoS Genet 2013;9:e1003660.
689 [85] Saujet L, Pereira FC, Serrano M, Soutourina O, Monot M, Shelyakin PV, et al., Genome-
690 wide analysis of cell type-specific gene transcription during spore formation in Clostridium
691 difficile. PLoS Genet 2013;9:e1003756.
692 [86] Kirk DG, Palonen E, Korkeala H, Lindstrom M, Evaluation of normalization reference
693 genes for RT-qPCR analysis of spo0A and four sporulation sigma factor genes in Clostridium
694 botulinum Group I strain ATCC 3502. Anaerobe 2014;26:14-19.
695 [87] Sarker MR, Carman RJ, McClane BA, Inactivation of the gene (cpe) encoding Clostridium
696 perfringens enterotoxin eliminates the ability of two cpe-positive C. perfringens type A human
697 gastrointestinal disease isolates to affect rabbit ileal loops. Molecular microbiology 1999;33:946-
698 958.
31 699 [88] Czeczulin JR, Collie RE, McClane BA, Regulated expression of Clostridium perfringens
700 enterotoxin in naturally cpe-negative type A, B, and C isolates of C. perfringens. Infect Immun
701 1996;64:3301-3309.
702 [89] Zhao Y, Melville SB, Identification and characterization of sporulation-dependent
703 promoters upstream of the enterotoxin gene (cpe) of Clostridium perfringens. J Bacteriol
704 1998;180:136-142.
705 [90] Ohtani K, Hirakawa H, Paredes-Sabja D, Tashiro K, Kuhara S, Sarker MR, et al., Unique
706 regulatory mechanism of sporulation and enterotoxin production in Clostridium perfringens. J
707 Bacteriol 2013;195:2931-2936.
708 [91] Li J, Chen J, Vidal JE, McClane BA, The Agr-like quorum-sensing system regulates
709 sporulation and production of enterotoxin and beta2 toxin by Clostridium perfringens type A
710 non-food-borne human gastrointestinal disease strain F5603. Infect Immun 2011;79:2451-2459.
711 [92] Steiner E, Scott J, Minton NP, Winzer K, An agr quorum sensing system that regulates
712 granulose formation and sporulation in Clostridium acetobutylicum. Appl Environ Microbiol
713 2012;78:1113-1122.
714 [93] Cooksley CM, Davis IJ, Winzer K, Chan WC, Peck MW, Minton NP, Regulation of
715 neurotoxin production and sporulation by a Putative agrBD signaling system in proteolytic
716 Clostridium botulinum. Appl Environ Microbiol 2010;76:4448-4460.
717 [94] Eichenberger P, Jensen ST, Conlon EM, van Ooij C, Silvaggi J, Gonzalez-Pastor JE, et al.,
718 The σE regulon and the identification of additional sporulation genes in Bacillus subtilis. J Mol
719 Biol 2003;327:945-972.
720 [95] Wang ST, Setlow B, Conlon EM, Lyon JL, Imamura D, Sato T, et al., The forespore line of
721 gene expression in Bacillus subtilis. J Mol Biol 2006;358:16-37.
32 722 [96] Fawcett P, Eichenberger P, Losick R, Youngman P, The transcriptional profile of early to
723 middle sporulation in Bacillus subtilis. Proc Natl Acad Sci U S A 2000;97:8063-8068.
724 [97] Traag BA, Pugliese A, Eisen JA, Losick R, Gene conservation among endospore-forming
725 bacteria reveals additional sporulation genes in Bacillus subtilis. J Bacteriol 2013;195:253-260.
726 [98] Paredes-Sabja D, Sarker N, Setlow B, Setlow P, Sarker MR, Roles of DacB and Spm
727 proteins in Clostridium perfringens spore resistance to moist heat, chemicals, and UV radiation.
728 Appl Environ Microbiol 2008;74:3730-3738.
729 [99] Paredes-Sabja D, Setlow B, Setlow P, Sarker MR, Characterization of Clostridium
730 perfringens spores that lack SpoVA proteins and dipicolinic acid. J Bacteriol 2008;190:4648-
731 4659.
732 [100] Setlow P, Resistance of spores of Bacillus species to ultraviolet light. Environmental and
733 molecular mutagenesis 2001;38:97-104.
734 [101] Paredes-Sabja D, Raju D, Torres JA, Sarker MR, Role of small, acid-soluble spore proteins
735 in the resistance of Clostridium perfringens spores to chemicals. Int J Food Microbiol
736 2008;122:333-335.
737 [102] Setlow P, I will survive: DNA protection in bacterial spores. Trends in microbiology
738 2007;15:172-180.
739 [103] Raju D, Waters M, Setlow P, Sarker MR, Investigating the role of small, acid-soluble
740 spore proteins (SASPs) in the resistance of Clostridium perfringens spores to heat. BMC
741 Microbiol 2006;6:50.
742 [104] Raju D, Sarker MR, Production of small, acid-soluble spore proteins in Clostridium
743 perfringens nonfoodborne gastrointestinal disease isolates. Can J Microbiol 2007;53:514-518.
33 744 [105] Raju D, Setlow P, Sarker MR, Antisense-RNA-mediated decreased synthesis of small,
745 acid-soluble spore proteins leads to decreased resistance of Clostridium perfringens spores to
746 moist heat and UV radiation. Appl Environ Microbiol 2007;73:2048-2053.
747 [106] Leyva-Illades JF, Setlow B, Sarker MR, Setlow P, Effect of a small, acid-soluble spore
748 protein from Clostridium perfringens on the resistance properties of Bacillus subtilis spores. J
749 Bacteriol 2007;189:7927-7931.
750 [107] Sarker MR, Shivers RP, Sparks SG, Juneja VK, McClane BA, Comparative experiments to
751 examine the effects of heating on vegetative cells and spores of Clostridium perfringens isolates
752 carrying plasmid genes versus chromosomal enterotoxin genes. Appl Environ Microbiol
753 2000;66:3234-3240.
754 [108] Li J, McClane BA, A novel small acid soluble protein variant is important for spore
755 resistance of most Clostridium perfringens food poisoning isolates. PLoS Pathog
756 2008;4:e1000056.
757
758
759
34 760 Figure Legends
761 Figure 1. Proposed sporulation model in Bacillus and Clostridium species. The temporal
762 progression of sporulation is shown for B. subtilis, C. difficile, and C. perfringens. In the
763 phosphorelay system of B. subtilis, after receiving the respective signal(s), all orphan kinases
764 autophosphorylate and transfer the phosphoryl group from their phosphotransferase domain to an
765 aspartate residue in Spo0F, a single domain response regulator. Next, the phosphoryl group is
766 passed to a histidine residue in a phosphotransferase domain, Spo0B. Finally, Spo0B
767 phosphorylate the key sporulation regulator Spo0A. There is no evidence of the presence of
768 Spo0F in any Clostridium species. Phosphorylation of Spo0A leads to the activation of a σ factor
769 cascade that acts in both mother cell and forespore. Differential features of the sporulation
770 pathway in C. difficile include the post-translational activation of σG by σF and the absence of
771 proteolytic activation of σK. A notable difference in the C. perfringens sporulation regulatory
772 circuit is the early requirement of σK to generate sufficient active σE. Solid arrows in the putative
773 regulatory cascade indicate confirmed interactions, whereas dotted arrows indicate that the
774 regulatory relationship between the factors has not been tested.
35 775 Table 1: Putative orphan histidine kinase of different Clostridium species. 776 Species/strainsa No. of No. of ORFs of putative orphan histidine kinases Reference histidine orphan kinasesb histidine kinasesc
Clostridium acrtobutylicum ATCC 824 34 7 CAC0903, CAC0323, CAC2220, CAC0317, CAC3319, [2] CAC2730, CAC0437
Clostridium acrtobutylicum EA 2018 39 9 CEAG2234, CEAG0328, CEAG3322, CEAG2739, CEAG0448, This study CEAG0334, CEAG0915, CEAG2551, CEAG0430
Clostridium acrtobutylicum DSM 1731 36 9 SMBG2253, SMBG0325, SMBG3356, SMBG2765, SMBG0920, This study SMBG0446, SMBG0331, SMBG2573, SMBG0428 Clostridium botulinum A str. Hall 34 4 CLC0398, CLC1171, CLC2637, CLC0394 This study
Clostridium botulinum A str. ATCC 32 3 CBO2762, CBO1120, CBO0336 This study 3502
Clostridium botulinum A str. ATCC 34 4 CLB1159, CLB2704, CLB0379, CLB0383 This study 19397
Clostridium difficile 630 51 6 CD630_24920, CD630_14920, CD630_13520, CD630_19490, This study CD630_15790, CD630_09970
Clostridium difficile CD196 48 7 CD196_1365, CD196_1829, CD196_1501, CD196_1216, This study CD196_2338, CD196_2713, CD196_0520
Clostridium difficile BI1 48 7 CDBI1_07760, CDBI1_06975, CDBI1_06215, CDBI1_11090, This study CDBI1_09440, CDBI1_12120, CDBI1_14040
Clostridium perfringensstr. 13 27 8 CPE1757, CPE1512, CPE1316, CPE0207, CPE1986, CPE1987, [2] CPE0951, CPE0870
36 Clostridium perfringensATCC13124 30 10 CPF2640, CPF2010, CPF1764, CPF2241, CPF2242, CPF1195, This study CPF0198, CPF1243, CPF1523, CPF0863
Clostridium perfringensSM101 24 9 CPR1023, CPR1953, CPR1954, CPR1493, CPR1316, CPR0195, This study CPR1807, CPR1055, CPR1728 777 778 a Strains were selected for identifying putative orphan histidine kinases based on the available whole genome sequence (WGS) data and respective with the 779 importance of the strain in research purpose. To date, in total 3 WGS for C. acetobutylicum (C. acetobutylicum ATCC 824, C. acetobutylicum EA 2018, and C. 780 acetobutylicum DSM 1731), 13 for C. botulinum (C. botulinum A str. Hall, C. botulinum A str. ATCC 3502, C. botulinum A str. ATCC 19397, C. botulinum F 781 str. Langeland, C. botulinum B1 str. Okra, C. botulinum A3 str. Loch Maree, C. botulinum B str. Eklund 17B, C. botulinum E3 str. Alaska E43, C. botulinum Ba4 782 str. 657, C. botulinum A2 str. Kyoto, C. botulinum F str. 230613, C. botulinum BKT015925, C. botulinum H04402 065), 4 for C. difficile (C. difficile 630, C. 783 difficile CD 196, C. difficile 2007855, and C. difficile BI1), and 3 for C. perfringens (C. perfringens str. 13, C. perfringens ATCC 13124, and C. perfringens 784 SM101) are available. 785 b The total no. of kinases were determined by searching with key word ‘histidine kinase’ for each of the respective strain in NCBI database. 786 c Orphan histidine kinases were identified by identifying the kinases with no adjacent response regulatory protein. 787 788 789
37 790 Table 2: Putative sporulation proteins in different Clostridium species.
Species Orthologs of putative sporulation proteins in Clostridiuma
BkdR CwlD DapG YlxY YlyA YlzA YqhQ Clostridium acrtobutylicum #c + + + + +/- + +/- Clostridium asparagiforme +/- − +/- +/- + +/- − Clostridium bartlettii +/- + + + + +/- +/- Clostridium beijerinckii # + + + + +/- + +/- Clostridium bolteae + + + +/- + − + Clostridium botulinum # + + + + + + + Clostridium butyricum + + + + +/- + +/- Clostridium cadaveris − − − − − − − Clostridium carboxidivorans + + + + +/- + +/- Clostridium cellulolyticum # − +/- + + +/- + + Clostridium cellulovorans # +/- +/- + + +/- + + Clostridium chauvaei + + + + +/- +/- +/- Clostridium clostridioforme + +/- + + +/- + + Clostridium colicanis +/- + + + +/- + +/- Clostridium difficile # + + + + +/- +/- + Clostridium hathewayi + + + + +/- + + Clostridium hylemonae − + + + +/- + + Clostridium innocuum +/- +/- − +/- +/- +/- − Clostridium kluyveri # + + + + +/- + +/- Clostridium leptum − +/- + + +/- +/- + Clostridium methylpentosum − +/- + +/- +/- + +/- Clostridium nexile − + + + +/- − + Clostridium novyi # + + + + +/- + +
38 Clostridium papyrosolvens + +/- + + +/- +/- +/- Clostridium paraputrificum − − − − − − − Clostridium perfringens# + + + + − + +/- Clostridium ramosum +/- +/- +/- +/- +/- +/- − Clostridium scatologenes − − − − − − − Clostridium scindens − +/- − + +/- +/- + Clostridium sordellii ATCC + + + + +/- + + 9714 Clostridium sporogenes + + + + +/- + +/- Clostridium sticklandii # − − − − − − − Clostridium tetani # + + + + +/- + +/- Clostridium thermocellum # +/- + + + +/- + + Clostridium tyrobutyricum + +/- + +/- +/- + +/- 791 792 a These 7 putative sporulation proteins were identified from the phylogenetic analysis in B. subtilis [12, 97]. From the pool of 127 Proteins, we have selected 793 these 7 proteins because 1) these are conserved among spore formers (mostly Bacillus and Clostridium strains) and mostly not found in other non-sporulating 794 bacteria, and 2) role of these proteins yet to be determined. 795 b Orthologs of sporulation proteins were identified by the BLASTP analyses with B. subtilis strain 168 genome and the presence (+) or absence (−) were listed for 796 different Clostridium species. (+/-) indicates the proteins those have very low identity or have different or unknown functions than it’s respective protein of B. 797 subtilis strain 168. The functions of the proteins in B. subtilis strain 168 are as follows: BkdR, transcriptional regulator; CwlD, N-acetylmuramoyl-L-alanine 798 amidase; DapG, aspartokinase I; YlxY, putative sugar deacetylase; YlyA, hypothetical protein; YlzA, hypothetical protein; and YqhQ, hypothetical protein. 799 c # after species name indicates the WGS are annotated and published for at least one of the strains for these species in NCBI database. The rest of the species do 800 not have any full-annotated genome available for any of their strains. 801 802 803 804 805 806 807 808
39 809
810 811 812
40 Figure 1
Fig. 1.