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
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Review article for publication in special issue: Genetics of toxigenic Clostridia
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Updates on the sporulation process in Clostridium species
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Prabhat K. Talukdar1, 2, Valeria Olguín-Araneda3, Maryam Alnoman1, 2, Daniel Paredes-Sabja1, 3,
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Mahfuzur R. Sarker1, 2.
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Microbiology, College of Science, Oregon State University, Corvallis, OR. U.S.A; 3Laboratorio
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de Mecanismos de Patogénesis Bacteriana, Departamento de Ciencias Biológicas, Facultad de
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Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile.
Department of Biomedical Sciences, College of Veterinary Medicine and 2Department of
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Running Title: Clostridium spore formation.
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Key Words: Clostridium, spores, sporulation, Spo0A, sigma factors
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Veterinary Medicine, Oregon State University, 216 Dryden Hall, Corvallis, OR 97331. Tel: 541-
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737-6918; Fax: 541-737-2730; e-mail: sarkerm@oregonstate.edu
Corresponding author: Dr. Mahfuzur Sarker, Department of Biomedical Sciences, College of
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Abstract
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Sporulation is an important strategy for certain bacterial species within the phylum Firmicutes to
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survive longer periods of time in adverse conditions. All spore-forming bacteria have two phases
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in their life; the vegetative form, where they can maintain all metabolic activities and replicate to
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increase numbers, and the spore form, where no metabolic activities exist. Although many
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essential components of sporulation are conserved among the spore-forming bacteria, there are
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differences in the regulation and the pathways among different genera, even at the species level.
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While we have gained much information from the most studied spore-forming bacterial genus,
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Bacillus, we still lack an in-depth understanding of spore-formation in the genus Clostridium.
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Clostridium and Bacillus share the master regulator of sporulation, Spo0A, and its downstream
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pathways, but there are differences in the activation of the Spo0A pathway. While Bacillus
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species use a multicomponent phosphorylation pathway for phosphorylation of Spo0A, termed
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phosphorelay, such a phosphorelay system is absent in Clostridium. On the other hand, a number
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of genes regulated by the different sporulation-specific transcription factors are conserved
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between different Clostridium and Bacillus species. In this review, we discuss the recent findings
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on Clostridium sporulation and compare the sporulation mechanism in Clostridium and Bacillus.
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1. Introduction
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Sporulation is an intriguing bacterial property of a certain low G+C group of Gram-
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positive bacteria, which have existed from ancient time (2.5 billion years ago) [1]. It is a
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complex developmental process, which leads to the generation of metabolically dormant spores
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from vegetative cells [2]. While the exact reason is not known for the decision of bacterial cell to
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form spores, it has been hypothesized that nutrient depletion or the presence of toxic compounds
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triggers the sporulation process [3]. Spore formation is a helpful strategy for the spore-forming
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bacteria leading to survival in unfavorable conditions in the environment or inside the hosts for
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prolonged periods of time, and transmission to other hosts or environments. Spores can
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withstand physical and chemical stresses, such as high temperatures, pressures, solvents,
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oxidizing agents, lytic enzymes, irradiation, acceleration, and antimicrobials [4, 5], which could
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rapidly destroy the vegetative form of the bacterium. In several instances, spores serve as an
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infective particle in human and animal diseases [6]. Each of these fascinating characteristics led
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microbiologists to engage their profound interest on dissecting spore structure and the
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mechanism of spore formation.
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Extensive studies have been conducted on the sporulation process of Bacillus species,
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especially Bacillus subtilis for many years, and thus it is regarded as the model organism for
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sporulation. Due to the availability of techniques for genetic manipulation, molecular
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microbiologists showed the most interest in this species to illustrate the sporulation mechanism.
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However, after gaining significant knowledge from B. subtilis, researchers have switched their
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focus to other spore-forming bacteria, especially Clostridium species. Although, it has been
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suggested that sporulation in Bacillus and Clostridium employ similar mechanisms based on
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their morphological similarities, studies have shown that there are some differences in the early
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stages of sporulation process in these two species [2, 7-9].
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Clostridium species are Gram-positive, anaerobic, spore-forming prokaryotes including
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strains of importance to human and animal health (C. tetani, C. perfringens, C. botulinum and C.
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difficile), cellulose degradation (C. phytofermentans and C. thermocellum), solvent production
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(C. acrtobutylicum and C. beijerinckii) and strains involved in bioremediation (C. cadavaris).
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This heterogeneous group of Clostridium is divided into 19 clusters [10]. These strains are
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widely distributed throughout the world in all sorts of environments, but most likely found in
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soils and animal intestines in the form of vegetative cells or dormant spores.
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It has been hypothesized that Bacilli and Clostridia were in the same class until about 2.5
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billion years ago when the rise of atmospheric oxygen occurred, also known as the ‘great
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oxidation’ event [1]. Bacilli diverged from the Clostridia as a separate class during that period.
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After that separation, Bacilli remain as an aerobic spore-former whereas Clostridia persisted as
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an anaerobic-spore former. Different environmental requirements for the growth of these two
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classes of bacteria may explain why there are differences in the molecular mechanism of
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sporulation, especially at the initial stages in sporulation. In contrast, signature sporulation genes
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are still conserved between these two classes long time after their separation indicating that both
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had originated from the same origin [11, 12].
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Despite having importance in the field of human and animal health and physiology,
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cellulose degradation, solvent production and bioremediation, the molecular events in
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Clostridium sporulation are not well understood, primarily as a result of limited genetic
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manipulation. Recent developments in molecular techniques such as, high-throughput genome
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sequencing, genome-wide transcriptional profiling, and directed or random mutagenesis
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techniques such as group II introns for insertional mutagenesis and transposons for random
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mutagenesis, have enabled researcher to find out the hints of molecular mechanism leads to the
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sporulation in Clostridium.
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In this review, we discuss the recent advancements in the sporulation study on four major
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Clostridium species including C. acetobutylicum, C. botulinum, C. difficile, and C. perfringens.
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We discuss how the components of the sporulation process differ between Clostridium than
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Bacillus species. Also, we compare the sporulation process among Clostridium species.
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2. Stages of sporulation and spore structure
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The morphological stages for spore formation are similar in all spore-forming bacteria. In
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every sporulation event, there are two forms present in the cell; the mother cell and the forespore.
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The total sporulation process can be divided into seven stages (stage I-VII) [13]. The first stage,
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called stage 0 is actually the growth of vegetative cells before the beginning of sporulation. In
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stage I and II, the cell DNA releases as an axial filament and the asymmetric cell division results
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in forming of two compartments, one with smaller prespore compartment and the other is larger
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mother cell compartment. Initially, one-third of the DNA material is deposited in the prespore,
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although the rest of the DNA is rapidly pumped into the prespore compartment via the action of
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the DNA translocase, SpoIIIE. During stage III, the prespore is engulfed by the mother cell and
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called forespore, which has inner and outer membranes surrounding and floating as a protoplast.
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In step IV, peptidoglycan (PG) layer synthesizes the primordial germ cell wall and the cortex in
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the space between inner and outer membranes surrounding the forespore. The outcome of stage
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V is the formation of the complex structure of proteins known as the spore coat, outside on the
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surface of the forespore. Despite these changes in the morphological structure of spores, there is
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one more stage to make the newly formed spore more durable. In stage VI, spore’s resistance to
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UV radiation and heat is established. After going through all these changes, mature spores are
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liberated from the mother cell into the environment during the stage VII of sporulation.
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The basic structure of spores and morphological stages are conserved among all spore-
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forming bacteria. The spore structures contribute to the dormant microorganism to sustain in a
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variety of environmental stresses like high temperature, pressure, extreme pH, and radiation until
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the spore finds itself in more favorable condition for vegetative growth. Usually, the bacterial
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genome is deposited inside the central compartment of the spore surrounded by the lipid bilayer
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covered with a layer of PG, which is known as the germ cell wall. This germ cell wall also serves
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as the cell wall of vegetative forms after the completion of spores germination. The Germ cell
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wall is wrapped in a thick layer of another layer of modified PG, termed the cortex. It is essential
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for the acquisition and the maintenance of the heat resistance [14, 15]. Finally, this cortex layer
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is encapsed by a multiprotein coat protecting it from the action of the PG-lytic enzymes. In
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several species such as B. anthracis, B. cereus and C. difficile, this multiprotein coat is further
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enclosed by another structure known as the exposporium [16-18]. If present, the exosporium or
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otherwise the coat serves as the interacting structure of the spore to the environment. The spore’s
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inner membrane contains the essential components for spore germination, including various
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germinant receptors, which interacts with small molecules that trigger germination and a return
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to the vegetative form [14, 19-21].
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3. Initiation of sporulation
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The full initiation pathway has been identified in the Bacillus species; however, the clear
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picture in Clostridium has yet to be elucidated. In Bacillus, a multi-component signal
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transduction system termed ‘phosphorelay’ is present [7]. Proper functioning of this system
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leads to the activation of a master regulator, Spo0A [22] (Fig. 1). At present, no such
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phosphorylation system has been found in Clostridium species. Another important component
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for the initiation of sporulation are kinases, termed orphans, because they lack the cognate
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response regulator [23]. These orphan kinases are involved in receiving different stimuli, both
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from the extracellular or intracellular and initiate the process of sporulation. In Bacillus, at least
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five orphan kinases (KinA - KinE) are present, but the KinA and KinB are the most efficient
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ones. Each of the kinases is able to respond to different stimuli [24].
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In Bacillus, all orphan kinases autophosphorylate upon receiving the respective signal(s)
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and transfer the phosphoryl group from their phosphotransferase domain to an aspartate residue
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in Spo0F, a single domain response regulator [24]. The phosphoryl group is then passed to a
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histidine residue in a phosphotransferase domain within Spo0B. Finally, Spo0B phosphorylates
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the key sporulation regulator, Spo0A, that eventually starts the second phase of the sporulation
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process (Fig. 1). There is no evidence for the presence of Spo0F in any Clostridium species [7].
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However, a homolog of Spo0B was found in C. tetani, but its function has yet to be known [25].
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There are more components involved in Bacillus sporulation and those can regulate the
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phosphorelay system either positively or negatively. An alternative sigma factor, SigH, is a
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transcription factor and activates Spo0A, Spo0F, KinA and KinE [26]. While Spo0A is activated
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directly by SigH, Spo0A increases the transcription of spo0H gene encoding SigH by repressing
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AbrB, a global repressor. In contrast to Bacillus, Clostridium spo0H is constitutively expressed
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at a low level, without any sign of increase at the onset of spore formation [9].
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In total, thirty-nine histdine kinases have been found in B. subtilis, of which nine are
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orphans [7, 24]. Among five sporulation specific histidine kinases, only three have been found to
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contain transmembrane domains [27], which means those three might response to the
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environmental signal. In an earlier review, Paredes et al [2] described the presence of putative
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orphan histidine kinases in three Clostridium species; C. acetobutylicum, C. perfringens, and C.
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tetani based on the completed genome sequences of Clostridium species at that time. These
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authors used a BLAST- based approach to identify 35 kinases in C. acetobutylicum and six of
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them are orphans (Table 1). Another study identified two different pathways for sporulation
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initiation in C. acetobutylicum [28], and by insertional inactivation of kinase genes (single and
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double mutants), they showed that CAC0903, CAC3319, and CAC0323 directly activate Spo0A.
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Our group then extended the search for putative orphan histidine kinases in other
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Clostridium species. We followed the strategy of Paredes et al [2], to find out the orphan
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histidine kinases from the Clostridium genomes in NCBI database. In C. perfringens Type-A
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food poisoning strain SM101, six putative orphan histidine kinases (ORFs CPR1953, CPR1493,
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CPR1316, CPR0195, CPR1055, and CPR1728) were identified based on the BLASTP analyses
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with kinases from B. subtilis. Knock-out mutations in two of these (CPR1055, and CPR1728)
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showed sporulation and germination defects suggesting their putative role in activating the
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Spo0A by phosphorylation (P. Udompijitkul and M. R. Sarker, unpublished).
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The phosphorelay system might explain why Bacillus is more environmentally versatile
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than Clostridium. In the process of evolution, Bacillus adopted themselves to the environmental
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changes by adding different functional genes for reacting to multiple signals. The absence of a
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phosphorelay system leaves remaining questions about how Clostridia initiate sporulation. One
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hypothesis is that, Clostridium Spo0A is activated by direct phosphorylation from the orphan
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histidine kinases. Another possibility is that there might be a different unknown phosphorelay
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system present in Clostridium.
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4. The master regulator, Spo0A
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The most widely studied and functionally characterized component of the sporulation
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machinery in spore-forming bacteria is the key regulator, Spo0A. It is a transcription factor that
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controls the transition of the bacterium into the spore form [2, 26]. The structure and function of
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Spo0A protein has been mostly studied in Bacillus species. In that system, Spo0A is activated by
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Spo0B [29-31]. When the number of phosphorylated Spo0A reaches a threshold [32], the protein
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binds directly to specific DNA sequences (TGNCGAA) upstream of several early sporulation
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genes and thus activates the sporulation process [33-35]. This particular DNA sequence is known
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as ‘0A box’ or the binding site for Spo0A. The structure of Spo0A is critical for its subsequent
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changes from de-phosphorylation to phosphorylation state and vice-versa. Spo0A has two
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functional domains, the N-terminal phosphorylation and dimerization domain (receiver), and the
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C-terminal DNA binding (effector) domain. These two domains are separated by a hinge region
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[36]. Phosphorylation leads to structural rearrangement that facilitates Spo0A dimerization [37,
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38], resulting in the disruption of transcription-inhibitory contacts between the receiver and
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effector domains. This disruption leads to the binding of DNA binding domains to the Spo0A
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protein. The crystal structure of the DNA binding domain confirms specific and non-specific
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contacts between Spo0A protein and the consensus sequence [38, 39]. Upon activation, Bacillus
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Spo0A directly activates 121 genes, including genes required for polar septum formation [34].
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Spo0A is conserved between both Bacillus and Clostridium [40, 41]. The inactivation of
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spo0A in several Clostridium species resulted in the blocking of sporulation and synthesis of
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sporulation-specific sigma factors [9, 42-44]. Transcriptomic and proteomic analyses identified
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C. dificile Spo0A as a global regulator that regulates metabolic and virulence factors outside the
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sporulation process [45]. Similarly as in B. subtilis [46], Spo0A has a role in biofilm formation in
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C. difficile [47, 48] and C. perfringens [49].
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One additional function of Spo0A is to regulate different Clostridial toxins [42, 50].
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Recent works have suggested that it may be responsible for the regulation of toxin A and toxin B
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production by C. difficile [35, 43, 51, 52]. Study demonstrated that Spo0A positively regulates
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toxin A and toxin B expression. The spo0A mutant in an erythromycin mutant strain of C.
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difficile, C. difficile strain 630 delta erm resulted in the productions of toxins [43]. However,
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another study showed a contradictory result; inactivation of spo0A in 630 delta erm strain had no
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affect on the toxin production [35]. Deakin et al [51] reported that a C. difficile R20291 spo0A
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mutant caused more severe disease in a murine model than the wild type strain. Recently, one
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study has shown the various affects of Spo0A on the toxin production in different C. difficile
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strains [52]. Spo0A has been implicated in virulence in mice models [43, 51]. In C.
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acetobutylicum, Spo0A as well as sporulation affect the solvent production [53].
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5. Sigma (σ) factors
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After the initial trigger of spore formation, the cell transitions through different
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morphological stages to form mature spores, facilitated by the contribution of different σ factors.
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These are the dissociable RNA polymerase subunits that alter the promoter specificity of the
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RNA polymerase complex under different environmental and growth phase-dependent
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conditions [26]. Four sporulation-specific σ factors were first identified in B. subtilis [13, 54-56].
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These σ factors are compartment specific; σ factor F (σF) and G (σG) are forespore specific and
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regulated by anti- σ factors and anti-anti- σ factors. On the other hand, σ factor E (σE) and K (σK)
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are mother cell specific, synthesized as precursor protein, which needs to be cleaved for the
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activation. Generally, σF is the first σ factor appeared in the sporulation process by controlling
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the early stages of the forespore followed by σE in the mother cells. Later, σG and σK have their
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actions in the formation of mature spores and vegetative cells, respectively (Fig. 1). The
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sequence similarities to all four σ factors have been identified in C. acetobutylicum [57] and
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confirmed by PCR based approach [41]. Similar results have also been observed in other
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Clostridium species; homologs of all four σ factors have been found in C. perfringens [58, 59].
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This has been confirmed by transcriptional and protein analyses [60, 61].
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5.1 σF
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In B. subtilis, the RNA polymerase σF is synthesized prior to the formation of polar
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septum and held inactive until septation is completed [26]. A similar pattern has been found in
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few Clostridium species (Fig. 1). In B. subtilis, σF encoding gene, spoIIAC, is transcribed by σH
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associated RNA polymerase during the initiation of sporulation [62, 63]. σF remains inactive in
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the pre-divisional cell by binding with anti- σ factor SpoIIB, until it is relieved by the anti-anti- σ
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factor SpoIIA. The functionality of SpoIIA becomes inactive by the phosphorylation with
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SpoIIAB, which is a kinase and anti- σ factor. In contrast, SpoIIA becomes active by the
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dephosphorylation with a phosphatase, SpoIIE. The non-phosphorylated SpoIIA interacts with
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the SpoIIB- σF complex and displace the σF. Released σF becomes active and directs gene
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expression via the control of different genes. The role of σF in sporulation of different
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Clostridium species has been demonstrated by gene-knock-out studies. Clostridium mutants
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lacking σF completely blocked for sporulation and this defect could be restored to nearly wild-
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type levels by complementation with wild-type sigF gene, indicating that σF is essential for spore
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formation in these organisms [61, 64, 65].
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5.2 σE
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In both Bacillus and Clostridium species, σE is synthesized as an inactive precursor
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protein (pro- σE) (Fig. 1), which is activated by the proteolysis event by the protease activity of
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SpoIIGA [66, 67]. Pro- σE is transcribed from spoIIG operon, in which sigE (earlier named as
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spoIIGB), is the second gene in the operon, initiated transcription before asymmetric septation
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and continues after septum formation, only in the mother cell [68]. This indicates that the σE is
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produced even after the septum formation. The expression of spoIIGA, the first gene of the
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spoIIG operon, requires the σF -controlled SpoIIR protein. This indicates that the mother-cell-
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specific σE is controlled by the forespore-specific σF -directed gene transcription and the presence
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of an intercellular gene transcription pathway [26, 64]. spoIIGA expression is also controlled by
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the Spo0A after asymmetric division [26, 69]. Like σF, σE mutant strains in C. perfringens and C.
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difficile showed sporulation defects in sporulation-inducing conditions [60, 64]. C.
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acetobutylicum σE mutant strain also blocks the sporulation before the asymmetric division [70].
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5.3 σG
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In B. subtilis, σG is synthesized in the pre-engulfment prespore, but is not activated until
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the end of stage III (complete engulfment of prespore by mother cell). Transcription of sigG
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(earlier named spoIIG) is dependent on σF [26, 71]. The products of spoIIIA and spoIIIJ are
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needed for the release of σG from inhibition in the forespore compartment. spoIIIA is selectively
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expressed in the mother cell under σE control, whereas spoIIIJ is expressed in the forespore [26,
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71]. Mutations in sigG blocks the sporulation in C. perfringens and C. difficile [61, 64]. In C.
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acetobutylicum, sigG mutant halted the sporulation during the maturation stage [70].
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5.4 σK
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σK is known as the late stage (stage IV) mother cell specific σ factor. It regulates spore
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coat formation during the late-stage of sporulation [72, 73]. An intervening element of 42-kb
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size, named skin (sigma K intervening) separates sigK into two parts in some Bacillus species
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[74]. Both halves are required, as mutations in either halves results in the failure of spore
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formation at late stage [75]. The absence of the skin element does not affect the growth or
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sporulation suggesting that this element does not contain any genes essential for survival [74].
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Inside the mother cell, the skin undergoes site-specific recombination to form sigK. Because it
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has no visible role in survival, it has been assumed that skin is a rudimentary element left from
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the ancestors [76]. Interestingly, this skin element has been found in C. difficile, and unlike B.
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subtilis, it is important for sporulation [77]. Although a 47-kb skin element, named skinCt has
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been identified in C. tetani [78], other known Clostridium genomes do not contain this element
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and have an uninterrupted sigK.
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The homolog of Bacillus σK has been found in the genomes of C. botulinum [79] and C.
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perfringens [58] and shown to be essential for early stage of sporulation [60, 80] (Fig. 1). Krik et
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al [80] constructed two sigK mutants in C. botulinum and demonstrated that σK also acts in the
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early stage of sporulation and plays as a transcriptional activator of Spo0A. σF transcript is also
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lower in σK muatnts. Also, σK was shown to have role in different stress tolerance such as cold
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and osmotic stress in C. botulinum ATCC3502 strain [81].
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5.5 Regulation of sigma factors in Clostridium
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Among the Clostridium species, the most studied organism for σ factor expression during
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sporulation is C. acetobutylicum [9, 41, 82]. In this species, the pattern of σ factor expression and
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solventogenesis for SpoIIA, σE, σG, and σK matched that in B. subtilis [9], although it was spread
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over a much longer time (35 h) than that seen in B. subtilis (8 h). Some σ factors have two
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separate developmental roles during sporulation. For example, in C. acetobutylicum, σK acts both
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early, even prior to Spo0A expression, and late stages of sporulation, past of σG activation [83].
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The expression and regulation of all four σ factors in C. perfringens has been evaluated in
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two separate studies. By introducing mutations in sigE and sigK genes of C. perfringens SM101,
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Harry et al [60] discovered some differences in expressions and functions of these σ factors
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during the sporulation of C. perfringens SM101 versus B. subtilis (Fig. 1). 1) Unlike B. subtilis,
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where σK is the last σ factor expressed during sporulation, normal production of sigF and sigE
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transcripts in sporulating SM101 cells is dependent on sigK. 2) sigF transcript production was
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delayed in a SM101 sigE-mutant, but sigF is the first transcript in B. subtilis. 3) sigG transcripts
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were detected in SM101-sigE and -sigK mutants while sigG transcription in B. subtilis requires
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both σE and σF. 4) Transcripts of all four σ factor genes were detected much earlier in C.
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perfringens SM101 than that reported for B. subtilis. Finally, unlike B. subtilis, where expression
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of spoIIID (a key mother cell specific transcription factor) requires σE-associated RNA
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polymerase, the transcription and translation of spoIIID in C. perfringens does not require either
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σK or σE.
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But these findings were questioned by another study conducted by Li and McClane [61].
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To investigate the role of σF and σG in sporulation and CPE production in C. perfringens SM101
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strain, they constructed isogenic sigF and sigG null mutants and corresponding complemented
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strains. By the Western blot analyses, they showed that there were little or no production of σ G
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σE, and σK in sigF mutant, which were completely restored in complementing strain [61]. These
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findings suggest that regulation of σ-factors in C. perfringens is similar to B. subtilis.
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In 2013, three extensive studies [64, 84, 85] were conducted on expression and regulation
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of σ factors in C. difficile. Saujet et al [85] identified 225 genes under the regulation of σ factors
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by genome-wide transcriptional analyses and promoter mapping. In a separate study, Fimlaid et
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al [84] has found almost the same number of genes (224) regulated by the sporulation specific σ
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factors. Pereira et al showed the intra-regulation of σ factors by disrupting each of the σ genes
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[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].
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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
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sigG were simultaneously high at the end of the exponential growth phase. Although very low
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sigK expression was detected during the exponential phase, this expression was peaked after 18
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h, means very late during the stationary phase.
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5.6 Role of σ factors in toxin production
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In addition to regulation of sporulation process, σ factors control the expression and
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production of some clostridial toxins. First example of sporulation and/or σ factors regulated
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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
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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
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21
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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
776
Table 1: Putative orphan histidine kinase of different Clostridium species.
Species/strainsa
No. of
histidine
kinasesb
No. of
orphan
histidine
kinasesc
ORFs of putative orphan histidine kinases
Reference
Clostridium acrtobutylicum ATCC 824
34
7
CAC0903, CAC0323, CAC2220, CAC0317, CAC3319,
CAC2730, CAC0437
[2]
Clostridium acrtobutylicum EA 2018
39
9
CEAG2234, CEAG0328, CEAG3322, CEAG2739, CEAG0448,
CEAG0334, CEAG0915, CEAG2551, CEAG0430
This study
Clostridium acrtobutylicum DSM 1731
36
9
This study
Clostridium botulinum A str. Hall
34
4
SMBG2253, SMBG0325, SMBG3356, SMBG2765, SMBG0920,
SMBG0446, SMBG0331, SMBG2573, SMBG0428
CLC0398, CLC1171, CLC2637, CLC0394
Clostridium botulinum A str. ATCC
3502
32
3
CBO2762, CBO1120, CBO0336
This study
Clostridium botulinum A str. ATCC
19397
34
4
CLB1159, CLB2704, CLB0379, CLB0383
This study
Clostridium difficile 630
51
6
CD630_24920, CD630_14920, CD630_13520, CD630_19490,
CD630_15790, CD630_09970
This study
Clostridium difficile CD196
48
7
CD196_1365, CD196_1829, CD196_1501, CD196_1216,
CD196_2338, CD196_2713, CD196_0520
This study
Clostridium difficile BI1
48
7
CDBI1_07760, CDBI1_06975, CDBI1_06215, CDBI1_11090,
CDBI1_09440, CDBI1_12120, CDBI1_14040
This study
Clostridium perfringensstr. 13
27
8
CPE1757, CPE1512, CPE1316, CPE0207, CPE1986, CPE1987,
CPE0951, CPE0870
[2]
36
This study
777
778
779
780
781
782
783
784
785
786
787
788
789
Clostridium perfringensATCC13124
30
10
CPF2640, CPF2010, CPF1764, CPF2241, CPF2242, CPF1195,
CPF0198, CPF1243, CPF1523, CPF0863
This study
Clostridium perfringensSM101
24
9
CPR1023, CPR1953, CPR1954, CPR1493, CPR1316, CPR0195,
CPR1807, CPR1055, CPR1728
This study
a
Strains were selected for identifying putative orphan histidine kinases based on the available whole genome sequence (WGS) data and respective with the
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.
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
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
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.
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
SM101) are available.
b
The total no. of kinases were determined by searching with key word ‘histidine kinase’ for each of the respective strain in NCBI database.
c
Orphan histidine kinases were identified by identifying the kinases with no adjacent response regulatory protein.
37
790
Table 2: Putative sporulation proteins in different Clostridium species.
Species
Orthologs of putative sporulation proteins in Clostridiuma
Clostridium acrtobutylicum #
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 #
c
BkdR
CwlD
DapG
YlxY
YlyA
YlzA
YqhQ
+
+/+/+
+
+
+
−
+
−
+
+
+
+
+
−
+
+/+
+
+
+
+
−
+
+/+
+
+/+
+
−
+/+
+
+/+
+
+/−
+
+/+/+
−
+
+
−
+/−
+/+/+
+
+/−
+
−
+/+
+
+/+
+
−
+/+
−
−
+
+/+/+
+/+
+
+
+
+/+
+/+/+
+
+
+
+
+
+
+
+
+
+
−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+/+
+
+/+
+
+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/-
+
+
+
+/+
+
+/+
+
+/+
+/+
−
+
+/+
+
+/+
+/+
+
+
−
+/+
+/+
+
−
+
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
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
+
−
+/−
+
−
+
−
+/−
+/−
+/−
+
+/−
−
+
+/−
+
+/−
+
+/−
−
+/−
+
+/−
+/−
−
+/-
−
+
+/-
+/-
+
+
+
+
+
+/-
+
+
+
−
+
−
+
−
+
−
+/−
+
−
+/−
+
+/+
+
+
+/-
+
+
+
+
+
+/-
+/+/+/-
+
+
+
+/+
+/-
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
these 7 proteins because 1) these are conserved among spore formers (mostly Bacillus and Clostridium strains) and mostly not found in other non-sporulating
bacteria, and 2) role of these proteins yet to be determined.
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
different Clostridium species. (+/-) indicates the proteins those have very low identity or have different or unknown functions than it’s respective protein of B.
subtilis strain 168. The functions of the proteins in B. subtilis strain 168 are as follows: BkdR, transcriptional regulator; CwlD, N-acetylmuramoyl-L-alanine
amidase; DapG, aspartokinase I; YlxY, putative sugar deacetylase; YlyA, hypothetical protein; YlzA, hypothetical protein; and YqhQ, hypothetical protein.
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
not have any full-annotated genome available for any of their strains.
39
809
810
811
812
40
Figure 1
Fig. 1.