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