Diversity of the Germination Apparatus in Clostridium botulinum Groups I, II, III, and IV

Abstract

Clostridium botulinum is a highly dangerous pathogen that forms very resistant endospores that are ubiquitous in the environment, and which, under favorable conditions germinate to produce vegetative cells that multiply and form the exceptionally potent botulinum neurotoxin. To improve the control of botulinum neurotoxin-forming clostridia, it is important to understand the mechanisms involved in spore germination. Here we present models for spore germination in C. botulinum based on comparative genomics analyses, with C. botulinum Groups I and III sharing similar pathways, which differ from those proposed for C. botulinum Groups II and IV. All spores germinate in response to amino acids interacting with a germinant receptor, with four types of germinant receptor identified [encoded by various combinations of gerA, gerB, and gerC genes (gerX)]. There are three gene clusters with an ABC-like configuration; ABC [gerX1], ABABCB [gerX2] and ACxBBB [gerX4], and a single CA-B [gerX3] gene cluster. Subtypes have been identified for most germinant receptor types, and the individual GerX subunits of each cluster show similar grouping in phylogenetic trees. C. botulinum Group I contained the largest variety of gerX subtypes, with three gerX1, three gerX2, and one gerX3 subtypes, while C. botulinum Group III contained two gerX1 types and one gerX4. C. botulinum Groups II and IV contained a single germinant receptor, gerX3 and gerX1, respectively. It is likely that all four C. botulinum Groups include a SpoVA channel involved in dipicolinic acid release. The cortex-lytic enzymes present in C. botulinum Groups I and III appear to be CwlJ and SleB, while in C. botulinum Groups II and IV, SleC appears to be important.

Genetic Characterization of the Exceptionally High Heat Resistance of the Non-toxic Surrogate Clostridium sporogenes PA 3679

“…This is not the first reported incidence of multiple spoVA operons in an endospore former. At least one group IV Clostridium species (Brunt et al, 2016), several species of Geobacillus and Bacillus cereus , as well as several of the more heat resistant Bacilli have multiple spoVA loci. Adding additional copies of spoVA in a mobile Tn 1546 transposon ( spoVA mob , Figure 2A) creates an additive resistance effect, greatly increasing the concentration of DPA in the Bacillus subtilis spore core (a D 112.5°C increase from 0.2 min to 25.6 min with three copies; Berendsen et al, 2016).…”

“…The presence of additional spoVA operons in Clostridium species has not been previously explored, though it is a phenomenon that occurs not just in PA 3679, but also in several closely related C. botulinum species (Figure 2B) and at least one C. argentinese strain (Brunt et al, 2016). This might seem paradoxical as C. botulinum is generally considered to have lower spore heat resistance than C. sporogenes .…”

Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219

“…Brunt et al . 20 recently made an inventory of the Ger gene clusters in groups I-IV of C. botulinum , based on in silico analysis of 148 C. botulinum and 8 C. sporogenes genomes 20 . The latter were included because of their close relatedness to gICb.…”

Analysis of the Germination of Individual Clostridium sporogenes Spores with and without Germinant Receptors and Cortex-Lytic Enzymes

“…In contrast, CaDPA-less spores of Bacillales species are very unstable and generally germinate spontaneously either within the sporulating cell or soon after release from the sporangium. Consequently, we would expect that C. sporogenes spores that lack CaDPA would also be unstable and rapidly germinate spontaneously as they indeed do ( Brunt et al, 2016 ).…”

“…Rather the latter spores contain the CLE SleC present in spores as an inactive zymogen, pro-SleC, which is activated by proteolytic cleavage early in germination. In contrast, C. sporogenes and Group I C. botulinum spores contain CwlJ and SleB, and at least SleB is needed for optimal germination and viability of Group I C. botulinum spores ( Meaney et al, 2015 ; Brunt et al, 2016 ). Spores of these two Clostridium species also contain IM GRs.…”