The orphan germinant receptor protein GerXAO (but not GerX3b) is essential for L-alanine induced germination in Clostridium botulinum Group II

Abstract

Clostridium botulinum is an anaerobic spore forming bacterium that produces the potent botulinum neurotoxin that causes a severe and fatal neuro-paralytic disease of humans and animals (botulism). C. botulinum Group II is a psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance and is a particular hazard in minimally heated chilled foods. Spore germination is a fundamental process that allows the spore to transition to a vegetative cell and typically involves a germinant receptor (GR) that responds to environmental signals. Analysis of C. botulinum Group II genomes shows they contain a single GR cluster (gerX3b), and an additional single gerA subunit (gerXAO). Spores of C. botulinum Group II strain Eklund 17B germinated in response to the addition of L-alanine, but did not germinate following the addition of exogenous Ca2+-DPA. Insertional inactivation experiments in this strain unexpectedly revealed that the orphan GR GerXAO is essential for L-alanine stimulated germination. GerX3bA and GerX3bC affected the germination rate but were unable to induce germination in the absence of GerXAO. No role could be identified for GerX3bB. This is the first study to identify the functional germination receptor of C. botulinum Group II.

Phage lysin that specifically eliminates Clostridium botulinum Group I cells

“…C. botulinum ATCC3502 spores were prepared as described 55 and fixed into a microfluidic plate using CellASIC ONIX2 microfluidic system (Merck Millipore) according to the manufacturer's instructions in an anaerobic workstation. TPGY medium was perfused into the microfluidic chamber with the pressure of 13.8 kPa for 6 h to enable sufficient spore germination.…”

CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research

“…The mechanism of a biphasic medium supporting efficient sporulation remains unclear, but it is possible that attachment to a solid surface induces sporulation ( Vlamakis et al, 2008 ). Liquid phases of nutrient-rich broths likely support growth better than a water phase, and thus we expected nutrient-rich liquid phases primarily to yield a larger number of sporulation-prone cells than water ( Peck et al, 1992 ; Braconnier et al, 2001 ; Brunt et al, 2018 ).…”

“…Peck (1992) used a biphasic sporulation medium consisting of a solid phase of CMM supplemented with agar and glucose, and a liquid phase of sterile water, to collect Group II spores for purification and heat inactivation assays (Peck et al, 1992). Other biphasic media with different solid phases and a liquid phase of sterile water have also been shown to support sporulation of Group II strains (Perkins, 1965;Bruch et al, 1968;Peck et al, 1992). The mechanism of a biphasic medium supporting efficient sporulation remains unclear, but it is possible that attachment to a solid surface induces sporulation (Vlamakis et al, 2008).…”

“…Several studies have shown evidence that biphasic media consisting of a solid agar phase and a liquid water phase can support efficient sporulation of Group II strains. However, the solid phase is prone to crumbling and thus makes sampling for many downstream applications difficult (Bruch et al, 1968;Peck et al, 1992). In order to facilitate spore biology research in C. botulinum Group II, a medium triggering efficient sporulation and enabling easy sampling is urgently needed.…”

Pan-Genomic Analysis of Clostridium botulinum Group II (Non-Proteolytic C. botulinum) Associated with Foodborne Botulism and Isolated from the Environment

“…This is the most comprehensive comparative genomic study of C. botulinum Group II undertaken to date. Additionally, the total of 208 genome sequences represent a valuable resource for the research community-for example, contributing to understanding the evolution of C. botulinum Group II and its neurotoxin genes, to the dissection of key aspects of the biology of C. botulinum Group II important for food chain transmission, such as spore formation, spore germination [52,53], psychrotrophy [54], neurotoxin formation [55][56][57], and in the targeting of PCR assays to detect and distinguish strains. Such information will contribute to more comprehensive microbiological risk assessments, the safe development of novel "fresh-like" foods, and the prevention of foodborne botulism.…”