Functional Characterisation of Germinant Receptors in <i>Clostridium botulinum</i> and <i>Clostridium sporogenes</i> Presents Novel Insights into Spore Germination Systems

<div><p><i>Clostridium botulinum</i> is a dangerous pathogen that forms the highly potent botulinum toxin, which when ingested causes a deadly neuroparalytic disease. The closely related <i>Clostridium sporogenes</i> is occasionally pathogenic, frequently associated with food spoilage and regarded as the non-toxigenic equivalent of Group I <i>C. botulinum</i>. Both species form highly resistant spores that are ubiquitous in the environment and which, under favourable growth conditions germinate to produce vegetative cells. To improve the control of botulinum neurotoxin-forming clostridia, it is imperative to comprehend the mechanisms by which spores germinate. Germination is initiated following the recognition of small molecules (germinants) by a specific germinant receptor (GR) located in the spore inner membrane. The present study precisely defines clostridial GRs, germinants and co-germinants. Group I <i>C. botulinum</i> ATCC3502 contains two tricistronic and one pentacistronic GR operons, while <i>C. sporogenes</i> ATCC15579 has three tricistronic and one tetracistronic GR operons. Insertional knockout mutants, allied with characterisation of recombinant GRs shows for the first time that amino acid stimulated germination in <i>C. botulinum</i> requires two tri-cistronic encoded GRs which act in synergy and cannot function individually. Spore germination in <i>C. sporogenes</i> requires one tri-cistronic GR. Two other GRs form part of a complex involved in controlling the rate of amino-acid stimulated germination. The suitability of using <i>C. sporogenes</i> as a substitute for <i>C. botulinum</i> in germination studies and food challenge tests is discussed.</p></div

Mutation of specific receptors precludes amino acid stimulated germination.

<p>Spores were incubated in 20 mM Tris buffer, pH 7.4, amino acid (100 mM) + L-lactate (50 mM) and NaHCO₃ (50 mM) at 30°C for 20 hours with L-alanine, L-cysteine, L-methionine, L-serine, L-phenylalanine, glycine (<i>C. botulinum</i> only) or in TY + L-lactate (50 mM). (a) <i>C. botulinum</i> single insertional knockout mutants and wild type spore germination. (b) <i>C. sporogenes</i> single insertional knockout mutants, triple insertional knockout mutants, quadruple insertional knockout GR mutant and wild type spore germination. * L-cysteine is a relatively insoluble amino acid and precipitates out of solution after 2 hours. Due to the 4 hour delay in germination of the mutant <i>gerXA4</i>-02140⁻ cysteine precipitation caused OD₆₀₀ readings to be unrepresentative and therefore germination was confirmed by microscopy. (c) Alanine induced germination rates of single insertional knockout GR mutants for <i>C. sporogenes</i> were determined using spores generated from the wild type (<i>C. sporogenes</i> ATCC15579) and mutants <i>gerXA1</i>-00838⁻, <i>gerXA4</i>-02140⁻, <i>gerXA3</i>-02217⁻, <i>gerXA2</i>-03006⁻. WT spores incubated in buffer only (see above), were included as a negative control (WT Buffer). Data labels (right) refer to percentage germination observed by phase contrast microscopy at the end of the experiment. Error bars in (a–c) represent the standard deviation of 3 independent experiments. Spore germination was confirmed by phase contrast microscopy.</p

Minimum D-amino acid concentration required to prevent^* germination by its equivalent L-amino acid.

<p>Tests were conducted in 20 mM Tris buffer, pH 7.4, L-amino acid (1–10 mM), D-amino acid (10–100 mM) + L-lactate (50 mM) and NaHCO₃ (50 mM) at 30°C. NT = D-phenylalanine was not inhibitory at 20 mM and could not be tested at a higher concentration due to low solubility.</p><p>*Defined as a <10% fall in OD₆₀₀, equating to <1% germination observed microscopically.</p><p>Minimum D-amino acid concentration required to prevent^{<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004382#nt102" target="_blank">*</a>} germination by its equivalent L-amino acid.</p

Constructed mutants and plasmids utilised in this study.

<p>− indicates the gene is absent; + indicates that only that gene is functional; ^no− describes the number of genes knocked out. a = antisense orientation insertion site; s = sense orientation insertion site. The designation of “<i>X</i>” before the letter A is used in clostridia as unlike <i>Bacillus</i>, GRs in these species have not yet been attributed to specific germinants.</p><p>Constructed mutants and plasmids utilised in this study.</p

The effect of spore production environment on subsequent germination of <i>C. sporogenes</i> spores.

<p>For germination tests, spores were incubated in 20 mM Tris buffer, pH 7.4, L-alanine (100 mM), L-lactate (50 mM) and NaHCO₃ (50 mM) at 30°C. Spores were produced in CMB at different temperatures; 15°C, 20°C, 28°C, 30°C, 37°C. Spores were also produced in TY broth at 37°C and on RCM+SM plates at 37°C. Spores produced at 37°C in CMB and incubated in 20 mM Tris, pH 7.4, L-lactate (50 mM) and NaHCO₃ (50 mM) only, were included as a negative control (WT Buffer). Spore germination was confirmed by phase contrast microscopy. Data labels (right) refer to percentage germination observed by phase contrast microscopy at the end of the experiment. Error bars represent the standard deviation of 3 independent experiments.</p

Alignment of <i>C. botulinum</i> and <i>C. sporogenes</i> germinant receptor proteins.

<p>Homologues of <i>C. botulinum</i> (strain ATCC3502) GRs were identified by BLASTp analyses using the draft un-assembled genome of <i>C. sporogenes</i> (strain ATCC15579). (a) Tree calculated (using Jalview <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004382#ppat.1004382-Waterhouse1" target="_blank">[76]</a>) from the pairwise sequence distances between GerXA only (determined from % sequence identities) of <i>C. sporogenes</i> (CLOSPO_number.) and <i>C. botulinum</i> (CBOnumber.) GRs, using the UPGMA algorithm <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004382#ppat.1004382-Waterhouse1" target="_blank">[76]</a>; average distances between GerXA (green) are shown on the branches. GerXB (red) and GerXC (yellow) are shown on the same tree (UPGMA produced identical-topology trees for each of the GerXB, GerXC proteins; distances not shown). (black triangle) Position of insertion sites of retargeted introns for mutations (in equivalent DNA sequence). Small green coloured region of CBO1974 represents a small protein fragment (CBO1973A), the coding sequence of which overlaps that of CBO1974, with homology to the C terminus of a GerXA protein. Blue square; 20 amino acid section that is deleted in its <i>C. sporogenes</i> homologue, CLOSPO_00838. (b) More detailed version of part of the above tree, showing the amino acid sequence encoded by a region in CBO0123 that is deleted from its <i>C. sporogenes</i> homologue, CLOSPO_00838.</p

Germination rates of complemented GR mutants for <i>C. botulinum</i> and <i>C. sporogenes</i>.

<p>Spores were incubated in 20 mM Tris buffer, pH 7.4, amino acid (100 mM) + L-lactate (50 mM) + NaHCO₃ (50 mM) at 30°C with L-cysteine (<i>C. botulinum</i>), L-alanine (<i>C. sporogenes</i>). (a) <i>C. botulinum</i> mutant <i>gerXA1</i>-0123⁻ complemented with plasmid pMTL8315esp (<i>gerXA1</i>-0123⁻esp) or plasmid pMTL8315fdx (<i>gerXA1</i>-0123⁻fdx). (b) <i>C. sporogenes</i> mutant <i>gerXA3</i>-02217⁻ complemented with plasmid pMTL8315esp (<i>gerXA3</i>-02217⁻esp) or plasmid pMTL8315fdx (<i>gerXA3</i>-02217⁻fdx). There were two negative controls. Firstly, the uncomplemented mutant (<i>gerXA1</i>-0123⁻ or <i>gerXA3</i>-02217⁻), secondly WT spores incubated in 20 mM Tris buffer (pH 7.4) + L-lactate (50 mM) + NaHCO₃ (50 mM) only (WT Buffer). Spore germination was confirmed by phase contrast microscopy. Error bars represent the standard deviation of 3 independent experiments. Data labels (right) refer to percentage germination observed by phase contrast microscopy at the end of the experiment.</p

Capacity of WT or mutant spores to germinate and form colonies.

<p><i>C. botulinum</i> and <i>C. sporogenes</i> wild type and mutant spore suspensions were enumerated using a haemocytometer and adjusted to a final concentration of ∼1×10⁸ spores/ml. Spores were heat activated (80°C, 15 min), serially diluted in 0.85% saline, and plated in triplicate on to TY agar before anaerobic incubation (37°C, 48 hrs), after which colonies were enumerated. Data presented represent the mean log₁₀ colony-forming units/ml from triplicate plates, with error bars representing the standard deviation of the mean.</p