Figure 1.
Rate of C. botulinum and C. sporogenes spore germination in the presence of selected amino acids.
Effect of L-alanine, L-cysteine, L-methionine, L-serine, L-phenylalanine and glycine (C. botulinum only) at 100 mM on germination of (a) C. botulinum ATCC3502 and (b) C. sporogenes ATCC15579. The effect of increasing L-alanine concentrations (0.5 mM–100 mM) on germination was also tested on (c) C. botulinum ATCC3502 and (d) C. sporogenes ATCC15579. Tests were conducted in 20 mM Tris buffer, pH 7.4, L-lactate (50 mM) and NaHCO3 (50 mM) at 30°C. Buffer only controls contained 20 mM Tris, pH 7.4, L-lactate (50 mM) and NaHCO3 (50 mM). 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.
Figure 2.
The effect of spore production environment on subsequent germination of C. sporogenes spores.
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.
Table 1.
Minimum D-amino acid concentration required to prevent* germination by its equivalent L-amino acid.
Figure 3.
Alignment of C. botulinum and C. sporogenes germinant receptor proteins.
Homologues of C. botulinum (strain ATCC3502) GRs were identified by BLASTp analyses using the draft un-assembled genome of C. sporogenes (strain ATCC15579). (a) Tree calculated (using Jalview [76]) from the pairwise sequence distances between GerXA only (determined from % sequence identities) of C. sporogenes (CLOSPO_number.) and C. botulinum (CBOnumber.) GRs, using the UPGMA algorithm [76]; 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 C. sporogenes 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 C. sporogenes homologue, CLOSPO_00838.
Figure 4.
Mutation of specific receptors precludes amino acid stimulated germination.
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 (C. botulinum only) or in TY + L-lactate (50 mM). (a) C. botulinum single insertional knockout mutants and wild type spore germination. (b) C. sporogenes 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 gerXA4-02140− 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 C. sporogenes were determined using spores generated from the wild type (C. sporogenes ATCC15579) and mutants gerXA1-00838−, gerXA4-02140−, gerXA3-02217−, gerXA2-03006−. 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.
Figure 5.
Germination rates of complemented GR mutants for C. botulinum and C. sporogenes.
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 (C. botulinum), L-alanine (C. sporogenes). (a) C. botulinum mutant gerXA1-0123− complemented with plasmid pMTL8315esp (gerXA1-0123−esp) or plasmid pMTL8315fdx (gerXA1-0123−fdx). (b) C. sporogenes mutant gerXA3-02217− complemented with plasmid pMTL8315esp (gerXA3-02217−esp) or plasmid pMTL8315fdx (gerXA3-02217−fdx). There were two negative controls. Firstly, the uncomplemented mutant (gerXA1-0123− or gerXA3-02217−), 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.
Figure 6.
Capacity of WT or mutant spores to germinate and form colonies.
C. botulinum and C. sporogenes 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.
Table 2.
Constructed mutants and plasmids utilised in this study.