Table 1.
Strains used in this study.
Fig 1.
Phylogenetic tree of C. botulinum Group I and C. sporogenes Isolates.
A maximum likelihood phylogeny inferred from core genome single nucleotide polymorphisms (SNPs) to show the strains analyzed in this study in the context of other C. botulinum Group I and C. sporogenes strains. Strains used in this study are highlighted in blue boxes.
Fig 2.
Principal component analysis reveals growth phase as a major factor in protein expression patterns.
Cell mass protein expression data (post-filtering) from all strains, including that at all time points and in both medium types, was analyzed by PCA. (A) Separation on PC1 (x-axis) demonstrates that a major influence on protein expression patterns is the growth phase from which cells were harvested, as datasets from the early time points (OD600 = 0.4 and 0.8; red and green, respectively) segregate from those taken at the late time point (5 days; blue). While samples clearly separate into early and late growth phase “groups” in part (A), (B) medium type shows a slight differentiation in data (mostly from the early time points). A similar grouping is not observed when data are colored by (C) strain identity. The same plot is shown in parts A-C, and has been colored by different factors for visualization.
Fig 3.
Proteins involved in growth and metabolic pathways are differentially expressed during early and late growth phases.
Expression of proteins from many metabolic pathways/processes in early and late growth phases were significantly different by t-test. For visualization, proteins with significantly different expression profiles from six pathways/processes were selected: A) ribosomal proteins, B) translation/transcription elongation and initiation factors, C) RNA Polymerase subunits and sigma factor, D) tRNA synthetases, E) glycolytic enzymes, and F) enzymes of the glycine reductase system. Clusters A-E demonstrate higher expression during early time points and lower expression during late phase growth, while the reverse is true for cluster F. Data included in this analysis were z-score transformed spectral count values for selected proteins in cell fractions from all strains. Samples are represented across the horizontal axis and proteins along the vertical axis. Clustering of samples across the top separated early and late time point samples as noted. Heat map coloring is from blue (low expression) to yellow (high expression).
Fig 4.
Growth phase-dependent expression of enzymes involved in acetate and butyrate fermentation.
Proteins involved in the fermentation pathways shown were identified in this study, with enrichment of acetate fermentation (A) during exponential phase growth, and butyrate fermentation (B) during stationary phase growth. All enzymes in these pathways were identified across all strains and in both medium types examined, and expression patterns are shown in the heat map on the right hand side for each enzyme identified in the pathways. Blue shading indicates lower protein expression, yellow shading represents higher protein expression, and gray indicates that the protein was not observed in the sample. Protein expression data from all cell fraction samples are included in the heat map, shown from left to right as columns in the heat map.
Fig 5.
Metabolic pathways related to protein and amino acid metabolism.
A subset of proteins were identified which are involved in the breakdown of proteins and amino acids for energy. This partial metabolic network shows how these metabolic features are influenced by growth phase. Red text indicates enriched observation of the protein during exponential phase, while green indicates enrichment at later stationary phase. Black indicates mixed observation during different growth phases.
Fig 6.
Botulinum neurotoxin and toxin-associated protein expression is highest in late phase growth.
C. botulinum (A) ATCC 3502, (B) ATCC 19397, and (C) ATCC 17841 express botulinum neurotoxin, non-toxin non-hemagglutinin (NTNH), and three hemagglutinin proteins (HA-17, HA-34, and HA-70), while (D) ATCC 35415 expresses botulinum neurotoxin, NTNH, and a different complex of associated proteins, including P-47, OrfX-2, and OrfX-3. The additional OrfX-1 protein was not detected in our experiments. Average protein abundance (spectral counts) of toxin complex proteins in cell and SN fractions are presented. Error bars denote standard deviation. Early time points (including OD600 = 0.4 and 0.8) are shown in white bars for cell fraction and gray bars for SN (note that all values were zero for early SN samples). The late time point (5 days) is represented in black bars for the cellular fraction and striped pattern for the SN fraction.
Table 2.
Protein relative abundance ratios as signatures of cultivation conditions.