The origins of 168, W23, and other Bacillus subtilis legacy strains

Abstract

Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three--NCIB 3610T, PY79, and SMY--we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more "W23 islands" in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains.

FIG. 1.

DNA sequence heterogeneity in the trp region of B. subtilis legacy strains. The arrows show reading frames in the B. subtilis 168 genome, while the axis indicates the corresponding position in the genome sequence. For each strain, boxes indicate the sizes and positions of contiguous DNA sequences determined in this study. Open boxes show sequences that are essentially identical to that of strain 168, while hatched boxes show sequences identical to that of W23. The upward-facing triangle (▴) shows the position of the trpC2 deletion, while the inverted triangle (▾) shows the position of the gudB duplication. The strain group labeled “168,3610^T” also includes ATCC 6051^T and the Burkholder and Giles mutants 122, 160, and 166.

FIG. 2.

Visualization of DNA sequence heterogeneity revealed by genome resequencing of B. subtilis strains SMY (A) and PY79 (B). For each figure, four panels are shown aligned to the position in the B. subtilis genome sequence. Bar lines in the first row indicate called SNPs, as follows: black bars, nonsense SNPs; blue bars, missense SNPs; light blue bars, silent (no amino acid change) SNPs; turquoise bars, SNPs within intergenic region. Blue bars in the second row show uncalled ROI. Gold lines in the third row show fully resequenced residues, and red lines in the fourth row show the net difference (arbitrary units) in hybridization intensity between the two tested strains, with the indicated “peaks” indicating large hybridization differences. For PY79, the positions of areas 1 to 6 are marked.

FIG. 3.

SNPs shared among B. subtilis 168, SMY, and PY79. Numbers indicate which SNPs are unique to a strain, which are shared between two strains, and which are shared among all three strains. The wild-type Marburg strain NCIB3610^T was chosen as the root sequence for this comparison. Residues falling within “W23 islands” were excluded from the comparison.

FIG. 4.

Correlation between sequence divergence and enzyme activity of PanB. The top panel shows the positions of the amino acid differences between the PanB enzymes from 168 and PY79; the bent arrow represents the wild-type sigma A promoter that controls transcription of panB. The bottom panel compares the activities of the His₆-tagged PanB enzymes isolated from B. subtilis 168 and PY79 (with the latter containing the 29-kb B. subtilis W23-derived DNA island surrounding the trp locus). The values are means for three independent experiments.

FIG. 5.

Model describing the history of early B. subtilis Trp⁺ legacy strains. The genomic heritage of individual strains is represented by shading, as follows: white cells, 168-like genomes; black cells, W23-like genomes; gray cells, 168-W23 hybrid genomes. The event leading to the isolation of a legacy strain is represented by a straight arrow (transformation, transduction, domestication, contamination, or renaming), zigzag arrow (mutagenesis through radiation), or dashed arrow (inferred but undocumented event). Rectangular boxes surround isolation events that are documented in the publications indicated.