Whole-Genome Sequencing and Phenotypic Analysis of Bacillus subtilis Mutants following Evolution under Conditions of Relaxed Selection for Sporulation ▿

Little is known about how genetic variation at the nucleotide level contributes to competitive fitness within species. During a 6,000-generation study of Bacillus subtilis evolved under relaxed selection for sporulation, a new strain, designated WN716, emerged with significantly different colony and cell morphologies; loss of sporulation, competence, acetoin production, and motility; multiple auxotrophies; and increased competitive fitness (H. Maughan and W. L. Nicholson, Appl. Environ. Microbiol. 77:4105–4118, 2011). The genome of WN716 was analyzed by OpGen optical mapping, whole-genome 454 pyrosequencing, and the CLC Genomics Workbench. No large chromosomal rearrangements were found; however, 34 single-nucleotide polymorphisms (SNPs) and +1 frameshifts were identified in WN716 that resulted in amino acid changes in coding sequences of annotated genes, and 11 SNPs were located in intergenic regions. Several classes of genes were affected, including biosynthetic pathways, sporulation, competence, and DNA repair. In several cases, attempts were made to link observed phenotypes of WN716 with the discovered mutations, with various degrees of success. For example, a +1 frameshift was identified at codon 13 of sigW, the product of which (SigW) controls a regulon of genes involved in resistance to bacteriocins and membrane-damaging antibiotics. Consistent with this finding, WN716 exhibited sensitivity to fosfomycin and to a bacteriocin produced by B. subtilis subsp. spizizenii and exhibited downregulation of SigW-dependent genes on a transcriptional microarray, consistent with WN716 carrying a knockout of sigW. The results suggest that propagation of B. subtilis for less than 2,000 generations in a nutrient-rich environment where sporulation is suppressed led to rapid initiation of genomic erosion