Increased Fitness and Alteration of Metabolic Pathways during Bacillus subtilis Evolution in the Laboratory

Five batch cultures of Bacillus subtilis were subjected to evolution in the laboratory for 6,000 generations under conditions repressing sporulation in complex liquid medium containing glucose. Between generations 1,000 and 2,000, variants with a distinct small-colony morphology arose and swept through four of the five populations that had been previously noted for their loss of sporulation (H. Maughan et al., Genetics 177: 937-948, 2007). To better understand the nature of adaptation in these variants, individual strains were isolated from one population before (WN715) and after (WN716) the sweep. In addition to colony morphology, strains WN715 and WN716 differed in their motility, aerotaxis, and cell morphology. Competition experiments showed that strain WN716 had evolved a distinct fitness advantage over the ancestral strain and strain WN715 during growth and the transition to the postexponential growth phase, which was more pronounced when WN715 was present in the coculture. Microarray analyses revealed candidate genes in which mutations may have produced some of the observed phenotypes. For example, loss of motility in WN716 was accompanied by decreased transcription of all flagellar, motility, and chemotaxis genes on the microarray. Transcription of alsS and alsD was also lower in strain WN716, and the predicted loss of acetoin production and enhanced acetate production was confirmed by high-performance liquid chromatography (HPLC) analysis. The results suggested that the derived colony morphology of strain WN716 was associated with increased fitness, the alteration of several metabolic pathways, and the loss of a typical postexponential-phase response.