Experimental evolution of gene essentiality in bacteria.

Essential gene products carry out fundamental cellular activities in interaction with other components. However, the lack of essential gene mutants and appropriate methodologies to link essential gene functions with their partners poses significant challenges. Here, we have generated deletion mutants in 32 genes previously identified as essential, with 23 mutants showing extremely slow growth in the SK36 strain of Streptococcus sanguinis . Whole-genome sequencing of 243 independently evolved populations of these mutants has identified >1000 spontaneous suppressor mutations in experimental evolution, many of which define new gene and pathway relationships, such as F1Fo-ATPase/V1Vo-ATPase/TrkA1-H1. Patterns of spontaneous mutations occurring in essential gene mutants differed from those found in wildtype. While gene duplications occurred rarely and appeared most often at later stages of evolution, substitutions, deletions, and insertions were prevalent in evolved populations. These essential gene deletion mutants and spontaneous mutations fixed in the mutant populations during evolution establish a foundation for understanding gene essentiality and the interaction of essential genes in networks.