Abstract
Many bacterial species are represented by a pan-genome, whose genetic repertoire far outstrips that of any single bacterial genome. Here we investigate how a bacterial pan-genome might influence gene essentiality and whether essential genes that are initially critical for the survival of an organism can evolve to become non-essential. By using Transposon insertion sequencing (Tn-seq), whole-genome sequencing and RNA-seq on a set of 36 clinical Streptococcus pneumoniae strains representative of >68% of the species' pan-genome, we identify a species-wide 'essentialome' that can be subdivided into universal, core strain-specific and accessory essential genes. By employing 'forced-evolution experiments', we show that specific genetic changes allow bacteria to bypass essentiality. Moreover, by untangling several genetic mechanisms, we show that gene essentiality can be highly influenced by and/or be dependent on: (1) the composition of the accessory genome, (2) the accumulation of toxic intermediates, (3) functional redundancy, (4) efficient recycling of critical metabolites and (5) pathway rewiring. While this functional characterization underscores the evolvability potential of many essential genes, we also show that genes with differential essentiality remain important antimicrobial drug target candidates, as their inactivation almost always has a severe fitness cost in vivo.