The large majority of intergenic sites in bacteria are selectively constrained, even when known regulatory elements are excluded

There are currently no broad estimates of the overall strength and direction of selection operating on intergenic variation in bacteria. Here we address this using large whole genome sequence datasets representing six diverse bacterial species; Escherichia coli, Staphylococcus aureus, Salmonella enterica, Streptococcus pneumoniae, Klebsiella pneumoniae, and Mycobacterium tuberculosis. Excluding M. tuberculosis, we find that a high proportion (62%-79%; mean 70%) of intergenic sites are selectively constrained, relative to synonymous sites. Non-coding RNAs tend to be under stronger selective constraint than promoters, which in turn are typically more constrained than rho-independent terminator