Silencing by H-NS Potentiated the Evolution of Salmonella

Abstract

The bacterial H-NS protein silences expression from sequences with higher AT-content than the host genome and is believed to buffer the fitness consequences associated with foreign gene acquisition. Loss of H-NS results in severe growth defects in Salmonella, but the underlying reasons were unclear. An experimental evolution approach was employed to determine which secondary mutations could compensate for the loss of H-NS in Salmonella. Six independently derived S. Typhimurium hns mutant strains were serially passaged for 300 generations prior to whole genome sequencing. Growth rates of all lineages dramatically improved during the course of the experiment. Each of the hns mutant lineages acquired missense mutations in the gene encoding the H-NS paralog StpA encoding a poorly understood H-NS paralog, while 5 of the mutant lineages acquired deletions in the genes encoding the Salmonella Pathogenicity Island-1 (SPI-1) Type 3 secretion system critical to invoke inflammation. We further demonstrate that SPI-1 misregulation is a primary contributor to the decreased fitness in Salmonella hns mutants. Three of the lineages acquired additional loss of function mutations in the PhoPQ virulence regulatory system. Similarly passaged wild type Salmonella lineages did not acquire these mutations. The stpA missense mutations arose in the oligomerization domain and generated proteins that could compensate for the loss of H-NS to varying degrees. StpA variants most able to functionally substitute for H-NS displayed altered DNA binding and oligomerization properties that resembled those of H-NS. These findings indicate that H-NS was central to the evolution of the Salmonellae by buffering the negative fitness consequences caused by the secretion system that is the defining characteristic of the species.

Author Summary

H-NS is an abundant DNA-binding protein found in enteric bacteria including the important pathogens Escherichia, Salmonella, Vibrio, and Yersinia, that plays a primary role in defending the bacterial genome by silencing AT-rich foreign genes. H-NS has been hypothesized to facilitate the evolution of bacterial species by acting as a buffer against the negative consequences that can occur when new genes are incorporated into pre-existing genetic landscapes. Here experimental evolution and whole-genome sequencing were employed to determine the factors underlying the severe growth defects displayed by Salmonella strains lacking H-NS. Through tracking the evolution of several independently derived mutant lineages, we find that compensatory mutations arise quickly and that they occur in loci related to virulence. A frequent outcome was loss of the Salmonella Pathogenicity Island-1, the defining genetic island of the genus Salmonella. Among other things these findings demonstrate that H-NS has enabled the birth of a new and important bacterial pathogen by buffering the fitness consequences caused by overexpression of SPI-1. These findings are likely generalizable to pathogens such as E. coli, Yersinia, Shigella, and Vibrio cholerae, all of which maintain a pool of “expensive” AT-rich virulence genes that are repressed by H-NS.