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.

Highlights

  • Horizontal gene transfer (HGT) has profoundly shaped the course of bacterial speciation and diversification

  • 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 HNS has enabled the birth of a new and important bacterial pathogen by buffering the fitness consequences caused by overexpression of SPI-1

  • The alleviating effect of rpoS mutations in the hns mutants may be due to the fact that loss of H-NS dramatically improves the stability of RpoS [58], which may cause the inappropriate overexpression of stationary-phase genes and interfere with the expression of housekeeping genes controlled by RpoD

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Summary

Introduction

Horizontal gene transfer (HGT) has profoundly shaped the course of bacterial speciation and diversification. SPI-1 is a 40 kb genomic island encoding a Type 3 Secretion System (TTSS) required for triggering inflammation and for invasion of cells lining the intestinal mucosa [9,10,11,12]. Together these systems enable Salmonella to outcompete other microbes in the mammalian gut where SPI-1 induces a potent oxidative inflammation that generates tetrathionate, which serves as a terminal electron acceptor for anaerobic respiration that is available solely to Salmonella but not other gut microbes [7]

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