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Abstract

Although enteric bacteria normally reside within the animal intestine, the ability to persist extraintestinally is an essential part of their overall lifestyle, and it might contribute to transmission between hosts. Despite this potential importance, few genetic determinants of extraintestinal growth and survival have been identified, even for the best-studied model, Escherichia coli. In this work, we thus used a genome-wide library of barcoded transposon insertions to systematically identify functional clusters of genes that are crucial for E. coli fitness in lake water. Our results revealed that inactivation of pathways involved in maintaining outer membrane integrity, nucleotide biosynthesis, and chemotaxis negatively affected E. coli growth or survival in this extraintestinal environment. In contrast, inactivation of another group of genes apparently benefited E. coli growth or persistence in filtered lake water, resulting in higher abundance of these mutants. This group included rpoS, which encodes the general stress response sigma factor, as well as genes encoding several other global transcriptional regulators and RNA chaperones, along with several poorly annotated genes. Based on this co-enrichment, we identified these gene products as novel positive regulators of RpoS activity. We further observed that, despite their enhanced growth, E. coli mutants with inactive RpoS had reduced viability in lake water, and they were not enriched in the presence of the autochthonous microbiota. This highlights the duality of the general stress response pathway for E. coli growth outside the host.