Laboratory-based evolution and whole-genome sequencing can link genotype and phenotype. We used evolution of acid resistance in exponential phase Escherichia coli to study resistance to a lethal stress. Iterative selection at pH 2.5 generated five populations that were resistant to low pH in early exponential phase. Genome sequencing revealed multiple mutations, but the only gene mutated in all strains was evgS, part of a two-component system that has already been implicated in acid resistance. All these mutations were in the cytoplasmic PAS domain of EvgS, and were shown to be solely responsible for the resistant phenotype, causing strong upregulation at neutral pH of genes normally induced by low pH. Resistance to pH 2.5 in these strains did not require the transporter GadC, or the sigma factor RpoS. We found that EvgS-dependent constitutive acid resistance to pH 2.5 was retained in the absence of the regulators GadE or YdeO, but was lost if the oxidoreductase YdeP was also absent. A deletion in the periplasmic domain of EvgS abolished the response to low pH, but not the activity of the constitutive mutants. On the basis of these results we propose a model for how EvgS may become activated by low pH.
Nematodes belong to one of the most diverse animal phyla. However, functional genomic studies in nematodes, other than in a few species, have often been limited in their reliability and success. Here we report that by combining liposome-based technology with microinjection, we were able to establish a wide range of genomic techniques in the newly described nematode genus Auanema. The method also allowed heritable changes in dauer larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As proof of concept for potential functional studies in other nematode species, we also induced RNAi in the free-living nematode Pristionchus pacificus and targeted the human parasite Strongyloides stercoralis.
3Nematodes belong to one of the most diverse animal phyla. However, functional genomic 1 4 studies in nematodes, other than in a few species, have often been limited in their reliability 1 5 and success. Here we report that by combining liposome-based technology with 1 6 microinjection, we were able to establish a wide range of genomic techniques in the newly 1 7 described nematode genus Auanema. The method also allowed heritable changes in dauer 1 8 larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As 1 9 3 1 the door to mechanistic studies in non-model systems. This allows us to ask questions that 3 2 cannot be addressed with well-established genetic model organisms 1,2 . 3 3 3 4
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