EscherichiaEscherichia colicoli responses to acid-stress: signal transduction and gene regulation

Microbial lab-based evolution is a technique to study evolutionary theory. It is a method which can provide insights into the ability of a microbe to adapt to a biological process such as low pH. To investigate pathways that could lead to an acid resistant phenotype in E. coli, we evolved six independent lines or populations of E. coli K-12 MG1655 by iterative growth and dilution experiments for approximately 740 generations at pH 4.5. Clones isolated from evolved populations were significantly fitter than the ancestor at pH 4.5. Five of the six evolved strains had acquired an identical mutation in rpoA, and mutations in cytR in addition to other mutations. PCR analysis of the fossil record of the evolved populations showed that the arcA mutations always arose first followed by the rpoA mutations. Investigating the genetic basis of adaptation showed that the mutations in arcA were loss of function in nature and conferred caused an intermediate increase in fitness. Transcriptional analysis showed a global change in their transcriptional signatures with significant upregulation of the arcA regulon. Our study showed that loss of function of ArcA caused an increase in the RpoS activity of the acid evolved strains leading to a general stress resistant phenotype

Structural and functional analysis of the Escherichia coli acid-sensing histidine kinase EvgS

The EvgS/EvgA two-component system of Escherichia coli is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in E. coli and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this