Experimental Evolution of Escherichia coli K-12 at High pH and with RpoS Induction

Abstract

Experimental evolution of K-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS knockout of (encoding the positive regulator of the phosphate regulon). A:: knockout increased growth at high pH. mutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations in [poly(A) polymerase] also increased growth at high pH. Three out of four populations showed deletions of , an inhibitor of TorR, which activates expression of (trimethylamine -oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma factor RpoS, either in the coding gene or in genes for regulators of RpoS expression. RpoS is required for survival at extremely high pH. In our microplate assay, deletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadaptors that block degradation (IraM, IraD, and IraP). Other genes with mutations after high-pH evolution encode regulators, such as those encoded by () (PhoPQ regulator), (nitrogen starvation sigma factor),, and , as well as envelope proteins, such as those encoded by and Overall, evolution at high pH selects for mutations in key transcriptional regulators, including and the stationary-phase sigma factor RpoS. in its native habitat encounters high-pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter the function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms.

Acid Experimental Evolution of the Extremely Halophilic Archaeon Halobacterium sp. NRC-1 Selects Mutations Affecting Arginine Transport and Catabolism

“…Our findings support previous reports of the importance of ISH elements in haloarchaeal 267 evolution (46), and the observations in Sulfolobus that large deletions and loss of function 268 mutations are fitness tradeoffs for surviving in stressful environments (55). Large deletions and 269 IS insertions are also common in experimental evolution of bacteria (20,21,26,54). We also 270 find evidence for accumulation of ploidy changes for the shorter minichromosome, pNRC100 271 (47).…”

“…A membrane-permeant acid, benzoic acid induces glutamate decarboxylase and drug 70 resistance regulons; yet these systems are lost or downregulated during experimental evolution 71 (Moore et al 2019 (20). At high external pH, E. coli survival requires the stress 72 sigma RpoS; yet generations of growth at high pH selects against RpoS expression and activity 73 (26). It was of interest to see whether similar patterns of reversal are found in archaea.…”

CCCP experimental evolution ofEscherichia coliselects for mutations that increase EmrA activity or that downregulate other PMF-driven drug efflux pumps

“…This result is thus consistent with the enhanced fitness shown by cecR mutation found in the C-B11 lineage. The mgrB gene is acid-inducible, via PhoPQ response (23) and shows mutations selected after high-pH evolution (11). The gene lptD forms part of the lipopolysaccharide transport slide (24), which could be relevant for CCCP membrane interactions.…”

“…Evolved strains show small pH growth effects independent of CCCP. Serial culture at low or high pH is known to shift the growth range (8,11). Thus, it was important to test the effect of pH alone during CCCP evolution at low or high pH.…”

“…Evolution experiments show how E. coli evolve over generations of pH-related stress and increase fitness under low pH, high pH, and benzoic acid exposure (8)(9)(10)(11). Such experiments often reveal surprising fitness tradeoffs.…”