Small multidrug resistance protein EmrE reduces host pH and osmotic tolerance to metabolic quaternary cation osmoprotectants

Abstract

The small multidrug resistance (SMR) transporter protein EmrE in Escherichia coli is known to confer resistance to toxic antiseptics classified as quaternary cation compounds (QCCs). Naturally derived QCCs synthesized during metabolic activities often act as osmoprotectants, such as betaine and choline, and participate in osmotic homoestasis. The goal of this study was to determine if EmrE proteins transport biological QCC-based osmoprotectants. Plasmid-encoded copies of E. coli emrE and the inactive variant emrE-E14C (emrE with the E → C change at position 14) were expressed in various E. coli strains grown in either rich or minimal media at various pHs (5 to 9) and under hypersaline (0.5 to 1.0 M NaCl and KCl) conditions to identify changes in growth phenotypes induced by osmoprotectant transport. The results demonstrated that emrE expression reduced pH tolerance of E. coli strains at or above neutral pH and when grown in hypersaline media at or above NaCl or KCl concentrations of 0.75 M. Hypersaline growth conditions were used to screen QCC osmoprotectants betaine, choline, l-carnitine, l-lysine, l-proline, and l-arginine. The study identified that betaine and choline are natural QCC substrates of EmrE.

Fig 1

pH susceptibility of E. coli BW25113 strains transformed with SMR plasmids. OD₆₀₀ values after 16 h of growth in either LB (A) or M9 (B) media at pH values ranging from 5 to 9 are shown. The results for E. coli BW25113 transformed with pMS119EH (black), pEmrE (gray), and active site mutant pEmrE-E14C (dark gray) after 16 h of growth in LB (A) or M9 (B) medium are shown in a bar chart format. Asterisks indicate statistically significant differences (P < 0.01) compared to either the untransformed strain without plasmid or the strain transformed with the empty control vector (pMS119EH). Panels C and D show pH susceptibility growth (OD₆₀₀) curve experiments with E. coli BW25113 transformed with pMS119EH (circles), pEmrE (squares), and pEmrE-E14C (triangles) grown at 37°C in either LB (C) or M9 (D) medium at pH 7.0 over 24 h.

Fig 2

Hypersaline susceptibility of E. coli BW25113 transformed with SMR plasmids. All panels show mean OD₆₀₀ values from plasmid-transformed E. coli BW25113 strains after 16 h of growth in either M9 (A and B) or LB (C and D) medium at hypersaline concentrations of NaCl (A and C) or KCl (B and D) ranging from 0.0 to 1.0 M salt. In each panel, the mean OD₆₀₀ values for E. coli BW25113 transformed with pMS119EH (black), pEmrE (gray), and pEmrE-E14C (dark gray) are provided in a bar chart format. Asterisks indicate statistically significant differences (P < 0.01) compared to either the untransformed strain lacking a plasmid (data not shown) or the strain transformed with the empty control vector (pMS119EH).

Fig 3

Growth phenotype screens of plasmid-transformed E. coli BW25113 in hypersaline M9 medium in the presence of various osmoprotectants. In all panels, the fold change in OD₆₀₀ (growth) after osmoprotectant addition was determined after 16 h of growth at 37°C. The fold changes in OD₆₀₀ after osmoprotectant addition in panels A and B are shown for E. coli transformed with plasmids pMS119EH (black), pEmrE (gray), and pEmrE-E14C (dark gray). All panels represent the fold changes in the growth of the culture in M9 medium at 1.0 M salt (NaCl or KCl) with 10 mM osmoprotectant divided from its mean OD₆₀₀ value in M9 medium at 1.0 M salt only, as indicated on the x axis. Panel A demonstrates the osmoprotection conferred by the addition of 10 mM osmoprotectant (refer to x axis) based on the growth of pMS119EH-transformed E. coli control strains in hypersaline (either 1.0 M NaCl or KCl) M9 medium. The area below the dashed line on this chart indicates any osmoprotectants that failed to exceed the 2.5-fold-cutoff value statistically determined to identify valid osmoprotectants for hypersaline EmrE osmoprotection screens shown in panel B. Panel B shows the results of hypersaline EmrE osmoprotection screens as the fold change in growth (OD₆₀₀) of E. coli BW25113(pEmrE) (gray) and BW25113(pEmrE-E14C) (dark gray) caused by the addition of 10 mM osmoprotectant (refer to x axis) to M9 medium containing either 1.0 M NaCl or KCl.

Fig 4

Mean fold change in growth (OD₆₀₀) of emrE plasmid-transformed E. coli BW25113 in the presence of betaine and choline. The fold change in OD₆₀₀ (growth) after osmoprotectant addition is shown on the y axis of both charts and represents the fold change in growth of plasmid-transformed E. coli BW25113 in hypersaline medium after the addition of 10 mM osmoprotectant (refer to x axis) at 16 h of growth at 37°C. In both panels, the fold change in growth after osmoprotectant addition is provided for E. coli BW25113 transformed with pMS119EH (black), pEmrE (gray), or pEmrE-E14C (dark gray) in hypersaline M9 medium at 0.5, 0.75, and 1.0 M NaCl or KCl in the presence and absence of 10 mM betaine (A) or choline (B). Asterisks indicate statistically significant differences (P < 0.01) compared to either the untransformed strain lacking a plasmid (data not shown) or the strain transformed with the empty control vector (pMS119EH).

Fig 5

Hypersaline tolerance of emrE plasmid-transformed E. coli JW0303 and JW0304 strains grown in the presence of betaine and choline. On the y axis, both panels show the fold change in OD₆₀₀ (growth) after osmoprotectant addition to the medium after 16 h of growth in either LB (A) medium or M9 (B) medium at a 1.0 M concentration of NaCl or KCl and 10 mM betaine or choline osmoprotectants. E. coli strains JW0303 (ΔbetA) and JW0304 (ΔbetB) transformed with pMS119EH (black), pEmrE (gray), and active site mutant pEmrE-E14C (dark gray) are shown on the x axis. The three-letter abbreviations provided on the x axes of both panels A and B indicate the culture medium compositions and are defined below. Panel A shows results for plasmid-transformed JW0303 and JW0304 after 16 h of growth in LB medium containing NaCl with betaine (LSB) or choline (LSC) and LB medium containing KCl with betaine (LKB) or choline (LKC). Panel B shows the fold change in growth after osmoprotectant addition to plasmid-transformed JW0303 and JW0304 after 16 h of growth in M9 medium containing NaCl with betaine (MSB) or choline (MSC) and M9 medium containing KCl with betaine (MKB) or choline (MKC). Asterisks indicate statistically significant differences (P < 0.01) compared to either the untransformed strain without plasmid or the strain transformed with the empty control vector (pMS119EH).