The UvrA-like protein Ecm16 requires ATPase activity to render resistance against echinomycin

Abstract

Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic-producing bacteria. In this work, we characterize the self-resistance protein Ecm16 encoded by echinomycin-producing bacteria. Ecm16 is a structural homolog of the nucleotide excision repair protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 binds DNA (double-stranded and single-stranded) using a nucleotide-independent binding mode. Ecm16's binding affinity for DNA increased by 1.7-fold when the DNA is intercalated with echinomycin. Ecm16 can render resistance against echinomycin toxicity independently of the nucleotide excision repair system. Similar to UvrA, Ecm16 has ATPase activity, and this activity is essential for Ecm16's ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each other's function. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin.

Keywords: DNA intercalator; Ecm16; SOS; UvrA; antibiotic resistance; echinomycin; nucleotide excision repair.

FIGURE 1

Expression of ecm16 gives resistance against echinomycin. (a) Structure of echinomycin. (b) Optical density after 6 h. Exposure to echinomycin results in reduction of E. coli growth in liquid media. Maximum optical densities were determined after 6 h exposure to echinomycin concentrations ranging from 1 μM to 20 μM. Echinomycin was added at time zero to cultures at 0.2 OD_600nm. E. coli K12 strains with pBAD vector‐control‐only (VCO) or pBAD‐ecm16 (p(ecm16) were used for comparison. Error bars represent standard error for duplicate replicates of one trial, analysis is representative of three independent trials. (c) Plots of optical density and corresponding colony forming units (CFUs) of K12/p(VCO) and K12/p(ecm16) strains. Overnight cultures were diluted to 0.1 OD₆₀₀ and grown over a 6‐hour period in LB containing 0, 1, 5, or 10 μM echinomycin. Samples taken every hour were serially diluted and plated on LB agar plates. The count (log CFU/ml) was determined from plates grown overnight at 37°C using MicrobeJ.

FIGURE 2

E. coli cells expressing ecm16 do not filament after echinomycin treatment. (a) Phase contrast microscopy of strains K12/p(VCO) (top panels) and K12/p(ecm16) (bottom panels) grown in the presence of inducer (arabinose 0.2%) supplemented with or without 5 μM echinomycin (ech). Cells were grown in the presence of echinomycin for 5 h and subsequently spotted on 1% agarose minimal media pads for imaging. Scale bar = 10 μm. (b) Scatter dot plots of cell size distribution of strains K12/p(VCO) and K12/p(ecm16) under wild‐type (WT), DsulA, and DrecA background supplemented with or without 5 μM echinomycin (ech). (c) Average cell length and standard error for 100 cells per condition are shown. 3% of cells displayed cell lengths over 25 μm (not included in plot). Cell lengths were measured using the software MicrobeJ. Representatives of three independent replicates are plotted.

FIGURE 3

DNA binding activity of Ecm16. (a) Reaction mixtures contained 0.25 nM DNA substrate in the absence (lane 1) or presence of 5 to 60 nM Ecm16 with increment of 5 nM (lanes 2–13), (a) ssDNA; (b) dsDNA; (c) echinomycin‐DNA. (b) Fraction of ssDNA (panel (a)), dsDNA (panel (b)), echinomycin‐DNA (panel (c)) bound to Ecm16 is plotted against the indicated amounts of Ecm16. ssDNA (filled circles); dsDNA (filled squares); echinomycin‐DNA (filled triangles). Each point on the curves represents the mean of three separate experiments. (c) DNA binding activity of Ecm16 in presence of 100% GC and AT, 75%/25% and 50%/50% GC/AT composition 0.25 nM DNA substrates in the absence (lane 1) or presence of 5 to 30 nM Ecm16 with increment of 5 nM (lanes 2–7).

FIGURE 4

ATPase activity of Ecm16. (a) Specific activity of 0.2 μM purified Ecm16 in the presence or absence of 1 μM DNA and DNA‐echinomycin substrates. Error bars represent standard error of three independent experiments. The reaction mixture contained 2‐amino‐6‐mercapto‐7‐methylpurine (MESG) and purine nucleoside phosphorylase, 1 mM ATP and 10 mM MgCl_2. A phosphate standard was measured to calibrate the UV absorbance signal to the amount of inorganic phosphate release. (b) Streak plates containing E. coli cultures containing a plasmid with WT ecm16 (p[emc16]), ecm16 ATP‐binding variant (p[emc16‐K526A]), or vector control (p(VCO)). Cultures were grown overnight and normalized to 0.5 OD₆₀₀ before streaking on LB ampicillin plates containing 0.2% arabinose or 1 μM echinomycin (with 0.2% arabinose) and were incubated at 37°C overnight. Plates are representative of 3 independent trials.

FIGURE 5

Ecm16 is not associated with classic NER function. (a) Exponential state growth curves (n = 3) of E. coli K12 strains with different components of the native nucleotide excision repair system (NER) knocked out (ΔuvrA, ΔuvrB, ΔuvrC, or ΔuvrD) and carrying vector‐control‐only or vector encoding ecm16. Cultures (2 ml) were set to OD_600nm ~ 0.2 in rich media (LB) supplemented with the inducer (0.2% arabinose) and varying concentrations of echinomycin (0–10 μM). Growth was monitored by measuring the absorbance at 600 nm every 30 min. E. coli K12 NER knockouts show similar patterns of resistance to echinomycin in the presence of inducer for the expression of ecm16. Results are shown for exponential growth phase with exponential trend line, error bars represent SEM of duplicate replicates. All results shown are representative of three independent replicates. (b) Colony forming unit (CFU) assays after UV radiation. 5 μl of cultures grown to OD_600nm ~ 0.2 were serial diluted (dilution factor = 5 × 10⁻¹ to 10⁻⁶) and spotted on LB plates supplemented with 0.2% arabinose. Cultures included the following strains: Control sample (top row) are wild‐type cells with vector‐control‐only, test samples (middle 2nd and 3rd rows) are strains with the native E. coli's uvrA gene knocked out and with vector‐control‐only or vector encoding ecm16, complementation control strain (bottom row) encodes E. coli's native uvrA gene in the same pBAD vector. Freshly spotted plates were exposed to UV radiation and then incubated at 37°C for 18 h prior to imaging. The data shown are a representative of three independent replicates.