Mechanisms of resistance to an amino acid antibiotic that targets translation

Abstract

Structural and functional diversity among the aminoacyl-tRNA synthetases prevent infiltration of the genetic code by noncognate amino acids. To explore whether these same features distinguish the synthetases as potential sources of resistance against antibiotic amino acid analogues, we investigated bacterial growth inhibition by S-(2-aminoethyl)-L-cysteine (AEC). Wild-type lysyl-tRNA synthetase (LysRS) and a series of active site variants were screened for their ability to restore growth of an Escherichia coli LysRS null strain at increasing concentrations of AEC. While wild-type E. coli growth is completely inhibited at 5 microM AEC, two LysRS variants, Y280F and F426W, provided substantial resistance and allowed E. coli to grow in the presence of up to 1 mM AEC. Elevated resistance did not reflect changes in the kinetics of amino acid activation or tRNA (Lys) aminoacylation, which showed at best 4-6-fold improvements, but instead correlated with the binding affinity for AEC, which was decreased approximately 50-fold in the LysRS variants. In addition to changes in LysRS, AEC resistance has also been attributed to mutations in the L box riboswitch, which regulates expression of the lysC gene, encoding aspartokinase. The Y280F and F426W LysRS mutants contained wild-type L box riboswitches that responded normally to AEC in vitro, indicating that LysRS is the primary cellular target of this antibiotic. These findings suggest that the AEC resistance conferred by L box mutations is an indirect effect resulting from derepression of lysC expression and increased cellular pools of lysine, which results in more effective competition with AEC for binding to LysRS.

Figure 1

Model of L-lysine recognition by the active site of E. coli LysRS (40). H-bonds are shown as dashed lines. The carbon backbone for the substrate lysine is gold, while the carbon backbone of the LysRS2 active site residues is gray. Oxygen and nitrogen are colored red and blue, respectively, for enzyme and substrate.

Figure 2

Growth of E.coli PALΔSΔUTR derivatives on MM supplemented with 5 μM AEC. Cells contained the vector pXLysSK1 encoding either wild-type LysRS or active site variants as indicated. Each time point represents an average reading derived from three independent cultures grown in 96 well microtiter plates.

Figure 3

Growth of E. coli PALΔSΔUTR derivatives in the presence of AEC. Cells containing the vector pXLysSK1 encoding LysRS2 Y280F (a) or F426W (b) were grown on MM supplemented with different concentrations of AEC (μM) as indicated. Each time point represents an average reading derived from three independent cultures grown in 96 well microtiter plates.

Figure 4

LysRS in E. coli PALΔSΔUTR derivatives. E. coli PALΔSΔUTR cells producing wild-type (WT), Y280F, and Y280F4 LysRS were grown in different media as indicated. Total cell protein from late log phase (A₆₀₀ = 0.8) cells grown on LB, MM supplemented with 2 mM lysine (MM), or MM supplemented with 5 μM AEC (AEC) were separated by SDS–PAGE, transferred to membranes, and detected by using anti-LysRS2 polyclonal antibodies. Total protein concentrations loaded were 20 μg (lanes 2 and 6), 10 μg (lanes 3 and 7), 1 μg (lanes 4 and 8), and 0.1 μg (lanes 5 and 9). Purified LysRS (10 ng, lane 1) was loaded as control.

Figure 5

RNase H cleavage of the E. coli lysC leader RNA. Labeled RNAs generated in the presence of increasing concentrations of lysine (a) or AEC (b) were incubated with a DNA oligonucleotide complimentary to the 3′ side of helix 1 to allow hybridization, which was followed by digestion of DNA/RNA hybrids with RNase H. FL indicates full-length transcripts; C indicates RNase H cleavage products. Lysine or AEC concentrations (mM) used in each transcription are shown above each lane. c) Quantitation of protection in response to lysine (■) or AEC (▲).

Figure 6

Model of L-lysine recognition by LysRS variants Y280F and F426W. Modeling of lysine binding at the active site of E. coli LysRS (40) for the Y280F (a) and F426W (b) variants. Residues were modified appropriately, and the structures were energy minimized using Swiss-Pdb Viewer v 3.7. The resulting models were visualized using PyMOL (41). H-bonds are shown as dashed lines. The carbon backbone for the substrate lysine is gold, while the carbon backbone of the LysRS2 active site residues is gray. Oxygen and nitrogen are colored red and blue, respectively, for enzyme and substrate.