Nonorthologous replacement of lysyl-tRNA synthetase prevents addition of lysine analogues to the genetic code

Abstract

Insertion of lysine during protein synthesis depends on the enzyme lysyl-tRNA synthetase (LysRS), which exists in two unrelated forms, LysRS1 and LysRS2. LysRS1 has been found in most archaea and some bacteria, and LysRS2 has been found in eukarya, most bacteria, and a few archaea, but the two proteins are almost never found together in a single organism. Comparison of structures of LysRS1 and LysRS2 complexed with lysine suggested significant differences in their potential to bind lysine analogues with backbone replacements. One such naturally occurring compound, the metabolic intermediate S-(2-aminoethyl)-L-cysteine, is a bactericidal agent incorporated during protein synthesis via LysRS2. In vitro tests showed that S-(2-aminoethyl)-L-cysteine is a poor substrate for LysRS1, and that it inhibits LysRS1 200-fold less effectively than it inhibits LysRS2. In vivo inhibition by S-(2-aminoethyl)-L-cysteine was investigated by replacing the endogenous LysRS2 of Bacillus subtilis with LysRS1 from the Lyme disease pathogen Borrelia burgdorferi. B. subtilis strains producing LysRS1 alone were relatively insensitive to growth inhibition by S-(2-aminoethyl)-L-cysteine, whereas a WT strain or merodiploid strains producing both LysRS1 and LysRS2 showed significant growth inhibition under the same conditions. These growth effects arising from differences in amino acid recognition could contribute to the distribution of LysRS1 and LysRS2 in different organisms. More broadly, these data demonstrate how diversity of the aminoacyl-tRNA synthetases prevents infiltration of the genetic code by noncanonical amino acids, thereby providing a natural reservoir of potential antibiotic resistance.

Fig. 1.

Quantitation of LysRS1 protein levels by immunoblot analysis. Aliquots of total cell protein were separated by SDS/PAGE, transferred to membranes, and detected by using an anti-LysRS1 polyclonal antibody. The amounts of total cell protein loaded were as follows: B. subtilis strain 168, 20 μg; strain BCJ118.49, 20 μg; strain BCJ140.1 without xylose inducer, 20 μg; strain BCJ140.1 with xylose inducer, 1 μg; and strain BCJ157.1, 0.125 and 0.25 μg. The amount of B. burgdorferi total cell lysate loaded was 3 μg. Note that the level of LysRS1 present in this bacterium is significantly higher than that found in strain BCJ118.49 or strain BCJ140.1 (induced).

Fig. 2.

Determination of the B. subtilis tRNA^Lys charging level by using Northern analysis. Two micrograms of total RNA from each of the strains B. subtilis 168, BCJ143.3, and BCJ157.1 grown on rich (LB) media, was separated by electrophoresis, transferred to membrane, and probed with a B. subtilis-specific tRNA^Lys probe as detailed in Materials and Methods. Two micrograms of an RNA sample similarly prepared but treated under deacylation conditions was also loaded (deacyl.tRNA). The percentage charging for each tRNA sample is shown under each lane.

Fig. 3.

Determination of the level of B. burgdorferi tRNA^Lys charging by Northern analysis. Twenty micrograms of total RNA from each of the strains B. subtilis with vector only (168 VO), B. subtilis expressing tRNA^Lys1 (168 Lys), BCJ157.1 with vector only (157.1 VO), and BCJ157.1 expressing tRNA^Lys1 (157.1 Lys) was separated by electrophoresis, transferred to membrane, and probed with a B. burgdorferi tRNA^Lys1-specific probe as detailed in Materials and Methods. Twenty micrograms of an RNA sample was treated under deacylation conditions before loading (deacyl.tRNA). The percentage charging of tRNA^Lys1 is shown under each lane.

Fig. 4.

Analysis of growth inhibition by thialysine by using disk assay. Sixty milligrams of thialysine was loaded onto each disk, which was placed in the center of an agar Petri dish that had been lawned with the appropriate bacterial strain. The area of the bactericidal zone is shown at the bottom.

Fig. 5.

Aminoacylation of tRNA^Lys1 with thialysine by LysRS1. Aminoacylation reactions were performed as described by using either 3 mM thialysine (▪) or 3 mM lysine (•), in the presence of 2 μM tRNA^Lys1 and 100 nM B. burgdorferi LysRS1.