Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi

Abstract

Lysyl-tRNAs are essential for protein biosynthesis by ribosomal mRNA translation in all organisms. They are synthesized by lysyl-tRNA synthetases (EC 6.1.1.6), a group of enzymes composed of two unrelated families. In bacteria and eukarya, all known lysyl-tRNA synthetases are subclass IIc-type aminoacyl-tRNA synthetases, whereas some archaea have been shown to contain an unrelated class I-type lysyl-tRNA synthetase. Examination of the preliminary genomic sequence of the bacterial pathogen Borrelia burgdorferi, the causative agent of Lyme disease, indicated the presence of an open reading frame with over 55% similarity at the amino acid level to archaeal class I-type lysyl-tRNA synthetases. In contrast, no coding region with significant similarity to any class II-type lysyl-tRNA synthetase could be detected. Heterologous expression of this open reading frame in Escherichia coli led to the production of a protein with canonical lysyl-tRNA synthetase activity in vitro. Analysis of B. burgdorferi mRNA showed that the lysyl-tRNA synthetase-encoding gene is highly expressed, confirming that B. burgdorferi contains a functional class I-type lysyl-tRNA synthetase. The detection of an archaeal-type lysyl-tRNA synthetase in B. burgdorferi and other pathogenic spirochetes, but not to date elsewhere in bacteria or eukarya, indicates that the gene that encodes this enzyme has a common origin with its orthologue from the archaeal kingdom. This difference between the lysyl-tRNA synthetases of spirochetes and their hosts may be readily exploitable for the development of anti-spirochete therapeutics.

Figure 1

Structure of the archaeal-type lysS ORF of B. burgdorferi. A putative σ⁷⁰-dependent −35/−10 promoter region is presented [the consensus sequence is shown above the boxed regions (38)]; a Shine–Delgano (SD) motif is indicated, as are the putative start (GTG) and stop (TAA) codons.

Figure 2

Alignment of class I-type LysRS amino acid sequences. The sequences were aligned by using the clustal w program (26). The sequences shown are from Archaeoglobus fulgidus (AF), B. burgdorferi (BB), M. jannaschii (MJ), M. maripaludis (MM), M. thermoautotrophicum (MT), and T. pallidum (TP).

Figure 3

Purification of B. burgdorferi His₆-LysRS. Protein purification was monitored by SDS/PAGE followed by staining with silver nitrate. Lane 1, S100 total protein extract from E. coli DH5α/pET15b-BBlysS; lane 2, S100 total protein extract from E. coli BL21(DE3)/pET15b-BBlysS following induction with isopropyl thiogalactoside; lane 3, purified His₆-LysRS; and lane M, molecular mass standards (sizes shown in kDa). The predicted molecular mass of His₆-LysRS is 63.2 kDa.

Figure 4

Aminoacylation of E. coli tRNA^Lys (1,400 pmol/A₂₆₀) by purified B. burgdorferi His₆-LysRS. Aminoacylation reactions were performed as described (20-μl samples) in the presence of 80 nM enzyme, 2 μM tRNA, and the following amino acids: •, 20 μM [¹⁴C]lysine; ○, 20 μM [¹⁴C]lysine and 800 μM unlabeled lysine.

Figure 5

Northern blot analysis of lysS transcripts in B. burgdorferi. Total RNA from B. burgdorferi strain B31 was isolated from cultures grown at 23°C and after a shift from 23°C to 35°C, separated on an agarose gel, blotted to a nylon membrane, and hybridized with a lysS probe (Upper). Mobilities of size standards (kb) are indicated on the left. The blot was subsequently hybridized with a flagellin probe to ensure equivalent loading of RNA in each lane (Lower).

Figure 6

Chromosomal location of the lysS gene in Borrelia. Total undigested DNA from B. hermsii strain HS1 (lane 1), B. burgdorferi strain B31 (lane 2), B. garinii strains G2 (lane 3) and IP2 (lane 4), and B. afzelii strain H2 (lane 5) were separated by field inversion electrophoresis on an agarose gel and stained with ethidium bromide (EtBr). Uncut and HindIII-digested λ DNA were included as size standards, as indicated to the left. The positions of the borrelial circular plasmids (cp), linear chromosome (lc), and linear plasmids (lp) are identified at the right. Duplicate Southern blots prepared from the same gel were hybridized at low stringency (2× SSC, 45°C) with probes specific for the lysS (KRS) and flagellin (FLA) genes. The relative positions of the λ size standards are indicated to the left of each blot.

Figure 7

Phylogenetic tree based on small subunit ribosomal RNA sequences (adapted from refs. and 40). The occurrence of lysS genes (10) encoding orthologues of either class I-type (indicated by 1) or class-II type (indicated by 2) LysRSs has been superimposed. The positioning of mitochondria within the bacterial lineage is after Barns et al. (39).