Direct glutaminyl-tRNA biosynthesis and indirect asparaginyl-tRNA biosynthesis in Pseudomonas aeruginosa PAO1

Abstract

The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA. We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS). The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P. aeruginosa PAO1, with the homologous tRNA as the substrate. The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P. aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNA(Gln), that AspRS is nondiscriminating, and that its Asp-tRNA(Asn) product is transamidated by AdT. On the other hand, tRNA(Gln) is directly glutaminylated by GlnRS. These results show that P. aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway. The essential role of AdT in the formation of Asn-tRNA in P. aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen. Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria.

FIG. 1.

Multiple-sequence alignments of P. aeruginosa PAO1 GluRS, GlnRS, GatC, GatA, GatB, and AspRS with a few respective orthologs from other bacterial species, created by using the Pileup program. The organisms were as follows: P_aeru, P. aeruginosa PAO1; T_ther, T. thermophilus; B_subt, B. subtilis; E_coli, E. coli; D_radi, Deinococcus radiodurans; C_trac, C. trachomatis. The GluRS sequences used were as follows: P_aeru, PA3134; T_ther, P27000; B_subt, P222450; E_coli, P04805. The aligned residues Arg358 of T. thermophilus GluRS-D, Arg358 of P. aeruginosa PAO1, and GluRS and Gln358 of B. subtilis GluRS-ND are boxed. The GlnRS sequences used were as follows: P_aeru, Q9I2U8; D_radi, P56926; E_coli, BAA35328. The GatC, GatA, and GatB sequences used were, respectively, as follows: P_aeru, AAG07870, AAG07871, AAG07872; C_trac, NP_219504, NP_219505, NP_219506; B_subt, O06492, CAB12488, O30509. The AspRS sequences used were as follows: P_aeru, NP_249654; C_trac, O84546; D_radi_D, NP_295070; T_ther_D, P36419; E_coli_D, NP_288303. Residues which were identical in all sequences of each multiple alignment are printed in white on black, and those conserved in at least three of the four GluRS sequences, two of three sequences (GlnRS, GatC, GatA, and GatB), or three or four of the five AspRS sequences are shaded.

FIG. 1.

Multiple-sequence alignments of P. aeruginosa PAO1 GluRS, GlnRS, GatC, GatA, GatB, and AspRS with a few respective orthologs from other bacterial species, created by using the Pileup program. The organisms were as follows: P_aeru, P. aeruginosa PAO1; T_ther, T. thermophilus; B_subt, B. subtilis; E_coli, E. coli; D_radi, Deinococcus radiodurans; C_trac, C. trachomatis. The GluRS sequences used were as follows: P_aeru, PA3134; T_ther, P27000; B_subt, P222450; E_coli, P04805. The aligned residues Arg358 of T. thermophilus GluRS-D, Arg358 of P. aeruginosa PAO1, and GluRS and Gln358 of B. subtilis GluRS-ND are boxed. The GlnRS sequences used were as follows: P_aeru, Q9I2U8; D_radi, P56926; E_coli, BAA35328. The GatC, GatA, and GatB sequences used were, respectively, as follows: P_aeru, AAG07870, AAG07871, AAG07872; C_trac, NP_219504, NP_219505, NP_219506; B_subt, O06492, CAB12488, O30509. The AspRS sequences used were as follows: P_aeru, NP_249654; C_trac, O84546; D_radi_D, NP_295070; T_ther_D, P36419; E_coli_D, NP_288303. Residues which were identical in all sequences of each multiple alignment are printed in white on black, and those conserved in at least three of the four GluRS sequences, two of three sequences (GlnRS, GatC, GatA, and GatB), or three or four of the five AspRS sequences are shaded.

FIG. 1.

Multiple-sequence alignments of P. aeruginosa PAO1 GluRS, GlnRS, GatC, GatA, GatB, and AspRS with a few respective orthologs from other bacterial species, created by using the Pileup program. The organisms were as follows: P_aeru, P. aeruginosa PAO1; T_ther, T. thermophilus; B_subt, B. subtilis; E_coli, E. coli; D_radi, Deinococcus radiodurans; C_trac, C. trachomatis. The GluRS sequences used were as follows: P_aeru, PA3134; T_ther, P27000; B_subt, P222450; E_coli, P04805. The aligned residues Arg358 of T. thermophilus GluRS-D, Arg358 of P. aeruginosa PAO1, and GluRS and Gln358 of B. subtilis GluRS-ND are boxed. The GlnRS sequences used were as follows: P_aeru, Q9I2U8; D_radi, P56926; E_coli, BAA35328. The GatC, GatA, and GatB sequences used were, respectively, as follows: P_aeru, AAG07870, AAG07871, AAG07872; C_trac, NP_219504, NP_219505, NP_219506; B_subt, O06492, CAB12488, O30509. The AspRS sequences used were as follows: P_aeru, NP_249654; C_trac, O84546; D_radi_D, NP_295070; T_ther_D, P36419; E_coli_D, NP_288303. Residues which were identical in all sequences of each multiple alignment are printed in white on black, and those conserved in at least three of the four GluRS sequences, two of three sequences (GlnRS, GatC, GatA, and GatB), or three or four of the five AspRS sequences are shaded.

FIG. 1.

Multiple-sequence alignments of P. aeruginosa PAO1 GluRS, GlnRS, GatC, GatA, GatB, and AspRS with a few respective orthologs from other bacterial species, created by using the Pileup program. The organisms were as follows: P_aeru, P. aeruginosa PAO1; T_ther, T. thermophilus; B_subt, B. subtilis; E_coli, E. coli; D_radi, Deinococcus radiodurans; C_trac, C. trachomatis. The GluRS sequences used were as follows: P_aeru, PA3134; T_ther, P27000; B_subt, P222450; E_coli, P04805. The aligned residues Arg358 of T. thermophilus GluRS-D, Arg358 of P. aeruginosa PAO1, and GluRS and Gln358 of B. subtilis GluRS-ND are boxed. The GlnRS sequences used were as follows: P_aeru, Q9I2U8; D_radi, P56926; E_coli, BAA35328. The GatC, GatA, and GatB sequences used were, respectively, as follows: P_aeru, AAG07870, AAG07871, AAG07872; C_trac, NP_219504, NP_219505, NP_219506; B_subt, O06492, CAB12488, O30509. The AspRS sequences used were as follows: P_aeru, NP_249654; C_trac, O84546; D_radi_D, NP_295070; T_ther_D, P36419; E_coli_D, NP_288303. Residues which were identical in all sequences of each multiple alignment are printed in white on black, and those conserved in at least three of the four GluRS sequences, two of three sequences (GlnRS, GatC, GatA, and GatB), or three or four of the five AspRS sequences are shaded.

FIG. 2.

Putative pathways for Gln-tRNA^Gln and Asn-tRNA^Asn synthesis in P. aeruginosa PAO1 based on the genes identified in its complete genomic sequence and without excluding the possibility that an atypical AsnRS is present.

FIG. 3.

P. aeruginosa PAO1 genes amplified by PCR and inserted into the multiple cloning site of the E. coli-P. aeruginosa shuttle vector pUCPSK or pUCPKS, which differ only by the orientation of their multiple cloning sites (36). The resulting vectors, identified on the right, express the inserted gene(s) from a proximal T7 promoter. The letters K, H, X, E, and S represent the KpnI, HindIII, XbaI, EcoRI, and SacI restriction sites, respectively. Upstream of gatB and gatC, we inserted by PCR the Shine-Dalgarno sequence AGGAGG frequently found in P. aeruginosa, 8 His codons (CAC), and a sequence encoding the factor Xa digestion site Arg-Glu-Gly-Arg (with codons preferentially used in P. aeruginosa). Downstream of gltX and aspS, we inserted a sequence encoding the factor Xa digestion site, 6 His codons, and two stop codons.

FIG. 4.

SDS-PAGE characterization of P. aeruginosa PAO1 GluRS (A), the three subunits of the heterotrimeric AdT (B), and AspRS (C) overproduced in P. aeruginosa ADD1976 and purified by affinity chromatography on Ni-NTA. (A) Lanes: 1, protein standard; 2 to 8, wash with 30 mM imidazole; 9 to 10, GluRS elution with 90 mM imidazole. (B) Lanes: 1 to 9, amidotransferase elution with 100 mM imidazole, after an initial wash with 30 mM imidazole; 10, protein standard. (C) Lanes: 1, protein standard; 2, AspRS elution with 85 mM imidazole, after an initial wash with 30 mM imidazole; 3 and 4, contaminant and pure AspRS, respectively, removed after Superdex 200 chromatography. Numbers indicate the molecular mass (in kilodaltons) of protein standards. The gels (8% polyacrylamide) are stained with Coomassie blue.

FIG. 5.

Aminoacylation of unfractionated tRNA, pure tRNA^Gln, tRNA^Asp, and tRNA^Asn from P. aeruginosa PAO1. The reactions shown in panels A, C, and E were conducted in the presence of unfractionated tRNA and catalyzed by partially purified GlnRS, pure GluRS, and pure AspRS from P. aeruginosa PAO1, respectively. Panel B shows the glutaminylation of pure tRNA^Gln by partially purified GlnRS, and panel F shows the aspartylation of pure tRNA^Asp and tRNA^Asn by pure AspRS. (D) Identification by TLC (see Methods) of the amino acid charged on tRNA by GluRS in the reaction shown in panel C (lane 1) and by GlnRS in the reaction shown in panel A (lane 2). Shown are complete reactions (filled circles) and controls without either tRNA (X) or enzyme (empty circles).

FIG. 6.

Activity of P. aeruginosa PAO1 heterotrimeric AdT. (A) P. aeruginosa Glu-tRNA. Lanes: 1, with (+) AdT; 2, without (−) AdT; 3 and 4, glutamine and glutamate, respectively, as standards. (B) B. subtilis Glu-tRNA. Lanes: 1, with AdT; 2, without AdT. (C) P. aeruginosa Asp-tRNA. Lanes: 1, with AdT for 30 min; 2, with AdT for 15 min; 3, without AdT. These panels show phosphorimages of TLC of the ¹⁴C-labeled amino acids after their removal from these tRNAs (see Materials and Methods) or free glutamine and glutamate as standards (panel A, lanes 3 and 4).