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Journal of Bacteriology, February 2004, p. 767-776, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.767-776.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Direct Glutaminyl-tRNA Biosynthesis and Indirect Asparaginyl-tRNA Biosynthesis in Pseudomonas aeruginosa PAO1

Pierre-Marie Akochy, Dominic Bernard, Paul H. Roy, and Jacques Lapointe*

Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Québec, Canada G1K 7P4

Received 18 July 2003/ Accepted 20 October 2003

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 tRNAGln, that AspRS is nondiscriminating, and that its Asp-tRNAAsn product is transamidated by AdT. On the other hand, tRNAGln 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.

* Corresponding author. Mailing address: Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Pavillon Marchand, Local 3211, Québec G1K 7P4, Canada. Phone: (418) 656-2131, ext. 3411. Fax: (418) 656-3664. E-mail: Jacques.Lapointe{at}bcm.ulaval.ca.

Journal of Bacteriology, February 2004, p. 767-776, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.767-776.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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