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Journal of Bacteriology, January 2004, p. 8-14, Vol. 186, No. 1
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.1.8-14.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cysteinyl-tRNACys Formation in Methanocaldococcus jannaschii: the Mechanism Is Still Unknown

Benfang Ruan,1,{dagger} Hiroaki Nakano,1,{dagger} Masashi Tanaka,2 Jonathan A. Mills,3,{ddagger} Joseph A. DeVito,3,§ Bokkee Min,1 K. Brooks Low,4 John R. Battista,2 and Dieter Söll1,5*

Departments of Molecular Biophysics and Biochemistry,1 Chemistry,5 Therapeutic Radiology, Yale University, New Haven, Connecticut 06520-8114,4 Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803,2 DuPont Pharmaceuticals Company, Wilmington, Delaware 198013

Received 31 July 2003/ Accepted 2 October 2003

Most organisms form Cys-tRNACys, an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS). However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS. It was reported that M. jannaschii prolyl-tRNA synthetase (C. Stathopoulos, T. Li, R. Longman, U. C. Vothknecht, H. D. Becker, M. Ibba, and D. Söll, Science 287:479-482, 2000; R. S. Lipman, K. R. Sowers, and Y. M. Hou, Biochemistry 39:7792-7798, 2000) or the M. jannaschii MJ1477 protein (C. Fabrega, M. A. Farrow, B. Mukhopadhyay, V. de Crécy-Lagard, A. R. Ortiz, and P. Schimmel, Nature 411:110-114, 2001) provides the "missing" CysRS activity for in vivo Cys-tRNACys formation. These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene. Here we show that E. coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the Km for cysteine and a ninefold reduction in the kcat for ATP. While transformants of E. coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E. coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium. An E. coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D. radiodurans DR0705 gene. Construction of a D. radiodurans DR0705 deletion strain showed this gene to be dispensable. However, attempts to delete D. radiodurans cysS failed, suggesting that this is an essential Deinococcus gene. These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNACys synthesis in M. jannaschii. Thus, the mechanism of Cys-tRNACys formation in M. jannaschii still remains to be discovered.

* Corresponding author. Mailing address: Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208114, 266 Whitney Ave., New Haven, CT 06520-8114. Phone: (203) 432-6200. Fax: (203) 432-6202. E-mail: soll{at}trna.chem.yale.edu.

{dagger} B.R. and H.N. contributed equally to the work described in this report.

{ddagger} Present address: Symyx Technologies, Santa Clara, CA 95051.

§ Present address: Rib-X Pharmaceuticals, Inc., New Haven, CT 06511.

Journal of Bacteriology, January 2004, p. 8-14, Vol. 186, No. 1
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.1.8-14.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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