Abstract
Site-directed mutagenesis were done in Haloarcula marismortui using the strategy that Khorana and coworkers devised for deleting the bacteriorhodopsin gene from Halobacterium halobium [Krebs et al. Proc Natl Acad Sci USA 90:1987–1991 (1993)]. Strains have been prepared from H. marsimortui, which normally has three rRNA operons, that are missing either its rrnB operon or both its rrnB and rrnC operons. In rich media, both strains grow at about the same rate as wild type. The G2099 in the 23S rRNA gene of the single operon strain was changed to A, and a three amino acid deletion was introduced into the gene for ribosomal protein L22 of the wild-type organism. The structural consequences of these and other such mutations can be determined with unusual accuracy because crystals of the large ribosomal subunit of H. marismortui diffract to atomic resolution.
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Abbreviations
- rrnA:
-
ribosomal RNA operon A
- rrnB:
-
ribosomal RNA operon B
- rrnC:
-
ribosomal RNA operon C
- rRNA:
-
ribosomal RNA
- Mevr:
-
Mevinolin resistant
References
Arndt E (1990) Nucleotide sequence of four genes encoding ribosomal proteins from the ‘S10 and spectinomycin’ operon equivalent region in the archaebacterium Halobacterium marismortui. FEBS Lett 267:193–198
Arndt E (1992) The genes for ribosomal protein L15 and the protein equivalent to secY in the archaebacterium Haloarcula (Halobacterium) marismortui. Biochim Biophys Acta 1130:113–116
Arndt E, Kromer W, Hatakeyama T (1990) Organization and nucleotide sequence of a gene cluster coding for eight ribosomal proteins in the archaebacterium Halobacterium marismortui. J Biol Chem 265:3034–3039
Arndt E, Weigel C (1990) Nucleotide sequence of the genes encoding the L11, L1, L10 and L12 equivalent ribosomal proteins from the archaebacterium Halobacterium marismortui. Nucleic Acids Res 18:1285
Baliga NS, Bonneau R, Facciotti MT, Pan M, Glusman G, Deutsch EW, Shannon P, Chiu Y, Weng RS, Gan RR, Hung P, Date SV, Marcotte E, Hood L, Ng WV (2004) Genome sequence of Haloarcula marismortui: a halophilic archaeon from the Dead Sea. Genome Res 14:2221–2234
Ban N, Freeborn B, Nissen P, Penczek P, Grassucci RA, Sweet R, Frank J, Moore PB, Steitz TA (1998) A 9 Å resolution X-ray crystallographic map of the large ribosomal subunit. Cell 93:1105–1115
Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (2000) The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289:905–920
Chang S, Cohen SN (1979) High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet 168:111–115
Charlebois RL, Lam WL, Cline SW, Doolittle WF (1987) Characterization of pHV2 from Halobacterium volcanii and its use in demonstrating transformation of an archaebacterium. Proc Natl Acad Sci USA 84:8530–8534
Chittum HS, Champney WS (1994) Ribosomal protein gene sequence changes in erythromycin-resistant mutants of Escherichia coli. J Bacteriol 176:6192–6198
Cline SW, Doolittle WF (1987) Efficient transfection of the archaebacterium Halobacterium halobium. J Bacteriol 169:1341–1344
Cline SW, Doolittle WF (1992) Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons. J Bacteriol 174:1076–1080
Cline SW, Lam WL, Charlebois RL, Schalkwyk LC, Doolittle WF (1989) Transformation methods for halophilic archaebacteria. Can J Microbiol 35:148–152
Dennis PP, Ziesche S, Mylvaganam S (1998) Transcription analysis of two disparate rRNA operons in the halophilic archaeon Haloarcula marismortui. J Bacteriol 180:4804–4813
Dyall-Smith M (2003) The Halohandbook: protocols for halobacterial genetics. http://www.microbiol.unimelb.edu.au/staff/mds/HaloHandbook/ (accessed March 2005)
Hansen JL, Ippolito JA, Ban N, Nissen P, Moore PB, Steitz TA (2002) The structures of four macrolide antibiotics bound to the large ribosomal subunit. Mol Cell 10:117–128
Kita T, Brown MS, Goldstein JL (1980) Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in livers of mice treated with mevinolin, a competitive inhibitor of the reductase. J Clin Invest 66:1094–1100
Klebe RJ, Harriss JV, Sharp ZD, Douglas MG (1983) A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25:333–341
Kraft A, Lutz C, Lingenhel A, Grobner P, Piendl W (1999) Control of ribosomal protein L1 synthesis in mesophilic and thermophilic archaea. Genetics 152:1363–1372
Krebs MP, Mollaaghababa R, Khorana HG (1993) Gene replacement in Halobacterium halobium and expression of bacteriorhodopsin mutants. Proc Natl Acad Sci USA 90:1987–1991
Lam WL, Doolittle WF (1989) Shuttle vectors for the archaebacterium Halobacterium volcanii. Proc Natl Acad Sci USA 86:5478–5482
Mevarech M, Hirsch-Twizer S, Goldman S, Yakobson E, Eisenberg H, Dennis PP (1989) Isolation and characterization of the rRNA gene clusters of Halobacterium marismortui. J Bacteriol 171:3479–3485
Ramírez C, Köpke AKE, Yang D-C, Boeckh T, Matheson AT (1993) The structure, function and evolution of archaeal ribosomes. In: Kates M, Kushner DJ, Matheson AT (eds) The biochemistry of archaea (archaebacteria), vol 26. Elsevier Science Publishers BV, Amsterdam, The Netherlands, pp439–466
Scholzen T, Arndt E (1991) Organization and nucleotide sequence of ten ribosomal protein genes from the region equivalent to the spectinomycin operon in the archaebacterium Halobacterium marismortui. Mol Gen Genet 228:70–80
Stern S, Moazed D, Noller HF (1988) Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. In: Harry F, Noller J, Moldave K (eds) Methods in enzymology, vol 164. Academic Press Inc., San Diego, pp 481–489
Tu D, Blaha G, Moore PB, Steitz TA (2005) Structures of MLS B K antibiotics bound to mutated large ribosomal subunits provide a structural explanation for resistance. Cell 121:257–270
Unge J, berg A, Al-Kharadaghi S, Nikulin A, Nikonov S, Davydova N, Nevskaya N, Garber M, Liljas A (1998) The crystal structure of ribosomal protein L22 from Thermus thermophilus: insights into the mechanism of erythromycin resistance. Structure 6:1577–1586
Weisblum B (1995) Erythromycin resistance by ribosome modification. Antimicrob Agents Chemother 39:577–585
Acknowledgements
We thank W. Ford Doolittle for providing pWL102, and Patrick P. Dennis for providing pHC8 and pHH10. Sequences from H. marismortui found at (http://zdna2.umbi.umd.edu, Version 2) were used in designing the rrnC knockout construct. This research was supported by National Institutes of Health grant GM022778 to T.A.S. and P.B.M., by a grant from the Agouron Institute to T.A.S. and P.B.M.
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Communicated by J.N. Reeve
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Tu, D., Blaha, G., Moore, P.B. et al. Gene replacement in Haloarcula marismortui: construction of a strain with two of its three chromosomal rRNA operons deleted. Extremophiles 9, 427–435 (2005). https://doi.org/10.1007/s00792-005-0459-y
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DOI: https://doi.org/10.1007/s00792-005-0459-y