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Genetics and Molecular Biology |
Departments of 1Microbiology, Box 357242, and 2Chemical Engineering, Box 351750, University of Washington, Seattle, WA 98195-1750, USA
Author for correspondence: Mary E. Lidstrom. Tel: +1 206 616 5282. Fax: +1 206 616 5721. e-mail: lidstrom{at}u.washington.edu
Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. One bacterium for which new genetic tools are needed is the methylotroph Methylobacterium extorquens AM1. IncQ and small IncP vectors were shown to be unsuitable for use in this bacterium, but a spontaneous mutant of a small IncP plasmid was isolated that functioned efficiently in M. extorquens AM1. This plasmid was sequenced and used as a base for developing improved broad-host-range cloning vectors. These vectors were found to replicate in a wide variety of bacterial species and have the following advantages: (1) high copy number in Escherichia coli; (2) small size (7·2 and 8·0 kb); (3) complete sequences; (4) variety of unique restriction sites; (5) blue–white screening via lacZ
; (6) conjugative mobilization between bacterial species; and (7) readily adaptable into species-specific promoter-probe and expression vectors. Two low-background promoter-probe vectors were constructed based on these cloning vectors with either lacZ or xylE as reporter genes; these were shown to report gene expression effectively in M. extorquens AM1. Specific expression vectors were developed for use in M. extorquens AM1, which were shown to express foreign genes at significant levels, and a simple strategy is outlined to develop specific expression vectors for other bacteria. The strong mxaF promoter was used for expression, since E. coli lac-derived promoters were expressed at very low levels. This suite of genetic tools will enable a more sophisticated analysis of the physiology of M. extorquens AM1, and these vectors should also be valuable tools in the study of a variety of bacterial species.
Keywords: plasmids, cloning vectors, expression vectors, promoter-probe vectors, Methylobacterium extorquens AM1
Abbreviations: Ap, ampicillin; ßGal, ß-galactosidase; bhr, broad-host-range; Km, kanamycin; LB, Luria–Bertani (medium); oligo, oligodeoxyribonucleotide; ori, origin(s) of DNA replication; P, promoter; Rif, rifamycin; Sm, streptomycin; t, terminator of transcription; Tc, tetracycline; wt, wild-type
The GenBank accession numbers for the sequences reported in this paper are AF327711, AF327712, AF327713, AF327714, AF327715, AF327716, AF327717, AF327718, AF327719 and AF327720.
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C. J. Marx, L. Chistoserdova, and M. E. Lidstrom Formaldehyde-Detoxifying Role of the Tetrahydromethanopterin-Linked Pathway in Methylobacterium extorquens AM1 J. Bacteriol., December 15, 2003; 185(24): 7160 - 7168. [Abstract] [Full Text] [PDF]
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C. J. Marx, M. Laukel, J. A. Vorholt, and M. E. Lidstrom Purification of the Formate-Tetrahydrofolate Ligase from Methylobacterium extorquens AM1 and Demonstration of Its Requirement for Methylotrophic Growth J. Bacteriol., December 15, 2003; 185(24): 7169 - 7175. [Abstract] [Full Text] [PDF]
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M. Zhang and M. E. Lidstrom Promoters and transcripts for genes involved in methanol oxidation in Methylobacterium extorquens AM1 Microbiology, April 1, 2003; 149(4): 1033 - 1040. [Abstract] [Full Text] [PDF]
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C. Morris, C. M. C. Yip, I. S. M. Tsui, D. K.-H. Wong, and J. Hackett The Shufflon of Salmonella enterica Serovar Typhi Regulates Type IVB Pilus-Mediated Bacterial Self-Association Infect. Immun., March 1, 2003; 71(3): 1141 - 1146. [Abstract] [Full Text] [PDF]
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S. J. Van Dien, Y. Okubo, M. T. Hough, N. Korotkova, T. Taitano, and M. E. Lidstrom Reconstruction of C3 and C4 metabolism in Methylobacterium extorquens AM1 using transposon mutagenesis Microbiology, March 1, 2003; 149(3): 601 - 609. [Abstract] [Full Text] [PDF]
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M. G. Kalyuzhnaya and M. E. Lidstrom QscR, a LysR-Type Transcriptional Regulator and CbbR Homolog, Is Involved in Regulation of the Serine Cycle Genes in Methylobacterium extorquens AM1 J. Bacteriol., February 15, 2003; 185(4): 1229 - 1235. [Abstract] [Full Text] [PDF]
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N. Korotkova, L. Chistoserdova, and M. E. Lidstrom Poly-{beta}-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11, gap20, and phaR J. Bacteriol., November 15, 2002; 184(22): 6174 - 6181. [Abstract] [Full Text] [PDF]
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A. Studer, C. McAnulla, R. Buchele, T. Leisinger, and S. Vuilleumier Chloromethane-Induced Genes Define a Third C1 Utilization Pathway in Methylobacterium chloromethanicum CM4 J. Bacteriol., July 1, 2002; 184(13): 3476 - 3484. [Abstract] [Full Text] [PDF]
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M. F. Kayser, Z. Ucurum, and S. Vuilleumier Dichloromethane metabolism and C1 utilization genes in Methylobacterium strains Microbiology, June 1, 2002; 148(6): 1915 - 1922. [Abstract] [Full Text] [PDF]
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N. Korotkova, L. Chistoserdova, V. Kuksa, and M. E. Lidstrom Glyoxylate Regeneration Pathway in the Methylotroph Methylobacterium extorquens AM1 J. Bacteriol., March 15, 2002; 184(6): 1750 - 1758. [Abstract] [Full Text] [PDF]
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