Establishment of a gene transfer system for Rhodothermus marinus

• Published in: Applied microbiology and biotechnology 2005-03, Vol.66 (6), p.675-682
• Main Authors: Bjornsdottir, S. H, Thorbjarnardottir, S. H, Eggertsson, G
• Format: Article
• Language: English
• Subjects: Bacteria; Biological and medical sciences; Biotechnology; DNA Restriction Enzymes - analysis; DNA, Bacterial - chemistry; E coli; Electroporation; Escherichia coli; Fundamental and applied biological sciences. Psychology; gene transfer; Gene Transfer Techniques; Gene Transfer, Horizontal; genes; Genes, Bacterial; Genetic engineering; Genetic technics; genetic transformation; Genetic Vectors; Methods. Procedures. Technologies; Molecular Sequence Data; mutants; Plasmids; Rhodothermus; Rhodothermus - genetics; Rhodothermus marinus; Selection, Genetic; Sequence Analysis, DNA; Transformation, Bacterial; Tryptophan - biosynthesis; Tryptophan - genetics; Vectors (cloning, transfer, expression). Insertion sequences and transposons
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-004-1730-3

Genetic manipulation of Rhodothermus marinus has been hampered by the lack of a selection system for gene transfer. We report construction of a Rhodothermus/Escherichia coli shuttle plasmid, containing the R. marinus trpB gene, based on pUC18 and the cryptic R. marinus plasmid pRM21. A plasmid-less R. marinus recipient strain was selected on the basis of growth characteristics and absence of restriction activity. The shuttle plasmid, pRM100, was successfully introduced into a TrpB⁻ mutant of the recipient strain using electroporation and was found to transform it to prototrophy. No loss or rearrangement of pRM100 was observed after growth for 80 generations in non-selective medium. The relative copy numbers of pRM100 and of the parental plasmid, pRM21, were determined as 7±1 and 42±4, respectively. The shuttle plasmid was used to optimize an electroporation protocol, and the maximal number of transformants obtained was 4.3±0.7×10⁶ cfu/μg DNA at 22.5 kV/cm, 200 Ω and 25 μF. Transformation failed, however, after chemical preparation of cells according to several protocols. This is the first report of genetic transformation in the genus Rhodothermus.