Establishment of a gene transfer system for Rhodothermus marinus.
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+/8-0.7x10(6) cfu/microg DNA at 22.5 kV/cm, 200 Omega and 25 microF. Transformation failed, however, after chemical preparation of cells according to several protocols. This is the first report of genetic transformation in the genus Rhodothermus.
Bjornsdottir
S H
SH
Laboratory of Molecular Genetics, Institute of Biology, University of Iceland, Reykjavik, Iceland.
Thorbjarnardottir
S H
SH
Eggertsson
G
G
D004262:DNA Restriction Enzymes / Q000032:analysis; D004269:DNA, Bacterial / Q000737:chemistry; D018274:Electroporation; D004926:Escherichia coli; D018014:Gene Transfer Techniques; D022761:Gene Transfer, Horizontal; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D045245:Rhodothermus / Q000235:genetics*; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial; D014364:Tryptophan / Q000096:biosynthesis / Q000235:genetics
D004269:DNA, Bacterial; D014364:Tryptophan; D004262:DNA Restriction Enzymes