9 – 5.0 ms. The competent cells were subsequently frozen in liquid nitrogen and stored at -80°C. Under these conditions see more cells can be stored for about 3 weeks, except of R. denitrificans, which was viable only for a maximum of 1 week. We used 25 ng and 50 ng plasmid-DNA (selleck screening library pBBR1MCS), both resulting in similar transformation rates. Different
pulse intensities were tested (1.5 – 3.0 kV). An intensity of 2.5 kV revealed the best results and was used for further experiments. The electroporation method was successful for all tested strains, although transformation rates differed between them. A maximum of 1 × 103 cfu/μg plasmid-DNA were observed for P. inhibens and R. litoralis. Slightly higher efficiencies of 1 × 104 cfu/μg plasmid-DNA were observed for D. shibae and R. denitrificans. Good efficiencies were observed for P. gallaeciensis with 1 × 105 cfu/μg plasmid- DNA and O. indolifex with an efficiency of 1 × 107 cfu/μg plasmid-DNA. Recently, an optimized electroporation method was described for the Gram-negative P. aeruginosa resulting
in transformation efficiencies ranging from 107 to 1011 cfu/μg plasmid-DNA . These results are comparable with the efficiencies obtained in O. indolifex, indicating that our protocol is sufficient for the members of the Roseobacter clade. Although BIRB 796 research buy the transformation efficiencies are much less for most of the tested Roseobacter strains, this technique can be used as a fast and easy method to transfer plasmids into Roseobacter cells. Efficient conjugal transformation of Roseobacter clade bacteria
Biparental mating using E. coli S17-1 as donor strain was described for plasmid transfer into S. pomeroyi and Sulfitobacter before [21, 23]. Thereby, the use of spontaneous emerged antibiotic-resistant mutants of the recipient strains is one of the principles used to counter-select against the E. coli donor strain after conjugation [e.g. [23, 41]]. It is well known that such mutations may also cause indirect pleiotropic effects that might influence the general physiology of the target strain. Changes in growth behaviour, uracil sensitivity and bacteriophage sensitivities were reported for spontaneous rifampicin-resistant mutants [42, 43]. A second approach utilises auxotrophic donor strains. Here, we used E. coli ST18 as donor strain for unless the conjugation procedure, which is a hemA mutant of E. coli S17 λ-pir . This strain cannot synthesize the general tetrapyrrole precursor aminolevulinic acid (ALA). Hence, to complement the lethal mutation ALA has to be added to the medium for growth. Consequently, for the selection of plasmid-containing Roseobacter recipients after conjugation hMB agar plates without ALA were used to inhibit growth of the E. coli donor cells. Several conditions of the conjugation procedure were varied including medium composition and conjugation time (for details see Methods section).