Even conjugation times below
24 h might be sufficient for the fast growing Phaeobacter strains and O. indolifex. Only two of the tested growth media provided appropriate Selleck Ilomastat conditions for donor and recipient strains (see above). Therefore, conjugation was carried out at 30°C on hMB and LB+hs agar plates supplemented with ALA. Media composition revealed a significant effect on conjugation efficiency. ALA supplemented hMB resulted in higher conjugation efficiencies. Various ratios of donor to recipient, related to the optical density of the cultures, were tested (1:1, 2:1, 5:1, 10:1). Best conjugation efficiencies were obtained with ratios of 5:1 and 10:1, ranged between 1 × 10-6 and 2.4 × 10-2 (Table 3). The lowest efficiencies were observed for the Phaeobacter and Roseobacter strains. Table 3 Conjugation efficiency determined with the vector pBBR1MCS. Strains Conjugants/viable cells Conjugants/ml P. inhibens
1.0 × 10-6 1.0 × 105 P. gallaeciensis 2.0 × 10-4 3.0 × 103 O. indolifex 2.7 × 10-2 5.0 × 105 R. litoralis 5.0 × 10-4 1.0 × 103 R. denitrificans 2.0 × 10-4 2.0 × 103 D. shibae 2.4 × 10-2 2.0 × 106 aThe recipient Roseobacter strains were cultivated for 18 h in MB at 30°C and the donor E. coli ST18 was grown up to the logarithmic phase (OD578 = 0.5-0.6) in LB supplemented with 50 μg/ml ALA at 37°C. BIIB057 molecular weight Mating mixtures were incubated on hMB supplemented with 50 μg/ml ALA over 24 h at 30°C in a donor:recipient ratio 10:1. Afterwards, the cells were resuspended in 1 ml MB, diluted serially in 1.7% (w/v) sea salt solution and plated on hMB with and without Selleckchem A-1155463 antibiotics, respectively, to determine the number of conjugants and viable cells. A donor:recipient
ratio of 5:1 revealed the same results. The results represent the mean of three independent experiments performed in duplicate. Several plasmids were tested for transfer via conjugation. These plasmids were successfully used for homologous expression of genes to complement gene knockouts in trans in other Gram-negative bacteria before. The IncP-plasmids pFLP2, pLAFR3 and pUCP20T were not transferable or not stable in the tested Roseobacter strains (see below). In contrast, the IncQ-plasmids Sclareol pRSF1010, pMMB67EH and the tested pBBR1MCS derivates were transferable. They were recovered from exconjugants by plasmid-DNA preparation and subsequently visualized via gel electrophoresis. Plasmid Stability There is only one report about homologous gene expression in Roseobacter clade bacteria using the vector pRK415 . This vector was widely used for a broad range of Gram-negative species, including R. sphaeroides [e.g. [44, 45]]. However, the small numbers of restriction enzyme sites available for cloning and the use of tetracycline as selective marker represent major drawbacks for its use.