E1 ΔBR mutants were grown in media amended with 25 μg mL−1 kanamycin. Dietzia sp. E1 cells were cultivated in 100-mL Erlenmeyer flasks containing 50 mL of MNPS minimal medium [0.1 M sodium-phosphate
buffer, 5 g L−1 (NH4)2SO4, 5 g L−1 KCl, 0.2 g L−1 MgSO4·7H2O, 0.05 g L−1 CaCl2·2H2O, 10 mL L−1 trace element solution SL-4 (http://www.dsmz.de/microorganisms/media_list.php, see Medium 14 and 27), pH 8.0] supplemented with 1 g L−1 of different individual n-alkanes or 2.9 g L−1 sodium acetate. Solid n-alkanes were weighed in Erlenmeyer flasks before autoclaving, while presterilized liquid n-alkanes were pipetted in the inoculated Dabrafenib datasheet media. For the inoculation, overnight GPY-grown E1 starter cultures were applied. The centrifuged (16 000g, 5 min) cells were resuspended in MNPS minimal broth and were diluted to a starting cell number of 106 mL−1. Flasks were incubated at 37 °C at 200 r.p.m. for 16–60 h. Microbial growth was monitored via the increases in OD600 nm and microscopically counted total cell number. All measurements were performed in triplicate. Plasmid DNA was isolated with the EZ-10 Spin Column Plasmid DNA MiniPreps Kit (Bio Basic Inc.). Chromosomal DNA from Dietzia Obeticholic Acid spp. was prepared as described previously (Szvetnik et al., 2010). Southern blot analysis was performed on genomic DNA digested with various restriction enzymes (BamHI, NotI, PstI, SalI
and SacI), using a 518-bp SacI/PstI Dietzia sp. E1 alkB fragment probe (Bihari et al., 2010). Nonradioactive DNA probe labelling, Southern hybridization and detection
were performed according to the manufacturer’s instructions (DIG DNA Labeling and Detection Kit, Roche). Plasmid pKAlkB518 was constructed by cloning the 518-bp SacI/PstI alkB fragment into pK18 (Pridmore, 1987). The construct was maintained in E. coli DH5α and introduced into competent Dietzia sp. E1 cells by electroporation (Szvetnik et al., 2010). Integration of the plasmid resulted in a kanamycin-resistant disruption mutant designated Dietzia sp. E1 ΔBR. The genomic DNA of this mutant strain was purified, digested with Olopatadine NotI or MunI and self-ligated. After propagation in DH5α, the two rescue plasmids obtained were sequenced partially by a combination of subcloning and walking primers. On the basis of the sequence data, outer alkBPromF and rubCFLAG oligonucleotides priming to the 5′ and 3′ flanking regions of the 518-bp alkB fragment were designed (Table 1). PCR reactions were carried out using these primers on the genomic DNA template of the ΔBR mutant and also that of each wild-type Dietzia strain. For this purpose, KOD Hot Start DNA Polymerase (Novagen) was applied according to the manufacturer’s instructions, except that long initial denaturation was performed and 10% dimethyl sulphoxide was present in all reactions. For PCRs, the following program was used: 10 min at 95 °C; 35 cycles of 0.5 min at 95 °C, 0.5 min at 55 °C and 2.5 min at 70 °C; and 5 min at 70 °C.
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