Cefotaxim-Empfindlichkeit von Bacteroidaceae

Physiology and genetics of ethanologenesis in the acetogenic bacterium Acetobacterium woodii

“…In the plasmids pMTL84151_JM_dadh3 and pMTL84151_JM_dadh6, 1000 bp of UFR and DFR of adh3 (Awo_c06180) and adh6 (Awo_c25410) were cloned into the multiple cloning sites. Since these plasmids possess a catP marker for chloramphenicol/thiamphenicol resistance from C. perfringens (Werner et al, 1977) and the pyrE from C. acetobutylicum (Westphal et al, 2018) as a counter selectable marker, the first selection was achieved in an agar plate with complex medium supplemented with 20 mM fructose and 30 ng μl À1 thiamphenicol after transformation of plasmids into A. woodii ΔpyrE strain by electroporation (625 V, 25 μF, 600 Ω, in 1 mm cuvettes). Then, the thiamphenicol-resistant colonies were further plated onto an agar plate with minimal medium (Westphal et al, 2018) supplemented with 20 mM fructose, 1 μg ml À1 uracil and 1 mg ml À1 5-FOA for disintegration.…”

“…To this end, suicide plasmids pMTL84151_JM_dadh3 and pMTL84151_JM_dadh6 were constructed, which carry each 1000 bp of upstream and downstream flanking regions (UFR and DFR) of the adh3 or adh6 gene leaving 3 bp behind the start codon and 3 bp in front of stop codon. For selection, the plasmids contained the pyrE gene from Clostridium acetobutylicum (Westphal et al, 2018) and the chloramphenicol/ thiamphenicol resistance cassette (catP) from Clostridium perfringens (Werner et al, 1977). First, the plasmids were integrated into the chromosome of A. woodii ΔpyrE at one flanking region under antibiotic pressure with thiamphenicol and subsequently, disintegration was forced by counter-selection with 5-fluoroorotate.…”