Subcloning vectors were double digested

with the prevailing added recognitions site for restriction enzymes. The flanking regions were excised, purified and ligated via a three-piece-ligation into the suicide selleck inhibitor vector pK19mobsacB [64]. Sequencing of the obtained plasmids pK19mobsacBΔldi and pK19mobsacBΔgeoA was performed to ensure correct sequence of the flanking regions including the start and stop codons of the deleted genes. Construction of complementation plasmids For construction of the in trans vector both, the ldi and the geoA was amplified from genomic DNA of C. defragrans 65Phen with primer pair encompassing the entired ORF, i.e. for the ldi primer pair ldi_EcoRI & ldi_BglII, and for geoA geoA_XbaI_F & geoA_HindIII_R (Table  4). Via the added restriction enzyme recognition sites the amplicon was inserted into the multiple cloning HMPL-504 datasheet site of two different derivatives of the broad-host range vector pBBR1MCS [69]. For confirmation of correct gene insertion the obtained plasmids pBBR1MCS-4ldi and pBBR1MCS-2geoA was sequenced. Conjugational plasmid transfer The donor strain, an overnight culture of E. coli S17-1 carrying the appropriate plasmid, and the recipient selleck chemical C. defragrans RIF were grown to late exponential phase and were mixed in several ratios (1:1, 1:5, 1:10) in a total volume of 20 μL and spread as a single drop on minimal agar. After incubation for 24 h

at 28°C under oxic conditions Molecular motor the bacteria were resuspended in 1 mL liquid minimal medium. Dilution-to-extinction series were streaked out onto solid minimal medium supplemented with kanamycin and rifampicin and anaerobically incubated at 28°C for four days. Preparation of cell-free extracts and determination of enzyme activities Soluble extract preparations of C. defragrans strains 65Phen, ΔgeoA and ΔgeoAcomp were performed as described [46]. The geraniol dehydrogenase activity was monitored in a standard assay following the reduction of NAD+ to NADH at 340 nm as described [47]. Equal total protein amounts were applied as certified in a 200-μl aliquot by the method of Bradford [70]

with BSA as standard protein; concentrations were corrected for the unusual high binding of the Coomassie stain to albumin [71]. Chemical analyses of biomass, educts and products Nitrate and nitrite was measured by HPLC as described by [72]. Based on the fact that protein accounts for 50% of the cell mass, the Bradford assay was applied in duplicates with two different dilutions to determine the total biomass yield [72]. Geranic acid formation was assayed in liquid cultures of C. defragrans strains after confirmed nitrate depletion (Merckoquant® test strips (Merck, Darmstadt, Germany)). 10 mL cell culture was acidified with H3PO4 (final concentration 0.1 M) and extracted with tert-butyl methyl ether in a 1:0.4 ratio (two biological replicates per strain). The ether extract was extracted with 0.