For the purposes of chromosomal and plasmid DNA isolation, E col

For the purposes of chromosomal and plasmid DNA isolation, E. coli was grown aerobically in Erlenmeyer flasks filled to maximally 10% of their volume with LB medium on a rotary shaker (250 rpm) and incubated at 37°C. Anaerobic growths were performed at 37°C in sealed bottles filled with anaerobic medium and under a nitrogen gas atmosphere. Cultures for determination of hydrogenase processing or for enzyme activity measurements were grown either in buffered TGYEP medium (1%

w/v Small molecule library tryptone, 0.8% w/v glucose, 0.5% w/v yeast extract, 0.1 M potassium phosphate buffer) pH 6,5 [15] supplemented with 15 mM formate or in M9 minimal medium [26] containing 0.8% (w/v) glucose as carbon source, all standard amino acids at a final concentration of 0,04 mg/ml and 0.3 μM thiamine. When used for growth and screening for hydrogen metabolism mutants M9-glucose was supplemented with 0.29 mM citrulline, 0.89 mM uracil and was solidified with 1.5% (w/v) agar. All media were supplemented with 0.1% (v/v) SLA trace element solution [27] except when different iron sources were tested in which case FeCl3 was omitted from

SLA and was replaced by the appropriate iron source at the concentration indicated. Dipyridyl was added at a final concentration of 300 μM. All growth media included 0.1 μM NiCl2. The antibiotics kanamycin, ampicillin, and Selleck INCB024360 chloramphenicol, when required, were added to the medium at the final concentrations of 50, 100, and 12.5 μg per ml, respectively. When indicated next anhydrotetracycline (AHT) was added at the final concentration of 0.2 μg per ml. Construction of hyaA’-'lacZ,

hybO’-'lacZ and hycA’-'lacZ translational fusions The translational fusions to hyaA and hybO were constructed by amplifying the respective promotor regions and the nucleotides coding for the first 14 or 13 amino acids, respectively, by PCR using Phusion DNA polymerase (Finnzymes, Germany) and the oligonucleotides hya_regulat_up 5′-GCG GGA TCC GCG CAG AGA TTC GAA CTC TG-3′, hya_regulat_down 5′-GCG GGA TCC TGA CGC CGC ATG GCC TGG TA-3′, hybO_-217 5′-CTC GGA TCC TAT GGC CGG TTA TCG CCT C-3′ and hybO_+38 5′-CTC GGA TCC ATG CCG TGA GAA TGG ATG A-3′. The resulting respective 565 bp and 274 bp fragments were digested with BamHI and ligated into pRS552 [20], which had been digested with BamHI and dephosphorylated with shrimp alkaline phosphatase (Roche, Germany). This procedure delivered plasmids phyaA552 and phybO552, respectively. The DNA sequence was verified by sequencing (Seqlab, Germany) and the insert transferred to λRS45 [20]. In a similar manner the hycA’-'lacZ fusion was constructed using plasmid pTL101 [28]. The resulting Φ(hyaA’-'lacZ), Φ(hybO’-'lacZ) and Φ(hycA’-'lacZ) protein fusions were introduced in single copy into the lambda attachment site of the respective mutants as indicated in Table 6.

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