cv. Frisson) seeds were surface-disinfected, pregerminated on agar plates, sown in Leonard jar-type assemblies, and inoculated with R. leguminosarum bv. viciae strains, as previously described [45]. Plants were grown for 21 days under bacteriologically controlled conditions with a nitrogen-free plant nutrient solution in a greenhouse adjusted to 18/25°C(night/day) temperatures. Nitrogen-free plant nutrient solution was supplemented with 170 μM NiCl2 on day 10 after seedling inoculation.

Bacteroid suspensions were obtained from nodules as previously described [40]. Hydrogenase activity assays Hydrogenase activity in bacteroid suspensions and Cell Cycle inhibitor in free-living microaerobic cell cultures was measured by an amperometric method using a Clark-type electrode with oxygen as PRI-724 electron acceptor [45]. find more Hydrogenase activity in vegetative cells was induced in 40-ml cultures grown under continuous bubbling with a gas mixture containing O2 concentrations of 1 or 3% in N2. Strains were aerobically grown

in YMB medium to an optical density at 600 nm (OD600) of ca. 0.4. From these cultures a 1:4 dilution was made in fresh YMB medium. Flasks were capped with a stoppered-tube system adapted to continuous flushing with 1% or 3% O2 on N2, and incubated at 28°C for 20 h. For HupL stability studies, bacteri-al cultures were maintained in a bottle with continuous bubbling with either 1% O2 or air

during 3 hours after standard microaerobic induction (1% O2). Cell cultures were centrifuged and suspended in 5 ml Dixon buffer (32 mM K2HPO4, 24 mM KH2PO4 and 0.24 mM MgCl2) before amperometric determinations. To prevent dam-age of hydrogenase due to O2 exposition, extracts were bubbled with argon during preparation. Protein contents of vegetative cells and bacteroids were determined by the bicinchoninic acid method SPTBN5 [46] after alkaline digestion of cells at 90°C in NaOH for 10 min, with bovine serum albumin as the standard. DNA manipulation techniques and mutant construction DNA manipulations, including purification, restriction, ligation, agarose gel electrophoresis, PCR amplification, and transformation into E. coli cells were carried out by standard methods [47]. In-frame deletions of hupF, hupK and hypC genes were generated in plasmid pALPF1 as described by Manyani et al. [19], resulting in plasmids pALPF5, pALPF10, and pALPF14, respectively. Primers used for deletions and plasmid generation are included in Table  4.