1a and b). When looking through the channel, the substituted isoleucine residue appears to extend further into the channel, potentially obstructing the passage of PD-0332991 chemical structure substrate to the active site (Fig. 1c and d). Site-directed mutants were constructed as indicated in Table 2 in a plasmid containing genes nifB2S2U2H2D2K2 using the Quikchange Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer’s instructions. The plasmids were sequenced to confirm that the desired mutations were present and that no other mutations were introduced, and a c. 5.5-kb fragment from pRL2948a containing the mobilization site, oriT, and the sacB gene (for

sucrose selection of double recombinants) was inserted to create mobilizable

plasmids. These were conjugated into A. variabilis strain JE21, a nif2 region deletion mutant in which the nifU2H2D2 region, including the NifD2 α-75 and α-76 residues, was replaced with a neomycin resistance gene (NmR) cassette (Fig. 2b) (Thiel et al., 1997). Double recombinants were selected by plating on AA media Osimertinib supplemented with 10% sucrose (Cai & Wolk, 1990). DNA sequencing of PCR products amplified from the nif2 region of the putative double-recombinant strains using primers NifD2seq38 and NifD2seq10 (Table 2) showed a wild-type version of the nif2 region with the exception of the designed point mutations (Fig. 2). Attempts to amplify the NmR cassette via PCR in the double-recombinant replacement strains PW350, PW253, and PW357 yielded no product, indicating that the replacement had

fully segregated and no copies of the parental JE21 genome remained (data not shown). Proton, acetylene, and dinitrogen reduction activities were analyzed for the wild-type and mutant strains. Cultures were grown in AA/8 medium supplemented with 5.0 mM fructose, 5.0 mM NH4Cl and 10 mM N-Tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid, pH 7.2, at 30 °C with illumination of 90–100 μE m−2 s−1 as described previously (Thiel et al., 1995). Cells were washed three times in AA/8 and resuspended in AA/8+50 mM fructose and 50 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to inhibit oxygen production from photosystem Cytidine deaminase II. Cells (10 mL) at an OD720 nm between 0.2 and 0.3 in capped, 18-mL Hungate tubes (Bellco) were sparged for 10 min with either argon or nitrogen using a 3-in hypodermic needle as an inlet port, with a second, smaller needle as an outlet port, and shaken at 30 °C with illumination at 90–100 μE m−2 s−1. The Nif2 nitrogenase was induced within 2 h of nitrogen step down, reaching maximal activity within 4–5 h (data not shown). At 5.5 and 7 h, 250-μL samples of headspace gas were analyzed for H2 as described previously (Weyman et al., 2008).