Primers M13universal and GlnKdelR (5′ AAGCC CTCGAG TTCAGTCACGGT 3′, Xho I AZD2281 cell line restriction site is underlined) were used to amplify a 180 bp region upstream of glnK and the first 107 bp of the glnK gene. The primers M13reverse and GlnKdelD (5′ GGACCTG CTCGAG GTGATCCGT 3′, Xho I restriction site is underlined) were used to amplify the last 58 bp of the glnK gene and the first 180 bp of amtB. The amplified fragments were joined by the Xho I sites. This fragment containing glnK deleted of 192 bp was then used as template for a PCR reaction with the primers M13reverse and M13universal. The resulting PCR product was

digested with Bam HI and Pst I and inserted into pUC18 to give pUCglnKdel. This fragment was then subcloned into pSUP202, yielding the plasmid pSUPglnKdel. A sacB -KmR cassette excised with Bam HI from pMH1701 CHIR-99021 cost [35] was inserted into the vector region of the Bam HI-cut pSUPglnKdel plasmid. The resulting plasmid (pSUPglnKdelsacB) was conjugated into H. seropedicae SmR1 using

E. coli strain S17.1 as the donor. Recombinant colonies were selected for kanamycin and chloramphenicol resistance. One mutant strain was selected, and grown overnight in liquid NFbHP medium supplemented with ammonium chloride (20 mmol/L) and 80 μg/mL streptomycin. One microliter of the culture was plated on solid NFbHP medium supplemented with 20 mmol/L NH4Cl, 5% sucrose and 80 μg/mL Methane monooxygenase streptomycin. Sucrose is toxic to Dinaciclib purchase bacteria containing the sacB gene in the chromosome, therefore only strains that lost the sacB -KmR cassette by

a second homologous recombination event would grow. The selected strains were analyzed by PCR with the primers GlnKF1 (5′TGTCCAAGACCTTCGACG3′) and GlnKR1 (5′CATGCTCATTAGAGTTCC3′) which were homologous to the glnK flanking 5′- and 3′- regions, confirming the deletion of the 192 bp glnK fragment (data not shown). This in-frame glnK strain (ΔglnK) was named LNglnKdel. Construction of plasmid pLNΔNifA An Eco47III/SacI DNA fragment containing the nifA gene promoter region of H. seropedicae was excised from the plasmid pEMS301[36] and sub-cloned into the SmaI/SacI-cut vector pDK6 [37], yielding plasmid pDK6pnifA. An Xba I DNA fragment encoding for the central and C-terminal region of NifA protein (ΔN-NifA) of H. seropedicae was excised from the plasmid pRAM2T7 and sub-cloned into the XbaI-cut pDK6pnifA, in the same orientation as the nifA promoter, yielding plasmid pDK6nifACT. Finally, a SacI/HindIII DNA fragment containing the nifA 5′-truncated gene was excised from pDK6nifACT and sub-cloned into pLAFR3.18Cm digested with Sac I and Hin dIII. The generated plasmid was named pLNΔNifA and encodes for the central and C-terminal domains of NifA under control of the nifA promoter. Construction of the plasmid pACB210 A 1.