This was digested with ApaI and NotI, and then the DNA fragment c

This was digested with ApaI and NotI, and then the DNA fragment containing the truncated ndvB fragment and the spectinomycin resistance Ω interposon was transferred to the suicide vector pJQ200SK (Quandt & Hynes, 1993) using the same restriction sites, generating pGF03. Finally, a tri-parental mating procedure with the helper plasmid pRK2013 (Figurski & Helinski, 1979) was used to transfer pGF03 into NGR234. Growth on TY agar plates supplemented with sucrose (5% w/v), and spectinomycin allowed selection for the ndvB mutant (named NGRΔndvB). The ndvB promoter region was amplified using the following primer pair: 5′-GCGAATTCATCAGCGAGCAGGT-3′ and 5′-TTTCTAGACACGGTCATGTGTCCC-3′. The resulting

fragment was digested with EcoRI and XbaI to enable cloning into pBluescript CSF-1R inhibitor pSK+ resulting in pALQ09. The ndvB promoter region of pALQ09 was then transferred into the PstI and ClaI sites of pBDG116 creating pALQ12. In turn, ndvB promoter was inserted into the HindIII restriction site of pPROBE-GT′ (generating

pALQ27). The flaC promoter region was amplified by PCR using the following primer pair: 5′-CGGAATTCTGGTGCGCTCCTTC-3′ and 5′-GGTCTAGATGCGGTTCTGCG-3′, digested using EcoRI–XbaI and cloned into pBluescript selleck chemical pSK+ generating pALQ24. The insert was transferred into the KpnI-SacI sites of pPROBE-GT-producing pALQ28. All constructed plasmids were sequenced to confirm PCR fidelity. The final constructs containing the ndvB and

the flaC promoters fused to the GFP-encoding gene (pALQ27 and pALQ28, respectively), or empty vectors were mobilized into recipient strains using tri-parental mating as described previously. To generate GFP-tagged strains, the broad host-range vector pHC60 (Cheng & Walker, 1998) which constitutively expresses GFP was mobilized very into NGR234 and the ndvB mutant by tri-parental mating. Extractions of CβGs were performed using the following protocol, based on a method developed by Inon de Iannino et al. (1998). Briefly, strains were cultivated in 50 mL TY for 2 days to a stationary growth phase (i.e., a final OD600 nm of 2.0–2.5). Cells were centrifuged for 10 min at 10 000 g, 10 °C and washed twice with water. Pellets were resuspended in 1 mL of 70% ethanol, incubated for 1 h at 37 °C, and further centrifuged for 2 min at 9000 g. The supernatants were finally desiccated by speed-vacuum and resuspended in 20 μL of 70% ethanol. Aliquots (5 μL) of each extract were separated by thin-layer chromatography (Cromatofolios AL TLC – Silicagel 60F) using n-butanol–ethanol-dH2O (v/v/v of 5 : 5 : 4), and CβGs were visualized by spraying the plates with 5% sulfuric acid in ethanol, followed by heating at 120 °C 10 min. Swimming plates were produced by adding 0.2% agar to GYM medium supplemented with various amounts of NaCl.

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