Transconjugants arising from a single cross-over LY2606368 event were selected as KmrSmr colonies in MM and simultaneously verified to retain sacarose sensitivity in TY agar (10% sucrose). KmrSmrSacs bacteria from an isolated colony were further cultured in TY broth and 106 cells from this culture were finally plated on TY agar containing 10% sucrose to select double cross-over
events (i.e. excision of pK18mobsacB). Deletion of the hfq gene in the mutant bacteria was checked by colony PCR with oligonucleotides 5HfqMut/3HfqMut followed by HindIII restriction of the PCR products. To express Hfq under the control of its own CYT387 cell line promoter for complementation of the mutants an 842-bp DNA fragment containing the Hfq coding sequence along with 571 nt of the upstream region was PCR amplified with Pfu using primers 5Hfq_C/3Hfq_C and pGEMhfq as the template. The PCR product was inserted into pGEM®-T Easy yielding INCB28060 pGEMHfq and finally cloned into the low copy plasmid vector pJB3Tc19 as an EcoRI fragment generating pJBHfq which was conjugated into the S. meliloti hfq mutant derivatives by triparental matings. Modification of the chromosomal hfq gene to express a C-terminal epitope-tagged Hfq protein was done as follows. A dsDNA fragment encoding 3 tandem FLAG epitopes (3 × FLAG; Sigma-Aldrich) was first generated by annealing
of the 3 × Flag and 3 × Flag-i 69mer oligonucleotides which were designed to leave 5′-end overhangs complementary to XbaI and HindIII recognition sequences. The resulting DNA fragment was then inserted between these two restriction sites in pBluescript II KS+ giving pKS3 × Flag. The full-length Hfq coding sequence (without the TGA stop codon) along with 655 bp of its upstream genomic region was PCR
amplified from pGEMhfq with the primers pair 5HfqTag/3HfqTag both carrying XbaI sites at the 5′-end. The resulting PCR product was cloned into pGEM®-T Easy and retrieved as an XbaI DNA fragment pheromone which was gel purified and inserted at the XbaI site of pKS3 × Flag yielding pKS3 × Flag5. A second 873-bp DNA fragment containing the stop codon for the translation of the epitope-tagged Hfq protein was generated by PCR amplification of the hfq downstream region from pGEMhfq using the primers pair 5FlxTag/3FlxTag which incorporates HindIII sites at both ends of the resulting fragment. The amplification product was inserted into pGEM®-T Easy, recovered as a HindIII fragment, gel purified and finally cloned into the HindIII site of pKS3 × Flag5 to obtain pKSHfq3 × Flag. This plasmid was used as template to amplify an 1,839-bp DNA fragment with a variant of primers 5HfqTag and 3FlxTag in which the XbaI and HindIII sites were replaced by EcoRI and SphI sites, respectively.