The resulting PCR fragment was digested with XbaI and ApaI, and the 3,054-bp fragment generated was cloned into pKS bluescript to give plasmid pMntREupR. Subsequently, an HpaI or HindIII recognition site was introduced in mntR or eupR respectively, using the PCR-based QuikChange Site-Directed Mutagenesis Kit (Stratagene) and the following oligonucleotides: MntRHpa_fw:

5′ CCGAATTGGTCGAGGACTATGTTAACGAGATTGCGCATTTGC-3′, MntRHpa_rv: 5′-GCAAATGCGCAATCTCGTTAACATAGTCCTCGACCAATTCGG-3′, EupRHind_fw: 5′-GCACGGCGCACCACCGGCGAAGCTTCGCTTCCCCAGATGACC-3′, and EupRHind_rv: 5′- GGTCATCTCGGGAAGCGAAGCTTCGCCGGTGGTGCGCCGTGC-3′, Smoothened Agonist clinical trial that were modified (residues in bold) to introduce the corresponding restriction sites. The resultant plasmids, pHpaIMntR and pHindIIIEupR were linearized with the enzyme HpaI or HindIII and ligated to 2-kb SmaI or HindIII fragments from pHP45-Ω [50] or pHP45-Ωaac [51], containing the Ω interposons for insertional mutagenesis (Smr or Gnr). The resulting RAD001 price plasmids were named pΩMntR and pΩEupR. To recombine the mntR or eupR mutations into the C. salexigens chromosome, 5-kb XbaI-ApaII fragments from pΩMntR or pΩEupR were cloned into the suicide vector pJQSK200 (Gmr) [52] to give plasmids pJQMntR and pJQEupR, which were mobilized into the C. salexigens wild type strain by triparental mating. Mutant strains resulting from a double homologous recombination

event were identified as Smr Gms, or Gnr Gms colonies Histidine ammonia-lyase on SW-2 plates containing 10% sucrose. Two of these colonies were purified for further analysis and were named CHR161 (mntR::Ω) and CHR183 (eupR::Ωaac). Insertions of the omega cassette in CHR161 and CHR183 were confirmed by PCR and sequencing. Determination of sensitivity to Mn To determine the sensitivity of C. salexigens strains to Mn, we used fresh plates of a modified SW-2 medium containing less than 1 mM of SO4Mg (to avoid interference of Mg2+ with Mn2+), which was additioned with 0.5 to 2.5 mM MnCl2. An overnight culture of each strain (100 μl) was spread onto the

assay plate and growth was observed after incubation at 37°C for 48 h. Determination of ectoine uptake Cells grown overnight in SW-2 were subcultured at a 1:100 dilution in glucose M63 medium containing 0.75, 1.5 or 2.5 M of NaCl, and grown up to exponential phase (OD600 ca. 0.5). Transport was initiated by adding [14C]-ectoine to 0.2 ml of bacterial suspensions and incubating the cultures at room temperature. The [14C]-ectoine (5.5 MBq mM) was prepared biologically from Brevibacterium linens as described [53] and was added at a final concentration of 87 μM. During 2 min, 50 μl of samples were taken at 30-s intervals, and transport was terminated by rapid filtration through Whatman GF/F discs (Fisher Bioblock, Illkirch, France). The cells were quickly washed twice with 2 ml of isotonic M63 medium.

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