RNA obtained was treated with 0.6 U of RQ1 DNase (Promega) for P505-15 datasheet 30 min at 37°C, followed by phenol extraction and ethanol Silmitasertib mouse precipitation, in order to eliminate contaminating genomic DNA. The RNA integrity was assessed by agarose/formaldehyde gel electrophoresis and quantified in a Nanodrop 2000

device (Thermo Scientific). The reactions were performed using primers RND3 and RND4 (located within the coding region of CCNA_02805 and CCNA_02806, respectively). cDNA was synthesized from 0.25 μg of RNA using Super Script™ First Strand Synthesis System (Life Technologies) in a 20 μl final volume, following the manufacturer’s instructions. PCR amplification was performed using 1.2 μg of cDNA as template, 10 pmol each primer, 5% DMSO in a final volume of 25 μl using Taq DNA polymerase (Fermentas). The PCR conditions were: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 1 min, with a final cycle at 72°C

for 5 minutes. A negative control reaction was performed as described above, without the addition of reverse transcriptase. The PCR products were analyzed on 1% agarose gel electrophoresis. Construction of the czrA and nczA mutant and complemented strains In-frame deletions were constructed by allelic exchange using the pNPTS138 suicide vector and C. crescentus NA1000 strain. Two genomic regions upstream and downstream of the gene to be deleted were amplified by PCR using pfx Platinum DNA polymerase (Life Technologies) and primers RND7/RND8 (785 bp, HindIII/EcoRI) and RND9/RND10 (752 bp, EcoRI/MluI) to czrA gene and 3-MA order primers RND11/RND12 (870 bp, HindIII/BamHI) and RND13/RND14 (654 bp, BamHI/MluI) to nczA gene. A terminal adenine was added with Taq DNA Polymerase (Life Technologies) and subsequently the fragments were cloned into vector pGEM-T Easy (Promega). The fragments were cloned in tandem into the pNPTS138 vector, the plasmids were transferred to C. crescentus strain NA1000 by selleckchem conjugation with E. coli S17-1, and recombinant

colonies were selected in PYE-kanamycin plates. A colony containing the integrated plasmid was inoculated in PYE medium without antibiotics for 48 hours, and loss of the plasmid was selected in PYE media containing 3% sucrose. The deletions were confirmed by PCR. Double mutant ΔczrAΔnczA was obtained by introducing the pNPTS138 vector containing the 5′ and 3′-flanking regions of czrA into the ΔnczA strain. PCR products using primers RND15/RND16 (3243 bp) and RND17/RND18 (3132 bp), containing the coding regions of czc1 and czc2 genes respectively, were used to generate complemented strains. Each fragment was cloned into the suicide vector pNPT228XNE, and the plasmid was inserted into the mutant strains by conjugation with E. coli S17-1. The insertion of the recombinant vector occurs at the xylose utilization locus, and expression of the cloned genes is induced with 0.2% xylose. Growth assays in the presence of metals Initial cultures at OD600 = 0.