The pSL487 plasmid expressing the GST–SpiA Selleck GSI-IX fusion protein was constructed by ligating the BamHI–XhoI fragment from pSL487 into the pGEX-4T3 vector. The pSL494 plasmid expressing the His6–WhcA fusion protein was constructed via the amplification of the whcA gene using the primers 5′-CCCAAGCTTTCATGACGTCTGTGATT-3′ and 5′-CCCAAGCTTTTAAACCCCGGC-3′, and by subsequently digesting the fragment with HindIII and ligating the DNA with the HindIII-digested pET28a vector. Corynebacterium

glutamicum (100 μL) genomic DNA (2 μg μL−1), isolated as described by Tomioka et al. (1981), was partially digested with 0.195 U of SauIIIA1 for 1 h at 37 °C. DNA fragments 1–3 kb in size were isolated and JQ1 solubility dmso inserted into the BamHI-digested pTRG vector. The recombinants were introduced into E. coli cells and plasmids were isolated and pooled from approximately 10 000 transformants. The BacterioMatch II Two-Hybrid System (Agilent Technology) was used according to the manufacturer’s instructions. Briefly, the

two plasmids, pBT and pTRG, containing the ‘bait’ and target genes, respectively, were used to simultaneously transform E. coli. Protein–protein interactions were screened based on expression of his3 and aadA, which confer histidine prototrophy (His+) and streptomycin resistance (Str+), respectively.

For screening, 50 ng of each pBT-whcA and target library DNA was introduced into reporter cells and spread onto the selective media (His− and Str+). Colonies were isolated and the plasmids in the growing cells were analyzed. Total RNA was prepared with the NucleoSpin RNA II Kit (Macherey-Nagel, Germany). cDNA conversion was carried out with DyNAmo™ cDNA Synthesis Kit (FinnZymes, Finland). Real-time quantitative PCR (RT-qPCR) was performed using a CFX96™ Real-Time PCR Detection System (Bio-Rad). Different gene expressions were normalized to the levels of 16S rRNA gene transcripts. The degree Dolutegravir mw of change in expression was calculated with the method using cfx™manager software (Bio-Rad). Primers used for the quantification of the reporter gene his3 were as follows: sense primer 5′-CGCTAATCGTTGAGTGCATTG-3′; antisense primer 5′-CGCAAATCCTGATCCAAACC-3′. 16S rRNA gene transcripts were amplified with sense primer 5′-TGGGAACTGCATCTGATACTGGCA-3′ and antisense primer 5′-TCTACGCATTTCACCGCTACACCT-3′. The GST–SpiA fusion protein was expressed and purified using the GSTrap™ FF column (GE Healthcare), in accordance with the manufacturer’s instructions. Pull-down experiments were performed with purified recombinant proteins.