TABLE 1.
Construction of pART2 and pART3 vectors and their derivatives
| Plasmid | Description and cloning strategy | Source |
|---|---|---|
| pAO1 | PCR template for amplification of the 6-d-hydroxynicotine oxidase gene (hdnO) promoter, the 2,6-dihydroxypyridine 3-hydroxylase (dhpH) gene, and the hdnO repressor gene (hnoR); purified pAO1 plasmid was used | 8 |
| p25435 | Carries the 1.9-kb DNA fragment of the cryptic plasmid pCG100 from C. glutamicum ATCC 13058, which allows independent replication in Arthrobacter species, and the Kanr gene from Tn903 used for the construction of pART vectors | 22 |
| pQEtriSystem-PhdnO | The 171-bp hdnO promoter/operator (hdnOp) was amplified by PCR using the primers 5′-TATATACCATGGTCTTGACAAGG-3′ and 5′-TATATAGGATCCATTTCCAACTCC-3′, restricted with NdeI/BamHI, and ligated into the corresponding sites of pQEtriSystem (Qiagen) | This work |
| pQEtriSystem-PhdnO-bla | The 861-bp bla gene was amplified by PCR from pET21a(+) (Novagen) using the primers 5′-ATATATGGATCCGAGTATTCAACATTTCC-3′ and 5′-ATATATCTCGAGCCAATGCTTAATCAG-3′, followed by BamHI/XhoI restriction and ligation into the BamHI/XhoI sites of pQEtriSystem-PhdnO | This work |
| pLigI | 1.97-kb E. coli vector encoding hdnOp-driven Ampr gene; 1.29-kb hdnOp-bla-His8 DNA fragment was excised with NdeI/NcoI from pQEtriSytem-PhdnO-bla and ligated to the 693-bp ColE1 ori restricted with NcoI/NdeI; ColE1 ori was amplified by PCR with the primers 5′-TATATACCATGGAGCGTCAGACC-3′ and 5′-ATATATCATATGTGAGCAAAAGGCC-3′ and with pThioA (Invitrogen) as a template | This work |
| pART1 | 5.44-kb E. coli-A. nicotinovorans shuttle vector; the NcoI-restricted pLigI plasmid was ligated to the 3.47-kb pCG100 Kanr DNA fragment, excised with NcoI from p25435 | This work |
| pART1A | The same as pART1, except that the XhoI (1041) site was changed to XbaI by site-directed mutagenesis with the primers 5′-GATTAAGCATTGGTCTAGACACCACCATC-3′ and 5′-GATGGTGGTGTCTAGACCAATGCTTAATC-3′ | This work |
| pART2 | 4.63-kb E. coli-A. nicotinovorans shuttle plasmid for hdnOp-driven constitutive expression; a BamHI-AatII-SalI-DraI-AvrII-SpeI-KpnI-PstI-XbaI fragment derived from annealing of two complementary, overhanging 5′-phosphorylated oligonucleotides (5′-GATCCGACGTCGTCGACTTTAAACCTAGGACTAGTGGTACCCTGCAGT-3′ and 5′-CTAGACTGCAGGGTACCACTAGTCCTAGGTTTAAAGTCGACGACGTCG-3′) was ligated into the BamHI/XbaI-restricted pART1A vector | This work |
| pART2-gfp | The 720-bp gfp gene was amplified by PCR from pEGFP-N1 (Clontech), using the primers 5′-CATGGATCCCAAGGGCGAGG-3′ and 5′-CGCGGCCTCTAGACTTGTACAGC-3′, restricted with BamHI/XbaI, and ligated into the corresponding sites of the pART2 vector | This work |
| pART2-dhpH | The 1.2-kb dhpH gene of A. nicotinovorans was amplified by PCR with the primers 5′-CACTAAGGAAGATCTCATGAGTCCCAC-3′ and 5′-GTTTTTCCTTATCTAGAATTAGTGAC-3′, restricted with BglII/XbaI, and ligated into compatible sites (BamHI/XbaI) of the pART2 vector | This work |
| pART3 | 5.4-kb E. coli-A. nicotinovorans shuttle plasmid for nicotine-inducible, hdnOp-driven gene expression; the hdnO repressor gene (hnoR) was amplified by PCR with the primers 5′-CTTTGTGGATCCGCCACCTGGGATGCC-3′ and 5′-AAAGGCGGATCCCCATAAGGAGCAAGG-3′, restricted with BamHI, and ligated into a compatible BglII site of the pART2 plasmid | This work |
| pART3-gfp | Same cloning strategy and primers as for pART2-gfp | This work |
| pART3-dhpH | Same cloning strategy and primers as for pART2-dhpH | This work |