Article Figures & Data
Figures
- FIG. 1.
Schematic diagram of the complementation vector (pTL2; Table 1) used in T. denitrificans. Plasmid construction is described in Materials and Methods. The following features are shown: oriV, the RK2 minimal vegetative origin of replication; oriT, the origin of transfer; trfA, encodes the RK2 replication initiation protein; bla, the beta-lactamase gene encoding ampicillin resistance; PKan, the 110-bp promoter of the kanamycin resistance gene from pTnMod-OKm′; and relevant restriction sites. The HpaI restriction site is unique and allows placement of genes for complementation. KpnI and HindIII bracket the original MCS from pRR10.
- FIG. 2.
(A) Electropherogram of PCR products from wild-type (WT) T. denitrificans, the hynL mutant, and the complemented (Compl.) hynL mutant, as well as digested plasmid DNA from the complemented mutant. Lane 1, HyperLadder III, Bioline; lane 2, wild-type DNA, primers hynL-2-f and -r; lane 3, hynL mutant (strain TL001) DNA, primers hynL-2-f and -r; lane 4, complemented-mutant genomic DNA, primers hynL-2-f and -r; lane 5, complemented-mutant plasmid DNA, primers hynL-2-r and pUC19-r; lane 6, Hi-Lo Marker, Bionexus (the arrow indicates 8 kb); lane 7, complemented-mutant pTL3 plasmid DNA, NdeI digested. (B) Maps of primer positions and amplicon sizes corresponding to lanes 2 to 5 in panel A. Note that the hynL-2-f primer anneals with genomic DNA upstream of the hynL gene, whereas the pUC19-r primer anneals with pTL3 plasmid DNA, rendering these primers specific to the T. denitrificans genome and the complementation plasmid pTL3, respectively.
- FIG. 3.
Hydrogen oxidation (measured as in vivo benzyl viologen reduction) versus time for wild-type T. denitrificans, strain TL001 (hynL mutant), strain TL002 (hynL hydA double mutant), and the complemented hynL mutant (TL001/pTL3). Negative controls (averaged results for controls with no H2 and controls with no benzyl viologen) are also shown. Each datum point represents the average of duplicate or triplicate assays. Linear-regression fits are plotted.
Tables
- TABLE 1.
Strains and plasmids used in this study
Strain, plasmid, or transposon Genotype or markers; characteristics and uses Source or reference Strains Escherichia coli TOP10 F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7967 galU galK rpsL (Strr) endA1 nupG Invitrogen Thiobacillus denitrificans ATCC 25259 Wild type ATCCa TL001 hynL::kan This work TL002 hynL::kan hydA::gent This work Plasmids pUC19 pMB1, Ampr; cloning vector 24 pPA20 pMB1, IncP Ampr Kanr; shuttle vector 1 pANT4 IncQ, Ampr Kanrmob+ 15 pTnMod-OCm pMB1, Camrmob+ Tn5 tnp 11 pTnMod-OGm pMB1, Gentrmob+ Tn5 tnp 11 pTnMod-OKm′ pMB1, Kanrmob+ Tn5 tnp 11 pTnMod-SmO pMB1, Spcmr Strrmob+ Tn5 tnp 11 pTnMod-OTc pMB1, Tetrmob+ Tn5 tnp 11 pRR10 IncP, Amprmob+lacZα 19 pTL1 IncP, Ampr, pRR10 with PKan inserted at MCS This work pTL2 IncP, Gentr Amps, pTL1 with bla::gent; T. denitrificans expression vector This work pTL3 IncP, Gentr, pTL2 with hynL-hupE inserted next to PKan allowing for expression This work EZ-Tn5 pMOD-2<MCS> ColE1, Ampr; transposon construction vector EPICENTRE Biotechnologies pMOD-2<gent> ColE1, Ampr Gentr with SacI fragment from pTnMod-OGm containing aacC1 inserted at MCS of pMOD-2 This work pUC19-hynL pMB1, Ampr, pUC19 with hynL inserted at MCS This work pUC19-hynL::kan pMB1, Ampr Kanr, pUC19 with hynL::kan inserted at MCS This work pUC19-hydA pMB1, Ampr, pUC19 with hydA inserted at MCS This work pUC19-hydA::gent pMB1, Ampr Gentr, pUC19 with hydA::gent inserted at MCS This work Transposons Tn-kan Kanr, EZ-Tn5<KAN-2> DNA fragment with kanamycin resistance selection marker located between Mosaic End Tn5 transposase recognition sequences EPICENTRE Biotechnologies Tn-gent Gentr, EZ-Tn5 pMOD-2<gent> DNA fragment with gentamicin resistance selection marker located between Mosaic End Tn5 transposase recognition sequences This work ↵ a ATCC, American Type Culture Collection, Manassas, VA.
- TABLE 2.
Sequences of primers used in this study
Primer Sequencea (5′ → 3′) hynL-1-f GAATTCTAGATGGCAACAACACAGCGCGTC hynL-1-r GATAGCATGCTTATTTGACCTTCACCTGCACG pUC19-f GCCAGGGTTTTCCCAGTCACGA pUC19-r GAGCGGATAACAATTTCACACAGG kanP-f AAAGCCACGTTGTGTCTC kanP-r GAATGTTAACACCCCTTGTATTACTG hynL-hupE-f phos-ATGGCAACAACACAGCGC hynL-hupE-r phos-TCACAGCCCGAGGATGAGG EZ-Tn5-fb ACCTACAACAAAGCTCTCATCAACC EZ-Tn5-rb GCAATGTAACATCAGAGATTTTGAG pMOD2-PCR-fb ATTCAGGCTGCGCAACTGGT pMOD2-PCR-rb GTCAGTGAGCGAGGAAGCGGAAG pMOD2-Seq-fb GCCAACGACTACGCACTAGCCAAC pMOD2-Seq-rb GAGCCAATATGCGAGAACACCCGAGAA hynL-2-f TCATGGCGGAGCATCTGGAG hynL-2-r CGTATTTCGGAACATGGGTGG hydA-f GAATGGTACCCAGTTCAACATGCTCTACCT hydA-r GATAGGTACCTTGAACGGCGCGTCGAACTG
