Development of a genetic system for the chemolithoautotrophic bacterium Thiobacillus denitrificans

Abstract

Thiobacillus denitrificans is a widespread, chemolithoautotrophic bacterium with an unusual and environmentally relevant metabolic repertoire, which includes its ability to couple denitrification to sulfur compound oxidation; to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV); and to oxidize mineral electron donors. Recent analysis of its genome sequence also revealed the presence of genes encoding two [NiFe]hydrogenases, whose role in metabolism is unclear, as the sequenced strain does not appear to be able to grow on hydrogen as a sole electron donor under denitrifying conditions. In this study, we report the development of a genetic system for T. denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. The antibiotic sensitivity of T. denitrificans was characterized, and a procedure for transformation with foreign DNA by electroporation was established. Insertion mutations were generated by in vitro transposition, the mutated genes were amplified by the PCR, and the amplicons were introduced into T. denitrificans by electroporation. The IncP plasmid pRR10 was found to be a useful vector for complementation. The effectiveness of the genetic system was demonstrated with the hynL gene, which encodes the large subunit of a [NiFe]hydrogenase. Interruption of hynL in a hynL::kan mutant resulted in a 75% decrease in specific hydrogenase activity relative to the wild type, whereas complementation of the hynL mutation resulted in activity that was 50% greater than that of the wild type. The availability of a genetic system in T. denitrificans will facilitate our understanding of the genetics and biochemistry underlying its unusual metabolism.

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; P_Kan, 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.