A Genetic System for the Thermophilic Acetogenic Bacterium Thermoanaerobacter kivui

Abstract

Thermoanaerobacter kivui is one of the very few thermophilic acetogenic microorganisms. It grows optimally at 66°C on sugars but also lithotrophically with H₂ + CO₂ or with CO, producing acetate as the major product. While a genome-derived model of acetogenesis has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in carbon and energy metabolism have been carried out. To address this issue, we developed a method for targeted markerless gene deletions and for integration of genes into the genome of T. kivui The strain naturally took up plasmid DNA in the exponential growth phase, with a transformation frequency of up to 3.9 × 10^-6 A nonreplicating plasmid and selection with 5-fluoroorotate was used to delete the gene encoding the orotate phosphoribosyltransferase (pyrE), resulting in a ΔpyrE uracil-auxotrophic strain, TKV002. Reintroduction of pyrE on a plasmid or insertion of pyrE into different loci within the genome restored growth without uracil. We subsequently studied fructose metabolism in T. kivui The gene fruK (TKV_c23150) encoding 1-phosphofructosekinase (1-PFK) was deleted, using pyrE as a selective marker via two single homologous recombination events. The resulting ΔfruK strain, TKV003, did not grow on fructose; however, growth on glucose (or on mannose) was unaffected. The combination of pyrE as a selective marker and the natural competence of the strain for DNA uptake will be the basis for future studies on CO₂ reduction and energy conservation and their regulation in this thermophilic acetogenic bacterium.IMPORTANCE Acetogenic bacteria are currently the focus of research toward biotechnological applications due to their potential for de novo synthesis of carbon compounds such as acetate, butyrate, or ethanol from H₂ + CO₂ or from synthesis gas. Based on available genome sequences and on biochemical experiments, acetogens differ in their energy metabolism. Thus, there is an urgent need to understand the carbon and electron flows through the Wood-Ljungdahl pathway and their links to energy conservation, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Unfortunately, genetic systems have been reported for only a few acetogenic bacteria. Here, we demonstrate proof of concept for the genetic modification of the thermophilic acetogenic species Thermoanaerobacter kivui The genetic system will be used to study genes involved in biosynthesis and energy metabolism, and may further be applied to metabolically engineer T. kivui to produce fuels and chemicals.

Keywords: DNA uptake; Thermoanaerobacter kivui; acetogenesis; fructose metabolism; genetic system.

FIG 1

Growth of T. kivui in solid medium and natural competence for DNA uptake. (A) Sealed metal jar for anaerobic incubation of T. kivui at 65°C. (B) T. kivui embedded in agar medium formed yellow to brownish disc-like colonies. (C) Natural competence of T. kivui during growth on complex medium with glucose at 65°C. One-ml subsamples of two growing cultures were incubated with 250 ng of plasmid pMU131 (16), and then embedded in complex medium with or without 200 μg · ml⁻¹ kanamycin. Squares, representative growth curve; triangles, corresponding transformants per CFU.

FIG 2

Deletion of pyrE. (A) T. kivui was transformed with plasmid pMBTkv002b, which was constructed for the deletion of the pyrE gene via double homologous recombination using 1-kbp upstream and downstream flanking regions (UFR and DFR). After transformation, T. kivui was plated in the presence of 5 mM 5-fluoroorotic acid (5-FOA). (B) The loss of pyrE (573 bp) was verified by PCR using primers binding outside the flanking regions (blue arrows in panel A). Shown is the electrophoretic separation of the DNA fragments from the PCRs using genomic DNA of T. kivui DSM2030 (wild type, WT) and of two 5-FOA-resistant isolates.

FIG 3

Physiology of the T. kivui ΔpyrE strain TKV002. (A) Growth of the wild type (DSM 2030, filled triangles), T. kivui TKV002 (open circles), and T. kivui TKV002 containing plasmid pKOM1 encoding pyrE under the control of P_kan (filled diamonds), without the addition of uracil. (B) Growth of T. kivui TKV002 with P_kan-pyrE reinserted into the genome in between open reading frames (ORFs) TKV_c24500 and TKV_c24520 (filled squares), and growth of T. kivui TKV002 in the presence of 40 μM uracil (closed circles). All experiments were performed in minimal medium with glucose (25 mM) at 65°C. Shown is one representative experiment out of three independent biological replicates.

FIG 4

Deletion of fruK. (A) Strategy for deletion of fruK using plasmid pMBTkv021 via two independent homologous recombination events. (B) DNA fragments separated by agarose gel electrophoresis after PCR amplification of the fruK locus from genomic DNA of wild type (WT) T. kivui and of uracil-auxotrophic isolates after selection against uracil auxotrophs in the first round of selection and (C) 5-FOA resistant isolates after the second round of selection. Putative plasmid integration, orange arrow; wild type allele, blue arrow; fruK deletion (black arrow). (D) Southern blot analysis of the fruK locus with NsiI-digested DNA from T. kivui DSM2030 (wild type, WT; expected fragment size, 2,668 bp) and the ΔfruK strain (expected fragment size, 1,735 bp).

FIG 5

FruK is essential for fructose metabolism. (A) Growth of the T. kivui strain ΔfruK strain TKV003 (closed squares) and the wild-type strain DSM 2030 (open circles) in the presence of 40 μM uracil. Growth of T. kivui TKV003 with plasmid pKOM3 (with fruK under the control of P_kan) in the absence of uracil (closed triangles). All experiments were performed on complex medium with 25 mM fructose at 65°C. (B) Growth of T. kivui TKV003 (closed squares) in the presence of uracil and the wild-type strain DSM 2030 (open circles) on complex medium with 25 mM glucose. Shown are data from one representative experiment out of three independent biological replicates.