Genetics Techniques for Thermococcus kodakarensis

Abstract

Thermococcus kodakarensis (T. kodakarensis) has emerged as a premier model system for studies of archaeal biochemistry, genetics, and hyperthermophily. This prominence is derived largely from the natural competence of T. kodakarensis and the comprehensive, rapid, and facile techniques available for manipulation of the T. kodakarensis genome. These genetic capacities are complemented by robust planktonic growth, simple selections, and screens, defined in vitro transcription and translation systems, replicative expression plasmids, in vivo reporter constructs, and an ever-expanding knowledge of the regulatory mechanisms underlying T. kodakarensis metabolism. Here we review the existing techniques for genetic and biochemical manipulation of T. kodakarensis. We also introduce a universal platform to generate the first comprehensive deletion and epitope/affinity tagged archaeal strain libraries.

Keywords: Thermococcales; archaea; genetics; hyperthermophilic; recombination; transcription.

Figure 1

Integration of a selection cassette into the Thermococcus kodakarensis genome provides a mechanism for genomic modification. A general scheme for integration of a selectable marker cassette (gene providing positive selection, gray; promoter, bent arrow) into the genome of a recipient strain. The resultant genome of the transformant results from two homologous recombination events (dashed lines) between flanking regions contained in both the donor DNA and targeted genomic locus. For simplicity, a single gene replacement of a target locus (yellow) by the selectable marker is shown, although different genomic alterations including allelic exchanges, promoter alterations, and additions of epitopes can similarly be introduced using the same technique. Each marker in Table 1, with the exception of 6MP, can be employed to generate strains that retain the marker at or near the modified locus.

Figure 2

Markerless-modification of the Thermococcus kodakarensis genome via sequential positive selection and subsequent counter-selection. Donor DNA, a hypothetical target locus in the recipient genome, the resultant intermediate, and final genomes are shown. The scheme diagrammed generates an intermediate strain wherein both the selectable (gray) and counter-selectable marker (TK0664; orange) are flanked by a direct repeat (cyan), and the intermediate strain is deleted for the target gene. Alternative donor DNAs can result in intermediate strains that retain the target gene, necessitating excision of the selectable markers in addition to the target gene to via recombination between direct repeats to generate the desired final markerless-deletion strain. The same protocol can be used with the uracil (pyrF) cassette that will serve as both the positive and counter-selective marker. Any combination of a positive selection cassette (Table 1) and TK0664-based 6MP counter-selection can be employed in a two-gene mechanism to introduce markerless modifications to the T. kodakarensis genome.

Figure 3

Replicative vectors provide platforms for exogenous and ectopic expression in Thermococcus kodakarensis. T.k-E. coli shuttle vectors are the result of merging the entire Thermococcus nautilus-derived pTN1 sequence with a common E. coli plasmid (pCR2.1-Topo; Invitrogen). The marker additions provide the means to select T. kodakarensis transformants on any media, and the expression cassette provides a mechanism to ectopically express any gene of choice in T. kodakarensis.

Figure 4

Construction of foundation plasmids by a ligation-independent technique. (Left) Each target gene as well as ∼500–700 bp of both upstream and downstream adjacent DNA is amplified from T. kodakarensis chromosomal DNA with a pair of primers that introduce unique 13 bp terminal extensions to the amplicon. Incubation of the purified amplicon with T4 DNA polymerase (DNAP) and only dGTP leads to a 3′-5′ exonuclease recession of the 3′ ends to produce non-complementary 12-nt sticky ends. (Right) pTS700 is linearized through a unique SwaI site, producing 3′ ends that are similarly recessed with T4 DNAP and dCTP to yield sticky ends complementary to the sticky ends of each amplicon permitting directional cloning of the amplicon in a ligase-independent reaction. Amplicons for each T. kodakarensis gene, regardless of gene orientation, gene length, or sequence can be cloned in an identical manner to yield foundation plasmids (A-plasmids). The TATA-box of P_hmtB is boxed and the site of transcription initiation marked with a bent arrow; the Shine–Dalgarno sequence (SD) is underlined; the SwaI recognition sequence is shown in gray; the selectable and counter-selectable expression cassettes, TK0149 and TK0664, are shown in pink and orange, respectively; the plasmid origin of replication (oriC) and E. coli resistance cassette (bla) are shown in gray. The color scheme is conserved in Figures 4–7.

Figure 5

Construction of deletion- and affinity-plasmids from the foundation plasmids. A-plasmids serve as templates for PCR (QuikChange; Agilent) wherein essentially the entire plasmid is replicated to generate B,C-plasmids respectively. B-plasmids result from primer pairs that are complementary, both upstream and downstream, of the target gene (yellow), whereas C-plasmids result from primer pairs that introduce 45 additional nucleotides, encoding both a His₆ and HA epitope tag, in-frame and immediately prior to the normal stop codon of the target gene. A simplified diagram of the plasmids is used for clarity only and each plasmid retains all the components highlighted in Figure 4. The larger green arrow depicts the sequence encoding the His₆-HA tag.

Figure 6

Use of deletion plasmids (B-plasmids) to generate markerless knockouts in the genome of Thermococcus kodakarensis. A hypothetical region of the genome of T. kodakarensis strain TS559 is shown at top, with the left and right panels depicting the two possible integration events yielding agmatine-prototrophic intermediate strains from the diagramed B-plasmid. Both intermediate strains #1 and #2 contain direct repeats flanking the target locus, and dependent on recombination responsible for excision upon counter-selection with 6MP, either the original TS559 genome can be restored or the desired deletion genome generated.

Figure 7

Use of epitope/affinity tag plasmids (C-plasmids) to generate markerlessly tagged-strains of Thermococcus kodakarensis. A hypothetical region of the genome of T. kodakarensis strain TS559 is shown at top, with the left and right panels depicting the two possible integration events yielding agmatine-prototrophic intermediate strains from the diagramed C-plasmid. Both intermediate strains #1 and #2 contain direct repeats flanking the target locus, and dependent on recombination responsible for excision upon counter-selection with 6MP, either the original TS559 genome can be restored or the desired tag- and epitope-containing genome generated.