Genetic manipulations of the hyperthermophilic piezophilic archaeon Thermococcus barophilus

Abstract

In this study, we developed a gene disruption system for Thermococcus barophilus using simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single- and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-type T. barophilus MP cells. Initially, a pyrF deletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of the hisB gene was also constructed in the UBOCC-3256 (ΔpyrF) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functional pyrF or hisB allele from T. barophilus was inserted into the chromosome of UBOCC-3256 (ΔpyrF) or UBOCC-3260 (ΔpyrF ΔhisB), allowing homologous complementation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea.

FIG 1

Construction of pUDH and pUFH plasmids. (A) Primers SalI-HMG-CoA-Up and KpnI-HMG-CoA-Do were used to amplify hmg_Pf from the vector pLC70 (20). pUD and HMG-CoA were digested by SalI (S) and BamHI (B) and were then ligated to obtain the plasmid pUDH. (B) Primers XhoI-pyrF-TB-Up and XmaISmaI-pyrF-TB-Do were used to amplify the T. barophilus pyrF gene. pUDH and pyrF(TB) were digested by XhoI (X) and SmaI (S) and were then ligated to obtain the plasmid pUFH. In pUDH and pUFH, the restriction enzyme sites BamHI (B) and KpnI (K) were conserved to enable cloning of the homologous regions in these plasmids.

FIG 2

Deletion pathway of pyrF and hisB genes. (A) Two suicide vectors were constructed to ligate homologous-region amplification (TERMP_01289, TERMP_01291, TERMP_00436, and TERMP_00438) with pUFH or pUDH. The plasmids used were pUDH-1 and pUFH-1. After transformation, the plasmid was integrated into the genome by a first crossover event in the homologous-region fragment. The second step was the pop-out recombination (or excision) event. There are two possibilities: a recombination between the other homologous fragments, resulting in the deletion of the targeted gene, or a recombination between the same homologous fragments of the first recombination, which gives the WT genotype. pyrF was deleted from strain UBOCC-3107 (WT), and hisB was deleted from strain UBOCC-3256 (ΔpyrF). (B) To verify the different genotype configurations, PCR amplification was performed with the primers matching the HR1 and HR2 regions: XhoI-pyrF-TB-Up and SmaI-pyrF-TB-Do for pyrF deletion and Verif-hisB-Up and Verif-hisB-Do for hisB deletion (data not shown).

FIG 3

Characterization of UBOCC-3256 (ΔpyrF) and UBOCC-3260 (ΔpyrF ΔhisB) mutants at 0.1 MPa and 40 MPa. Growth assays were carried out in TAA medium at 85°C, without uracil (■), with uracil (●), and with uracil and histidine (▲). These growth experiments were carried out at hydrostatic pressures of 0.1 MPa and 40 MPa.

FIG 4

Complementation of pyrF and hisB mutations. Growth assays of strains UBOCC-3107 (WT) (●), UBOCC-3256 (ΔpyrF) (■), and UBOCC-3262 (ΔpyrF TERMP_00006::pUFH-2) (▲) (A) and of strains UBOCC-3107 (WT) (●), UBOCC-3260 (ΔpyrF-ΔhisB) (■), and UBOCC-3265 (ΔpyrF ΔhisB TERMP_00004::pUHH) (▲) (B) were carried out at 85°C in TAA medium without (A) or with (B) uracil. These growth experiments were performed at 0.1 MPa.