Induction of a toxin-antitoxin gene cassette under high hydrostatic pressure enables markerless gene disruption in the hyperther

The discovery of hyperthermophiles has dramatically changed our understanding of the habitats in which life can thrive. However, the extreme high temperatures in which these organisms live have severely restricted the development of genetic tools. The archaeon Pyrococcus yayanosii A1 is a strictly anaerobic and piezophilic hyperthermophile that is an ideal model for studies of extreme environmental adaptation. In the present study, we identified a high hydrostatic pressure (HHP)inducible promoter (Phhp) that controls target gene expression under HHP. We developed an HHP-inducible toxin-antitoxin cassette (HHP-TAC) containing (i) a counterselectable marker in which a gene encoding a putative toxin (virulenceassociated protein C [PF0776 {VapC}]) controlled by the HHP-inducible promoter was used in conjunction with the gene encoding antitoxin PF0775 (VapB), which was fused to a constitutive promoter (PhmtB), and (ii) a positive marker with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-encoding gene from P. furiosus controlled by the constitutive promoter Pgdh. The HHP-TAC was constructed to realize markerless gene disruption directly in P. yayanosii A1 in rich medium. The pop-out recombination step was performed using an HHP-inducible method. As proof, the PYCH_13690 gene, which encodes a 4-␣-glucanotransferase, was successfully deleted from the strain P. yayanosii A1. The results showed that the capacity for starch hydrolysis in the Δ1369 mutant decreased dramatically compared to that in the wild-type strain. The inducible toxin-antitoxin system developed in this study greatly increases the genetic tools available for use in hyperthermophiles. IMPORTANCE Genetic manipulations in hyperthermophiles have been studied for over 20 years. However, the extremely high temperatures under which these organisms grow have limited the development of genetic tools. In this study, an HHPinducible promoter was used to control the expression of a toxin. Compared to sugar-inducible and cold-shock-inducible promoters, the HHP-inducible promoter rarely has negative effects on the overall physiology and central metabolism of microorganisms, especially piezophilic hyperthermophiles. Previous studies have used auxotrophic strains as hosts, which may interfere with studies of adaptation and metabolism. Using an inducible toxin-antitoxin (TA) system as a counterselectable marker enables the generation of a markerless gene disruption strain without the use of auxotrophic mutants and counterselection with 5-fluoroorotic acid. TA systems are widely distributed in bacteria and archaea and can be used to overcome the limitations of high growth temperatures and dramatically extend the selectivity of genetic tools in hyperthermophiles.

Analysis of the gene disruption strain (Δ1369). (A) PCR analysis of fragments from the upstream and downstream regions of the PYCH_13690 gene. Lane M, 1-kb marker; lane 1, A1; lanes 2 to 5, the intermediate strain. (B) PCR analysis of fragments from the upstream and downstream regions of the PYCH_13690 gene. Lane M, 1-kb marker; lane 1, A1; lane 2, Δ1369 mutant. (C) PCR analysis of internal fragments of the PYCH_13690 gene. Lane M, 1-kb marker; lane 1, A1; lane 2, Δ1369 mutant. (D) Measurement of residual starch via Lugol’s iodine method after 36 h at 95°C. Lane 1, TRM with 2‰ (wt/vol) soluble starch; lane 2, strain A1 cultured in TRM with 2‰ (wt/vol) soluble starch; lane 3, Δ1369 mutant cultured in TRM with 2‰ (wt/vol) soluble starch; lane 4, TRM.

Reference:

Qinghao Song, Zhen Li, Rouke Chen, Xiaopan Ma, Xiang Xiao, Jun Xu. (2019). Induction of a toxin-antitoxin gene cassette under high hydrostatic pressure enables markerless gene disruption in the hyperthermophilic archaeon Pyrococcus yayanosii. Applied and Environmental Microbiology 85(4), e02662-02618.

Link: journals.asm.org/doi/full/10.1128/AEM.02662-18