ORIGINAL RESEARCH published: 03 July 2019 doi: 10.3389/fmicb.2019.01256 A Genetic System for Methanocaldococcus jannaschii: An Evolutionary Deeply Rooted Hyperthermophilic Methanarchaeon Dwi Susanti1, Mary C. Frazier1 and Biswarup Mukhopadhyay1,2,3* 1Department of Biochemistry, Virginia Tech, Blacksburg, VA, United States, 2 Biocomplexity Institute, Virginia Tech, Blacksburg, VA, United States, 3 Virginia Tech Carilion School of Medicine, Virginia Tech, Blacksburg, VA, United States Edited by: Phylogenetically deeply rooted methanogens belonging to the genus of Thomas J. Santangelo, Methanocaldococcus living in deep-sea hydrothermal vents derive energy exclusively from Colorado State University, United States hydrogenotrophic methanogenesis, one of the oldest respiratory metabolisms on Earth. Reviewed by: These hyperthermophilic, autotrophic archaea synthesize their biomolecules from inorganic Dina Grohmann, substrates and perform high temperature biocatalysis producing methane, a valuable fuel University of Regensburg, Germany Adam Guss, and potent greenhouse gas. The information processing and stress response systems Oak Ridge National Laboratory of archaea are highly homologous to those of the eukaryotes. For this broad relevance, (DOE), United States Methanocaldococcus jannaschii, the first hyperthermophilic chemolithotrophic organism Janet Westpheling, University of Georgia, United States that was isolated from a deep-sea hydrothermal vent, was also the first archaeon and *Correspondence: third organism for which the whole genome sequence was determined. The research that Biswarup Mukhopadhyay followed uncovered numerous novel information in multiple fields, including those described [email protected] above. M. jannaschii was found to carry ancient redox control systems, precursors of Specialty section: dissimilatory sulfate reduction enzymes, and a eukaryotic-like protein translocation system. This article was submitted to It provided a platform for structural genomics and tools for incorporating unnatural amino Microbial Physiology and Metabolism, a section of the journal acids into proteins. However, the assignments of in vivo relevance to these findings or Frontiers in Microbiology interrogations of unknown aspects of M. jannaschii through genetic manipulations Received: 18 November 2018 remained out of reach, as the organism was genetically intractable. This report presents Accepted: 20 May 2019 tools and methods that remove this block. It is now possible to knockout or modify a Published: 03 July 2019 gene in M. jannaschii and genetically fuse a gene with an affinity tag sequence, thereby Citation: Susanti D, Frazier MC and allowing facile isolation of a protein with M. jannaschii-specific attributes. These tools have Mukhopadhyay B (2019) A Genetic helped to genetically validate the role of a novel coenzyme F420-dependent sulfite reductase System for Methanocaldococcus jannaschii: An Evolutionary Deeply in conferring resistance to sulfite inM. jannaschii and to demonstrate that the organism Rooted Hyperthermophilic possesses a deazaflavin-dependent system for neutralizing oxygen. Methanarchaeon. Front. Microbiol. 10:1256. Keywords: genetics, ancient archaea, methanogen, hyperthermophile, anaerobe, Methanocaldococcus doi: 10.3389/fmicb.2019.01256 jannaschii, gene knockout, protein overexpression Frontiers in Microbiology | www.frontiersin.org 1 July 2019 | Volume 10 | Article 1256 Susanti et al. A Genetic System for Methanocaldococcus jannaschii INTRODUCTION that is key to the function of numerous gut microbial systems has roots in the hydrothermal vent life of ancient organisms Methanocaldococcus jannaschii is the first known such as M. jannaschii (Mukhopadhyay et al., 2000; Johnson hyperthermophilic methanogen (Jones et al., 1983). This and Mukhopadhyay, 2005; Susanti and Mukhopadhyay, 2012; phylogenetically deeply rooted archaeon is also the first Susanti et al., 2014, 2016). hyperthermophilic chemolithotrophic organism that was isolated Most of the above-mentioned post-genome studies have from a deep-sea hydrothermal vent (Jones et al., 1983) where utilized recombinant proteins generated in Escherichia coli and the environmental conditions mimic those of early Earth (Jones few utilized cellular parts of M. jannaschii or direct physiological et al., 1983; Jannasch and Mottl, 1985). M. jannaschii derives measurements in M. jannaschii cultures. The latter efforts were energy solely from hydrogenotrophic methanogenesis facilitated by a method for a bioreactor-based cultivation of (4H2 + CO2 → CH4 + 2H2O), which is one of the most ancient the organism under controlled conditions, which was developed respiratory metabolisms on Earth and developed most likely soon after the sequencing of M. jannaschii genome 3.49 billion years ago (Jones et al., 1983; Jannasch and Mottl, (Mukhopadhyay et al., 1999). Several valuable inferences on 1985; Ueno et al., 2006). It also generates the entire cell from the in vivo relevance of these discoveries have been made inorganic nutrients (Jones et al., 1983) and, therefore, represents using a surrogate system, Methanococcus maripaludis, a a minimal requirement for life to exist independent of other mesophilic marine organism that is closely related to living systems. M. jannaschii and genetically tractable (Tumbula et al., 1997; Due to the special features mentioned above, M. jannaschii Moore and Leigh, 2005). Similar benefit has also come from was the first archaeon and third organism for which the whole work with Methanosarcina species, which are amenable to genome sequence was determined (Bult et al., 1996). Analysis sophisticated genetic analysis (Metcalf et al., 1997; Nayak and of the sequence data revealed many novel metabolic features Metcalf, 2017). However, an interrogation of M. jannaschii as well as genomic basis for known special features of the genes with unknown or tentatively assigned functions in the archaea (Bult et al., 1996). However, for 60% of the genes, organism itself remained unattainable, as this archaeon has even a predicted function could not be assigned (Bult et al., been intractable genetically. In this communication, we report 1996). These findings and the option of making genome-based that we have filled this gap by developing tools and methods inquiries catalyzed vigorous research with M. jannaschii that for genetic analysis of M. jannaschii, which provide two important yielded major breakthroughs. genome manipulation capabilities: to construct in-frame gene Work with purified cellular parts and recombinant forms deletion and enable homologous overexpression of a protein of proteins of M. jannaschii provided robust validation of with an affinity tag. The former will allow finer analysis of previous observation that archaeal DNA replication, transcription, gene functions. The latter would allow facile purification of translation, and stress management machineries are simpler M. jannaschii proteins produced in the most physiologically forms of respective eukaryotic systems (Olsen and Woese, 1996; relevant location, in the organism itself, for in vitro studies. Koonin et al., 1997; Reich et al., 2001; Grohmann and Werner, 2011). Similar parallels were found for eukaryotic protein translocation systems (Van den Berg et al., 2004; Suloway et al., MATERIALS AND METHODS 2012). A structural genomics program leveraged M. jannaschii proteins for the discovery of new protein folds and molecular Materials, Organisms, and Reagents functions of newly identified proteins (Kim et al., 2003; Shin M. jannaschii JAL-1 (Jones et al., 1983) was from the laboratory et al., 2007). The organism provided a platform for the elucidation stock (Johnson and Mukhopadhyay, 2005), which was derived of the pathways for the biosynthesis of coenzymes that are from a culture that was a gift of Prof. Ralph S. Wolfe from specific or key to methanogenesis (Graham and White, 2002). the University of Illinois at Urbana-Champaign (Urbana, IL). A novel tRNA-based cysteine biosynthesis system was discovered M. jannaschii DSM 2661 was obtained from Leibniz-Institut in an effort to understand how M. jannaschii generates this DSMZ-Deutsche Sammlung von Mikroorganismen und amino acid (Sauerwald et al., 2005), and specific tRNA-aminoacyl- Zellkulturen GmbH or DSMZ (Braunschweig, Germany). tRNA synthetase pairs from M. jannaschii assisted the effort Methanocaldococcus strain FS.406-22 (MFS.406-22) (Mehta and to introduce unnatural amino acids and imparting new functions Baross, 2006) was a gift from Prof. Robert Blankenship of to proteins (Wang and Schultz, 2001). the Washington University (St Louis, MO). Escherichia coli Genome-guided physiological studies led to novel hydrogen Stellar™ was obtained from Takara Bio (Mountain View, CA) and redox-controlled systems of ecological and evolutionary and DNA oligonucleotides and synthetic DNA were from biology relevance in M. jannaschii (Mukhopadhyay et al., 2000; Integrated DNA Technologies, Inc. (Coralville, IA). The Johnson and Mukhopadhyay, 2005; Susanti and Mukhopadhyay, antibiotics and base analogs were obtained from the following 2012; Susanti et al., 2014, 2016). In particular, the results of sources: puromycin, neomycin, simvastatin, novobiocin, these studies led to the hypotheses that methanogenesis and 6-methylpurine, 6-thioguanine, 6-azauracil, and 5-fluorouracil dissimilatory sulfate reduction system, another ancient respiratory (MilliporeSigma, Burlington, MA); 8-azahypoxanthine (TCI metabolism on Earth (Shen et al., 2001), have intertwined