A bioluminescent reporter for the halophilic archaeon Haloferax volcanii
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ORIGINAL PAPER
A bioluminescent reporter for the halophilic archaeon Haloferax volcanii Chris R. Davis1 · Carl H. Johnson2 · J. Brian Robertson1 Received: 28 May 2020 / Accepted: 21 July 2020 © Springer Japan KK, part of Springer Nature 2020
Abstract Haloarchaea have evolved to thrive in hypersaline environments. Haloferax volcanii is of particular interest due to its genetic tractability; however, few in vivo reporters exist for halophiles. Haloarchaeal proteins evolved characteristics that promote proper folding and function at high salt concentrations, but many mesophilic reporter proteins lack these characteristics. Mesophilic proteins that acquire salt-stabilizing mutations, however, can lead to proper function in haloarchaea. Using laboratory-directed evolution, we developed and demonstrated an in vivo luciferase that functions in the hypersaline cytosol of H. volcanii. Keywords Luciferase · Bioluminescence · Reporter · Halophile Abbreviations CDS Coding sequence CBG Click Beetle Green luciferase CCD Charge-coupled Device
Introduction Understanding the physiology and gene regulation of extremophile archaea has proven difficult in the past due to both the environments required to culture these organisms and developing tools for alteration and analysis at the genetic level. However, an increasing number of methods and protocols for exploiting Haloferax volcanii, along with the relative ease to culture the species in a laboratory setting, grant this species status as a model organism for investigating the halophilic archaea and use in biotechnology (Allers and Ngo Communicated by A. Oren. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00792-020-01193-x) contains supplementary material, which is available to authorized users. * J. Brian Robertson [email protected] 1
Department of Biology, Middle Tennessee State University, Murfreesboro, TN, USA
Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA
2
2003; Allers et al. 2004; Haque et al. 2020; Hartman et al. 2010; Reuter and Maupin-Furlow 2004). Developing additional tools for genetic regulation studies in H. volcanii can provide insights into it and similar halophiles. To date, several reporter systems have been developed for measuring gene regulation in halophilic microbes. Antibiotic resistance genes have been used as crude reporters since the 1990s to correlate colony presence/size with levels of promoter activity driving antibiotic resistance gene expression when grown in the presence of antibiotic; however, precise quantitation of promoter activity required extraction and measurement of the reporter’s mRNA abundance (Danner and Soppa 1996; Lam and Doolittle 1992). A halophilic beta-galactosidase assay was developed in the early 2000s which allowed colony-based colorimetric screening; however, accurate quantitation of the reporter enzyme from liquid culture still required cells to be collected and lysed (Patenge et al. 2000). The need to lyse halophilic microb
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