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<p>The identification and characterization of promoters and regulatory elements are commonly assessed using reporter genes. However, very few of these are available for haloarchaea. In this study, we describe the construction and validation of a reporter system for the haloarchaeon <i>Haloferax volcanii</i> based on a modified and stable version of the carotenoid biosynthesis enzyme phytoene synthase (PSY). This tool enables the analysis of a target gene expression through direct visualization of cell pigmentation and/or the extraction and quantification of carotenoid content. The modified <i>crtB</i> gene encoding PSY was cloned into the pTA963 vector under two regulatable promoters previously characterized in <i>H. volcanii</i>: <i>PtnaA</i> and <i>Pxyl</i>, inducible with tryptophan and xylose, respectively. The construct was introduced into and expressed in a non-pigmented <i>H. volcanii</i> strain (Δ<i>crtB</i>) under varying inducer concentrations. For both promoters, a clear increase in pigmentation was visually observed in cultures and cell pellets with increasing inducer levels. These observations were corroborated by carotenoid extraction and quantification, as well as by Western blot analysis of PSY protein levels. This work presents a robust, easy-to-use, and versatile reporter system for investigating gene expression in <i>H. volcanii</i>, expanding the toolkit for genetic studies in haloarchaea.</p>

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A colorful new reporter system to evaluate gene expression in the archaeon Haloferax volcanii

  • 期刊论文

  • 发表日期：

    2025年6月27日

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  • Sofía D. Frecha,
  • María I. Giménez,
  • Roberto A. Paggi,
  • Rosana E. De Castro,
  • Micaela Cerletti

摘要

The identification and characterization of promoters and regulatory elements are commonly assessed using reporter genes. However, very few of these are available for haloarchaea. In this study, we describe the construction and validation of a reporter system for the haloarchaeon Haloferax volcanii based on a modified and stable version of the carotenoid biosynthesis enzyme phytoene synthase (PSY). This tool enables the analysis of a target gene expression through direct visualization of cell pigmentation and/or the extraction and quantification of carotenoid content. The modified crtB gene encoding PSY was cloned into the pTA963 vector under two regulatable promoters previously characterized in H. volcanii: PtnaA and Pxyl, inducible with tryptophan and xylose, respectively. The construct was introduced into and expressed in a non-pigmented H. volcanii strain (ΔcrtB) under varying inducer concentrations. For both promoters, a clear increase in pigmentation was visually observed in cultures and cell pellets with increasing inducer levels. These observations were corroborated by carotenoid extraction and quantification, as well as by Western blot analysis of PSY protein levels. This work presents a robust, easy-to-use, and versatile reporter system for investigating gene expression in H. volcanii, expanding the toolkit for genetic studies in haloarchaea.