Development of a thermostable β‐glucuronidase‐based reporter system for monitoring gene expression in hyperthermophiles

Abstract

Mesophilic glucuronidases are the most widely used reporters of gene expression in plants, but unsuitable as reporters in (hyper‐)thermophiles due their insufficient thermal stability. Here we present the native 66.8 kDa thermostable β‐glucuronidase of Sulfolobus solfataricus. The enzyme activity is characterized in a wide temperature range ideal for, but not limited to, in vivo genetic study of hyperthermophiles. As a proof of concept, we demonstrate its use as a reporter of gene expression in Sulfolobus, by monitoring a promoter fusion created with the β‐glucuronidase coding gene gusB and a copper‐responsive promoter. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.

The development of a new reporter system based on the β‐glucuronidase gene, gusB, from the hyperthermophile Sulfolobus solfataricus is presented. The activity of the enzyme, characterized under different conditions, is optimal at 75°C, and is maintained after prolonged exposure to elevated temperatures. This work demonstrates the effectiveness of this reporter for in vivo promoter studies in Sulfolobus, by monitoring a reporter plasmid carrying a fusion between a copper‐responsive promoter and gusB.