A sweet new set of inducible and constitutive promoters for haloarchaea

Abstract

Inducible promoters are one of the most important technical tools for cellular and molecular biology. The ability to deplete, replete, and overexpress genes on demand is the foundation of most functional studies. Here, we develop and characterize a new xylose-responsive promoter (Pxyl), the second inducible promoter system for the model haloarcheon Haloferax volcanii. Generating RNA-seq datasets from cultures in the presence of four historically used inducers (arabinose, xylose, maltose, and IPTG), we mapped upregulated genomic regions largely re-pressed in the absence of the above inducers. The most upregulated promoter was found to control the expression of the xacEA (HVO_B0027-28) operon in the pHV3 chromosome. To characterize this promoter region, we cloned msfGFP (monomeric superfold green fluorescent protein) under the control of two different 5' UTR fragments into a modified pTA962 vector: the first 250 bp (P250) and the whole 750 bp intergenic region (P750). The P250 region showed to express msfGFP constitutively and its expression does not respond to the presence or ab-sence of xylose. However, the P750 promoter showed not only to be repressed in the absence of xylose but also expressed higher levels of msfGFP in the presence of the inducer. Finally, we validated our new inducible Pxyl promoter by reproducing morphological phenotypes al-ready described in the literature. By overexpressing the tubulin-like FtsZ1 or FtsZ2, we ob-served similar, but slightly more pronounced morphological defects compared to the trypto-phan-inducible promoter PtnaA. FtsZ1 overexpression created larger, deformed cells, whereas cells overexpressing FtsZ2 were smaller but mostly retained their shape. In summary, this work contributes with a new xylose-inducible promoter Pxyl that can be used simultaneously with the well-established PtnaA in functional studies in H. volcanii.