A sweet new set of inducible and constitutive promoters in Haloferax volcanii

Abstract

Inducible promoters are one of cellular and molecular biology's most important technical tools. The ability to deplete, replete, and overexpress genes on demand is the foundation of most functional studies. Here, we developed and characterized 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 primarily repressed in the absence of the above inducers. We found a highly upregulated promoter that controls 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 upstream regions into a modified pTA962 vector: the first 250 bp (P250) and the whole 750 bp intergenic fragments (P750). The P250 sequence drove the expression of msfGFP constitutively, and its expression did not respond to the presence or absence of xylose. However, the P750 promoter showed not only to be repressed in the absence of xylose but also expressed higher levels of msfGFP than the previously described inducible promoter PtnaA in the presence of the inducer. Finally, we validated the inducible Pxyl promoter by reproducing morphological phenotypes already described in the literature. By overexpressing the tubulin-like FtsZ1 and FtsZ2, we observed similar but slightly more pronounced morphological defects than the tryptophan-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 a new xylose-inducible promoter that could be used simultaneously with the well-established PtnaA in functional studies in H. volcanii in the future.

Keywords: Haloferax volcanii; archaea; constitutive promoters; haloarchaea; inducible promoters; xylose-inducible promoter.

Figure 1

RNA-seq map of inducer-responsive promoters in H. volcanii. (A) Growth curves of H. volcanii DS2 cells under different xylose concentrations. (B) Cell area measurements at different xylose concentrations by phase contrast microscopy from mid-exponential cultures. Datapoints (and means) were colored (pink, yellow, and blue), indicating different biological replicates. (C) Gene expression ratios mapped across the H. volcanii genome from RNA-seq datasets of mid-exponential cultures with and without 10 mM arabinose (orange), xylose (blue), maltose (green), and IPTG (pink). Numbers 1 and 2 indicate genomic regions where gene expression increased above 5-fold. Arrowheads indicate promising genomic regions that did not satisfy our arbitrary 5-fold cutoff. (D) Locus organization of regions 1 and 2. (E) Expression map (transcripts per million) of genomic regions from RNA-seq datasets without inducers (left) and the relative increase in expression (right) from uninduced (Cab), arabinose (Ara), and xylose (Xyl).

Figure 2

Pxyl constructs can be either strong inducible or constitutive promoters. (A) Fragments of the 5′ intergenic region of xacE were used to clone into pAL vectors and map the P250 and P750 promoters tested in this work. (B) Phase-contrast and epifluorescence images of different constructs expressing the msfGFP fluorescent protein. (C) Mean msfGFP fluorescence measurements per cell from images shown in panel B. Each replicate means, and data points are independently labeled with different colors (pink, yellow, and blue). Datapoints (and means) were colored (pink, yellow, and blue), indicating different biological replicates. (D) Comparative background expression from constructs with and without glucose repression. Datapoints (and means) were colored (pink, yellow, and blue), indicating different biological replicates. (E) The dynamic range of PtnaA and Pxyl promoters across different inducer concentrations in raw numbers (top) and normalized (bottom) fold-changes. Shades indicate the 95% confidence interval from triplicate datasets.

Figure 3

P750 shows different induction profiles in different media, at stationary phase, and upon adding arabinose. Epifluorescence microscopy quantification from exponential growing cells in Hv-Cab medium and Hv-YPC medium induced with 10 mM xylose (first and second plots). Expression of msfGFP was also recorded in cells grown to exponential phase in Hv-Cab medium with 10 mM arabinose (third plot). Cells grown in Hv-Cab to stationary phase and induced with 10 mM xylose (fourth plot).

Figure 4

Overexpression of ftsZ1 and ftsZ2 under the Pxyl promoter. (A) Phase-contrast microscopy showing wild-type cells and cells overexpressing ftsZ1 or ftsZ2 under the Pxyl (5 mM xylose) or PtnaA (2 mM tryptophan) promoters. (B) Representative cell bridge phase-contrast and epifluorescence images. DNA was stained with ethidium bromide. (C) Cell area and circularity measurements from cells overexpressing ftsZ1 and ftsZ2 with the Pxyl (5 mM xylose) and the PtnaA (2 mM tryptophan) promoters. Datapoints (and means) were colored (pink, yellow, and blue), indicating different biological replicates.