A conserved hexanucleotide motif is important in UV-inducible promoters in Sulfolobus acidocaldarius

Abstract

Upon DNA damage, Sulfolobales exhibit a global gene regulatory response resulting in the expression of DNA transfer and repair proteins and the repression of the cell division machinery. Because the archaeal DNA damage response is still poorly understood, we investigated the promoters of the highly induced ups operon. Ups pili are involved in cellular aggregation and DNA exchange between cells. With LacS reporter gene assays we identified a conserved, non-palindromic hexanucleotide motif upstream of the ups core promoter elements to be essential for promoter activity. Substitution of this cis regulatory motif in the ups promoters resulted in abolishment of cellular aggregation and reduced DNA transfer. By screening the Sulfolobus acidocaldarius genome we identified a total of 214 genes harbouring the hexanucleotide motif in their respective promoter regions. Many of these genes were previously found to be regulated upon UV light treatment. Given the fact that the identified motif is conserved among S. acidocaldarius and Sulfolobus tokodaii promoters, we speculate that a common regulatory mechanism is present in these two species in response to DNA-damaging conditions.

Fig. 1.

Defining the minimal size of the upsX promoter by promoter deletion analysis. (a) Schematic overview of the ups operon of S. acidocaldarius. The cluster encodes a protein of unknown function, UpsX (Saci_1493), a secretion ATPase UpsE (Saci_1494), an integral membrane protein, UpsF (Saci_1495) and two pilin subunits, UpsA and B (Saci_1496 and Saci_1497, respectively). The transcription start sites are indicated by black arrows. (b) Upper panel: specific β-galactosidase activity from the reporter plasmids containing the truncated upsX promoters assayed under non-UV conditions and 3 h after UV treatment. Negative control (Neg. ctrl) is the pCMal LacS plasmid from which the malE promoter was removed. Lower panel: truncation of upsX promoters in the reporter plasmids. The numbers above each construct indicate the 5′ end of each promoter fragment in respect to the transcription start site.

Fig. 2.

Investigation of the region from −46 to −39 of the upsX promoter by promoter substitution analysis. Upper panel: specific β-galactosidase activity of the reporter plasmids containing the mutated promoter. Negative control (Neg. ctrl) was the pCmalLacS plasmid of which the malE promoter was removed. Lower panel: the construction of the substituted promoters and the relative promoter activity of each construction in comparison to the upsX promoter 3 h after UV stress. P_upsX is the longest fragment of upsX promoter (D-339 in Fig. 1), others are promoter mutants with information about the location and number of nucleotides which were replaced. Substitutions are indicated by lower case letters and dashed lines representing identical nucleotides. On the right: relative promoter activity in comparison to the upsX promoter after UV induction.

Fig. 3.

(a) Alignment of promoters of upsX, upsE and upsA; the hexanucleotide motif is boxed, the TATA boxes are highlighted in red; the transcription start sites are underlined with arrows above, and the start codons are shown in cyan. (b) Specific β-galactosidase activity of the plasmid containing the substituted promoter of upsE assayed without and 3 h after UV stress. The original upsE promoter (P_upsE) was used as a positive control. Negative control is the pCmalLacS, from which the malE promoter was removed. (c) Conservation of promoter elements in UV-inducible genes in S. acidocaldarius: the TATA box, BRE and the hexanucleotide motif are marked with bars below the logo sequence. Promoters of upsX, upsE, upsA, tfb3, saci_1302, saci_1225 and saci_0951 were used to create the sequence logo.

Fig. 4.

The role of the 5′-A(N)TTTC-3′ motif in transcription of genes in the ups operon. (a) Transcription levels of upsX, upsE and upsA 3 h after UV stress in the upsX promoter substitution strain (P_{upsX sub} strain). Change in transcription levels of genes 3 h after UV treatment (75 J m⁻²) was measured by qRT-PCR. Differences are displayed as log2-fold changes. (b) Transcription levels of upsX, upsE and upsA in the P_{upsX sub} strain in comparison to that of MW001. Changes in the transcription levels of genes were measured by qRT-PCR. Differences are displayed as log2-fold changes. (c) Aggregation assay with the upsX and upsE substitution promoter strains (P_{upsX sub} and P_{upsE sub}, respectively). Non-UV treated cells (−UV) and cells 3 h after induction with UV light (75 J m⁻²) (+UV) were visualized using phase-contrast microscopy. MW001 was used as a positive control and ΔupsE as a negative control (scale bar, 10 µm). (d) Quantification of cellular aggregation of non-UV-treated cells (−UV) and cells 3 h after UV treatment (+UV). Single and aggregated cells (n>3) were counted. Average numbers of cells in aggregates were calculated from three individual experiments.

Fig. 5.

DNA transfer assays using upsX deletion mutant as well as upsX promoter substitution mutant (P_{upsX sub}). Two different background strains (MW001 and JDS22) contained mutations in the pyrE gene (involved in de novo synthesis of uracil). Two auxotrophic strains were treated with (UV) or without (C) UV light and mixed and plated on selective media to obtain prototrophic colonies (Pyr+). Bars represent the average of at least three independent mating experiments each. Every experiment was normalized to JDS22 (UV) * MW001 (UV) as 100 %.