Response of the hyperthermophilic archaeon Sulfolobus solfataricus to UV damage

Abstract

In order to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeote Sulfolobus solfataricus to UV damage, we used high-density DNA microarrays which covered 3,368 genetic features encoded on the host genome, as well as the genes of several extrachromosomal genetic elements. While no significant up-regulation of genes potentially involved in direct DNA damage reversal was observed, a specific transcriptional UV response involving 55 genes could be dissected. Although flow cytometry showed only modest perturbation of the cell cycle, strong modulation of the transcript levels of the Cdc6 replication initiator genes was observed. Up-regulation of an operon encoding Mre11 and Rad50 homologs pointed to induction of recombinational repair. Consistent with this, DNA double-strand breaks were observed between 2 and 8 h after UV treatment, possibly resulting from replication fork collapse at damaged DNA sites. The strong transcriptional induction of genes which potentially encode functions for pilus formation suggested that conjugational activity might lead to enhanced exchange of genetic material. In support of this, a statistical microscopic analysis demonstrated that large cell aggregates formed upon UV exposure. Together, this provided supporting evidence to a link between recombinational repair and conjugation events.

FIG. 1.

Representative growth curve of S. solfataricus PH1 after UV treatment. A UV dose of approximately 75 J/m² (254 nm) was used for the treatment, and the cultures were recultivated at time point 0 h. The time points of sampling for DNA and RNA extractions are indicated by the symbols.

FIG. 2.

Aggregation of S. solfataricus cells after UV treatment. (A) Micrographs (phase contrast) of S. solfataricus cells fixed on a gelrite-coated microscope slide. Representative pictures of cells and/or aggregates at 3 h, 6 h, and 8 h after UV treatment are shown. C, control culture without UV treatment. An increasing amount of cell aggregates was observed in UV-treated cultures after 3 h. (B) Top: typical cell aggregates, as observed around 3 to 6 h after UV treatment. Bottom: typical maximum cell aggregate, mostly discovered at 6 h after UV treatment; these data are excluded from the results shown in panel C, because the number of cells was uncountable. (C) Quantitative analysis of cell aggregate formation at different time points after UV treatment. The pre-UV culture was split into a UV-treated culture (dark gray) and a control culture (light gray). The amount of cells in and out of aggregates was counted until 1,000 single cells were found in total. Means and standard deviations (error bars) are shown from three independent UV experiments. The amount of cells found in aggregates is an underestimate, because cells in the large aggregates were not countable (see panel B description in the legend).

FIG. 3.

Flow cytometry analysis of S. solfataricus cells after UV treatment (strain PH1). Cells were fixed in 80% ice-cold ethanol, and the DNA contents were measured by fluorescence [see Fig. S1 for data for strain PH1(SSV1)]. Samples from the UV-treated culture (post-UV) and the mock-treated control culture (control) were analyzed from 0 h to 8.5 h. A chromosome content of 1N (“1” on the x axis) is found in G₁ phase, between 1N and 2N represents S phase (DNA synthesis), and 2N is the G₂ phase of the cell cycle (20).

FIG. 4.

PFGE analysis of total DNA from S. solfataricus PH1 after UV treatment to determine the extent of DSB. DNA from UV-treated (+) and control (-) cells was analyzed from 0 h to 14 h after UV treatment and pre-UV. Fragmentation of DNA is visible as a smear in the area of the gel below the compression zone (at 600 kb), from 2 h to 8 h, mostly in the UV-treated samples.

FIG. 5.

Expression profiles of the general transcriptional response after UV treatment of strain PH1. The curves display the means of the three identified UV-dependent regulated gene groups as displayed in Table 2: the highly induced group of 19 genes (red curve), the induced gene group of 14 genes (orange), and the down-regulated group of 22 genes (green). The blue dashed curve was generated from the averages of 11 genes but represents qualitatively the pattern of approximately 400 gene that are mostly involved in the transcription and translation processes. Errors bars do not represent standard deviations but express the range of gene expression of the different genes that are “summarized” in each line.

FIG. 6.

Expression profiles of the three cdc6 genes in both strains PH1 (sleek lines) and PH1(SSV1) (lines with triangles) showed a strong up-regulation of the potential repressor of replication cdc6-2 (red), shortly after UV treatment, while the potential main initiator of replication cdc6-1 (green) is repressed. The data represent means of two to three experiments, but the display of standard deviations has been omitted for clarity.

FIG. 7.

Expression profile of a strongly UV-induced operon encoding homologs of a putative pilus or secretion system (type II/IV). All five genes show a high induction in both strains PH1 (A) and PH1(SSV1) (B). The genes flanking the operon (SSO0122 and SSO5209) showed no effect after UV light exposure (not shown). Predicted gene functions are based on a bioinformatics analysis (Table 2).

FIG. 8.

Expression profile of the archaeal rad50/mre11 operon after UV treatment. The transcriptional activity was first detected in S. acidocaldarius. herA, archaeal helicase, encodes a new class of bipolar DNA helicases (6); mre11, single-stranded DNA endonuclease and 3′-to-5′ double-stranded DNA exonuclease; rad50, ATPase; nurA, nuclease of archaea, a 5′-to-3′ exonuclease. The four genes, which are supposedly involved in homologous recombination as part of the putative recombination repair system, show a weak but significant UV-dependent response, while radA (SSO0250; blue curve) follows the pattern of highly transcribed genes (see the blue line in Fig. 5, above). (A) Strain PH1; (B) strain PH1(SSV1).