New components of the community-based DNA-repair mechanism in Sulfolobales

Abstract

After exposure to ultraviolet (UV) light, Sulfolobus acidocaldarius cells aggregate in a species-specific manner to exchange DNA and repair double-strand breaks via homologous recombination. The formation of cell-cell interactions is mediated by Ups pili. DNA exchange subsequently occurs through the Crenarchaeal system for exchange of DNA (Ced), which imports DNA. To identify novel players in these processes, we investigated that several genes upregulated after UV exposure, by creating in-frame deletion mutants and performing cell aggregation and DNA exchange assays. This led to the identification of two novel components involved in the Ups and Ced systems: UpsC, a minor pilin of the Ups pili, and CedD, a VirD4-like ATPase essential for DNA import. Altogether, these findings provide new insights into the DNA damage response mechanisms in Sulfolobales.

Keywords: Crenarchaeota; DNA damage; DNA exchange; Sulfolobales; homologous recombination; type IV pili; type IV secretion.

Figure 1.

(a) Genetic loci of the ced and ups genes of S. acidocaldarius and A. pernix, as well as the ted genes of P. caldifontis . (b) AlphaFold predictions of S. acidocaldarius proteins, including: the putative pilin subunits CedA1 and CedA2 [compared to atomic models of CedA1 from A. pernix, PDB: 8DFU; TedC from P. caldifontis, PDB: 8DFT; and VirB2 from A. tumefaciens, PDB: 8EXH (Beltran et al. 2023)]; a hexamer of CedD; as well as pilin subunits UpsA, UpsB, and UpsC from S. acidocaldarius. The hexamer of CedD was modeled using the AlphaFold server and has a predicted pore diameter of around 28 Å (left). Positive and negative charges are represented in blue and red, respectively, revealing a positively charged inner pore (right).

Figure 2.

UV-induced cellular aggregation of S. acidocaldarius MW001, ΔupsC, and complementation strains expressing upsC and upsC-HA from a plasmid. (a) Percentages of aggregated cells following UV light exposure and in the nonexposed control. Error bars represent standard deviation. (b) Representative phase-contrast images of MW001 and ΔupsC with and without UV treatment. Scale bars = 5 µm. (c) Electron microscopy images of MW501 (lacking Aap pili and archaella) and MW1351 (ΔupsC in MW501) strains. Ups pili are visible in both strains, demonstrating that pilus formation is reduced but not abolished in the absence of UpsC. Arrows indicate a putative additional structure at the tip of the Ups pili in MW501. Scale bars = 200 nm.

Figure 3.

DNA exchange assays using upsC, cedD, upsE, and cedB deletion mutants. Two different mutants (either in JDS22 or MW001 backgrounds), exposed to UV light (UV) or left untreated (C), were mixed in various combinations and plated on selective media. Both background strains carried mutations in the pyrE gene, which is involved in uracil biosynthesis. The mutations were located at different genomic positions, allowing recombination between the strains to restore the wild-type pyrE phenotype. Bars represent the average of at least three independent mating experiments, with each experiment normalized to the JDS22 (UV) × MW001 (UV) condition, which was set at 100%. Error bars indicate standard deviation.

Figure 4.

Current model of DNA transport in Sulfolobales via the Ced system. Sulfolobus cells aggregate after UV-stress, a process facilitated by the Ups pili system. UpsC, a minor pilin subunit, may function either at the base or tip of the Ups-pilus, with a potential role in initial cell adherence if positioned at the tip. The Ced system enables the import of DNA from neighboring cells, which is mediated by CedA, a polytopic membrane protein. CedA1 and CedA2, likely functioning as pseudopilins, may form a channel through which DNA is transferred between cells. CedB, a membrane-bound VirB4-like ATPase, and CedD, a VirD4-like membrane-bound ATPase, collaborate to power the DNA transport process. CedB likely energizes the assembly of the DNA transport machinery, while CedD functions as a coupling protein, processing incoming DNA and guiding it through the channel formed by CedA, CedA1, and CedA2. CedD may also act as a gatekeeper, controlling the opening of the translocation channel. It remains unclear whether the Ced system transports single-stranded or double-stranded DNA. Once inside the recipient cell, the imported DNA is used for DNA repair via homologous recombination.