Species-Specific Recognition of Sulfolobales Mediated by UV-Inducible Pili and S-Layer Glycosylation Patterns

Abstract

The UV-inducible pili system of Sulfolobales (Ups) mediates the formation of species-specific cellular aggregates. Within these aggregates, cells exchange DNA to repair DNA double-strand breaks via homologous recombination. Substitution of the Sulfolobus acidocaldarius pilin subunits UpsA and UpsB with their homologs from Sulfolobus tokodaii showed that these subunits facilitate species-specific aggregation. A region of low conservation within the UpsA homologs is primarily important for this specificity. Aggregation assays in the presence of different sugars showed the importance of N-glycosylation in the recognition process. In addition, the N-glycan decorating the S-layer of S. tokodaii is different from the one of S. acidocaldarius Therefore, each Sulfolobus species seems to have developed a unique UpsA binding pocket and unique N-glycan composition to ensure aggregation and, consequently, also DNA exchange with cells from only the same species, which is essential for DNA repair by homologous recombination.IMPORTANCE Type IV pili can be found on the cell surface of many archaea and bacteria where they play important roles in different processes. The UV-inducible pili system of Sulfolobales (Ups) pili from the crenarchaeal Sulfolobales species are essential in establishing species-specific mating partners, thereby assisting in genome stability. With this work, we show that different Sulfolobus species have specific regions in their Ups pili subunits, which allow them to interact only with cells from the same species. Additionally, different Sulfolobus species have unique surface-layer N-glycosylation patterns. We propose that the unique features of each species allow the recognition of specific mating partners. This knowledge for the first time gives insights into the molecular basis of archaeal self-recognition.

Keywords: DNA exchange; Sulfolobus; archaea; glycosylation; species-specific recognition; type IV pili.

FIG 1

S. acidocaldarius upsAB mutants and their aggregation behavior. (A) Schematic overview of genes encoding pilin subunits upsA and upsB and chimera mutants that were created; (parts of) upsA and B from S. acidocaldarius (MW501, green) were replaced with the same regions from S. tokodaii (red), resulting in MW135 (exchange from start codon of upsA until stop codon of upsB) and MW137 (exchange of amino acid 84 to 98 in S. acidocaldarius upsA with amino acid 80 to 101 of S. tokodaii upsA) (see Fig. S2A for an alignment of UpsA from different species). (B) Quantitative analysis of UV-induced cellular aggregation of mutants shown in A. Percentage of cells in aggregates 3 h after induction with or without 75 J/m² UV (dark or light gray, respectively). (C) Aggregation behavior of mixtures of S. tokodaii (red) with different S. acidocaldarius mutants (green) after treatment with UV light (UV). Untreated cells were used as a control. Mutants used for this experiment were MW501 (wild-type [WT] upsAB), MW143 (ΔupsAB), MW135, and MW137. FISH-labeled cells were visualized with fluorescence microscopy. Scale bar, 10 μm.

FIG 2

UV-induced aggregation of S. acidocaldarius MW001 upon addition of 20 mM mannose, glucose, or N-acetylglucosamine. (A) Percentage of cells in aggregates. (B) Average sizes of formed aggregates. Light gray bars represent noninduced cells, and dark gray bars represent cells induced with 75 J/m² UV.

FIG 3

High cell density (HCD) MS² spectra of heptasaccharide (m/z, 1,651.7) (Fig. S4a) released from the S-layer proteins from S. tokodaii by hydrazinolysis.

FIG 4

Structure of the glycan trees present on the S-layer of S. tokodaii compared with those from S. acidocaldarius (39) and S. solfataricus (42).

FIG 5

UV-induced cellular aggregation of S. acidocaldarius ΔupsAB complementation strains. A S. acidocaldarius ΔupsAB mutant (MW143) was complemented with maltose-inducible plasmids carrying upsAB or upsAB with a D85A, N87, N94A, or Y96A mutation in UpsA (see also Fig. S2A). Percentage of cells in aggregates 3 h after induction with or without 75 J/m² UV (dark or light gray, respectively).

FIG 6

Proposed model of species-specific interactions between Ups pili and N-glycosylated S-layer of Sulfolobales. Ups pili of S. acidocaldarius (green) only form interactions with the N-glycan of the same species and not with that of other species (S. tokodaii, red).