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

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.

showed that these subunits facilitate species-specific aggregation. A region of low 23 conservation within the UpsA homologs is primarily important for this specificity. 24 Aggregation assays in the presence of different sugars showed the importance of N-25 glycosylation in the recognition process. In addition, the N-glycan decorating the S-layer of S. 26 tokodaii is different from the one of S. acidocaldarius. Therefore, each Sulfolobus species 27 seems to have developed a unique UpsA binding pocket and unique N-glycan composition to 28 ensure aggregation and consequently also DNA exchange with cells from only the same 29 species, which is essential for DNA repair by homologous recombination. 30 31 Importance 32 Type IV pili can be found on the cell surface of many archaea and bacteria where they play 33 important roles in different processes. The Ups-pili from the crenarchaeal Sulfolobales 34 species are essential in establishing species-specific mating partners, ensuring genome 35 stability. With this work, we show that different Sulfolobus species have species-specific 36 regions in their Ups-pilin subunits, which allow them to interact only with cells from the 37 same species. Additionally, different Sulfolobus species all have unique S-layer N-38 glycosylation patterns. We propose that the unique features of each species allow the 39 recognition of specific mating partners. This knowledge for the first time gives insights into 40 the molecular basis of archaeal self-recognition. 41

Introduction 44
Type IV pili (T4P) are cell surface appendages that can be found on the cell-surfaces of many 45 bacteria and archaea (1, 2). They have been implicated in motility, secretion, DNA 46 transformation, adhesion to surfaces and the formation of intercellular associations (3, 4). In 47 bacteria, many examples of T4P with cellular-binding properties have been described. The 48 major pilin subunit PilE from Neisseria T4P, was shown to bind endothelial cells and 49 hemagglutinate erythrocytes whereas the Neisseria minor pilin PilV is essential for adherence 50 to host cells (5-10). Additionally, major pilin PilA from Myxococcus xanthus binds to self-51 produced exopolysacharides, subsequent retraction of the T4P allows gliding motility and 52 fruiting body formation (11,12). T4P also form intercellular connections that are essential for 53 conjugational exchange of DNA. For instance, PAPI-1 encoded T4P bring Pseudomonas 54 aeruginosa cells in close proximity by binding to lipopolysaccharides of the recipient cells 55 and thereby promote exchange of PAPI-1 DNA (13,14). 56 In archaea, several gene clusters have been found to encode T4P-like structures (4, 15-57 19). The best-characterized archaeal T4P-like structure is the archaellum, which is essential 58 for swimming motility (4, 19-21). However, little is known about the role and mode of 59 action of archaeal non-archaellum T4P in attachment to biotic or abiotic surfaces. T4P from 60 the thermophilic crenarchaeon Sulfolobus acidocaldarius (Aap: archaeal adhesive pili) and 61 the euryarchaea Haloferax volcanii and Methanococcus maripaludis were shown to be 62 involved in attachment to surfaces (22-27). However, their exact mode of binding has not 63 been studied. Next to Aap-pili, Ups-pili can be found in Sulfolobales (UV inducible pili of 64 Sulfolobales) (28-31). These T4P assemble upon treatment of the cells with UV-stress and 65 other DNA double strand break inducing agents. They are crucial in cellular self-interactions 66 thereby mediating the formation of species-specific cellular aggregates (32, 33). Within these 67 aggregates, cells are able to exchange chromosomal DNA using the Ced-system 68 (Crenarchaeal exchange of DNA), suggesting a community based DNA repair system via 69 homologous recombination (32, 34). Interestingly, the Ced-system was found to function 70 independently of the Ups-pili, even though both systems are essential for DNA transport (35). 71 The ups-operon encodes two pilin subunits with a class III signal peptide: UpsA and 72 UpsB (29). Deletion mutants of either upsA or upsB still form pili (though less and smaller), 73 but do not aggregate after UV induction. The pilins are therefore both suggested to be major 74 subunits forming mixed Ups-pili (31, 32) . While the importance of Ups-pili in cellular 75 recognition is known, the underlying molecular mechanism of the species-specific cellular 76 aggregation of Sulfolobus species has not been determined. 77 In this study, we investigated the role of Ups-pili in species-specific aggregation on a 78 molecular level. To this end, in vivo chimera mutants were constructed in which we 79 exchanged (parts of) the genes encoding the pilin subunits UpsA and UpsB of S. 80 acidocaldarius and S. tokodaii. By using these strains in aggregation assays and fluorescence 81 in situ hybridization (FISH) experiments, we were able to assign a specific region of UpsA to 82 be required for species-specific cell aggregation of archaeal cells. Furthermore, aggregation 83 assays in the presence of different sugars suggested a role of N-glycosylation in cellular 84 recognition. Glycan analysis on the thus far unstudied S. tokodaii S-layer showed a different 85 N-glycan composition compared to that of other Sulfolobus species. Based on these 86 experiments, we propose that a specific region of UpsA forms a binding site to bind species-87 specific N-glycan chains of S-layer components, thereby allowing species-specific cell 88 aggregation and subsequent DNA exchange. 89 90 91

Results 92
The role of pilin subunits in species-specificity 93 To study the role of the Ups-pilin subunits (UpsA and UpsB) in species-specific recognition 94 of Sulfolobus cells, a S. acidocaldarius strain was constructed in which the genomic region 95 from the start codon of upsA until the stop codon of upsB was exchanged with the 96 orthologous region from S. tokodaii (resulting in strain MW135; Figure 1A, Table 1). Upon 97 UV-induction S. acidocaldarius MW135 was still found to produce Ups-pili ( Figure S3), 98 however, interestingly, it showed little to no cellular aggregation ( Figure 1B). In order to test 99 if this S. acidocaldarius Ups-hybrid strain was able to recognize and therefore aggregate with To find putative species-specific regions in the pilin subunits involved in species-110 specific recognition, alignments were made using UpsA and UpsB amino acid sequences, 111 from several Sulfolobales ( Figure S1). Additionally, the relationship between UpsA and 112 UpsB homologs was studied by creating a phylogenetic tree ( Figure S2, Supplementary  113 results and methods). A region with low conservation was revealed in UpsA ( Figure S1, 114 amino acid 84-98 for S. acidocaldarius, red box). To test whether this region plays a role in 115 cell-cell recognition, a strain was constructed in which only the region of low conservation in 116 S. acidocaldarius UpsA (amino acid 84-98) was exchanged with the corresponding part from 117 S. tokodaii UpsA (amino acid 80-101) (MW137 Figure 1A, Table 1). Similar to what was 118 observed for the S. acidocaldarius mutant in which upsA and upsB were exchanged 119 completely (MW135), S. acidocaldarius MW137 showed pili formation ( Figure S3) but 120 showed little to no aggregation with itself ( Figure 1B). Instead, it was found to aggregate 121 with S. tokodaii ( Figure 1C, fourth panel). This observation strongly suggests that the non-122 conserved region (exchanged in MW137) defines the species-specificity during cellular 123 aggregation. 124

The role of glycosylation in species-specificity 125
The fact that Sulfolobus Ups wild-type strains are able to form mating pairs with Ups-deletion 126 strains (32), suggests that factors, other than Ups-pili, play a role in species-specific 127 recognition. The surface proteins of Sulfolobales are heavily glycosylated (36, 37). We 128 therefore suggested that Ups-pili might recognize glycosylated proteins and thereby initiate 129 cellular interactions. To confirm this hypothesis, UV induced aggregation assays were 130 performed in the presence of monosaccharides that are also part of the S. acidocaldarius N-131 glycan chain (Glc 1 Man 2 GlcNAc 2 QuiS, containing glucose, mannose, N-acetylglucosamine 132 and the Sulfolobus-specific sulfoquinovose residues) (37) (Figure 2). The addition of N-133 acetylgucosamine or glucose did not result in altered cellular aggregation (Figure 2A  During this study, we have examined the role that Ups-pili play in the formation of 185 Sulfolobus mating partners. In particular, we focused on the role that pilin subunit UpsA 186 plays in cell-recognition. 187 The Ups-pilus is formed by two pilin subunits UpsA and UpsB, which are both thought 188 to be major pilin subunits that build up mixed pili structures (31). We revealed that UpsA is 189 involved in species-specific cellular interactions and we were able to alter this specificity by 190 exchanging (parts of) the pilin subunit with that of another species (Figure 1). The binding of 191 bacterial T4P to other cells is often based on pilin-sugar interactions. Surface exposed 192 glycans can be found on cells from all domains of life where they display an enormous range 193 of different structures that are often highly specific to certain species (40). Glycans are 194 therefore perfect anchors to bind specific host-or partner cells. In Saccharomyces cerevisiae, 195 surface exposed lectins can bind to surface exposed sugars in a calcium-dependent manner, This study has given molecular insights in the cellular recognition mechanism of the 230 previously described crenarchaeal Ups-system (29, 31, 32). Our current model suggests that 231 upon DNA damage, Ups-pili are formed; the UpsA pilin subunits contain a species-specific 232 glycan binding pocket in pilin subunit UpsA that can only bind glycans presented on cells 233 from the same species (Figure 6). This system allows the formation of species-specific 234 cellular connections prior to DNA-exchange via the Ced-system (35). In that way, only DNA 235 from the same species is exchanged and used for DNA repair via efficient homologous 236 recombination. This proposed cellular recognition mechanism in Sulfolobales restricts 237 exchange of genomic DNA to cells from the same species, thereby playing an important in 238 role genome integrity and the maintenance of species. Co-evolution of N-glycosylation and 239 pilin subunit UpsA might play an important role in speciation. 240

Experimental procedures 241
Culture conditions. 242 Sulfolobus acidocaldarius strains and derived mutants ( Table 2) Table S1.  (Table S1). The PCR product and synthetic 257 DNA fragments were subsequently cloned into pSVA406, resulting in the plasmids listed in 258 and B was amplified using primers listed in Table S1 and cloned into pSVA1450 under 269 control of a maltose inducible promoter resulting in plasmid pSVA1855 (Table S1). This 270 plasmid was subsequently used to as a template to introduce point mutations into upsA 271 (D85A, N87A, N94A and Y96A) (Table 1) using two overlapping primers per mutation 272 (Table S1) Table S1: Plasmids and primers used during this study. 514 Wildtype and mutated upsAB genes (black squares in Figure S1) were expressed in a 533 ΔupsAB/∆flaI/∆aapF strain (MW143). The following maltose-inducible expression plasmids 534 were used: pSVA1855 expressing wildtype upsAB; pSVA1860, expressing upsAB with a 535 D85A mutation in upsA; pSVA1860, expressing upsAB with a D85A mutation in upsA. 536 pSVA1861, expressing upsAB with a N87A mutation in upsA; pSVA1862, expressing upsAB 537 with a N94A mutation in upsA; pSVA1863, expressing upsAB with a Y96A mutation in 538 upsA. Scale bar 100 nm. 539