Article Figures & Data
Figures
- 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).
Tables
- TABLE 2
List of N-linked glycans released from S-layer glycoprotein from S. tokodaii detected by MALDI-TOF-MSa
FIG S1
(A) UV-induced cellular aggregation of S. acidocaldarius MW501. Representative differential inference contrast (DIC) microscopy images of noninduced cells (C) and cells 3 hours after UV induction (UV) with 75 J/m2 UV. Scale bar, 5 μm. (B) Transmission electron micrographs of UV-induced S. acidocaldarius mutants (top panel) and expression strains (bottom panel). Top panel: Ups pili of S. acidocaldarius MW501 (ΔflaI/ΔaapF), MW135 (exchange S. acidocaldarius upsAB genes with those of S. tokodaii from start codon of upsA until stop codon of upsB), and MW137 (Saci upsA aa 84 to 98::ST upsA aa 80 to 101). Bottom panel: UV-induced S. acidocaldarius expression strains. Wild-type and mutated upsAB genes (black squares in Fig. S2A) were expressed in a ΔupsAB/ΔflaI/ΔaapF strain (MW143). The following maltose-inducible expression plasmids were used: pSVA1855 expressing wild-type upsAB; pSVA1860, expressing upsAB with a D85A mutation in upsA; pSVA1860, expressing upsAB with a D85A mutation in upsA; pSVA1861, expressing upsAB with a N87A mutation in upsA; pSVA1862, expressing upsAB with a N94A mutation in upsA; and pSVA1863, expressing upsAB with a Y96A mutation in upsA. Scale bar, 100 nm. Download FIG S1, TIF file, 2.3 MB.
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FIG S2
(A) Alignments of UpsA from different Sulfolobales. The class III cleavage site, cleaved by PibD, is depicted by a red line. The red box in UpsA indicates the less conserved region. Shown are UpsA amino acid sequences from Sulfolobus acidocaldarius DSM 639 (Saci), Sulfolobus tokodaii strain 7 (ST), Sulfolobus solfataricus P2 (Sso), Stygiolobus azoricus (Staz), Metallosphaera cuprina Ar-4 (Mcup), Metallosphaera hakonensis DSM 7519 (Mhak), Metallosphaera sedula DSM5348 (Msed), and Metallosphaera yellowstonensis MK1 (Myel). (B) Maximum-likelihood phylogenetic tree of UpsA and UpsB homologs from different archaeal species (Sulfolobus acidocaldarius strains DSM 639, N8, Ron12/I, and SUSAZ; Sulfolobus solfataricus strains P2 and 98/2, P1; Sulfolobus islandicus strains REY15A, HVE10/4, and M.16.4; Sulfolobus tokodaii strain 7; Stygiolobus azoricus; Metallosphaera sedula DSM5348; Metallosphaera cuprina Ar-4; Metallosphaera hakonensis DSM 7519; and Metallosphaera yellowstonensis MK1). Branch numbers represent bootstrap values above 80% (100 replicates). Download FIG S2, TIF file, 2.8 MB.
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TEXT S1
Describing the phylogenetic analysis of UpsA and UpsB from different Sulfolobales and the N-glycan analysis of the glycosylated S-layer of S. tokodaii S-layer proteins SlaA and SlaB. Download Text S1, DOCX file, 0.03 MB.
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FIG S3
Effect of mannose on transcript levels of upsA genes in S. acidocaldarius MW001 after treatment with UV (3 hours after treatment with 75 J/m2 UV) (right) or not (left) as measured by reverse transcription-quantitative PCR (qRT-PCR). Differences are displayed as log2 fold changes. Used primers are summarized in Table S1. Download FIG S3, TIF file, 0.9 MB.
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FIG S4
(A) MALDI MS spectra of N-glycans released from the S-layer protein from S. tokodaii by hydrazinolysis observed (positive ion mode). (B) MALDI MS spectra of N-glycans released from the S-layer protein from S. tokodaii by hydrazinolysis observed (negative ion mode). Structures are assigned based on MS2 analysis.*, During hydrazinolysis, a fraction of glycans gets derivatized by hydrazine reagent. Download FIG S4, TIF file, 0.7 MB.
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FIG S5
Glycosyl linkages of monosaccharides of N-glycans from the S-layer proteins of S. tokodaii were determined by gas chromatography (GC)-MS analysis using the partially methylated alditol acetate (PMAA) method. Download FIG S5, TIF file, 1.8 MB.
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FIG S6
Different possible glycoforms of N-glycans identified on the S-layer proteins from S. tokodaii. Multiple isomers of each glycoforms were also observed. The structure, branching, and linkage of N-glycans were characterized by MSn fragmentation by electrospray ionization (ESI)-MSn and linkage analysis by GC-MS. Download FIG S6, TIF file, 1.0 MB.
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FIG S7
N-linked glycosylation sites identified from SlaA of S. tokodaii by LC-MS2 analysis (tryptic digestion and semispecific cleavage search using Byonic software). (A) HCD MS2 spectra of glycopeptide 1175IYYN[SuphoQuinovose1Hex4HexNAc2] ATSGR1183 from SlaA. (B) HCD MS2 spectra of glycopeptide 1188NVYGQVVLN[SuphoQuinovose1Hex4HexNAc2]AS GN1200 from SlaA. (C) HCD MS2 spectra of glycopeptide 1222AVLPN[SuphoQuinovose1Hex4HexNAc2]NTLTTL TFNK1236 from SlaA. (D) HCD MS2 spectra of glycopeptide 1298IIPAN[SuphoQuinovose1Hex4HexNAc2]ITPIR1307 from SlaA. (E) HCD MS2 spectra of glycopeptide 1362EGVN[SuphoQuinovose1Hex4HexNAc2]ASVTSPV VYYSYQAV VAK1383 from SlaA. (F) HCD MS2 spectra of glycopeptide 1421AVGPAISEYPVNLVFTN[SuphoQuinovose1Hex4 HexNAc2]VT VEK1442 from SlaA. Download FIG S7, TIF file, 1.9 MB.
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TABLE S1
Plasmids and primers used during this study. Download Table S1, DOCX file, 0.02 MB.
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FIG S8
N-linked glycosylation sites identified from SlaB of S. tokodaii by LC-MS2 analysis (tryptic digestion and semispecific cleavage search using Byonic software). (A) HCD MS2 spectra of glycopeptide 200GN[SuphoQuinovose1Hex4HexNAc2]QTISLTLK209 from SlaB. (B) HCD MS2 spectra of glycopeptide 347EIETVN[SuphoQuinovose1Hex4HexNAc2]QTVYTL MNEIK363 from SlaB. (C) HCD MS2 spectra of glycopeptide 364SLN[SuphoQuinovose1Hex4HexNAc2]ASISQLSTTL SSTTTEITTLE NDIK392 from SlaB. Download FIG S8, TIF file, 1.0 MB.
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