This is the first description of a [beta]-d-GalNAc-Thr linkage in glycoproteins.
Key words: Aneurinibacillus thermoaerophilus/surface layer (S-layer)/prokaryotic glycoprotein/O-glycan/structure determination
Extraction plants of beet sugar factories have been recognized as sources for isolation of aerobic and anaerobic thermophilic bacterial strains (Dubourg and Devillers, 1953; Klaushofer and Hollaus, 1970; Hollaus and Klaushofer, 1973). Many of the aerobic organisms obtained from extraction juice samples belong to the species Bacillus stearothermophilus (Messner et al., 1984) and Bacillus smithii (Messner et al., 1997).
In our systematic survey of prokaryotic glycoproteins (for review, see Messner, 1997) we have isolated pure cultures from extraction juice samples of an Austrian beet sugar factory and have elucidated the structures of their surface layer (S-layer) glycoprotein glycans. Aneurinibacillus (formerly Bacillus) thermoaerophilus represents a new species of thermophilic Gram-positive bacteria (Meier-Stauffer et al., 1996; Heyndrickx et al., 1997) covered by a glycosylated S-layer protein lattice (Messner, 1996; Sleytr, 1997). Recently, the repeating unit structure of the S-layer glycoprotein from the type strain of this species, A.thermoaerophilus L420-91T, had been determined to be a hexasaccharide with the constituents d-rhamnose and 3-acetamido-3,6-dideoxy-d-galactose (3-N-acetyl-d-fucosamine; Kosma et al., 1995a).
In this communication the taxonomic affiliation of the new isolate GS4-97 in relation to the type strain of the species Aneurinibacillus thermoaerophilus is described. Further, we report on the complete glycan structure of the GS4-97 S-layer glycoprotein, including the connecting core oligosaccharide and its linkage to the S-layer polypeptide. The saccharide moiety is linked by an O-glycosidic linkage from 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine) to threonine which is common among eukaryotic glycoproteins (for review, see Vliegenthart and Montreuil, 1995) but has never been observed before in bacterial S-layer glycoproteins (Messner, 1997). However, in contrast to the common [alpha]-d-GalNAc-Ser/Thr linkages in eukaryotic glycoproteins (for review, see Vliegenthart and Montreuil, 1995), here we describe for the first time a [beta]-d-GalNAc-Ser/Thr linkage.
Morphological and taxonomic characterization
From the extraction juice sample four different pure isolates, designated GS1-97, GS2-97, GS3-97, and GS4-97, were obtained by serial dilutions and recultivation on agar plates. On the basis of similarities in protein band patterns on SDS-PAGE gels, lattice dimensions of the S-layers determined by freeze-fracture electron microscopy, chemotaxonomic and genetic analyses such as polar lipid analysis, FACE analysis, RAPD analysis, and partial 16S rDNA sequencing, the taxonomic affiliations of the new isolates were assessed. The results (not shown) demonstrated that two of them (GS1-97 and GS2-97) possess either nonglycosylated or weakly glycosylated S-layer proteins. These organisms showed high similarities to the strains Bacillus stearothermophilus S65-67 (Messner et al., 1984) and L407-91 (Meier-Stauffer et al., 1996), respectively. In contrast, by partial 16S rDNA sequence analysis, the isolates GS3-97 and GS4-97 revealed 100% similarity to each other and 99.8% similarity to the type strain of the newly described species Aneurinibacillus thermoaerophilus L420-91T (DSM 10154; Meier-Stauffer et al., 1996; Heyndrickx et al., 1997). All other chemotaxonomic and genetic characterizations confirmed this result (for example, see Figure
Figure 1. RAPD analysis of the extraction juice isolates. As a typical example the banding patterns obtained with Biomol primer no. 3 are shown (for details see Materials and methods). Lanes: 1, Bacillus stearothermophilus DSM 22T; 2, GS1-97; 3, GS2-97; 4, GS3-97; 5, GS4-97; 6, Aneurinibacillus thermoaerophilus DSM 10154T. Chemical characterization
In the present study we characterized composition and structure of the S-layer glycan chain of isolate GS4-97. The square S-layer lattice with center-to-center spacings between the morphological units of ~10 nm consists of identical glycoprotein monomers with an apparent molecular mass of 109,000, each. After thorough proteolytic degradation of purified S-layer glycoprotein by pronase E and purification of the reaction mixture by gel permeation, chromato-focusing and reversed-phase chromatography, a homogeneous glycopeptide fraction was obtained. Monosaccharide analysis by HPAEC-PED revealed rhamnose and 3-N-acetylfucosamine in a molar ratio of approximately 2:1 as main constituents of the S-layer glycan chain of GS4-97 together with small amounts of N-acetylgalactosamine (Figure
Figure 2. HPAEC-PED chromatogram of the S-layer glycopeptide of A.thermoaerophilus GS4-97 showing the constituents Fuc3NAc, Rha,and GalNAc. NMR measurements
The glycan structure of the glycopeptide was derived from one- and two-dimensional NMR data recorded in D2O at 323 K. The repeating unit was shown to be identical to that of A.thermoaerophilus L420-91T (Kosma et al., 1995a) established by means of the major signal 13C NMR chemical shift values (Figure
In previous studies evidence was obtained that even closely related bacterial strains may possess S-layers with different ultrastructure and chemical composition (Messner et al., 1984, 1993, 1997). Even within the species Aneurinibacillus thermoaerophilus different S-layer glycan compositions have been reported (Kosma et al., 1995a,b). Type strain A.thermoaerophilus L420-91T (DSM 10154) possesses a hexasaccharide repeating unit with the constituents d-rhamnose and 3-N-acetyl-d-fucosamine (Kosma et al., 1995a), whereas the disaccharide repeating unit of strain A.thermoaerophilus DSM 10155 is composed of l-rhamnose and d-glycero-d-manno-heptose residues (Kosma et al., 1995b). The latter sugar is a typical constituent of lipopolysaccharides of Gram-negative bacteria (Raetz, 1990). The new isolate GS4-97 is closely related to the type strain L420-91T; it showed 99.8% identity by 16S rDNA sequencing and a high degree of homology by different chemotaxonomic and genetic analyses (see Figure
Table I.
Figure 3. 13C NMR spectrum of the S-layer glycopeptide of A.thermoaerophilus GS4-97.
The identification of the terminal unit was facilitated by the O-methyl group at chemical shifts of [delta]C = 57.5 ppm and[delta]H = 3.44 ppm. The O-methyl substituent was found to be linked to C-3 of [alpha]-rhamnose at 80.4 ppm in the long-range HSQC spectrum. Due to the isolated position of this 13C signal it was possible to trace the signals of the rhamnose residue in the HSQC-TOCSY spectrum from C-1 up to C-5 as well as to C-1 of 3-N-acetylfucosamine, linked to C-2 of the terminal rhamnose (using long-range HSQC). As can be seen from the chemical shift values (72.5 ppm and 70.3 ppm for C-4 and C-5), C-4 of this rhamnose residue is not substituted. Since only the C-1 signal of the 3-N-acetylfucosamine residue, linked to the terminal rhamnose, can be distinguished from all other fucosamine signals, the O-methyl group terminates the polysaccharide, as it was also observed for other bacterial glycoproteins (Gerwig et al., 1989, 1991; Bock et al., 1994). The occurrence of a terminating O-methyl group at the glycan chain also supports the concept of a blockwise assembly of the polysaccharides with hexasaccharide units of the described structure (Kosma et al., 1995a, and this study).
Figure 4. Anomeric part of the HSQC C-H correlation spectrum without proton decoupling during acquisition. I indicates C-1 of the noncleaved [beta]-GalNAc residue in the glycopeptide presenting its 1JC,H at 161 Hz. I[prime] and I[prime][prime] correspond to [alpha] and [beta] C-1 values (1JC,H, 170 and 161 Hz, respectively) of GalNAc residues from the hydrolyzed carbohydrate-protein linkage GalNAc-Thr.
Due to the small content of core sugars in the glycopeptide the determination of the saccharide structure at the reducing end was more complicated than at the terminal end. The 13C NMR signals of the amino acids were assigned using model compound data (Keim et al., 1973) but their sequence was derived from N-terminal sequencing. Thus, Asx from amino acid analysis was identified to represent an Asp residue. Further, only one of the two Thr residues was glycosylated and the Ser residue was not glycosylated. The unusual low frequency shift of the methyl 13C signal of the substituted Thr (17.0 ppm as compared to 19.6 ppm of the unsubstituted one) indicates substitution by a [beta]-GalNAc residue (Dill et al., 1981). Thus, the only [beta]-anomeric sugar present in the glycopeptide (Figure
Combined results from HPAEC-PED and NMR integration corresponding to the constituting monosaccharides of the S-layer glycan suggested an average of 15 hexasaccharide repeats. Additional signals of rhamnose residues can be assigned to the core linkage region between the S-layer glycan repeats and the polypeptide-associated [beta]-GalNAc residue. Three of them present their C-1 at 102.7 to 102.9 ppm, which is the usual magnitude for [alpha]-l-rhamnose residues, not substituted in position 2, if linked to C-3 either of d-galactose or l-rhamnose (Bock et al., 1994; Messner et al., 1995). The fourth rhamnose presents its C-1 at 97.1 ppm, which is common for an [alpha]-d-manno system linked to C-3 either of an l-manno or a d-galacto system (Shaskov et al., 1988). Since the rhamnose residues of the repeating unit were determined to belong to the d-series (Kosma et al., 1995a) two core structures are possible. One possibility is a core with the structure ->2)-[alpha]-d-Rha-(1]n->[3)-[alpha]-d-Rha-(1]n=0-2 ->3)-[alpha]-d-Rha-(1->3)-[beta]-d-GalNAc-(1->O showing three rhamnose C-1 signals with non-stoichiometric intensities at 103 ppm and one signal at 97 ppm (in that order), and the other structure is ->2)-[alpha]-d-Rha-(1]n->[3)-[alpha]-l-Rha-(1]n=0-2 ->3)-[alpha]-l-Rha-(1->3/4)-[beta]-d-GalNAc-(1->O, presenting the signals in reversed order. Support for an [alpha]1,3-linked core structure comes from the 13C NMR data showing C-6 of the GalNAc residue at 62 ppm to be unsubstituted. Comparable core structures have been reported previously in other S-layer glycoproteins (Bock et al., 1994; Messner et al., 1995). Additionally, the Smith degradation experiment of a fluorescent-labeled S-layer glycopeptide of strain A.thermoaerophilus DSM 10155 showed resistance of the core rhamnoses against oxidation by periodate and the presence of a mixture of core sugars (T. Wugeditsch, N. E. Zachara, M. Puchberger, P. Kosma, A. A. Gooley, and P. Messner, unpublished observations). Based on linkage models for both l-Rha-(1->3)-[beta]-d-GalNAc and the l-Rha-(1->4)-[beta]-d-GalNAc (Oxley and Wilkinson, 1987, 1990) an [alpha]-l-rhamnose linkage to C-4 of GalNAc is excluded. Evidence for a core consisting exclusively of d-rhamnose residues is also derived from 13C-shift value measurement. Although the observed 13C chemical shift of 97.1 ppm perfectly fits to the value expected for an [alpha]-d-manno to C-3 of an l-manno linkage (Christian et al., 1988), an additional upfield shift should have been observed, if C-2 of rhamnose is substituted (Poszsgay et al., 1987), as is the case with repeating unit rhamnoses of this organism. Thus, the absence of a 13C signal close to 95 ppm (e.g., 95.0 ppm in a similar system; Christian et al., 1993) strongly indicates that all rhamnoses belong to the same stereochemical series. The chemical shift value of 97.1 ppm now can be assigned to rhamnose C-1 of the d-Rha-(1->3)-[beta]-d-GalNAc part. This signal corresponds to the value found in an analogous system (Oxley and Wilkinson, 1988). The C-1 signal of the 2-substituted repeating unit, however, is hidden within the bulk of other signals.
From these data we propose the structure in Figure Growth of bacteria
Serial dilutions of the cultures from sugar beet extraction juice samples in 0.85% NaCl were grown overnight in modified SVIII medium (Messner et al., 1984), supplemented with 1.2 g of glucose instead of sucrose (SVIII/glc), and plated onto agar plates containing SVIII/glc medium. After incubation overnight at 55°C single colonies were isolated and grown again in SVIII/glc medium. This procedure was repeated until pure cultures were obtained. Cultures of each isolate were rapidly frozen and stored under liquid nitrogen.
For analysis of the S-layer glycoprotein the organism was grown in continuous culture in the medium described before in a Braun Biostat E 10-l fermenter (B. Braun AG, Melsungen, Germany). Strain GS4-97 was grown at 60°C in 10 l of SVIII/glc medium at a pH value of 7.0 with a dilution rate of 0.3 h-1. Oxygen saturation (pO2) was maintained at a value above 30% by manual adjustment of aeration and stirring speed. The process was controlled by measuring optical density at 600 nm, pH value, redox potential, and pO2. Representative samples were collected at various times and analyzed by SDS-PAGE as described previously (Messner et al., 1997). Cells were collected by centrifugation in a Heraeus model 17RS contifuge, washed once with distilled water, and stored at -20°C until use. Analytical methods
SDS-PAGE of whole-cell protein, polar lipid analysis, RAPD, and FACE analyses were carried out as described previously (Messner et al., 1997). Carbohydrate and amino acid analyses and electron microscopy were performed according to Bock et al. (1994). Optical rotation measurement was performed on a Perkin-Elmer model 243B polarimeter (Perkin-Elmer, Norwalk, CT). Isolation of S-layer glycoprotein and S-layer glycopeptide
The S-layer glycoprotein was isolated as published previously (Kosma et al., 1995a). After exhaustive pronase digestion glycopeptide fractions were purified by a combination of gel permeation and cation exchange chromatography. The material of interest was subjected to chromatofocusing (Bock et al., 1994). Final purification was achieved by RP-HPLC on a Nucleosil 120 3C18 (8.0 × 120 mm; Macherey & Nagel, Düren, Germany) column equipped with a Nucleosil 120 3C18 guard column(8.0 × 40 mm, Macherey & Nagel) equilibrated with 0.1% trifluoroacetic acid in MilliQ water. Glycopeptides were eluted with the following gradient: 0-30 min, 0-10% acetonitrile; 30-35 min, 10-100% acetonitrile; 35-40 min, 100% acetonitrile. Appropriate fractions were combined, lyophilized, and stored at -20°C.
Figure 5. Proposed structure of the S-layer glycopeptide of A.thermoaerophilus, strain GS4-97. NMR experiments
Solutions of glycopeptide (~ 80 mg. ml-1) in 99.95% 2H2O (Uetikon) at pH 3.8 (nonbuffered) were used. The NMR sample was lyophilized twice in 99.95% 2H2O to reduce the residual 2HOH signal. Spectra were recorded at 323 K non-spinning in 5 mm tubes at 500.13 MHz for 1H and 125.77 MHz for 13C with a Bruker Avance DRX 500 spectrometer using a 5 mm TXI 13C probe with triple axis gradient coils. In order to compare to previous work (Kosma et al., 1995a) the two N-acetyl signals of the glycan chain were referenced to 2.066/2.070 ppm for 1H and to 22.90/22.96 ppm for 13C spectra. Typical 90° hard pulse durations were 12.5 µs (1H) and 17.5 µs (13C); 90° pulses in decoupling sequences were set to 100 µs (1H) and 80 µs (13C). Repetition intervals were 2 s and gradient durations were 1.5 ms.
High resolution double quantum filtered homonuclear 2D correlated spectra (2QF-COSY) were acquired using pulsed field gradients for coherence selection (Davies et al., 1991) and TPPI (Marion and Wuethrich, 1983) to achieve phase sensitive detection. The 2K × 8K (F1 × F2) real acquisition data matrix was zero filled and Fourier transformed to 2K × 4K complex data points after multiplication with a Gauss-Lorentz window function (LB1 = LB2-2.4, GB1 = GB2 = 0.1). For NOESY (80 ms mixing interval with a 7G cm-1 homospoil pulse) and ROESY (offset compensated ROESY by Griesinger et al. [1987], 80 ms spin lock at B1 = 3 kHz, carrier at 3.325 ppm) the States et al. (1982) method was used to obtain pure absorptive spectra of the same dimensions. Proton detected heteronuclear correlated 2D-NMR spectra using pulsed field gradients for coherence selection and the echo/anti-echo procedure (Davies et al., 1992) to obtain phase sensitive data (HSQC without or with proton decoupling and sensitivity improvement (Kay et al., 1992), HSQC-NOESY (Jahnke et al., 1995), HSQC-TOCSY (de Beer et al., 1994) with a 12 kHz spin-lock field), were performed in the proton detected form using the States method recording 2 × 1024(F1) × 2048(F2) complex data points. As suggested by Reynolds et al. (1997) the data matrix was extended to 4096 × 4096 by means of zero filling in the F2 and linear prediction data filling in the F1 dimension. For the linear prediction 32 autoregressive coefficients obtained with the covariance method were used (Stephenson, 1988).
We thank Sonja Zayni for carbohydrate and amino acid analyses. Protein sequence analysis was performed by Dr. Karola Vorauer, Institut für Angewandte Mikrobiologie, Universität für Bodenkultur Wien, and partial 16S rDNA sequencing was performed by Dr. Cathrin Spröer, Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH., Braunschweig, Germany. The work was supported by the Austrian Science Fund, project S7201-MOB (to P.M.) and the Federal Ministry of Science and Transportation. Financial support by the Stadt Linz and the Land Oberösterreich is acknowledged by N.M.
Asp, aspartic acid; COSY, correlated spectroscopy; FACE, fluorophore-assisted carbohydrate electrophoresis; FID, free induction decay; d-Fuc3NAc, 3-N-acetyl-d-fucosamine (3-acetamido-3,6-dideoxy-d-galactose); GalNAc, N-acetyl-d-galactosamine (2-acetamido-2-deoxy-d-galactose); Gly, glycine; HPAEC-PED, high-performance anion exchange chromatography with pulsed electrochemical detection; HSQC, heteronuclear single quantum correlation spectroscopy; NMR, nuclear magnetic resonance spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; RAPD, random amplified polymorphic DNA; Rha, rhamnose; ROESY, rotating frame Overhauser spectroscopy; RP-HPLC, reversed-phase high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Ser, serine; S-layer, crystalline bacterial cell surface layer; Thr, threonine; TPPI, time proportional phase increment.
Discussion
Carbon/Proton
1
2
3
4
5
6
7
8
Residue
A[prime]
100.85
52.17
175.16
22.87
(171
141)a
5.084
3.805
4.185
B[prime]
101.49
74.90
80.43
71.95
70.28
57.53b
(171
150
145
146
141
144)
5.354
4.307
3.596
3.657
3.856
3.443
(1
3.5
10
10.5)c
D'
101.51
78.82
77.71
(172
150
143)
5.105
4.115
4.052
A
101.04
67.18
52.34
71.18
67.98
16.10
175.21
22.97
(170
147
140
150
143
127
129)
5.153
3.824
4.198
3.770
4.124
1.187
2.062
B
101.62
78.66
77.30
73.16
70.17
17.40
(172
149
143
143
147
127)
5.236
4.107
3.948
3.764
3.764
1.315
C
101.15
67.28
52.25
71.18
67.98
16.10
175.10
22.92
(170
147
140
150
143
127
129)
5.112
3.824
4.195
3.770
4.124
1.187
2.072
D
101.49
78.66
77.51
72.95
70.38
17.32
(172
150
143
146
142
127)
5.095
4.103
4.002
3.782
3.784
1.313
E
101.59
78.66
70.94
73.30
70.10
17.59
(172
150
143
143
145
127)
5.169
4.172
3.916
3.487
3.727
1.292
F
101.46
78.44
70.99
73.16
70.03
17.54
(172
149
143
143
144
127)
5.205
4.055
3.865
3.491
3.773
1.307
F[prime]
101.30
79.06
70.88
(171
150
143)
5.126
4.055
3.911
G1
102.65
70.60
79.28
(171
150
145)
4.985
4.136
3.813
G2
102.83
70.60
78.25
(172
150)
5.133
4.226
G3
102.87
(172)
5.102
4.174
H
97.09
70.80
78.36
72.48
(170
150
144
147)
5.015
4.139
3.865
3.606
I
100.75
51.67
74.32
64.49
75.57
62.00
174.80
23.20
(161
142
141
146
143
145
129)
4.595
3.983
3.613
4.187
3.842
3.926/3.797
2.062
(8.1
9.5
4
2)
I'd
92.67
51.82
(170
140)
5.219
4.178
(4
11.5)
I"
97.65
55.75
(161
148)
4.643
3.931
(9.5
10.4
3.5)
Ser
171.64
55.57
61.75
(147
143)
4.272
3.882/3.805
Thr*e
172.04
61.11
75.62
17.053
(147
141
127)
4.033
3.660
1.216
Gly
174.19
43.50
(140)
4.009
Asp
175.02
50.96
36.42
173.25
(141
131)
4.781
2.959
Thr
168.98
61.926
59.142
19.65
(145
142
127)
4.033
4.544
1.350
Materials and methods
Acknowledgments
Abbreviations
3To whom correspondence should be sent at: Dr. Paul Messner, Zentrum für Ultrastrukturforschung, Universität für Bodenkultur, Gregor-Mendel-Strasse 33, A-1180 Wien, Austria