18
[1] W.L. van Veen, E.G. Mulder, M.H. Deinema, The Sphaerotilus-Leptothrix group of bacteria,
19
20
21
22
23
24
25
26
Microbiol. Rev. 42 (1978) 329−356.
[2] D. Jenkins, M.G. Richard, G.T. Daigger, Manual on the causes and control of activated sludge
bulking and forming, second ed., Lewis Publishers, Chelsea, Michigan, 1993.
[3] A.M. Martins, K. Pagilla, J.J. Heijnen, M.C. van Loosdrecht, Filamentous bulking sludge—a
critical review, Water Res. 38 (2004) 793−817.
[4] A.H. Romano, J.P. Peloquin, Composition of the sheath of Sphaerotilus natans, J. Bacteriol. 86
(1963) 252−258.
[5] K. Kondo, M. Takeda, W. Ejima, Y. Kawasaki, T. Umezu, M. Yamada, J. Koizumi, T. Mashima,
19
M. Katahira, Study of a novel glycoconjugate, thiopeptidoglycan, and a novel polysaccharide
lyase, thiopeptidoglycan lyase, Int. J. Biol. Macromol. 48 (2011) 256−262.
[6] A.H. Romano, D.J. Geason, Pattern of sheath synthesis in Sphaerotilus natans, J. Bacteriol. 88
(1964) 1145−1150.
[7] M. Takeda, T. Umezu, Y. Kawasaki, S. Shimura, K. Kondo, J. Koizumi, A spatial relationship
between sheath elongation and cell proliferation in Sphaerotilus natans, Biosci. Biotechnol.
Biochem. 76 (2012) 2357−2359.
[8] P.L. Siering, W.C. Ghiorse, Phylogeny of the Sphaerotilus-Leptothrix group inferred from
morphological comparisons, genomic fingerprinting, and 16S ribosomal DNA sequence analyses,
10
Int. J. Syst. Bacteriol. 46 (1996) 173−182.
11
[9] P.L. Siering, W.C. Ghiorse, Development and application of 16S rRNA-targeted probes for
12
detection of iron- and manganese-oxidizing sheathed bacteria in environmental samples, Appl.
13
Environ. Microbiol. 63 (1997) 644−651.
14
[10] M. Takeda, Y. Kawasaki, T. Umezu, S. Shimura, M. Hasegawa, J. Koizumi, Patterns of sheath
15
elongation, cell proliferation, and manganese(II) oxidation in Leptothrix cholodnii, Arch.
16
Microbiol. 194 (2012) 667−673.
17
[11] E. Gridneva, E. Chernousova, G. Dubinina, V. Akimov, J. Kuever, E. Detkova, M. Grabovich,
18
Taxonomic investigation of representatives of the genus Sphaerotilus: descriptions of
19
Sphaerotilus montanus sp. nov., Sphaerotilus hippei sp. nov., Sphaerotilus natans subsp. natans
20
subsp. nov. and Sphaerotilus natans subsp. sulfidivorans subsp. nov., and an emended
21
description of the genus Sphaerotilus, Int. J. Syst. Evol. Microbiol. 61 (2011) 916−925.
22
23
[12] M.A. Nott, H.E. Driscoll, M. Takeda, M. Vangala, S.R. Corsi, S. W. Tighe, Advanced biofilm
analysis in streams receiving organic deicer runoff. PLoS One. 15 (2020) e0227567.
24
[13] T. Suzuki, T. Kanagawa, Y. Kamagata, Identification of a gene essential for sheathed structure
25
formation in Sphaerotilus natans, a filamentous sheathed bacterium, Appl. Environ. Microbiol.
26
68 (2002) 365–371.
20
[14] M. Takeda, K. Nishina, Y. Hanaoka, M. Higo, M. Terui, I. Suzuki, J. Koizumi, A novel gene
encoding an enzyme that degrades a polysaccharide from the sheath of Sphaerotilus natans,
Biosci. Biotechnol. Biochem. 67 (2003) 2300−2303.
[15] M. Takeda, K. Iohara, S. Shinmaru, I. Suzuki, J. Koizumi, Purification and properties of an
enzyme capable of degrading the sheath of Sphaerotilus natans, Appl. Environ. Microbiol. 66
(2000) 4998−5004.
[16] M. Takeda, Y. Kamagata, S. Shinmaru, T. Nishiyama, J. Koizumi, Paenibacillus koleovorans
sp. nov., able to grow on the sheath of Sphaerotilus natans, Int. J. Syst. Evol. Microbiol. 52
(2002) 1597−1601.
10
11
[17] M. Takeda, F. Nakano, T. Nagase, K. Iohara, J. Koizumi, Isolation and chemical composition of
the sheath of Sphaerotilus natans, Biosci. Biotechnol. Biochem. 62 (1998) 1138−1143.
12
[18] R.L. Taylor, H. E. Conrad, Stoichiometric depolymerization of polyuronides and
13
glycosaminoglycuronans to monosaccharides following reduction of their carbodiimide-
14
activated carboxyl groups, Biochemistry 11 (1972) 1383−1388.
15
[19] R.K. Merkle, I. Poppe, Carbohydrate composition analysis of glycoconjugates by gas-liquid
16
chromatography/mass spectrometry, in: W.J. Lennarz, G.W. Hart (Eds.), Methods in Enzymology
17
Vol. 230, Academic Press, San Diego, 1994, pp. 1–15.
18
19
[20] T.M. Filisetti-Cozzi, N.C. Carpita, Measurement of uronic acids without interference from
neutral sugars, Anal. Biochem. 197 (1991) 157−162.
20
[21] G.J. Gerwig, J.P. Kamerling, J.F.G. Vliegenthart, Determination of the D and L configuration of
21
neutral monosaccharides by high-resolution capillary G.L.C., Carbohydr. Res. 62 (1978)
22
349−357.
23
[22] G.J. Gerwig, J.P. Kamerling, J.F.G. Vliegenthart, Determination of the absolute configuration of
24
monosaccharides in complex carbohydrates by capillary G.L.C., Carbohydr. Res. 77 (1979) 1−7.
25
[23] E.H. Armbruster, Improved technique for isolation and identification of Sphaerotilus, Appl.
26
Microbiol. 17 (1969) 320–321.
21
[24] M. Takeda, T. Nakamori, M. Hatta, H. Yamada, J. Koizumi J, Structure of the polysaccharide
isolated from the sheath of Sphaerotilus natans, Int. J. Biol. Macromol. 33 (2003) 245−250.
[25] M. Takeda, K. Kondo, R. Tominaga, H. Mori, M. Kato, R. Usami, T. Murakami, K. Ueda, I.
Suzuki, M. Katahira, Aggregability of β(1→4)-linked glucosaminoglucan originating from a
sulfur-oxidizing bacterium Sphaerotilus natans, Biosci. Biotechnol. Biochem. 84 (2020)
2085−2095.
10
[26] J.F. Hoeniger, H.D. Tauschel, J.L. Stokes, The fine structure of Sphaerotilus natans, Can. J.
Microbiol. 19 (1973) 309−313.
[27] D. Emerson, W.C. Ghiorse, Ultrastructure and chemical composition of the sheath of Leptothrix
discophora SP-6, J. Bacteriol. 175 (1993) 7808−7818.
11
[28] T. Kunoh, K. Morinaga, S. Sugimoto, S. Miyazaki, M. Toyofuku, K. Iwasaki, N. Nomura, A.S
12
Utada, Polyfunctional nanofibril appendages mediate attachment, filamentation, and filament
13
adaptability in Leptothrix cholodnii, ACS Nano 14 (2020) 5288−5297.
14
[29] M. Takeda, K. Kondo, M. Yamada, J. Koizumi, T. Mashima, A. Matsugami, M. Katahira,
15
Solubilization and structural determination of a glycoconjugate which is assembled into the
16
sheath of Leptothrix cholodnii, Int. J. Biol. Macromol. 46 (2010) 206−211.
17
18
[30] D. Emerson, W.C. Ghiorse, Role of disulfide bonds in maintaining the structural integrity of the
sheath of Leptothrix discophora SP-6. J. Bacteriol. 175 (1993) 7819−7827.
19
[31] D.J. Little, G. Li., C. Ing, B.R. DiFrancesco, N.C. Bamford, H Robinson, M. Nitz, R. Pomès,
20
P.L. Howell, Modification and periplasmic translocation of the biofilm exopolysaccharide poly-
21
β-1,6-N-acetyl-D-glucosamine, PNAS 111 (2014) 11013–11018.
22
23
[32] M. Pauly, V. Ramírez, New insights into wall polysaccharide O-acetylation, Front. Plant Sci. 9
(2018) 1210.
24
22
Table 1. 1H and 13C resonance assignments (δ in ppm)
Residue
α-GalNAc (A)
α-GalNAc (B)
β-GlcA (C)
β-GalNAc (D)
β-Glc (E)
Nucleus
6 (6′)
NAc
4.00
4.05
3.95
3.65 (3.69)
2.05
96.0
52.4
69.9
79.3
74.6
62.8
24.0, 177.4
4.93a
4.17
4.16
4.23
4.37
3.62 (3.86)
2.07
101.2
53.0
70.1
78.2
73.0
63.3
24.0, 177.0
4.78
3.53
4.39
3.85
4.20
102.9
72.8
69.6
75.0
76.7
178.3
4.76
4.03
3.93
4.16
3.69
3.77 (3.81)
2.01
104.9
54.3
82.7
70.9
77.4
63.8
24.0, 177.6
4.52
3.35
3.63
3.62
3.55
3.81 (3.91)
106.8
75.4
76.8
81.3
77.4
62.8
13
4.29
13
5.10
13
13
1 (3J1,2, Hz)
13
(8.4)
(6.6)
(7.2)
A minor signal was detected at 4.98 ppm.
23
Table 2. 1H and 13C resonance assignments of ABEE derivatives (δ in ppm)
Derivative I
Sugar
Nucleus
residue
β-ΔGlcA
13
(dC)
β-Glc
(E)
β-GalNAc
(D)
α-GalNAc
(B)
α-GalNAc
(Ar)
Nonsugar
residue
ABEE
6 (6′)
NAc
OAc
5.16
3.86
4.34
5.71
101.5
67.9
65.1
110.3
147.2
ND
4.58
3.55
4.94
4.06
3.60
3.93 (3.79)
2.09
106.8
74.0
77.8
75.3
77.7
62.7
23.2, 176.7
4.70
3.99
3.89
4.18
3.65
3.80 (3.80)
1.98
104.6
54.1
83.0
70.6
77.3
63.5
25.1, 177.6
5.04
4.13
3.84
4.00
3.87
3.69 (3.66)
2.04
101.4
52.7
70.3
78.0
74.0
64.0
24.7, 177.1
3.59
3.70
4.09
3.91
3.96
3.65 (3.65)
45.3
53.5
71.6
80.8
73.8
64.8
13
13
13
13
Nucleus
CH2
CH3
C=O
2, 6
3, 5
4.35
1.37
7.89
6.81
7.91
64.5
16.3
171.9
121.1
115.0
134.3
154.7
13
Derivative II
Sugar
Nucleus
residue
β-ΔGlcA
13
(dC)
β-Glc
(E)
β-GalNAc
(D)
α-GalNAc
(B)
α-GalNAc
(Ar)
Nonsugar
residue
ABEE
6 (6′)
NAc
OAc
5.17
3.88
4.35
5.82
101.8
67.8
65.0
112.3
145.6
170.0
4.60
3.53
4.94
4.04
3.59
3.92 (3.79)
2.08
106.6
74.0
77.9
75.6
77.5
62.7
23.2, 176.7
4.48
3.89
4.01
4.18
3.59
3.77 (3.67)
2.01
104.5
54.3
82.2
70.2
77.0
63.5
25.0, 177.6
5.06
4.36
4.90
3.96
3.81
3.68 (3.60)
1.97
2.14
101.1
50.7
72.6
76.1
73.9
63.6
24.5, 176.8
22.9, 175.9
3.61
3.73
4.11
3.94
3.97
3.64 (3.64)
44.9
53.3
70.6
80.5
73.6
64.8
13
13
13
13
Nucleus
CH2
CH3
C=O
2, 6
3, 5
4.32
1.35
7.89
6.82
7.92
4.32
64.4
16.4
172.0
121.4
115.2
134.5
154.4
13
ND: Not detected.
24
Figure legends
Fig. 1. Micrographs of the filament (a, c, e) and purified sheath (b, d, f) of S. montanus. S. montanus
filaments grown on glucose-free medium were observed using phase-contrast microscopy (a). The
suspension of the sheath was observed using phase-contrast microscopy (b). The membrane filter
attached to the filament (c) or sheath (b) was fixed and metal coated for observation using scanning
electron microscopy. The filament (e) or sheath (f) airdried on a silicon wafer was subjected to
scanning probe microscopy.
10
11
Fig. 2. 13C cross polarization/magic angle spinning spectra of the Sphaerotilus sheaths (a, b) and the
12
derivatives of the S. montanus sheath (c, d, e). The lyophilized samples were subjected to analysis at
13
25 °C. The spectra of the purified sheaths of S. montanus (a) and S. natans (b) are compared in the
14
left column. In the right column, the spectra of de-O-acetylated (c), de-O-N-acetylated (d), and N-
15
acetylated (e) derivatives of the S. montanus sheath are shown. Important signals are indicated by
16
C=O (carbonyl carbon signal), Anomeric (anomeric carbon signal) and Ac (methyl carbon signal due
17
to acetyl group).
18
19
Fig. 3. 1D-1H NMR spectrum of the N-acetylated derivative of the S. montanus sheath. The solution
20
(approximately 5 mg/mL) of the N-acetylated derivative was subjected to analysis at 30 °C. Important
21
signals are indicated by arrows. Note that a weak unidentified signal (X-H1) was detected in the
22
anomeric proton region. Relative intensities are indicated in the parentheses.
23
24
Fig. 4. 1D-1H NMR spectra of the ABEE derivatives. The solutions (approximately 5 mg/mL) of the
25
ABEE derivatives (I and II) purified by HPLC were subjected to 1D-1H NMR analysis. Whole spectra
26
(a) and partial spectra of the acetyl proton region (b) of both derivatives are shown. Note that three
27
and four major signals are detected in the acetyl proton region of derivatives I and II, respectively.
25
Fig. 5. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry spectra
derivatives I (a) and II (b). Spectra were acquired using a DHB matrix solution in reflectron mode
(positive). Possible ions for major signals are indicated.
Fig. 6. Edited heteronuclear single quantum coherence spectroscopy (a) and heteronuclear multiple
bond correlation (b) spectra of derivative I. The solution (approximately 5 mg/mL) of derivative I
was subjected to analysis using 3-(trimethylsilyl)propionic acid and acetone as internal standards.
Positive and negative heteronuclear single quantum coherence spectroscopy signals are indicated by
10
red and green contour lines, respectively. The crosspeaks identified are designated as dC1 (correlation
11
between C1 and H1 of unsaturated residue C), etc. The heteronuclear multiple bond correlation
12
signals within the carbonyl carbon (C=O) region are displayed separately. The crosspeaks identified
13
are designated as C=O/E(OAc) (correlation between C=O and O-acetyl protons of residue E), etc.
14
15
Fig. 7. Chemical structures of derivative I (a), derivative II (b), the sheath-forming polymer of S.
16
montanus (c), and the sheath-forming polymer of S. natans (d). The arrows indicate the linkage
17
cleaved by thiopeptidoglycan lyase.
18
19
Fig. 8 Comparative phase-contrast (left), epifluorescent (middle), and merged (right) images of
20
immunostained filaments of N-biotinylated S. montanus. S. montanus was N-biotinylated and then
21
cultivated. The bacterial filaments were recovered at 0 h (a) and 3 h (b) of cultivation and
22
immunostained for visualization of the sheath. The edges of the sheath are not closed (a). A cultured
23
(3 h) filament exhibited fluorescence only in the middle region (b).
24
26
10
10 μm
10 μm
11
12
13
1 μm
1 μm
14
15
16
17
18
19
2 μm
2 μm
20
21
22
Fig. 1 - Takeda - International Journal of Biological Macromolecules
23
27
Ac
C1
C1
10
11
C=O
C1
C=O
12
C1
C=O
15
18
Ac
C1
14
17
Ac
13
16
Ac
C=O
C=O
180
160
140
120 100 80
60
Chemical shift (ppm)
40
20
160
120
80
40
Chemical shift (ppm)
19
20
21
22
Ac
Fig. 2 - Takeda - International Journal of Biological Macromolecules
23
28
Acetyl signals (9.09)
HDO
Anomeric (H1)
signals
B-H1
X-H1
A-H1
(1.00)
D-H1
C-H1
5.0
E-H1
4.5
4.0
3.5
Chemical shift (ppm)
3.0
2.5
10
Fig. 3 - Takeda - International Journal of Biological Macromolecules
11
12
29
2.0
1119.4
+Na+ (m/z 1119.4), +K+ (m/z 1135.4)
ΔGlcA-Glc-GalNAc-GalNAc-GalNr-ABEE
Ac
+Na+
(m/z 961.4),
+K+
961.4
1135.4
977.3
(m/z 977.3)
m/z
1161.4
1177.4
+Na+ (m/z 1161.4), +K+ (m/z 1177.4)
ΔGlcA-Glc-GalNAc-GalNAc-GalNr-ABEE
Ac
+Na+
(m/z 1003.4),
+K+
Ac
(m/z 1019.4)
1003.4
1019.4
m/z
Fig. 4 - Takeda - International Journal of Biological Macromolecules
30
Derivative I
HDO
Olefinic signal
Methyl signal
(ABEE)
Aryl signals (ABEE)
Derivative II
HDO
Olefinic signal
Methyl signal
(ABEE)
Aryl signals (ABEE)
10
11
12
Acetyl signals
13
14
15
16
17
Derivative I
18
19
20
Derivative II
21
22
23
24
25
Fig. 5 - Takeda - International Journal of Biological Macromolecules
26
31
Fig. 6 - Takeda - International Journal of Biological Macromolecules
32
10
11
12
13
β-GlcA
β-Glc
β-GalNAc
α-GalNAc
α-GalN
β-GlcA
β-Glc
β-GalNAc
α-GalNAc
α-GalN
14
15
16
17
Gly
18
Cys
19
20
21
22
Fig. 7 - Takeda - International Journal of Biological Macromolecules
23
33
5 μm
5 μm
20 μm
20 μm
5 μm
10
11
12
Fig. 8 - Takeda - International Journal of Biological Macromolecules
13
34
20 μm
...
論文の公開元へ
