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Structural and genetic studies of the O-antigen of Enterobacter cloacae G2277
Carbohydrate Research 387 (2014) 10–13
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
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Structural and genetic studies of the O-antigen of Enterobacter cloacae G2277 Andrei V. Perepelov a,⇑, Min Wang b,c, Andrei V. Filatov a, Xi Guo c, Alexander S. Shashkov a, Lei Wang b,c, Yuriy A. Knirel a a
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, PR China c Tianjin Key Laboratory for Microbial Functional Genomics, TEDA College, Nankai University, TEDA, Tianjin 300457, PR China b
a r t i c l e
i n f o
Article history: Received 22 November 2013 Received in revised form 6 January 2014 Accepted 7 January 2014 Available online 14 January 2014 Keywords: Enterobacter cloacae Lipopolysaccharide O-Polysaccharide O-Antigen gene cluster Bacterial polysaccharide structure
a b s t r a c t The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Enterobacter cloacae G2277 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the linear pentasaccharide repeating unit was established, where a galacturonic acid (GalA) residue is mono-O-acetylated at position either 2 or 3:
→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→3)-α-D-GlcpNAc-(1→ 2/3 | OAc ~30/60 % The O-antigen gene cluster of E. cloacae G2277 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the O-polysaccharide structure. Ó 2014 Elsevier Ltd. All rights reserved.
Nosocomial infections caused by Enterobacter cloacae remain problematic1,2 and are complicated by the emergence of multidrug resistant strains.3 In 1983, a serotyping scheme for E. cloacae including 28 O-serogroups was developed based on O-antigens (O-specific polysaccharides).4 Until recently, O-polysaccharide structure has been reported for only one strain, E. cloacae NCTC 11579, belonging to serogroup O10.5 Recently, we have established the O-polysaccharide structure of another E. cloacae strain, C6285, and found it to contain a higher sugar, di-N-acetyllegionaminic acid (paper submitted for publication). In this work, we established a new structure of the O-polysaccharide of E. cloacae G2277. A high-molecular mass O-polysaccharide (OPS) was obtained by mild acid degradation of the lipopolysaccharide isolated from dried bacterial cells by the phenol–water procedure. Sugar analysis of the O-polysaccharide by GLC of the alditol acetates revealed the presence of rhamnose (Rha) and GlcNAc. GLC of the acetylated (S)-2-octyl glycosides indicated that rhamnose is L and GlcNAc is D. Further NMR spectroscopic studies showed that the polysaccharide also included galacturonic acid (GalA), and its configuration ⇑ Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328. E-mail address: [email protected] (A.V. Perepelov). http://dx.doi.org/10.1016/j.carres.2014.01.001 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
was established using known regularities in the effects of glycosylation on 13C NMR chemical shifts: a relatively small effect (+0.2 ppm) on C-4 of GlcNAc in the a-D-GalA-(1?3)-a-D-GlcNAc disaccharide indicated the same absolute configuration of the constituent monosaccharides, that is, the D configuration of GalA (compare published data6 0.1 and 1.3 ppm for the same and different absolute configurations, respectively). The 13C NMR spectrum of the OPS (Fig. 1A) contained signals of different intensities, most likely, owing to non-stoichiometric O-acetylation as there were two signals for O-acetyl groups at d 21.8 and 22.1 (CH3). The 1H NMR spectrum of the OPS showed signals for two O-acetyl and one N-acetyl groups at d 2.22, 2.16, and 1.99 in the ratios 0.3:0.6:1.0, respectively. To simplify the NMR spectra the OPS was subjected to O-deacetylation with aqueous ammonia, and the resultant O-deacetylated polysaccharide (DPS) was found to be regular. Its 13 C NMR spectrum (Fig. 1B) showed signals for five anomeric carbons in the region d 96.5–102.1, three C-CH3 groups (C-6 of Rha) in the region d 17.8–18.0, one C-CO2H group (C-6 of GalA) at d 175.5, one HOCH2-C group (C-6 of GlcN) at d 61.4, one nitrogen-bearing carbon (C-2 of GlcN) at d 53.2, 19 oxygen-bearing non-anomeric carbons in the region d 69.5–81.0, and one N-acetyl
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A. V. Perepelov et al. / Carbohydrate Research 387 (2014) 10–13
Figure 1. 13C NMR spectra of the OPS (A) and DPS (B) from E. cloacae G2277. Numbers refer to carbons in sugar residues denoted as shown in Table 1. Peak annotations for 2-O-acetylated GalA are shown in regular, and those for 3-O-acetylated GalA in italics.
group at d 23.1 (CH3) and 175.7 (CO). Accordingly, the 1H NMR spectrum of the DPS displayed signals for five anomeric protons at d 5.01–5.31, three C-CH3 groups (H-6 of Rha) in the region d 1.24–1.31, other sugar protons in the region d 3.41–4.38, and one N-acetyl group at d 2.01. Therefore, the OPS has a pentasaccharide repeat (O-unit) containing three residues of L-Rha (denoted as units A–C) and one residue each of D-GalA and D-GlcNAc (units D and E, respectively). The 1H and 13C NMR spectra of the DPS were analyzed using 2D homonuclear 1H,1H COSY, TOCSY, ROESY, and heteronuclear 1H,13C HSQC and HMBC experiments. Based on intra-residue H,H and H,C correlations and coupling constant values, spin systems were assigned to residues of A–E (Table 1), all being in the pyranosidic form. The spin system for unit E was distinguished from others
showed correlations between anomeric protons and linkage carbons and vice versa (data not shown). Positions of the O-acetyl groups were determined by a 1H,13C HSQC experiment on the OPS. As compared with the HSQC spectrum of the DPS, a smaller part of the H-2,C-2 cross-peak and a larger part of the H-3,C-3 cross-peak of unit D shifted from d 3.99/69.5 and 3.98/71.6 to 4.99/71.8 and 5.21/73.8, respectively. These displacements were due to a deshielding effect of the O-acetyl groups and indicated partial (by 30% and 60%) mono-O-acetylation of unit D at position 2 or 3, respectively. The O-acetylation pattern was confirmed by upfield shifts by 2.9 and 3.2 ppm of parts of the C-1 and C-3 signals (for GalA2OAc) and upfield shift by 2.0 and 1.7 ppm of parts of the C-2 and C-4 signals (for GalA3OAc) of unit D (b-effects of O-acetylation, see Fig. 1A).
A B C D E →2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→3)-α-D-GlcpNAc-(1→ 2/3 | OAc ~30/60 %
by a correlation between the proton at the nitrogen-bearing carbon (H-2) and the corresponding carbon (C-2) at d 4.04/53.2. Relatively large J1,2 coupling constant values of 7 Hz showed that units D and E are b-linked. The position of the C-5 signals at d 70.1–70.7 indicated that units A–C are a-linked (compare published data d 69.5 and 73.2 for a- and b-Rhap, respectively6). The signals for C-2 of units A, B, and C, C-3 of unit E and C-4 of unit D shifted significantly downfield, as compared with their positions in the corresponding non-substituted monosaccharides.6,7 These data demonstrated the linear character of the OPS chain and defined the glycosylation pattern in the O-unit. The ROESY spectrum of the DPS showed the following correlations between anomeric protons and protons at the linkage carbons: A H-1, B H-2; B H-1,C H-2; C H-1,D H-4; D H-1,E H-3, and E H-1,A H-2 at d 5.02/4.07, 5.10/4.04, 5.35/4.38, 5.31/3.95, 5.01/4.09, respectively. The monosaccharide sequence thus established was confirmed by a heteronuclear 1H,13C HMBC experiment, which
Therefore, the OPS of E. cloacae G2277 has the following structure:
Table 1 H and 13C NMR chemical shifts (d, ppm) of the DPS of E. cloacae G2277
1
Sugar residue
H-1 C-1
H-2 C-2
H-3 C-3
H-4 C-4
H-5 C-5
H-6 (6a, 6b) C-6
?2)-a-L-Rhap-(1? A ?2)-a-L-Rhap-(1? B ?2)-a-L-Rhap-(1? C ?4)-a-D-GalpA-(1? D ?3)-a-D-GlcpNAc-(1? E
5.02 100.2 5.10 101.9 5.35 100.5 5.31 102.1 5.01 96.5
4.09 76.3 4.07 79.5 4.04 79.4 3.99 69.5 4.04 53.2
3.91 70.6 3.89 71.2 3.90 71.3 3.98 71.6 3.95 81.0
3.56 73.1 3.45 73.4 3.41 73.5 4.38 77.5 3.73 71.6
3.75 70.7 3.73 70.5 3.80 70.1 4.16 72.7 4.04 72.9
1.31 18.0 1.28 17.9 1.24 17.8 175.5 3.81; 3.84 61.4
The chemical shifts for the N-acetyl group are dH 2.01 (Me), dC 23.1 (Me), and 175.7 (CO).
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A. V. Perepelov et al. / Carbohydrate Research 387 (2014) 10–13
Figure 2. Organization of the O-antigen gene cluster of E. cloacae G2277. Glycosyltransferase genes and O-antigen processing genes are shown in black and gray, respectively.
This structure is closely related to the O-polysaccharide structure of Plesiomonas shigelloides strains 22074 and 12254, which differs only in the linkage between GlcNAc and Rha A (1?3 vs 1?2).8 As the O-polysaccharides in question are GlcNAc-initiated (see below), the GlcNAc?Rha linkage forms upon polymerization of the preassembled O-units, which are thus identical in all these bacteria. The O-antigen gene cluster of E. cloacae strain G2277 was found between the housekeeping genes galF and gnd. Nine open reading frames (orfs) excluding galF and gnd were identified in the gene cluster, all of which have the same transcriptional direction from galF to gnd (Fig. 2). All orfs were assigned functions based on their similarity to related genes from the available databases and taking into account the E. cloacae G2277 antigen structure. Genes for synthesis of nucleotide precursors of common sugars, such as D-GlcNAc, are located outside the O-antigen gene cluster.9 Two genes, ugd and gla, responsible for the synthesis of UDP-D-GalA from UDP-D-Glc are absent from the O-antigen gene cluster too; this occurs in some other members of Enterobacteriaceae, where these genes map downstream gnd.10,11 Orf1-4 shared high-level identities to many known RmlB, D, A, and C proteins, and the rmlBDAC gene set responsible for the synthesis of dTDP12 L-Rha has been well characterized. Therefore, orf1-4 were identified as rmlBDAC and named accordingly. In GlcNAc-initiated O-antigens, wecA mediates transfer of the first sugar to a lipid carrier and is located outside the O-antigen gene cluster.13 Apart from wecA, four glycosyltransferase genes were expected for synthesis of the E. cloacae G2277 pentasaccharide O-unit. Orf7 showed 53.8% identity to Orf10 of Escherichia coli O116, whose O-antigen also has the a-D-GalpA-(1?3)-D-GlcpNAc linkage.14 Therefore, Orf7 was suggested to be glycosyltransferase for transfer of a-D-GalA to make the (1?3)-linkage to D-GlcpNAc, and the encoding gene was named wfiQ. Orf6 is a homolog of many rhamnosyltransferases and named wfiP. In the assembly of O-units that contain a sequence of three or more L-Rha residues, for example, in E. coli O13, O99, O129, O135, O139, and O150,15–18 one rhamnosyltransferase may be responsible for transfer of two L-Rha residues to make the same or a different a-L-Rhap?L-Rhap linkage. Therefore, it was suggested that WfiP catalyzes formation of two a-L-Rhap-(1?2)-LRhap linkages. As for the a-L-Rhap-(1?4)-D-GalpA linkage, this can be made with the help of WfiP too provided that this rhamnosyltransferase is trifunctional and able to transfer L-Rha to different acceptors. Alternatively, the missing glycosyltransferase may have a different origin compared to the genes in the O-antigen gene cluster and be located, for example, in a bacteriophage (recently, a similar situation has been reported in E. coli O12019). orf8 was identified as acyltransferase gene responsible for Oacetylation of GalA, and named wfiR. Orf5 and Orf9 are the only two proteins with predicted transmembrane segments, and hence the encoding genes are wzx (for O-unit flippase) and wzy (for O-antigen polymerase), respectively.20 1. Experimental 1.1. Bacterial strain and isolation of lipopolysaccharide E. cloacae G2277 is strain ATCC 7256, which was originally isolated from well water. The restriction fragment length polymor-
phism (RFLP) analysis of the O-antigen gene cluster showed that it has a distinct O pattern. Bacteria were grown to late log phase in 8 L of Luria-Bertani broth using a 10-L BIOSTAT C-10 fermentor (B. Braun Biotech Int., Germany) under constant aeration at 37 °C and pH 7.0. Bacterial cells were washed and dried as described.21 The lipopolysaccharide was isolated in a yield 17.5% from dried cells by the phenol–water method,22 the crude extract was dialyzed without separation of the layers and freed from nucleic acids and proteins by treatment with 50% aq CCl3CO2H to pH 2. The supernatant was dialyzed and lyophilized. 1.2. Isolation of OPS and preparation of DPS Delipidation of the lipopolysaccharide (200 mg) was performed with 2% aq HOAc at 100 °C until precipitation of lipid A. The precipitate was removed by centrifugation (13,000g, 20 min), and the supernatant was fractionated by GPC on a column (56 2.6 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer pH 5.5, monitored with a differential refractometer (Knauer, Germany). A high-molecular-mass OPS was obtained in a yield of 12.7% of the lipopolysaccharide mass. An OPS sample (11 mg) was treated with aq 12.5% ammonia at 37 °C for 16 h, ammonia was removed with a stream of air, the remaining solution was desalted on a column (90 2.5 cm) of TSK HW-40 (S) (Merck, Germany) in water and freeze-dried to give the DPS (9 mg). 1.3. Monosaccharide analysis The OPS was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were identified by GLC of the alditol acetates on an Agilent 7820A chromatograph (USA) equipped with an HP-5 column (0.32 mm 30 m) and a temperature program of 160 (1 min) to 290 °C at 7 °C min1. The absolute configurations of the monosaccharides were determined by GLC of the acetylated (S)-2-octyl glycosides as described.23 1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying from D2O and then examined as solution in 99.95% D2O. NMR spectra were recorded on a Bruker Avance II 600 spectrometer (Germany) at 25 °C and pH 9 using internal TSP (dH 0) and acetone (dC 31.45) as reference. 2D NMR spectra were obtained using standard Bruker software, and Bruker TopSpin 2.1 program was used to acquire and process the NMR data. A mixing time of 100 and 150 ms was used in TOCSY and ROESY experiments, respectively. 1.5. Sequencing and analysis of genes Chromosomal DNA was prepared as described.24 Long-range PCR was performed with the Expand Long Templated PCR system (Roche) using primers WL_35686(50 -AGG GCA GGT GAG CCG BAT CGT TGA GTT-30 ) and WL_35687(50 -TCA CGG TTA CGA CGA ATG GTG TCS TGG-30 ) based on the galF and gnd genes, respectively.25 The PCR cycles used were as follows: denaturation at 94 °C for 10 s, annealing at 60 °C for 45 s, and extension at 68 °C for 15 min. The PCR products were digested with DNase I, and the resulting DNA fragments were cloned into pGEM-T Easy to produce a bank as described.26 Sequencing was carried out using an ABI 3730 automated DNA sequencer (Applied Biosystems), and the sequence of the O-antigen gene clusters was analyzed as described.27
A. V. Perepelov et al. / Carbohydrate Research 387 (2014) 10–13
Acknowledgements This work was supported by the Russian Foundation for Basic Research (14-04-00663), the National Key Program for Infectious Diseases of China (2013ZX10004216-001-001); the National 973 program of China (2011CB504901); National Natural Science Foundation of China (NSFC) Program (31030002, 81171524); Research Project of Chinese Ministry of Education (No. 113015A). References 1. Dalben, M.; Varkulja, G.; Basso, M.; Krebs, V. L.; Gibelli, M. A.; van der Heijden, I.; Rossi, F.; Duboc, G.; Levin, A. S.; Costa, S. F. J. Hosp. Infect. 2008, 70, 7–14. 2. Fernández, A.; Pereira, M. J.; Suárez, J. M.; Poza, M.; Treviño, M.; Villalón, P.; Sáez-Nieto, J. A.; Regueiro, B. J.; Villanueva, R.; Bou, G. J. Clin. Microbiol. 2011, 49, 822–828. 3. Bush, K. Curr. Opin. Microbiol. 2010, 13, 558–564. 4. Gaston, M. A.; Bucher, C.; Pitt, T. L. J. Clin. Microbiol. 1983, 18, 1079–1083. 5. Moule, A. L.; Kuhl, P. M. D.; Galbraith, L.; Wilkinson, S. G. Carbohydr. Res. 1989, 186, 287–293. 6. Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175, 59–75. 7. Jansson, P.-E.; Kenne, L.; Widmalm, G. Carbohydr. Res. 1989, 188, 169–191. 8. Linnerborg, M.; Widmalm, G.; Weintraub, A.; Albert, M. J. Eur. J. Biochem. 1995, 231, 839–844. 9. Samuel, G.; Reeves, P. Carbohydr. Res. 2003, 338, 2503–2519.
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10. Liu, B.; Knirel, Y. A.; Feng, L.; Perepelov, A. V.; Senchenkova, S. N.; Wang, Q.; Reeves, P. R.; Wang, L. FEMS Microbiol. Rev. 2008, 32, 627–653. 11. Sun, Y.; Wang, M.; Wang, Q.; Cao, B.; He, X.; Li, K.; Feng, L.; Wang, L. Appl. Environ. Microbiol. 2012, 78, 3966–3974. 12. Hao, Y.; Lam, J. S. In Bacterial Lipopolysaccharides: Structure, Chemical Synthesis, Biogenesis and Interaction with Host Cells; Knirel, Y. A., Valvano, M. A., Eds.; Springer: Wien, 2011; pp 195–235. 13. Alexander, D. C.; Valvano, M. A. J. Bacteriol. 1994, 176, 7079–7084. 14. Leslie, M. R.; Parolis, H.; Parolis, L. A. S. Carbohydr. Res. 1999, 321, 246–256. 15. Wang, L.; Liu, B.; Kong, Q.; Steinruck, H.; Krause, G.; Beutin, L.; Feng, L. Vet. Microbiol. 2005, 111, 181–190. 16. Perepelov, A. V.; Han, W.; Senchenkova, S. N.; Shevelev, S. D.; Shashkov, A. S.; Feng, L.; Liu, B.; Knirel, Y. A.; Wang, L. Carbohydr. Res. 2007, 342, 648–652. 17. Perepelov, A. V.; Li, D.; Liu, B.; Senchenkova, S. N.; Guo, D.; Shevelev, S. D.; Shashkov, A. S.; Guo, X.; Feng, L.; Knirel, Y. A.; Wang, L. FEMS Immunol. Med. Microbiol. 2009, 57, 80–87. 18. Perepelov, A. V.; Shevelev, S. D.; Liu, B.; Senchenkova, S. N.; Shashkov, A. S.; Feng, L.; Knirel, Y. A.; Wang, L. Carbohydr. Res. 2010, 345, 1594–1599. 19. Perepelov, A. V.; Wang, Q.; Senchenkova, S. N.; Gong, Y.; Shashkov, A. S.; Wang, L.; Knirel, Y. A. Carbohydr. Res. 2012, 353, 106–110. 20. Valvano, M. A. Biochemistry (Moscow) 2011, 76, 729–735. 21. Robbins, P. W.; Uchida, T. Biochemistry 1962, 1, 323–335. 22. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91. 23. Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87–89. 24. Bastin, D. A.; Reeves, P. R. Gene 1995, 164, 17–23. 25. Feng, L.; Wang, W.; Tao, J.; Guo, H.; Krause, G.; Beutin, L.; Wang, L. J. Clin. Microbiol. 2004, 42, 3799–3804. 26. Wang, L.; Reeves, P. R. Infect. Immun. 1998, 66, 3545–3551. 27. Feng, L.; Senchenkova, S. N.; Yang, J.; Shashkov, A. S.; Tao, J.; Guo, H.; Zhao, G.; Knirel, Y. A.; Reeves, P.; Wang, L. J. Bacteriol. 2004, 182, 383–392.
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Note
Structural and genetic studies of the O-antigen of Enterobacter cloacae G2277 Andrei V. Perepelov a,⇑, Min Wang b,c, Andrei V. Filatov a, Xi Guo c, Alexander S. Shashkov a, Lei Wang b,c, Yuriy A. Knirel a a
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, PR China c Tianjin Key Laboratory for Microbial Functional Genomics, TEDA College, Nankai University, TEDA, Tianjin 300457, PR China b
a r t i c l e
i n f o
Article history: Received 22 November 2013 Received in revised form 6 January 2014 Accepted 7 January 2014 Available online 14 January 2014 Keywords: Enterobacter cloacae Lipopolysaccharide O-Polysaccharide O-Antigen gene cluster Bacterial polysaccharide structure
a b s t r a c t The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Enterobacter cloacae G2277 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the linear pentasaccharide repeating unit was established, where a galacturonic acid (GalA) residue is mono-O-acetylated at position either 2 or 3:
→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→3)-α-D-GlcpNAc-(1→ 2/3 | OAc ~30/60 % The O-antigen gene cluster of E. cloacae G2277 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the O-polysaccharide structure. Ó 2014 Elsevier Ltd. All rights reserved.
Nosocomial infections caused by Enterobacter cloacae remain problematic1,2 and are complicated by the emergence of multidrug resistant strains.3 In 1983, a serotyping scheme for E. cloacae including 28 O-serogroups was developed based on O-antigens (O-specific polysaccharides).4 Until recently, O-polysaccharide structure has been reported for only one strain, E. cloacae NCTC 11579, belonging to serogroup O10.5 Recently, we have established the O-polysaccharide structure of another E. cloacae strain, C6285, and found it to contain a higher sugar, di-N-acetyllegionaminic acid (paper submitted for publication). In this work, we established a new structure of the O-polysaccharide of E. cloacae G2277. A high-molecular mass O-polysaccharide (OPS) was obtained by mild acid degradation of the lipopolysaccharide isolated from dried bacterial cells by the phenol–water procedure. Sugar analysis of the O-polysaccharide by GLC of the alditol acetates revealed the presence of rhamnose (Rha) and GlcNAc. GLC of the acetylated (S)-2-octyl glycosides indicated that rhamnose is L and GlcNAc is D. Further NMR spectroscopic studies showed that the polysaccharide also included galacturonic acid (GalA), and its configuration ⇑ Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328. E-mail address: [email protected] (A.V. Perepelov). http://dx.doi.org/10.1016/j.carres.2014.01.001 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
was established using known regularities in the effects of glycosylation on 13C NMR chemical shifts: a relatively small effect (+0.2 ppm) on C-4 of GlcNAc in the a-D-GalA-(1?3)-a-D-GlcNAc disaccharide indicated the same absolute configuration of the constituent monosaccharides, that is, the D configuration of GalA (compare published data6 0.1 and 1.3 ppm for the same and different absolute configurations, respectively). The 13C NMR spectrum of the OPS (Fig. 1A) contained signals of different intensities, most likely, owing to non-stoichiometric O-acetylation as there were two signals for O-acetyl groups at d 21.8 and 22.1 (CH3). The 1H NMR spectrum of the OPS showed signals for two O-acetyl and one N-acetyl groups at d 2.22, 2.16, and 1.99 in the ratios 0.3:0.6:1.0, respectively. To simplify the NMR spectra the OPS was subjected to O-deacetylation with aqueous ammonia, and the resultant O-deacetylated polysaccharide (DPS) was found to be regular. Its 13 C NMR spectrum (Fig. 1B) showed signals for five anomeric carbons in the region d 96.5–102.1, three C-CH3 groups (C-6 of Rha) in the region d 17.8–18.0, one C-CO2H group (C-6 of GalA) at d 175.5, one HOCH2-C group (C-6 of GlcN) at d 61.4, one nitrogen-bearing carbon (C-2 of GlcN) at d 53.2, 19 oxygen-bearing non-anomeric carbons in the region d 69.5–81.0, and one N-acetyl
11
A. V. Perepelov et al. / Carbohydrate Research 387 (2014) 10–13
Figure 1. 13C NMR spectra of the OPS (A) and DPS (B) from E. cloacae G2277. Numbers refer to carbons in sugar residues denoted as shown in Table 1. Peak annotations for 2-O-acetylated GalA are shown in regular, and those for 3-O-acetylated GalA in italics.
group at d 23.1 (CH3) and 175.7 (CO). Accordingly, the 1H NMR spectrum of the DPS displayed signals for five anomeric protons at d 5.01–5.31, three C-CH3 groups (H-6 of Rha) in the region d 1.24–1.31, other sugar protons in the region d 3.41–4.38, and one N-acetyl group at d 2.01. Therefore, the OPS has a pentasaccharide repeat (O-unit) containing three residues of L-Rha (denoted as units A–C) and one residue each of D-GalA and D-GlcNAc (units D and E, respectively). The 1H and 13C NMR spectra of the DPS were analyzed using 2D homonuclear 1H,1H COSY, TOCSY, ROESY, and heteronuclear 1H,13C HSQC and HMBC experiments. Based on intra-residue H,H and H,C correlations and coupling constant values, spin systems were assigned to residues of A–E (Table 1), all being in the pyranosidic form. The spin system for unit E was distinguished from others
showed correlations between anomeric protons and linkage carbons and vice versa (data not shown). Positions of the O-acetyl groups were determined by a 1H,13C HSQC experiment on the OPS. As compared with the HSQC spectrum of the DPS, a smaller part of the H-2,C-2 cross-peak and a larger part of the H-3,C-3 cross-peak of unit D shifted from d 3.99/69.5 and 3.98/71.6 to 4.99/71.8 and 5.21/73.8, respectively. These displacements were due to a deshielding effect of the O-acetyl groups and indicated partial (by 30% and 60%) mono-O-acetylation of unit D at position 2 or 3, respectively. The O-acetylation pattern was confirmed by upfield shifts by 2.9 and 3.2 ppm of parts of the C-1 and C-3 signals (for GalA2OAc) and upfield shift by 2.0 and 1.7 ppm of parts of the C-2 and C-4 signals (for GalA3OAc) of unit D (b-effects of O-acetylation, see Fig. 1A).
A B C D E →2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→3)-α-D-GlcpNAc-(1→ 2/3 | OAc ~30/60 %
by a correlation between the proton at the nitrogen-bearing carbon (H-2) and the corresponding carbon (C-2) at d 4.04/53.2. Relatively large J1,2 coupling constant values of 7 Hz showed that units D and E are b-linked. The position of the C-5 signals at d 70.1–70.7 indicated that units A–C are a-linked (compare published data d 69.5 and 73.2 for a- and b-Rhap, respectively6). The signals for C-2 of units A, B, and C, C-3 of unit E and C-4 of unit D shifted significantly downfield, as compared with their positions in the corresponding non-substituted monosaccharides.6,7 These data demonstrated the linear character of the OPS chain and defined the glycosylation pattern in the O-unit. The ROESY spectrum of the DPS showed the following correlations between anomeric protons and protons at the linkage carbons: A H-1, B H-2; B H-1,C H-2; C H-1,D H-4; D H-1,E H-3, and E H-1,A H-2 at d 5.02/4.07, 5.10/4.04, 5.35/4.38, 5.31/3.95, 5.01/4.09, respectively. The monosaccharide sequence thus established was confirmed by a heteronuclear 1H,13C HMBC experiment, which
Therefore, the OPS of E. cloacae G2277 has the following structure:
Table 1 H and 13C NMR chemical shifts (d, ppm) of the DPS of E. cloacae G2277
1
Sugar residue
H-1 C-1
H-2 C-2
H-3 C-3
H-4 C-4
H-5 C-5
H-6 (6a, 6b) C-6
?2)-a-L-Rhap-(1? A ?2)-a-L-Rhap-(1? B ?2)-a-L-Rhap-(1? C ?4)-a-D-GalpA-(1? D ?3)-a-D-GlcpNAc-(1? E
5.02 100.2 5.10 101.9 5.35 100.5 5.31 102.1 5.01 96.5
4.09 76.3 4.07 79.5 4.04 79.4 3.99 69.5 4.04 53.2
3.91 70.6 3.89 71.2 3.90 71.3 3.98 71.6 3.95 81.0
3.56 73.1 3.45 73.4 3.41 73.5 4.38 77.5 3.73 71.6
3.75 70.7 3.73 70.5 3.80 70.1 4.16 72.7 4.04 72.9
1.31 18.0 1.28 17.9 1.24 17.8 175.5 3.81; 3.84 61.4
The chemical shifts for the N-acetyl group are dH 2.01 (Me), dC 23.1 (Me), and 175.7 (CO).
12
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Figure 2. Organization of the O-antigen gene cluster of E. cloacae G2277. Glycosyltransferase genes and O-antigen processing genes are shown in black and gray, respectively.
This structure is closely related to the O-polysaccharide structure of Plesiomonas shigelloides strains 22074 and 12254, which differs only in the linkage between GlcNAc and Rha A (1?3 vs 1?2).8 As the O-polysaccharides in question are GlcNAc-initiated (see below), the GlcNAc?Rha linkage forms upon polymerization of the preassembled O-units, which are thus identical in all these bacteria. The O-antigen gene cluster of E. cloacae strain G2277 was found between the housekeeping genes galF and gnd. Nine open reading frames (orfs) excluding galF and gnd were identified in the gene cluster, all of which have the same transcriptional direction from galF to gnd (Fig. 2). All orfs were assigned functions based on their similarity to related genes from the available databases and taking into account the E. cloacae G2277 antigen structure. Genes for synthesis of nucleotide precursors of common sugars, such as D-GlcNAc, are located outside the O-antigen gene cluster.9 Two genes, ugd and gla, responsible for the synthesis of UDP-D-GalA from UDP-D-Glc are absent from the O-antigen gene cluster too; this occurs in some other members of Enterobacteriaceae, where these genes map downstream gnd.10,11 Orf1-4 shared high-level identities to many known RmlB, D, A, and C proteins, and the rmlBDAC gene set responsible for the synthesis of dTDP12 L-Rha has been well characterized. Therefore, orf1-4 were identified as rmlBDAC and named accordingly. In GlcNAc-initiated O-antigens, wecA mediates transfer of the first sugar to a lipid carrier and is located outside the O-antigen gene cluster.13 Apart from wecA, four glycosyltransferase genes were expected for synthesis of the E. cloacae G2277 pentasaccharide O-unit. Orf7 showed 53.8% identity to Orf10 of Escherichia coli O116, whose O-antigen also has the a-D-GalpA-(1?3)-D-GlcpNAc linkage.14 Therefore, Orf7 was suggested to be glycosyltransferase for transfer of a-D-GalA to make the (1?3)-linkage to D-GlcpNAc, and the encoding gene was named wfiQ. Orf6 is a homolog of many rhamnosyltransferases and named wfiP. In the assembly of O-units that contain a sequence of three or more L-Rha residues, for example, in E. coli O13, O99, O129, O135, O139, and O150,15–18 one rhamnosyltransferase may be responsible for transfer of two L-Rha residues to make the same or a different a-L-Rhap?L-Rhap linkage. Therefore, it was suggested that WfiP catalyzes formation of two a-L-Rhap-(1?2)-LRhap linkages. As for the a-L-Rhap-(1?4)-D-GalpA linkage, this can be made with the help of WfiP too provided that this rhamnosyltransferase is trifunctional and able to transfer L-Rha to different acceptors. Alternatively, the missing glycosyltransferase may have a different origin compared to the genes in the O-antigen gene cluster and be located, for example, in a bacteriophage (recently, a similar situation has been reported in E. coli O12019). orf8 was identified as acyltransferase gene responsible for Oacetylation of GalA, and named wfiR. Orf5 and Orf9 are the only two proteins with predicted transmembrane segments, and hence the encoding genes are wzx (for O-unit flippase) and wzy (for O-antigen polymerase), respectively.20 1. Experimental 1.1. Bacterial strain and isolation of lipopolysaccharide E. cloacae G2277 is strain ATCC 7256, which was originally isolated from well water. The restriction fragment length polymor-
phism (RFLP) analysis of the O-antigen gene cluster showed that it has a distinct O pattern. Bacteria were grown to late log phase in 8 L of Luria-Bertani broth using a 10-L BIOSTAT C-10 fermentor (B. Braun Biotech Int., Germany) under constant aeration at 37 °C and pH 7.0. Bacterial cells were washed and dried as described.21 The lipopolysaccharide was isolated in a yield 17.5% from dried cells by the phenol–water method,22 the crude extract was dialyzed without separation of the layers and freed from nucleic acids and proteins by treatment with 50% aq CCl3CO2H to pH 2. The supernatant was dialyzed and lyophilized. 1.2. Isolation of OPS and preparation of DPS Delipidation of the lipopolysaccharide (200 mg) was performed with 2% aq HOAc at 100 °C until precipitation of lipid A. The precipitate was removed by centrifugation (13,000g, 20 min), and the supernatant was fractionated by GPC on a column (56 2.6 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer pH 5.5, monitored with a differential refractometer (Knauer, Germany). A high-molecular-mass OPS was obtained in a yield of 12.7% of the lipopolysaccharide mass. An OPS sample (11 mg) was treated with aq 12.5% ammonia at 37 °C for 16 h, ammonia was removed with a stream of air, the remaining solution was desalted on a column (90 2.5 cm) of TSK HW-40 (S) (Merck, Germany) in water and freeze-dried to give the DPS (9 mg). 1.3. Monosaccharide analysis The OPS was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were identified by GLC of the alditol acetates on an Agilent 7820A chromatograph (USA) equipped with an HP-5 column (0.32 mm 30 m) and a temperature program of 160 (1 min) to 290 °C at 7 °C min1. The absolute configurations of the monosaccharides were determined by GLC of the acetylated (S)-2-octyl glycosides as described.23 1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying from D2O and then examined as solution in 99.95% D2O. NMR spectra were recorded on a Bruker Avance II 600 spectrometer (Germany) at 25 °C and pH 9 using internal TSP (dH 0) and acetone (dC 31.45) as reference. 2D NMR spectra were obtained using standard Bruker software, and Bruker TopSpin 2.1 program was used to acquire and process the NMR data. A mixing time of 100 and 150 ms was used in TOCSY and ROESY experiments, respectively. 1.5. Sequencing and analysis of genes Chromosomal DNA was prepared as described.24 Long-range PCR was performed with the Expand Long Templated PCR system (Roche) using primers WL_35686(50 -AGG GCA GGT GAG CCG BAT CGT TGA GTT-30 ) and WL_35687(50 -TCA CGG TTA CGA CGA ATG GTG TCS TGG-30 ) based on the galF and gnd genes, respectively.25 The PCR cycles used were as follows: denaturation at 94 °C for 10 s, annealing at 60 °C for 45 s, and extension at 68 °C for 15 min. The PCR products were digested with DNase I, and the resulting DNA fragments were cloned into pGEM-T Easy to produce a bank as described.26 Sequencing was carried out using an ABI 3730 automated DNA sequencer (Applied Biosystems), and the sequence of the O-antigen gene clusters was analyzed as described.27
A. V. Perepelov et al. / Carbohydrate Research 387 (2014) 10–13
Acknowledgements This work was supported by the Russian Foundation for Basic Research (14-04-00663), the National Key Program for Infectious Diseases of China (2013ZX10004216-001-001); the National 973 program of China (2011CB504901); National Natural Science Foundation of China (NSFC) Program (31030002, 81171524); Research Project of Chinese Ministry of Education (No. 113015A). References 1. Dalben, M.; Varkulja, G.; Basso, M.; Krebs, V. L.; Gibelli, M. A.; van der Heijden, I.; Rossi, F.; Duboc, G.; Levin, A. S.; Costa, S. F. J. Hosp. Infect. 2008, 70, 7–14. 2. Fernández, A.; Pereira, M. J.; Suárez, J. M.; Poza, M.; Treviño, M.; Villalón, P.; Sáez-Nieto, J. A.; Regueiro, B. J.; Villanueva, R.; Bou, G. J. Clin. Microbiol. 2011, 49, 822–828. 3. Bush, K. Curr. Opin. Microbiol. 2010, 13, 558–564. 4. Gaston, M. A.; Bucher, C.; Pitt, T. L. J. Clin. Microbiol. 1983, 18, 1079–1083. 5. Moule, A. L.; Kuhl, P. M. D.; Galbraith, L.; Wilkinson, S. G. Carbohydr. Res. 1989, 186, 287–293. 6. Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175, 59–75. 7. Jansson, P.-E.; Kenne, L.; Widmalm, G. Carbohydr. Res. 1989, 188, 169–191. 8. Linnerborg, M.; Widmalm, G.; Weintraub, A.; Albert, M. J. Eur. J. Biochem. 1995, 231, 839–844. 9. Samuel, G.; Reeves, P. Carbohydr. Res. 2003, 338, 2503–2519.
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