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Structure and gene cluster of the O-antigen of Escherichia coli O133


Carbohydrate Research 430 (2016) 82–84

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Carbohydrate Research
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Structure and gene cluster of the O-antigen of Escherichia coli O133
Alexander S. Shashkov a, Yuanyuan Zhang b, Qiangzheng Sun c, Xi Guo b, Sof’ya N. Senchenkova a, Andrei V. Perepelov a,*, Yuriy A. Knirel a
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457 Tianjin, China State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, China
b c a

A R T I C L E

I N F O

A B S T R A C T

Article history: Received 3 April 2016 Accepted 29 April 2016 Available online 3 May 2016 Keywords: Escherichia coli O-speci?c polysaccharide structure O-antigen biosynthesis gene cluster Glycosyltransferase trifunctional

The O-speci?c polysaccharide (O-antigen) of Escherichia coli O133 was obtained by mild acid hydrolysis of the lipopolysaccharide of E. coli O133. The structure of the hexasaccharide repeating unit of the polysaccharide was elucidated by 1H and 13C NMR spectroscopy, including a two-dimensional 1H–1H ROESY experiment:

Functions of genes in the O-antigen gene cluster were putatively identi?ed by comparison with sequences in the available databases and, particularly, an encoded predicted multifunctional glycosyltransferase was assigned to three α-l-rhamnosidic linkages. ? 2016 Elsevier Ltd. All rights reserved.

Escherichia coli is the predominant facultative anaerobe of the colonic ?ora of many mammals, including humans, and has both commensal and pathogenic forms. The O-speci?c polysaccharide (OPS) or O-antigen, a part of the lipopolysaccharide in the outer membrane, consists of oligosaccharide repeats (O-units) containing up to eight residues from a broad range of common or rarely occurring sugars and their derivatives. The O-antigen is the most variable cell constituent with variations in the types of sugars present, their arrangement, and the linkages within and between O-units.1,2 The diversity of the O-antigen forms is mostly due to genetic variations in the O-antigen gene cluster,3 which in E. coli is located between the conserved galF and gnd genes. These are genes for synthesis of nucleotide sugar precursors, glycosyl transferase genes for the assembly of the O-unit on an undecaprenyl phosphate carrier, and O-unit processing genes, including those for ?ippase responsible for the translocation of the preassembled lipidlinked O-unit through the cytoplasmic membrane and O-antigen polymerase. Currently, more than 180 O-antigen forms have been recognized (http://www.ssi.dk/English/SSI%20Diagnostica/Products%20

* Corresponding author. N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation. Tel.: +7 499 1376148; fax: +7 499 1355328. E-mail address: perepel@ioc.ac.ru (A.V. Perepelov).
http://dx.doi.org/10.1016/j.carres.2016.04.028 0008-6215/? 2016 Elsevier Ltd. All rights reserved.

from%20SSI%20Diagnostica/Bacterial%20strains/E%20coli.aspx). Sequences of their O-antigen gene clusters have been reported4 and >140 OPS structures elucidated.2 In this work, we established the structure of the OPS of E. coli O133, which has not been studied earlier. The O133-antigen gene cluster was analyzed and functions of the genes were putatively assigned. Structure elucidation of the O-polysaccharide. Lipopolysaccharide was obtained from cells of E. coli O133 by phenol–water extraction5 and degraded with mild acid to give a high-molecular mass OPS, which was isolated by gel-permeation chromatography on Sephadex G-50. Sugar analysis by GLC of the acetylated alditols revealed rhamnose (Rha), glucose, and N-acetylglucosamine in the ratio ~ 3:1:0.6 (detector response). Further NMR spectroscopic studies showed that the OPS also contains galacturonic acid. Analyses of the content of the O-antigen gene cluster (see below) and glycosylation effects in the 13 C NMR spectrum 6 showed that Rha has the l con?guration, and Glc and GlcNAc have the d con?guration. The 1H and 13C NMR spectra of the OPS (Table 1) demonstrated a hexasaccharide O-unit. The 13C NMR spectrum showed signals for six anomeric carbons at δ 95.6–103.4, two HOCH2–C groups of Glc and GlcNAc at δ 61.4 and 61.8, three CH3–C groups of Rha at δ 17.8– 17.9, one HO2C–C group of GalA at δ 175.0, one nitrogen-bearing carbon of GlcNAc at δ 53.4, other sugar-ring carbons at δ 68.0– 80.9, and one N-acetyl group at δ 23.3 (CH3) and 175.7 (CO). The 1 H NMR spectrum contained, inter alia, signals for six anomeric

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Table 1 1 H and 13C NMR chemical shifts (δ, ppm) of the OPS from E. coli O133. The chemical shifts for the N-acetyl group are δH 1.99; δS 23.3 (CH3), and 175.7 (CO) Sugar residue →3)-α-d-GlcpNAc-(1→ A →3,4)-α-d-GalpA-(1→ B →2)-α-l-Rhap-(1→ C →2)-α-l-Rhap-(1→ D →3)-α-l-Rhap-(1→ E α-d-Glcp-(1→ F H(1) C(1) 5.03 95.6 5.37 102.1 5.62 98.9 5.30 101.1 4.97 103.4 5.15 97.9 H(2) C(2) 4.06 53.4 4.10 68.6 4.05 76.9 4.07 80.0 4.22 68.0 3.59 72.5 H(3) C(3) 3.99 80.9 4.07 77.7 3.86 72.0 3.88 71.0 3.83 76.5 3.64 74.2 H(4) C(4) 3.77 71.6 4.61 72.3 3.41 73.4 3.44 73.4 3.53 71.6 3.44 70.7 H(5) C(5) 4.04 72.9 4.22 72.9 3.72 70.4 3.79 70.4 3.76 70.5 3.96 73.2 H(6) (a, b) C(6) 3.79; 3.81 61.4 175.0 1.25 17.8 1.31 17.9 1.29 17.9 3.76; 3.83 61.8

Fig. 2. Organization of genes involved in biosynthesis of the O-antigen of E. coli O133. O-antigen processing genes are shown in black, genes for glycosyltransferases in dark gray, and genes for synthesis of sugar nucleotide precursors in light gray.

protons at δ 4.97–5.62, three CH3–C groups of Rha at δ 1.25–1.31, and one N-acetyl group at δ 1.99. The NMR spectra were assigned using a set of shift-correlated two-dimensional NMR experiments, including 1H–1H COSY, 1H–1H TOCSY, 1H–1H ROESY, and 1H–13C HSQC, and spin-systems for six sugar residues were revealed, which were designated units A–F (Table 1). GlcNAc (unit A) was distinguished by a correlation between proton at the nitrogen-bearing carbon H(2) and the corresponding carbon C(2) at δ 4.06/53.4. Rha residues (units C–E) were recognized by the presence in the spin systems of CH3 protons. GalA (unit B) was characterized by a low-?eld position at δ 4.61 and 4.22 and a narrow singlet shape of the H(4) and H(5) signals, respectively. The following cross-peaks between anomeric protons and protons at the linkage carbons were observed in the ROESY spectrum of the OPS (Fig. 1) and de?ned the monosaccharide sequence in the O-unit: A H(1)/E H(3), B H(1)/A H(3), C H(1)/B H(4), D H(1)/C H(2), E H(1)/D H(2), and F H(1)/B H(3) at δ 5.03/3.83, 5.37/3.99, 5.62/4.61, 5.30/ 4.05, 4.97/4.07, and 5.15/4.07, respectively. These correlations were in agreement with the 13C NMR chemical shifts of the signals for the linkage carbons (Table 2), which were shifted signi?cantly

down?eld by glycosylation7,8 as compared with those of the corresponding non-substituted monosaccharides. Additional intense interresidue cross-peaks were between H(1) of unit A and H(2) of unit E at δ 5.03/4.22, H(1) of unit F and H(4) of unit B at δ 5.15/ 4.61, H(1) of unit C and H(5) of unit D at δ 5.62/3.79, and H(1) of unit D and H(5) of unit E at δ 5.30/3.76. These correlations were characteristic of the corresponding linkages and further con?rmed the sequence of the monosaccharides. The data obtained indicated that the OPS of E. coli O133 has the following structure:

Fig. 1. Part of a two-dimensional ROESY spectrum the polysaccharides form E. coli O133. The corresponding parts of the 1H NMR spectrum are displayed along the axes. Arabic numerals refer to protons in sugar residues denoted by letters as shown in Table 1. Interresidue cross-peaks are annotated in bold face.

Characterization of the O-antigen gene cluster. Genes involved in the synthesis of the E. coli O133-antigen have been sequenced (GenBank accession No. AB812057).4 There are 8 genes between the conserved galF and gnd genes (orfs 1–10), all having the same transcriptional direction from galF to gnd, and 3 genes downstream of gnd (Fig. 2). orfs 1–4 were identi?ed as rmlB, rmlD, rmlA, and rmlC, respectively, based on their high level identity (81–98%) to known rml genes from a number of other E. coli strains. This set of genes is responsible for the synthesis of dTDP-l-rhamnose,3 the nucleotide precursor of l-Rha that is present in the O133 OPS. d-Glc and d-GlcNAc are common sugars in bacteria, and genes for synthesis of their precursors UDP-d-Glc and UDP-d-GlcNAc are located outside the O-antigen gene cluster. Typically of d-GalAcontaining E. coli O-antigens,4,9 ugd and gla genes that are involved in the synthesis of the nucleotide precursor of this monosaccharide, UDP-d-GalA,3 map downstream of gnd. They are followed by wzz gene encoding a protein that is responsible for the O-antigen chain length regulation.10 The O-antigen biosynthesis is initiated by the transfer of d-GlcNAc 1-phosphate from UDP-d-GlcNAs to the undecaprenyl phosphate carrier. wecA gene for the initiating transferase is located in the gene cluster for synthesis of the enterobacterial common antigen11 rather than in the O-antigen gene cluster. There are two putative genes for glycosyltransferases, orf6 and orf7. The predicted glycosyltransferase Orf7 shares 51% and 53% identity and 69% and 71% similarity with Orf10 and WcmT of E. coli O116 and O158, whose O-antigens include the α-d-GalpA-(1→3)-dGlcpNAc12 and α-d-GalpA-(1→3)-d-GalpNAc13 linkage, respectively. Therefore, it was proposed that Orf7 is responsible for the formation of the former linkage (B→A) in E. coli O133. Orf6 is 36% and 33% identical (52% and 50% similar) to WbgF (RfbF) of E. coli O13 and WfbY of E. coli O147, which are bifunctional α-l-rhamnosyltransferases.14 Both WbgF and WfbY form the α-l-Rhap-(1→2)-l-Rhap linkage and, in addition, the former makes the α-l-Rhap-(1→3)-l-Rhap linkage and the latter the α-l-Rhap(1 → 4)- d -Gal p A linkage. Therefore, we propose that Orf6 is a multifunctional α-l-rhamnosyltransferase that is responsible for the formation of two α-l-Rhap-(1→2)-l-Rhap linkages (E→D and D→C) and one α-l-Rhap-(1→4)-d-GalpA linkage (C→B). A homologous multifunctional rhamnosyltransferase WcnY that shares 31% identity and 48% similarity with Orf6 is predicted to make two

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α- l -Rha p -(1 → 2)- l -Rha p linkages and one α- l -Rha p -(1 → 3)- l Rhap linkage in E. coli O35, which possesses the α-l-Rhap-(1→2)α-l-Rhap-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap O-antigen fragment.15 No gene for adding the side-chain α-d-Glc residue was found between galF and gnd , and it is evident that an additional glycosyltransferase is present outside the O-antigen gene cluster. Most likely, it has a bacteriophage origin, as reported for a group of E. coli and Salmonella enterica O-antigens.16 Finally, the presence of genes for ?ippase Wzx ( orf5 ) and O-antigen polymerase Wzy (orf8), as well as the O-antigen chain length regulator Wzz, indicate that the OPS of E. coli O133 is synthesized by the Wzx/Wzy-dependent pathway.10 Therefore, the content of the O-antigen gene cluster is consistent with the O133 OPS structure established in this work. 1. Experimental 1.1. Bacterial strain and isolation of the lipopolysaccharide E. coli O133 (laboratory stock number G1272) was obtained from the Institute of Medical and Veterinary Science, Adelaide, Australia (IMVS). Bacteria were grown to late log phase in 8 L of Luria broth using a 10-L fermentor (BIOSTAT C-10, B. Braun Biotech International, Germany) under constant aeration at 37 °C and pH 7.0. Bacterial cells were washed and dried as described.17 The lipopolysaccharide was isolated in a yield of 5.5% from dried bacterial cells by the phenol–water method,5 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 at 4 °C. The supernatant was dialyzed and lyophilized. 1.2. Isolation of the O-speci?c polysaccharide Mild acid degradation of the LPS (50 mg) was performed with aq 2% HOAc at 100 °C until precipitation of lipid (1 h). The precipitate was removed by centrifugation (13,000 × g, 20 min), and the supernatant was fractionated by GPC on a column (56 × 2.6 cm) of Sephadex G-50 Super?ne (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer pH 4.5 monitored using a differential refractometer (Knauer, Germany) to give a polysaccharide (9 mg). 1.3. Monosaccharide analysis A polysaccharide sample (0.5 mg) was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were identi?ed by GLC of the alditol acetates on a Maestro (Agilent 7820) chromatograph

(Interlab, Russia) equipped with an HP-5 column (0.32 mm × 30 m) and a temperature program of 160 °C (1 min) to 290 °C at 7 °C min?1. 1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying from 99.9% D2O and then examined as solution in 99.95% D2O. NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer (Germany) at 30 °C for the polysaccharides and 24 °C for Leg5Ac7R, using sodium 3-trimethylsilylpropanoate-2,2,3,3-d4 (δH 0, δC ?1.6) as the internal 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. Acknowledgements This work was supported by the Russian Foundation for Basic Research (project 14-04-01709-a), the International Science & Technology Cooperation Program of China (2012DFG31680 and 2013DFR30640), the National Key Program for Infectious Diseases of China (2013ZX10004216-001-001 and 2013ZX10004221-003), the National Natural Science Foundation of China (NSFC) Program (31371259, 81271788, and 81471904), and the Research Project of Chinese Ministry of Education (NO. 113015A). References
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