Construction of shotgun cloning gene bank.

Chromosomal DNAs were prepared as previously described (4). The O-antigen gene cluster was amplified by PCR (MasterAmp extra-long PCR kit; Epicenter, Madison, Wis.) with primers based on galF and hisI as previously described (26). A total of 10 PCR products were pooled to minimize the effect of PCR errors. After gel purification, the PCR products were sheared by nebulizers (Invitrogen) and cloned into the pCR4Blunt-TOPO vector to generate a bank (280 colonies) according to the instruction manual of the TOPO shotgun cloning kit (Invitrogen).

Sequencing and analysis.

Sequencing was carried out using an ABI 3730 automated DNA sequencer. Sequence data were assembled using Phred/Phrap package from the Genome Center of the University of Washington. The program Artemis was used for gene annotation (24). BLAST and PSI-BLAST (1) were used for searching databases including the GenBank and Pfam protein motif databases (5). Sequence alignment and comparison were done using the program Clustal W (28).

Deletion of wzy and wzz genes from E. coli O86:B7 and O86:H2.

The wzy and wzz genes from the two O86 strains were each replaced by the chloramphenicol acetyltransferase (CAT) gene using the RED recombination system of phage lambda (9, 33). The CAT gene was PCR amplified from plasmid pKK232-8 using primers binding to the 5′ and 3′ ends of the gene, and each primer also carried 40 bp flanking the target genes. The PCR products were transformed into O86 carrying pKD20, and chloramphenicol-resistant transformants were selected after induction of the RED genes according to the protocol described by Datsenko and Wanner (9). PCR assays using primers specific to the CAT gene and DNA flanking the wzy and wzz genes were carried out to confirm the deletion.

Cloning of wzy and wzz genes from E. coli O86:B7 and O86:H2.

To complement the mutants, wzz genes from both O86 strains and the wzy gene from O86:H2 were PCR amplified and cloned into the NcoI and BamHI sites of pTRC99A to make the plasmids pTR101, pTR102, and pTR103. Expression of the cloned genes was induced by 0.3 mM IPTG (isopropyl-β-d-thiogalactopyranoside).

Analysis of LPS.

E. coli strains were grown for 16 h at 37°C in LB containing appropriate antibiotics. Small-scale samples were prepared from whole-cell lysate by the proteinase K method as described by Hitchcock and Brown (12). After electrophoresis on a 12.5% polyacrylamide gel, LPS was detected by a silver staining method as described before (30).

Serum resistance assay.

Serum resistance assays were performed with pooled normal human serum, serum plus EGTA, and heat-inactivated serum. The concentration of serum in assays was 80%. Metal chelation inactivation of the classical complement pathway was achieved by adding EGTA and MgCl₂ to final concentrations of 10 and 5 mM, respectively. Heat inactivation was performed by incubating the serum at 56°C for 30 min. A bacterial culture that had been allowed to grow overnight was diluted 1:100 in LB and grown to mid-log phase (~3 × 10⁸ cells per ml). The bacteria were then diluted 1:5 in pooled normal human serum, serum plus EGTA, or heat-inactivated serum and incubated at 37°C. After 0, 1, 2, or 3 h, survival of the strains was tested by plating an aliquot on LB agar plates containing the appropriate antibiotic. If the viable counts dropped to less than 1% of the initial value, the strain was termed sensitive, and if more than 90% survived after 3 h, the strain was considered serum resistant.

(i) Nucleotide sugar biosynthesis genes.

The O86:B7 O unit contains five sugar residues (Fig. ​(Fig.1):1): one fucose (Fuc) and two each of N-acetylgalactosamine (GalNAc) and galactose (Gal). As genes for the synthesis of the UDP-Gal are located elsewhere (22), only genes for UDP-GalNAc and GDP-L-Fuc were expected in the O-antigen gene cluster.

The deduced protein sequence of orf1 showed 57 and 22% identity to identified Gne proteins of Yersinia enterocolitica O:8 and Pseudomonas aeruginosa O6, respectively (6, 14). Gne catalyzes the conversion of UDP-GlcNAc to UDP-GalNAc, so orf1 was identified as gne for the biosynthesis of UDP-GalNAc in E. coli O86:B7.

Orf3 to Orf7 shared 95, 99, 94, 85, and 92% identity to the putative GDP-L-Fuc pathway enzymes Gmd, Fcl, Gmm, ManC, and ManB, respectively, of E. coli O128 (25). GDP-L-Fuc is synthesized from GDP-mannose by two enzymes: GDP-mannose 4,6-dehydratase (Gmd) and GDP-L-Fuc synthetase (Fcl). ManA (phosphomannose isomerase), ManB (phosohomannomutase), and ManC (GDP-mannose pyrophosphorylase) are three additional enzymes needed in the synthesis of GDP-mannose from fructose 6-phosphate. ManA is also a member of the mannose metabolism pathway in E. coli, and the gene is located elsewhere on the chromosome (18). Gmm (GDP-mannose mannosyl hydrolase) has been suggested to regulate cell wall biosynthesis by influencing the concentration of GDP-mannose in the cell (11).

(ii) Sugar transferase genes.

Five glycosyltransferases are required to transfer all five sugar residues in O86:B7 (Fig. ​(Fig.1).1). The first glycosyltransferase (WecA) to transfer UDP-GalNAc is located outside the O-antigen gene cluster (2); four glycosyltransferase genes were expected in the E. coli O86:B7 O-antigen gene cluster.

Glycosyltransferases can be classified into 78 distinct sequence-based protein families as described by Campbell et al. (8) (http://afmb.cnrs-mrs.fr/CAZY/). WbwH belongs to glycosyltransferase family 1 (PF00534, E = 2.0 × e⁻¹⁶) and also shows 68 and 62% similarity to putative glycosyltransferases of Yersinia enterocolitica and Edwardsiella ictaluri, respectively (Table ​(Table2).2). WbwI belongs to glycosyltransferase family 6 (PF03414, E = 1.0×e⁻¹¹); it also shares high similarity with WbgM from E. coli O55, which has been proposed to encode α-1,3-galactosyltransferase (29). Therefore, we tentatively designated WbwI asan α-1,3-galactosyltransferase that makes the Gal-α-1,3-Gal linkage. WbwJ belongs to glycosyltransferase family 2 (PF00535, E = 5.0 × e⁻¹⁶). WbwK belongs to glycosyltransferase family 11 (PF01531, E = 1.0 × e⁻¹⁵), which only consists of several fucosyltransferases from different organisms (http://afmb.cnrs-mrs.fr/CAZY). It also shares 32% identity with the identified fucosyltransferase of E. coli O128 (25). It is likely that wbwK encodes the fucosyltransferase that links a fucose to the O-antigen unit. Consequently, WbwI, WbwJ, WbwK, and WbwH are the four additional glycosyltransferases involved in the E. coli O86:B7 sugar transfer.

(iii) O-antigen processing genes.

Wzx and Wzy are the only two membrane proteins with more than eight transmembrane segments encoded in the O-antigen gene cluster. The protein encoded by orf8 has 12 transmembrane segments. When searching the Pfam database, Orf8 belongs to polysaccharide biosynthesis protein family (E = 0.0037), members of which are involved in the production of polysaccharide, including RfbX of the O-antigen biosynthesis operon. Therefore, orf8 was named wzx.

Orf10 has 10 transmembrane segments with a very large periplasmic loop made up of 75 amino acids, which is a characteristic of Wzy. Although it showed low similarity with other putative Wzy proteins, it is one of only two proteins with many transmembrane segments. We tentatively designated it wzy. The function of the wzy gene was further confirmed by comparison of LPS phenotypes between the wild type and the wzy mutant strain in which the wzy gene was replaced by a CAT gene. While the wild-type O86:B7 strain (see Fig. ​Fig.5A,5A, lane 3) produced smooth LPS, which consists of lipid A, core, and modal distributed O antigen, the wzy mutant O86:B7 (see Fig. ​Fig.5A,5A, lane 4) produced semirough LPS which contained lipid A-core and only one O unit. Therefore, we believed that orf10 was the wzy gene and named it accordingly.

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FIG. 5.

Effect of wzy mutation on O86:B7 and O86:H2 LPS phenotypes. (A) Silver-stained SDS-PAGE analysis of LPS. Lanes: 1, G102 (E. coli O86:H2); 2, G123 (G102 missing wzy gene); 3, G101 (E. coli O86:B7); 4, G121 (G101 missing wzy gene); 5, H104 (G121 with plasmid pTR-103). wt, wild type. (B) Scheme for hypothesis of existence of βwzy in H2.

Orf15 belongs to the WzzB family (COG 3765, E = 2.0 × e⁻⁷³), which consists of chain length determinant proteins involved in lipopolysaccharide biosynthesis. It also shares 98% amino acid identity with the identified Wzz protein from Shigella flexneri (16). Analyzed with the TMpred program (13), Orf15 exhibited a topology of two transmembrane segments located at the amino terminus and carboxy terminus and a large hydrophilic loop in the periplasm, which is characteristic of Wzz proteins. Accordingly, orf15 was believed to encode Wzz protein and named wzz.

LPS of E. coli O86:B7 exhibited a very short O-antigen modality.

Comparison of the Wzz proteins from O86:B7 and O86:H2 showed that they are 90% identical. Although quite similar, they impart significantly different modal chain lengths to the LPSs in their wild-type strains. LPS of O86:B7 (Fig. ​(Fig.3,3, lane 1) exhibited a modal distribution of LPS bands with relative short O-antigen chains attached to lipid A-core. Most LPS molecules contain 1 to 4 O units; the longest LPS molecule consists of 11 O units. This is a novel observation, since all previously reported wild-type smooth LPSs in E. coli contain relatively long O-antigen chains. The LPS from the O86:H2 strain (Fig. ​(Fig.3,3, lane 3) exhibits intermediate modal distribution of LPS bands (10 to 18 O units). In an attempt to understand the genetic basis of O-antigen chain length regulation, we transferred a cloned wzz gene from O86:H2 into the O86:B7 wild-type strain. The recombinant strain produced bimodal distribution of LPS (Fig. ​(Fig.3,3, lane 2), with modal chain length imparted by the two Wzz proteins. It is further proof that two Wzz proteins can coexist in one E. coli strain and function independently.

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FIG. 3.

SDS-PAGE analysis of E. coli O86 LPS. Lanes: 1, G101 (E. coli O86:B7); 2, H102 (G101 with plasmid pTR-102); 3, G102 (E. coli O86:H2); 4, G124 (G102 missing wzz gene); 5, H105 (G124 with plasmid pTR-101); 6, G122 (G101 missing wzz gene); 7, H101 (G122 with plasmid pTR-101); 8, H103 (G122 with plasmid pTR-102).

To better understand this new type of wzz gene from O86:B7, it was replaced with the CAT gene to make the mutant strain. The LPS profile showed that the wzz mutant strain exhibited nonmodal distribution (Fig. ​(Fig.3,3, lane 6), and much longer LPS molecules composed of more than 15 O units were detected. After the construct containing the wzz gene from O86:B7 was transferred to the mutant via electroporation, the complemented strain (Fig. ​(Fig.3,3, lane 7) regained modal distribution of LPS similar to that of the wild type. This confirmed that the O-antigen chain length was regulated by the Wzz protein.

To correlate the chain length regulation with different wzz genes, plasmids pTR101 and pTR102 expressing respective wzz genes from O86:B7 and O86:H2 were transferred to wzz mutants O86:H2 and O86:B7. Compared to the O86:H2 wild-type strain, the wzz mutant O86:H2 strain (Fig. ​(Fig.3,3, lane 4) showed a nonmodal distribution of the LPS phenotype. As expected, the wzz mutant O86:H2 strain containing pTR101 (Fig. ​(Fig.3,3, lane 5) produced a modal chain length LPS similar to that of the O86:B7 wild type, only short LPS molecules were detected. At the same time, wzz mutant O86:B7 containing pTR102 (Fig. ​(Fig.3,3, lane 8) exhibited modal distribution of LPS quite similar to that of the O86:H2 wild-type strain. In summary, when the wzz genes from O86:B7 and O86:H2 were expressed in wzz mutant O86 stains, the length of the complemented strain corresponded well to the chain lengths of the donor strain.

P.G.W. acknowledges support from an endowed professorship of the Ohio Eminent Scholar program on macromolecular structure and function in the Department of Biochemistry at The Ohio State University and financial support (R01 AI44040) from the National Institutes of Health.