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Journal of Bacteriology, September 2005, p. 6242-6247, Vol. 187, No. 17
0021-9193/05/$08.00+0     doi:10.1128/JB.187.17.6242-6247.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

OpsX from Haemophilus influenzae Represents a Novel Type of Heptosyltransferase I in Lipopolysaccharide Biosynthesis

Sabine Gronow,^1* Werner Brabetz,^1, Buko Lindner,² and Helmut Brade¹

Divisions of Medical and Biochemical Microbiology,¹ Biophysics, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany²

Received 1 April 2005/ Accepted 26 May 2005

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The inner core region of the lipopolysaccharide (LPS) of Haemophilus influenzae is characterized by the presence of a phosphorylated 3-deoxy--D-manno-octulosonic acid (Kdo). In this study, we show that the heptosyltransferase I adding the first L-glycero-D-manno-heptose residue to this acceptor is encoded by the gene opsX, which differs in substrate specificity from the other heptosyltransferase I, known as WaaC.

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In gram-negative bacteria, the inner core region of the outer membrane macromolecule lipopolysaccharide (LPS) is highly conserved (Fig. 1). In most LPS structures elucidated so far, the backbone of the lipid A moiety is substituted at position 6' with a 24-linked disaccharide of 3-deoxy--D-manno-octulosonic acid (Kdo). During LPS biosynthesis in Enterobacteriaceae, two Kdo residues are attached to a tetraacylated lipid A precursor followed by the transfer of two nonhydroxylated fatty acids. The resulting molecule serves as an acceptor for the transfer of the first heptose residue to position 5 of the first Kdo residue, which is accomplished by the heptosyltransferase I WaaC (14, 31). Heptosyltransferase I genes in Salmonella enterica, Escherichia coli, Klebsiella pneumoniae (29), and Serratia marcescens (8, 9, 31), as well as genes in Pseudomonas aeruginosa (11), Neisseria spp. (32, 39), Campylobacter spp. (25), and Burkholderia cepacia (15), were characterized. They display a high degree of sequence identity to one another and were all termed waaC. In several species of Gammaproteobacteria, like Haemophilus influenzae, Vibrio cholerae, Pasteurella haemolytica, and Shewanella putrefaciens, as well as in Bordetella spp. which belong to the Betaproteobacteria, the Kdo which links the core region to the lipid A is substituted with a phosphate residue at position 4 (17, 36). All wild-type bacteria synthesizing a Kdo phosphate (Kdo-P) carry a heptose on position 5 of the Kdo. However, the H. influenzae strain I69 is a spontaneous mutant that contains only the Kdo-P (16) and thus represents the minimal LPS structure to enable survival in this species. The phosphorylation of Kdo is carried out by the enzyme Kdo kinase (37). Although the kinase is not essential for the growth of bacteria containing a complete LPS (20), we showed that it is essential in a deep-rough mutant of E. coli harboring the Kdo transferase of H. influenzae, which transfers only a single Kdo residue to lipid A. This mutant can survive only if the Kdo kinase is also expressed (3). The genome sequence of H. influenzae (12) lacks the gene waaC, but sequence similarity to the opsX gene of Xanthomonas campestris, which is considered to be involved in LPS biosynthesis, was found (20, 24). Accordingly, the gene from H. influenzae was also named opsX. Hood and coauthors proposed that opsX encodes a heptosyltransferase I homolog (20), and Cody and coauthors assigned this gene that function (10). However, no experimental data on the enzymatic activity of the OpsX protein are available. The core structure of the LPS of X. campestris contains a Kdo-P (A. Molinaro, personal communication).

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FIG. 1. General scheme of the most common LPS structure in the inner core region, with R1 representing the outer core and R2 representing a Kdo or phosphate residue. Hep, L-glycero-D-manno-heptose. Dotted lines indicate the defects leading to the LPS chemotypes Rd1, Rd2, and Re.

Heptosyltransferases are interesting targets for the design of novel antibiotics, since deep-rough mutants, defective in heptose addition, synthesize a truncated LPS which contains only the Kdo region and lipid A. The inhibition of heptosyltransferase I could allow the bacteria to survive, but the truncated LPS results in the destabilization of the outer membrane, which makes such bacteria more susceptible to the action of antibacterial peptides, complement, bile salts, and phagocytosis, rendering them vulnerable to the host defense systems. The OpsX protein is a novel type of heptosyltransferase I, and data collected about this enzyme will broaden our understanding of heptosyltransferases in general.

Cloning and sequence analysis of opsX from H. influenzae. The open reading frame HI0261 (12), which has high sequence similarity to the opsX gene of Xanthomonas campestris (24), was amplified from chromosomal DNA of H. influenzae DSM11121by PCR using the primer pair HIOPSX1 (5'-GATAAAGGATCCTCAATGCCACTTTTTACC-3', where the BamHI site is underlined and the start codon is in bold) and HIOPSX2 (5'-GTTTTGGTCGACAGATTATTTTTTCAAAATAACCC-3', where the SalI site is underlined and the stop codon is in bold). The amplicon of 1,082 bp was digested with BamHI and SalI, ligated into the corresponding sites of pCB20 (2), and transformed into E. coli XL1-Blue. The resultant plasmid was termed pJKB89. The opsX gene was confirmed by sequence analysis. For in vivo complementation analysis, the putative heptosyltransferase gene was cloned into plasmid pSU2718 for comparison with similar constructs harboring heptosyltransferase I (WaaC) from E. coli (4). The 1,074-bp BamHI-SalI fragment pJKB89 was ligated between the corresponding restriction sites of plasmid pSU2718 (28) to give pJKB90.

In vivo complementation studies of opsX from H. influenzae. To elucidate the role of opsX from H. influenzae, the LPS chemotypes of a number of recombinant E. coli strains were compared (Fig. 2). Recombinant E. coli strains (all bacterial strains and plasmids used are listed in Table 1) were cultivated for 24 h at 30°C in 8 liters of LB medium supplemented with the appropriate antibiotics. The cells were dried as described previously (3), and the LPSs were extracted with phenol-chloroform-light petroleum ether (13). Analyses of glucosamine (GlcN), Kdo, and phosphate were performed as described previously (23).

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FIG. 2. Characterization of LPS from recombinant E. coli strains that express WaaC or OpsX as heptosyltransferase I by using monoclonal antibodies. LPS was isolated from E. coli strains WBB01 (lane 1), WBB01/pWSB31 (lane 2), WBB01/pJKB90 (lane 3), WBB22 (lane 4), WBB22/pWSB31 (lane 5), and WBB22/pJKB90 (lane 6) and separated by SDS-polyacrylamide gel electrophoresis. Visualization was achieved by staining with alkaline silver nitrate (A) or after blotting (B through E) and development with MAb A20 (recognizing a terminal Kdo residue) (B), MAb S42-21 (recognizing Kdo-4-P) (C), MAb S36-20 (recognizing Rd2-type LPS [heptose-Kdo2-lipid A]) (D), or rabbit antiserum K236 (obtained after immunization with Rd2-type LPS) (E).

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TABLE 1. Strains and plasmids used in this study

Strain WBB01, lacking both heptosyltransferases WaaC and WaaF (3), served as a control and as a recipient for plasmids pWSB31, encoding WaaC from E. coli (4), and pJKB90, encoding OpsX from H. influenzae. Strain WBB22, lacking both heptosyltransferases, carrying the Kdo transferase (WaaA) and the Kdo kinase (KdkA) from H. influenzae (3), was used as a control and as a recipient for plasmids pWSB31 and pJKB90.

Since both recipient E. coli strains display a deep-rough LPS phenotype, WBB01 synthesizing two Kdo residues and WBB22 synthesizing a Kdo-P, they are suitable test systems to compare the substrate specificities of heptosyltransferases from different origins. LPS was prepared from all six strains, WBB01, WBB01/pWSB31, WBB01/pJKB90, WBB22, WBB22/pWSB31, and WBB22/pJKB90, and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels, followed by staining with alkaline silver nitrate (35) or, after blotting, with specific monoclonal antibodies (MAbs). As expected, all samples showed electrophoretic mobilities similar to that of Re LPS from WBB01 (Fig. 2A). When Western blots were developed with MAbs of known specificities (all MAbs and the antiserum used in this study are listed in Table 2) (5), the following information was obtained (Fig. 2B through E). LPS of reference strain WBB01 reacted only with MAb A20, recognizing a terminal Kdo residue (Fig. 2B, lane 1) (6), whereas LPS of strain WBB22 reacted only with MAb S42-21, which recognizes a Kdo-4-phosphate (Fig. 2C, lane 4) (30). When WaaC was expressed in both strains, LPS from WBB01/pWSB31 showed reactivity with MAb S36-20, specific for Salmonella enterica serovar Minnesota LPS of the Rd₂ chemotype (33), carrying two Kdo residues and one heptose (Fig. 2D, lane 2), whereas LPS of strain WBB22/pWSB31 did not react with S36-20 (Fig. 2D, lane 5). However, a positive reaction was obtained with polyclonal rabbit antiserum K236, which was obtained after immunization with S. enterica serovar Minnesota Rd₂ LPS (34) and has a broader specificity than MAb S36-20, indicating that WaaC can transfer a heptose residue also to Kdo-P (Fig. 2E, lane 5). The expression of OpsX from H. influenzae in strain WBB01 resulted in an LPS with the same reaction profile as that of the parent strain (Fig. 2, lane 3). Thus, no heptose was transferred to the Re-type LPS acceptor by OpsX. In contrast, LPS of strain WBB22/pJKB90 showed strong reactivity with K236, demonstrating that OpsX is a heptosyltransferase I that uses a phosphorylated Kdo as an acceptor (Fig. 2E, lane 6). The reactivities of LPSs from WBB22/pWSB31 and WBB22/pJKB90 with MAb S42-21 showed that complementation was not complete, especially in the case of WBB22/pWSB31 (Fig. 2C, lane 5).

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TABLE 2. Antisera and MAb used in this study

Similar results were obtained when S. enterica strain SA1377, carrying the waaC630 mutation and expressing Re-LPS, was complemented with plasmids pWSB31 and pJKB90. The parent strain presented a typical deep-rough LPS phenotype on SDS-polyacrylamide gels. Transformation with plasmid pWSB31 led to complementation to a smooth LPS with a ladder pattern; however, transformation with plasmid pJKB90 did not alter the LPS phenotype of the parent strain, indicating that the parental LPS is not an acceptor for OpsX.

Mass spectrometric analysis of recombinant LPSs. LPSs were isolated from WBB01, WBB22, and the mutant strains WBB01/pWSB31, WBB01/pJKB90, WBB22/pWSB31, and WBB22/pJKB90 as described previously (13). To reduce the structural complexity due to the heterogeneity of the acylation pattern, the LPSs were O-deacylated by mild hydrazinolysis (19) before being subjected to high-resolution electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (7 T Apex II; Bruker Daltonics, Billerica, MA). Mass spectra were acquired in the negative-ion mode using standard experimental sequences as provided by the manufacturer. Samples were dissolved at a concentration of 10 ng/µl in a mixture of 2-propanol, water, and triethylamine, which was added stepwise not to exceed pH 9 (50:50:0.001 [vol/vol/vol]), and sprayed at a flow rate of 2 µl/min. Capillary entrance voltage was set to 3.8 kV and dry gas temperature to 150°C. All spectra were charge deconvoluted, and the mass numbers given refer to the neutral monoisotopic peaks. The mass spectrum of LPS obtained from strain WBB01/pWSB31, expressing WaaC (Fig. 3A), exhibited an abundant molecular ion at 1,584.65 amu consistent with O-deacylated LPS carrying an additional heptose (calculated mass, 1,584.647 amu), proving that heptosyltransferase WaaC effectively transfers a heptose on Kdo-Kdo-lipid A. Interestingly, there is also a mass peak of minor intensity at 1,364.59 amu corresponding to a molecule lacking one Kdo. Whether this molecular species is an artifact produced during the isolation procedure or a natural product has to be elucidated. The similarity between the mass spectra obtained from the LPS of WBB01/pJKB90, expressing OpsX (Fig. 3B), and that obtained from strain WBB01 (data not shown) was in agreement with the immunoblot analysis and demonstrated that OpsX could not use the Kdo disaccharide as the substrate for the transfer of a heptose. Both spectra contained prominent molecular ions at 1,392.59 and 952.47 amu corresponding to the expected LPS consisting of two residues of Kdo, two molecules of GlcN, two hydroxytetradecanoic acid molecules, and two phosphates (calculated monoisotopic mass [m_cal] = 1,392.584 amu), and O-deacylated lipid A comprised two molecules of GlcN, two hydroxytetradecanoic acid molecules, and two phosphates, (m_cal = 952.467 amu), respectively. The mass spectrum of strain WBB22/pWSB31 (Fig. 3C) exhibited the O-deacylated lipid A mass peak at 952.47 amu, a prominent O-deacylated LPS peak at 1,252.50 amu representing one Kdo-P, two molecules of GlcN, two hydroxytetradecanoic acid molecules, and two phosphates (m_cal = 1,252.492 amu), and a mass peak at 1,444.58 amu corresponding to LPS with an additional heptose. This demonstrates that WaaC can use Kdo-P and Kdo disaccharide as a substrate with preference of the latter (Fig. 3A). However, the mass spectrum of O-deacylated LPS from strain WBB22/pJKB90 revealed a predominant peak at 1,444.57 amu (Fig. 3D) whereas the peak at 1,252.50 amu was of only minor intensity, indicating that OspX effectively transferred heptose to Kdo-P. The mass spectrum of LPS obtained from strain WBB22 comprised only peaks at 952.47 amu and 1,252.50 amu (data not shown).

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FIG. 3. Negative-ion high-resolution electrospray ionization-Fourier transform ion cyclotron resonance mass spectra of O-deacylated LPS from E. coli strains WBB01/pWSB31 (A), WBB01/pJKB90 (B), WBB22/pWSB31 (C), and WBB22/pJKB90 (D). Spectra are charge deconvoluted, and the mass numbers given refer to the neutral monoisotopic peaks. +22 and + 38 indicate sodium and potassium adduct ions, respectively.

In vitro characterization of OpsX in comparison to WaaC. Corynebacterium glutamicum R163 was transformed with construct pJKB89, which harbors the opsX gene from H. influenzae on a shuttle vector for expression of the enzyme. WaaC from E. coli was obtained from C. glutamicum R163/pJKB10 (14). Cell extracts of both recombinant strains were prepared and subjected to in vitro assays to test the activity of the two heptosyltransferases as described previously (14). The heptose donor ADP-heptose was provided by a partially purified cell extract of the heptosyltransferase-deficient E. coli strain WBB06 (4) as described previously (14). In most assays, purified LPS from E. coli F515 (Re type) or H. influenzae I69 (Re type with Kdo-P) was used as the lipid acceptor at a concentration of 100 µM. Separation on thin-layer chromatography (TLC) plates, blotting, and immunological identification of the reaction products were performed as described previously (14), by using MAb S36-20 (33) or rabbit antiserum K236 (34).

To elucidate the structural requirements of the enzymes, LPS preparations from E. coli Re and H. influenzae I69 were used as lipid acceptors. Rd₂-type LPS isolated from S. enterica serovar Minnesota served as a control (Fig. 4, lane 1). As expected, WaaC converted the Re-type LPS from E. coli to a product that reacted with MAb S36-20 (Fig. 4, lane 4), confirming the transfer of a single heptose residue to form an Rd₂-type LPS. In contrast, incubation of E. coli Re-type LPS with OpsX did not give a product reacting in immunoblots (Fig. 4, lane 5). When LPS from H. influenzae I69 was used as an acceptor, OpsX yielded a strong band which stained with MAb S36-20 (Fig. 4, lane 3) whereas WaaC led to a product which was not detected with MAb S36-20 (Fig. 4, lane 2) but was seen when developed with the polyclonal antiserum K236 (data not shown). Obviously, small amounts of product were synthesized, and the affinity of MAb S36-20 was too low for its detection. These results showed that OpsX had a strict acceptor specificity for phosphorylated Kdo, whereas WaaC was able to transfer heptose to 24-linked Kdo disaccharide and Kdo-P, however, with preference for the former.

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FIG. 4. In vitro activity of heptosyltransferases WaaC and OpsX on different acceptors. Reaction products were separated by TLC and immunologically detected after blotting. Rd2-type LPS (lane 1) of S. enterica serovar Minnesota strain R4 served as a control, and either Re-type LPS from H. influenzae I69 was used as an acceptor for WaaC (lane 2) or OpsX (lane 3) or Re-type LPS from E. coli F515 was used as an acceptor for WaaC (lane 4) or OpsX (lane 5). Bands were visualized with MAb S36-20 against Rd2 LPS.

Coupled reactions (so-called one-pot reactions, since the reagents for different enzymatic reactions are all mixed in one reaction vial) were carried out as described previously (14) using the synthetic tetraacylated and bisphosphorylated lipid A precursor of E. coli compound 406 (21) and Kdo transferase of either E. coli isolated from C. glutamicum R163/pJKB16 (2) or Kdo transferase of H. influenzae isolated from C. glutamicum R163/pCB23 (2) in addition to a heptosyltransferase. Precursor 406 was used as an acceptor for Kdo transferase from either E. coli (Fig. 5, lanes 3 and 4) or H. influenzae (lanes 1 and 2) in combination with WaaC from E. coli (lane 2 and 4) or OpsX from H. influenzae (lane 1 and 3) as heptosyltransferase. The combination of both E. coli enzymes yielded a product reacting strongly with MAb S36-20 and antiserum K236 (Fig. 5A and B, lane 4), whereas that of both H. influenzae enzymes converted the acceptor to a product migrating faster than those of E. coli, which showed strong reactivity with K236 (Fig. 5B, lane 1) but only faint reactivity with MAb S36-20 (Fig. 5A, lane 1). The missing side chain Kdo residue was obviously responsible for this weak reaction. When Kdo transferase of E. coli was combined with OpsX, no heptose-containing product was obtained, independent of whether MAb or antiserum was used (Fig. 5, lane 3). The faint signal shown in Fig. 5B was due to a cross-reactivity of the antiserum with the intermediate reaction product containing two Kdo residues. The combination of Kdo transferase of H. influenzae with WaaC yielded a small amount of product reacting with K236 (Fig. 5B, lane 2). This last result indicated that a lipid A acceptor with only one Kdo was not suitable for WaaC, whereas OpsX was capable of transferring a heptose on such a molecule. To confirm the in vitro results obtained by immunostaining, the material from the coupled reaction of Kdo transferase and OpsX from H. influenzae with the synthetic lipid A precursor 406 (compare Fig. 5, lane 1) was subjected to mass spectrometry analysis (data not shown). Due to the complex composition of the mixture (proteins and degradation products), the mass spectrum comprised several mass peaks which were not identified. However, peaks for the substrate compound 406 and the reaction products Kdo-406 and Hep-Kdo-406 were detected as predicted by the immunoblot (Fig. 5, lane 1).

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FIG. 5. Coupled in vitro assays using different Kdo transferases and heptosyltransferases on synthetic acceptor 406. Reaction products were separated by TLC and immunologically detected after blotting. Synthetic lipid A precursor 406 was incubated with extracts from R163/pCB23 and R163/pJKB89 (lane 1), R163/pCB23 and R163/pJKB10 (lane 2), R163/pJKB16 and R163/pJKB89 (lane 3), and R163/pJKB16 and R163/pJKB10 (lane 4). Bands were visualized with MAb S36-20 (A) or rabbit antiserum K236 (B). Kdo-T, Kdo transferase; Hep-T, heptosyltransferase; H.i., H. influenzae; E.c., E. coli.

Taken together, our results are in accordance with the fact that OpsX shows the highest sequence identity to WaaQ (heptosyltransferase III) among the heptosyltransferases. In E. coli, WaaQ also requires a phosphorylated acceptor, since it incorporates the third heptose residue only if the first heptose is phosphorylated (38). Thus, it differs considerably from WaaC and WaaF (heptosyltransferases I and II, respectively) since they use nonphosphorylated acceptors. The data published for Bordetella pertussis are quite different. Although this species contains, as does H. influenzae, a single phosphorylated Kdo in its LPS core (18), the degree of sequence identity of its heptosyltransferase I to WaaC from E. coli is significantly higher than that to OpsX from H. influenzae (44% versus 16%, respectively, by using ClustalW). Furthermore, S. enterica strain SA1377 could be complemented with waaC from B. pertussis to express smooth LPS (22), which is not possible with opsX from H. influenzae. Allen and coworkers failed to construct a knockout of the waaC gene in B. pertussis. The mutant with the shortest LPS had a defect in waaF (1). This result is also in contrast to H. influenzae where mutant I69 was shown to harbor only a phosphorylated Kdo attached to the lipid A (16). Investigations of the heptosyltransferase I of B. pertussis will be interesting in learning whether this enzyme is equivalent to WaaC of E. coli or if it displays a kind of hybrid function of WaaC and OpsX. Further comparative investigations about heptosyltransferases are necessary to complete the characterization of the different types of enzymes.

 
We thank S. Kusomoto, Osaka, Japan, for providing synthetic lipid A precursor 406, P. Kosma, Vienna, Austria, for Kdo, and I. J. von Cube, H. Lüthje, and V. Susott for technical assistance.

We thank the Deutsche Forschungsgemeinschaft for financial support (SFB 470/A1 to S.G.).

 
* Corresponding author. Mailing address: Division of Medical and Biochemical Microbiology, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany. Phone: 49 4537 188469. Fax: 49 4537 188419. E-mail: sgronow{at}fz-borstel.de.

Present address: Biotype AG, Moritzburger Weg 67, D-01109 Dresden, Germany.

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Journal of Bacteriology, September 2005, p. 6242-6247, Vol. 187, No. 17
0021-9193/05/$08.00+0     doi:10.1128/JB.187.17.6242-6247.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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