3-Deoxy-d-manno-oct-2-ulosonic Acid (Kdo) Transferase of Legionella pneumophila Transfers Two Kdo Residues to a Structurally Different Lipid A Precursor of Escherichia coli

Abstract

The 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) transferase gene of Legionella pneumophila was cloned and sequenced. Despite remarkable structural differences in lipid A, the gene complemented a corresponding Escherichia coli mutant and was shown to encode a bifunctional enzyme which transferred 2 Kdo residues to a lipid A acceptor of E. coli.

Lipopolysaccharides (LPS) of gram-negative bacteria consist of a lipid component, termed lipid A, and a covalently bound polysaccharide which is subdivided into a lipid A proximal core oligosaccharide and, in smooth-type bacteria, a lipid A distal O antigen (21). The LPS of Legionella pneumophila, the causative agent of Legionnaires' disease (8), shows unique structural characteristics such as unusually long fatty acids attached to the lipid A and extremely hydrophobic sugar residues within the outer core oligosaccharide and the O antigen (14–16, 26). Due to these peculiarities of the LPS, L. pneumophila possesses a very hydrophobic cell surface which may contribute to the spread and virulence of this microorganism (19, 26).

The inner core oligosaccharide of L. pneumophila LPS is characterized by a 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) disaccharide [α-Kdo-(2→4)-α-Kdo-(2→6)] linked to lipid A which is conserved within many gram-negative bacteria and is essential for microbial growth (4, 21, 25). Remarkably, as shown for Escherichia coli (3), Acinetobacter spp. (6), and Chlamydiaceae (5, 18, 20), there is only one enzyme necessary for the transfer of all Kdo residues, even in different linkages. Therefore, these transferases are multifunctional.

The carbohydrate backbone of the lipid A of L. pneumophila is composed of a biphosphorylated β(1→6)-linked disaccharide of 2,3-diamino-2,3-dideoxy-d-glucose substituting for glucosamine, which is present in the majority of structurally characterized LPS (21). Consequently, all fatty acids are amide bound to positions 2, 2′, 3, and 3′ in the lipid A of L. pneumophila (26). To date, Kdo transferases from bacteria containing this structural element have not been studied.

Bacterial strains and growth conditions.

L. pneumophila serogroup 1 (strain Philadelphia 1, ATCC 33152) was grown at 37°C on buffered charcoal yeast extract agar (Oxoid). E. coli strains and Corynebacterium glutamicum R163 (17) were cultivated at 37 or 30°C, respectively, in 2× YT (23) supplemented with the appropriate antibiotics (20 mg of kanamycin/liter and 100 mg of ampicillin/liter, the latter applied only to E. coli).

Cloning and sequence analysis of the waaA gene of L. pneumophila.

The three degenerate primers +880-1 (5′-GTNCCNMGNCAYNYNGAAA-3′), +880-2 (5′-GTNCCNMGNCAYNYNGAAG-3′), and −1010 (5′-TCNARNRRRTTRTGNCCNC-3′) were designed based on two stretches of identical amino acids between known WaaA sequences and were used in PCR with chromosomal DNA from L. pneumophila as described previously (6). A corresponding DNA fragment of 146 bp was obtained, blunt ended, and ligated into the SrfI site of pCR-ScriptAmpSC(+) (Stratagene) to produce plasmid pLPO1. The plasmid was transformed into E. coli XL1BlueMRF′Kan^r (Stratagene), and its insert was sequenced. The DNA fragment was then used as a probe in Southern experiments to clone a 3.5-kb StuI fragment from the chromosome of L. pneumophila into the SmaI site of pMBL19 (22). The corresponding plasmid was termed pLPO28, and both strands of the DNA insert were sequenced.

Computer analysis (GeneWorks; Intelligenetics) of the nucleotide sequence of the cloned insert revealed two complete open reading frames, oriented in opposite directions (Fig. 1). These open reading frames were homologous to waaA and djlA, respectively, as shown by a TBLASTN (1) search at the National Center for Biotechnology Information. The Kdo transferase was encoded by a DNA segment of 1,260 bp starting with an AUG and terminating with a UAG codon. Its deduced amino acid sequence was aligned with all known complete WaaA proteins by using the programs ClustalX 1.8 (24) and GeneDoc 2.5 (K. B. Nicholas and H. B. Nicholas, Jr., 1997 [http://www.cris.com/∼Ketchup/genedoc.shtml]) (Fig. 2). All sequences shared in total 5% identical and 9.8% similar (according to the Dayhoff-PAM250 matrix) amino acids, respectively. More than 50% of the conserved residues matched into a segment of approximately 140 amino acids within the C-terminal half of all Kdo transferases which defines a glycosyltransferase group 1 domain (Pfam database accession number PF00534) (2).

FIG. 1.

Cloned and sequenced DNA fragment of L. pneumophila serogroup 1. The positions (nucleotide numbers in parentheses) of some restriction sites are shown. waaA, Kdo transferase gene; djlA, dnaJ-like A gene; orf1′ and orf2′, putative open reading frames which showed no homology to known genes.

FIG. 2.

Comparative analysis of the amino acid sequences of WaaA proteins. On top, the consensus of all aligned complete amino acid sequences of Kdo transferases is diagrammed to scale (with the N terminus on the left). Thick segments correspond to more-conserved regions of the protein. The dashed line represents an insertion of 41 amino acids which is present only in the N terminus of WaaA from Rickettsia prowazekii. Vertical lines mark the positions of highly conserved amino acids. A glycosyltransferase group 1 domain (Pfam database accession number PF00534) is shaded, and the amino acid sequence alignment of the central part of this region is shown in detail at the bottom. Dashes represent gaps introduced to optimize the alignment. Amino acids which are identical for at least 75% of all sequences are shaded. Horizontal arrows indicate the regions which were used to design the totally degenerate primers. Abbreviations of species names (with GenBank accession numbers of the corresponding DNA sequences given in parentheses) are as follows: Lpn, L. pneumophila (AJ011775); Eco, E. coli (M60670); Sma, Serratia marcescens (U52844); Kpn, Klebsiella pneumoniae (AF146532); Hin, H. influenzae (U32748); Aba, Acinetobacter baumannii (Z96925); Aha, Acinetobacter haemolyticus (Z96927); Bbr, Bordetella bronchiseptica (AJ007747); Bpe, Bordetella pertussis (X90711); Chs, Chlamydophila psittaci (X69476); Cha, Chlamydophila abortus (AF111203); Chp, Chlamydophila pneumoniae (Z31593); Ctr, Chlamydia trachomatis (Z22659); Hpy, Helicobacter pylori (AE000604); Aeo, Aquifex aeolicus (AE000684); Rpr, R. prowazekii (AJ235269).

Subcloning of waaA.

The waaA gene was amplified from pLPO28 using Pfu DNA polymerase and the specific oligonucleotides LP1 (5′-TGTGTTTGGATCCTGTTTATGC-3′ [the BamHI site is underlined and the start codon is boldfaced]) and LP2 (5′-GAATTTGTCGACAAGCTAATATCCCAA-3′ [the SalI site is underlined, and the stop codon is boldfaced]), cut with BamHI and SalI, and ligated between the corresponding restriction sites of the E. coli-C. glutamicum expression vector pCB20 (6) to produce plasmid pLPO10. The kanamycin resistance gene of pLPO10 was further replaced by an ampicillin gene. For this purpose a 1,280-bp SmaI-PstI fragment from pLPO10, a 1,190-bp BglI-PstI fragment from pBTac2 (11), and a 2,042-bp BglI-BalI fragment from pBR322 (10) were ligated to produce plasmid pLPO11.

Complementation of an E. coli waaA mutant.

E. coli CJB26 (4), which harbors a knockout insertion of a kanamycin resistance gene within the essential chromosomal waaA and is complemented by an intact copy of the gene encoded on a temperature-sensitive plasmid, was transformed with pLPO11 and selected at 44°C with ampicillin and kanamycin. One positive clone, termed E. coli WBB54, was confirmed by Southern hybridization and PCR (data not shown) and showed the same growth behavior and similar LPS on a silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (6) as strain CJB26 (data not shown).

Activity of the cloned Kdo transferase.

pLPO10 was transformed into the gram-positive bacterium C. glutamicum R163 (17), which made it possible to study the enzymatic activity of the cloned transferase without interference by host cell enzymes (6, 18). Cell extracts were prepared from recombinant strains, and the Kdo transferase assay was performed under standard conditions, as described previously (6). Briefly, the enzyme reaction mixture (20 μl) contained Tris-HCl (50 mM; pH 7.5), MgCl₂ (10 mM), Triton X-100 (3.2 mM), Kdo (2 mM), CTP (5 mM), the synthetic biphosphorylated tetraacyl lipid A precursor 406 (0.1 mM) (13), CMP-Kdo synthetase (1.67 pkat; prepared from C. glutamicum R163/pJKB14 [6]), and cell extracts (40 μg of protein). The in vitro test mixtures were incubated for 30 to 60 min at 37°C, and reactions were stopped by spotting 5 μl onto silica-60 thin-layer chromatography (TLC) plates (Merck). TLC plates were developed with a solvent consisting of chloroform-pyridine-88% formic acid-water in a ratio of 30:70:16:10 (by volume). Radioactive [4′-³²P]-labeled 406 was synthesized from the monophosphorylated tetraacyl lipid A precursor 405 as described previously (9) by using lipid A 4′-kinase prepared from E. coli BLR(DE3)/pLysS/pJK2 (12). The specific activity was adjusted with unlabeled 406 to approximately 15,000 to 20,000 cpm nmol⁻¹. Radioactive products were detected and quantified with a PhosphorImager and ImageQuant software (Molecular Dynamics).

The cloned Kdo transferase from L. pneumophila was able to transfer up to 2 Kdo residues to compound 406 (Fig. 3A). The enzyme activity clearly depended on the presence of Kdo and CMP-Kdo synthetase (Fig. 3A, lanes 5 to 7). CTP could be provided, but in limiting amounts, from cell-free lysates of recombinant C. glutamicum (Fig. 3A, lane 8). A complete reaction mixture with a cell extract from C. glutamicum R163/pCB20 (6) was used as a negative control, and no conversion of [4′-³²P]-radiolabeled 406 was observed in this case (data not shown). The reaction products of the cloned WaaA had the same R_f values as isolated, radioactively labeled standards of Kdo-406 and Kdo₂-406 (Fig. 3A) (reaction products of in vitro assays with the recombinant Kdo transferases of E. coli and Haemophilus influenzae were purified from TLC plates as described in reference 9). In addition, the transfer of Kdo could be confirmed by Western blot analysis of the same TLC plate (6, 18) by using the Kdo-specific monoclonal antibody A20 (7) (Fig. 3B). Furthermore, this technique clearly allowed us to distinguish between a nonspecific impurity present in the radioactively labeled acceptor preparation (see Fig. 3A, lanes 1, 6, and 7) and Kdo-406, which also differed slightly in its R_f value. In addition, 5-μl samples were withdrawn from a reaction mixture (40 μl) at different time points and reactions were stopped with 10 μl of ice-cold ethanol. Aliquots (12 μl) of the inactivated samples were then separated by TLC, and reaction products were detected (Fig. 4A) and quantified (Fig. 4B) as described above. A continuous increase in Kdo₂-406, the major product of the enzyme reaction, was observed. The specific activity for its formation within the corynebacterial cell extract was calculated as 1.1 nmol min⁻¹ mg of protein⁻¹ at 37°C. In addition, Kdo-406 was formed in small amounts during the first 5 min until a constant level was achieved. A reaction product with 1 Kdo residue was also reported by Belunis et al. (3) as a minor in vitro by-product of the bifunctional WaaA of E. coli, and similar reactivities were obtained for a cell extract of a recombinant C. glutamicum strain which expressed the cloned enzyme of E. coli (reference 6 and data not shown).

FIG. 3.

In vitro activity of the cloned Kdo transferase of L. pneumophila. The enzyme assay was performed with cell extracts of C. glutamicum/pLPO10 as described in the text. Detection of the radioactively labeled substrates and reaction products was performed after TLC with a PhosphorImager (A) or after blotting from the TLC plate onto a nitrocellulose membrane by immunostaining with the Kdo-specific monoclonal antibody A20 (B). The positions of the detected compounds are indicated on the right. Lane 1, isolated compound 406; lane 2, mixture of isolated compounds 406 and Kdo-406; lane 3, isolated Kdo-406; lane 4, isolated Kdo₂-406; lane 5, complete reaction mixture with recombinant WaaA of L. pneumophila; lane 6, assay without Kdo; lane 7, assay without CMP-Kdo synthetase; lane 8, assay without CTP.

FIG. 4.

Time course of the enzyme reaction with the cloned Kdo transferase of L. pneumophila. Kinetic experiments were performed as described in the text. The reaction products were separated by TLC and were detected (A) and quantified (B) by using a PhosphorImager and ImageQuant software (Molecular Dynamics). The specific enzyme activity for the formation of Kdo₂-406 within cell extracts of recombinant C. glutamicum strains was determined as 1.1 nmol · min⁻¹ · mg of protein⁻¹. The positions of the detected compounds in panel A are indicated on the right. Symbols in panel B: ○, compound 406; ▴, compound Kdo-406; ●, compound Kdo₂-406.

In conclusion, waaA of L. pneumophila encodes a bifunctional Kdo transferase that is able to complement a corresponding E. coli mutant and to transfer 2 Kdo residues to compound 406. The overall enzyme reaction did not depend on specific structural prerequisites of the lipid A of L. pneumophila (26). Neither 2,3-diamino-2,3-dideoxy-d-glucose, which is present in lipid A of L. pneumophila and substitutes for glucosamine in compound 406, nor 2,3-dihydroxy-12-methyl tridecanoic acid, 2,3-dihydroxytetradecanoic acid, and 3-hydroxyoctadecanoic acid, which were identified in amide linkages in lipid A of L. pneumophila and substitute for 3-hydroxytetradecanoic acid in compound 406, were required for the enzyme activity. However, differences in the relative reactivity of the enzyme between the applied substrate and the native lipid A acceptor of L. pneumophila, which is not yet available, may exist. The results presented in this study further support the hypothesis that, in general, Kdo transferases share a common acceptor motif for catalytic activity, which is located in the carbohydrate backbone of lipid A and comprises at least a β(1→6)-linked disaccharide of a gluco-configured aminosugar with a phosphate group in position 4′ of the nonreducing sugar moiety (3, 6, 9, 18). This basic structure could serve as a starting point for the rational design of new chemotherapeutics which may inhibit all LPS-specific Kdo transferases.

Nucleotide sequence accession number.

The sequence of the cloned chromosomal DNA fragment from L. pneumophila has been submitted to the EMBL nucleotide database and is available under accession number AJ011775.