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.

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).

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.