Comparative analyses of secondary gene products of 3‐deoxy‐D‐manno‐oct‐2‐ulosonic acid transferases from Chlamydiaceae in Escherichia coli K‐12

Abstract

The waaA gene encoding the essential, lipopolysaccharide (LPS)-specific 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) transferase was inactivated in the chromosome of a heptosyltransferase I and II deficient Escherichia coli K-12 strain by insertion of gene expression cassettes encoding the waaA genes of Chlamydia trachomatis, Chlamydophila pneumoniae or Chlamydophila psittaci. The three chlamydial Kdo transferases were able to complement the knockout mutation without changing the growth or multiplication behaviour. The LPS of the mutants were serologically and structurally characterized in comparison to the LPS of the parent strain using compositional analyses, high performance anion exchange chromatography, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry and specific monoclonal antibodies. The data show that chlamydial Kdo transferases can replace in E. coli K-12 the host's Kdo transferase and retain the product specificities described in their natural background. In addition, we unequivocally proved that WaaA from C. psittaci transfers predominantly four Kdo residues to lipid A, forming a branched tetrasaccharide with the structure a-Kdo-(238)-[a-Kdo-(234)]-a-Kdo-(234)-a-Kdo.

WaaA of the Hyperthermophilic Bacterium Aquifex aeolicus Is a Monofunctional 3-Deoxy-d-manno-oct-2-ulosonic Acid Transferase Involved in Lipopolysaccharide Biosynthesis

“…aeolicus waaA Encodes a Monofunctional Kdo Transferase That Can Complement a ⌬waaA Mutation in E. coli-In vivo examination of Kdo transferases of different origin has usually been performed in E. coli to gain insight into the structure of the resulting LPS. In consideration of the fact that viability of wild-type E. coli cells is normally dependent upon the presence of a functional waaA copy (55), several strategies have been developed to reduce or even avoid LPS complexity stemming from co-expression of the heterologous and the host-specific Kdo transferase, such as cloning of Kdo transferases in an E. coli Re mutant (56,57), plasmid-encoded expression of WaaA in an E. coli waaA null mutant by displacement under nonpermissive conditions of a functional waaA ECO copy provided in trans (9,15,55), or homologous recombination of WaaA expression cassettes into the waaA locus of a recBC sbcBC E. coli strain (12,14). Here we have constructed the ⌬waaA suppressor strain KPM56 as a novel host for expression and in vivo analysis of exclusively plasmidborne Kdo transferases.…”

“…Thus, WaaA is capable of catalyzing the formation of two different glycosidic bonds, tolerating acceptor molecules with varying extents of acylation but strictly depending on the 4Ј-phosphate group of the tetraacyldisaccharide 1,4Ј-bisphosphate intermediate (6). In chlamydiae, however, which express an LPS composed of a Kdo trisaccharide with an unusual ␣-(238)-linkage between the second and a third Kdo residue (11), the Kdo transferases were shown to display at least trifunctional activity (12). The LPS of Chlamydophila psittaci consists of up to four Kdo residues of the structure ␣-Kdo-(234)-[␣-Kdo-(238)]-␣-Kdo-(234)-␣-Kdo (13), and heterologous expression of the waaA gene in E. coli was found to be sufficient for synthesis of the complete chlamydial Kdo structure (12).…”

“…In chlamydiae, however, which express an LPS composed of a Kdo trisaccharide with an unusual ␣-(238)-linkage between the second and a third Kdo residue (11), the Kdo transferases were shown to display at least trifunctional activity (12). The LPS of Chlamydophila psittaci consists of up to four Kdo residues of the structure ␣-Kdo-(234)-[␣-Kdo-(238)]-␣-Kdo-(234)-␣-Kdo (13), and heterologous expression of the waaA gene in E. coli was found to be sufficient for synthesis of the complete chlamydial Kdo structure (12). Finally, the Kdo transferases of Haemophilus influenzae and Bordetella pertussis were shown to be monofunctional (14,15), consistent with the presence of a single phosphorylated Kdo residue in their respective LPS (16,17).…”

Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis

“…The incorporation of Kdo is catalyzed by the membrane-embedded Kdo transferase WaaA, which transfers Kdo from the donor substrate CMP-Kdo to a 4′-phosphorylated lipid A precursor at the cytoplasmic face of the inner membrane. Remarkably, Kdo transferases of different bacteria are mono-, bi-, tri-, or even tetrafunctional (2,11), typically consistent with the number of Kdo residues present in the LPS core oligosaccharide.…”

3-Deoxy-d-manno-oct-2-ulosonic Acid (Kdo) Transferase (WaaA) and Kdo Kinase (KdkA) of Haemophilus influenzae Are Both Required to Complement a waaAKnockout Mutation of Escherichia coli

“…This strategy allowed us to characterize LPS that were synthesized in vivo by cloned Kdo transferases without interfering activity of the essential host-specific enzyme (18). Using this approach, we now show that the cloned monofunctional waaA from H. influenzae is not able to complement a knockout mutation within the corresponding gene of E. coli; however, cloning of both waaA and kdkA from a wild type or the I69 strain of H. influenzae, respectively, we are able to complement the mutation.…”