Priyanka D. Abeyrathne

Functional Characterization of WaaL, a Ligase Associated with Linking O-Antigen Polysaccharide to the Core of Pseudomonas aeruginosa Lipopolysaccharide

The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of D-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.

WaaL of Pseudomonas aeruginosa utilizes ATP in in vitro ligation of O antigen onto lipid A-core.

waaL has been implicated as the gene that encodes the O‐antigen ligase. To date, in vitro biochemical evidence to prove that WaaL possesses ligase activity has been lacking due to the difficulty of purifying WaaL and unavailability of substrates. Here we describe the purification of WaaL, a membrane protein with 11 potential transmembrane segments from Pseudomonas aeruginosa, and the development of an in vitro O‐antigen ligase assay. WaaL was expressed in a P. aeruginosa wbpL knockout strain, which is defective in its initial glycosyltransferase for O‐antigen biosynthesis. This approach allowed the purification of WaaL without contaminating O‐antigen‐undecaprenol‐phosphate (Und‐P) molecules. Purified WaaL resolved to a monomer (35 kDa) and a dimer (70 kDa) band in SDS‐PAGE. The substrates for the O‐antigen ligase assay, O‐antigen‐Und‐P and lipid A‐core were prepared from a waaL mutant. ATP at 2–4 mM is optimum for the O‐ligase activity, and ATP hydrolysis by WaaL follows Michaelis–Menten kinetics. Site‐directed mutagenesis analysis indicated that the periplasmic loop region of WaaL is important for ligase activity. A waaL mutant of P. aeruginosa could not be cross‐complemented by waaL of Escherichia coli, which suggested that each of these proteins has specificity for its cognate core oligosaccharide.

Oligomeric structure and functional characterization of the urea transporter from Actinobacillus pleuropneumoniae.

Urea transporters (UTs) facilitate urea permeation across cell membranes in prokaryotes and eukaryotes. Bacteria use urea as a means to survive in acidic environments and/or as a nitrogen source. The UT from Actinobacillus pleuropneumoniae, ApUT, the pathogen that causes porcine pleurisy and pneumonia, was expressed in Escherichia coli and purified. Analysis of the recombinant protein using cross-linking and blue-native gel electrophoresis established that ApUT is a dimer in detergent solution. Purified protein was reconstituted into proteoliposomes and urea efflux was measured by stopped-flow fluorometry to determine the urea transport kinetics of ApUT. The measured urea flux was saturable, could be inhibited by phloretin, and was not affected by pH. Two-dimensional crystals of the biologically active ApUT show that it is also dimeric in a lipid membrane and provide the first structural information on a member of the UT family.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

Biochemical Characterization of MsbA from Pseudomonas aeruginosa

Lipopolysaccharide of Pseudomonas aeruginosa is a major constituent of the outer membrane, and it is composed of three distinct regions: lipid A, core oligosaccharide, and O antigen. Lipid A and core oligosaccharides (OS) are synthesized and assembled at the cytoplasmic side of the inner membrane and then translocated to the periplasmic side of the membrane where lipid A-core becomes the acceptor of the O antigens. Here we show that MsbA encoded by pA4997 of the P. aeruginosa genome is a member of the ABC transporter family, but this protein has distinctive features when compared with other MsbA proteins. msbA is an essential gene in this organism since mutation in this gene is lethal to the bacterium. Disruption of the chromosomal msbA was achieved only when a functional copy of the gene was provided in trans. msbA from Escherichiacoli (msbAEc) could not cross complement the msbA merodiploid cells of P. aeruginosa. MsbA was expressed and purified, and the kinetic of its ATPase activity is vastly different than that of MsbAEc. The activity of MsbA could be selectively stimulated by different truncated versions of core OS of P. aeruginosa LPS. Specifically, phosphate substituents in the lipid A-core are important for stimulating ATPase activity of MsbA. Expression of MsbAEc but not MsbAPa conferred resistance to erythromycin in P. aeruginosa.

Coexistence of Two Distinct Versions of O-Antigen Polymerase, Wzy-Alpha and Wzy-Beta, in Pseudomonas aeruginosa Serogroup O2 and Their Contributions to Cell Surface Diversity

Assembly of B-band lipopolysaccharide (LPS) in Pseudomonas aeruginosa follows a Wzy-dependent pathway, requiring the O-antigen polymerase Wzy and other proteins. The peptide sequences of the wzy(alpha) product from strains of serotypes O2, O5, and O16 are identical, but the O units in O5 are alpha-glycosidically linked, while those in O2 and O16 are beta-linked. We hypothesized that a derivative of the D3 bacteriophage wzy(beta) is present in the chromosomes of O2 and O16 and that this gene is responsible for the beta-linkage. By a combination of PCR and primer walking, wzy(beta) genes of both serotypes have been amplified and cloned. They are identical but share only 87.42% sequence identity with their xenolog in D3. A chromosomal knockout mutant of O16 wzy(beta) was made, and it produces semirough LPS devoid of B-band O antigen. The cloned wzy(beta) is capable of complementing the O16 wzy(beta) mutant, as well as cross-complementing a wzy(alpha) knockout mutant. However, in the latter case, the restored O antigen was beta-linked. Using reverse transcription-PCR, we showed that wzy(alpha) was transcribed in O2 and O16 strains and was functional, since both of these genes could complement the wzy(alpha) mutant of O5. With the coexistence of wzy(alpha) and wzy(beta) in O2 and O16 and the B-band O polysaccharides in these being beta-linked, we hypothesized that iap, an inhibitor of the alpha-polymerase gene, must be present in these serotypes. Indeed, through PCR, TOPO-cloning, and nucleotide-sequencing results, we verified the presence of iap in both O2 and O16 serotypes.