Joseph S. Lam

Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di‐rhamnolipid biosynthesis

Pseudomonas aeruginosa is an opportunistic pathogen capable of producing a wide variety of virulence factors, including extracellular rhamnolipids and lipopolysaccharide. Rhamnolipids are tenso‐active glycolipids containing one (mono‐rhamnolipid) or two (di‐rhamnolipid) l‐rhamnose molecules. Rhamnosyltransferase 1 (RhlAB) catalyses the synthesis of mono‐rhamnolipid from dTDP‐l‐rhamnose and β‐hydroxydecanoyl‐β‐hydroxydecanoate, whereas di‐rhamnolipid is produced from mono‐rhamnolipid and dTDP‐l‐rhamnose. We report here the molecular characterization of rhlC, a gene encoding the rhamnosyltransferase involved in di‐rhamnolipid (l‐rhamnose‐l‐rhamnose‐β‐hydroxydecanoyl‐β‐hydroxydecanoate) production in P. aeruginosa. RhlC is a protein consisting of 325 amino acids with a molecular mass of 35.9 kDa. It contains consensus motifs that are found in other glycosyltransferases involved in the transfer of l‐rhamnose to nascent polymer chains. To verify the biological function of RhlC, a chromosomal mutant, RTII‐2, was generated by insertional mutagenesis and allelic replacement. This mutant was unable to produce di‐rhamnolipid, whereas mono‐rhamnolipid was unaffected. In contrast, a null rhlA mutant (PAO1‐rhlA) was incapable of producing both mono‐ and di‐rhamnolipid. Complementation of mutant RTII‐2 with plasmid pRTII‐26 containing rhlC restored the level of di‐rhamnolipid production in the recombinant to a level similar to that of the wild‐type strain PAO1. The rhlC gene was located in an operon with an upstream gene (PA1131) of unknown function. A σ54‐type promoter for the PA1131–rhlC operon was identified, and a single transcriptional start site was mapped. Expression of the PA1131–rhlC operon was dependent on the P. aeruginosa rhl quorum‐sensing system, and a well‐conserved lux box was identified in the promoter region. The genetic regulation of rhlC by RpoN and RhlR was in agreement with the observed increasing RhlC rhamnosyltransferase activity during the stationary phase of growth. This is the first report of a rhamnosyltransferase gene responsible for the biosynthesis of di‐rhamnolipid.

Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production

Exopolysaccharides contribute significantly to attachment and biofilm formation in the opportunisitc pathogen Pseudomonas aeruginosa. The Psl polysaccharide, which is synthesized by the polysaccharide synthesis locus (psl), is required for biofilm formation in non‐mucoid strains that do not rely on alginate as the principal biofilm polysaccharide. In‐frame deletion and complementation studies of individual psl genes revealed that 11 psl genes, pslACDEFGHIJKL, are required for Psl production and surface attachment. We also present the first structural analysis of the psl‐dependent polysaccharide, which consists of a repeating pentasaccharide containing d‐mannose, d‐glucose and l‐rhamnose:

Review: Lipopolysaccharide biosynthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes serious nosocomial infections, and an important virulence factor produced by this organism is lipopolysaccharide (LPS). This review summarizes knowledge about biosynthesis of all three structural domains of LPS — lipid A, core oligosaccharide, and O polysaccharides. In addition, based on similarities with other bacterial species, this review proposes new hypothetical pathways for unstudied steps in the biosynthesis of P. aeruginosa LPS. Lipid A biosynthesis is discussed in relation to Escherichia coli and Salmonella, and the biosyntheses of core sugar precursors and core oligosaccharide are summarised. Pseudomonas aeruginosa attaches a Common Polysaccharide Antigen and O-Specific Antigen polysaccharides to lipid A-core. Both forms of O polysaccharide are discussed with respect to their independent synthesis mechanisms. Recent advances in understanding O-polysaccharide biosynthesis since the last major review on this subject, published nearly a decade ago, are highlighted. Since P. aeruginosa O polysaccharides contain unusual sugars, sugar-nucleotide biosynthesis pathways are reviewed in detail. Knowledge derived from detailed studies in the O5, O6 and O11 serotypes is applied to predict biosynthesis pathways of sugars in poorly-studied serotypes, especially O1, O4, and O13/O14. Although further work is required, a full understanding of LPS biosynthesis in P. aeruginosa is almost within reach.

The structural basis of the catalytic mechanism and regulation of glucose-1-phosphate thymidylyltransferase (RmlA)

The synthesis of deoxy‐thymidine di‐phosphate (dTDP)–L‐rhamnose, an important component of the cell wall of many microorganisms, is a target for therapeutic intervention. The first enzyme in the dTDP–L‐rhamnose biosynthetic pathway is glucose‐1‐phosphate thymidylyltransferase (RmlA). RmlA is inhibited by dTDP–L‐rhamnose thereby regulating L‐rhamnose production in bacteria. The structure of Pseudomonas aeruginosa RmlA has been solved to 1.66 Å resolution. RmlA is a homotetramer, with the monomer consisting of three functional subdomains. The sugar binding and dimerization subdomains are unique to RmlA‐like enzymes. The sequence of the core subdomain is found not only in sugar nucleotidyltransferases but also in other nucleotidyltransferases. The structures of five distinct enzyme substrate–product complexes reveal the enzyme mechanism that involves precise positioning of the nucleophile and activation of the electrophile. All the key residues are within the core subdomain, suggesting that the basic mechanism is found in many nucleotidyltransferases. The dTDP–L‐rhamnose complex identifies how the protein is controlled by its natural inhibitor. This work provides a platform for the design of novel drugs against pathogenic bacteria.

Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B‐band lipopolysaccharide gene cluster

Pseudomonas aeruginosa co‐expresses A‐band lipopolysaccharide (LPS), a homopolymer of rhamnose, and B‐band LPS, a heteropolymer with a repeating unit of 2–5 sugars which is the serotype‐specific antigen. The gene clusters for A‐ and B‐band biosynthesis in P. aeruginosa O5 (strain PAO1) have been cloned previously. Here we report the DNA sequence and molecular analysis of the B‐band O‐antigen biosynthetic cluster. Sixteen open reading frames (ORFs) thought to be involved in synthesis of the O5 O antigen were identified, including wzz (rol), wzy (rfc), and wbpA–wbpN. A further 3 ORFs not thought to be involved with LPS synthesis were identified (hisH, hisF, and uvrB). Most of the wbp genes are found only in serotypes O2, O5, O16, O18, and O20, which form a chemically and structurally related O‐antigen serogroup. In contrast, wbpM and wbpN are common to all 20 serotypes of P. aeruginosa. Although wbpM is not serogroup‐specific, knockout mutations confirmed it is necessary for O5 O‐antigen biosynthesis. A novel insertion sequence, IS1209, is present at the junction between the serogroup‐specific and non‐specific regions. We have predicted the functions of the proteins encoded in the wbp cluster based on their homologies to those in the databases, and provide a proposed pathway of P. aeruginosa O5 O‐antigen biosynthesis.

Absolute Quantitation of Bacterial Biofilm Adhesion and Viscoelasticity by Microbead Force Spectroscopy

Bacterial biofilms are the most prevalent mode of bacterial growth in nature. Adhesive and viscoelastic properties of bacteria play important roles at different stages of biofilm development. Following irreversible attachment of bacterial cells onto a surface, a biofilm can grow in which its matrix viscoelasticity helps to maintain structural integrity, determine stress resistance, and control ease of dispersion. In this study, a novel application of force spectroscopy was developed to characterize the surface adhesion and viscoelasticity of bacterial cells in biofilms. By performing microbead force spectroscopy with a closed-loop atomic force microscope, we accurately quantified these properties over a defined contact area. Using the model gram-negative bacterium Pseudomonas aeruginosa, we observed that the adhesive and viscoelastic properties of an isogenic lipopolysaccharide mutant wapR biofilm were significantly different from those measured for the wild-type strain PAO1 biofilm. Moreover, biofilm maturation in either strain also led to prominent changes in adhesion and viscoelasticity. To minimize variability in force measurements resulting from experimental parameter changes, we developed standardized conditions for microbead force spectroscopy to enable meaningful comparison of data obtained in different experiments. Force plots measured under standard conditions showed that the adhesive pressures of PAO1 and wapR early biofilms were 34 +/- 15 Pa and 332 +/- 47 Pa, respectively, whereas those of PAO1 and wapR mature biofilms were 19 +/- 7 Pa and 80 +/- 22 Pa, respectively. Fitting of creep data to a Voigt Standard Linear Solid viscoelasticity model revealed that the instantaneous and delayed elastic moduli in P. aeruginosa were drastically reduced by lipopolysaccharide deficiency and biofilm maturation, whereas viscosity was decreased only for biofilm maturation. In conclusion, we have introduced a direct biophysical method for simultaneously quantifying adhesion and viscoelasticity in bacterial biofilms under native conditions. This method could prove valuable for elucidating the contribution of genetic backgrounds, growth conditions, and environmental stresses to microbial community physiology.

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.

Involvement of the rml locus in core oligosaccharide and O polysaccharide assembly in Pseudomonas aeruginosa

L-Rhamnose (L-Rha) is a component of the lipopolysaccharide (LPS) core, several O antigen polysaccharides, and the cell surface surfactant rhamnolipid of Pseudomonas aeruginosa. In this study, four contiguous genes (rmlBDAC) responsible for the synthesis of dTDP-L-Rha in P. aeruginosa have been cloned and characterized. Non-polar chromosomal rmlC mutants were generated in P. aeruginosa strains PAO1 (serotype O5) and PAK (serotype O6) and LPS extracted from the mutants was analysed by SDS-PAGE and Western immunoblotting. rmlC mutants of both serotype O5 and serotype O6 synthesized a truncated core region which was unable to act as an attachment point for either A-band or B-band O antigen. A rmd rmlC PAO1 double mutant (deficient in biosynthesis of both D-Rha and L-Rha) was constructed to facilitate structural analysis of the mutant core region. This strain has an incomplete core oligosaccharide region and does not produce A-band O antigen. These results provide the genetic and structural evidence that L-Rha is the receptor on the P. aeruginosa LPS core for the attachment of O polysaccharides. This is the first report of a genetically defined mutation that affects the synthesis of a single sugar in the core oligosaccharide region of P. aeruginosa LPS, and provides further insight into the mechanisms of LPS biosynthesis and assembly in this bacterium.

Three rhamnosyltransferases responsible for assembly of the A-band D-rhamnan polysaccharide in Pseudomonas aeruginosa: a fourth transferase, WbpL, is required for the initiation of both A-band and B-band lipopolysaccharide synthesis

The Pseudomonas aeruginosa A‐band lipopolysaccharide (LPS) molecule has an O‐polysaccharide region composed of trisaccharide repeat units of α1 → 2, α1 → 3, α1 → 3 linked D‐rhamnose (Rha). The A‐band polysaccharide is assembled by the α‐D‐rhamnosyltransferases, WbpX, WbpY and WbpZ. WbpZ probably transfers the first Rha residue onto the A‐band accepting molecule, while WbpY and WbpX subsequently transfer two α1 → 3 linked Rha residues and one α1 → 2 linked Rha respectively. The last two transferases are predicted to be processive, alternating in their activities to complete the A‐band polymer. The genes coding for these transferases were identified at the 3′ end of the A‐band biosynthetic cluster. Two additional genes, psecoA and uvrD, border the 3′ end of the cluster and are predicted to encode a co‐enzyme A transferase and a DNA helicase II enzyme respectively. Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A‐band LPS, while B‐band synthesis is unaffected. WbpL, a transferase encoded within the B‐band biosynthetic cluster, was previously proposed to initiate B‐band biosynthesis through the addition of Fuc2NAc (2‐acetamido‐2,6‐dideoxy‐D‐galactose) to undecaprenol phosphate (Und‐P). In this study, chromosomal wbpL mutants were generated that did not express A band or B band, indicating that WbpL initiates the synthesis of both LPS molecules. Cross‐complementation experiments using WbpL and its homologue, Escherichia coli WecA, demonstrates that WbpL is bifunctional, initiating B‐band synthesis with a Fuc2NAc residue and A‐band synthesis with either a GlcNAc (N‐acetylglucosamine) or GalNAc (N‐acetylgalactosamine) residue. These data indicate that A‐band polysaccharide assembly requires four glycosyltransferases, one of which is necessary for initiating both A‐band and B‐band LPS synthesis.

Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide.

This study reports the organization of the wbp gene cluster and characterization of a number of genes that are essential for B-band O antigen biosynthesis in the clinically prevalent Pseudomonas aeruginosa serotype 06. Twelve genes were identified that share homology with other LPS and polysaccharide biosynthetic genes. This cluster contains homologues of wzx (encoding the O antigen flippase/translocase) and wzz (which modulates O antigen chain length distribution) genes, typical of a wzy-dependent pathway. However, a complete wzy gene (encoding the O-polymerase) was not found within the cluster. Four biosynthetic genes, wbpO, wbpP, wbpV and wbpM, and four putative glycosyltransferase genes, wbpR, wbpT, wbpU and wbpL, were identified in the cluster. To characterize their roles in LPS biosynthesis, null mutants of wbpO, wbpP, wbpV, wbpL and wbpM were generated using a gene-replacement strategy. Mutations in each of these genes caused deficiency in B-band synthesis. The wbpL mutant was deficient in both A-band and B-band LPS. WbpL(O6) is a bi-functional enzyme which could initiate B-band synthesis through the addition of QuiNAc to undecaprenol phosphate, and A-band synthesis by transferring either a GalNAc or a GlcNAc residue. Another approach used to assign function to the wbp(O6) genes was by complementation analysis. Two genes from Salmonella typhi, wcdA and wcdB, responsible for the synthesis of a homopolymer of GalNAcA called Vi antigen were used in complementation experiments to verify the functions of wbpO and wbpP. wcdA and wcdB restored B-band synthesis in wbpO and wbpP mutants respectively, implying that wbpO and wbpP are involved in UDP-GalNAcA synthesis. Although wbpV has homology to wbpK of the serotype O5 B-band LPS synthesis cluster, complementation analysis using the respective null mutants showed that the genes are not interchangeable. A knockout mutation of wbpN (located downstream of wbpM) did not abrogate LPS synthesis in either 05 or 06; therefore, it has been renamed orf48.5. These results establish the organization of genes involved in P. aeruginosa B-band O antigen synthesis and provide the evidence to assign functions to a number of LPS biosynthetic genes.