Biomedical subjects

J S Lam

Publications and source records attributed to J S Lam.

At least 19 recordsLinked to original sources

PubMed2001

Use of prostatic stents for the treatment of benign prostatic hyperplasia in high-risk patients.

Benign prostatic hyperplasia (BPH) is a frequent disease in men and a major cause of lower urinary tract symptoms (LUTS). Transurethral resection of the prostate (TURP) or open surgery remains the gold standard of treatment for symptomatic BPH. However, 10% to 15% of patients with BPH cannot undergo surgery due to grave concomitant diseases. For patients presenting with contraindications to surgery or anesthesia, several minimally invasive alternative treatment modalities are available. One such therapeutic alternative is prostatic stenting, which can serve as a temporary or permanent solution for bladder outlet obstruction caused by BPH. Although not a new concept, this is a relatively new treatment modality in the United States, primarily because of the strict regulatory forces governing the use of these devices. Prostatic urethral stents have been widely demonstrated to be safe and effective for the treatment of symptomatic BPH. In addition to being minimally invasive, prostatic stenting is generally rapid, easy to perform, immediately effective, and has a low cost compared with conventional surgical treatment. Prostatic stents are therefore well suited to treat the frail elderly patient who would not be able to withstand the stress of undergoing surgery. This report reviews the current use of prostatic urethral stents in the treatment of high-risk surgical patients with BPH.

Age Factors↗

PubMed2001

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 beta-hydroxydecanoyl-beta-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-beta-hydroxydecanoyl-beta-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 sigma54-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.

Bacterial Proteins↗

PubMed2001

Topological and functional characterization of WbpM, an inner membrane UDP-GlcNAc C6 dehydratase essential for lipopolysaccharide biosynthesis in Pseudomonas aeruginosa.

WbpM is essential for the biosynthesis of B-band lipopolysaccharide (LPS) in many serotypes of Pseudomonas aeruginosa. Homologues that can functionally complement a wbpM null mutant and that are also necessary for virulence have been identified in numerous pathogenic bacteria. WbpM and most of its homologues are large membrane proteins, which has long hampered the elucidation of their biochemical function. This paper describes the detailed characterization of WbpM using both in vivo and in vitro approaches. LacZ and PhoA fusion experiments showed that WbpM was anchored to the inner membrane via four N-terminal transmembrane domains, whereas the C-terminal catalytic domain resided in the cytoplasm. Although the membrane domains did not have any catalytic activity, complementation experiments suggested that they were important for the polymerization of high-molecular-weight B-band LPS. The biochemical characterization of a soluble truncated form of WbpM, His-S262, showed that WbpM was a C6 dehydratase specific for UDP-GlcNAc. It exhibited unusual low temperature (25-30 degrees C) and high pH (pH 10) optima. Although WbpM possessed an altered catalytic triad composed of SMK as opposed to SYK commonly found in other dehydratases, its catalysis was very efficient, with a kcat of 168 min(-1) and a kcat/Km of 58 mM(-1) min(-1). These unusual physico-kinetic properties suggested a potentially different mechanism of C6 dehydration for WbpM and its large homologues. His-S262 is now a precious tool for further structure-function studies.

Bacterial Proteins↗

PubMed2001

Structural analysis of the carbohydrate components of the outer membrane of the lipopolysaccharide-lacking cellulolytic ruminal bacterium Fibrobacter succinogenes S85.

The polysaccharides from the outer membrane of the Gram-negative ruminal bacterium Fibrobacter succinogenes were isolated by phenol/water extraction and separated by size-exclusion chromatography in the presence of deoxycholate detergent into a lower-molecular-mass fraction designated 'glycolipid' and a high-molecular-mass 'capsular polysaccharide' fraction. Both fractions lacked typical lipopolysaccharide components including 2-keto-3-deoxyoctulosonic acid and 3-hydroxy fatty acids. Carbohydrate components of these fractions were represented by two polysaccharides and one oligosaccharide (possibly glycolipid) with the following structures: : : where HEAEP is N-(2-hydroxyethyl)-2-aminoethylphosphonic acid, found for the first time in natural compounds. The polysaccharides contained pentadecanoic acid and anteisopentadecanoic acid, possibly present as the acyl components. All constituent monosaccharides except L-rhamnose had a D-configuration. In addition to having a structural role in the outer membrane, these polysaccharides may provide protection for this lipopolysaccharide-less bacterium in the highly competitive ruminal environment, as phosphonic acids covalently linked to membrane polymers have in the past been attributed the function of stabilizing membranes in the presence of phosphatases and lipases.

Animals↗

PubMed2001

Three-component-mediated serotype conversion in Pseudomonas aeruginosa by bacteriophage D3.

Bacteriophage D3 is capable of lysogenizing Pseudomonas aeruginosa PAO1 (serotype O5), converting the O-antigen from O5 to O16 and O-acetylating the N-acetylfucosamine moiety. To investigate the mechanism of lysogenic conversion, a 3.6 kb fragment from the D3 genome was isolated capable of mediating serotypic conversion identical to the D3 lysogen strain (AK1380). The PAO1 transformants containing this 3.6 kb of D3 DNA exhibited identical lipopolysaccharide (LPS) banding patterns to serotype O16 in silver-stained SDS-PAGE gels and displayed reactivity to an antibody specific for O-acetyl groups. Further analysis led to the identification of three open reading frames (ORFs) required for serotype conversion: an alpha-polymerase inhibitor (iap); an O-acetylase (oac); and a beta-polymerase (wzybeta). The alpha-polymerase inhibitor (Iap) is capable of inhibiting the assembly of the serotype-specific O5 B-band LPS and allows the phage-encoded beta-polymerase (Wzybeta) to form new beta-linked B-band LPS. The D3 phage also alters the LPS by the addition of O-acetyl groups to the FucNAc residue in the O-antigen repeat unit by the action of the D3 O-acetylase (Oac). These three components form a simple yet elegant system by which bacteriophage D3 is capable of altering the surface of P. aeruginosa PAO1.

Acetyltransferases↗

PubMed2000

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 A 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.

Amino Acid Sequence↗

PubMed2000

FlaA1, a new bifunctional UDP-GlcNAc C6 Dehydratase/ C4 reductase from Helicobacter pylori.

FlaA1 is a small soluble protein of unknown function in Helicobacter pylori. It has homologues that are essential for the virulence of numerous medically relevant bacteria. FlaA1 was overexpressed as a histidine-tagged protein and purified to homogeneity by nickel chelation and cation exchange chromatography. Spectrophotometric assays, capillary electrophoresis, and mass spectrometry analyses showed that FlaA1 is a novel bifunctional C(6) dehydratase/C(4) reductase specific for UDP-GlcNAc. It converts UDP-GlcNAc into a UDP-4-keto-6-methyl-GlcNAc intermediate, which is stereospecifically reduced into UDP-QuiNAc. Substrate conversions as high as 80% were obtained at equilibrium. The K(m) and V(max) for UDP-GlcNAc were 159 microm and 65 pmol/min, respectively. No exogenous cofactor was required to obtain full activity of FlaA1. Additional NADH was only used with poor efficiency for the reduction step. The biochemical characterization of FlaA1 is important for the elucidation of biosynthetic pathways that lead to the formation of 2,6-deoxysugars in medically relevant bacteria. It establishes unambiguously the first step of the pathway and provides the means of preparing the substrate UDP-QuiNAc, which is necessary for the study of downstream enzymes.

Bacterial Proteins↗

PubMed2000

WbpO, a UDP-N-acetyl-D-galactosamine dehydrogenase from Pseudomonas aeruginosa serotype O6.

WbpO is associated with B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa serotype O6. This protein is thought to catalyze the enzymatic conversion of UDP-N-acetyl-d-galactosamine (UDP-GalNAc) to UDP-N-acetyl-d-galactosaminuronic acid (UDP-GalNAcA). WbpO was overexpressed with a C-terminal hexahistidine tag. The soluble form of expressed WbpO (WbpO(Sol)) exhibited a secondary structure with 29.2% alpha-helix and 20.1% beta-strand. However, no enzymatic activity could be detected using either high performance anion exchange chromatography or capillary electrophoresis-mass spectrometry analysis. An insoluble form of expressed WbpO was purified in the presence of guanidine hydrochloride by immobilized metal ion affinity chromatography. After refolding, this preparation of WbpO (designated as WbpO(Rf)) exhibited stable secondary structure at pH 7.5 to 8.2, and it was enzymatically active. Capillary electrophoresis-mass spectrometry and tandem mass spectrometry analysis showed that WbpO(Rf) catalyzed the conversion of UDP-GalNAc to UDP-GalNAcA. 26 and 22% of the substrate could be converted to UDP-GalNAcA in the presence of NAD(+) and NADP(+) as the cofactors, respectively. The K(m) values of WbpO(Rf) for UDP-GalNAc, NAD(+), and NADP(+) were 7.79, 0.65, and 0.44 mm, respectively. WbpO(Rf) can also catalyze the conversion of UDP-GlcNAc to UDP-GlcNAcA. In conclusion, this is the first report of the overexpression, purification, and biochemical characterization of an NAD(+)/NADP(+)-dependent UDP-GalNAc dehydrogenase. Our results also complete the biosynthetic pathway for GalNAcA that is part of the O-antigen of P. aeruginosa serotype O6 lipopolysaccharide.

Amino Acid Sequence↗

PubMed2000

Pseudomonas aeruginosa B-band lipopolysaccharide genes wbpA and wbpI and their Escherichia coli homologues wecC and wecB are not functionally interchangeable.

The O antigen unit of Pseudomonas aeruginosa serotype O5 is a complex trisaccharide containing 2-acetamido-3-acetiminido-2, 3-dideoxy-beta-D-mannuronic acid, 2-acetimido-3-acetimido-2, 3-dideoxy-beta-D-mannuronic acid, and 2-acetimido-2, 6-deoxy-beta-D-galactosamine. Specific knockout mutations in the putative UDP-D-N-acetylglucosamine (UDP-D-GlcNAc) epimerase gene, wbpI, or the putative UDP-D-N-acetylmannosamine dehydrogenase gene, wbpA, resulted in strains that no longer produced B-band lipopolysaccharide, confirming the essential roles of these genes in B-band O antigen synthesis. Despite approximately 50% similarity of wbpI and wbpA to the Escherichia coli genes wecB (rffE) and wecC (rffD) involved in enterobacterial common antigen synthesis, cross-complementation experiments were not successful. These results imply that the P. aeruginosa UDP-D-GlcNAc precursor may be di-N-acetylated prior to further modification, preventing the E. coli enzymes from recognizing it as a substrate.

Escherichia coli↗

PubMed2000

Expression, purification, and biochemical characterization of WbpP, a new UDP-GlcNAc C4 epimerase from Pseudomonas aeruginosa serotype O6.

B-band lipopolysaccharide is an important virulence factor of the opportunistic pathogen Pseudomonas aeruginosa. WbpP is an enzyme essential for B-band lipopolysaccharide production in serotype O6. Sequence analysis suggests that it is involved in the formation of N-acetylgalacturonic acid. To test this hypothesis, overexpression and biochemical characterization of WbpP were performed. By using spectrophotometric assays and capillary electrophoresis, we show that WbpP is a UDP-GlcNAc C4 epimerase. The K(m) for UDP-GlcNAc and UDP-GalNAc are 197 and 224 micrometer, respectively. At equilibrium, 70% of UDP-GalNAc is converted to UDP-GlcNAc, whereas the yield of the reverse reaction is only 30%. The enzyme can also catalyze the inter-conversion of non-acetylated substrates, although the efficiency of catalysis is significantly lower. Only 15 and 40% of UDP-Glc and UDP-Gal, respectively, are converted at equilibrium. WbpP contains tightly bound NAD(H) and does not require additional cofactors for activity. It exists as a dimer in its native state. This paper is the first report of expression and characterization of a C4 UDP-GlcNAc epimerase at the biochemical level. Moreover, the characterization of the enzymatic function of WbpP will help clarify ambiguous surface carbohydrate biosynthetic pathways in P. aeruginosa and other organisms where homologues of WbpP exist.

Amino Acid Sequence↗

PubMed2000

Lipopolysaccharide core phosphates are required for viability and intrinsic drug resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for its intrinsic drug resistance. We have used chemical and genetic techniques to characterize three putative kinase genes that are involved in the addition of phosphate to the inner core region of P. aeruginosa lipopolysaccharide. The first gene is a waaP homologue, whereas the other two (wapP and wapQ) are unique to P. aeruginosa. Repeated attempts using a variety of membrane-stabilizing conditions to generate waaP:Gm (Gm, gentamicin) or wapP:Gm mutants were unsuccessful. We were able to generate a chromosomal waaP mutant that had a wild-type copy of either waaPPa or waaPEc in trans, but were unable to cure this plasmid-borne copy of the gene. These results are consistent with the fact that P. aeruginosa mutants lacking inner core heptose (Hep) or phosphate have never been isolated and demonstrate the requirement of Hep-linked phosphate for P. aeruginosa viability. A wapQ:Gm mutant was isolated and it had an unaltered minimum inhibitory concentration (MIC) for novobiocin and only a small decrease in the MIC for sodium dodecyl sulphate (SDS), suggesting that the loss of a phosphate group transferred by WapQ may only be having a small impact on outer-membrane permeability. Nuclear magnetic resonance and methylation linkage analysis showed that WaaPPa could add one phosphate to O4 of HepI in a Salmonella typhimurium waaP mutant. The expression of WaaPPa increased the outer-membrane integrity of these complemented mutants, as evidenced by 35-fold and 75-fold increases in the MIC for novobiocin and SDS respectively. The S. typhimurium waaP mutant transformed with both waaP and wapP had over 250-fold and 1000-fold increases, respectively, in these MICs. The inner core phosphates of P. aeruginosa appear to be playing a key role in the intrinsic drug resistance of this bacterium.

Carbohydrate Sequence↗

PubMed2000

Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5.

The point of attachment of the O-chain in the outer core region of Pseudomonas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined following a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O-chain repeating unit, present in both the wild-type strain PAO1 and O-chain deficient mutant strains AK1401 and PAO-rfc. The structure of the extended core oligosaccharide was determined by various mass spectrometric methods as well as one-dimensional and two-dimensional NMR spectroscopy. Furthermore, the one-dimensional analogues of NOESY and TOCSY experiments were applied to confirm the structure of the outer core region in the O-chain polysaccharide. In both the extended core oligosaccharide and the core of the smooth LPS, a loss of one of the beta-glucosyl residues and the translocation of the alpha-rhamnosyl residue, followed by the attachment of the first O-chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with alpha-1, 6-linkage specificity and another with alpha-1,3-linkage specificity. It is also plausible that an alpha-1,3 rhamnosyltransferase facilitates the addition of the 'new' alpha-rhamnosyl residue that will act as a receptor for the attachment of the single O-antigen repeating unit in the LPS of the semi-rough mutant. The 2-amino-2-deoxy-fucosyl residue of the first O-chain repeating unit directly attached to the core was found to have a beta-anomeric configuration instead of an alpha configuration, characteristic for this residue as a component of the O-chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O-chain containing LPS, differing from the O-chain deficient rough LPS.

Bacterial Capsules↗

PubMed2000

Mucosal IL-12 is more effective than systemic IL-12 in augmenting IFN-gamma expression and inhibiting allergic lung eosinophilia in murine lungs.

The relative efficacy of mucosal (intratracheal) and systemic (intraperitoneal) delivery of interleukin (IL)-12 was evaluated in a mouse model of allergic lung eosinophilia. Mucosal administration of IL-12 achieved 100- to 600-fold higher bronchoalveolar lavage (BAL) levels of IL-12, but 2- to 10-fold lower serum levels compared to systemic administration. Whereas both mucosal and systemic IL-12 inhibited BAL eosinophil recruitment at high doses (100-1000 ng), only mucosal IL-12 was effective at low doses (1-10 ng). Mucosal, but not systemic, administration of 1000 ng of IL-12 increased interferon (IFN)-gamma expression in BAL cells. In a model of ongoing eosinophilic inflammation, when mucosal or systemic IL-12 doses were initiated prior to peak eosinophilia, further eosinophil recruitment was inhibited. However, when IL-12 treatment was initiated after peak eosinophil recruitment occurred, recovery from eosinophilic inflammation was not facilitated. Our findings are the first to demonstrate that locally administered IL-12 inhibits eosinophil recruitment at 100-fold lower doses than systemic IL-12. The most likely mechanism of this enhanced inhibitory activity is a sustained increase in lung levels of IL-12 that augments IFN-gamma production from BAL cells. We suggest that future studies should evaluate the efficacy of low doses of nebulized IL-12 in inhibiting eosinophilic lung inflammation in asthma.

Adjuvants, Immunologic↗

PubMed2000

The purification, crystallization and preliminary structural characterization of glucose-1-phosphate thymidylyltransferase (RmlA), the first enzyme of the dTDP-L-rhamnose synthesis pathway from Pseudomonas aeruginosa.

Glucose-1-phosphate thymidylyltransferase (RmlA; E.C. 2.7.7.24) is the first of four enzymes involved in the biosynthesis of dTDP-L-rhamnose, the precursor of L-rhamnose, a key component of the cell wall of many pathogenic bacteria. RmlA catalyses the condensation of thymidine triphosphate (dTTP) and alpha-D-glucose-1-phosphate (G1P), yielding dTDP-D-glucose. RmlA from Pseudomonas aeruginosa has been overexpressed and purified. Crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with PEG 6000 and lithium sulfate as precipitant. Several diffraction data sets of single frozen crystals were collected to a resolution of 1.66 A. Crystals belonged to space group P1, with unit-cell parameters a = 71.5, b = 73.1, c = 134.7 A, alpha = 89.9, beta = 80.9, gamma = 81.1 degrees. The asymmetric unit contains eight monomers in the form of two RmlA tetramers with a solvent content of 51%. Selenomethionine-labelled protein has been obtained and crystallized.

Chromatography, High Pressure Liquid↗

PubMed2000

Functional conservation of the polysaccharide biosynthetic protein WbpM and its homologues in Pseudomonas aeruginosa and other medically significant bacteria.

WbpM is a highly conserved protein involved in synthesis of the O antigens of Pseudomonas aeruginosa. Homologues of this protein have been identified in a large number of bacteria, and they can be divided into two subfamilies: subfamily 1, including WbpM, contains large proteins ( approximately 600 amino acids), while subfamily 2, typified by HP0840 (FlaA1) of Helicobacter pylori, contains smaller proteins ( approximately 350 amino acids) homologous to the C termini of proteins in subfamily 1. Analysis of knockout mutants of wbpM in P. aeruginosa serotypes O3, O10, O15, and O17 showed that although all 20 serotypes of P. aeruginosa possess wbpM, it is not universally required for O-antigen biosynthesis. Homologous genes from Bordetella pertussis (wlbL), Staphylococcus aureus (cap8D), and H. pylori (flaA1) complemented a P. aeruginosa O5 wbpM mutant to various degrees. These conserved proteins may represent interesting targets for the design of inhibitors of bacterial exopolysaccharide biosynthesis.

Bacterial Proteins↗

PubMed1999

Genetic and biochemical characterization of an operon involved in the biosynthesis of 3-deoxy-D-manno-octulosonic acid in Pseudomonas aeruginosa.

A Pseudomonas aeruginosa serotype O5 (PAO1) genomic DNA fragment that was able to complement a temperature-sensitive mutation in the 3-deoxy-D-manno-octulosonic acid (Kdo) 8-P synthase gene (kdsA) of Salmonella enterica serovar typhimurium was cloned. Nucleotide sequence analysis revealed the presence of a potential operon with the gene order pyrG, kdsA, eno. PyrG catalyzes the synthesis of the nucleotide cytidine triphosphate, while Eno catalyzes the formation of phosphoenolpyruvate from phosphoglycerate during glycolysis. Phosphoenolpyruvate is one of the substrates for Kdo-8-P biosynthesis by KdsA and cytidine triphosphate is the nucleotide used to activate Kdo prior to its transfer to lipid A. pyrG and eno are important for many metabolic pathways and it is interesting to find them linked to kdsA. A sigma 70-like promoter was found upstream of pyrG and evidence was provided to show that this promoter was responsible for the initiation of transcription of the genes in this operon. These genes mapped to 28.2-29.9 min on the 75-min PAO1 chromosome, unlinked to other lipopolysaccharide biosynthetic gene clusters.

Aldehyde-Lyases↗

PubMed1999

Effect of wzx (rfbX) mutations on A-band and B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa O5.

The wbp cluster of Pseudomonas aeruginosa O5 encodes a number of proteins involved in biosynthesis of the heteropolymeric and Wzy-dependent B-band O antigen, including Wzy, the O-antigen polymerase, and Wzz, the regulator of O-antigen chain length. A gene (formerly wbpF), contiguous with wzy in the wbp cluster, is predicted to encode a highly hydrophobic protein with multiple membrane-spanning domains. This secondary structure is consistent with that of Wzx (RfbX), the putative O-antigen unit translocase or "flippase." Insertion of a Gmr cassette at two separate sites within the putative wzx gene led in both cases to the loss of B-band lipopolysaccharide (LPS) O-antigen production. To our knowledge, this is the first report of the successful generation of chromosomal wzx gene replacement mutations. Surprisingly, inactivation of wzx also led to a marked delay in production of the ATP-binding cassette-transporter-dependent, D-rhamnose homopolymer, A-band LPS. This effect on A-band LPS synthesis was alleviated by supplying multiple copies of WbpL in trans. WbpL, a WecA (Rfe) homologue, was shown recently to be essential for the initiation of both A-band and B-band LPS synthesis in P. aeruginosa O5 (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, Mol. Microbiol. 28:1103-1119, 1998). These results suggest that the delay in A-band LPS production may arise from insufficient access to WbpL when the completed B-band O unit is not successfully translocated to the periplasm. Without adequate WbpL, A-band LPS synthesis is delayed. A subset of wzx mutants appeared to have accumulated second-site mutations which either restored the normal expression of A-band LPS or abolished A-band expression completely. Complementation studies showed that all of the additional mutations affecting LPS synthesis that were characterized in this study were located within the B-band LPS genes.

Bacterial Proteins↗

PubMed1999

Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa.

Pathogenic bacteria produce an elaborate assortment of extracellular and cell-associated bacterial products that enable colonization and establishment of infection within a host. Lipopolysaccharide (LPS) molecules are cell surface factors that are typically known for their protective role against serum-mediated lysis and their endotoxic properties. The most heterogeneous portion of LPS is the O antigen or O polysaccharide, and it is this region which confers serum resistance to the organism. Pseudomonas aeruginosa is capable of concomitantly synthesizing two types of LPS referred to as A band and B band. The A-band LPS contains a conserved O polysaccharide region composed of D-rhamnose (homopolymer), while the B-band O-antigen (heteropolymer) structure varies among the 20 O serotypes of P. aeruginosa. The genes coding for the enzymes that direct the synthesis of these two O antigens are organized into two separate clusters situated at different chromosomal locations. In this review, we summarize the organization of these two gene clusters to discuss how A-band and B-band O antigens are synthesized and assembled by dedicated enzymes. Examples of unique proteins required for both A-band and B-band O-antigen synthesis and for the synthesis of both LPS and alginate are discussed. The recent identification of additional genes within the P. aeruginosa genome that are homologous to those in the A-band and B-band gene clusters are intriguing since some are able to influence O-antigen synthesis. These studies demonstrate that P. aeruginosa represents a unique model system, allowing studies of heteropolymeric and homopolymeric O-antigen synthesis, as well as permitting an examination of the interrelationship of the synthesis of LPS molecules and other virulence determinants.

Amino Acid Sequence↗