Pseudomonas aeruginosa PAO1 ceases to express serotype-specific lipopolysaccharide at 45 degrees C.

Most Pseudomonas aeruginosa strains are able to produce two distinct lipopolysaccharide (LPS) O-polysaccharide types, A-band (common-antigen) and B-band (serotype-specific) LPSs. The relative expression levels of these two LPS types in P. aeruginosa PAO1 (O5 serotype) at various growth temperatures were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining or Western blotting (immunoblotting) with monoclonal antibodies specific for each O polysaccharide. A-band and B-band LPSs were expressed concurrently when the cells grew at 15, 25, and 35 degrees C; however, growth at 45 degrees C resulted in a surface deficiency in B-band LPS as determined by immunoblotting and agglutination with B-band-specific monoclonal antibody. Transfer of these cells (expressing A-band LPS but deficient in B-band LPS) [A+B-]) to a lower temperature (at which the division time was comparable) resulted in a rapid resumption of normal A-band and B-band expression. B-band LPS was detectable by immunoblotting before measurable growth of the culture had occurred.     

Presence or absence of lipopolysaccharide O antigens affects type III secretion by Pseudomonas aeruginosa

Pseudomonas aeruginosa is one of the major causative agents of mortality and morbidity in hospitalized patients due to a multiplicity of virulence factors associated with both chronic and acute infections. Acute P. aeruginosa infection is primarily mediated by planktonic bacteria expressing the type III secretion system (TTSS), a surface-attached needle-like complex that injects cytotoxins directly into eukaryotic cells, causing cellular damage. Lipopolysaccharide (LPS) is the principal surface-associated virulence factor of P. aeruginosa. This molecule is known to undergo structural modification (primarily alterations in the A- and B-band O antigen) in response to changes in the mode of life (e.g., from biofilm to planktonic). Given that LPS exhibits structural plasticity, we hypothesized that the presence of LPS lacking O antigen would facilitate eukaryotic intoxication and that a correlation between the LPS O-antigen serotype and TTSS-mediated cytotoxicity would exist. Therefore, strain PAO1 (A+ B+ O-antigen serotype) and isogenic mutants with specific O-antigen defects (A+ B-, A- B+, and A- B-) were examined for TTSS expression and cytotoxicity. A strong association existed in vitro between the absence of the large, structured B-band O antigen and increased cytotoxicity of these strains. In vivo, all three LPS mutant strains demonstrated significantly increased lung injury compared to PAO1. Clinical strains lacking the B-band O antigen also demonstrated increased TTSS secretion. These results suggest the existence of a cooperative association between LPS O-antigen structure and the TTSS in both laboratory and clinical isolates of P. aeruginosa.     

Molecular cloning of genes involved with expression of A-band lipopolysaccharide, an antigenically conserved form, in Pseudomonas aeruginosa.

Most strains of Pseudomonas aeruginosa can express two chemically and immunologically distinct types of lipopolysaccharide (LPS), an antigenically conserved form called A band and the serotype-specific form called B band. To study the molecular controls regulating expression of the A-band LPS antigen, we have cloned the genes involved with A-band LPS expression. Strain AK1401, a phage-resistant mutant of PAO1 which was shown previously to produce only A-band LPS and not the O-antigen-containing B-band LPS, was mutagenized by using ethyl methanesulfonate to generate an A-band-deficient mutant called rd7513. A cosmid clone bank of P. aeruginosa PAO1 whole genomic DNA was constructed in Escherichia coli. The gene bank was mobilized en masse into strain rd7513, and detection of complementation of synthesis of A band was done by screening transconjugants in a colony immunoblot assay with the A-band-specific monoclonal antibody N1F10. One recombinant cosmid, pFV3, complemented synthesis of A-band polysaccharide in rd7513. Silver-stained polyacrylamide gel and Western immunoblot analyses of LPS extracted from the transconjugant rd7513(pFV3) showed that the A band produced had a higher molecular weight than the A band of AK1401. Analysis of the plasmid pFV3 showed that it contained a chromosomal insert of 27 kb. Two subclones of pFV3, namely, pFV35 and pFV36, containing chromosomal inserts of 5.3 and 4.2 kb, respectively, also complemented A-band expression in rd7513. The LPS banding profile of rd7513(pFV35) was similar to that of AK1401, while the LPS profile of rd7513(pFV36) more closely resembled that of rd7513(pFV3). pFV3 complemented A-band expression in five of the six P. aeruginosa O serotypes which lack A band as well as in rough strain AK44 but failed to complement A-band expression in core mutants AK1012 and AK1282, suggesting that pFV3 contains genes for A-band expression and that synthesis of a complete core region in isogenic mutant strains is required for A-band synthesis.     

Identification and functional characterization of an ABC transport system involved in polysaccharide export of A-band lipopolysaccharide in Pseudomonas aeruginosa.

Pseudomonas aeruginosa coexpresses two distinct lipopolysaccharide (LPS) molecules known as A band and B band. B band is the serospecific LPS, while A band is the common LPS antigen composed of a D-rhamnose O-polysaccharide region. An operon containing eight genes responsible for A-band polysaccharide biosynthesis and export has recently been identified and characterized (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, unpublished data; H. L. Rocchetta, J. C. Pacan, and J. S. Lam, unpublished data). In this study, we report the characterization of two genes within the cluster, designated wzm and wzt. The Wzm and Wzt proteins have predicted sizes of 29.5 and 47.2 kDa, respectively, and are homologous to a number of proteins that comprise ABC (ATP-binding cassette) transport systems. Wzm is an integral membrane protein with six potential membrane-spanning domains, while Wzt is an ATP-binding protein containing a highly conserved ATP-binding motif. Chromosomal wzm and wzt mutants were generated by using a gene replacement strategy in P. aeruginosa PAO1 (serotype 05). Western blot analysis and immunoelectron microscopy using A-band- and B-band-specific monoclonal antibodies demonstrated that the wzm and wzt mutants were able to synthesize A-band polysaccharide, although transport of the polymer to the cell surface was inhibited. The inability of the polymer to cross the inner membrane resulted in the accumulation of cytoplasmic A-band polysaccharide. This A-band polysaccharide is likely linked to a carrier lipid molecule with a phenol-labile linkage. Chromosomal mutations in wzm and wzt were found to have no effect on B-band LPS synthesis. Rather, immunoelectron microscopy revealed that the presence of A-band LPS may influence the arrangement of B-band LPS on the cell surface. These results demonstrate that A-band and B-band O-antigen assembly processes follow two distinct pathways, with the former requiring an ABC transport system for cell surface expression.     

Atomic force microscopy study of the effect of lipopolysaccharides and extracellular polymers on adhesion of Pseudomonas aeruginosa

The roles of lipopolysaccharides (LPS) and extracellular polymers (ECP) on the adhesion of Pseudomonas aeruginosa PAO1 (expresses the A-band and B-band of O antigen) and AK1401 (expresses the A-band but not the B-band) to silicon were investigated with atomic force microscopy (AFM) and related to biopolymer physical properties. Measurement of macroscopic properties showed that strain AK1401 is more negatively charged and slightly more hydrophobic than strain PAO1 is. Microscopic AFM investigations of individual bacteria showed differences in how the biopolymers interacted with silicon. PAO1 showed larger decay lengths in AFM approach cycles, suggesting that the longer polymers on PAO1 caused greater steric repulsion with the AFM tip. For both bacterial strains, the long-range interactions we observed (hundreds of nanometers) were inconsistent with the small sizes of LPS, suggesting that they were also influenced by ECP, especially polysaccharides. The AFM retraction profiles provide information on the adhesion strength of the biopolymers to silicon (F_adh). For AK1401, the adhesion forces were only slightly lower (F_adh = 0.51 nN compared to 0.56 nN for PAO1), but the adhesion events were concentrated over shorter distances. More than 90% of adhesion events for AK1401 were at distances of <600 nm, while >50% of adhesion events for PAO1 were at distances of >600 nm. The sizes of the observed molecules suggest that the adhesion of P. aeruginosa to silicon was controlled by ECP, in addition to LPS. Steric and electrostatic forces each contributed to the interfacial interactions between P. aeruginosa and the silicon surface.     

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.     

Effect of O-Side-Chain-Lipopolysaccharide Chemistry on Metal Binding

Pseudomonas aeruginosa PAO1 produces two chemically distinct types of lipopolysaccharides (LPSs), termed A-band LPS and B-band LPS. The A-band O-side chain is electroneutral at physiological pH, while the B-band O-side chain contains numerous negatively charged sites due to the presence of uronic acid residues in the repeat unit structure. Strain PAO1 (A⁺ B⁺) and three isogenic LPS mutants (A⁺ B⁻, A⁻ B⁺, and A⁻ B⁻) were studied to determine the contribution of the O-side-chain portion of LPS to metal binding by the surfaces of gram-negative cells. Transmission electron microscopy with energy-dispersive X-ray spectroscopy was used to locate and analyze sites of metal deposition, while atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry were used to perform bulk quantitation of bound metal. The results indicated that cells of all of the strains caused the precipitation of gold as intracellular, elemental crystals with a d-spacing of 2.43 Å. This type of precipitation has not been reported previously for gram-negative cells and suggests that in the organisms studied gold binding is not a surface-mediated event. All four strains bound similar amounts of copper (0.213 to 0.222 μmol/mg [dry weight] of cells) at the cell surface, suggesting that the major surface metal-binding sites reside in portions of the LPS which are common to all strains (perhaps the phosphoryl groups in the core-lipid A region). However, significant differences were observed in the abilities of strains dps89 (A⁻ B⁺) and AK1401 (A⁺ B⁻) to bind iron and lanthanum, respectively. Strain dps89 caused the precipitation of iron (1.623 μmol/mg [dry weight] of cells) as an amorphous mineral phase (possibly iron hydroxide) on the cell surface, while strain AK1401 nucleated precipitation of lanthanum (0.229 μmol/mg [dry weight] of cells) as apiculate, surface-associated crystals. Neither iron nor lanthanum precipitates were observed on the cells of other strains, which suggests that the combination of A-band LPS and B-band LPS produced by a cell may result in a cell surface which promotes the formation of metal-rich precipitates. We therefore propose that the negatively charged sites located in the O-side chains are not directly responsible for the binding of metallic ions; however, the B-band LPS molecule as a whole may contribute to overall cell surface properties which favor the precipitation of distinct metal-rich mineral phases.     

Chromosomal mapping, expression and synthesis of lipopolysaccharide in Pseudomonas aeruginosa: a role for guanosine diphospho (GDP)-D-mannose

Pseudomonas aeruginosa can express two distinct forms of lipopolysaccharide (LPS), called A-band and B-band. As an attempt to understand the molecular biology of the synthesis and regulation of these LPS antigens, a recombinant plasmid, pFV3, containing genes for A-band expression was isolated previously. In the present study, P. aeruginosa strain PAO1 was mutagenized with transposon Tn5-751 and yielded a B-band-deficient mutant, called ge6. This mutant was mated with a PAO1 genomic library, and transconjugants were screened for complementation of B-band using B-band-specific monoclonal antibody MF15-4. Recombinant plasmid pFV100 was subsequently isolated by its ability to complement B-band expression in ge6. SDS-PAGE analysis of LPS from ge6 and ge6(pFV100) revealed that ge6 was deficient in expression of B-band, while ge6(pFV100) had an LPS profile similar to that of the parent strain PA01. With A-band and B-band genes cloned in separate plasmids, pFV3 and pFV100 respectively, we were able to determine the map location of these LPS genes on the P. aeruginosa PAO1 chromosome using pulsed-field gel electrophoresis. A-band genes mapped at 5.75 to 5.89 Mbp (Spel fragment SpK; Dpnl fragment DpF₂), while genes involved with expression of B-band LPS mapped at 1.9 Mbp (Spel fragments SpC, Spl and SpAl; Dpnl fragment DpD) on the 5.9 Mbp chromosome. We also performed initial characterization of a gene involved with synthesis of A-band present on pFV3. We previously reported that recombinant plasmid pFV3 and subcloned plasmid pFV36 complemented A-band synthesis in rd7513, an A^? mutant derived from A⁺ strain AK1401. pFV36 was mutagenized with transposon Tn1000 to reveal a one-kilobase region capable of complementing the expression of A-band in the A^? strain rd7513. This region was subcloned as a 1.6 kb Kpnl fragment into plasmid vector pAK1900 and the resulting clone named pFV39. Labelling of proteins encoded by pAK1900 and pFV39 in Escherichia coli maxicells revealed a single unique polypeptide of approximately 37kDa expressed by pFV39. Supernatants from disrupted cells of rd7513(pFV39) and AK1401 converted ¹⁴C-labelled-guanosine diphospho (GDP)-D-mannose to GDP-rhamnose, while supernatants from rd7513 did not show synthesis of GDP-rhamnose. The data therefore suggest that conversion of GDP-D-mannose to GDP-rhamnose is required for synthesis of A-band LPS, and that a 37kDa protein is involved in this conversion.     

The Pseudomonas aeruginosa algC gene encodes phosphoglucomutase, required for the synthesis of a complete lipopolysaccharide core.

We have constructed strains of Pseudomonas aeruginosa with mutations in the algC gene, previously shown to encode the enzyme phosphomannomutase. The algC mutants of a serotype O5 strain (PAO1) and a serotype O3 strain (PAC1R) did not express lipopolysaccharide (LPS) O side chains or the A-band (common antigen) polysaccharide. The migration of LPS from the algC mutant strains in Tricine-sodium dodecyl sulfate-polyacrylamide gels was similar to that of LPS from a PAO1 LPS-rough mutant, strain AK1012, and from a PAC1R LPS-rough mutant, PAC605, each previously shown to be deficient in the incorporation of glucose onto the LPS core (K. F. Jarrell and A. M. Kropinski, J. Virol. 40:411-420, 1981, and P. S. N. Rowe and P. M. Meadow, Eur. J. Biochem. 132:329-337, 1983). We show that, as expected, the algC mutant strains had no detectable phosphomannomutase activity and that neither algC strain had detectable phosphoglucomutase (PGM) activity. To confirm that the PGM activity was encoded by the algC gene, we transferred the cloned, intact P. aeruginosa algC gene to a pgm mutant of Escherichia coli and observed complementation of the pgm phenotype. Our finding that the algC gene product has PGM activity and that strains with mutations in this gene produce a truncated LPS core suggests that the synthesis of glucose 1-phosphate is necessary in the biosynthesis of the P. aeruginosa LPS core. The data presented here thus demonstrate that the algC gene is required for the synthesis of a complete LPS core in two strains with different LPS core and O side chain structures.     

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, IS 1209 , 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.     

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.     

Characterization of polyagglutinating and surface antigens in Pseudomonas aeruginosa

Mutants of Pseudomonas aeruginosa PAC1R (serotype O:3) which were resistant to bacteriophage D were isolated and shown to react with O:5d, O:9 and O:13 antisera as well as O:3. Antisera to the parent strain and to the three polyagglutinating (PA) mutants also showed cross-reactions. The mutants differed from the parent strain in their lipopolysaccharide (LPS) composition. The LPS from two of the three mutants yielded high molecular weight polysaccharide fractions. Although the high molecular weight fraction from one of the mutants contained the amino sugars and other components characteristic of the O:3 serotype strains, its mobility on Sephadex G75 was different from that of the parent strain. The high molecular weight material from the second mutant lacked the O-antigenic determinants but these were present in a semi-rough LPS fraction. The third mutant appeared rough and completely lacked the O-antigenic components. These three mutants were compared with the parent strain and with a non-agglutinating LPS-defective mutant which lacked both O-antigenic side chains and all neutral sugars in the outer core. Agglutination with absorbed sera and haemagglutination using purified LPS and ELISA detection suggested that wall components other than LPS were responsible for some of the cross-reactions observed. The components responsible were detected after SDS-PAGE of crude outer membrane fractions by a combination of Coomassie blue and silver-staining and antigenic components were detected by immunoelectrophoresis and ELISA-linked immunoblotting of the gels. The main antigenic determinants detected by antiserum to the parent strain were in the high molecular weight O-polysaccharide fractions and in the semirough fractions of the LPS, with some activity due to the H protein of the outer membrane. O:5d antisera reacted with unidentified high molecular weight polysaccharide fractions. Cross-reactions with the O:9 antiserum appeared to be due mainly to the F porin and, to a lesser extent, to the G and E proteins of the outer membrane. O:13 antiserum reacted with high molecular weight polysaccharide fractions but also with the rough core and F and H protein. Cross-reactivity of the other three mutant antisera could largely be interpreted in terms of the outer membrane components exposed in each strain. One reacted strongly with the F porin and the rough core, while the others reacted with a number of protein and LPS-derived fractions.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Common antigen lipopolysaccharide from Pseudomonas aeruginosa AK1401 as a receptor for bacteriophage A7.

A-band, a D-rhamnose-containing common lipopolysaccharide antigen isolated from Pseudomonas aeruginosa AK1401, was found to be a receptor for bacteriophage A7. The phage-borne rhamnanase was capable of hydrolyzing the A-band to expose core-lipid A containing only two or three rhamnose repeats. Interaction of the hydrolyzed A-band with core- or lipid A-specific monoclonal antibodies revealed that common epitopes exist in the inner core and lipid A regions, while the outer core of A-band appears to be different from that of the serotype-specific (B-band) lipopolysaccharide.     

Pseudomonas aeruginosa B-band O-antigen chain length is modulated by Wzz (Ro1).

The wbp gene cluster, encoding the B-band lipopolysaccharide O antigen of Pseudomonas aeruginosa serotype O5 strain PAO1, was previously shown to contain a wzy (rfc) gene encoding the O-antigen polymerase. This study describes the molecular characterization of the corresponding wzz (rol) gene, responsible for modulating O-antigen chain length. P. aeruginosa O5 Wzz has 19 to 20% amino acid identity with Wzz of Escherichia coli, Salmonella enterica, and Shigella flexneri. Knockout mutations of the wzz gene in serotypes O5 and O16 (which has an O antigen structurally related to that of O5) yielded mutants expressing O antigens with a distribution of chain lengths differing markedly from that of the parent strains. Unlike enteric wzz mutants, the P. aeruginosa wzz mutants continued to display some chain length modulation. The P. aeruginosa O5 wzz gene complemented both O5 and O16 wzz mutants as well as an E. coli wzz mutant. Coexpression of E. coli and P. aeruginosa wzz genes in a rough strain of E. coli carrying the P. aeruginosa wbp cluster resulted in the expression of two populations of O-antigen chain lengths. Sequence analysis of the region upstream of wzz led to identification of the genes rpsA and himD, encoding 30S ribosomal subunit protein S1 and integration host factor, respectively. This finding places rpsA and himD adjacent to wzz and the wbp cluster at 37 min on the PAO1 chromosomal map and completes the delineation of the O5 serogroup-specific region of the wbp cluster.     

Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.     

Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses.

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.     

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.     

Evidence that WapB is a 1,2-glucosyltransferase of Pseudomonas aeruginosa involved in Lipopolysaccharide outer core biosynthesis

Pseudomonas aeruginosa is an important opportunistic pathogen infecting debilitated individuals. One of the major virulence factors expressed by P. aeruginosa is lipopolysaccharide (LPS), which is composed of lipid A, core oligosaccharide (OS), and O-antigen polysaccharide. The core OS is divided into inner and outer regions. Although the structure of the outer core OS has been elucidated, the functions and mechanisms of the glycosyltransferases involved in core OS biogenesis are currently unknown. Here, we show that a previously uncharacterized gene, pa1014, is involved in outer core biosynthesis, and we propose to rename this gene wapB. We constructed a chromosomal mutant, wapB::Gm, in a PAO1 (O5 serotype) strain background. Characterization of the LPS from the mutant by Western immunoblotting showed a lack of reactivity to PAO1 outer core-specific monoclonal antibody (MAb) 5c-101. The chemical structure of the core OS of the wapB mutant was elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry techniques and revealed that the core OS of the wapB mutant lacked the terminal β-1,2-linked-d-glucose residue. Complementation of the mutant with wapB in trans restored the core structure to one that is identical to that of the wild type. Eleven of the 20 P. aeruginosa International Antigenic Typing Scheme (IATS) serotypes produce LPSs that lack the terminal d-glucose residue (Glc(IV)). Interestingly, expressing wapB in each of these 11 serotypes modifies each of their outer core OS structures, which became reactive to MAb 5c-101 in Western immunoblotting, suggesting the presence of a terminal d-glucose in these core OS structures. Our results strongly suggested that wapB encodes a 1,2-glucosyltransferase.