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.     

Characterization of the lipopolysaccharide from a wbjE mutant of the serogroup O11 Pseudomonas aeruginosa strain, PA103

The lipopolysaccharide (LPS) of a wbjE mutant of Pseudomonas aeruginosa PA103, a serogroup O11 strain consists of both high and low molecular weight (HMW and LMW) LPSs. The HMW LPS consisted exclusively of rhamnan A-band LPS and no B-band LPS was detected in the wbjE mutant. Interestingly, the LMW LPS from the wbjE mutant showed that it contained a variety of oligosaccharides, each with two or three phosphate groups present as mono- or pyrophosphates. These oligosaccharides consisted of the complete core octasaccharide. The GalN residue was present as an N-acetylated residue in all of these oligosaccharides except the tetrasaccharide in which it is present as an N-alanylated residue. None of these oligosaccharides contained either a d- or l-FucpNAc residue. These results are discussed with regard to the role of wbjE in the biosynthesis of P. aeruginosa PA103 B-band LPS.     

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.     

Three highly conserved proteins catalyze the conversion of UDP-N-acetyl-D-glucosamine to precursors for the biosynthesis of O antigen in Pseudomonas aeruginosa O11 and capsule in Staphylococcus aureus type 5. Implications for the UDP-N-acetyl-L-fucosamine biosynthetic pathway

N-Acetyl-l-fucosamine is a constituent of surface polysaccharide structures of Pseudomonas aeruginosa and Staphylococcus aureus. The three P. aeruginosa enzymes WbjB, WbjC, and WbjD, as well as the S. aureus homologs Cap5E, Cap5F, and Cap5G, involved in the biosynthesis of N-acetyl-l-fucosamine have been overexpressed and purified to near homogeneity. Capillary electrophoresis (CE), mass spectroscopy (MS), and nuclear magnetic resonance spectroscopy have been used to elucidate the biosynthesis pathway, which proceeds in five reaction steps. WbjB/Cap5E catalyzed 4,6-dehydration of UDP-N-acetyl-d-glucosamine and 3- and 5-epimerization to yield a mixture of three keto-deoxy-sugars. The third intermediate compound was subsequently reduced at C-4 to UDP-2-acetamido-2,6-dideoxy-l-talose by WbjC/Cap5F. Incubation of UDP-2-acetamido-2,6-dideoxy-l-talose (UDP-TalNAc) with WbjD/Cap5G resulted in a new peak separable by CE that demonstrated identical mass and fragmentation patterns by CE-MS/MS to UDP-TalNAc. These results are consistent with WbjD/Cap5G-mediated 2-epimerization of UDP-TalNAc to UDP-FucNAc. A nonpolar gene knockout of wbjB, the first of the genes associated with this pathway, was constructed in P. aeruginosa serotype O11 strain PA103. The corresponding mutant produced rough lipopolysaccharide devoid of B-band O antigen. This lipopolysaccharide deficiency could be complemented with P. aeruginosa wbjB or with the S. aureus homolog cap5E. Insertional inactivation of either the cap5G or cap5F genes abolished capsule polysaccharide production in the S. aureus strain Newman. Providing the appropriate gene in trans, thereby complementing these mutants, fully restored the capsular polysaccharide phenotype.     

Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis

Natriuretic peptides of various forms are present in animals and plants, and display structural similarities to cyclic antibacterial peptides. Pretreatment of Pseudomonas aeruginosa PAO1 with brain natriuretic peptide (BNP) or C-type natriuretic peptide (CNP) increases bacterium-induced glial cell necrosis. In eukaryotes, natriuretic peptides act through receptors coupled to cyclases. We observed that stable analogs of cAMP (dibutyryl cAMP) and cGMP (8-bromo-cGMP) mimicked the effect of brain natriuretic peptide and CNP on bacteria. Further evidence for the involvement of bacterial cyclases in the regulation of P. aeruginosa PAO1 cytotoxicity by natriuretic peptides is provided by the observed doubling of intrabacterial cAMP concentration after exposure to CNP. Lipopolysaccharide (LPS) extracted from P. aeruginosa PAO1 treated with both dibutyryl cAMP and 8-bromo-cGMP induces higher levels of necrosis than LPS extracted from untreated bacteria. Capillary electrophoresis and MALDI-TOF MS analysis have shown that differences in LPS toxicity are due to specific differences in the structure of the macromolecule. Using a strain deleted in the vfr gene, we showed that the Vfr protein is essential for the effect of natriuretic peptides on P. aeruginosa PAO1 virulence. These data support the hypothesis that P. aeruginosa has a cyclic nucleotide-dependent natriuretic peptide sensor system that may affect virulence by activating the expression of Vfr and LPS biosynthesis.     

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.     

The structure of the lipopolysaccharide from a galU mutant of Pseudomonas aeruginosa serogroup-O11

The lipopolysaccharide (LPS) of a galU mutant of Pseudomonas aeruginosa PA103, a serogroup O11 strain, was sequentially extracted with phenol–chloroform–petroleum ether (PCP) followed by hot phenol–water extraction of the bacterial pellet remaining after PCP extraction. LPS was found in both the PCP extract as well as in the water phase of the hot phenol–water extract. Analysis of the carbohydrate portion released by mild acid hydrolysis of both LPS preparations, both before and after removal of all phosphate groups by treatment with aqueous HF, was performed by glycosyl composition and linkage analyses as well as by NMR and mass spectrometric analyses. The results showed that the carbohydrate portion of these two LPS extracts contained the same structure: namely, -GalN(Ala)-(1→3)--(7-Cm)HepII-(1→3)--HepI-(1→5)--Kdo-(2→. The oligosaccharide preparation from PCP-extracted LPS consisted of a variety of structures containing up to six phosphate groups present as mono-, pyro-, and possibly triphosphate, primarily located on the HepI residue with some molecules having a monophosphate on HepII. The oligosaccharide preparation from the hot phenol–water-extracted LPS contained a similar variety of structures, but with an additional structure in which HepI contained a PPEA group at O-2. In addition, PAGE immunoblot analysis of the crude cellular extract with anti-A-antibodies revealed the presence of A-band material in both PA103 and the galU mutant. The A-band material was purified and characterized by glycosyl composition and linkage analyses, as well as by NMR spectroscopy, which confirmed that the A-band rhamnan polysaccharide was present but not as typical LPS since lipid-A or LPS core oligosaccharide components were not detected.     

MucR, a Novel Membrane-Associated Regulator of Alginate Biosynthesis in Pseudomonas aeruginosa

Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3′-5′)-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic ΔmucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44.     

Control of Pyrimidine Biosynthesis in Pseudomonas aeruginosa

The pathway of pyrimidine biosynthesis in Pseudomonas aeruginosa has been shown to be the same as in other bacteria. Twenty-seven mutants requiring uracil for growth were isolated and the mutant lesions were identified. Mutants lacking either dihydroorotic acid dehydrogenase, orotidine monophosphate pyrophosphorylase, orotidine monophosphate decarboxylase, or aspartic transcarbamylase were isolated; none lacking dihydroorotase were found. By using transduction and conjugation, four genes affecting pyrimidine biosynthetic enzymes have been identified and shown to be unlinked to each other. The linkage of pyrB to met-28 and ilv-2 was shown by contransduction. Repression by uracil alone or by broth could not be demonstrated for any enzymes of this pathway, in contrast to the situation in Escherichia coli and Serratia marcescens. In addition, derepression of these enzymes could not be demonstrated. A low level of feedback inhibition of aspartic transcarbamylase was found to occur. It is suggested that the control of such constitutive biosynthetic enzymes in P. aeruginosa may be related to the comprehensive metabolic activities of this organism.     

Analysis of a common-antigen lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from Pseudomonas aeruginosa PAO1 (O5 serotype) was separated into two antigenically distinct fractions. A minor fraction, containing shorter polysaccharide chains, reacted with a monoclonal antibody to a P. aeruginosa common antigen but did not react with antibodies specific to O5-serotype lipopolysaccharide. In contrast, fractions containing long polysaccharide chains reacted only with the O5-specific monoclonal antibodies. The shorter, common-antigen fraction lacked phosphate and contained stoichiometric amounts of sulfate, and the fatty acid composition of this fraction was similar to that of the O-antigen-specific fraction. The lipid A derived from the serotype-specific lipopolysaccharide cross-reacted with monoclonal antibodies against lipid A from Escherichia coli, while the lipid A derived from the common antigen did not react. We propose that many serotypes of P. aeruginosa produce two chemically and antigenically distinct lipopolysaccharide molecules, one of which is a common antigen with a short polysaccharide and a unique core-lipid A structure.     

A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis

Yersinia enterocolitica serotype O:3 strain 6471/76-c (YeO3-c) was sensitive to bacteriophage φR1-37 when grown at 37°C but not when grown at 22°C because of steric hindrance by abundant lipopolysaccharide (LPS) O-side chain (O-antigen) expressed at 22°C. The transposon library of YeO3-c was grown at 37°C and screened for phage φR1-37-resistant transposon insertion mutants. Three types of mutant were isolated: (i) phage receptor mutants expressing O-antigen (LPS-smooth), (ii) phage receptor mutants not expressing O-antigen (LPS-rough), and (iii) LPS-smooth mutants with the phage receptor constitutively sterically blocked. Mutant type (i) was characterized in detail; the transposon insertion inactivates an operon, named the trs operon. The main findings based on this mutant are: (i) the trs operon is involved in the biosynthesis of the LPS outer core in YeO3-c; the nucleotide sequence of the trs operon revealed eight novel genes showing similarity to known polysaccharide biosynthetic genes of various Gram-negative bacteria as well as to capsule biosynthesis genes of Staphylococcus aureus ; (ii) the biosynthesis of the core of YeO3-c involves at least two genetic loci; (iii) the trs operon is required for the biosynthesis of the bacteriophage φR1-37 receptor structures; (iv) the homopolymeric O-antigen of YeO3-c is ligated to the inner core in Y. enterocolitica O:3; (v) the trs operon is located between the adk—hemH and galE—gsk gene pairs in the Y. enterocolitica chromosome; and (vi) the phage φR1-37 receptor is present in many but not in all Y. enterocolitica serotypes. The results also allow us to speculate that the trs operon is a relic of the ancestral rfb region of Y. enterocolitica O:3 carrying genes indispensable for the completion of the core polysaccharide biosynthesis.     

Biosynthesis of exopolysaccharide by Pseudomonas aeruginosa.

In batch cultures of Pseudomonas aeruginosa, the maximum rate of exopolysaccharide synthesis occurred during exponential growth. In nitrogen-limited continuous culture, the specific rate of exopolysaccharide synthesis increased from 0.27 g g of cell-1 h-1 at a dilution rate (D) of 0.05 h-1 to 0.44 g g of cells h-1 at D=0.1 H-1. The yield of exopolysaccharide on the basis of glucose used was in the range of 56 to 64%. Exopolysaccharide was also synthesized in carbon-limited cultures at 0.19 g g of cell-1 h-1 at D=0.05 h-1 in a 33% yield. Nonmucoid variants appeared after seven generations in continuous culture and rapidly increased in proportion to the total number of organisms present.     

The chemical composition of the lipopolysaccharide of Pseudomonas aeruginosa

1. Lipopolysaccharide was isolated from both cell walls and acetone-dried whole cells of Pseudomonas aeruginosa (N.C.T.C. 1999). 2. Closely similar products are obtained, although that from whole cells cannot be completely freed from small amounts (2-7%) of residual nucleic acids. 3. The lipid moiety (23-33%) has a similar amino sugar backbone to that of lipids of enterobacterial lipopolysaccharides, but contains different hydroxy acids (2- and 3-hydroxydodecanoic acid and 3-hydroxydecanoic acid). 3-Hydroxytetradecanoic acid is absent, and 3-hydroxydodecanoic acid is the main N-acylating acid. No clear evidence permitting a distinction between the possibilities that phosphodiester or glycosidic linkages exist between the glucosamine residues was obtained. 4. Identifiable sugars (glucose, rhamnose, 3-deoxy-2-octulonic acid and heptose) account for less than 20% of the lipopolysaccharide, and alanine, galactosamine and fucosamine are apparently components of the polysaccharide moiety. 5. The polysaccharide moiety is unusual in that it is not readily obtained from the lipopolysaccharide by treatment with dilute acetic acid, which does, however, solubilize much of the phosphorus of the lipopolysaccharide. 6. The ;polysaccharide' fraction (approx. 21%) obtained by treatment with dilute acetic acid contains only a small proportion of the total polysaccharide components, and in one case only 45% of the fraction was accountable for in terms of identifiable components. 7. Evidence suggests that unidentified nitrogenous components are concentrated in the residual material after removal of both the lipid and the ;polysaccharide' fraction from the lipopolysaccharide.     

Isolation and characterization of two genes, waaC (rfaC) and waaF (rfaF), involved in Pseudomonas aeruginosa serotype O5 inner-core biosynthesis.

Recent studies have provided evidence to implicate involvement of the core oligosaccharide region of Pseudomonas aeruginosa lipopolysaccharide (LPS) in adherence to host tissues. To better understand the role played by LPS in the virulence of this organism, the aim of the present study was to clone and characterize genes involved in core biosynthesis. The inner-core regions of P. aeruginosa and Salmonella enterica serovar Typhimurium are structurally very similar; both contain two main chain residues of heptose linked to lipid A-Kdo2 (Kdo is 3-deoxy-D-manno-octulosonic acid). By electrotransforming a P. aeruginosa PAO1 library into Salmonella waaC and waaF (formerly known as rfaC and rfaF, respectively) mutants, we were able to isolate the homologous heptosyltransferase I and II genes of P. aeruginosa. Two plasmids, pCOREc1 and pCOREc2, which restored smooth LPS production in the waaC mutant, were isolated. Similarly, plasmid pCOREf1 was able to complement the Salmonella waaF mutant. Sequence analysis of the DNA insert of pCOREc2 revealed one open reading frame (ORF) which could code for a protein of 39.8 kDa. The amino acid sequence of the deduced protein exhibited 53% identity with the sequence of the WaaC protein of S. enterica serovar Typhimurium. pCOREf1 contained one ORF capable of encoding a 38.4-kDa protein. The sequence of the predicted protein was 49% identical to the sequence of the Salmonella WaaF protein. Protein expression by the Maxicell system confirmed that a 40-kDa protein was encoded by pCOREc2 and a 38-kDa protein was encoded by pCOREf1. Pulsed-field gel electrophoresis was used to determine the map locations of the cloned waaC and waaF genes, which were found to lie between 0.9 and 6.6 min on the PAO1 chromosome. Using a gene-replacement strategy, we attempted to generate P. aeruginosa waaC and waaF null mutants. Despite multiple attempts to isolate true knockout mutants, all transconjugants were identified as merodiploids.