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

2005 ◽  
Vol 187 (9) ◽  
pp. 3002-3012 ◽  
Author(s):  
Priyanka D. Abeyrathne ◽  
Craig Daniels ◽  
Karen K. H. Poon ◽  
Mauricia J. Matewish ◽  
Joseph S. Lam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Functional Characterization ◽  
Wild Type ◽  
The Core ◽  
O Antigen ◽  
Repeat Units ◽  
Cytoplasmic Face ◽  
O Antigens

ABSTRACT 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.

scholarly journals Functional Characterization of MigA and WapR: Putative Rhamnosyltransferases Involved in Outer Core Oligosaccharide Biosynthesis of Pseudomonas aeruginosa

2008 ◽  
Vol 190 (6) ◽  
pp. 1857-1865 ◽  
Author(s):  
Karen K. H. Poon ◽  
Erin L. Westman ◽  
Evgeny Vinogradov ◽  
Shouguang Jin ◽  
Joseph S. Lam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Functional Characterization ◽  
Outer Core ◽  
Resonance Spectroscopy ◽  
Core Oligosaccharide ◽  
Strain Pao1 ◽  
The Core ◽  
O Antigen ◽  
Knockout Mutants

ABSTRACT Pseudomonas aeruginosa lipopolysaccharide (LPS) contains two glycoforms of core oligosaccharide (OS); one form is capped with O antigen through an α-1,3-linked l-rhamnose (l-Rha), while the other is uncapped and contains an α-1,6-linked l-Rha. Two genes in strain PAO1, wapR (PA5000) and migA (PA0705), encode putative glycosyltransferases associated with core biosynthesis. We propose that WapR and MigA are the rhamnosyltransferases responsible for the two linkages of l-Rha to the core. Knockout mutants with mutations in both genes were generated. The wapR mutant produced LPS lacking O antigen, and addition of wapR in trans complemented this defect. The migA mutant produced LPS with a truncated outer core and showed no reactivity to outer core-specific monoclonal antibody (MAb) 5C101. Complementation of this mutant with migA restored reactivity of the LPS to MAb 5C101. Interestingly, LPS from the complemented migA strain was not reactive to MAb 18-19 (specific for the core-plus-one O repeat). This was due to overexpression of MigA in the complemented strain that caused an increase in the proportion of the uncapped core OS, thereby decreasing the amount of the core-plus-one O repeat, indicating that MigA has a regulatory role. The structures of LPS from both mutants were elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry. The capped core of the wapR mutant was found to be truncated and lacked α-1,3-l-Rha. In contrast, uncapped core OS from the migA mutant lacked α-1,6-l-Rha. These results provide evidence that WapR is the α-1,3-rhamnosyltransferase, while MigA is the α-1,6-rhamnosyltransferase.

scholarly journals Interactions of Pulmonary Collectins with Bordetella bronchiseptica and Bordetella pertussis Lipopolysaccharide Elucidate the Structural Basis of Their Antimicrobial Activities

2004 ◽  
Vol 72 (12) ◽  
pp. 7124-7130 ◽  
Author(s):  
Lyndsay M. Schaeffer ◽  
Francis X. McCormack ◽  
Huixing Wu ◽  
Alison A. Weiss
Keyword(s):  
Bordetella Pertussis ◽  
Lipid A ◽  
Antimicrobial Activities ◽  
Innate Immune ◽  
Bordetella Bronchiseptica ◽  
Structural Basis ◽  
Wild Type ◽  
Core Oligosaccharide ◽  
The Core ◽  
O Antigen

ABSTRACT Surfactant proteins A (SP-A) and D (SP-D) play an important role in the innate immune defenses of the respiratory tract. SP-A binds to the lipid A region of lipopolysaccharide (LPS), and SP-D binds to the core oligosaccharide region. Both proteins induce aggregation, act as opsonins for neutrophils and macrophages, and have direct antimicrobial activity. Bordetella pertussis LPS has a branched core structure and a nonrepeating terminal trisaccharide. Bordetella bronchiseptica LPS has the same structure, but lipid A is palmitoylated and there is a repeating O-antigen polysaccharide. The ability of SP-A and SP-D to agglutinate and permeabilize wild-type and LPS mutants of B. pertussis and B. bronchiseptica was examined. Previously, wild-type B. pertussis was shown to resist the effects of SP-A; however, LPS mutants lacking the terminal trisaccharide were susceptible to SP-A. In this study, SP-A was found to aggregate and permeabilize a B. bronchiseptica mutant lacking the terminal trisaccharide, while wild-type B. bronchiseptica and mutants lacking only the palmitoyl transferase or O antigen were resistant to SP-A. Wild-type B. pertussis and B. bronchiseptica were both resistant to SP-D; however, LPS mutants of either strain lacking the terminal trisaccharide were aggregated and permeabilized by SP-D. We conclude that the terminal trisaccharide protects Bordetella species from the bactericidal functions of SP-A and SP-D. The O antigen and palmitoylated lipid A of B. bronchiseptica play no role in this resistance.

scholarly journals Lipopolysaccharide O-Antigen Chain Length Regulation in Pseudomonas aeruginosa Serogroup O11 Strain PA103

2007 ◽  
Vol 190 (8) ◽  
pp. 2709-2716 ◽  
Author(s):  
Erica Kintz ◽  
Jennifer M. Scarff ◽  
Antonio DiGiandomenico ◽  
Joanna B. Goldberg
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Chain Length ◽  
Gene Mutations ◽  
Side Chain ◽  
Wild Type ◽  
O Antigen ◽  
Length Regulation ◽  
O Antigens ◽  
Chain Lengths ◽  
Long Chain

ABSTRACT The Wzz proteins are important for determining the length of the O-antigen side chain attached to lipopolysaccharide (LPS). Several bacteria, including Pseudomonas aeruginosa strain PAO1 (serogroup O5), produce two such proteins responsible for the preference of two different chain lengths on the surface. Our group has previously identified one wzz gene (wzz1) within the O-antigen locus of P. aeruginosa strain PA103 (serogroup O11). In this study we have identified the second wzz gene (wzz2), located in the same region of the genome and with 92% similarity to PAO1's wzz2 gene. Mutations were generated in both wzz genes by interruption with antibiotic resistance cassettes, and the effects of these mutations were characterized. Wild-type PA103 prefers two O-antigen chain lengths, referred to as long and very long. The expression of the long O-antigen chain length was reduced in the wzz1 mutant, indicating the Wzz1 protein is important for this chain length preference. The wzz2 mutant, on the other hand, was missing O-antigens of the very long chain length, indicating the Wzz2 protein is responsible for the production of very long O-antigen. The effects of the wzz mutations on virulence were also investigated. In both serum sensitivity assays and a mouse pneumonia model of infection, the wzz1 mutants exhibited greater defects in virulence compared to either wild-type PA103 or the wzz2 mutant, indicating the long chain length plays a greater role during these infectious processes.

scholarly journals Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

2005 ◽  
Vol 187 (18) ◽  
pp. 6479-6487 ◽  
Author(s):  
Bradley L. Reuhs ◽  
Biserka Relić ◽  
L. Scott Forsberg ◽  
Corinne Marie ◽  
Tuula Ojanen-Reuhs ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Outer Core ◽  
Nitrogen Fixing ◽  
Primary Sequence ◽  
O Antigen ◽  
Duration Of Infection ◽  
Acidic Sugars ◽  
Biosynthesis Activation ◽  
O Antigens

ABSTRACT Rhizobium (Sinorhizobium) sp. strain NGR234 contains three replicons, the smallest of which (pNGR234a) carries most symbiotic genes, including those required for nodulation and lipo-chito-oligosaccharide (Nod factor) biosynthesis. Activation of nod gene expression depends on plant-derived flavonoids, NodD transcriptional activators, and nod box promoter elements. Nod boxes NB6 and NB7 delimit six different types of genes, one of which (fixF) is essential for the formation of effective nodules on Vigna unguiculata. In vegetative culture, wild-type NGR234 produces a distinct, flavonoid-inducible lipopolysaccharide (LPS) that is not produced by the mutant (NGRΩfixF); this LPS is also found in nitrogen-fixing bacteroids isolated from V. unguiculata infected with NGR234. Electron microscopy showed that peribacteroid membrane formation is perturbed in nodule cells infected by the fixF mutant. LPSs were purified from free-living NGR234 cultured in the presence of apigenin. Structural analyses showed that the polysaccharide portions of these LPSs are specialized, rhamnose-containing O antigens attached to a modified core-lipid A carrier. The primary sequence of the O antigen is [-3)-α-l-Rhap-(1,3)-α-l-Rhap-(1,2)-α-l-Rhap-(1-]n, and the LPS core region lacks the acidic sugars commonly associated with the antigenic outer core of LPS from noninduced cells. This rhamnan O antigen, which is absent from noninduced cells, has the same primary sequence as the A-band O antigen of Pseudomonas aeruginosa, except that it is composed of l-rhamnose rather than the d-rhamnose characteristic of the latter. It is noteworthy that A-band LPS is selectively maintained on the P. aeruginosa cell surface during chronic cystic fibrosis lung infection, where it is associated with an increased duration of infection.

Structural characterization of the lipid A component of Pseudomonas aeruginosa wild-type and rough mutant lipopolysaccharides

1991 ◽  
Vol 198 (3) ◽  
pp. 697-704 ◽  
Author(s):  
Vladimir A. KULSHIN ◽  
Ulrich ZAHRINGER ◽  
Buko LINDNER ◽  
Karl-Erich JAGER ◽  
Boris A. DMITRIEV ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Structural Characterization ◽  
Lipid A ◽  
Wild Type ◽  
Rough Mutant

Functional characterization of the H+/K+ ATPase in wild type and gastrin knock-out mice

2007 ◽  
Vol 45 (05) ◽  
Author(s):  
A Schnur ◽  
P Hegyi ◽  
V Venglovecz ◽  
Z Rakonczay ◽  
I Ignáth ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Wild Type ◽  
Knock Out ◽  
Knock Out Mice

An alkaline phosphatase mutant of Pseudomonas aeruginosa. 1. Effects of regulatory, structural, and environmental shifts on enzyme function

1978 ◽  
Vol 24 (4) ◽  
pp. 427-432 ◽  
Author(s):  
M. L. Marceau-Day ◽  
D. F Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Core Region ◽  
Significant Activity ◽  
Wild Type ◽  
Glycerol Phosphate ◽  
The Core ◽  
Nitrophenyl Phosphate ◽  
Mutant Cells

An alkaline phosphatase mutant of Pseudomonas aeruginosa exhibiting both regulatory and catalytic changes was isolated. Under repression conditions (i.e. high inorganic phosphate (Pi)) the mutant culture produced an alkaline phosphatase (APase) displaying significant activity against both β-glycerol phosphate (βGP) and p-nitrophenyl phosphate (pNPP), while the wild type displayed no activity directed towards these substrates under the same conditions. In vivo, the mutant enzyme's ratio of specific activities was 45:1 in favour of βGP versus pNPP, whereas this ratio was reversed to 1:9 βGP versus pNPP for the same enzyme isolated from mutant cells. In addition, the kinetic parameters and stability requirements for the mutant-derived enzyme was altered in comparison with those of the wild type. A study of lipopolysaccharide(LPS) preparations from both the mutant and wild type indicated the mutant to be deficient in the core region of its LPS. The authors propose that the modifications in the catalytic activity of the mutant enzyme, demonstrated in vivo, are due to a change in the enzyme's microenvironment.

scholarly journals A Novel 3-Deoxy-d-manno-Octulosonic Acid (Kdo) Hydrolase That Removes the Outer Kdo Sugar of Helicobacter pylori Lipopolysaccharide

2005 ◽  
Vol 187 (10) ◽  
pp. 3374-3383 ◽  
Author(s):  
Christopher Stead ◽  
An Tran ◽  
Donald Ferguson ◽  
Sara McGrath ◽  
Robert Cotter ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Lipid A ◽  
In Vitro Assay ◽  
Spectrometric Analysis ◽  
Vitro Assay ◽  
The Core ◽  
H Pylori

ABSTRACT The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-d-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IVA. In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.

Sign in / Sign up

Export Citation Format

Share Document