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.

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 Identification of the Pseudomonas aeruginosa O17 and O15 O-Specific Antigen Biosynthesis Loci Reveals an ABC Transporter-Dependent Synthesis Pathway and Mechanisms of Genetic Diversity

2020 ◽  
Vol 202 (19) ◽  
Author(s):  
Steven M. Huszczynski ◽  
Youai Hao ◽  
Joseph S. Lam ◽  
Cezar M. Khursigara
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Abc Transporter ◽  
Specific Antigen ◽  
Opportunistic Pathogen ◽  
Content Type ◽  
O Antigen ◽  
O Antigens ◽  
Dependent Pathway

ABSTRACT Many bacterial cell surface glycans, such as the O antigen component of lipopolysaccharide (LPS), are produced via the so-called Wzx/Wzy- or ABC transporter-dependent pathways. O antigens are highly diverse polysaccharides that protect bacteria from their environment and engage in important host-pathogen interactions. The specific structure and composition of O antigens are the basis of classifying bacteria into O serotypes. In the opportunistic pathogen Pseudomonas aeruginosa, there are currently 20 known O-specific antigen (OSA) structures. The clusters of genes responsible for 18 of these O antigens have been identified, all of which follow the Wzx/Wzy-dependent pathway and are located at a common locus. In this study, we located the two unidentified O antigen biosynthesis clusters responsible for the synthesis of the O15 and the O17 OSA structures by analyzing published whole-genome sequence data. Intriguingly, these clusters were found outside the conserved OSA biosynthesis locus and were likely acquired through multiple horizontal gene transfer events. Based on data from knockout and overexpression studies, we determined that the synthesis of these O antigens follows an ABC transporter-dependent rather than a Wzx/Wzy-dependent pathway. In addition, we collected evidence to show that the O15 and O17 polysaccharide chain lengths are regulated by molecular rulers with distinct and variable domain architectures. The findings in this report are critical for a comprehensive understanding of O antigen biosynthesis in P. aeruginosa and provide a framework for future studies. IMPORTANCE P. aeruginosa is a problematic opportunistic pathogen that causes diseases in those with compromised host defenses, such as those suffering from cystic fibrosis. This bacterium produces a number of virulence factors, including a serotype-specific O antigen. Here, we identified and characterized the gene clusters that produce the O15 and O17 O antigens and show that they utilize a pathway for synthesis that is distinct from that of the 18 other known serotypes. We also provide evidence that these clusters have acquired mutations in specific biosynthesis genes and have undergone extensive horizontal gene transfer within the P. aeruginosa population. These findings expand on our understanding of O antigen biosynthesis in Gram-negative bacteria and the mechanisms that drive O antigen diversity.

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.

Importance of the O-antigen, core-region and lipid A of rhizobial lipopolysaccharides for the induction of systemic resistance in potato to Globodera pallida

Nematology ◽  
2002 ◽  
Vol 4 (1) ◽  
pp. 73-79 ◽  
Author(s):  
Martina Reitz ◽  
Peter Oger ◽  
Stephen Farrand ◽  
Johannes Hallmann ◽  
Andreas Meyer ◽  
...  
Keyword(s):  
Lipid A ◽  
Resistance Mechanism ◽  
Spatial Separation ◽  
Core Region ◽  
Nematode Infection ◽  
Globodera Pallida ◽  
Systemic Resistance ◽  
O Antigen ◽  
Split Root System ◽  
O Antigens

AbstractLipopolysaccharides (LPS) of the rhizobacterium Rhizobium etli strain G12 are involved in the induction of systemic resistance in potato roots towards the potato cyst nematode Globodera pallida. In the present study, the wild type strain G12 and three LPS-mutant strains with truncated O-antigens all reduced nematode infection of potato plants to about 30% that of untreated controls. Therefore, the O-antigen of the bacterial LPS cannot be the sole inducer of the resistance mechanism. In further search of the mode of action, the isolated core-region and lipid A-fraction of the LPS of one O-antigen-mutant strain R. etli #4-153 were tested for elicitor activity. Using a split-root system, which provides spatial separation of the inducing agent from the parasite, it was shown that both the entire LPS and its core-region systemically reduced G. pallida infection to 45% that of the untreated control. In contrast, the lipid A-fraction was less effective; the 20% reduction in nematode infection was not different from the control nor from the LPS and core region treatments. It is concluded that the oligosaccharides of the core-region are the main trigger of systemic resistance in potato roots towards G. pallida infection.

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 Temperature-Induced Changes in the Lipopolysaccharide of Yersinia pestis Affect Plasminogen Activation by the Pla Surface Protease

2010 ◽  
Vol 78 (6) ◽  
pp. 2644-2652 ◽  
Author(s):  
Marjo Suomalainen ◽  
Leandro Araujo Lobo ◽  
Klaus Brandenburg ◽  
Buko Lindner ◽  
Ritva Virkola ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Yersinia Pestis ◽  
Lipid A ◽  
Three Dimensional ◽  
Outer Core ◽  
Plasminogen Activation ◽  
Protein Motif ◽  
The Difference ◽  
O Antigens ◽  
Induced Changes

ABSTRACT The Pla surface protease of Yersinia pestis activates human plasminogen and is a central virulence factor in bubonic and pneumonic plague. Pla is a transmembrane β-barrel protein and member of the omptin family of outer membrane proteases which require bound lipopolysaccharide (LPS) to be proteolytically active. Plasminogen activation and autoprocessing of Pla were dramatically higher in Y. pestis cells grown at 37°C than in cells grown at 20°C; the difference in enzymatic activity by far exceeded the increase in the cellular content of the Pla protein. Y. pestis modifies its LPS structure in response to growth temperature. We purified His6-Pla under denaturing conditions and compared various LPS types for their capacity to enhance plasmin formation by His6-Pla solubilized in detergent. Reactivation of His6-Pla was higher with Y. pestis LPSs isolated from bacteria grown at 37°C than with LPSs from cells grown at 25°C. Lack of O antigens and the presence of the outer core region as well as a lowered level of acylation in LPS were found to enhance the Pla-LPS interaction. Genetic substitution of arginine 138, which is part of a three-dimensional protein motif for binding to lipid A phosphates, decreased both the enzymatic activity of His6-Pla and the amount of Pla in Y. pestis cells, suggesting the importance of the Pla-lipid A phosphate interaction. The temperature-induced changes in LPS are known to help Y. pestis to avoid innate immune responses, and our results strongly suggest that they also potentiate Pla-mediated proteolysis.

Pseudomonas aeruginosa O-antigen chain length is determined before ligation to lipid A core

2002 ◽  
Vol 4 (12) ◽  
pp. 883-897 ◽  
Author(s):  
Craig Daniels ◽  
Corrie Griffiths ◽  
Bryony Cowles ◽  
Joseph S. Lam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Chain Length ◽  
Lipid A ◽  
O Antigen
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