scholarly journals Coexistence of Two Distinct Versions of O-Antigen Polymerase, Wzy-Alpha and Wzy-Beta, in Pseudomonas aeruginosa Serogroup O2 and Their Contributions to Cell Surface Diversity

2007 ◽  
Vol 189 (11) ◽  
pp. 4141-4152 ◽  
Author(s):  
Katarina Kaluzny ◽  
Priyanka D. Abeyrathne ◽  
Joseph S. Lam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Surface ◽  
Nucleotide Sequencing ◽  
Polymerase Gene ◽  
Knockout Mutant ◽  
Sequence Identity ◽  
O Antigen ◽  
Reverse Transcription Pcr ◽  
Topo Cloning ◽  
Dependent Pathway

ABSTRACT Assembly of B-band lipopolysaccharide (LPS) in Pseudomonas aeruginosa follows a Wzy-dependent pathway, requiring the O-antigen polymerase Wzy and other proteins. The peptide sequences of the wzy α product from strains of serotypes O2, O5, and O16 are identical, but the O units in O5 are α-glycosidically linked, while those in O2 and O16 are β-linked. We hypothesized that a derivative of the D3 bacteriophage wzy β is present in the chromosomes of O2 and O16 and that this gene is responsible for the β-linkage. By a combination of PCR and primer walking, wzy β genes of both serotypes have been amplified and cloned. They are identical but share only 87.42% sequence identity with their xenolog in D3. A chromosomal knockout mutant of O16 wzy β was made, and it produces semirough LPS devoid of B-band O antigen. The cloned wzy β is capable of complementing the O16 wzy β mutant, as well as cross-complementing a wzy α knockout mutant. However, in the latter case, the restored O antigen was β-linked. Using reverse transcription-PCR, we showed that wzy α was transcribed in O2 and O16 strains and was functional, since both of these genes could complement the wzy α mutant of O5. With the coexistence of wzy α and wzy β in O2 and O16 and the B-band O polysaccharides in these being β-linked, we hypothesized that iap, an inhibitor of the alpha-polymerase gene, must be present in these serotypes. Indeed, through PCR, TOPO-cloning, and nucleotide-sequencing results, we verified the presence of iap in both O2 and O16 serotypes.

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 Membrane Topology Mapping of the O-Antigen Flippase (Wzx), Polymerase (Wzy), and Ligase (WaaL) from Pseudomonas aeruginosa PAO1 Reveals Novel Domain Architectures

mBio ◽  
2010 ◽  
Vol 1 (3) ◽  
Author(s):  
Salim T. Islam ◽  
Véronique L. Taylor ◽  
Meng Qi ◽  
Joseph S. Lam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Opportunistic Pathogen ◽  
Membrane Topology ◽  
Inner Membrane ◽  
Content Type ◽  
O Antigen ◽  
Dependent Pathway

ABSTRACTBiosynthesis of B-band lipopolysaccharide (LPS) inPseudomonas aeruginosafollows the Wzy-dependent pathway, requiring the integral inner membrane proteins Wzx (O-antigen [O-Ag] flippase), Wzy (O-Ag polymerase), and WaaL (O-Ag ligase). For an important first step in deciphering the mechanisms of LPS assembly, we set out to map the membrane topology of these proteins. Random and targeted 3′wzx,wzy, andwaaLtruncations were fused to aphoA-lacZαdual reporter capable of displaying both alkaline phosphatase and β-galactosidase activity. The results from truncation fusion expression and the corresponding differential enzyme activity ratios allowed for the assignment of specific regions of the proteins to cytoplasmic, transmembrane (TM), or periplasmic loci. Protein orientation in the inner membrane was confirmed via C-terminal fusion to green fluorescent protein. Our data revealed unique TM domain properties in these proteins, particularly for Wzx, indicating the potential for a charged pore. Novel periplasmic and cytoplasmic loop domains were also uncovered, with the latter in Wzy and WaaL revealing tracts consistent with potential Walker A/B motifs.IMPORTANCEThe opportunistic pathogenPseudomonas aeruginosasynthesizes its virulence factor lipopolysaccharide via the Wzy-dependent pathway, requiring translocation, polymerization, and ligation of lipid-linked polysaccharide repeat units by the integral inner membrane proteins Wzx, Wzy, and WaaL, respectively. However, structural evidence to help explain the function of these proteins is lacking. Since membrane proteins are difficult to crystallize, topological mapping is an important first step in identifying exposed and membrane-embedded domains. We mapped the topologies of Wzx, Wzy, and WaaL fromP. aeruginosaPAO1 by use of truncation libraries of a randomly fused C-terminal reporter capable of different enzyme activities in the periplasm and cytoplasm. Topology maps were created based directly on residue localization data, eliminating the bias associated with reliance on multiple topology prediction algorithms for initial generation of consensus transmembrane domain localizations. Consequently, we have identified novel periplasmic, cytoplasmic, and transmembrane domain properties that would help to explain the proposed functions of Wzx, Wzy, and WaaL.

scholarly journals Cell surface hydrophobicity determines Pseudomonas aeruginosa aggregate assembly

2021 ◽  
Author(s):  
Sheyda Azimi ◽  
Jacob Thomas ◽  
Sara E. Cleland ◽  
Jennifer E. Curtis ◽  
Joanna B. Goldberg ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Cell Surface ◽  
Spatial Organization ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Population Heterogeneity ◽  
Chronic Infections ◽  
O Antigen ◽  
The Impact

AbstractIt is now well established that bacteria live in structured aggregates during chronic infections, where they evolve to adapt to the host environment in order to evade host immune responses and therapeutic interventions. Despite recent developments on how the physical properties of polymers impact on aggregate formation, changes in bacterial properties to overcome this have been overlooked. Here we show that even with physical entropic forces imposed by polymers in a sputum medium environment, lipopolysaccharide (LPS) plays a crucial role in aggregate assembly in Pseudomonas aeruginosa by altering the hydrophobicity of the cell surface. Our findings highlight that in chronic infections such as the polymer rich (eDNA and mucin) airways in cystic fibrosis (CF) lungs, O-antigen can dictate the type of aggregate assembly allowing the cells to overcome entropic forces, and sheds new light on the benefits or loss of O-antigen in polymer rich environments such as CF lungs.ImportanceDuring chronic infection, several factors contribute to the biogeography of microbial communities. Heterogeneous populations of Pseudomonas aeruginosa form aggregates in cystic fibrosis airways, however, the impact of this population heterogeneity on spatial organization and aggregate assembly is not well understood. In this study we found that changes in O-antigen structure determine the spatial organization of P. aeruginosa cells by altering the relative cell surface hydrophobicity.

scholarly journals Variability in Pseudomonas aeruginosa Lipopolysaccharide Expression during Crude Oil Degradation

2002 ◽  
Vol 68 (10) ◽  
pp. 5096-5103 ◽  
Author(s):  
R. Sean Norman ◽  
Roberto Frontera-Suau ◽  
Pamela J. Morris
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Surface ◽  
Crude Oil ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Surface Hydrophobicity ◽  
Colony Morphology ◽  
Oil Degradation ◽  
O Antigen ◽  
Crude Oil Degradation

ABSTRACT Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation.

scholarly journals lfnA from Pseudomonas aeruginosa O12 and wbuX from Escherichia coli O145 Encode Membrane-Associated Proteins and Are Required for Expression of 2,6-Dideoxy-2-Acetamidino-l-Galactose in Lipopolysaccharide O Antigen

2007 ◽  
Vol 190 (5) ◽  
pp. 1671-1679 ◽  
Author(s):  
Jerry D. King ◽  
Erin F. Mulrooney ◽  
Evgeny Vinogradov ◽  
Bernd Kneidinger ◽  
Kristen Mead ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Opportunistic Pathogen ◽  
Multidrug Resistant ◽  
Nuclear Magnetic Resonance Analysis ◽  
O Antigen ◽  
O Antigens

ABSTRACT The rare sugar 2,6-dideoxy-2-acetamidino-l-galactose (l-FucNAm) is found only in bacteria and is a component of cell surface glycans in a number of pathogenic species, including the O antigens of Pseudomonas aeruginosa serotype O12 and Escherichia coli O145. P. aeruginosa is an important opportunistic pathogen, and the O12 serotype is associated with multidrug-resistant epidemic outbreaks. O145 is one of the classic non-O157 serotypes associated with Shiga toxin-producing, enterohemorrhagic E. coli. The acetamidino (NAm) moiety of l-FucNAm is of interest, because at neutral pH it contributes a positive charge to the cell surface, and we aimed to characterize the biosynthesis of this functional group. The pathway is not known, but expression of NAm-modified sugars coincides with the presence of a pseA homologue in the relevant biosynthetic locus. PseA is a putative amidotransferase required for synthesis of a NAm-modified sugar in Campylobacter jejuni. In P. aeruginosa O12 and E. coli O145, the pseA homologues are lfnA and wbuX, respectively, and we hypothesized that these genes function in l-FucNAm biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and nuclear magnetic resonance analysis of the lfnA mutant O-antigen structure indicated that the mutant expresses 2,6-dideoxy-2-acetamido-l-galactose (l-FucNAc) in place of l-FucNAm. The mutation could be complemented by expression of either His6-tagged lfnA or wbuX in trans, confirming that these genes are functional homologues and that they are required for NAm moiety synthesis. Both proteins retained their activity when fused to a His6 tag and localized to the membrane fraction. These data will assist future biochemical investigation of this pathway.

scholarly journals Biosynthesis and function of cell-surface polysaccharides in the social bacterium Myxococcus xanthus

2020 ◽  
Vol 401 (12) ◽  
pp. 1375-1387 ◽  
Author(s):  
María Pérez-Burgos ◽  
Lotte Søgaard-Andersen
Keyword(s):  
Cell Surface ◽  
Social Life ◽  
Myxococcus Xanthus ◽  
Spore Coat ◽  
Polysaccharide Biosynthesis ◽  
O Antigen ◽  
The Social ◽  
Surface Polysaccharides ◽  
And Function ◽  
Dependent Pathway

AbstractIn bacteria, cell-surface polysaccharides fulfill important physiological functions, including interactions with the environment and other cells as well as protection from diverse stresses. The Gram-negative delta-proteobacterium Myxococcus xanthus is a model to study social behaviors in bacteria. M. xanthus synthesizes four cell-surface polysaccharides, i.e., exopolysaccharide (EPS), biosurfactant polysaccharide (BPS), spore coat polysaccharide, and O-antigen. Here, we describe recent progress in elucidating the three Wzx/Wzy-dependent pathways for EPS, BPS and spore coat polysaccharide biosynthesis and the ABC transporter-dependent pathway for O-antigen biosynthesis. Moreover, we describe the functions of these four cell-surface polysaccharides in the social life cycle of M. xanthus.

scholarly journals Effects of Antibiotics on Quorum Sensing in Pseudomonas aeruginosa

2008 ◽  
Vol 52 (10) ◽  
pp. 3648-3663 ◽  
Author(s):  
Mette E. Skindersoe ◽  
Morten Alhede ◽  
Richard Phipps ◽  
Liang Yang ◽  
Peter O. Jensen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Virulence Factor ◽  
Dna Microarrays ◽  
Underlying Mechanism ◽  
Homoserine Lactone ◽  
Reverse Transcription Pcr ◽  
Animal Infection ◽  
Beneficial Action ◽  
Severe Infections

ABSTRACT During infection, Pseudomonas aeruginosa employs bacterial communication (quorum sensing [QS]) to coordinate the expression of tissue-damaging factors. QS-controlled gene expression plays a pivotal role in the virulence of P. aeruginosa, and QS-deficient mutants cause less severe infections in animal infection models. Treatment of cystic fibrosis (CF) patients chronically infected with P. aeruginosa with the macrolide antibiotic azithromycin (AZM) has been demonstrated to improve the clinical outcome. Several studies indicate that AZM may accomplish its beneficial action in CF patients by impeding QS, thereby reducing the pathogenicity of P. aeruginosa. This led us to investigate whether QS inhibition is a common feature of antibiotics. We present the results of a screening of 12 antibiotics for their QS-inhibitory activities using a previously described QS inhibitor selector 1 strain. Three of the antibiotics tested, AZM, ceftazidime (CFT), and ciprofloxacin (CPR), were very active in the assay and were further examined for their effects on QS-regulated virulence factor production in P. aeruginosa. The effects of the three antibiotics administered at subinhibitory concentrations were investigated by use of DNA microarrays. Consistent results from the virulence factor assays, reverse transcription-PCR, and the DNA microarrays support the finding that AZM, CFT, and CPR decrease the expression of a range of QS-regulated virulence factors. The data suggest that the underlying mechanism may be mediated by changes in membrane permeability, thereby influencing the flux of N-3-oxo-dodecanoyl-l-homoserine lactone.

scholarly journals Identification of cell-surface glycans that mediate motility-dependent binding and internalization of Pseudomonas aeruginosa by phagocytes

2020 ◽  
Author(s):  
Hector Sanchez ◽  
Daniel Hopkins ◽  
Sally Demirdjian ◽  
Cecilia Gutierrez ◽  
George A. O’Toole ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Surface ◽  
Cell Surface Glycans

scholarly journals Adaptation of Pseudomonas aeruginosa to Phage PaP1 Predation via O-Antigen Polymerase Mutation

2018 ◽  
Vol 9 ◽  
Author(s):  
Gang Li ◽  
Mengyu Shen ◽  
Yuhui Yang ◽  
Shuai Le ◽  
Ming Li ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
O Antigen
Sign in / Sign up

Export Citation Format

Share Document