Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP-d-Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3-d-Rhamnosyltransferase WbpZ

ABSTRACTThe opportunistic pathogenPseudomonas aeruginosaproduces two major cell surface lipopolysaccharides, characterized by distinct O antigens, called common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA contains a polymer ofd-rhamnose (d-Rha) in α1-2 and α1-3 linkages. Three putative glycosyltransferase genes,wbpX,wbpY, andwbpZ, are part of the CPA biosynthesis cluster. To characterize the enzymatic function of thewbpZgene product, we chemically synthesized the donor substrate GDP-d-Rha and enzymatically synthesized GDP-d-[3H]Rha. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that WbpZ transferred oned-Rha residue from GDP-d-Rha in α1-3 linkage to both GlcNAc- and GalNAc-diphosphate-lipid acceptor substrates. WbpZ is also capable of transferringd-mannose (d-Man) to these acceptors. Therefore, WbpZ has a relaxed specificity with respect to both acceptor and donor substrates. The diphosphate group of the acceptor, however, is required for activity. WbpZ does not require divalent metal ion for activity and exhibits an unusually high pH optimum of 9. WbpZ from PAO1 is therefore a GDP-d-Rha:GlcNAc/GalNAc-diphosphate-lipid α1,3-d-rhamnosyltransferase that has significant activity of GDP-d-Man:GlcNAc/GalNAc-diphosphate-lipid α1,3-d-mannosyltransferase. We used site-directed mutagenesis to replace the Asp residues of the two DXD motifs with Ala. Neither of the mutant constructs ofwbpZ(D172A or D254A) could be used to rescue CPA biosynthesis in the ΔwbpZknockout mutant in a complementation assay. This suggested that D172 and D254 are essential for WbpZ function. This work is the first detailed characterization study of ad-Rha-transferase and a critical step in the development of CPA synthesis inhibitors.IMPORTANCEThis is the first characterization of ad-rhamnosyltransferase and shows that it is essential inPseudomonas aeruginosafor the synthesis of the common polysaccharide antigen.

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Single-Nucleotide Polymorphisms Found in themigAandwbpXGlycosyltransferase Genes Account for the Intrinsic Lipopolysaccharide Defects Exhibited by Pseudomonas aeruginosa PA14

Journal of Bacteriology ◽

10.1128/jb.00337-15 ◽

2015 ◽

Vol 197 (17) ◽

pp. 2780-2791 ◽

Cited By ~ 8

Author(s):

Youai Hao ◽

Kathleen Murphy ◽

Reggie Y. Lo ◽

Cezar M. Khursigara ◽

Joseph S. Lam

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Specific Antigen ◽

Nucleotide Polymorphisms ◽

Core Oligosaccharide ◽

Polysaccharide Antigen ◽

Single Nucleotide ◽

Content Type ◽

Chain Lengths ◽

Long Chain

ABSTRACTPseudomonas aeruginosaPA14 is widely used by researchers in many laboratories because of its enhanced virulence over strain PAO1 in a wide range of hosts. Although lipopolysaccharide (LPS) is an important virulence factor of allP. aeruginosastrains, the LPS of PA14 has not been characterized fully. A recent study showed that the structure of its O-specific antigen (OSA) belongs to serotype O19. We found that the OSA gene cluster of PA14 shares ∼99% identity with those of the O10/O19 group. These two serotypes share the same O-unit structure, except for anO-acetyl substitution in one of the sugars in O10. Here we showed that both PA14 and O19 LPS cross-reacted with the O10-specific monoclonal antibody MF76-2 in Western blots. Analysis by SDS-PAGE and silver staining showed that PA14 LPS exhibited modal chain lengths that were different from those of O19 LPS, in that only “very long” and “short” chain lengths were observed, while “medium” and “long” chain lengths were not detected. Two other novel observations included the lack of the uncapped core oligosaccharide epitope and of common polysaccharide antigen (CPA) LPS. The lack of the uncapped core oligosaccharide was caused by point mutations in the glycosyltransferase genemigA, while the CPA-negative phenotype was correlated with a single amino acid substitution, G20R, in the glycosyltransferase WbpX. Additionally, we showed that restoring CPA biosynthesis in PA14 significantly stimulated mature biofilm formation after 72 h, while outer membrane vesicle production was not affected.IMPORTANCEP. aeruginosaPA14 is a clinical isolate that has become an important reference strain used by many researchers worldwide. LPS of PA14 has not been characterized fully, and hence, confusion about its phenotype exists in the literature. In the present study, we set out to characterize the O-specific antigen (OSA), the common polysaccharide antigen (CPA), and the core oligosaccharide produced by PA14. We present evidence that PA14 produces an LPS consisting of “very-long-chain” and some “short-chain” OSA belonging to the O19 serotype but is devoid of CPA and the uncapped core oligosaccharide epitope. These intrinsic defects in PA14 LPS were due to single-nucleotide polymorphisms (SNPs) in the genes that encode glycosyltransferases in the corresponding biosynthesis pathways. Since sugars in CPA and the uncapped core are receptors for different bacteriocins and pyocins, the lack of CPA and an intact core may contribute to the increased virulence of PA14. Restoring CPA production in PA14 was found to stimulate mature biofilm formation.

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N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Applied and Environmental Microbiology ◽

10.1128/aem.02881-15 ◽

2015 ◽

Vol 82 (4) ◽

pp. 1004-1014 ◽

Cited By ~ 23

Author(s):

Canfang Niu ◽

Huiying Luo ◽

Pengjun Shi ◽

Huoqing Huang ◽

Yaru Wang ◽

...

Keyword(s):

Acid Phosphatase ◽

Biochemical Characterization ◽

Animal Feed ◽

Glycosylation Site ◽

Site Directed Mutagenesis ◽

Acidic Ph ◽

Cleavage Sites ◽

Ph Optimum ◽

Content Type ◽

Histidine Acid Phosphatase

ABSTRACTN-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases fromYersinia kristensenii(YkAPPA) andYersinia rohdei(YrAPPA), each having anN-glycosylation motif, and one pepsin-sensitive HAP phytase fromYersinia enterocolitica(YeAPPA) that lacked anN-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering theN-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed inPichia pastorisfor biochemical characterization. Compared with those of theN-glycosylation site deletion mutants andN-deglycosylated enzymes, allN-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of theN-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of theN-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced inEscherichia colibut had no effect on the pepsin resistance ofN-glycosylated enzymes produced inP. pastoris. Thus,N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation ofN-glycosylation, for improvement of phytase properties for use in animal feed.

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Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

Journal of Bacteriology ◽

10.1128/jb.00040-15 ◽

2015 ◽

Vol 197 (9) ◽

pp. 1668-1680 ◽

Cited By ~ 11

Author(s):

Sarah Siu ◽

Anna Robotham ◽

Susan M. Logan ◽

John F. Kelly ◽

Kaoru Uchida ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Mass Spectroscopy ◽

Specific Antigen ◽

Mass Spectrometry Analysis ◽

Methanococcus Maripaludis ◽

Western Blots ◽

Bacterial Gene ◽

Content Type ◽

Spectrometry Analysis ◽

Type Iv

ABSTRACTMethanococcus maripaludishas two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354andmmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions inmmp0350,mmp0351,mmp0352, ormmp0353were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352strain confirmed a glycan with only GalNAc, suggestingmmp0350tommp0353were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr).M. maripaludismmp0350,mmp0351,mmp0352,mmp0353, andmmp0357are proposed to be functionally equivalent toPseudomonas aeruginosawbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of theP. aeruginosapathway withmmp0357supports this hypothesis.IMPORTANCEThis work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins ofMethanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway.

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Spatial Patterns of Carbonate Biomineralization in Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.01585-15 ◽

2015 ◽

Vol 81 (21) ◽

pp. 7403-7410 ◽

Cited By ~ 30

Author(s):

Xiaobao Li ◽

David L. Chopp ◽

William A. Russin ◽

Paul T. Brannon ◽

Matthew R. Parsek ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Important Process ◽

Mineral Precipitation ◽

Content Type ◽

Biofilm Architecture ◽

The Common ◽

Engineered Systems ◽

Over Time ◽

Carbonate Deposits

ABSTRACTMicrobially catalyzed precipitation of carbonate minerals is an important process in diverse biological, geological, and engineered systems. However, the processes that regulate carbonate biomineralization and their impacts on biofilms are largely unexplored, mainly because of the inability of current methods to directly observe biomineralization within biofilms. Here, we present a method forin situ, real-time imaging of biomineralization in biofilms and use it to show thatPseudomonas aeruginosabiofilms produce morphologically distinct carbonate deposits that substantially modify biofilm structures. The patterns of carbonate biomineralization producedin situwere substantially different from those caused by accumulation of particles produced by abiotic precipitation. Contrary to the common expectation that mineral precipitation should occur at the biofilm surface, we found that biomineralization started at the base of the biofilm. The carbonate deposits grew over time, detaching biofilm-resident cells and deforming the biofilm morphology. These findings indicate that biomineralization is a general regulator of biofilm architecture and properties.

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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

Journal of Bacteriology ◽

10.1128/jb.00347-20 ◽

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.

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Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies

Applied and Environmental Microbiology ◽

10.1128/aem.03172-16 ◽

2017 ◽

Vol 83 (7) ◽

Cited By ~ 8

Author(s):

Taeho Kim ◽

Robert Flick ◽

Joseph Brunzelle ◽

Alex Singer ◽

Elena Evdokimova ◽

...

Keyword(s):

Rational Design ◽

Molecular Mechanisms ◽

Substrate Binding ◽

Aromatic Aldehydes ◽

Site Directed Mutagenesis ◽

Substrate Selectivity ◽

Significant Activity ◽

Structural Determinants ◽

One Pot ◽

Content Type

ABSTRACT The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 from Pseudomonas aeruginosa showed the highest activity and was selected for comparative studies with STM2406 from Salmonella enterica serovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase in k cat/Km . Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates. IMPORTANCE In this study, we identified several aldo-keto reductases with significant activity in reducing 3-hydroxybutanal to 1,3-butanediol (1,3-BDO), an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate-binding residues, including the two structural determinants necessary for high activity in the biosynthesis of 1,3-BDO. This work expands our understanding of the molecular mechanisms of the substrate selectivity of aldo-keto reductases and demonstrates the potential for protein engineering of these enzymes for applications in the biocatalytic production of 1,3-BDO and other valuable chemicals.

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Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.00457-13 ◽

2013 ◽

Vol 79 (13) ◽

pp. 4072-4077 ◽

Cited By ~ 57

Author(s):

Xuguo Duan ◽

Jian Chen ◽

Jing Wu

Keyword(s):

Catalytic Efficiency ◽

Kinetic Studies ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Ph Optimum ◽

Widespread Application ◽

Content Type ◽

Major Factors

ABSTRACTPullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase fromBacillus deramificanswere selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that theKmvalues for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and theKcat/Kmvalues increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.

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Crystal Structure of the Mobile Metallo-β-Lactamase AIM-1 from Pseudomonas aeruginosa: Insights into Antibiotic Binding and the Role of Gln157

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00448-12 ◽

2012 ◽

Vol 56 (8) ◽

pp. 4341-4353 ◽

Cited By ~ 39

Author(s):

Hanna-Kirsti S. Leiros ◽

Pardha S. Borra ◽

Bjørn Olav Brandsdal ◽

Kine Susann Waade Edvardsen ◽

James Spencer ◽

...

Keyword(s):

Crystal Structure ◽

Pseudomonas Aeruginosa ◽

In Silico ◽

Mobile Genetic Element ◽

Catalytic Efficiency ◽

Drug Binding ◽

Site Directed Mutagenesis ◽

Gram Negative Bacteria ◽

Content Type

ABSTRACTMetallo-β-lactamase (MBL) genes confer resistance to virtually all β-lactam antibiotics and are rapidly disseminated by mobile genetic elements in Gram-negative bacteria. MBLs belong to three different subgroups, B1, B2, and B3, with the mobile MBLs largely confined to subgroup B1. The B3 MBLs are a divergent subgroup of predominantly chromosomally encoded enzymes. AIM-1 (AdelaideIMipenmase 1) fromPseudomonas aeruginosawas the first B3 MBL to be identified on a readily mobile genetic element. Here we present the crystal structure of AIM-1 and usein silicodocking and quantum mechanics and molecular mechanics (QM/MM) calculations, together with site-directed mutagenesis, to investigate its interaction with β-lactams. AIM-1 adopts the characteristic αβ/βα sandwich fold of MBLs but differs from other B3 enzymes in the conformation of an active site loop (residues 156 to 162) which is involved both in disulfide bond formation and, we suggest, interaction with substrates. The structure, together with docking and QM/MM calculations, indicates that the AIM-1 substrate binding site is narrower and more restricted than those of other B3 MBLs, possibly explaining its higher catalytic efficiency. The location of Gln157 adjacent to the AIM-1 zinc center suggests a role in drug binding that is supported by ourin silicostudies. However, replacement of this residue by either Asn or Ala resulted in only modest reductions in AIM-1 activity against the majority of β-lactam substrates, indicating that this function is nonessential. Our study reveals AIM-1 to be a subclass B3 MBL with novel structural and mechanistic features.

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Novel Role for PilNO in Type IV Pilus Retraction Revealed by Alignment Subcomplex Mutations

Journal of Bacteriology ◽

10.1128/jb.00220-15 ◽

2015 ◽

Vol 197 (13) ◽

pp. 2229-2238 ◽

Cited By ~ 25

Author(s):

Tiffany L. Leighton ◽

Neha Dayalani ◽

Liliana M. Sampaleanu ◽

P. Lynne Howell ◽

Lori L. Burrows

Keyword(s):

Pseudomonas Aeruginosa ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Type Iv Pili ◽

Site Directed Mutagenesis ◽

Twitching Motility ◽

Content Type ◽

The Core ◽

Type Iv ◽

Two Hybrid

ABSTRACTType IV pili (T4P) are dynamic protein filaments that mediate bacterial adhesion, biofilm formation, and twitching motility. The highly conserved PilMNOP proteins form an inner membrane alignment subcomplex required for function of the T4P system, though their exact roles are unclear. Three potential interaction interfaces for PilNO were identified: core-core, coiled coils (CC), and the transmembrane segments (TMSs). A high-confidence PilNO heterodimer model was used to select key residues for mutation, and the resulting effects on protein-protein interactions were examined both in a bacterial two-hybrid (BTH) system and in their nativePseudomonas aeruginosacontext. Mutations in the oppositely charged CC regions or the TMS disrupted PilNO heterodimer formation in the BTH assay, while up to six combined mutations in the core failed to disrupt the interaction. When the mutations were introduced into theP. aeruginosachromosome at thepilNorpilOlocus, specific changes at each of the three interfaces—including core mutations that failed to disrupt interactions in the BTH system—abrogated surface piliation and/or impaired twitching motility. Unexpectedly, specific CC mutants were hyperpiliated but nonmotile, a hallmark of pilus retraction defects. These data suggest that PilNO participate in both the extension and retraction of T4P. Our findings support a model of multiple, precise interaction interfaces between PilNO; emphasize the importance of studying protein function in a minimally perturbed context and stoichiometry; and highlight potential target sites for development of small-molecule inhibitors of the T4P system.IMPORTANCEPseudomonas aeruginosais an opportunistic pathogen that uses type IV pili (T4P) for host attachment. The T4P machinery is composed of four cell envelope-spanning subcomplexes. PilN and PilO heterodimers are part of the alignment subcomplex and essential for T4P function. Three potential PilNO interaction interfaces (the core-core, coiled-coil, and transmembrane segment interfaces) were probed using site-directed mutagenesis followed by functional assays in anEscherichia colitwo-hybrid system and inP. aeruginosa. Several mutations blocked T4P assembly and/or motility, including two that revealed a novel role for PilNO in pilus retraction, while other mutations affected extension dynamics. These critical PilNO interaction interfaces represent novel targets for small-molecule inhibitors with the potential to disrupt T4P function.

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Five New Genes Are Important for Common Polysaccharide Antigen Biosynthesis in Pseudomonas aeruginosa

mBio ◽

10.1128/mbio.00631-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 13

Author(s):

Youai Hao ◽

Jerry D. King ◽

Steven Huszczynski ◽

Dana Kocíncová ◽

Joseph S. Lam

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Surface ◽

Gene Cluster ◽

Opportunistic Pathogen ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Polysaccharide Antigen ◽

Content Type ◽

New Genes ◽

Cell Surface Structure

ABSTRACTCommon polysaccharide antigen (CPA) is a conserved cell surface polysaccharide produced byPseudomonas aeruginosa. It contains a rhamnan homopolymer and is one of the two forms of O polysaccharide attached toP. aeruginosalipopolysaccharide (LPS). Our laboratory has previously characterized an eight-gene cluster (pa5447-pa5454inP. aeruginosaPAO1) required for biosynthesis of CPA. Here we demonstrate that an adjacent five-gene clusterpa5455-pa5459is also involved. Using reverse transcriptase PCR (RT-PCR), we showed that the original eight-gene cluster and the new five-gene cluster are both organized as operons. We have analyzed the LPS phenotypes of in-frame deletion mutants made in each of the five genes, and the results verified that these five genes are indeed required for CPA biosynthesis, extending the CPA biosynthesis locus to contain 13 contiguous genes. By performing overexpression experiments of different sets of these biosynthesis genes, we were able to obtain information about their possible functions in CPA biosynthesis.IMPORTANCELipopolysaccharide (LPS) is an important cell surface structure of Gram-negative bacteria. The human opportunistic pathogenPseudomonas aeruginosasimultaneously produces an O-antigen-specific (OSA) form and a common polysaccharide antigen (CPA) form of LPS. CPA, the focus of this study, is composed of α-1-2, α1-3-linkedd-rhamnose sugars and has been shown to be important for attachment of the bacteria to human airway epithelial cells. Genome sequencing of this species revealed a new five-gene cluster that we predicted to be involved in CPA biosynthesis and modification. In this study, we have generated chromosomal knockouts by performing in-frame deletions and allelic replacements. Characterizing the function of each of the five genes is important for us to better understand CPA biosynthesis and the mechanisms of chain length termination and regulation of this unique D-rhamnan polysaccharide.

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