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

ABSTRACT The wbp cluster of Pseudomonas aeruginosaO5 encodes a number of proteins involved in biosynthesis of the heteropolymeric and Wzy-dependent B-band O antigen, including Wzy, the O-antigen polymerase, and Wzz, the regulator of O-antigen chain length. A gene (formerly wbpF), contiguous with wzy in the wbp cluster, is predicted to encode a highly hydrophobic protein with multiple membrane-spanning domains. This secondary structure is consistent with that of Wzx (RfbX), the putative O-antigen unit translocase or “flippase.” Insertion of a Gmr cassette at two separate sites within the putativewzx gene led in both cases to the loss of B-band lipopolysaccharide (LPS) O-antigen production. To our knowledge, this is the first report of the successful generation of chromosomalwzx gene replacement mutations. Surprisingly, inactivation of wzx also led to a marked delay in production of the ATP-binding cassette–transporter-dependent, d-rhamnose homopolymer, A-band LPS. This effect on A-band LPS synthesis was alleviated by supplying multiple copies of WbpL in trans. WbpL, a WecA (Rfe) homologue, was shown recently to be essential for the initiation of both A-band and B-band LPS synthesis in P. aeruginosa O5 (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, Mol. Microbiol. 28:1103–1119, 1998). These results suggest that the delay in A-band LPS production may arise from insufficient access to WbpL when the completed B-band O unit is not successfully translocated to the periplasm. Without adequate WbpL, A-band LPS synthesis is delayed. A subset of wzx mutants appeared to have accumulated second-site mutations which either restored the normal expression of A-band LPS or abolished A-band expression completely. Complementation studies showed that all of the additional mutations affecting LPS synthesis that were characterized in this study were located within the B-band LPS genes.

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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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Cloning and surface expression of Pseudomonas aeruginosa O antigen in Escherichia coli.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.22.10716 ◽

1992 ◽

Vol 89 (22) ◽

pp. 10716-10720 ◽

Cited By ~ 30

Author(s):

J. B. Goldberg ◽

K. Hatano ◽

G. S. Meluleni ◽

G. B. Pier

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Surface Expression ◽

O Antigen

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Faculty Opinions recommendation of WaaL of Pseudomonas aeruginosa utilizes ATP in in vitro ligation of O antigen onto lipid A-core.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1089396.542628 ◽

2007 ◽

Author(s):

Miguel Valvano

Keyword(s):

Pseudomonas Aeruginosa ◽

Lipid A ◽

O Antigen

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

Journal of Bacteriology ◽

10.1128/jb.01546-07 ◽

2008 ◽

Vol 190 (6) ◽

pp. 1857-1865 ◽

Cited By ~ 32

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.

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The O-Antigen Gene Cluster of Escherichia coli O55:H7 and Identification of a New UDP-GlcNAc C4 Epimerase Gene

Journal of Bacteriology ◽

10.1128/jb.184.10.2620-2625.2002 ◽

2002 ◽

Vol 184 (10) ◽

pp. 2620-2625 ◽

Cited By ~ 70

Author(s):

Lei Wang ◽

Sandy Huskic ◽

Adam Cisterne ◽

Deborah Rothemund ◽

Peter R. Reeves

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Gene Cluster ◽

Gene Clusters ◽

The Other ◽

Recombination Site ◽

E Coli ◽

Antigen Gene ◽

O Antigen ◽

Usual Location

ABSTRACT Escherichia coli O55 is an important antigen which is often associated with enteropathogenic E. coli clones. We sequenced the genes responsible for its synthesis and identified genes for O-antigen polymerase, O-antigen flippase, four enzymes involved in GDP-colitose synthesis, and three glycosyltransferases, all by comparison with known genes. Upstream of the normal O-antigen region there is a gne gene, which encodes a UDP-GlcNAc epimerase for converting UDP-GlcNAc to UDP-GalNAc and is essential for O55 antigen synthesis. The O55 gne product has only 20 and 26% identity to the gne genes of Pseudomonas aeruginosa and E. coli O113, respectively. We also found evidence for the O55 gene cluster's having evolved from another gene cluster by gain and loss of genes. Only three of the GDP-colitose pathway genes are in the usual location, the other two being separated, although nearby. It is thought that the E. coli O157:H7 clone evolved from the O55:H7 clone in part by transfer of the O157 gene cluster into an O55 lineage. Comparison of genes flanking the O-antigen gene clusters of the O55:H7 and O157:H7 clones revealed one recombination site within the galF gene and located the other between the hisG and amn genes. Genes outside the recombination sites are 99.6 to 100% identical in the two clones, while most genes thought to have transferred with the O157 gene cluster are 95 to 98% identical.

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