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Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control.

ABSTRACT: Lipopolysaccharides (LPS) are essential outer membrane glycolipids in most gram-negative bacteria. Biosynthesis of the O-antigenic polysaccharide (OPS) component of LPS follows one of three widely distributed strategies, and similar processes are used to assemble other bacterial surface glycoconjugates. This study focuses on the ATP-binding cassette (ABC) transporter-dependent pathway, where glycans are completed on undecaprenyl diphosphate carriers at the cytosol:membrane interface, before export by the ABC transporter. We describe Raoultella terrigena WbbB, a prototype for a family of proteins that, remarkably, integrates several key activities in polysaccharide biosynthesis into a single polypeptide. WbbB contains three glycosyltransferase (GT) modules. Each of the GT102 and GT103 modules characterized here represents a previously unrecognized GT family. They form a polymerase, generating a polysaccharide of [4)-?-Rhap-(1?3)-?-GlcpNAc-(1?] repeat units. The polymer chain is terminated by a ?-linked Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) residue added by a third GT module belonging to the recently discovered GT99 family. The polymerase GT modules are separated from the GT99 chain terminator by a coiled-coil structure that forms a molecular ruler to determine product length. Different GT modules in the polymerase domains of other family members produce diversified OPS structures. These findings offer insight into glycan assembly mechanisms and the generation of antigenic diversity as well as potential tools for glycoengineering.

SUBMITTER: Williams DM

PROVIDER: S-EPMC5321029 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control.

Williams Danielle M DM Ovchinnikova Olga G OG Koizumi Akihiko A Mainprize Iain L IL Kimber Matthew S MS Lowary Todd L TL Whitfield Chris C

Proceedings of the National Academy of Sciences of the United States of America 20170130 7

Lipopolysaccharides (LPS) are essential outer membrane glycolipids in most gram-negative bacteria. Biosynthesis of the O-antigenic polysaccharide (OPS) component of LPS follows one of three widely distributed strategies, and similar processes are used to assemble other bacterial surface glycoconjugates. This study focuses on the ATP-binding cassette (ABC) transporter-dependent pathway, where glycans are completed on undecaprenyl diphosphate carriers at the cytosol:membrane interface, before expo ...[more]

PMID: 28137848

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Project description:The outer leaflet of the outer membrane of nearly all Gram-negative bacteria contains lipopolysaccharide (LPS). The distal end of LPS may be capped with O antigen, a long polysaccharide that can range from a few to hundreds of sugars in length. The chain length of the polysaccharide has many implications for bacterial survival and consequently is tightly controlled. In the Wzx/Wzy-dependent route of O antigen synthesis, one or more Wzz proteins determine the chain length via an unknown mechanism. To gain insight into this mechanism, we identified and characterized important regions of two Wzz proteins in Pseudomonas aeruginosa serotype O13, which confer the production of "long" (Wzz1) and "very long" (Wzz2) chain lengths, respectively. We found that compared to Wzz1, Wzz2 has distinct amino acid insertions in the central α-helices (insα6 and insα7) and in membrane-distal (insL4) and -proximal (insIL) loops. When these regions were deleted in Wzz2, the mutant proteins conferred drastically shortened chain lengths. Within these regions we identified several conserved amino acid residues that were then targeted for site-directed mutagenesis. Our results implicate an RTE motif in loop 4 and a "hot spot" of charged and polar residues in insα7 in the function of Wzz2 We present evidence that the functionally important residues of insα7 are likely involved in stabilizing Wzz through coiled-coil interactions.IMPORTANCE O antigen is an important virulence factor presented on the cell surface of Gram-negative bacteria that is critical for bacterial physiology and pathogenesis. However, some aspects of O antigen biosynthesis, such as the mechanisms for determining polysaccharide chain length, are poorly understood. In this study, we identified unique regions in the O antigen chain length regulators (termed Wzz) of the problematic opportunistic pathogen Pseudomonas aeruginosa We show that these regions are critical for determining O antigen chain length, which provides new insight into the model of the Wzz mechanism. Ultimately, our work adds knowledge toward understanding an important step in the biosynthesis of this virulence factor, which is applicable to a wide range of Gram-negative pathogens.

Dataset Information

Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control.

Publications

Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control.

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