Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane

Yan Wang; Archana Andole Pannuri; Dongchun Ni; Haizhen Zhou; Xiou Cao; Xiaomei Lu; Tony Romeo; Yihua Huang

doi:10.1074/jbc.M115.711762

Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane*

From the ^‡National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China,
^§University of Chinese Academy of Sciences, Beijing 100049, China,
^¶Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611-0700,
^‖Department of Cardiovascular Diseases, Tianjin Xiqing Hospital, Tianjin 300380, China,
^**School of Life Sciences, Peking University, Beijing 100871, China, and
^‡‡Dongguan Institute of Pediatrics, the Eighth People's Hospital of Dongguan, Dongguan 523325, Guangdong Province, China

↵2 To whom correspondence may be addressed. Tel.: 86-10-64888789; E-mail: tromeo{at}ufl.edu.
↵3 To whom correspondence may be addressed. Tel.: 86-10-64888789; E-mail: yihuahuang{at}sun5.ibp.ac.cn.

↵1 Both authors contributed equally to this work.

Abstract

The partially de-N-acetylated poly-β-1,6-N-acetyl-d-glucosamine (dPNAG) polymer serves as an intercellular biofilm adhesin that plays an essential role for the development and maintenance of integrity of biofilms of diverse bacterial species. Translocation of dPNAG across the bacterial outer membrane is mediated by a tetratricopeptide repeat-containing outer membrane protein, PgaA. To understand the molecular basis of dPNAG translocation, we determined the crystal structure of the C-terminal transmembrane domain of PgaA (residues 513–807). The structure reveals that PgaA forms a 16-strand transmembrane β-barrel, closed by four loops on the extracellular surface. Half of the interior surface of the barrel that lies parallel to the translocation pathway is electronegative, suggesting that the corresponding negatively charged residues may assist the secretion of the positively charged dPNAG polymer. In vivo complementation assays in a pgaA deletion bacterial strain showed that a cluster of negatively charged residues proximal to the periplasm is necessary for biofilm formation. Biochemical analyses further revealed that the tetratricopeptide repeat domain of PgaA binds directly to the N-deacetylase PgaB and is critical for biofilm formation. Our studies support a model in which the positively charged PgaB-bound dPNAG polymer is delivered to PgaA through the PgaA-PgaB interaction and is further targeted to the β-barrel lumen of PgaA potentially via a charge complementarity mechanism, thus priming the translocation of dPNAG across the bacterial outer membrane.

Footnotes

↵* This work was supported by National Institutes of Health Grants GM059969 and GM066794 (to T. R.). This work was also supported by National Natural Science Foundation of China Grants 31470743 and 31170698 (to Y. H.), Ministry of Science and Technology (China) “973” Project Grants 2013CB910603 and 2012CB917302 (to Y. H.), Chinese Academy of Sciences Grant XDB08020302 (to Y. H.), and University of Florida CHRIS Project FLA-MCS-004949 (to T. R.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The atomic coordinates and structure factors (code 4Y25) have been deposited in the Protein Data Bank (http://wwpdb.org/).