AlgK is a TPR-containing protein and the periplasmic component of a novel exopolysaccharide secretin.

The opportunistic pathogen Pseudomonas aeruginosa causes chronic biofilm infections in cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate. Here we show that AlgK, a protein essential for production of high molecular weight alginate, is an outer membrane lipoprotein that contributes to the correct localization of the porin AlgE. Our 2.5 A structure shows AlgK is composed of 9.5 tetratricopeptide-like repeats, and three putative sites of protein-protein interaction have been identified. Bioinformatics analysis suggests that BcsA, PgaA, and PelB, involved in the production and export of cellulose, poly-beta-1,6-N-Acetyl-D-glucosamine, and Pel exopolysaccharide, respectively, share the same topology as AlgK/E. Together, our data suggest that AlgK plays a role in the assembly of the alginate biosynthetic complex and represents the periplasmic component of a new type of outer membrane secretin that differs from canonical bacterial capsular polysaccharide secretion systems.

[1]  D. Eisenberg,et al.  A combined algorithm for genome-wide prediction of protein function , 1999, Nature.

[2]  M. Jinek,et al.  The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin α , 2004, Nature Structural &Molecular Biology.

[3]  H. Ertesvåg,et al.  The dual roles of AlgG in C‐5‐epimerization and secretion of alginate polymers in Pseudomonas aeruginosa , 2003, Molecular microbiology.

[4]  G J Kleywegt,et al.  Phi/psi-chology: Ramachandran revisited. , 1996, Structure.

[5]  Paul Stoodley,et al.  Bacterial biofilms: from the Natural environment to infectious diseases , 2004, Nature Reviews Microbiology.

[6]  D. Higgins,et al.  T-Coffee: A novel method for fast and accurate multiple sequence alignment. , 2000, Journal of molecular biology.

[7]  Carrie-Lynn Keiski,et al.  Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas fluorescens AlgK. , 2007, Acta crystallographica. Section F, Structural biology and crystallization communications.

[8]  Dohyun Han,et al.  Crystal structure of PilF: functional implication in the type 4 pilus biogenesis in Pseudomonas aeruginosa. , 2006, Biochemical and biophysical research communications.

[9]  P. Howell,et al.  Expression, refolding, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgE. , 2009, Acta crystallographica. Section F, Structural biology and crystallization communications.

[10]  Daniel Kahne,et al.  Identification of a Multicomponent Complex Required for Outer Membrane Biogenesis in Escherichia coli , 2005, Cell.

[11]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[12]  M. Rohde,et al.  The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix , 2001, Molecular microbiology.

[13]  V. Deretic,et al.  Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. , 1996, Microbiological reviews.

[14]  B. Lugtenberg,et al.  Outer membranes of gram-negative bacteria. , 1987, Biochemical Society transactions.

[15]  S. Matsuyama,et al.  Deletion of lolB, Encoding an Outer Membrane Lipoprotein, Is Lethal for Escherichia coli and Causes Accumulation of Lipoprotein Localization Intermediates in the Periplasm , 2001, Journal of bacteriology.

[16]  Vincent T. Lee,et al.  The second messenger bis‐(3′‐5′)‐cyclic‐GMP and its PilZ domain‐containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa , 2007, Molecular microbiology.

[17]  Carlos C. Goller,et al.  Roles of pgaABCD Genes in Synthesis, Modification, and Export of the Escherichia coli Biofilm Adhesin Poly-β-1,6-N-Acetyl-d-Glucosamine , 2008, Journal of bacteriology.

[18]  J. Tommassen,et al.  Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties , 1994, Journal of bacteriology.

[19]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[20]  K. Henrick,et al.  Inference of macromolecular assemblies from crystalline state. , 2007, Journal of molecular biology.

[21]  B. Rehm,et al.  In Vitro Alginate Polymerization and the Functional Role of Alg8 in Alginate Production by Pseudomonas aeruginosa , 2006, Applied and Environmental Microbiology.

[22]  R. Hancock,et al.  Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier , 1978, Journal of bacteriology.

[23]  D. Wozniak,et al.  Role of polysaccharides in Pseudomonas aeruginosa biofilm development. , 2007, Current opinion in microbiology.

[24]  M. P. Gallagher,et al.  A novel gene, algK, from the alginate biosynthesis cluster of Pseudomonas aeruginosa. , 1997, Microbiology.

[25]  U. Römling.  Molecular biology of cellulose production in bacteria. , 2002, Research in microbiology.

[26]  J. Tropea,et al.  Structural characterization of the Yersinia pestis type III secretion system needle protein YscF in complex with its heterodimeric chaperone YscE/YscG , 2008, Journal of molecular biology.

[27]  B. Rehm,et al.  Alg44, a unique protein required for alginate biosynthesis in Pseudomonas aeruginosa , 2006, FEBS letters.

[28]  I. Henderson,et al.  Membrane protein architects: the role of the BAM complex in outer membrane protein assembly , 2009, Nature Reviews Microbiology.

[29]  A. Pugsley,et al.  Secretins take shape , 2006, Molecular microbiology.

[30]  Creg Darby.  Uniquely insidious: Yersinia pestis biofilms. , 2008, Trends in microbiology.

[31]  Jack Snoeyink,et al.  Nucleic Acids Research Advance Access published April 22, 2007 MolProbity: all-atom contacts and structure validation for proteins and nucleic acids , 2007 .

[32]  D. Amikam,et al.  Genetic organization of the cellulose synthase operon in Acetobacter xylinum. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[33]  R. Kolter,et al.  Exopolysaccharide Production Is Required for Development of Escherichia coli K-12 Biofilm Architecture , 2000, Journal of bacteriology.

[34]  J. Preston,et al.  The pgaABCD Locus of Escherichia coli Promotes the Synthesis of a Polysaccharide Adhesin Required for Biofilm Formation , 2004, Journal of bacteriology.

[35]  Rolf Apweiler,et al.  InterProScan - an integration platform for the signature-recognition methods in InterPro , 2001, Bioinform..

[36]  Lynne Regan,et al.  Sequence variation in ligand binding sites in proteins , 2005, BMC Bioinformatics.

[37]  S. Valla,et al.  AlgX Is a Periplasmic Protein Required for Alginate Biosynthesis in Pseudomonas aeruginosa , 2004, Journal of bacteriology.

[38]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[39]  P. Howell,et al.  Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa PelD. , 2012, Acta crystallographica. Section F, Structural biology and crystallization communications.

[40]  C. Sander,et al.  Protein structure comparison by alignment of distance matrices. , 1993, Journal of molecular biology.

[41]  Gerard J. Kleywegt,et al.  Crystallographic refinement of ligand complexes , 2006, Acta crystallographica. Section D, Biological crystallography.

[42]  Thomas C. Terwilliger,et al.  Automated MAD and MIR structure solution , 1999, Acta crystallographica. Section D, Biological crystallography.

[43]  Luis Moroder,et al.  Structure of TPR Domain–Peptide Complexes Critical Elements in the Assembly of the Hsp70–Hsp90 Multichaperone Machine , 2000, Cell.

[44]  Roberto Kolter,et al.  Biofilms: the matrix revisited. , 2005, Trends in microbiology.

[45]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[46]  D. Ohman,et al.  Role of an Alginate Lyase for Alginate Transport in Mucoid Pseudomonas aeruginosa , 2005, Infection and Immunity.

[47]  D. Ohman,et al.  Membrane topology and roles of Pseudomonas aeruginosa Alg8 and Alg44 in alginate polymerization. , 2008, Microbiology.

[48]  C. Chothia,et al.  Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure. , 2001, Journal of molecular biology.

[49]  M. Grütter,et al.  The crystal structure of Helicobacter cysteine-rich protein C at 2.0 A resolution: similar peptide-binding sites in TPR and SEL1-like repeat proteins. , 2004, Journal of molecular biology.

[50]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[51]  C. Whitfield.  Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. , 2006, Annual review of biochemistry.

[52]  R. Kolter,et al.  Biofilm formation as microbial development. , 2000, Annual review of microbiology.

[53]  Stavros J. Hamodrakas,et al.  PRED-TMBB: a web server for predicting the topology of ?barrel outer membrane proteins , 2004, Nucleic Acids Res..

[54]  D T Jones,et al.  Protein secondary structure prediction based on position-specific scoring matrices. , 1999, Journal of molecular biology.

[55]  J. O’Gara.  ica and beyond: biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus. , 2007, FEMS microbiology letters.

[56]  Thomas C. Terwilliger,et al.  Electronic Reprint Biological Crystallography Automated Main-chain Model Building by Template Matching and Iterative Fragment Extension , 2022 .

[57]  J. Koo,et al.  PilF Is an Outer Membrane Lipoprotein Required for Multimerization and Localization of the Pseudomonas aeruginosa Type IV Pilus Secretin , 2008, Journal of bacteriology.

[58]  J. Eswaran,et al.  Three's company: component structures bring a closer view of tripartite drug efflux pumps. , 2004, Current opinion in structural biology.

[59]  R. Kolter,et al.  Two Genetic Loci Produce Distinct Carbohydrate-Rich Structural Components of the Pseudomonas aeruginosa Biofilm Matrix , 2004, Journal of bacteriology.

[60]  B. Matthews,et al.  Use of differentially substituted selenomethionine proteins in X-ray structure determination. , 1999, Acta crystallographica. Section D, Biological crystallography.

[61]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[62]  Thomas R. Schneider,et al.  HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs , 2004 .

[63]  C. Whitfield,et al.  Periplasmic export machines for outer membrane assembly. , 2008, Current opinion in structural biology.

[64]  D. Ohman,et al.  Deletion of algK in Mucoid Pseudomonas aeruginosa Blocks Alginate Polymer Formation and Results in Uronic Acid Secretion , 1998, Journal of bacteriology.

[65]  Zukang Feng,et al.  Validation of protein structures for protein data bank. , 2003, Methods in enzymology.

[66]  J. Simorre,et al.  Structure of the heterotrimeric complex that regulates type III secretion needle formation , 2007, Proceedings of the National Academy of Sciences.

[67]  G. Harauz,et al.  Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30 , 2003, Journal of Biological Chemistry.

[68]  Martin Ester,et al.  Sequence analysis PSORTb v . 2 . 0 : Expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis , 2004 .