Pantoea stewartii WceF is a glycan biofilm-modifying enzyme with a bacteriophage tailspike-like fold

Pathogenic microorganisms often reside in glycan-based biofilms. Concentration and chain length distribution of these mostly anionic exopolysaccharides (EPS) determine the overall biophysical properties a biofilm result highly viscous environment. Bacterial communities regulate this state via intracellular small-molecule signaling to initiate EPS synthesis. Reorganization or degradation glycan matrix, however, requires action extracellular glycosidases. So far, were mainly described for bacteriophages that must degrade biofilms gaining access host bacteria. The plant pathogen Pantoea stewartii (P. stewartii) encodes protein WceF within its synthesis cluster. has homologs various forming pathogens Erwinia family. In work, we show is glycosidase active on stewartan, main P. component. remarkable structural similarity with bacteriophage tailspike proteins (TSPs). Crystal structure analysis showed native trimer right-handed parallel β-helices. Despite similar fold, lacks high stability found TSPs. stewartan hydrolase produces oligosaccharides, corresponding single repeat units. However, compared stewartan-specific origin, lectin-like autoagglutination resulting notably slower cleavage velocities. This emphasizes bacterial enzyme role matrix reorganization clearly different from exopolysaccharide depolymerase. Many produce matrices composed polymeric substances organize themselves microbial (1Flemming H.C. Wingender J. Szewzyk U. Steinberg Rice S.A. Kjelleberg S. Biofilms: An emergent form life.Nat. Rev. Microbiol. 2016; 14: 563-575Crossref PubMed Scopus (2050) Google Scholar, 2Lohse M.B. Gulati M. Johnson A.D. Nobile C.J. Development regulation single- multi-species Candida albicans biofilms.Nat. 2018; 16: 19-31Crossref (227) Scholar). production exerts multitude effects not only lifestyle embedded cells, but also their interactions environment pathogenicity. 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Stewartii.Mol. 2009; 74: 903-913Crossref (24) Additionally, 90% modified (6→1)-β-d-GlcVI. regulated cell-density-dependent controls gene systems (19Carlier Most located wce-I cluster Wzy-dependent pathway 20Raetz C.R. Lipopolysaccharide endotoxins.Annu. Biochem. 2002; 71: 635-700Crossref (3171) Glycosyltransferases encoded catalyze RU assembly. glycosyltransferase region (wceB, K, M, N) contains two genes, wceJ wceF. WceJ was be nonfunctional pyruvate-transferase, which virulence. wceF (formerly designated cpsH) conserved (Table S1 Fig. S1). It 80 kDa polypeptide (736 amino acids) following Tat-secretion signal. A function so far described. Mutations resulted increased size up 7.6 MDa suggested (21Schollmeyer Langlotz Huber Variations molecular masses amylovoran, pyrifolan stewartan.Int. Macromol. 2012; 50: 518-522Crossref (5) report crystal interaction purified exopolysaccharide. shows (TSPs) enzymatically proposing when mucoid, producing state. We recombinantly expressed, purified, crystallized lacking N-terminal 28 acids Tat-signal peptide Tat-pathway folded (22Cline Mechanistic aspects transport twin arginine translocase (tat).J. 16530-16538Abstract (49) sequences typically cleaved off signal peptidase once transported. therefore consider our recombinant construct (WceF residues 29–736) represents WceF. solved 2.55 Å resolution, using selenomethionine variant phasing S2). 34 736 resolved electron density. confirmed size-exclusion chromatography, homotrimer about 200 Each head domain, neck central β-helix domain followed short triple β-helix, β-sandwich, C-terminal stem 2). (residues 34–146) made β-sheets four antiparallel β-strands flanked α-helices. Electron density 25 residues. five-stranded Greek-key motif surrounding one α-helix then forms 166–229) body large three-stranded 236–514) eight complete rungs capped 252–265). At C-terminus makes β-hairpin turn 120° kinks turns 517–531). intertwined chains again separate individually β-sandwich jelly roll fold arranged four-stranded sheets 531–667). C terminus, smaller strands, respectively, connected loop 678–736). All regions intertwine structure. classified Conserved Domain Database (CCD) resemblance TSP P22 (23Steinbacher Miller Baxa Budisa N. Weintraub Seckler Phage protein: head-binding 2.3 A, fully refined endorhamnosidase 1.56 basis O-antigen recognition cleavage.J. Mol. 1997; 267: 865-880Crossref (149) 24Marchler-Bauer Bo Han L.Y. J.E. Lanczycki Lu S.N. Chitsaz Derbyshire M.K. Geer R.C. Gonzales N.R. Gwadz Hurwitz D.I. Marchler G.H. Song J.S. et al.CDD/SPARCLE: Functional classification subfamily architectures.Nucleic Acids 45: D200-D203Crossref (1492) comparison DALI P22TSP N terminus (Cα rmsd 2.6 Å) S3) (25Holm Rosenstrom Dali server: Conservation mapping 3D.Nucleic 2010; 38: W545-W549Crossref (2965) inserted between N-terminus superimposes very well (DALI: Cα 1.1 insert preceding tailspikes CBA120 (26Plattner Shneider M.M. Arbatsky N.P. Shashkov Chizhov A.O. Nazarov Prokhorov N.S. Taylor N.M.I. Buth Gambino Gencay Y.E. Brondsted Kutter E.M. Knirel Y.A. Leiman P.G. receptor-binding CBA120.J. 431: 3718-3739Crossref (28) HHPred identified (27Zimmermann Stephens Nam S.Z. Rau Kubler Lozajic Gabler Soding Lupas A.N. Alva completely reimplemented MPI bioinformatics toolkit new HHpred server core.J. 430: 2237-2243Crossref (934) best hits, TSPs viunalikevirus phage podovirus 28Adriaenssens Ackermann H.-W. Anany Blasdel B. Connerton I.F. Goulding Griffiths M.W. Hooton S.P. Kropinski A.M. Lee J.-H. Maes Pickard Ryu Sepehrizadeh Z. al.A genus: “Viunalikevirus”.Arch. Virol. 157: 2035-2046Crossref (57) 29Greenfield Shang X.R. Luo Zhou Heselpoth R.D. Nelson Herzberg O. machinery ORF212 E. coli O157:H7 (TSP3).Sci. Rep. 9: 7349Crossref (14) 30Seul Muller J.J. Andres Stettner Heinemann Bacteriophage tailspike: interdomain linker.Acta Crystallogr. D 70: 1336-1345Crossref (16) Among all hits many trimers parallel, β-helices structurally S3). Moreover, similarities diverse set degrading enzymes, polygalacturonases, 236–514). thermostable ensure infectivity even under harsh conditions (31Barbirz Becker Freiberg beta-solenoid carbohydrate binding materials.Macromol Biosci. 169-173Crossref Their trimeric assemblies kinetically stabilized, slow denaturation temperatures above 70 °C, presence detergents. contrast, spite assembly TSPs, rapidly denatured 2% (w/v) SDS room temperature 3B). migrated monomer SDS-PAGE, whereas HK620 remained SDS. shown before, monomers after heating 100 °C several minutes prior electrophoresis (32Miller Schuler Removal unmasks folding mutations native-state thermal stability.Protein 1998; 7: 2223-2232Crossref (36) Hence, less stable detergent origin. agreement calculated lower interface stabilization S9). WceF, areas found; markedly properties, predicted contribute interface. N-terminally truncated variants assess S4). Constructs P22TSP-homologous soluble aggregated, presumably because they impaired stabilization. more residues, i.e., cap insoluble. contrast P22TSP, dispensable (33Danner Fuchs Folding N-terminal, domain.Eur. 1993; 215: 653-661Crossref (59) Scholar), hence conclude Wcef domains. Depolymerization capsules, lipopolysaccharide essentially drive process (34Latka Maciejewska Majkowska-Skrobek G. Briers Drulis-Kawa Bacteriophage-encoded virion-associated overcome during process.Appl. Biotechnol. 101: 3103-3119Crossref (122) 35Broeker N.K. Roske Valleriani Stephan M.S. Koetz Time-resolved DNA release O-antigen-specific Salmonella contractile tail.J. 11751-11761Abstract right-handed, prokaryotic origin involved (36Bradley Cowen Menke King Berger BETAWRAP: Successful prediction beta -helices primary sequence association pathogens.Proc. 2001; 98: 14819-14824Crossref (90) tested major isolated three-day-old solution (14Dunsing mixed solutions lost 3 days S5). this, assumed analyzed WceF-stewartan mixtures reducing end 4A). Reducing ends slowly over time course than only, 3-methyl-2-benzothiazolinon-hydrazone (MBTH) method (37Zhang Y.Q. Wang Z.P. Chen C.G. Wu Q. L.L. X.Y. Quantitative determination chitinolytic lysozyme half-deacetylated chitosan substrate.Carbohydr. Polym. 85: 554-559Crossref (10) time, observed sudden, 30-fold increase velocity. alone did whole 300 h experiment. highest pH 5 4B) decreased increasing salt concentrations 4C). No absorption 235 nm experiment would characteristic double bond putative activity. stewartan. number bacteriophages. To compare enzyme, φEa1H. φEa1HTSP shares 25% identity (38Hartung Fulbright D.W. Klos E.J. Cloning expression amylovora.Mol. Microbe. 1988; 1: 87-93Crossref significantly higher velocity reached saturation 30 min 5). broad range 10 independent concentration S6). Also, had use tenfold (0.14 μM) experiments (1.50 able follow reaction. efficient earlier, used oligosaccharides units, respectively S7) (39Nimtz Qiu fire blight amylovora.Carbohydr. 287: 59-76Crossref (95) cleaves φ-1,3-glycosidic galactoses (cf. 1). standards capillary (CE) analyze products obtained either 6). Due glycosidic velocities oligosaccharide reaction times, 197 6 φEa1HTSP, respectively. CE elution profiles 1RU standard, depolymerized heptasaccharides (7mers). 2RU tetrakaidekasaccharides (14mers). Analytical chromatography MALDI-MS further fragments S7). propose like β-1,3 galactosidase. Concentrated mg ml−1 earlier notable hindrance Native (ca. kDa, cf. S2) may diffuse solutions. indeed exhibited initial phase 4), behavior ATTO 488-labeled fluorescence microscopy 7). absence slightly prone aggregation, indicated small fluorescent spots. much larger spots estimated diameter μm, indicating associated roughly spherical aggregates. Particles too freely 1 autoagglutinate while does mask sites φEa1HTSP. Free restored equally correlation spectroscopy Scholar) S8). Glycan-modifying regularly machines polysaccharides encapsulation formation. respect, cleave bonds seems counterintuitive, and, indeed, biochemical combined mutagenesis studies points multifaceted functional roles dep