title: Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis.
abstract: Together with fungi, saprophytic bacteria are central to the decomposition and recycling of biomass in forest environments. The Bacteroidetes phylum is abundant in diverse habitats, and several species have been shown to be able to deconstruct a wide variety of complex carbohydrates. The genus Chitinophaga is often enriched in hotspots of plant and microbial biomass degradation. We present a proteomic assessment of the ability of Chitinophaga pinensis to grow on and degrade mannan polysaccharides, using an agarose plate-based method of protein collection to minimise contamination with exopolysaccharides and proteins from lysed cells, and to reflect the realistic setting of growth on a solid surface. We show that select Polysaccharide Utilisation Loci (PULs) are expressed in different growth conditions, and identify enzymes that may be involved in mannan degradation. By comparing proteomic and enzymatic profiles, we show evidence for the induced expression of enzymes and PULs in cells grown on mannan polysaccharides compared with cells grown on glucose. In addition, we show that the secretion of putative biomass-degrading enzymes during growth on glucose comprises a system for nutrient scavenging, which employs constitutively produced enzymes.
  
  
  
  Chitinophaga pinensis belongs to a bacterial genus which is prominent in microbial communities in agricultural and forest environments, where plant and fungal biomass is intensively degraded. Such degradation is hugely significant in the recycling of carbon in the natural environment, and the enzymes responsible are of biotechnological relevance in emerging technologies involving the deconstruction of plant cell wall material. The bacterium has a comparatively large genome, which includes many uncharacterised carbohydrate-active enzymes. We present the first proteomic assessment of the biomass-degrading machinery of this species, focusing on mannan, an abundant plant cell wall hemicellulose. Our findings include the identification of several novel enzymes, which are promising targets for future biochemical characterisation. In addition, the data indicate the expression of specific Polysaccharide Utilisation Loci, induced in the presence of different growth substrates. We also highlight how a constitutive secretion of enzymes which deconstruct microbial biomass likely forms part of a nutrient scavenging process.
authors.affiliation: Norwegian University of Life Sciences, NMBU, Department of Chemistry, Biotechnology and Food Sciences, PO Box 5003, NO-1432 Ås, Norway; Wallenberg Wood Science Center, Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University, 412 96 Gothenburg, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Larsbrink
authors.affiliation: Norwegian University of Life Sciences, NMBU, Department of Chemistry, Biotechnology and Food Sciences, PO Box 5003, NO-1432 Ås, Norway.
authors.forename: Tina R
authors.initials: TR
authors.lastname: Tuveng
authors.affiliation: Norwegian University of Life Sciences, NMBU, Department of Chemistry, Biotechnology and Food Sciences, PO Box 5003, NO-1432 Ås, Norway.
authors.forename: Phillip B
authors.initials: PB
authors.lastname: Pope
authors.affiliation: Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, 106 91 Stockholm, Sweden; ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, Urrbrae, SA 5064, Australia.
authors.forename: Vincent
authors.initials: V
authors.lastname: Bulone
authors.affiliation: Norwegian University of Life Sciences, NMBU, Department of Chemistry, Biotechnology and Food Sciences, PO Box 5003, NO-1432 Ås, Norway.
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
authors.affiliation: Michael Smith Laboratories, Department of Chemistry, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4, Canada; Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, 106 91 Stockholm, Sweden; Wallenberg Wood Science Center, Teknikringen 56-58, SE-100 44 Stockholm, Sweden.
authors.forename: Harry
authors.initials: H
authors.lastname: Brumer
authors.affiliation: Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, 106 91 Stockholm, Sweden; Wallenberg Wood Science Center, Teknikringen 56-58, SE-100 44 Stockholm, Sweden. Electronic address: mckee@kth.se.
authors.forename: Lauren S
authors.initials: LS
authors.lastname: McKee
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D041963:Bacteroidetes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D018533:Biomass; D050260:Carbohydrate Metabolism; D002244:Carbon / Q000378:metabolism; D065928:Forests; D005947:Glucose / Q000378:metabolism; D008351:Mannans / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D008351:Mannans; D002244:Carbon; D005947:Glucose
keywords: CAZyme; Chitinophaga pinensis; Galactoglucomannan; Label-free quantification; Secretome