title: A polysaccharide utilization locus fromFlavobacterium johnsoniaeenables conversion of recalcitrant chitin.
abstract: BACKGROUND
  Chitin is the second most abundant polysaccharide on earth and as such a great target for bioconversion applications. The phylum Bacteroidetes is one of nature's most ubiquitous bacterial lineages and is essential in the global carbon cycle with many members being highly efficient degraders of complex carbohydrates. However, despite their specialist reputation in carbohydrate conversion, mechanisms for degrading recalcitrant crystalline polysaccharides such as chitin and cellulose are hitherto unknown.
  
  
  RESULTS
  Here we describe a complete functional analysis of a novel polysaccharide utilization locus (PUL) in the soil BacteroideteFlavobacterium johnsoniae, tailored for conversion of chitin. TheF. johnsoniaechitin utilization locus (ChiUL) consists of eleven contiguous genes encoding carbohydrate capture and transport proteins, enzymes, and a two-component sensor-regulator system. The key chitinase (ChiA) encoded by ChiUL is atypical in terms of known Bacteroidetes-affiliated PUL mechanisms as it is not anchored to the outer cell membrane and consists of multiple catalytic domains. We demonstrate how the extraordinary hydrolytic efficiency of ChiA derives from synergy between its multiple chitinolytic (endo- andexo-acting) and previously unidentified chitin-binding domains. Reverse genetics show that ChiA and PUL-encoded proteins involved in sugar binding, import, and chitin sensing are essential for efficient chitin utilization. Surprisingly, the ChiUL encodes two pairs of SusC/D-like outer membrane proteins. Ligand-binding and structural studies revealed functional differences between the two SusD-like proteins that enhance scavenging of chitin from the environment. The combined results from this study provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and reveal important novel aspects of the PUL paradigm.
  
  
  CONCLUSIONS
  By combining reverse genetics to map essential PUL genes, structural studies on outer membrane chitin-binding proteins, and enzymology, we provide insight into the mechanisms employed by Bacteroidetes to degrade recalcitrant polysaccharides and introduce a new saccharolytic mechanism used by the phylum Bacteroidetes. The presented discovery and analysis of the ChiUL will greatly benefit future enzyme discovery efforts as well as studies regarding enzymatic intramolecular synergism.
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway ; Wallenberg Wood Science Center, Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96 Gothenburg, Sweden.
authors.forename: Johan
authors.identifier: 0000-0001-8386-2914
authors.initials: J
authors.lastname: Larsbrink
authors.affiliation: Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201 USA.
authors.forename: Yongtao
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201 USA.
authors.forename: Sampada S
authors.initials: SS
authors.lastname: Kharade
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109 USA.
authors.forename: Kurt J
authors.initials: KJ
authors.lastname: Kwiatkowski
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway.
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109 USA.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Koropatkin
authors.affiliation: Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201 USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: McBride
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway.
authors.forename: Phillip B
authors.initials: PB
authors.lastname: Pope
keywords: Bacteroidetes; Chitin; Polysaccharide utilization locus; Recalcitrant polysaccharides