A polysaccharide utilization locus from Flavobacterium johnsoniae enables conversion of recalcitrant chitin

Abstract

BackgroundChitin 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.ResultsHere we describe a complete functional analysis of a novel polysaccharide utilization locus (PUL) in the soil Bacteroidete Flavobacterium johnsoniae, tailored for conversion of chitin. The F. johnsoniae chitin 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- and exo-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.ConclusionsBy 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0674-z) contains supplementary material, which is available to authorized users.

Identification of a major glucose transporter in Flavobacterium johnsoniae: Inhibition of F. johnsoniae colony spreading by glucose uptake

“…In this study, we found that: (i) the mfsA mutant showed no glucose uptake; (ii) the mfsA mutant did not utilize glucose for its growth; and (iii) the mfsA gene complemented the glucose‐negative phenotype of E. coli LJ141. The present findings in combination with previous findings strongly indicate that mfsA (Fjoh_4565, which is one of the eight above‐mentioned genes) encodes the sole glucose transporter in F. johnsoniae. Comparison with proteins in the MFS family using the International Union of Biochemistry and Molecular Biology (IUBMB) approved Transporter Classification Database (http://www.tcbd.org) revealed that a protein most similar to MfsA is glucose/galactose transporter Ggp (2.A.1.7.2) in Brucella abortus , which belongs to the fucose:H+ symporter (FHS) family (2.A.1.7) and that the top 13 proteins similar to MfsA belong to the FHS family.…”

“…A recent study by Larsbrink et al . identified a locus containing mfsA and 10 other genes that were involved in F. johnsoniae chitin utilization; mfsA was shown to be required for growth on glucose, N ‐acetylglucosamine, and chitin. Our results confirm and extend these findings.…”

“…2a). Fjoh_4565 was recently shown to be required for growth on glucose, N-acetyl-glucosamine, and chitin (28). We tentatively named this gene mfsA.…”

Polysaccharide Utilization Loci: Fueling Microbial Communities

“…Recently, the detailed genetic, biochemical, and enzyme structural characterization of a galactomannanspecific PUL from B. ovatus revealed the interplay of two mannan-specific SGBPs, two GH26 endo-␤-mannanases, and a GH36 exo-␣-galactosidase in the deconstruction of this plant cell wall polysaccharide (110,111). Among environmental bacteria, a complex chitin utilization locus from Flavobacterium johnsoniae has been extensively functionally characterized (112). Notable features of the system include two pairs of SusC/SusD homologs, a secreted chitinase composed of two GH18 modules separated by a chitin-binding module, and an intracellular glucosamine-6-phosphate deaminase.…”

Flavobacterium alkalisoli sp. nov., isolated from rhizosphere soil of Suaeda salsa

“…seawater, freshwater, wastewater, sediments, marine sediment, rhizosphere soil, alga, compost, glacier and plant [4], suggesting that Flavobacterium strains were ubiquitous in diverse terrestrial and aquatic environments. Some Flavobacterium strains were found to be able to degrade chitin [12], polysaccharide [13], diesel [14], and pesticide [15], as well as to suppress plant pathogen [16], suggesting that Flavobacterium strains play a vital role in the ecosystem. Therefore, the study of characterization for Flavobacterium strains isolated from saline-alkaline may help us to understand the roles of Flavobacterium in the ecosystem of saline soil.…”