A polysaccharide utilization locus from Chitinophaga pinensis simultaneously targets chitin and β-glucans found in fungal cell walls

Abstract

In nature, complex carbohydrates are rarely found as pure isolated polysaccharides. Instead, bacteria in competitive environments are presented with glycans embedded in heterogeneous matrices such as plant or microbial cell walls. Members of the Bacteroidota phylum thrive in such ecosystems because they are efficient at extracting nutrients from complex substrates, secreting consortia of synergistic enzymes to release metabolizable sugars. Carbohydrate-binding modules (CBMs) are used to target enzymes to substrates, enhancing reaction rate and product release. Additionally, genome organizational tools like polysaccharide utilization loci (PULs) ensure that the appropriate set of enzymes is produced when needed. In this study, we show that the soil bacterium Chitinophaga pinensis uses a PUL and several CBMs to coordinate the activities of enzymes targeting two distinct polysaccharides found in fungal cell walls. We describe the enzymatic activities and carbohydrate-binding behaviors of components of the fungal cell wall utilization locus (FCWUL), which uses multiple chitinases and one β-1,3-glucanase to hydrolyze two different substrates. Unusually, one of the chitinases is appended to a β-glucan-binding CBM, implying targeting to a bulk cell wall substrate rather than to the specific polysaccharide being hydrolyzed. Based on our characterization of the PUL's outer membrane sensor protein, we suggest that the FCWUL is activated by β-1,3-glucans, even though most of its enzymes are chitin-degrading. Our data showcase the complexity of polysaccharide deconstruction in nature and highlight an elegant solution for how multiple different glycans can be accessed using one enzymatic cascade. IMPORTANCE We report that the genome of the soil bacterium Chitinophaga pinensis encodes three multi-modular carbohydrate-active enzymes that work together to hydrolyze the major polysaccharide components found in fungal cell walls (FCWs). The enzymes are all encoded by one polysaccharide utilization locus and are co-expressed, potentially induced in the presence of β-1,3-glucans. We present biochemical characterization of each enzyme, including the appended carbohydrate-binding modules that likely tether the enzymes to a FCW substrate. Finally, we propose a model for how this so-called fungal cell wall utilization locus might enable C. pinensis to metabolize both chitin and β-1,3-glucans found in complex biomass in the soil.

Keywords: Bacteroidota; Chitinophaga; carbohydrate-binding module; chitin; glycoside hydrolase; polysaccharide utilization locus; β-1,3-glucan.

Fig 1

Putative PULs from selected Bacteroidota species. Top, the Chitinophaga pinensis FCWUL is proposed to span genes with locus tags Cpin_2184 to Cpin_2192, as shown. The colored genes in the C. pinensis locus have equivalents (i.e., the same CAZyme modularity or predicted function) in the other loci depicted, which are shown in the same color. The gene product of Cpin_2185 is a putative β/γ crystallin lysyl endopeptidase according to KEGG and NCBI automated annotations. Cpin_2189 and Cpin_2190 encode an ECF-σ and anti-σ factor, respectively. The arrows on each gene point in the direction of transcription. PULs from other species with full or partial predicted functional synteny to the FCWUL are shown, with color coding matched to the CAZyme architecture of the labeled components of the C. pinensis locus. Syntenic PULs were identified using the KEGG server gene cluster analysis tool together with manual NCBI searching.

Fig 2

Chitinase activity of CpChiA. Exo-chitinase activity of CpChiA_C (A, B) and CpChiA_C-CBM5 (C, D) is demonstrated via HPAEC-PAD analysis of hydrolytic products generated during incubation with β-chitin (A, C). GlcNAc and Chi2 are the major products from this substrate, and the product profile does not change over time, indicating an exo mode of hydrolysis. The same final products are released from the pentasaccharide substrate Chi5 (B, D). (E) Kinetic analysis of CpChiA_C and CpChiA_C-CBM5 against the 4MU-Chi3 fluorescent substrate.

Fig 3

Chitinase and chitin-binding activities of CpChiB. Exo-chitinase activity of CpChiB (A) and CpChiB-CBM6 (B) is demonstrated after HPAEC-PAD analysis of hydrolytic products released from β-chitin after incubation for 30 min or up to 8 h. The product profile does not change over time, indicating an exo mode of hydrolysis. (C) Kinetic analysis of CpChiB and CpChiB-CBM6.

Fig 4

Endo β-1,3-glucanase activity of CpGlu16A on curdlan. (A and B ) Endo-glucanase activity of CpGlu16A-CBM6 is demonstrated by HPAEC-PAD analysis of products released from curdlan after incubation from 15 min to 8 h. Longer oligosaccharides (L3–L6) are produced in the first few minutes and are then hydrolyzed, leading to the final products of Glc and L2. (C) Kinetic analysis of CpGlu16A-CBM6 activity.

Fig 5

Proposed pathway of synergistic action by the proteins of the FCWUL. Left panel: model of a fungal cell wall undergoing hydrolysis by FCWUL enzymes. Right panel: theoretical model of the FCWUL (OM, outer membrane of bacterial cell; IM, inner membrane of bacterial cell). Components are not shown to scale in either panel. All FCWUL enzymes are depicted as being extracellular, because they all carry standard bacterial SpI signal peptides (19 - 21) and (except for CpGlu16A) the C-terminal domain for secretion via the type IX secretion system, T9SS (22). Dark blue squares, GlcNAc; light blue circles, β-1,3-linked Glc; orange circles, β-1,6-linked Glc; white circles, terminal Glc; green circles, mannose found on glycosylated FCW proteins.