In Campylobacter jejuni, a new type of chaperone receives heme from ferrochelatase.

Intracellular heme formation and trafficking are fundamental processes in living organisms. Bacteria and archaea utilize three biogenesis pathways to produce iron protoporphyrin IX (heme b) that diverge after the formation of the common intermediate uroporphyrinogen III (uro'gen III). In this study, we identify and provide a detailed characterization of the enzymes involved in the transformation of uro'gen III into heme in Campylobacter jejuni, demonstrating that this bacterium utilizes the protoporphyrin-dependent (PPD) pathway. In general, limited knowledge exists regarding the mechanisms by which heme b reaches its target proteins after this final step. Specifically, the chaperones necessary for trafficking heme to prevent the cytotoxic effects associated with free heme remain largely unidentified. In C. jejuni, we identified a protein named CgdH2 that binds heme with a dissociation constant of 4.9 ± 1.0 µM, and this binding is impaired upon mutation of residues histidine 45 and 133. We demonstrate that C. jejuni CgdH2 establishes protein-protein interactions with ferrochelatase, suggesting its role in facilitating heme transfer from ferrochelatase to CgdH2. Furthermore, phylogenetic analysis reveals that C. jejuni CgdH2 is evolutionarily distinct from the currently known chaperones. Therefore, CgdH2 is the first protein identified as an acceptor of intracellularly formed heme, expanding our knowledge of the mechanisms underlying heme trafficking within bacterial cells.

The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome.

The Bacteroides thetaiotaomicron has developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RG-II depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of the module at the C-terminal domain, which we designated BT0996-C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical ß-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.