Metabolism of a hybrid algal galactan by members of the human gut microbiome.

Native porphyran is a hybrid of porphryan and agarose. As a common element of edible seaweed, this algal galactan is a frequent component of the human diet. Bacterial members of the human gut microbiota have acquired polysaccharide utilization loci (PULs) that enable the metabolism of porphyran or agarose. However, the molecular mechanisms that underlie the deconstruction and use of native porphyran remains incompletely defined. Here, we have studied two human gut bacteria, porphyranolytic Bacteroides plebeius and agarolytic Bacteroides uniformis, that target native porphyran. This reveals an exo-based cycle of porphyran depolymerization that incorporates a keystone sulfatase. In both PULs this cycle also works together with a PUL-encoded agarose depolymerizing machinery to synergistically reduce native porphyran to monosaccharides. This provides a framework for understanding the deconstruction of a hybrid algal galactan, and insight into the competitive and/or syntrophic relationship of gut microbiota members that target rare nutrients.

The structure of a family 110 glycoside hydrolase provides insight into the hydrolysis of α-1,3-galactosidic linkages in λ-carrageenan and blood group antigens.

α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1-3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure-function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1-3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1-3Gal revealed the parallel ß-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.

Clinical Mutations That Partially Activate the Stringent Response Confer Multidrug Tolerance in Staphylococcus aureus.

Antibiotic tolerance is an underappreciated antibiotic escape strategy that is associated with recurrent and relapsing infections, as well as acting as a precursor to resistance. Tolerance describes the ability of a bacterial population to survive transient exposure to an otherwise lethal concentration of antibiotic without exhibiting an elevated MIC. It is detected in time-kill assays as a lower rate of killing than a susceptible strain and can be quantified by the metric minimum duration for killing (MDK). The molecular mechanisms behind tolerance are varied, but activation of the stringent response (SR) via gene knockouts and/or chemical induction has long been associated with tolerance. More recently, two Gram-positive clinical isolates from persistent bacteremias were found to bear mutations in the SR controller, Rel, that caused elevated levels of the alarmone (p)ppGpp. Here, we show that introduction of either of these mutations into Staphylococcus aureus confers tolerance to five different classes of antibiotic as a result of (p)ppGpp-mediated growth defects (longer lag time and/or lower growth rate). The degree of tolerance is related to the severity of the growth defect and ranges from a 1.5- to 3.1-fold increase in MDK. Two classes of proposed SR inhibitor were unable to reverse or reduce this tolerance. Our findings reveal the significance of SR-activating mutations in terms of tolerance and clinical treatment failures. The panel of strains reported here provide a clinically relevant model of tolerance for further investigation of its link to resistance development, as well as potential validation of high-throughput tolerance screens.

Biochemical Reconstruction of a Metabolic Pathway from a Marine Bacterium Reveals Its Mechanism of Pectin Depolymerization.

Pectin is a complex uronic acid-containing polysaccharide typically found in plant cell walls, though forms of pectin are also found in marine diatoms and seagrasses. Genetic loci that target pectin have recently been identified in two phyla of marine bacteria. These loci appear to encode a pectin saccharification pathway that is distinct from the canonical pathway typically associated with phytopathogenic terrestrial bacteria. However, very few components of the marine pectin metabolism pathway have been experimentally validated. Here, we biochemically reconstructed the pectin saccharification pathway from a marine Pseudoalteromonas sp. in vitro and show that it results in the production of galacturonate and the key metabolic intermediate 5-keto-4-deoxyuronate (DKI). We demonstrate the sequential de-esterification and depolymerization of pectin into oligosaccharides and the synergistic action of glycoside hydrolases (GHs) to fully degrade these oligosaccharides into monosaccharides. Furthermore, we show that this pathway relies on enzymes belonging to GH family 105 to carry out the equivalent chemistry afforded by an exolytic polysaccharide lyase (PL) and KdgF in the canonical pectin pathway. Finally, we synthesize our findings into a model of marine pectin degradation and compare it with the canonical pathway. Our results underline the shifting view of pectin as a solely terrestrial polysaccharide and highlight the importance of marine pectin as a carbon source for suitably adapted marine heterotrophs. This alternate pathway has the potential to be exploited in the growing field of biofuel production from plant waste.IMPORTANCE Marine polysaccharides, found in the cell walls of seaweeds and other marine macrophytes, represent a vast sink of photosynthetically fixed carbon. As such, their breakdown by marine microbes contributes significantly to global carbon cycling. Pectin is an abundant polysaccharide found in the cell walls of terrestrial plants, but it has recently been reported that some marine bacteria possess the genetic capacity to degrade it. In this study, we biochemically characterized seven key enzymes from a marine bacterium that, together, fully degrade the backbone of pectin into its constituent monosaccharides. Our findings highlight the importance of pectin as a marine carbon source available to bacteria that possess this pathway. The characterized enzymes also have the potential to be utilized in the production of biofuels from plant waste.

Properties of a family 56 carbohydrate-binding module and its role in the recognition and hydrolysis of ß-1,3-glucan.

BH0236 from Bacillus halodurans is a multimodular ß-1,3-glucanase comprising an N-terminal family 81 glycoside hydrolase catalytic module, an internal family 6 carbohydrate-binding module (CBM) that binds the nonreducing end of ß-1,3-glucan chains, and an uncharacterized C-terminal module classified into CBM family 56. Here, we determined that this latter CBM, BhCBM56, bound the soluble ß-1,3-glucan laminarin with a dissociation constant (Kd ) of ∼26 µm and displayed higher affinity for insoluble ß-1,3-glucans with Kd values of ∼2-10 µm but lacked affinity for ß-1,3-glucooligosaccharides. The X-ray crystal structure of BhCBM56 and NMR-derived chemical shift mapping of the binding site revealed a ß-sandwich fold, with the face of one ß-sheet possessing the ß-1,3-glucan-binding surface. On the basis of the functional and structural properties of BhCBM56, we propose that it binds a quaternary polysaccharide structure, most likely the triple helix adopted by polymerized ß-1,3-glucans. Consistent with the BhCBM56 and BhCBM6/56 binding profiles, deletion of the CBM56 from BH0236 decreased activity of the enzyme on the insoluble ß-1,3-glucan curdlan but not on soluble laminarin; additional deletion of the CBM6 also did not affect laminarin degradation but further decreased curdlan hydrolysis. The pseudo-atomic solution structure of BH0236 determined by small-angle X-ray scattering revealed structural insights into the nature of avid binding by the BhCBM6/56 pair and how the orientation of the active site in the catalytic module factors into recognition and degradation of ß-1,3-glucans. Our findings reinforce the notion that catalytic modules and their cognate CBMs have complementary specificities, including targeting of polysaccharide quaternary structure.

A Fluorogenic Disaccharide Substrate for α-Mannosidases Enables High-Throughput Screening and Identification of an Inhibitor of the GH92 Virulence Factor from Streptococcus pneumoniae.

Trimming of host glycans is a mechanism that is broadly employed by both commensal and pathogenic microflora to enable colonization. Host glycan trimming by the opportunistic Gram-positive bacterium Streptococcus pneumoniae has been demonstrated to be an important mechanism of virulence. While S. pneumoniae employs a multitude of glycan processing enzymes, the exo-mannosidase SpGH92 has been shown to be an important virulence factor. Accordingly, SpGH92 is hypothesized to be a target for much-needed new treatments of S. pneumoniae infection. Here we report the synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→2)-ß-d-mannopyranoside (Manα1,2Manß-4MU) as a fluorogenic disaccharide substrate and development of an assay for SpGH92 that overcomes its requirement for +1 binding site occupancy. We miniaturize our in vitro assay and apply it to a high-throughput screen of >65 000 compounds, identifying a single inhibitory chemotype, LIPS-343. We further show that Manα1,2Manß-4MU is also a substrate of the human Golgi-localized α-mannosidase MAN1A1, suggesting that this substrate should be useful for assessing the activity of this and other mammalian α-mannosidases.

Sulfatases: Critical Enzymes for Algal Polysaccharide Processing.

Microbial sulfatases are important biocatalysts in the marine environment where they play a key role in the catabolic biotransformation of abundant sulphated algal polysaccharides. The sulphate esters decorating algal polysaccharides, such as carrageenan, fucoidan and ulvan, can constitute up to 40% of the biopolymer dry weight. The use of this plentiful carbon and energy source by heterotrophic microbes is enabled in part by the sulfatases encoded in their genomes. Sulfatase catalysed hydrolytic removal of sulphate esters is a key reaction at various stages of the enzymatic cascade that depolymerises sulphated polysaccharides into monosaccharides that can enter energy yielding metabolic pathways. As the critical roles of sulfatases in the metabolism of sulphated polysaccharides from marine algae is increasingly revealed, the structural and functional analysis of these enzymes becomes an important component of understanding these metabolic pathways. The S1 family of formylglycine-dependent sulfatases is the largest and most functionally diverse sulfatase family that is frequently active on polysaccharides. Here, we review this important sulfatase family with emphasis on recent developments in studying the structural and functional relationship between sulfatases and their sulphated algal polysaccharide substrates. This analysis utilises the recently proposed active site nomenclature for sulfatases. We will highlight the key role of sulfatases, not only in marine carbon cycling, but also as potential biocatalysts for the production of a variety of novel tailor made sulphated oligomers, which are useful products in, for example, pharmaceutical or cosmetic applications.

Insights into the κ/ι-carrageenan metabolism pathway of some marine Pseudoalteromonas species.

Pseudoalteromonas is a globally distributed marine-associated genus that can be found in a broad range of aquatic environments, including in association with macroalgal surfaces where they may take advantage of these rich sources of polysaccharides. The metabolic systems that confer the ability to metabolize this abundant form of photosynthetically fixed carbon, however, are not yet fully understood. Through genomics, transcriptomics, microbiology, and specific structure-function studies of pathway components we address the capacity of newly isolated marine pseudoalteromonads to metabolize the red algal galactan carrageenan. The results reveal that the κ/ι-carrageenan specific polysaccharide utilization locus (CarPUL) enables isolates possessing this locus the ability to grow on this substrate. Biochemical and structural analysis of the enzymatic components of the CarPUL promoted the development of a detailed model of the κ/ι-carrageenan metabolic pathway deployed by pseudoalteromonads, thus furthering our understanding of how these microbes have adapted to a unique environmental niche.

The Molecular Basis of Polysaccharide Sulfatase Activity and a Nomenclature for Catalytic Subsites in this Class of Enzyme.

Sulfatases play a biologically important role by cleaving sulfate groups from molecules. They can be identified on the basis of signature sequences within their primary structures, and the largest family, S1, has predictable features that contribute specifically to the recognition and catalytic removal of sulfate groups. However, despite advances in the prediction and understanding of S1 sulfatases, a major question regards the molecular determinants that drive substrate recognition beyond the targeted sulfate group. Here, through analysis of an endo-4S-ι-carrageenan sulfatase (PsS1_19A) from Pseudoalteromonas sp. PS47, particularly X-ray crystal structures in complex with intact substrates, we show that specific recognition of the substrate leaving group components, in this case carbohydrate, provides the enzyme with specificity for its substrate. On the basis of these results we propose a catalytic subsite nomenclature that we anticipate will form a general foundation for understanding and describing the molecular basis of substrate recognition by sulfatases.