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Pseudoalteromonas fuliginea sp. PS47 is a recently identified marine bacterium that has extensive enzymatic machinery to metabolize polysaccharides, including a locus that targets pectin‐like substrates. This locus contains a gene (locus tag EU509_03255) that encodes a pectin‐degrading lyase, called PfPL1, that belongs to polysaccharide lyase family 1 (PL1). The 2.2 Å resolution X‐ray crystal structure of PfPL1 reveals the compact parallel β‐helix fold of the PL1 family. The back side of the core parallel β‐helix opposite to the active site is a meandering set of five α‐helices joined by lengthy loops. A comparison of the active site with those of other PL1 enzymes suggests a catalytic mechanism that is independent of metal ions, such as Ca2+, but that substrate recognition may require metal ions. Overall, this work provides the first structural insight into a pectinase of marine origin and the first structure of a PL1 enzyme in subfamily 2. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae is a frequent colonizer of the upper airways; however, it is also an accomplished pathogen capable of causing life‐threatening diseases. To colonize and cause invasive disease, this bacterium relies on a complex array of factors to mediate the host–bacterium interaction. The respiratory tract is rich in functionally important glycoconjugates that display a vast range of glycans, and, thus, a key component of the pneumococcus–host interaction involves an arsenal of bacterial carbohydrate‐active enzymes to depolymerize these glycans and carbohydrate transporters to import the products. Through the destruction of host glycans, the glycan‐specific metabolic machinery deployed by S. pneumoniae plays a variety of roles in the host–pathogen interaction. Here, we review the processing and metabolism of the major host‐derived glycans, including N‐ and O‐linked glycans, Lewis and blood group antigens, proteoglycans, and glycogen, as well as some dietary glycans. We discuss the role of these metabolic pathways in the S. pneumoniae–host interaction, speculate on the potential of key enzymes within these pathways as therapeutic targets, and relate S. pneumoniae as a model system to glycan processing in other microbial pathogens. [ABSTRACT FROM AUTHOR]

Our current understanding of the temperature response of biological processes in soil is based on the Arrhenius equation. This predicts an exponential increase in rate as temperature rises, whereas in the laboratory and in the field, there is always a clearly identifiable temperature optimum for all microbial processes. In the laboratory, this has been explained by denaturation of enzymes at higher temperatures, and in the field, the availability of substrates and water is often cited as critical factors. Recently, we have shown that temperature optima for enzymes and microbial growth occur in the absence of denaturation and that this is a consequence of the unusual heat capacity changes associated with enzymes. We have called this macromolecular rate theory - MMRT (Hobbs et al., , ACS Chem. Biol. 8:2388). Here, we apply MMRT to a wide range of literature data on the response of soil microbial processes to temperature with a focus on respiration but also including different soil enzyme activities, nitrogen and methane cycling. Our theory agrees closely with a wide range of experimental data and predicts temperature optima for these microbial processes. MMRT also predicted high relative temperature sensitivity (as assessed by Q10 calculations) at low temperatures and that Q10 declined as temperature increases in agreement with data synthesis from the literature. Declining Q10 and temperature optima in soils are coherently explained by MMRT which is based on thermodynamics and heat capacity changes for enzyme-catalysed rates. MMRT also provides a new perspective, and makes new predictions, regarding the absolute temperature sensitivity of ecosystems - a fundamental component of models for climate change. [ABSTRACT FROM AUTHOR]

SUMMARYAntibiotic treatment failures in the absence of resistance are not uncommon. Recently, attention has grown around the phenomenon of antibiotic tolerance, an underappreciated contributor to recalcitrant infections first detected in the 1970s. Tolerance describes the ability of a bacterial population to survive transient exposure to an otherwise lethal concentration of antibiotic without exhibiting resistance. With advances in genomics, we are gaining a better understanding of the molecular mechanisms behind tolerance, and several studies have sought to examine the clinical prevalence of tolerance. Attempts have also been made to assess the clinical significance of tolerance through in vivo infection models and prospective/retrospective clinical studies. Here, we review the data available on the molecular mechanisms, detection, prevalence, and clinical significance of genotypic tolerance that span ~50 years. We discuss the need for standardized methodology and interpretation criteria for tolerance detection and the impact that methodological inconsistencies have on our ability to accurately assess the scale of the problem. In terms of the clinical significance of tolerance, studies suggest that tolerance contributes to worse outcomes for patients (e.g., higher mortality, prolonged hospitalization), but historical data from animal models are varied. Furthermore, we lack the necessary information to effectively treat tolerant infections. Overall, while the tolerance field is gaining much-needed traction, the underlying clinical significance of tolerance that underpins all tolerance research is still far from clear and requires attention.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a method in which a fluorophore is covalently attached to the reducing end of carbohydrates, thereby allowing high-resolution separation by electrophoresis and visualization. This method can be used for carbohydrate profiling and sequencing, as well as for determining the specificity of carbohydrate-active enzymes. Here we describe and demonstrate the use of FACE to separate and visualize the glycans released following digestion of oligosaccharides by glycoside hydrolases (GHs) using two examples: (i) the digestion of chitobiose by the streptococcal β-hexosaminidase GH20C and (ii) the digestion of glycogen by the GH13 member SpuA., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (Copyright © 2018 American Society for Microbiology.)

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a method in which a fluorophore is covalently attached to the reducing end of carbohydrates, thereby allowing visualization following high-resolution separation by electrophoresis. This method can be used for carbohydrate profiling and sequencing, as well as for the determination of the specificity of carbohydrate-active enzymes. Here, we describe and demonstrate the use of FACE to separate and visualize the glycans released following digestion of oligosaccharides by glycoside hydrolases (GHs) using two examples: (1) the digestion of chitobiose by the streptococcal β-hexosaminidase GH20C, and (2) the digestion of glycogen by the GH13 member SpuA.

Our current understanding of the temperature response of biological processes in soil is based on the Arrhenius equation. This predicts an exponential increase in rate as temperature rises, whereas in the laboratory and in the field, there is always a clearly identifiable temperature optimum for all microbial processes. In the laboratory, this has been explained by denaturation of enzymes at higher temperatures, and in the field, the availability of substrates and water is often cited as critical factors. Recently, we have shown that temperature optima for enzymes and microbial growth occur in the absence of denaturation and that this is a consequence of the unusual heat capacity changes associated with enzymes. We have called this macromolecular rate theory - MMRT (Hobbs et al., , ACS Chem. Biol. 8:2388). Here, we apply MMRT to a wide range of literature data on the response of soil microbial processes to temperature with a focus on respiration but also including different soil enzyme activities, nitrogen and methane cycling. Our theory agrees closely with a wide range of experimental data and predicts temperature optima for these microbial processes. MMRT also predicted high relative temperature sensitivity (as assessed by Q10 calculations) at low temperatures and that Q10 declined as temperature increases in agreement with data synthesis from the literature. Declining Q10 and temperature optima in soils are coherently explained by MMRT which is based on thermodynamics and heat capacity changes for enzyme-catalysed rates. MMRT also provides a new perspective, and makes new predictions, regarding the absolute temperature sensitivity of ecosystems - a fundamental component of models for climate change., (© 2014 John Wiley & Sons Ltd.)