A Broad-Spectrum α-Glucosidase of Glycoside Hydrolase Family 13 from Marinovum sp., a Member of the Roseobacter Clade.

Glycoside hydrolases (GHs) are a diverse group of enzymes that catalyze the hydrolysis of glycosidic bonds. The Carbohydrate-Active enZymes (CAZy) classification organizes GHs into families based on sequence data and function, with fewer than 1% of the predicted proteins characterized biochemically. Consideration of genomic context can provide clues to infer possible enzyme activities for proteins of unknown function. We used the MultiGeneBLAST tool to discover a gene cluster in Marinovum sp., a member of the marine Roseobacter clade, that encodes homologues of enzymes belonging to the sulfoquinovose monooxygenase pathway for sulfosugar catabolism. This cluster lacks a gene encoding a classical family GH31 sulfoquinovosidase candidate, but which instead includes an uncharacterized family GH13 protein (MsGH13) that we hypothesized could be a non-classical sulfoquinovosidase. Surprisingly, recombinant MsGH13 lacks sulfoquinovosidase activity and is a broad-spectrum α-glucosidase that is active on a diverse array of α-linked disaccharides, including maltose, sucrose, nigerose, trehalose, isomaltose, and kojibiose. Using AlphaFold, a 3D model for the MsGH13 enzyme was constructed that predicted its active site shared close similarity with an α-glucosidase from Halomonas sp. H11 of the same GH13 subfamily that shows narrower substrate specificity.

Defining the molecular architecture, metal dependence, and distribution of metal-dependent class II sulfofructose-1-phosphate aldolases.

Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) is a sulfosugar that is the anionic head group of plant, algal, and cyanobacterial sulfolipids: sulfoquinovosyl diacylglycerols. SQ is produced within photosynthetic tissues, forms a major terrestrial reservoir of biosulfur, and is an important species within the biogeochemical sulfur cycle. A major pathway for SQ breakdown is the sulfoglycolytic Embden-Meyerhof-Parnas pathway, which involves cleavage of the 6-carbon chain of the intermediate sulfofructose-1-phosphate (SFP) into dihydroxyacetone and sulfolactaldehyde, catalyzed by class I or II SFP aldolases. While the molecular basis of catalysis is understood for class I SFP aldolases, comparatively little is known about class II SFP aldolases. Here, we report the molecular architecture and biochemical basis of catalysis of two metal-dependent class II SFP aldolases from Hafnia paralvei and Yersinia aldovae. 3D X-ray structures of complexes with substrate SFP and product dihydroxyacetone phosphate reveal a dimer-of-dimers (tetrameric) assembly, the sulfonate-binding pocket, two metal-binding sites, and flexible loops that are implicated in catalysis. Both enzymes were metal-dependent and exhibited high KM values for SFP, consistent with their role in a unidirectional nutrient acquisition pathway. Bioinformatic analysis identified a range of sulfoglycolytic Embden-Meyerhof-Parnas gene clusters containing class I/II SFP aldolases. The class I and II SFP aldolases have mututally exclusive occurrence within Actinobacteria and Firmicutes phyla, respectively, while both classes of enzyme occur within Proteobacteria. This work emphasizes the importance of SQ as a nutrient for diverse bacterial phyla and the different chemical strategies they use to harvest carbon from this sulfosugar.

Conservation, abundance, glycosylation profile, and localization of the TSP protein family in Cryptosporidium parvum.

Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling cryptosporidiosis. At present, the only approved vaccine against any apicomplexan parasite targets a conserved adhesin possessing a thrombospondin repeat domain. C. parvum possesses 12 orthologous thrombospondin repeat domain-containing proteins known as CpTSP1-12, though little is known about these potentially important antigens. Here, we explore the architecture and conservation of the CpTSP protein family, as well as their abundance at the protein level within the sporozoite stage of the life cycle. We examine the glycosylation states of these proteins using a combination of glycopeptide enrichment techniques to demonstrate that these proteins are modified with C-, O-, and N-linked glycans. Using expansion microscopy, and an antibody against the C-linked mannose that is unique to the CpTSP protein family within C. parvum, we show that these proteins are found both on the cell surface and in structures that resemble the secretory pathway of C. parvum sporozoites. Finally, we generated a polyclonal antibody against CpTSP1 to show that it is found at the cell surface and within micronemes, in a pattern reminiscent of other apicomplexan motility-associated adhesins, and is present both in sporozoites and meronts. This work sheds new light on an understudied family of C. parvum proteins that are likely to be important to both parasite biology and the development of vaccines against cryptosporidiosis.

Cuantificación de bacterias diazótrofas aisladas de suelos cacaoteros (Theobroma cacao L.), por la técnica de Número Más Probable (NMP) / Quantification of diazotrophs bacteria isolated from cocoa soils (Theobroma cacao L.), by the technique of Most Probable Number (MPN)

Esta investigación tuvo como objetivo cuantificar bacterias diazótrofas y comparar fisicoquímicamente suelos rizosféricos de tres cacaotales (Theobroma cacao L.) del Departamento Norte de Santander, Colombia; para lo cual se caracterizaron, diferenciándose en área cultivada, manejo agronómico y edad del cultivo. A partir de diluciones seriadas de las muestras y empleando la técnica de Número Más Probable (NMP), en medios de cultivo semiselectivos (NFb, JMV, LGI, JNFb) semisólidos, se cuantificaron las diazótrofas, evaluando como positivo la formación de una película subsuperficial en el medio contenido en viales sellados; muestras pares se enviaron al laboratorio Bioambiental (UNET) para los análisis fisicoquímicos. Como resultado, las muestras evaluadas mostraron deficiencias en el porcentaje de materia orgánica y elementos como Potasio, Fósforo y Magnesio. Se reportaron estadísticamente diferencias altamente significativas en NMP. La mayor cuantificación de diazótrofas se reportó en la finca Florilandia, que se caracterizó por tener riego por goteo. La mayor cuantificación de diazotrófas se registró en los medios NFb y JMV, demostrándose una mayor presencia de los presuntos géneros Azospirillum sp. y Burkholderia sp. los cuales son fácilmente aislados de suelos rizosféricos, a diferencia de los géneros Herbaspirillum sp. y Gluconacetobacter sp. que por su carácter endófito suelen ser menos predominantes en este tipo de muestras. Se concluye además, que las características fisicoquímicas del suelo, la humedad y las relaciones climáticas al momento de la toma de las muestras, condicionan la cantidad de exudados de las raíces y por tanto son factores que condicionaron la presencia de diazótrofas en las muestras.

The objective of this research was to quantify diazotrophic bacteria and compare physicochemically rhizospheric soils of three cocoa plantations (Theobroma cacao L.) in Norte de Santander Department, Colombia; for which they were characterized, differing in cultivated area, agronomic management and crop age. From serial dilutions of the samples and using the technique of Most Probable Number (MPN), In semisolid culture media (NFb, JMV, LGI, JNFb), the diazotrophs were quantified, evaluating as positive the formation of a subsurface film in the medium contained in sealed vials; equal samples were sent to the Bioambiental laboratory (UNET) for physicochemical analyzes. As a result, the evaluated samples showed deficiencies in the percentage of organic matter and elements such as Potassium, Phosphorus and Magnesium. Statistically highly significant differences in MPN were reported. The highest quantification of diazotrophs was reported in the Florilandia farm, which was characterized by drip irrigation. The highest quantification of diazotrophs was recorded in the media NFb and JMV, demonstrating a greater presence of the presumed genera Azospirillum sp. and Burkholderia sp. which are easily isolated from rhizospheric soils, unlike the genera Herbaspirillum sp. and Gluconacetobacter sp. which by their endophytic character tend to be less predominant in this type of samples. It is also concluded that the physicochemical characteristics of the soil, humidity and climatic relationships at the moment of sampling, condition the amount of root exudates and therefore are factors that conditioned the presence of diazotrophs in the samples.