RESUMEN
Laminarin, a ß(1,3)-glucan, serves as a storage polysaccharide in marine microalgae such as diatoms. Its abundance, water solubility and simple structure make it an appealing substrate for marine bacteria. Consequently, many marine bacteria have evolved strategies to scavenge and decompose laminarin, employing carbohydrate-binding modules (CBMs) as crucial components. In this study, we characterized two previously unassigned domains as laminarin-binding CBMs in multimodular proteins from the marine bacterium Christiangramia forsetii KT0803T, thereby introducing the new laminarin-binding CBM families CBM102 and CBM103. We identified four CBM102s in a surface glycan-binding protein (SGBP) and a single CBM103 linked to a glycoside hydrolase module from family 16 (GH16_3). Our analysis revealed that both modular proteins have an elongated shape, with GH16_3 exhibiting greater flexibility than SGBP. This flexibility may aid in the recognition and/or degradation of laminarin, while the constraints in SGBP could facilitate the docking of laminarin onto the bacterial surface. Exploration of bacterial metagenome-assembled genomes (MAGs) from phytoplankton blooms in the North Sea showed that both laminarin-binding CBM families are widespread among marine Bacteroidota. The high protein abundance of CBM102- and CBM103-containing proteins during phytoplankton blooms further emphasizes their significance in marine laminarin utilization.
Asunto(s)
Proteínas Bacterianas , Glucanos , Fitoplancton , Glucanos/metabolismo , Fitoplancton/metabolismo , Fitoplancton/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Bacteroidetes/metabolismo , Bacteroidetes/genética , Eutrofización , Diatomeas/metabolismo , Diatomeas/genética , Receptores de Superficie CelularRESUMEN
The carrageenophyte red alga Chondrus crispus produces three family 16 glycoside hydrolases (CcGH16-1, CcGH16-2, and CcGH16-3). Phylogenetically, the red algal GH16 members are closely related to bacterial GH16 homologs from subfamilies 13 and 14, which have characterized marine bacterial ß-carrageenase and ß-porphyranase activities, respectively, yet the functions of these CcGH16 hydrolases have not been determined. Here, we first confirmed the gene locus of the ccgh16-3 gene in the alga to facilitate further investigation. Next, our biochemical characterization of CcGH16-3 revealed an unexpected ß-porphyranase activity, since porphyran is not a known component of the C. crispus extracellular matrix. Kinetic characterization was undertaken on natural porphyran substrate with an experimentally determined molecular weight. We found CcGH16-3 has a pH optimum between 7.5 and 8.0; however, it exhibits reasonably stable activity over a large pH range (pH 7.0-9.0). CcGH16-3 has a KM of 4.0 ± 0.8 µM, a kcat of 79.9 ± 6.9 s-1, and a kcat/KM of 20.1 ± 1.7 µM-1 s-1. We structurally examined fine enzymatic specificity by performing a subsite dissection. CcGH16-3 has a strict requirement for D-galactose and L-galactose-6-sulfate in its -1 and +1 subsites, respectively, whereas the outer subsites are less restrictive. CcGH16-3 is one of a handful of algal enzymes characterized with a specificity for a polysaccharide unknown to be found in their own extracellular matrix. This ß-porphyranase activity in a carrageenophyte red alga may provide defense against red algal pathogens or provide a competitive advantage in niche colonization.
Asunto(s)
Chondrus , Rhodophyta , Chondrus/genética , Rhodophyta/genética , Polisacáridos , Glicósido Hidrolasas , BiologíaRESUMEN
Marine flavobacteria possess dedicated Polysaccharide Utilization Loci (PULs) enabling efficient degradation of a variety of algal polysaccharides. The expression of these PULs is tightly controlled by the presence of the substrate, yet details on the regulatory mechanisms are still lacking. The marine flavobacterium Zobellia galactanivorans DsijT digests many algal polysaccharides, including alginate from brown algae. Its complex Alginate Utilization System (AUS) comprises a PUL and several other loci. Here, we showed that the expression of the AUS is strongly and rapidly (<30 min) induced upon addition of alginate, leading to biphasic substrate utilization. Polymeric alginate is first degraded into smaller oligosaccharides that accumulate in the extracellular medium before being assimilated. We found that AusR, a GntR family protein encoded within the PUL, regulates alginate catabolism by repressing the transcription of most AUS genes. Based on our genetic, genomic, transcriptomic and biochemical results, we propose the first model of regulation for a PUL in marine bacteria. AusR binds to promoters of AUS genes via single, double or triple copies of operator. Upon addition of alginate, secreted enzymes expressed at a basal level catalyze the initial breakdown of the polymer. Metabolic intermediates produced during degradation act as effectors of AusR and inhibit the formation of AusR/DNA complexes, thus lifting transcriptional repression.
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Alginatos/metabolismo , Proteínas Bacterianas/metabolismo , Flavobacteriaceae/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/metabolismo , Flavobacteriaceae/metabolismo , Regiones Promotoras GenéticasRESUMEN
Alginate is a major compound of brown macroalgae and as such an important carbon and energy source for heterotrophic marine bacteria. Despite the rather simple composition of alginate only comprising mannuronate and guluronate units, these bacteria feature complex alginolytic systems that can contain up to seven alginate lyases. This reflects the necessity of large enzyme systems for the complete degradation of the abundant substrate. Numerous alginate lyases have been characterized. They belong to different polysaccharide lyase (PL) families, but only one crystal structure of a family 17 (PL17) alginate lyase has been reported to date, namely Alg17c from the gammaproteobacterium Saccharophagus degradans. Biochemical and structural characterizations are helpful to link sequence profiles to function, evolution of functions and niche-specific characteristics. Here, we combined detailed biochemical and crystallographic analysis of AlyA3, a PL17 alginate lyase from the marine flavobacteria Zobellia galactanivorans DsijT, providing the first structure of a PL17 in the Bacteroidetes phylum. AlyA3 is exo-lytic and highly specific of mannuronate stretches. As part of an "alginate utilizing locus", its activity is complementary to that of other characterized alginate lyases from the same bacterium. Structural comparison with Alg17c highlights a common mode of action for exo-lytic cleavage of the substrate, strengthening our understanding of the PL17 catalytic mechanism. We show that unlike Alg17c, AlyA3 contains an inserted flexible loop at the entrance to the catalytic groove, likely involved in substrate recognition, processivity and turn over.
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Flavobacteriaceae/enzimología , Polisacárido Liasas/química , Polisacárido Liasas/metabolismo , Biocatálisis , Polisacárido Liasas/genética , Conformación ProteicaRESUMEN
Sea lettuce (Ulva) is recognised for its potential in food, pharmaceutical, nutraceutical, biorefinery and bioremediation industries and is increasingly being cultivated. The requirements of those industries vary widely in terms of biomass composition. Ulva biomass composition and growth is known to be directly influenced by environmental factors, e.g., temperature, light, salinity, nutrient availability as well as by genetic factors and likely by microbiome composition. In order to select for the highest yielding strains in a given environment, we tested the suitability of common-garden experiments, i.e., the co-cultivation of different strains grown under shared conditions. Fifteen strains from six different foliose Ulva species were grown together under two different salinities, 35 ppt and 15 ppt. After 32 days, only U. australis strains remained at both salinities. If selection at low salinity was mostly based on survival, the selection process at seawater salinity was driven by competition, largely based on growth performance. Growth rates after a month were very similar at both salinities, suggesting the U. australis strains cope equally well in either condition. However, the composition of the biomass produced in both environments varied, with the content of all organic compounds being higher at low salinity, and the ash content being reduced in average by 66%. To summarize, this study provides an established bulk-selection protocol for efficiently screening large numbers of locally-sourced strains and highlights the potential of low salinity treatments for increased organic matter content, particularly in carbohydrates. Supplementary Information: The online version contains supplementary material available at 10.1007/s10811-024-03222-0.
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Ulvan is a green macroalgal cell wall polysaccharide that has tremendous potential for valorisation due to its unique composition of sulphated rhamnose, glucuronic acid, iduronic acid and xylose. Several potential applications such as production of biofuels, bioplastics and other value-added products necessitate the breakdown of the polysaccharide to oligomers or monomers. Research on ulvan saccharifying enzymes has been continually increasing over the last decade, with the increasing focus on valorisation of seaweed biomass for a biobased economy. Lyases are the first of several enzymes that are involved in saccharifying the polysaccharide and several ulvan lyases have been structurally and biochemically characterised to enable their effective use in the valorisation processes. This study investigates the whole genome of Vibrio sp. FNV38, an ulvan metabolising organism and biochemical characteristics of a PL24 ulvan lyase that it possesses. The genome of Vibrio sp. FNV38 has a diverse CAZy profile with several genes involved in the metabolism of ulvan, cellulose, agar, and alginate. The enzyme exhibits optimal activity at pH 8.5 in 100 mM Tris-HCl buffer and 30 °C. However, its thermal stability is poor with significant loss of activity after 2 h of incubation at temperatures above 25 °C. Breakdown product analysis reveals that the enzyme depolymerised the polysaccharide predominantly to disaccharides and tetrasaccharides. Supplementary Information: The online version contains supplementary material available at 10.1007/s10811-023-03136-3.
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In this review, we summarize the current state of knowledge on the biosynthesis of carrageenan by exploring both the enzyme activities and their localizations. Genomic data, with the sequencing of the genome of Chondrus crispus and the first transcriptomic study into the life cycle stages of this organism, as well as fine carbohydrate structural determination of matrix glycans, provide leads in the study of carrageenan anabolism. Comparison to related carbohydrate-active enzymes, detailed phylogenies alongside classic histochemical studies and radioactivity assays, help predict the localization of the carrageenan-related enzyme biochemistries. Using these insights, we provide an updated model of carrageenan biosynthesis which contributes to understanding the ancestral pathway of sulfated polysaccharide biosynthesis in eukaryotes.
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Sulfated fucans from brown algae are a heterogeneous group of biologically active molecules. To learn more on their structure and to analyze and exploit their biological activities, there is a growing need to develop reliable and cost effective protocols for their preparation. In the present study, a brown alga Pelvetia canaliculata (Linnaeus) was used as a rich source of sulfated fucans. Sulfated fucan preparation methods included neutral and acidic extractions followed by purification with activated charcoal (AC), polyvinylpolypyrrolidone (PVPP), or cetylpyridinium chloride (CPC). Final products were compared in terms of yield, purity, monosaccharide composition and molecular weight. Acidic extractions provided higher yields compared to neutral ones, whereas the AC purification provided sulfated fucan products with the highest purity. Mass spectrometry analyses were done on oligosaccharides produced by the fucanase MfFcnA from the marine bacterium Mariniflexille fucanivorans. This has provided unique insight into enzyme specificity and the structural characteristics of sulfated fucans.
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Phaeophyceae , Peso Molecular , Oligosacáridos/química , Phaeophyceae/química , Polisacáridos/químicaRESUMEN
The hydroxy protons of κ- and κ/µ-hybrid carrageenan oligosaccharides have been studied by NMR spectroscopy in 85% H(2)O/15% acetone-d(6). Hydration and hydrogen bonding interactions in di- (κ), tetra- (κκ), hexa (κκκ), and octa- (κκκκ) κ-oligosaccharides and hexa- (κµκ), octa- (κµµκ), and deca- (κµµµκ) κ/µ-oligosaccharides have been investigated by measuring the chemical shifts, temperature coefficients, and chemical exchange of the hydroxy protons. These NMR parameters indicate that no strong and persistent intramolecular hydrogen bonds involving hydroxy protons stabilize the structure of κ-carrageenan oligosaccharides in aqueous solution. In the κ/µ-oligosaccharides, the presence of chemical exchange between OH3 of α-d-Gal-6-sulfate (D6S) and OH2 of ß-d-Gal-4-sulfate (G4S) across the ß-d-Gal-4-S-(1â4)-α-d-Gal-6-S linkage reveals the existence of a weak hydrogen bond interaction between the two hydroxyl groups. The smaller temperature coefficients of OH2_D6S and OH3_D6S indicate reduced hydration, interpreted as spatial proximity to the 4-sulfate group and O5 ring oxygen of the neighboring G4S residues, respectively. These first experimental results on the conformation of κ/µ-carrageenan oligosaccharides shine light on the potential role of "kinks" in the properties of the three-dimensional carrageenan gel network.
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Carragenina/química , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética/métodos , Conformación de Carbohidratos , Geles , Soluciones , Agua/químicaRESUMEN
A better understanding of the biological roles of carbohydrates requires the use of tools able to provide efficient and rapid structural information. Unfortunately, highly acidic oligomers-such as polysulfated oligosaccharides-are very challenging to characterize because of their high polarity, structural diversity, and sulfate lability. These features pose special problems for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis because polysulfated carbohydrates exhibit poor ionization efficiency and usually do not produce any signal. The present report demonstrates how MALDI-MS can be used to derive structural and compositional information from pure and mixed fractions of polysulfated oligosaccharides. Indeed, pyrenemethylguanidine (pmg, a derivatizing agent and ionization efficiency enhancer) was used for the analysis of di- to decasaccharides, carrying from two to nine sulfate groups. The method is applied to various highly sulfated chondroitin and carrageenan oligosaccharides as well as to the analysis of mixtures of compounds. In the mass spectra, the observation of a unique pmg-complexed ladder of peaks in both ionization modes allows an easy and rapid determination of both the number of sulfate groups carried by the analyte and its molecular weight. Moreover, we have developed a software tool for the rapid and automatic structural elucidation of carrageenans based on the mass spectra obtained.
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Metilguanidina/química , Oligosacáridos/química , Pirenos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Sulfatos/química , Carragenina/análisis , Carragenina/aislamiento & purificación , Sulfatos de Condroitina/análisis , Eucariontes/química , Programas Informáticos , Sacarosa/análogos & derivados , Sacarosa/análisisRESUMEN
Standard Eucheuma denticulatum iota-carrageenan was degraded with the Alteromonas fortis iota-carrageenase. The most abundant products, the neo-iota-carratetraose and neo-iota-carrahexaose were purified by permeation gel chromatography, and their corresponding (1)H and (13)C NMR spectra were fully assigned.
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Carragenina/química , Alteromonas/enzimología , Proteínas Bacterianas/metabolismo , Secuencia de Carbohidratos , Carragenina/metabolismo , Cromatografía en Gel , Glicósido Hidrolasas/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Molecular , Rhodophyta/químicaRESUMEN
Hybrid iota-/nu-carrageenan was water-extracted from Eucheuma denticulatum and incubated with Alteromonas fortis iota-carrageenase. The degradation products were then separated by anion-exchange chromatography. The three most abundant fractions of hybrid iota-/nu-carrageenan oligosaccharides were purified and their structures were analyzed by NMR. The smallest hybrid was an octasaccharide with a iota-iota-nu-iota structure. The second fraction was composed of two decasaccharides with iota-iota-iota-nu-iota and iota-[iota/nu]-iota-iota structures. The third fraction was a mixture of dodecasaccharides which contained at least a iota-iota-iota-iota-nu-iota oligosaccharide. The carbon and proton NMR spectra of the octasaccharides were completely assigned, thereby completely attributing the nu-carrabiose moiety for the first time.