Unlocking bacterial potential to reduce farmland N2O emissions.

Farmed soils contribute substantially to global warming by emitting N2O (ref. 1), and mitigation has proved difficult2. Several microbial nitrogen transformations produce N2O, but the only biological sink for N2O is the enzyme NosZ, catalysing the reduction of N2O to N2 (ref. 3). Although strengthening the NosZ activity in soils would reduce N2O emissions, such bioengineering of the soil microbiota is considered challenging4,5. However, we have developed a technology to achieve this, using organic waste as a substrate and vector for N2O-respiring bacteria selected for their capacity to thrive in soil6-8. Here we have analysed the biokinetics of N2O reduction by our most promising N2O-respiring bacterium, Cloacibacterium sp. CB-01, its survival in soil and its effect on N2O emissions in field experiments. Fertilization with waste from biogas production, in which CB-01 had grown aerobically to about 6 × 109 cells per millilitre, reduced N2O emissions by 50-95%, depending on soil type. The strong and long-lasting effect of CB-01 is ascribed to its tenacity in soil, rather than its biokinetic parameters, which were inferior to those of other strains of N2O-respiring bacteria. Scaling our data up to the European level, we find that national anthropogenic N2O emissions could be reduced by 5-20%, and more if including other organic wastes. This opens an avenue for cost-effective reduction of N2O emissions for which other mitigation options are lacking at present.

Metagenomic insights into the dynamic degradation of brown algal polysaccharides by kelp-associated microbiota.

Marine bacteria play important roles in the degradation and cycling of algal polysaccharides. However, the dynamics of epiphytic bacterial communities and their roles in algal polysaccharide degradation during kelp decay are still unclear. Here, we performed metagenomic analyses to investigate the identities and predicted metabolic abilities of epiphytic bacterial communities during the early and late decay stages of the kelp Saccharina japonica. During kelp decay, the dominant epiphytic bacterial communities shifted from Gammaproteobacteria to Verrucomicrobia and Bacteroidetes. In the early decay stage of S. japonica, epiphytic bacteria primarily targeted kelp-derived labile alginate for degradation, among which the gammaproteobacterial Vibrionaceae (particularly Vibrio) and Psychromonadaceae (particularly Psychromonas), abundant in alginate lyases belonging to the polysaccharide lyase (PL) families PL6, PL7, and PL17, were key alginate degraders. More complex fucoidan was preferred to be degraded in the late decay stage of S. japonica by epiphytic bacteria, predominantly from Verrucomicrobia (particularly Lentimonas), Pirellulaceae of Planctomycetes (particularly Rhodopirellula), Pontiellaceae of Kiritimatiellota, and Flavobacteriaceae of Bacteroidetes, which depended on using glycoside hydrolases (GHs) from the GH29, GH95, and GH141 families and sulfatases from the S1_15, S1_16, S1_17, and S1_25 families to depolymerize fucoidan. The pathways for algal polysaccharide degradation in dominant epiphytic bacterial groups were reconstructed based on analyses of metagenome-assembled genomes. This study sheds light on the roles of different epiphytic bacteria in the degradation of brown algal polysaccharides.IMPORTANCEKelps are important primary producers in coastal marine ecosystems. Polysaccharides, as major components of brown algal biomass, constitute a large fraction of organic carbon in the ocean. However, knowledge of the identities and pathways of epiphytic bacteria involved in the degradation process of brown algal polysaccharides during kelp decay is still elusive. Here, based on metagenomic analyses, the succession of epiphytic bacterial communities and their metabolic potential were investigated during the early and late decay stages of Saccharina japonica. Our study revealed a transition in algal polysaccharide-degrading bacteria during kelp decay, shifting from alginate-degrading Gammaproteobacteria to fucoidan-degrading Verrucomicrobia, Planctomycetes, Kiritimatiellota, and Bacteroidetes. A model for the dynamic degradation of algal cell wall polysaccharides, a complex organic carbon, by epiphytic microbiota during kelp decay was proposed. This study deepens our understanding of the role of epiphytic bacteria in marine algal carbon cycling as well as pathogen control in algal culture.

The catabolic specialization of the marine bacterium Polaribacter sp. Q13 to red algal ß1,3/1,4-mixed-linkage xylan.

Catabolism of algal polysaccharides by marine bacteria is a significant process of marine carbon cycling. ß1,3/1,4-Mixed-linkage xylan (MLX) is a class of xylan in the ocean, widely present in the cell walls of red algae. However, the catabolic mechanism of MLX by marine bacteria remains elusive. Recently, we found that a marine Bacteroidetes strain, Polaribacter sp. Q13, is a specialist in degrading MLX, which secretes a novel MLX-specific xylanase. Here, the catabolic specialization of strain Q13 to MLX was studied by multiomics and biochemical analyses. Strain Q13 catabolizes MLX with a canonical starch utilization system (Sus), which is encoded by a single xylan utilization locus, XUL-Q13. In this system, the cell surface glycan-binding protein SGBP-B captures MLX specifically, contributing to the catabolic specificity. The xylanolytic enzyme system of strain Q13 is unique, and the enzymatic cascade dedicates the stepwise hydrolysis of the ß1,3- and ß1,4-linkages in MLX in the extracellular, periplasmic, and cytoplasmic spaces. Bioinformatics analysis and growth observation suggest that other marine Bacteroidetes strains harboring homologous MLX utilization loci also preferentially utilize MLX. These results reveal the catabolic specialization of MLX degradation by marine Bacteroidetes, leading to a better understanding of the degradation and recycling of MLX driven by marine bacteria.IMPORTANCERed algae contribute substantially to the primary production in marine ecosystems. The catabolism of red algal polysaccharides by marine bacteria is important for marine carbon cycling. Mixed-linkage ß1,3/1,4-xylan (MLX, distinct from hetero-ß1,4-xylans from terrestrial plants) is an abundant red algal polysaccharide, whose mechanism of catabolism by marine bacteria, however, remains largely unknown. This study reveals the catabolism of MLX by marine Bacteroidetes, promoting our understanding of the degradation and utilization of algal polysaccharides by marine bacteria. This study also sets a foundation for the biomass conversion of MLX.

Direct Degradation of Fresh and Dried Macroalgae by Agarivorans albus B2Z047.

Marine macroalgae are increasingly recognized for their significant biological and economic potential. The key to unlocking this potential lies in the efficient degradation of all carbohydrates from the macroalgae biomass. However, a variety of polysaccharides (alginate, cellulose, fucoidan, and laminarin), are difficult to degrade simultaneously in a short time. In this study, the brown alga Saccharina japonica was found to be rapidly and thoroughly degraded by the marine bacterium Agarivorans albus B2Z047. This strain harbors a broad spectrum of carbohydrate-active enzymes capable of degrading various polysaccharides, making it uniquely equipped to efficiently break down both fresh and dried kelp, achieving a hydrolysis rate of up to 52%. A transcriptomic analysis elucidated the presence of pivotal enzyme genes implicated in the degradation pathways of alginate, cellulose, fucoidan, and laminarin. This discovery highlights the bacterium's capability for the efficient and comprehensive conversion of kelp biomass, indicating its significant potential in biotechnological applications for macroalgae resource utilization.

A repertoire of alginate lyases in the alginate polysaccharide utilization loci of marine bacterium Wenyingzhuangia fucanilytica: biochemical properties and action pattern.

BACKGROUND: Alginate lyases are important tools for alginate biodegradation and oligosaccharide production, which have great potential in food and biofuel fields. The alginate polysaccharide utilization loci (PUL) typically encode a series of alginate lyases with a synergistic action pattern. Exploring valuable alginate lyases and revealing the synergistic effect of enzymes in the PUL is of great significance. RESULTS: An alginate PUL was discovered from the marine bacterium Wenyingzhuangia fucanilytica CZ1127T , and a repertoire of alginate lyases within it was cloned, expressed and characterized. The four alginate lyases in PUL demonstrated similar optimal reaction conditions: maximum enzyme activity at 35-50 °C and pH 8.0-9.0. The results of action pattern indicated that they were two PL7 endolytic bifunctional enzymes (Aly7A and Aly7B), a PL6 exolytic bifunctional enzyme (Aly6A) and a PL17 exolytic M-specific enzyme (Aly17A). Ultra-performance liquid chromatography-mass spectrometry was employed to reveal the synergistic effect of the four enzymes. The end products of Aly7A were further degraded by Aly7B and eventually generated oligosaccharides, from disaccharide to heptasaccharide. The oligosaccharide products were completely degraded to monosaccharides by Aly6A, but it was unable to directly degrade alginate. Aly17A could also produce monosaccharides by cleaving the M-blocks of oligosaccharide products, as well as the M-blocks of polysaccharides. The combination of these enzymes resulted in the complete degradation of alginate to monosaccharides. CONCLUSION: A new alginate PUL was mined and four novel alginate lyases in the PUL were expressed and characterized. The four cooperative alginate lyases provide novel tools for alginate degradation and biological fermentation. © 2023 Society of Chemical Industry.

Characterization and structural identification of a family 16 carbohydrate-binding module (CBM): First structural insights into porphyran-binding CBM.

Porphyran is a favorable functional polysaccharide widely distributed in Porphyra. It displays a linear structure majorly constituted by alternating 1,4-linked α-l-galactopyranose-6-sulfate (L6S) and 1,3-linked ß-d-galactopyranose (G) units. Carbohydrate-binding modules (CBMs) are desired tools for the investigation and application of polysaccharides, including in situ visualization, on site and specific assay, and functionalization of biomaterials. However, only one porphyran-binding CBM has been hitherto reported, and its structural knowledge is lacking. Herein, a novel CBM16 family domain from a marine bacterium Aquimarina sp. BL5 was discovered and expressed. The recombinant protein AmCBM16 exhibited the desired specificity for porphyran. Bio-layer interferometry assay revealed that the protein binds to porphyran tetrasaccharide (L6S-G)2 with an association constant of 1.3 × 103 M-1. The structure of AmCBM16 was resolved by the X-ray crystallography, which displays a ß-sandwich fold with two antiparallel ß-sheets constituted by 10 ß-strands. Site-directed mutagenesis analysis demonstrated that the residues Gly-30, Trp-31, Lys-88, Lys-123, Phe-125, and Phe-127 play dominant roles in AmCBM16 binding. This study provides the first structural insights into porphyran-binding CBM.

Particle-attached bacteria act as gatekeepers in the decomposition of complex phytoplankton polysaccharides.

BACKGROUND: Marine microalgae (phytoplankton) mediate almost half of the worldwide photosynthetic carbon dioxide fixation and therefore play a pivotal role in global carbon cycling, most prominently during massive phytoplankton blooms. Phytoplankton biomass consists of considerable proportions of polysaccharides, substantial parts of which are rapidly remineralized by heterotrophic bacteria. We analyzed the diversity, activity, and functional potential of such polysaccharide-degrading bacteria in different size fractions during a diverse spring phytoplankton bloom at Helgoland Roads (southern North Sea) at high temporal resolution using microscopic, physicochemical, biodiversity, metagenome, and metaproteome analyses. RESULTS: Prominent active 0.2-3 µm free-living clades comprised Aurantivirga, "Formosa", Cd. Prosiliicoccus, NS4, NS5, Amylibacter, Planktomarina, SAR11 Ia, SAR92, and SAR86, whereas BD1-7, Stappiaceae, Nitrincolaceae, Methylophagaceae, Sulfitobacter, NS9, Polaribacter, Lentimonas, CL500-3, Algibacter, and Glaciecola dominated 3-10 µm and > 10 µm particles. Particle-attached bacteria were more diverse and exhibited more dynamic adaptive shifts over time in terms of taxonomic composition and repertoires of encoded polysaccharide-targeting enzymes. In total, 305 species-level metagenome-assembled genomes were obtained, including 152 particle-attached bacteria, 100 of which were novel for the sampling site with 76 representing new species. Compared to free-living bacteria, they featured on average larger metagenome-assembled genomes with higher proportions of polysaccharide utilization loci. The latter were predicted to target a broader spectrum of polysaccharide substrates, ranging from readily soluble, simple structured storage polysaccharides (e.g., laminarin, α-glucans) to less soluble, complex structural, or secreted polysaccharides (e.g., xylans, cellulose, pectins). In particular, the potential to target poorly soluble or complex polysaccharides was more widespread among abundant and active particle-attached bacteria. CONCLUSIONS: Particle-attached bacteria represented only 1% of all bloom-associated bacteria, yet our data suggest that many abundant active clades played a pivotal gatekeeping role in the solubilization and subsequent degradation of numerous important classes of algal glycans. The high diversity of polysaccharide niches among the most active particle-attached clades therefore is a determining factor for the proportion of algal polysaccharides that can be rapidly remineralized during generally short-lived phytoplankton bloom events. Video Abstract.

In situ community transcriptomics illuminates CO2-fixation potentials and supporting roles of phagotrophy and proton pump in plankton in a subtropical marginal sea.

Lineage-wise physiological activities of plankton communities in the ocean are important but challenging to characterize. Here, we conducted whole-assemblage metatranscriptomic profiling at continental shelf and slope sites in the South China Sea to investigate carbon fixation potential in different lineages. RuBisCO expression, the proxy of Calvin carbon fixation (CCF) potential, was mainly contributed by Bacillariophyta, Chlorophyta, Cyanobacteria, and Haptophyta, which was differentially affected by environmental factors among lineages. CCF potential exhibited positive or negative correlations with phagotrophy gene expression, suggesting phagotrophy possibly enhances or complements CCF. Our data also reveal significant non-Calvin carbon fixation (NCF) potential, as indicated by the active expression of genes in all five currently recognized NCF pathways, mainly contributed by Flavobacteriales, Alteromonadales, and Oceanospirillales. Furthermore, in Flavobacteriales, Alteromonadales, Pelagibacterales, and Rhodobacterales, NCF potential was positively correlated with proton-pump rhodopsin (PPR) expression, suggesting that NCF might be energetically supported by PPR. The novel insights into the lineage-differential potential of carbon fixation, widespread mixotrophy, and PPR as an energy source for NCF lay a methodological and informational foundation for further research to understand carbon fixation and the trophic landscape in the ocean.IMPORTANCEMarine plankton plays an important role in global carbon cycling and climate regulation. Phytoplankton and cyanobacteria fix CO2 to produce organic compounds using solar energy and mainly by the Calvin cycle, whereas autotrophic bacteria and archaea may fix CO2 by non-Calvin cycle carbon fixation pathways. How active individual lineages are in carbon fixation and mixotrophy, and what energy source bacteria may employ in non-Calvin carbon fixation, in a natural plankton assemblage are poorly understood and underexplored. Using metatranscriptomics, we studied carbon fixation in marine plankton with lineage resolution in tropical marginal shelf and slope areas. Based on the sequencing results, we characterized the carbon fixation potential of different lineages and assessed Calvin- and non-Calvin- carbon fixation activities and energy sources. Data revealed a high number of unigenes (4.4 million), lineage-dependent differential potentials of Calvin carbon fixation and responses to environmental conditions, major contributors of non-Calvin carbon fixation, and their potential energy source.

Hydrogen-Bonding and Hydrophobic Interaction Networks as Structural Determinants of Microbial Rhodopsin Function.

Microbial pump rhodopsins are highly versatile light-driven membrane proteins that couple protein conformational dynamics with ion translocation across the cell membranes. Understanding how microbial pump rhodopsins use specific amino acid residues at key functional sites to control ion selectivity and ion pumping direction is of general interest for membrane transporters, and could guide site-directed mutagenesis for optogenetics applications. To enable direct comparisons between proteins with different sequences we implement, for the first time, a unique numbering scheme for the microbial pump rhodopsin residues, NS-mrho. We use NS-mrho to show that distinct microbial pump rhodopsins typically have hydrogen-bond networks that are less conserved than anticipated from the amino acid residue conservation, whereas their hydrophobic interaction networks are largely conserved. To illustrate the role of the hydrogen-bond networks as structural elements that determine the functionality of microbial pump rhodopsins, we performed experiments, atomic-level simulations, and hydrogen bond network analyses on GR, the outward proton pump from Gloeobacter violaceus, and KR2, the outward sodium pump from Krokinobacter eikastus. The experiments indicate that multiple mutations that recover KR2 amino acid residues in GR not only fail to convert it into a sodium pump, but completely inactivate GR by abolishing photoisomerization of the retinal chromophore. This observation could be attributed to the drastically altered hydrogen-bond interaction network identified with simulations and network analyses. Taken together, our findings suggest that functional specificity could be encoded in the collective hydrogen-bond network of microbial pump rhodopsins.

Multi-omics analysis of antiviral interactions of Elizabethkingia anophelis and Zika virus.

The microbial communities residing in the mosquito midgut play a key role in determining the outcome of mosquito pathogen infection. Elizabethkingia anophelis, originally isolated from the midgut of Anopheles gambiae possess a broad-spectrum antiviral phenotype, yet a gap in knowledge regarding the mechanistic basis of its interaction with viruses exists. The current study aims to identify pathways and genetic factors linked to E. anophelis antiviral activity. The understanding of E. anophelis antiviral mechanism could lead to novel transmission barrier tools to prevent arboviral outbreaks. We utilized a non-targeted multi-omics approach, analyzing extracellular lipids, proteins, metabolites of culture supernatants coinfected with ZIKV and E. anophelis. We observed a significant decrease in arginine and phenylalanine levels, metabolites that are essential for viral replication and progression of viral infection. This study provides insights into the molecular basis of E. anophelis antiviral phenotype. The findings lay a foundation for in-depth mechanistic studies.

Systematic screening for the biocatalytic hydration of fatty acids from different oily substrates by Elizabethkingia meningoseptica oleate hydratase through a Design-of-experiments approach.

The edible plant oils production is associated with the release of different types of by-products. The latter represent cheap and available substrates to produce valuable compounds, such as flavours and fragrances, biologically active compounds and bio-based polymers. Elizabethkingia meningoseptica Oleate hydratases (Em_OhyA) can selectively catalyze the conversion of unsaturated fatty acids, specifically oleic acid, into hydroxy fatty acids, which find different industrial applications. In this study, Design-of-experiment (DoE) strategy was used to screen and identify conditions for reaching high yields in the reaction carried out by Escherichia coli whole-cell carrying the recombinant enzyme Em_OhyA using Waste Cooking Oils (WCO)-derived free fatty acids (FFA) as substrate. The identified reaction conditions for high oleic acid conversion were also tested on untreated triglycerides-containing substrates, such as pomace oil, sunflower oil, olive oil and oil mill wastewater (OMW), combining the triglyceride hydrolysis by the lipase from Candida rugosa and the E. coli whole-cell containing Em_OhyA for the production of hydroxy fatty acids. When WCO, sunflower oil and OMW were used as substrate, the one-pot bioconversion led to an increase of oleic acid conversion compared to the standard reaction. This work highlights the efficiency of the DoE approach to screen and identify conditions for an enzymatic reaction for the production of industrially-relevant products.