Mannitol Dehydrogenase Research Articles

Dioscorea alata tuber proteome analysis shows over thirty dioscorin isoforms and novel tuber proteins

In Dioscorea, dioscorin (31 kDa) is the major storage protein constituting 85% of the total tuber proteins. An integrated proteomic and biochemical approach was used to understand the physiological role of dioscorin in the two contrasting growth stages (germinating and mature tuber). HPLC analysis showed 3 fold reduction in mannitol and 12.88 and 1.24 fold increase in sucrose and maltose in the germinating tuber. A 1.8 and 3 fold increase in sucrose phosphate synthase and mannitol dehydrogenase activity respectively was observed in the germinating tuber while a 2 fold higher invertase probably lowers the sucrose accumulation in the mature tuber. SDS-PAGE and 2-D maps of the mature and germinating tubers confirmed depletion (more than 50%) of dioscorin on germination. Dioscorin was purified using ion exchange and gel filtration chromatography with 43.32 fold purification and 38.16 yield. Out of a trail of 35 spots at 31 kDa only 12 spots (identified as dioscorin isoforms) were present in the 2D gel of the purified fraction. To search for other tuber proteins besides dioscorin, the unbound fractions of DEAE column were analysed by 2DGE. DREB 1A, caffeic acid 3-O-methyltransferase and Rab-1 small GTP binding protein were identified perhaps for the first time in the Dioscorea proteome. The interactome analysis revealed these to be involved in oxidative stress, carotenoid synthesis and vesicular transport. This is perhaps the first attempt to identify tuber proteome (although limited) and to understand the physiological significance of these proteins.

Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex.

Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.

Characterization of a thermostable mannitol dehydrogenase from hyperthermophilic Thermotoga neapolitana DSM 4359 with potential application in mannitol production

Mannitol-2-dehydrogenase (MtDH) (E.C. 1.1.1.67) gene was cloned from Thermotoga neapolitana DSM 4359 and expressed in Escherichia coli BL21. The purified enzyme showed a predicted clear band of 36kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), native molecular mas was 135kDa. Km and Vmax values for reduction of D-fructose to D-mannitol were 20mM and 200U mg-1 respectively. kcat for reduction direction was 180s−1 and kcat/Km were 9mM−1s−1. The enzyme showed optimal pH at 6.5 and the optimum temperature was 90°C with 100% relative activity. The purified enzyme was quite stable at 75°C and had half of initial activity after 1h of incubation at 90°C. (TnMtDH) showed no activity with xylitol, inositol, sorbitol, rahmanose, mannose and xylose, and with NADPH and NADP+ as co factors. The presence of some divalent metals in the reaction enhanced the enzyme activity. The enzyme might be utilizing to produce mannitol without other sugar conformation under high temperature.

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans.

Gluconobacter (G.) oxydans is able to incompletely oxidize various sugars and polyols for the production of biotechnologically important compound. Recently, we have shown that the organism produces and accumulates mannitol as compatible solute under osmotic stress conditions. The present study describes the role of two cytoplasmic mannitol dehydrogenases for osmotolerance of G. oxydans. It was shown that Gox1432 is a NADP+-dependent mannitol dehydrogenase (EC 1.1.1.138), while Gox0849 uses NAD+ as cofactor (EC 1.1.1.67). The corresponding genes were deleted and the mutants were analyzed for growth under osmotic stress and non-stress conditions. A severe growth defect was detected for Δgox1432 when grown in high osmotic media, while the deletion of gox0849 had no effect when cells were exposed to 450mM sucrose in the medium. Furthermore, the intracellular mannitol content was reduced in the mutant lacking the NADP+-dependent enzyme Gox1432 in comparison to the parental strain and the Δgox0849 mutant under stress conditions. In addition, transcriptional analysis revealed that Gox1432 is more important for mannitol production in G. oxydans than Gox0849 as the transcript abundance of gene gox1432 was 30-fold higher than of gox0849. In accordance, the activity of the NADH-dependent enzyme Gox0849 in the cell cytoplasm was 10-fold lower in comparison to the NADPH-dependent mannitol dehydrogenase Gox1432. Overexpression of gox1432 in the corresponding deletion mutant restored growth of the cells under osmotic stress, further strengthening the importance of the NADP+-dependent mannitol dehydrogenase for osmotolerance in G. oxydans. These findings provide detailed insights into the molecular mechanism of mannitol-mediated osmoprotection in G. oxydans and are helpful engineering strains with improved osmotolerance for biotechnological applications.

Transgenic peanut overexpressing mtlD gene confers enhanced salinity stress tolerance via mannitol accumulation and differential antioxidative responses

Globally, peanut is an important oilseed crop, which is cultivated under different agro-climatic zones. Soil salinity is one of the major constraints in peanut cultivation. Therefore, to understand the physio-biochemical mechanisms imparting salinity stress, four transgenic peanut lines (cv. GG20) already developed and confirmed by our lab, having bacterial mannitol dehydrogenase gene (mtlD), were subjected to different levels of salinity stresses (1, 2 and 3 dS m−1) in pots under containment facility. Further, these lines were also characterized for various physio-biochemical parameters at flowering, pegging and pod formation stages. All the transgenic lines recorded significantly higher mannitol dehydrogenase (MTD) activity and mannitol accumulation than the wild type (WT). Under salinity stress, significantly higher levels of superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase activities, while significantly lower levels of H2O2 and malondialdehyde contents, were recorded in the transgenics compared to WT. Similarly, significantly higher ascorbic acid and relative water content (RWC) were recorded in transgenic lines. The MTD activity showed positive correlation with various antioxidant enzymes, growth parameters and RWC, while negative correlation was recorded with H2O2 and malondialdehyde content at most of the plant growth stages. The mtlD transgenic peanut lines under pot conditions were found maintaining lower oxidative injuries, indicating amelioration of salinity-induced oxidative stress by enhanced protection mechanisms via mannitol accumulation and antioxidative responses. The best lines identified (MTD1 and MTD4) may be used further as pre-breeding source for imparting salinity stress tolerance in peanut. Besides, these lines may also be tested under open-field trials for release as salt-tolerant variety.

Polyol production during heterofermentative growth of the plant isolate Lactobacillus florum 2F.

This study examined the fermentative growth and polyol production of Lactobacillus florum and other plant-associated lactic acid bacteria (LAB). Sugar consumption and end-product production were measured for Lact.florum 2F in the presence of fructose, glucose and both sugars combined. The genome of Lact.florum was examined for genes required for mannitol and erythritol biosynthesis. The capacity for other plant-associated LAB to synthesize polyols was also assessed. Lactobacillus florum exhibited higher growth rates and cell yields in the presence of both fructose and glucose. Lactobacillus florum 2F produced lactate, acetate and ethanol as well as erythritol and mannitol. Lactobacillus florum 2F synthesized mannitol during growth on fructose and erythritol during growth on glucose. Gene and protein homology searches identified a mannitol dehydrogenase in the Lact.florum 2F genome but not the genes responsible for erythritol biosynthesis. Lastly, we found that numerous other heterofermentative LAB species synthesize erythritol and/or mannitol. Lactobacillus florum is a recently identified, plant-associated, fructophilic LAB species. Our results show that Lact.florum growth rates and heterofermentation end-products differ depending on the sugar substrates present and growth yields can be improved when combinations of sugars are provided. Lactobacillus florum 2F produces erythritol and mannitol, two polyols that are relevant to foods and potentially also in plant environments. The capacity for polyol biosynthesis appears to be common among plant-associated, LAB species.

Identification of proteins associated with the production of melanin and with pathogenicity in maize pathogen Curvularia lunata

Curvularia lunata produces 1,8-dihydroxynaphthalene (DHN) melanin. Previously, we have successfully cloned and silenced the Brn1 gene, which is involved in DNH melanin production and pathogenicity. However, we hypothesise that other proteins may also be related to melanin production or pathogenicity. In this study, a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry techniques was used to identify the specific proteins associated with Brn1 expression in C. lunata wild-type CX-3 and Brn1-silenced mutant T5. One up-regulated and seven down-regulated proteins were successfully identified in T5. Whilst transaldolase, NADP-dependent mannitol dehydrogenase, formate dehydrogenase, and UTP-glucose-1-phosphate uridylyltransferase are involved in energy metabolism, elongation factor 3 facilitates the synthesis of important proteins required for the production of melanin and stress tolerance-related proteins (HSP30 and HSP70) and catalase (CAT) contribute to the pathogenicity of C. lunata. Of these proteins, CAT was further analysed as a protein of interest. Bioinformatics analysis showed that CAT consists of 748 amino acids and clustered into one branch with Dothideomycete CAT homologues in a phylogenetic tree. Further research on the exact role of the CAT gene is underway.

Mannitol metabolism during pathogenic fungal–host interactions under stressed conditions

Numerous plants and fungi produce mannitol, which may serve as an osmolyte or metabolic store; furthermore, mannitol also acts as a powerful quencher of reactive oxygen species (ROS). Some phytopathogenic fungi use mannitol to stifle ROS-mediated plant resistance. Mannitol is essential in pathogenesis to balance cell reinforcements produced by both plants and animals. Mannitol likewise serves as a source of reducing power, managing coenzymes, and controlling cytoplasmic pH by going about as a sink or hotspot for protons. The metabolic pathways for mannitol biosynthesis and catabolism have been characterized in filamentous fungi by direct diminishment of fructose-6-phosphate into mannitol-1-phosphate including a mannitol-1-phosphate phosphatase catalyst. In plants mannitol is integrated from mannose-6-phosphate to mannitol-1-phosphate, which then dephosphorylates to mannitol. The enzyme mannitol dehydrogenase plays a key role in host–pathogen interactions and must be co-localized with pathogen-secreted mannitol to resist the infection.

The camelliagenin from defatted seeds of Camellia oleifera as antibiotic substitute to treat chicken against infection of Escherichia coli and Staphylococcus aureus.

BackgroundEscherichia coli and Staphylococcus aureus are the main pathogens infectious to poultry, and their resistances against antibiotics have become troublesome currently. Biofilm formation is an important reason for drug resistance. Our previous research has found that the extract of Camellia oleifera seeds has lots of pharmacological effects. In order to find the substitute for antibiotics, the saponin was isolated from the defatted C. oleifera seeds with structural identification. Its efficacy was evaluated by the inhibition on amoxicillin-resistant E. coli and erythromycin-resistant S. aureus and therapeutic effect on chicks infected by the two bacteria.ResultsThe bacterial growth inhibition rate increased and the bacterial count in vivo decreased significantly in dose dependence after administration of the saponin and its combination with amoxicillin or erythromycin, suggesting its antibacterial effect. The saponin identified as camelliagenin shows significant inhibition on the biofilm of E. coli and S. aureus, and it is related to the decrease of mannitol dehydrogenase (MDH) activity and extracellular DNA (eDNA) content. Molecular simulation reveals the strong interaction existing between the saponin and MDH or eDNA.ConclusionsThe mechanism of camelliagenin’s improvement on antibiotic effects is its interaction with MDH and eDNA in biofilm. The saponin is a prospective substitute of antibiotics, and molecular simulation is a convenient alternative method to find out hopeful candidates of antibiotics substitute.

A Genomic View of Lactobacilli and Pediococci Demonstrates that Phylogeny Matches Ecology and Physiology.

Lactobacilli are used widely in food, feed, and health applications. The taxonomy of the genus Lactobacillus, however, is confounded by the apparent lack of physiological markers for phylogenetic groups of lactobacilli and the unclear relationships between the diverse phylogenetic groups. This study used the core and pan-genomes of 174 type strains of Lactobacillus and Pediococcus to establish phylogenetic relationships and to identify metabolic properties differentiating phylogenetic groups. The core genome phylogenetic tree separated homofermentative lactobacilli and pediococci from heterofermentative lactobacilli. Aldolase and phosphofructokinase were generally present in homofermentative but not in heterofermentative lactobacilli; a two-domain alcohol dehydrogenase and mannitol dehydrogenase were present in most heterofermentative lactobacilli but absent in most homofermentative organisms. Other genes were predominantly present in homofermentative lactobacilli (pyruvate formate lyase) or heterofermentative lactobacilli (lactaldehyde dehydrogenase and glycerol dehydratase). Cluster analysis of the phylogenomic tree and the average nucleotide identity grouped the genus Lactobacillus sensu lato into 24 phylogenetic groups, including pediococci, with stable intra- and intergroup relationships. Individual groups may be differentiated by characteristic metabolic properties. The link between phylogeny and physiology that is proposed in this study facilitates future studies on the ecology, physiology, and industrial applications of lactobacilli.

Analyzing redox balance in a synthetic yeast platform to improve utilization of brown macroalgae as feedstock

Macroalgae have high potential to be an efficient, and sustainable feedstock for the production of biofuels and other more valuable chemicals. Attempts have been made to enable the co-fermentation of alginate and mannitol by Saccharomyces cerevisiae to unlock the full potential of this marine biomass. However, the efficient use of the sugars derived from macroalgae depends on the equilibrium of cofactors derived from the alginate and mannitol catabolic pathways. There are a number of strong metabolic limitations that have to be tackled before this bioconversion can be carried out efficiently by engineered yeast cells.An analysis of the redox balance during ethanol fermentation from alginate and mannitol by Saccharomyces cerevisiae using metabolic engineering tools was carried out. To represent the strain designed for conversion of macroalgae carbohydrates to ethanol, a context-specific model was derived from the available yeast genome-scale metabolic reconstructions. Flux balance analysis and dynamic simulations were used to determine the flux distributions. The model indicates that ethanol production is determined by the activity of 4-deoxy-l-erythro-5-hexoseulose uronate (DEHU) reductase (DehR) and its preferences for NADH or NADPH which influences strongly the flow of cellular resources. Different scenarios were explored to determine the equilibrium between NAD(H) and NADP(H) that will lead to increased ethanol yields on mannitol and DEHU under anaerobic conditions. When rates of mannitol dehydrogenase and DehRNADH tend to be close to a ratio in the range 1–1.6, high growth rates and ethanol yields were predicted. The analysis shows a number of metabolic limitations that are not easily identified through experimental procedures such as quantifying the impact of the cofactor preference by DEHU reductase in the system, the low flux into the alginate catabolic pathway, and a detailed analysis of the redox balance. These results show that production of ethanol and other chemicals can be optimized if a redox balance is achieved. A possible methodology to achieve this balance is presented. This paper shows how metabolic engineering tools are essential to comprehend and overcome this limitation.

Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis

The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica.

Genome sequence analysis of potential probiotic strain Leuconostoc lactis EFEL005 isolated from kimchi.

Leuconostoc lactis EFEL005 (KACC 91922) isolated from kimchi showed promising probiotic attributes; resistance against acid and bile salts, absence of transferable genes for antibiotic resistance, broad utilization of prebiotics, and no hemolytic activity. To expand our understanding of the species, we generated a draft genome sequence of the strain and analyzed its genomic features related to the aforementioned probiotic properties. Genome assembly resulted in 35 contigs, and the draft genome has 1,688,202 base pairs (bp) with a G+C content of 43.43%, containing 1,644 protein-coding genes and 50 RNA genes. The average nucleotide identity analysis showed high homology (≥ 96%) to the type strain L. lactis KCTC3528, but low homology (≤ 95%) to L. lactis KCTC3773 (formerly L. argentinum). Genomic analysis revealed the presence of various genes for sucrose metabolism (glucansucrases, invertases, sucrose phosphorylases, and mannitol dehydrogenase), acid tolerance (F1F0 ATPases, cation transport ATPase, branched-chain amino acid permease, and lysine decarboxylase), vancomycin response regulator, and antibacterial peptide (Lactacin F). No gene for production of biogenic amines (histamine and tyramine) was found. This report will facilitate the understanding of probiotic properties of this strain as a starter for fermented foods.

Acquired Resistance to Mefenoxam in Sensitive Isolates of Phytophthora infestans.

The systemic fungicide mefenoxam has been important in the control of late blight disease caused by Phytophthora infestans. This phenylamide fungicide has a negative effect on the synthesis of ribosomal RNA; however, the genetic basis for inherited field resistance is still not completely clear. We recently observed that a sensitive isolate became tolerant after a single passage on mefenoxam-containing medium. Further analyses revealed that all sensitive isolates tested (in three diverse genotypes) acquired this resistance equally quickly. In contrast, isolates that were "resistant" to mefenoxam in the initial assessment (stably resistant) did not increase in resistance upon further exposure. However, there appeared to be a cost associated with acquired resistance in the initially sensitive isolates, in that isolates with acquired resistance grew more slowly on mefenoxam-free medium than did the same isolates that had never been exposed to mefenoxam. The acquired resistance of the sensitive isolates declined slightly with subsequent culturing on medium free of mefenoxam. To investigate the mechanism of acquired resistance, we employed strand-specific RNA sequencing. Many differentially expressed genes were genotype specific, but one set of genes was differentially expressed in all genotypes. Among these were several genes (a phospholipase "Pi-PLD-like-3," two ATP-binding cassette superfamily [ABC] transporters, and a mannitol dehydrogenase) that were up-regulated and whose function might contribute to a resistance phenotype.

From respiratory sensitization to food allergy: Anaphylactic reaction after ingestion of mushrooms (Agaricus bisporus)

We report a case of a 38-year-old mold-allergic patient who developed episodes of generalized urticaria and systemic anaphylactic shock immediately after ingesting button mushrooms. A manganese-dependent superoxide dismutase (MnSOD) and a NADP-dependent mannitol dehydrogenase (MtDH) from Agaricus bisporus mushroom were identified as patient-specific IgE-binding proteins. Cross-reactivity between A. bisporus MnSOD and mold aeroallergens was confirmed. We conclude that prior sensitization to mold aeroallergens might explain severe food reactions to cross-reacting homologs mushroom proteins.

Activity of select dehydrogenases with Sepharose-immobilized N6-carboxymethyl-NAD

N6-carboxymethyl-NAD (N6-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N6-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N6-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N6-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N6-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N6-amine group on NAD.

Progeny of Selfed Plants from Tomato Breeding Line ‘NC1 Grape’ Overexpressing Mannitol Dehydrogenase (MTD) Have Increased Resistance to the Early Blight Fungus, Alternaria solani

Reactive oxygen species (ROS) made by plants in response to pathogen infection not only initiate local and systemic defenses, they are also antimicrobial. A number of fungi are hypothesized to secrete the antioxidant mannitol to protect against this antimicrobial ROS during infection. This hypothesis is supported by reports that overexpression of the mannitol catabolic enzyme mannitol dehydrogenase (MTD) in plants increases resistance to mannitol-secreting pathogens like Botrytis cinerea and Alternaria alternata. To extend this hypothesis and test the general utility of this approach, we overexpressed celery MTD in a tomato breeding line (NC1 Grape) currently used in our program. Although we reported earlier that MTD overexpression provides resistance to Botrytis gray mold in a greenhouse tomato, this is the first report of overexpression in an elite breeding variety providing heritable, whole-plant resistance to A. solani (tomato early blight). In this study, progeny from a high-MTD-expressing line had infection rates <65% those of nontransformed plants, and transformants outgrew infection by 7 days post-inoculation. Finally, our results suggest that screening for higher innate MTD expression in plants, rather than screening solely for the presence of the Mtd gene, might be a more effective way to identify parental lines for use in conventional breeding of early blight resistance. Accepted for publication 7 August 2015. Published 14 August 2015.

Tomato Plants Overexpressing a Celery Mannitol Dehydrogenase (MTD) Have Decreased Susceptibility to <i>Botrytis cinerea</i>

The oxidative burst is a critical early event in plant-pathogen interactions that leads to a localized, programmed cell death (PCD) called the hypersensitive response (HR). The HR and associated PCD retard infection by biotrophic pathogens, but can, in fact, enhance infection by necrotrophic pa- thogens like Botrytis cinerea. In addition to signaling the induction of the HR, reactive oxygen spe- cies (ROS) produced during the oxidative burst are antimicrobial. We hypothesize that pathogens such as B. cinerea survive the antimicrobial effects of ROS, at least partially by secreting the anti- oxidant mannitol during infection. This is supported by the previous observation that overexpres- sion of the catabolic enzyme mannitol dehydrogenase (MTD) can decrease a plants susceptibility to mannitol-secreting pathogens like B. cinerea. To extend the above hypothesis, and test the gen- eral utility of this approach in an important horticultural crop, we overexpressed celery MTD in tomato (Solanum lycopersicum cv. Moneymaker). In these studies, we observed a significant in- crease (up to 90%) in resistance to B. cinerea in transgenic tomatoes expressing high amounts of MTD.

Synthesis and anti-biofilm activities of dihydro-pyrrol-2-one derivatives on Pseudomonas aeruginosa

Biofilm formation is an important reason for bacterial resistance to antimicrobials. Many compounds with dihydro-pyrrol-2-one (DPO) have antibacterial effects. It is prospective to base on DPO skeleton to design new compounds for biofilm inhibition. DPO was designed by a novel method of tandem cyclization between ethyl glyoxalate and amines, the series of DPO derivatives were synthesized by change of the amines. Their activities were evaluated by the inhibition of biofilm in Pseudomonas aeruginosa. The interaction of DPO derivatives with mannitol dehydrogenase (MDH) or extracellular DNA (eDNA) in the biofilm was simulated by molecular docking to reveal possible mechanism. 19 new DPO derivatives were synthesized and identified, 15 of them had antibacterial activities, but only 5 of them had more than 50% inhibition on biofilm of P. aeruginosa at 50μg/mL. The MDH activity and eDNA content in biofilm decreased significantly after treatment of the DPO derivatives in concentration dependence. The simulation reveals that strong interaction exists between the five DPO derivatives and MDH or eDNA, which are involved in anti-biofilm mechanism. The synthetic method of DPO derivatives is practical to provide effective anti-biofilm agents for P. aeruginosa, and they take effect through inhibition on MDH and eDNA of biofilm.

Niches of two polysaccharide-degrading Polaribacter isolates from the North Sea during a spring diatom bloom.

Members of the flavobacterial genus Polaribacter thrive in response to North Sea spring phytoplankton blooms. We analyzed two respective Polaribacter species by whole genome sequencing, comparative genomics, substrate tests and proteomics. Both can degrade algal polysaccharides but occupy distinct niches. The liquid culture isolate Polaribacter sp. strain Hel1_33_49 has a 3.0-Mbp genome with an overall peptidase:CAZyme ratio of 1.37, four putative polysaccharide utilization loci (PULs) and features proteorhodopsin, whereas the agar plate isolate Polaribacter sp. strain Hel1_85 has a 3.9-Mbp genome with an even peptidase:CAZyme ratio, eight PULs, a mannitol dehydrogenase for decomposing algal mannitol-capped polysaccharides but no proteorhodopsin. Unlike other sequenced Polaribacter species, both isolates have larger sulfatase-rich PULs, supporting earlier assumptions that Polaribacter take part in the decomposition of sulfated polysaccharides. Both strains grow on algal laminarin and the sulfated polysaccharide chondroitin sulfate. For strain Hel1_33_49, we identified by proteomics (i) a laminarin-induced PUL, (ii) chondroitin sulfate-induced CAZymes and (iii) a chondroitin-induced operon that likely enables chondroitin sulfate recognition. These and other data suggest that strain Hel1_33_49 is a planktonic flavobacterium feeding on proteins and a small subset of algal polysaccharides, while the more versatile strain Hel1_85 can decompose a broader spectrum of polysaccharides and likely associates with algae.