Genome-wide analysis of glutamate receptor gene family in allopolyploid Brassica napus and its diploid progenitors.

Ionotropic glutamate receptors are ligand-gated nonselective cation channels that mediate neurotransmission in the central nervous system of animals. Plants possess homologous proteins called glutamate receptor-like channels (GLRs) which are involved in vital physiological processes including seed germination, long-distance signaling, chemotaxis, Ca2+ signaling etc. Till now, a comprehensive genome-wide analysis of the GLR gene family members in different economically important species of Brassica is missing. Considering the origin of allotetraploid Brassica napus from the hybridization between the diploid Brassica oleracea and Brassica rapa, we have identified 11, 27 and 65 GLR genes in B. oleracea, B. rapa and B. napus, respectively showing an expansion of this gene family in B. napus. Chromosomal locations revealed several tandemly duplicated GLR genes in all the three species. Moreover, the gene family expanded in B. napus after allopolyploidization. The phylogenetic analysis showed that the 103 GLRs are classified into three main groups. The exon-intron structures of these genes are not very conserved and showed wide variation in intron numbers. However, protein sequences are much conserved as shown by the presence of ten short amino acid sequence motifs. Predicted cis-acting elements in 1 kb promoters of GLR genes are mainly involved in light, stress and hormone responses. RNA-seq analysis showed that in B. oleracea and B. rapa, some GLRs are more tissue specific than others. In B. napus, some GLRs are downregulated under cold stress, while others are upregulated. In summary, this bioinformatic study of the GLR gene family of the three Brassica species provides evidence for the expansion of this gene family in B. napus and also provided useful information for in-depth studies of their biological functions in Brassica.

Isorhamnetin exerts anti-tumor activity in DEN + CCl4-induced HCC mice.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer and the main cause of cancer death globally. The use of medicinal herbs as chemotherapeutic agents in cancer treatment is receiving attention as they possess no or minimum side effects. Isorhamnetin (IRN), a flavonoid, has been under attention for its anti-inflammatory and anti-proliferative properties in a number of cancers, including colorectal, skin, and lung cancers. However, the in vivo mechanism of isorhamnetin to suppress liver cancer has yet to be explored. METHODS AND RESULT: HCC was induced by N-diethylnitrosamine (DEN) and carbon tetrachloride (CCL4) in Swiss albino mice. Isorhamnetin (100 mg/kg body weight) was given to examine its anti-tumor properties in HCC mice model. Histological analysis and liver function assays were performed to assess changes in liver anatomy. Probable molecular pathways were explored using immunoblot, qPCR, ELISA, and immunohistochemistry techniques. Isorhamnetin inhibited various pro-inflammatory cytokines to suppress cancer-inducing inflammation. Additionally, it regulated Akt and MAPKs to suppress Nrf2 signaling. Isorhamnetin activated PPAR-γ and autophagy while suppressing cell cycle progression in DEN + CCl4-administered mice. Additionally, isorhamnetin regulated various signaling pathways to suppress cell proliferation, metabolism, and epithelial-mesenchymal transition in HCC. CONCLUSION: Regulating diverse cellular signaling pathways makes isorhamnetin a better anti-cancer chemotherapeutic candidate in HCC. Importantly, the anti-TNF-α properties of isorhamnetin could prove it a valuable therapeutic agent in sorafenib-resistant HCC patients. Additionally, anti-TGF-ß properties of isorhamnetin could be utilized to reduce the EMT-inducing side effects of doxorubicin.

Histidine-Containing Amphiphilic Peptide-Based Non-Cytotoxic Hydrogelator with Antibacterial Activity and Sustainable Drug Release.

A histidine-based amphiphilic peptide (P) has been found to form an injectable transparent hydrogel in phosphate buffer solution over a pH range from 7.0 to 8.5 with an inherent antibacterial property. It also formed a hydrogel in water at pH = 6.7. The peptide self-assembles into a nanofibrillar network structure which is characterized by high-resolution transmission electron microscopy, field-emission scanning electron microscopy, atomic force microscopy, small-angle X-ray scattering, Fourier-transform infrared spectroscopy, and wide-angle powder X-ray diffraction. The hydrogel exhibits efficient antibacterial activity against both Gram-positive bacteria Staphylococcus aureus (S. aureus) and Gram-negative bacteria Escherichia coli (E. coli). The minimum inhibitory concentration of the hydrogel ranges from 20 to 100 µg/mL. The hydrogel is capable of encapsulation of the drugs naproxen (a non-steroidal anti-inflammatory drug), amoxicillin (an antibiotic), and doxorubicin, (an anticancer drug), but, selectively and sustainably, the gel releases naproxen, 84% being released in 84 h and amoxicillin was released more or less in same manner with that of the naproxen. The hydrogel is biocompatible with HEK 293T cells as well as NIH (mouse fibroblast cell line) cells and thus has potential as a potent antibacterial and drug releasing agent. Another remarkable feature of this hydrogel is its magnification property like a convex lens.

Polycyclic aromatic hydrocarbon sequestration by intertidal phototrophic biofilms cultivated in hydrophobic and hydrophilic biofilm-promoting culture vessels.

Phototrophic biofilms collected from intertidal sediments of the world's largest tidal mangrove forest were cultured in two sets of a biofilm-promoting culture vessel having hydrophilic glass surface and hydrophobic polymethyl methacrylate surface wherein 16 priority polycyclic aromatic hydrocarbons (PAHs) were spiked. Biofilms from three locations of the forest were most active in sequestering 98-100% of the spiked pollutants. PAH challenge did not alter the biofilm phototrophic community composition; rather biofilm biomass production and synthesis of photosynthetic pigments and extracellular polymeric substances (EPS) were enhanced. Photosynthetic pigment and EPS synthesis were sensitive to vessel-surface property. The lowest mean residual amounts of PAHs in the liquid medium as well as inside the biofilm were recorded in the very biofilm cultivated in the hydrophobic flask where highest values of biofilm biomass, total chlorophyll, released polysaccharidic (RPS) carbohydrates, RPS uronic acids, capsular polysaccharidic (CPS) carbohydrates, CPS proteins, CPS uronic acids and EPS hydrophobicity were obtained. Ratios of released RPS proteins: polysaccharides increased during PAH sequestration whereas the ratios of CPS proteins: polysaccharides remained constant. Efficacious PAH removal by the overlying phototrophic biofilm will reduce the entry of these contaminants in the sediments underneath and this strategy could be a model for "monitored natural recovery".

Assessment of polycyclic aromatic hydrocarbon contamination in the Sundarbans, the world's largest tidal mangrove forest and indigenous microbial mixed biofilm-based removal of the contaminants.

The distribution of polycyclic aromatic hydrocarbons (PAHs) in the surface water and sediments in five regions of the Indian Sundarbans was assessed. The capability of microbial biofilm communities to sequester PAHs in a biofilm-promoting vessel was evaluated. The total PAH concentration of water and sediments ranged from undetectable to 125 ng ml-1 and 4880 to 2 × 104 ng g-1 dry weight respectively. The total PAHs concentration of sediments exceeded the Effects Range-Low value and the recommended Effects Range-Median values, implying the PAHs might adversely affect the biota of the Sundarbans. Pyrogenic and petrogenic sources of PAH contamination were identified in most of the sampling sites. Indigenous biofilms were cultivated in a patented biofilm-promoting culture vessel containing liquid media spiked with 16 priority PAHs. Biofilm-mediated 97-100% removal efficiency of 16 PAHs was attained in all media. There was no significant difference between the mean residual PAH from the liquid media collected from hydrophobic and hydrophilic flasks. Residual amounts of acenaphthene (Ace), anthracene (Ant), benzo(b)fluoranthene [B(b)F], benzo(a)pyrene [B(a)P] and benzo(g,h,i)perylene [B(g,h,i)P] showed differences when cultivated in hydrophobic and hydrophilic flasks. The mean residual amounts of total PAHs extracted from biofilm biomasses were variable. A biofilm obtained from a specific sampling site cultured in the hydrophobic flask showed higher PAH sequestration when compared to the removal attained in the hydrophilic flask. Relative abundances of different microbial communities in PAH-sequestering biofilms revealed bacterial phyla including Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Chloroflexi and Planctomycetes as well as members of Ascomycota phylum of fungi. The dominance of Candida tropicalis, Clostridium butyricum, Sphingobacterium multivorum and Paecilomyces fulvus were established.

Remarkably high Pb2+ binding capacity of a novel, regenerable bioremediator Papiliotrema laurentii RY1: Functional in both alkaline and neutral environments.

The ability of P. laurentii strain RY1 to remediate lead (Pb2+) from water was investigated in batch and column studies. The lead removal ability of non-viable biomass, non-viable biomass immobilised on agar-agar (biobeads) and agar-agar at different pH was compared in batch studies. It was found that among the three, biobeads have maximum ability to remove Pb2+ followed by biomass and agar-agar beads. Maximum and almost equal lead removal by biobeads was observed at both neutral and alkaline pH making it a novel and more applicable bioremediator as all other reported bioremediators have a single pH for optimum activity. Studies were performed to determine the optimum conditions for lead removal from aqueous solutions for biobeads. The physical and chemical characterization of the biobeads before and after Pb2+ biosorption was done by using S.E.M. and F.T.I.R. respectively. The adsorption of Pb2+ on biobeads obeyed the Langmuir adsorption isotherm and pseudo first order kinetics. These mean that the Pb2+ binding sites are identical, located on the surface of the adsorbant and the rate of Pb2+ removal from aqueous solution is directly proportional to the number of Pb2+ binding sites on the biobeads. The thermodynamics of the biosorption process is also investigated. The binding capacity of the biobeads in batch study was found to be 52.91mg/gm which is higher in comparison to other reported yeast bioremediators. The used biobeads can be desorbed using 0.1(M) CaCl2. The desorbed biobeads can be used subsequently for several cycles of lead removal making it cost-effective. Column studies were also performed for biobeads with the help of Thomas model for examining its suitability for industrial application. Maximum specific lead uptake of the biobeads when applied in the column was found to be 58.26mg/gm which being promising makes it suitable for application in industries involved in the treatment of wastewater contaminated with high amounts of lead. The high mass transfer co-efficient indicate that small sized column can be used effectively to remove high amounts of lead which makes the bioremediation process by the biobeads more economical and advantageous for industrial application. Several factors like effectiveness of the biobeads in Pb2+removal at both neutral and alkaline pH, reusability, high mass transfer co-efficient, regenerability and high binding capacity makes it a novel versatile, cost-effective and high utility bioremediator.

Anti-virulence activity of polyphenolic fraction isolated from Kombucha against Vibrio cholerae.

The use of traditional foods and beverages or their bioactive compounds as anti-virulence agents is a new alternative method to overcome the increased global emergence of antimicrobial resistance in enteric pathogens. In the present study, we investigated the anti-virulence activity of a polyphenolic fraction previously isolated from Kombucha, a 14-day fermented beverage of sugared black tea, against Vibrio cholerae O1. The isolated fraction was mainly composed of the polyphenols catechin and isorhamnetin. The fraction, the individual polyphenols and the combination of the individual polyphenols significantly inhibited bacterial swarming motility and expression of flagellar regulatory genes motY and flaC, even at sub-inhibitory concentrations. The polyphenolic compounds also decreased bacterial protease secretion and mucin penetration in vitro. In vivo study revealed that the polyphenolic fraction significantly inhibited V. cholerae induced fluid accumulation in the rabbit ileal loop model and intestinal colonization in suckling mice model. Therefore, the anti-virulence activity of the Kombucha polyphenolic fraction involved inhibition of motility and protease secretion of V. cholerae, thus preventing bacterial penetration through the mucin layer as well as fluid accumulation and bacterial colonization in the intestinal epithelial cells. The overall results implied that Kombucha might be considered as a potential alternative source of anti-virulence polyphenols against V. cholerae. To the best of our knowledge, this is the first report on the anti-virulence activity of Kombucha, mostly attributed to its polyphenolic content.

Removal of Pb (II), As (III), and Cr (VI) by nitrogen-starved Papiliotrema laurentii strain RY1.

Heavy metals such as lead, chromium, and metalloid like arsenic dominate the pinnacle in posing a threat to life. Being environment-friendly, elucidating the mechanism by which microorganisms detoxify such elements has always been an active field of research hitherto. In the present study, we have investigated the capability of nitrogen-deprived Papiliotrema laurentii strain RY1 toward enhanced tolerance and neutralizing toxic elements. There were biosorption and bioprecipitation of lead and chromium at the cell surfaces. Bioprecipitation mechanisms included the formation of lead phosphates and pyromorphites from lead, grimaldite from chromium. Transcripts such as metallothionein, aquaporins, and arsenical pump-driving ATPase have been surmised to be involved in the detoxification of elements. Furthermore, activation of antioxidant defense mechanisms for the cells for each of the elements should contribute towards yeast's propagation. The efficiency of removal of elements for live cells and immobilized cells were high for lead and chromium. To the best of our knowledge, this is the first report of such high tolerance of lead, arsenic, and chromium for any yeast. The yeast showed such varied response under dual stress due to nitrogen starvation and in the presence of respective elements. The yeast possesses promising potentials in nitrogen deprived and enriched environments to aid in bioremediation sectors.

DMSO strengthens chitin deacetylase-chitin interaction: Physicochemical, kinetic, structural and catalytic insights.

Chitin deacetylase, an enzyme isolated from Cryptococcus laurentii RY1, catalyzes the hydrolysis of acetamido group of N-acetyl-D-glucosamine unit of chitin. The primary objective of this study was to characterize and comprehend the activation of chitin deacetylase by DMSO. The secondary structure of the protein was determined by circular dichroism(CD).The interaction of protein with DMSO was evaluated by CD and tryptophan fluorescence spectroscopy which revealed that DMSO had no effect on overall secondary structure, but induced change in the tertiary structure of the enzyme. The interaction of chitin deacetylase with chitin in DMSO system when investigated by molecular dynamics simulation revealed stronger chitin deacetylase-chitin interaction involving several amino acid residues. The enhanced activity of the enzyme in presence of DMSO along with the fact that its kcat is highest of all other reported chitin deacetylases makes it a superior candidate in the industrial sector involved in chitosan production from chitin.

Nitrogen deprivation elicits dimorphism, capsule biosynthesis and autophagy in Papiliotrema laurentii strain RY1.

Stress response due to the lack of essential nutrient(s) for an organism has been a focal point of several scientific investigations. The present study investigates the cellular adaptations behind the ability of Papiliotrema laurentii strain RY1 to perpetuate without added nitrogen and propagate robustly in growth- limiting amount of nitrogen. We executed phenotypic (using scanning electron microscopy, differential interference contrast microscopy and transmission electron microscopy), microbiological and computational analyses to show multiple responses of dimorphism, capsule formation and autophagy as a survival strategy by the yeast upon nitrogen starvation. The roles of phosphomannose isomerase, phosphomannomutase and several autophagy-related transcripts aiding in such a response have been discussed.

U32 collagenase from Pseudoalteromonas agarivorans NW4327: Activity, structure, substrate interactions and molecular dynamics simulations.

A protease of the primary pathogen (Pseudoalteromonas agarivorans NW4327) of the disease affecting the Great Barrier Reef sponge Rhopaloeides odorabile was purified. Zymography demonstrated calcium-dependent collagenase and gelatinase activity of the purified protein. This metalloprotease was identified by matrix assisted laser desorption ionization time-of-flight mass spectrophotometry as a 52,509 Da U32 collagenase. Predicted tertiary structure of U32 collagenase (by Phyre2 fold recognition server) demonstrated 13% identity with known hydrolases establishing novelty of the enzyme. Molecular docking conceived two interacting loops of the collagenase that bound with collagen triple helices and two calcium ions remained centered between the loops. According to ConSurf multiple sequence alignment, the residues of loop1 of the collagenase were mostly conserved while variations among residues of loop2 were comparatively higher than loop1. Asp262, Glu263 of loop1 and Thr363, Lys364, Gln365 of loop2 participated in the interaction with Ca2+ and collagen. Root mean square deviation and root mean square fluctuation values signified higher stability of the collagen-Ca2+-collagenase complex and greater structural stability of the residues of the loops in the complex compared to apocollagenase. Observed properties of NW4327 U32 collagenase and its interaction with collagen were different from similar enzymes of thermophilic bacteria and terrestrial pathogens.

Investigation of conformational changes of levansucrase isolated from Acetobacter nitrogenifigens strain RG1 by mercuric and cadmium ion.

Levansucrase is a secretary enzyme of Acetobacter nitrogenifigens strain RG1. The enzyme shows enhanced activity in the presence of Hg2+ in spite of being inhibited by other heavy metal ion Cd2+. In this study the structural characterization of levansucrase in native state as well as in the presence of Hg2+ and Cd2+ by CD spectroscopy is done. The secondary structures of the native enzyme and the enzyme treated with Hg2+ and Cd2+ on comparison by their CD spectra revealed that their spectra showed no significant difference indicating that both Hg2+ as well as Cd2+ had no effect on the overall secondary structure of the protein. The respective CD spectra on analysis revealed that they have almost identical percentage of secondary structural elements. The interaction of levansucrase with Hg2+ as well as Cd2+ was studied further by tryptophan fluorescence spectroscopy which on analysis revealed static quenching indicating protein-heavy metal complex formation. A blue shift in the tryptophan fluorescence spectra of Hg2+ treated protein indicated that the tryptophan residues have moved to a more hydrophobic environment in the protein away from aqueous phase. The mechanism of interaction of enzyme with mercury and cadmium was determined from their tryptophan fluorescence spectra.

A new collagenase enzyme of the marine sponge pathogen Pseudoalteromonas agarivorans NW4327 is uniquely linked with a TonB dependent receptor.

The primary pathogen of the Great Barrier Reef sponge Rhopaloeides odorabile, recently identified as a novel strain (NW4327) of Pseudoalteromonas agarivorans, produced collagenase which degraded R. odorabile skeletal fibers. We now report the collagenase of P. agarivorans as a metalloprotease which required Ca2+ and Zn2+ as cofactors. The collagenase was a TonB dependent receptor (TBDR) having a carboxypeptidase regulatory like domain (CRLD) in the N-terminal along with an outer membrane (OM) channel superfamily domain. The genes for TBDR sub-components and collagenase formed one unified entity in the genome of P. agarivorans NW4327. This association of a collagenase with a TBDR distinguished it from all known functional collagenases till date and for the first time, established the enzymatic capability of TBDRs. Predicted TBDR model demonstrated only 15% identity with ferripyoverdin receptor and the CRLD displayed merely 24% identity with carboxypeptidase catalytic chain. Presence of signal peptide, lack of transmembrane helices, absence of N-terminal in the cytoplasmic side, extracellular localization and recovery from the culture supernatant implicated that the TBDR was secreted. Stronger binding of the collagenase with marine sponge type IV collagen than type I collagen, revealed through molecular docking, indicated higher specificity of the enzyme towards type IV collagen.

De novo RNA-Seq based transcriptome analysis of Papiliotrema laurentii strain RY1 under nitrogen starvation.

Nitrogen is a key nutrient for all cell forms. Most organisms respond to nitrogen scarcity by slowing down their growth rate. On the contrary, our previous studies have shown that Papiliotrema laurentii strain RY1 has a robust growth under nitrogen starvation. To understand the global regulation that leads to such an extraordinary response, we undertook a de novo approach for transcriptome analysis of the yeast. Close to 33 million sequence reads of high quality for nitrogen limited and enriched condition were generated using Illumina NextSeq500. Trinity analysis and clustered transcripts annotation of the reads produced 17,611 unigenes, out of which 14,157 could be annotated. Gene Ontology term analysis generated 44.92% cellular component terms, 39.81% molecular function terms and 15.24% biological process terms. The most over represented pathways in general were translation, carbohydrate metabolism, amino acid metabolism, general metabolism, folding, sorting, degradation followed by transport and catabolism, nucleotide metabolism, replication and repair, transcription and lipid metabolism. A total of 4256 Single Sequence Repeats were identified. Differential gene expression analysis detected 996 P-significant transcripts to reveal transmembrane transport, lipid homeostasis, fatty acid catabolism and translation as the enriched terms which could be essential for Papiliotrema laurentii strain RY1 to adapt during nitrogen deprivation. Transcriptome data was validated by quantitative real-time PCR analysis of twelve transcripts. To the best of our knowledge, this is the first report of Papiliotrema laurentii strain RY1 transcriptome which would play a pivotal role in understanding the biochemistry of the yeast under acute nitrogen stress and this study would be encouraging to initiate extensive investigations into this Papiliotrema system.

Homology modeling, molecular docking and molecular dynamics studies of the catalytic domain of chitin deacetylase from Cryptococcus laurentii strain RY1.

This study provides structural insights into chitin deacetylase, over-expressing under nitrogen limiting condition in Cryptococcus laurentii strain RY1. The enzyme converts chitin, the second most abundant natural biopolymer, to chitosan, which offers tremendous applications in diverse fields. To elucidate the structure-function relationship of this biologically and industrially important enzyme, a homology model of the catalytic domain was constructed. The stability of the structure was assessed by molecular dynamics simulation studies. Tryptophan 151 of the domain was identified to form hydrogen bond and stacking interaction with chitin upon docking. In Silico substitution of Tryptophan (W) to Alanine (A), Phenylalanine (F) and Aspartate (D) corroborated the importance of the Tryptophan residue in interaction with the substrate. This is the first report of unravelling the structural characteristics of chitin deacetylase from Cryptococcus and understanding the approach of the enzyme towards its substrate. Our results would be helpful to perform experimental validations and apply quantum mechanics/molecular mechanics techniques to determine the detailed catalytic mechanism and enhance the industrial potency of the enzyme.

Antibacterial Activity of Polyphenolic Fraction of Kombucha Against Enteric Bacterial Pathogens.

The emergence of multi-drug-resistant enteric pathogens has prompted the scientist community to explore the therapeutic potentials of traditional foods and beverages. The present study was undertaken to investigate the efficacy of Kombucha, a fermented beverage of sugared black tea, against enterotoxigenic Escherichia coli, Vibrio cholerae, Shigella flexneri and Salmonella Typhimurium followed by the identification of the antibacterial components present in Kombucha. The antibacterial activity was evaluated by determining the inhibition zone diameter, minimal inhibitory concentration and minimal bactericidal concentration. Kombucha fermented for 14 days showed maximum activity against the bacterial strains. Its ethyl acetate extract was found to be the most effective upon sequential solvent extraction of the 14-day Kombucha. This potent ethyl acetate extract was then subjected to thin layer chromatography for further purification of antibacterial ingredients which led to the isolation of an active polyphenolic fraction. Catechin and isorhamnetin were detected as the major antibacterial compounds present in this polyphenolic fraction of Kombucha by High Performance Liquid Chromatography. Catechin, one of the primary antibacterial polyphenols in tea was also found to be present in Kombucha. But isorhamnetin is not reported to be present in tea, which may thereby suggest the role of fermentation process of black tea for its production in Kombucha. To the best of our knowledge, this is the first report on the presence of isorhamnetin in Kombucha. The overall study suggests that Kombucha can be used as a potent antibacterial agent against entero-pathogenic bacterial infections, which mainly is attributed to its polyphenolic content.

Kombucha tea fermentation: Microbial and biochemical dynamics.

Kombucha tea, a non-alcoholic beverage, is acquiring significant interest due to its claimed beneficial properties. The microbial community of Kombucha tea consists of bacteria and yeast which thrive in two mutually non-exclusive compartments: the soup or the beverage and the biofilm floating on it. The microbial community and the biochemical properties of the beverage have so far mostly been described in separate studies. This, however, may prevent understanding the causal links between the microbial communities and the beneficial properties of Kombucha tea. Moreover, an extensive study into the microbial and biochemical dynamics has also been missing. In this study, we thus explored the structure and dynamics of the microbial community along with the biochemical properties of Kombucha tea at different time points up to 21 days of fermentation. We hypothesized that several biochemical properties will change during the course of fermentation along with the shifts in the yeast and bacterial communities. The yeast community of the biofilm did not show much variation over time and was dominated by Candida sp. (73.5-83%). The soup however, showed a significant shift in dominance from Candida sp. to Lachancea sp. on the 7th day of fermentation. This is the first report showing Candida as the most dominating yeast genus during Kombucha fermentation. Komagateibacter was identified as the single largest bacterial genus present in both the biofilm and the soup (~50%). The bacterial diversity was higher in the soup than in the biofilm with a peak on the seventh day of fermentation. The biochemical properties changed with the progression of the fermentation, i.e., beneficial properties of the beverage such as the radical scavenging ability increased significantly with a maximum increase at day 7. We further observed a significantly higher D-saccharic acid-1,4-lactone content and caffeine degradation property compared to previously described Kombucha tea fermentations. Our data thus indicate that the microbial community structure and dynamics play an important role in the biochemistry of the fermentation of the beverage. We envisage that combined molecular and biochemical analyses like in our study will provide valuable insights for better understanding the role of the microbial community for the beneficial properties of the beverage.

Expression of a chitin deacetylase gene, up-regulated in Cryptococcus laurentii strain RY1, under nitrogen limitation.

This study reports the identification of a chitin deacetylase gene in Cryptococcus laurentii strain RY1 over-expressing under nitrogen limitation by differential display. The up-regulation took place in robustly growing cells rather than in starving quiescent autophagic cells. Quantitative Real Time-PCR, enzyme activity in cell lysate and cell wall analysis corroborated the up-regulation of chitin deacetylase under nitrogen limitation. These results suggest chitin deacetylase might play a significant role in nitrogen limiting growth of Cryptococcus laurentii strain RY1.

Enhanced biofilm formation and melanin synthesis by the oyster settlement-promoting Shewanella colwelliana is related to hydrophobic surface and simulated intertidal environment.

A direct relationship between biofilm formation and melanogenesis in Shewanella colwelliana with increased oyster recruitment is already established. Previously, S. colwelliana was grown in a newly patented biofilm-cultivation device, the conico-cylindrical flask (CCF), offering interchangeable hydrophobic/hydrophilic surfaces. Melanization was enhanced when S. colwelliana was cultivated in a hydrophobic vessel compared with a hydrophilic vessel. In the present study, melanogenesis in the CCF was positively correlated with increased architectural parameters of the biofilm (mean thickness and biovolume obtained by confocal laser scanning microscopy) and melanin gene (melA) expression observed by densitometry. Niche intertidal conditions were mimicked in a process operated in an ultra-low-speed rotating disk bioreactor, which demonstrated enhanced biofilm formation, melanogenesis, exopolysaccharide synthesis and melA gene expression compared with a process where 12-h periodic immersion and emersion was prevented. The wettability properties of the settling plane as well as intermittent wetting and drying, which influenced biofilm formation and melA expression, may affect oyster settlement in nature.