Ecofriendly biosynthesis of copper nanoparticles from novel marine S. rhizophila species for enhanced antibiofilm, antimicrobial and antioxidant potential.

Marine microorganisms offer a promising avenue for the eco-friendly synthesis of nanoparticles due to their unique biochemical capabilities and adaptability to various environments. This study focuses on exploring the potential of a marine bacterial species, Stenotrophomonas rhizophila BGNAK1, for the synthesis of biocompatible copper nanoparticles and their application for hindering biofilms formed by monomicrobial species. The study begins with the isolation of the novel marine S. rhizophila species from marine soil samples collected from the West coast region of Kerala, India. The isolated strain is identified through 16S rRNA gene sequencing and confirmed to be S. rhizophila species. Biosynthesis of copper nanoparticles using S. rhizophila results in the formation of nanoparticles with size of range 10-50 nm. The nanoparticles exhibit a face-centered cubic crystal structure of copper, as confirmed by X-Ray Diffraction analysis. Furthermore, the synthesized nanoparticles display significant antimicrobial activity against various pathogenic bacteria and yeast. The highest inhibitory activity was against Staphylococcus aureus with a zone of 27 ± 1.00 mm and the least activity was against Pseudomonas aeruginosa with a zone of 22 ± 0.50 mm. The zone of inhibition against Candida albicans was 16 ± 0.60 mm. The antibiofilm activity against biofilm-forming clinical pathogens was evidenced by the antibiofilm assay and SEM images. Additionally, the copper nanoparticles exhibit antioxidant activity, as evidenced by their scavenging ability against DPPH, hydroxyl, nitric oxide, and superoxide radicals, as well as their reducing power in the FRAP assay. The study highlights the potential of the marine bacterium S. rhizophila BGNAK1 for the eco-friendly biosynthesis of copper nanoparticles with diverse applications. Synthesized nanoparticles exhibit promising antibiofilm, antimicrobial, and antioxidant properties, suggesting their potential utility in various fields such as medicine, wastewater treatment, and environmental remediation.

Marine Organisms as a Prolific Source of Bioactive Depsipeptides.

Depsipeptides, an important group of polypeptides containing residues of hydroxy acids and amino acids linked together by amide and ester bonds, have potential applications in agriculture and medicine. A growing body of evidence demonstrates that marine organisms are prolific sources of depsipeptides, such as marine cyanobacteria, sponges, mollusks, microorganisms and algae. However, these substances have not yet been comprehensively summarized. In order to enrich our knowledge about marine depsipeptides, their biological sources and structural features, as well as bioactivities, are highlighted in this review after an extensive literature search and data analysis.

Marine-derived microbes and molecules for drug discovery.

Increasing attention has been paid to marine-derived biomolecules as sources of therapeutics for autoimmune diseases. Nagasaki Prefecture has many islands and is surrounded by seas, straits, gulfs, bays, and coves, giving it the second longest coastline in Japan after Hokkaido. We have collected more than 20,000 marine microbes and have been preparing an original marine microbial extract library, which contains small and mid-size biomolecules that may penetrate cell membranes and interfere with the intracellular protein-protein interaction involved in the development of autoinflammatory diseases such as familial Mediterranean fever. In addition, we have been developing an indoor shark farming system to prepare shark nanobodies that could be developed as potential therapeutic agents for autoimmune diseases. Sharks produce heavy-chain antibodies, called immunoglobulin new antigen receptors (IgNARs), consisting of one variable domain (VNAR) and five constant domains (CNAR); of these, VNAR can recognize a variety of foreign antigens. A VNAR single domain fragment, called a nanobody, can be expressed in Escherichia coli and has the properties of an ideal therapeutic candidate for autoimmune diseases. Shark nanobodies contain complementarity-determining regions that are formed through the somatic rearrangement of variable, diversity, and joining segments, with the segment end trimming and the N- and P-additions, as found in the variable domains of mammalian antibodies. The affinity and diversity of shark nanobodies are thus expected to be comparable to those of mammalian antibodies. In addition, shark nanobodies are physically robust and can be prepared inexpensively; as such, they may lead to the development of highly specific, stable, effective, and inexpensive biotherapeutics in the future. In this review, we first summarize the history of the development of conventional small molecule drugs and monoclonal antibody therapeutics for autoimmune diseases, and then introduce our drug discovery system at Nagasaki University, including the preparation of an original marine microbial extract library and the development of shark nanobodies.

Structural and Biochemical Basis of a Marine Bacterial Glycoside Hydrolase Family 2 ß-Glycosidase with Broad Substrate Specificity.

Uronic acids are commonly found in marine polysaccharides and increase structural complexity and intrinsic recalcitrance to enzymatic attack. Glycoside hydrolase family 2 (GH2) includes proteins that target sugar conjugates with hexuronates and are involved in the catabolism and cycling of marine polysaccharides. Here, we report a novel GH2, AqGalA from a marine alga-associated Bacteroidetes organism with broad substrate specificity. Biochemical analyses revealed that AqGalA exhibits hydrolyzing activities against ß-galacturonide, ß-glucuronide, and ß-galactopyranoside via retaining mechanisms. We solved the AqGalA crystal structure in complex with galacturonic acid (GalA) and determined (via mutagenesis) that charge characteristics at uronate-binding subsites controlled substrate selectivity for uronide hydrolysis. Additionally, conformational flexibility of the AqGalA active-site pocket was proposed as a key component for broad substrate enzyme selectivity. Our AqGalA structural and functional data augment the current understanding of substrate recognition of GH2 enzymes and provide key insights into the bacterial use of uronic acid-containing polysaccharides. IMPORTANCE The decomposition of algal glycans driven by marine bacterial communities represents one of the largest heterotrophic transformations of organic matter fueling marine food webs and global carbon cycling. However, our knowledge on carbohydrate cycling is limited due to structural complexity of marine polysaccharides and the complicated enzymatic machinery of marine microbes. To degrade algal glycan, marine bacteria such as members of the Bacteroidetes produce a complex repertoire of carbohydrate-active enzymes (CAZymes) matching the structural specificities of the different carbohydrates. In this study, we investigated an extracellular GH2 ß-glycosidase, AqGalA from a marine Bacteroidetes organism, to identify the key components responsible for glycuronide recognition and hydrolysis. The broad substrate specificity of AqGalA against glycosides with diverse stereochemical substitutions indicates its potential in processing complex marine polysaccharides. Our findings promote a better understanding of microbially driven mechanisms of marine carbohydrate cycling.

Phthalic acid esters degradation by a novel marine bacterial strain Mycolicibacterium phocaicum RL-HY01: Characterization, metabolic pathway and bioaugmentation.

Phthalic acid esters (PAEs) are one of the most widely used plasticizers and the well-studied environmental pollutants with endocrine disrupting properties. Investigation about PAEs in terrestrial ecosystem has been extensively conducted while the fate of PAEs in marine environment remains underexplored. In this study, a novel di-(2-ethylhexyl) phthalate (DEHP) degrading marine bacterial strain, Mycolicibacterium phocaicum RL-HY01, was isolated and characterized from intertidal sediments. Strain RL-HY01 could utilize a range of PAE plasticizers as sole carbon source for growth. The effects of different environmental factors on the degradation of PAEs were evaluated and the results indicated that strain RL-HY01 could efficiently degrade PAEs under a wide range of pH (5.0 to 9.0), temperature (20 °C to 40 °C) and salinity (below 10%). Specifically, when Tween-80 was added as solubilizing agent, strain RL-HY01 could rapidly degrade DEHP and achieve complete degradation of DEHP (50 mg/L) in 48 h. The kinetics of DEHP degradation by RL-HY01 were well fitted with the modified Gompertz model. The metabolic intermediates of DEHP by strain RL-HY01 were identified by ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis and then the metabolic pathway of DEHP was deduced. DEHP was transformed into di-ethyl phthalate (DEP) via ß-oxidation and then DEP was hydrolyzed into phthalic acid (PA) by de-esterification. PA was further transformed into gentisate via salicylic acid and further utilized for cell growth. Bioaugmentation of strain RL-HY01 with marine samples was performed to evaluate its application potential and the results suggested that strain RL-HY01 could accelerate the elimination of DEHP in marine samples. The results have advanced our understanding of the fate of PAEs in marine ecosystem and identified an efficient bioremediation strategy for PAEs-polluted marine sites.

Homoseongomycin, a compound isolated from marine actinomycete bacteria K3-1, is a potent inhibitor of encephalitic alphaviruses.

Marine microorganisms have been a resource for novel therapeutic drugs for decades. In addition to anticancer drugs, the drug acyclovir, derived from a marine sponge, is FDA-approved for the treatment of human herpes simplex virus-1 infections. Most alphaviruses that are infectious to terrestrial animals and humans, such as Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV), lack efficient antiviral drugs and it is imperative to develop these remedies. To push the discovery and development of anti-alphavirus compounds forward, this study aimed to isolate and screen for potential antiviral compounds from cultured marine microbes originating from the marine environment. Compounds from marine microbes were of interest as they are prolific producers of bioactive compounds across the spectrum of human diseases and infections. Homoseongomycin, an actinobacteria isolated from a marine sponge displayed impressive activity against VEEV from a total of 76 marine bioactive products. The 50% effective concentration (EC50) for homoseongomycin was 8.6 µM for suppressing VEEV TC-83 luciferase reporter virus replication. Homoseongomycin was non-toxic up to 50 µM and partially rescued cells from VEEV induced cell death. Homoseongomycin exhibited highly efficient antiviral activity with a reduction of VEEV infectious titers by 8 log10 at 50 µM. It also inhibited EEEV replication with an EC50 of 1.2 µM. Mechanism of action studies suggest that homoseongomycin affects both early and late stages of the viral life cycle. Cells treated with 25 µM of homoseongomycin had a ~90% reduction in viral entry. In comparison, later stages showed a more robust reduction in infectious titers (6 log10) and VEEV extracellular viral RNA levels (4 log10), but a lesser impact on intracellular viral RNA levels (1.5 log10). In sum, this work demonstrates that homoseongomycin is a potential anti-VEEV and anti-EEEV compound due to its low cytotoxicity and potent antiviral activity.

Optimization of fermentation conditions and medium compositions for the production of chrysomycin a by a marine-derived strain Streptomyces sp. 891.

Chrysomycin A is one of the valuable drug leads used to treat infectious diseases such as tuberculosis and methicillin-resistant Staphylococcus aureus. In order to increase its yield, this work firstly focuses on optimization of fermentation conditions and medium compositions of a wild-type chrysomycin A-producing strain Streptomyces sp. 891 from marine sediment. By single-factor experiment, effects of fermentation conditions (fermentation time, seed age, initial pH, inoculum amount, liquid loading, shaking speed) and medium composition (carbon sources, nitrogen sources, inorganic salts) on the yield of chrysomycin A were carefully evaluated and analyzed followed by optimization at shake-flask level. The results indicated its optimal fermentation conditions for producing chrysomycin A were as follows: fermentation time 168 h, seed age 48 h, initial pH 6.5, inoculum amount 5.0%, liquid loading 30 mL in 250-mL Erlenmeyer flask and shaking speed 220 rpm. By orthogonal test, the optimal fermentation medium constitutes 40 g/L glucose, 20 g/L corn starch, 25 g/L hot-pressed soybean flour, 3 g/L CaCO3. Verification tests suggested the yield of chrysomycin A under optimized conditions reaches up to 3648 ± 119 mg/L, which is increased by almost 5 times. These findings definitely pave the way for scale-up preparation of chrysomycin A and application in the pharmaceutical industry.

Molecular Basis for Substrate Recognition and Catalysis by a Marine Bacterial Laminarinase.

Laminarin is an abundant algal polysaccharide that serves as carbon storage and fuel to meet the nutrition demands of heterotrophic microbes. Laminarin depolymerization catalyzed by microbial extracellular enzymes initiates remineralization, a key process in ocean biogeochemical cycles. Here, we described a glycoside hydrolase 16 (GH16) family laminarinase from a marine alga-associated Flavobacterium at the biochemical and structural levels. We found that the endolytic enzyme cleaved laminarin with a preference for ß-1,3-glycoside linkages and showed transglycosylation activity across a broad range of acceptors. We also solved and compared high-resolution crystal structures of laminarinase in the apo form and in complex with ß-1,3-tetrasaccharides, revealing an expanded catalytic cleft formed following substrate binding. Moreover, structure and mutagenesis studies identified multiple specific contacts between the enzyme and glucosyl residues essential for the substrate specificity for ß-1,3-glucan. These results provide novel insights into the structural requirements for substrate binding and catalysis of GH16 family laminarinase, enriching our understanding of bacterial utilization of algal laminarin.IMPORTANCE Heterotrophic bacterial communities are key players in marine biogeochemical cycling due to their ability to remineralize organic carbon. Processing of complex organic matter requires heterotrophic bacteria to produce extracellular enzymes with precise specificity to depolymerize substrates to sizes sufficiently small for uptake. Thus, extracellular enzymatic hydrolysis initiates microbe-driven heterotrophic carbon cycling. In this study, based on biochemical and structural analyses, we revealed the depolymerization mechanism of ß-1,3-glucan, a carbon reserve in algae, by laminarinase from an alga-associated marine Flavobacterium The findings provide new insights into the substrate recognition and catalysis of bacterial laminarinase and promote a better understanding of how extracellular enzymes are involved in organic matter cycling.

Marine Microbiome as a Source of Antimalarials.

It is important to discover novel antimalarial pharmacophores because of the widespread emergence of Plasmodium falciparum isolates resistant to the available drugs. Secondary metabolites derived from microbes associated with marine invertebrates are a valuable resource for the discovery of novel drug leads. However, the potential of marine microbes as a source of antimalarials has not been explored. We investigated the promise of marine microorganisms for the production of antimalarial activities by testing 2365 diverse microbial extracts using phenotypic screening of a multidrug resistant chloroquine resistant P. falciparum strain. We conducted counter screening against mammalian cells for the 317 active extracts that exhibited more than 70% inhibition at 1 µg/mL. The screen identified 17 potent bioactive leads from a broad range of taxa. Our results establish that the marine microbiome is a rich source of antiplasmodial compounds that warrants in depth exploration.

Proposal of Litorimonas haliclonae sp. nov., isolated from a marine sponge of the genus Haliclona.

A bright-orange-pigmented, Gram-stain-negative, motile, and rod-shaped bacterium, strain MAA42T, was isolated from a marine sponge of the genus Haliclona, which is in long-time culture in a marine aquarium system at the Justus Liebig University Giessen, Germany. The strain grew at 4-34 °C (optimum 28 °C), in the presence of 0.5-9.5 % (w/v) NaCl (optimum 3.5 %) and at pH 4.5-10.0 (optimum pH 7.5). Strain MAA42T shared the highest 16S rRNA gene sequence similarity (98.1 %) with the type strain of Litorimonas taeanensis. Sequence similarities to all other closely related type strains were below 97 %. DNA-DNA hybridization of strain MAA42T with L. taeanensis DSM 22008T resulted in values of 4.7 % (reciprocal 17.7 %). Major cellular fatty acids of strain MAA42T were C18 : 1ω7c (66.2 %), C18 : 1 2-OH (17.4 %), and C18 : 0 (14.1 %). Spermidine was predominant in the polyamine pattern, and ubiquinone Q-10 was the major respiratory quinone. The polar lipid profile contained the major compounds phosphatidylglycerol, monoglycosyldiglyceride, three unidentified phospholipids, and one unidentified glycolipid. Glucuronopyranosyldiglyceride was present as a minor compound. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The genomic DNA G+C content was 52.8 mol%. Based on the genotypic, chemotaxonomic, and phenotypic analyses, strain MAA42T represents a novel species of the genus Litorimonas, for which the name Litorimonas haliclonae is proposed. The type strain is MAA42T (=CCM 8709T=CIP 111178T=LMG 29765T).

Comparative Genomics of Methanopyrus sp. SNP6 and KOL6 Revealing Genomic Regions of Plasticity Implicated in Extremely Thermophilic Profiles.

Methanopyrus spp. are usually isolated from harsh niches, such as high osmotic pressure and extreme temperature. However, the molecular mechanisms for their environmental adaption are poorly understood. Archaeal species is commonly considered as primitive organism. The evolutional placement of archaea is a fundamental and intriguing scientific question. We sequenced the genomes of Methanopyrus strains SNP6 and KOL6 isolated from the Atlantic and Iceland, respectively. Comparative genomic analysis revealed genetic diversity and instability implicated in niche adaption, including a number of transporter- and integrase/transposase-related genes. Pan-genome analysis also defined the gene pool of Methanopyrus spp., in addition of ~120-Kb genomic region of plasticity impacting cognate genomic architecture. We believe that Methanopyrus genomics could facilitate efficient investigation/recognition of archaeal phylogenetic diverse patterns, as well as improve understanding of biological roles and significance of these versatile microbes.

Cytotoxic Natural Products from Marine Sponge-Derived Microorganisms.

A growing body of evidence indicates that marine sponge-derived microbes possess the potential ability to make prolific natural products with therapeutic effects. This review for the first time provides a comprehensive overview of new cytotoxic agents from these marine microbes over the last 62 years from 1955 to 2016, which are assorted into seven types: terpenes, alkaloids, peptides, aromatics, lactones, steroids, and miscellaneous compounds.

Biosynthesis of gold and silver nanoparticles using a novel marine strain of Stenotrophomonas.

The present study aims at exploiting marine microbial diversity for biosynthesis of metal nanoparticles and also investigates role of microbial proteins in the process of bio-mineralization of gold and silver. This is the first report for concurrent production of gold and silver nanoparticles (AuNPs and AgNPs) by extracellular secretion of a novel strain of Stenotrophomonas, isolated from Indian marine origin. This novel strain has faster rate kinetics for AgNPs synthesis than any other organism reported earlier. The nanoparticles were further characterized using UV-vis spectrophotometer, TEM, DLS and EDAX confirming their size ranging from 10-50 nm and 40-60 nm in dimensions for AuNPs and AgNPs, respectively. TEM analysis indicated formation of multi-shaped nanoparticles with heterogeneous size distribution in both the cases. Finally, the SDS-PAGE analysis of extracellular media supernatant suggested a potential involvement of certain low molecular weight secretory proteins in AuNPs and AgNPs biosynthesis.