Marine seaweed endophytic fungi-derived active metabolites promote reactive oxygen species-induced cell cycle arrest and apoptosis in human breast cancer cells.

BACKGROUND: Endophytic fungi have an abundant sources rich source of rich bioactive molecules with pivotal pharmacological properties. Several studies have found that endophytic fungi-derived bioactive secondary metabolites have antiproliferative, anti-oxidant, and anti-inflammatory properties, but the molecular mechanism by which they induce cell cycle arrest and apoptosis pathways is unknown. This study aimed to determine the molecular mechanism underlying the anticancer property of the endophytic fungi derived active secondary metabolites on human breast cancer cells. METHODS: In this study, we identified four endophytic fungi from marine seaweeds and partially screened its phytochemical properties by Chromatography-Mass Spectrometry (GC-MS) analysis. Moreover, the molecular mechanism underlying the anticancer property of these active secondary metabolites (FA, FB, FC and FE) on human breast cancer cells were examined on MCF-7 cells by TT assay, Apoptotic assay by Acridine orang/Ethidium Bromide (Dual Staining), DNA Fragmentation by DAPI Staining, reactive oxygen species (ROS) determination by DCFH-DA assay, Cell cycle analysis was conducted Flow cytometry and the apoptotic signalling pathway was evaluated by westernblot analysis. Doxorubicin was used as a positive control drug for this experiment. RESULTS: The GC-MS analysis of ethyl acetate extract of endophytic fungi from the marine macro-algae revealed the different functional groups and bioactive secondary metabolites. From the library, we observed the FC (76%), FB (75%), FA (73%) and FE (71%) have high level of antioxidant activity which was assessed by DPPH scavenging assay. Further, we evaluated the cytotoxic potentials of these secondary metabolites on human breast cancer MCF-7 cells for 24 h and the IC50 value were calculated (FA:28.62 ± 0.3 µg/ml, FB:49.81 ± 2.5 µg/ml, FC:139.42 ± µg/ml and FE:22.47 ± 0.5 µg/ul) along with positive control Doxorubicin 15.64 ± 0.8 µg/ml respectively by MTT assay. The molecular mechanism by which the four active compound induced apoptosis via reactive oxygen species (ROS) and cell cycle arrest in MCF-7 cells was determined H2DCFDA staining, DAPI staining, Acridine orange and ethidium bromide (AO/EtBr) dual staining, flowcytometry analysis with PI staining and apoptotic key regulatory proteins expression levels measured by westernblot analysis. CONCLUSION: Our findings, revealed the anticancer potential of endophytic fungi from marine seaweed as a valuable source of bioactive compounds with anticancer properties and underscore the significance of exploring marine-derived endophytic fungi as a promising avenue for the development of novel anticancer agents. Further investigations are necessary to isolate and characterize specific bioactive compounds responsible for these effects and to validate their therapeutic potential in preclinical and clinical settings.

Cultivable epiphytic bacteria of the Chlorophyta Ulva sp.: diversity, antibacterial, and biofilm-modulating activities.

AIMS: Macroalgae harbor a rich epiphytic microbiota that plays a crucial role in algal morphogenesis and defense mechanisms. This study aims to isolate epiphytic cultivable microbiota from Ulva sp. surfaces. Various culture media were employed to evaluate a wide range of cultivable microbiota. Our objective was to assess the antibacterial and biofilm-modulating activities of supernatants from isolated bacteria. METHODS AND RESULTS: Sixty-nine bacterial isolates from Ulva sp. were identified based on 16S rRNA gene sequencing. Their antibacterial activity and biofilm modulation potential were screened against three target marine bacteria: 45%, mostly affiliated with Gammaproteobacteria and mainly grown on diluted R2A medium (R2Ad), showed strong antibacterial activity, while 18% had a significant impact on biofilm modulation. Molecular network analysis was carried out on four bioactive bacterial supernatants, revealing new molecules potentially responsible for their activities. CONCLUSION: R2Ad offered the greatest diversity and proportion of active isolates. The molecular network approach holds promise for both identifying bacterial isolates based on their molecular production and characterizing antibacterial and biofilm-modulating activities.

Non-selective microbiota reduction after the elicitation of a seaweed's immune response.

Pattern-triggered immunity (PTI) is an integral part of the innate immune system of many eukaryotic hosts, assisting in the defence against pathogen invasions. In plants and animals, PTI exerts a selective pressure on the microbiota that can alter community composition. However, the effect of PTI on the microbiota for non-model hosts, including seaweeds, remains unknown. Using quantitative polymerase chain reaction complemented with 16S rRNA gene and transcript amplicon sequencing, this study profiled the impact that PTI of the red seaweed Gracilaria gracilis has on its microbiota. PTI elicitation with agar oligosaccharides resulted in a significant reduction in the number of bacteria (by >75% within 72 h after treatment). However, the PTI elicitation did not cause any significant difference in the community diversity or structure. These findings demonstrated that PTI can be non-selective, and this might help to maintain a stable microbiota by uniformly reducing bacterial loads.

Unveiling the culturable and non-culturable actinobacterial diversity in two macroalgae species from the northern Portuguese coast.

Actinomycetota, associated with macroalgae, remains one of the least explored marine niches. The secondary metabolism of Actinomycetota, the primary microbial source of compounds relevant to biotechnology, continues to drive research into the distribution, dynamics, and metabolome of these microorganisms. In this study, we employed a combination of traditional cultivation and metagenomic analysis to investigate the diversity of Actinomycetota in two native macroalgae species from the Portuguese coast. We obtained and taxonomically identified a collection of 380 strains, which were distributed across 12 orders, 15 families, and 25 genera affiliated with the Actinomycetia class, with Streptomyces making up approximately 60% of the composition. Metagenomic results revealed the presence of Actinomycetota in both Chondrus crispus and Codium tomentosum datasets, with relative abundances of 11% and 2%, respectively. This approach identified 12 orders, 16 families, and 17 genera affiliated with Actinomycetota, with minimal overlap with the cultivation results. Acidimicrobiales emerged as the dominant actinobacterial order in both macroalgae, although no strain affiliated with this taxonomic group was successfully isolated. Our findings suggest that macroalgae represent a hotspot for Actinomycetota. The synergistic use of both culture-dependent and independent approaches proved beneficial, enabling the identification and recovery of not only abundant but also rare taxonomic members.

Enrichable consortia of microbial symbionts degrade macroalgal polysaccharides in Kyphosus fish.

Coastal herbivorous fishes consume macroalgae, which is then degraded by microbes along their digestive tract. However, there is scarce genomic information about the microbiota that perform this degradation. This study explores the potential of Kyphosus gastrointestinal microbial symbionts to collaboratively degrade and ferment polysaccharides from red, green, and brown macroalgae through in silico study of carbohydrate-active enzyme and sulfatase sequences. Recovery of metagenome-assembled genomes (MAGs) from previously described Kyphosus gut metagenomes and newly sequenced bioreactor enrichments reveals differences in enzymatic capabilities between the major microbial taxa in Kyphosus guts. The most versatile of the recovered MAGs were from the Bacteroidota phylum, whose MAGs house enzyme collections able to decompose a variety of algal polysaccharides. Unique enzymes and predicted degradative capacities of genomes from the Bacillota (genus Vallitalea) and Verrucomicrobiota (order Kiritimatiellales) highlight the importance of metabolic contributions from multiple phyla to broaden polysaccharide degradation capabilities. Few genomes contain the required enzymes to fully degrade any complex sulfated algal polysaccharide alone. The distribution of suitable enzymes between MAGs originating from different taxa, along with the widespread detection of signal peptides in candidate enzymes, is consistent with cooperative extracellular degradation of these carbohydrates. This study leverages genomic evidence to reveal an untapped diversity at the enzyme and strain level among Kyphosus symbionts and their contributions to macroalgae decomposition. Bioreactor enrichments provide a genomic foundation for degradative and fermentative processes central to translating the knowledge gained from this system to the aquaculture and bioenergy sectors.IMPORTANCESeaweed has long been considered a promising source of sustainable biomass for bioenergy and aquaculture feed, but scalable industrial methods for decomposing terrestrial compounds can struggle to break down seaweed polysaccharides efficiently due to their unique sulfated structures. Fish of the genus Kyphosus feed on seaweed by leveraging gastrointestinal bacteria to degrade algal polysaccharides into simple sugars. This study reconstructs metagenome-assembled genomes for these gastrointestinal bacteria to enhance our understanding of herbivorous fish digestion and fermentation of algal sugars. Investigations at the gene level identify Kyphosus guts as an untapped source of seaweed-degrading enzymes ripe for further characterization. These discoveries set the stage for future work incorporating marine enzymes and microbial communities in the industrial degradation of algal polysaccharides.

Not all parents are the same: Diverse strategies of symbiont transmission in seaweeds.

Different marine seaweed species have been shown to harbour specific bacterial communities, however, the extent to which vertical symbiont transmission from parents to offspring contributes to host-specificity is unclear. Here we use fluorescence and electron microscopy as well as 16S rRNA gene-based community analysis to investigate symbiont transmission in members of the three major seaweed groups (green Chlorophyta, red Rhodophyta and brown Phaeophyceae). We found seaweeds employ diverse strategies to transfer symbionts to their progeny. For instance, the green Ulva australis does not appear to have the capacity for vertical transmission. In contrast, the brown Phyllospora comosa adopts a non-selective vertical transmission. The red Delisea pulchra demonstrates weak selectivity in symbiont transmission, while the brown Hormosira banksii exhibits a strongly selective symbiont transfer. Mucilage on the gametes appears to facilitate vertical transmission and transferred bacteria have predicted properties that could support early development of the seaweeds. Previous meta-analysis has indicated that vertical transmission is rare in aquatic compared to terrestrial environments, however, our results contribute to the growing evidence that this might not be the case and that instead vertical transmission with various degrees of symbiont selection occurs in the ecologically important group of seaweeds.

Epiphytic common core bacteria in the microbiomes of co-located green (Ulva), brown (Saccharina) and red (Grateloupia, Gelidium) macroalgae.

BACKGROUND: Macroalgal epiphytic microbial communities constitute a rich resource for novel enzymes and compounds, but studies so far largely focused on tag-based microbial diversity analyses or limited metagenome sequencing of single macroalgal species. RESULTS: We sampled epiphytic bacteria from specimens of Ulva sp. (green algae), Saccharina sp. (brown algae), Grateloupia sp. and Gelidium sp. (both red algae) together with seawater and sediment controls from a coastal reef in Weihai, China, during all seasons. Using 16S rRNA amplicon sequencing, we identified 14 core genera (consistently present on all macroalgae), and 14 dominant genera (consistently present on three of the macroalgae). Core genera represented ~ 0.7% of all genera, yet accounted for on average 51.1% of the bacterial abundances. Plate cultivation from all samples yielded 5,527 strains (macroalgae: 4,426) representing 1,235 species (685 potentially novel). Sequencing of selected strains yielded 820 non-redundant draft genomes (506 potentially novel), and sequencing of 23 sampled metagenomes yielded 1,619 metagenome-assembled genomes (MAGs), representing further 1,183 non-redundant genomes. 230 isolates and 153 genomes were obtained from the 28 core/dominant genera. We analyzed the genomic potential of phycosphere bacteria to degrade algal polysaccharides and to produce bioactive secondary metabolites. We predicted 4,451 polysaccharide utilization loci (PULs) and 8,810 biosynthetic gene clusters (BGCs). These were particularly prevalent in core/dominant genera. CONCLUSIONS: Our metabolic annotations and analyses of MAGs and genomes provide new insights into novel species of phycosphere bacteria and their ecological niches for an improved understanding of the macroalgal phycosphere microbiome. Video Abstract.

First report of Planomicrobium okeanokoites associated with Himantothallus grandifolius (Desmarestiales, Phaeophyta) from Southern Hemisphere.

Gram-positive, aerobic, motile, rod-shaped, mesophilic epiphytic bacterium Planomicrobium okeanokoites was isolated from the surface of endemic species Himantothallus grandifolius in Larsemann Hills, Eastern Antarctica. The diversity of epiphytic bacterial communities living on marine algae remains primarily unexplored; virtually no reports from Antarctic seaweeds. The present study used morpho-molecular approaches for the macroalgae and epiphytic bacterium characterization. Phylogenetic analysis was performed using mitochondrial genome encoded COX1 gene; chloroplast genome encodes rbcL; nuclear genome encoded large subunit ribosomal RNA gene (LSU rRNA) for Himantothallus grandifolius and ribosomal encoded 16S rRNA for Planomicrobium okeanokoites. Morphological and molecular data revealed that the isolate is identified as Himantothallus grandifolius, which belongs to Family Desmarestiaceae of Order Desmarestiales in Class Phaeophyceae showing 99.8% similarity to the sequences of Himantothallus grandifolius, from King George Island, Antarctica (HE866853). The isolated bacterial strain was identified on the basis of chemotaxonomic, morpho-phylogenetic, and biochemical assays. A phylogenetic study based on 16S rRNA gene sequences revealed that the epiphytic bacterial strain SLA-357 was closest related to the Planomicrobium okeanokoites showing 98.7% sequence similarity. The study revealed the first report of this species from the Southern Hemisphere to date. Also, there has been no report regarding the association between the Planomicrobium okeanokoites and Himantothallus grandifolius; however, there are some reports on this bacterium isolated from sediments, soils, and lakes from Northern Hemisphere. This study may open a gateway for further research to know about the mode of interactions and how they affect the physiology and metabolism of each other.

Effects of UV-B radiation on epiphytic bacterial communities on male and female Sargassum thunbergii.

The effects of increased UV-B radiation on macroalgae have been widely studied, but knowledge concerning the response of communities of algal epiphytic bacteria to increased UV-B radiation and differences between male and female algae is still lacking. Via 16S rDNA high-throughput sequencing technology, changes in the epiphytic bacterial communities on male and female S. thunbergii under increased UV-B radiation were studied in the lab. Under different UV-B radiation intensities, although the α diversity and community composition of epiphytic bacteria changed little, the ß diversity indicated that the community structure of bacteria on S. thunbergii was obviously clustered, and the relative abundance of dominant bacteria and indicator species changed considerably. There were unique bacteria in each experimental group, and the bacteria whose abundance obviously changed were members of groups related to environmental resistance or adaptability. The variation in the abundance of epiphytic bacteria was different in male and female S. thunbergii, and the bacteria whose abundance greatly changed were mainly related to algal growth and metabolism. The abundance of genes with predicted functions related to metabolism, genetic information processing, environmental adaptation and infectious diseases changed with increased UV-B radiation, and those variations differed between epiphytic bacteria on male and female S. thunbergii. This study found that the algal epiphytic bacteria were influenced by the increase in UV-B radiation and underwent certain adaptations through adjustments to community structure and function, and this response was also affected by the sex of the macroalgae. These results are expected to serve as experimental basis and provide reference for further understanding of the response of algae epiphytic bacteria to enhanced UV-B radiation caused by the thinning of the ozone layer and the resulting changes in the relationship between algae and bacteria, which may change the community of the marine ecosystem and affect important marine ecological process.

From model organism to application: Bacteria-induced growth and development of the green seaweed Ulva and the potential of microbe leveraging in algal aquaculture.

The marine green macroalga Ulva (Chlorophyta, Ulvales), also known as sea lettuce, coexists with a diverse microbiome. Many Ulva species proliferate in nature and form green algal blooms ("green tides"), which can occur when nutrient-rich wastewater from agricultural or densely populated areas is flushed into the sea. Bacteria are necessary for the adhesion of Ulva to its substrate, its growth, and the development of its blade morphology. In the absence of certain bacteria, Ulva mutabilis develops into a callus-like morphotype. However, with the addition of the necessary marine bacteria, the entire morphogenesis can be restored. Surprisingly, just two bacteria isolated from U. mutabilis are sufficient for inducing morphogenesis and establishing the reductionist system of a tripartite community. While one bacterial strain causes algal blade cell division, another causes the differentiation of basal cells into a rhizoid and supports cell wall formation because of a low concentration of the morphogen thallusin (below 10-10 mol/L). This review focuses on the research conducted on this topic since 2015, discusses how U. mutabilis has developed into a model organism in chemical ecology, and explores the questions that have already been addressed and the perspectives that a reductionist model system allows. In particular, the field of systems biology will achieve a comprehensive, quantitative understanding of the dynamic interactions between Ulva and its associated bacteria to better predict the behavior of the system as a whole. The reductionist approach has enabled the study of the bacteria-induced morphogenesis of Ulva. Specific questions regarding the optimization of cultivation conditions as well as the yield of raw materials for the food and animal feed industries can be answered in the laboratory and through applied science. Genome sequencing, the improvement of genetic engineering tools, and the first promising attempts to leverage macroalgae-microbe interactions in aquaculture make this model organism, which has a comparatively short parthenogenetic life cycle, attractive for both fundamental and applied research. The reviewed research paves the way for the synthetic biology of macroalgae-associated microbiomes in sustainable aquacultures.

Seaweed-associated heterotrophic Bacillus altitudinis MTCC13046: a promising marine bacterium for use against human hepatocellular adenocarcinoma.

Since the report of the antibiotic with anticancer properties, scientists have been focusing to isolate and characterize novel anti-microbial natural products possessing anticancer activities. The current study describes the production of seaweed-associated heterotrophic Bacillus altitudinis MTCC13046 with potential anticancer properties. The bacterium was screened for its capacity to diminish the cell proliferation of the human hepatocellular adenocarcinoma (HepG2) cell line, without upsetting the normal cells. The bacterial extract showed anticancer properties in a dose-reactive form against HepG2 (IC50, half maximal inhibitory concentration ~ 29.5 µg/ml) on tetrazolium bromide analysis with less significant cytotoxicity on common fibroblast (HDF) cells (IC50 ~ 77 µg/ml). The potential antioxidant ability of the organic extract of B. altitudinis MTCC13046 (IC90 133 µg/ml) could corroborate its capacity to attenuate the pathophysiology leading to carcinogenesis. The results of the apoptosis assay showed that the crude extracts of B. altitudinis maintained 68% viability in normal cells compared to 11% in the cancer cells (IC50 76.9 µg/ml). According to the findings, B. altitudinis MTCC13046 could be used to develop prospective anticancer agents.

Effects of Water Loss Stress under Tidal Effects on the Epiphytic Bacterial Community of Sargassum thunbergii in the Intertidal Zone.

Intertidal macroalgae face periodic water loss and rehydration caused by daily tidal changes. However, the effect of water loss stress on algal epiphytic bacteria has not yet been reported. In this study, the effects of water loss stress on the epiphytic bacteria community of Sargassum thunbergii were analyzed, and the different responses of epiphytic bacteria to water loss stress were compared between male and female algae. The results showed that after water loss stress, the diversity of the epiphytic bacterial community of S. thunbergii first decreased and then increased. Among the dominant taxa, the abundance of Cyanobacteria decreased significantly, whereas the abundance of Portibacter and Aquimarina first increased and then decreased. Additionally, the indicator species and the abundance of predicted functional genes related to carbon, nitrogen, and sulfur metabolism both changed significantly. More importantly, when the epiphytic bacteria were analyzed separately according to the algal sex, the changes in algal epiphytic bacterial community structure and indicator species were more significant, and there were sexual differences. Therefore, it was concluded that water loss stress has a significant effect on the community structure and function of the epiphytic bacteria on S. thunbergii. Meanwhile, the epiphytic bacteria community of two sexes of S. thunbergii differed in the response to water loss stress. IMPORTANCE Periodic water loss caused by the tide is an important environmental factor that is faced by intertidal macroalgae, but the impact of periodic water loss on the epiphytic bacterial communities associated with macroalgae is still unknown. Through this study, we found that the diversity, the relative abundance of dominant taxa, the indicator species, and the abundance of the predicted functional genes in the epiphytic bacteria on S. thunbergii changed with the time of water loss. Moreover, male and female S. thunbergii exhibited different responses to water loss stress. This study not only paves the way for the delineation of the interactions between S. thunbergii and its epiphytic bacteria but also provides new insights for the mechanisms of the adaptation and evolution of macroalgae in the intertidal zone.

Stereoselective Total Synthesis of (-)-Thallusin for Bioactivity Profiling.

Chemical mediators are key compounds for controlling symbiotic interactions in the environment. Here, we disclose a fully stereoselective total synthesis of the algae differentiation factor (-)-thallusin that utilizes sophisticated 6-endo-cyclization chemistry and effective late-stage sp2 -sp2 -couplings using non-toxic reagents. An EC50 of 4.8 pM was determined by quantitative phenotype profiling in the green seaweed Ulva mutabilis (Chlorophyte), underscoring this potent mediator's enormous, pan-species bioactivity produced by symbiotic bacteria. SAR investigations indicate that (-)-thallusin triggers at least two different pathways in Ulva that may be separated by chemical editing of the mediator compound structure.

Pharmacological potential of seaweed-associated heterotrophic Firmicutes.

Seaweed-associated bacterial symbionts are sources of potential pharmacological properties. The present study resulted in the culture-dependent isolation of bioactive heterotrophs belonging to the bacterial phylum Firmicutes, which were dominated more than 30% of the 127 cultivable isolates, amongst which 23 of them showed potential antimicrobial activities against a wide range of pathogens. The symbionts isolated from the seaweed Sargassum wightii showed significant bioactivity. Those were characterized as Bacillus safensis MTCC13040, B. valismortis MTCC13041, B. velezensis MTCC13044, B. methylotrophicus MTCC13042, Oceanobacillus profundus MTCC13045, B. tequilensis MTCC13043, and B. altitudinis MTCC13046. The organic extracts of the studied isolates showed potential antimicrobial properties against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci (minimum inhibitory concentration 6·25-12·5 µg ml-1 ). The organic extract of B. altitudinis MTCC13046 displayed significantly greater radical quenching ability (IC90 133 µg ml-1 , P < 0·05) other than attenuating hydroxymethyl glutaryl coenzyme A reductase (IC90 10·21 µg ml-1 , P < 0·05) and angiotensin-converting enzyme-1 (IC90 498 µg ml-1 , P < 0·05) relative to other studied heterotrophs. The organic extract of B. tequilensis MTCC13043 displayed significantly greater attenuation potential against pro-inflammatory 5-lipooxygenase (IC90 5·94 µg ml-1 , P < 0·05) and dipeptidyl peptidase-4 (IC90 271 µg ml-1 , P < 0·05). The seaweed-associated B. altitudinis MTCC13046 and B. tequilensis MTCC13043 could be used to develop promising pharmacological leads.

Seaweed-associated heterotrophic bacteria: are they future novel sources of antimicrobial agents against drug-resistant pathogens?

Emergence of multidrug-resistant microorganisms and requirements for novel antimicrobial compounds necessitate exploring newer habitats to develop potential bioactive leads. Culture-contingent analysis of heterotrophic bacterial flora from the seaweeds led to the isolation of bioactive strains possessing potential antibacterial properties against wide-ranging clinical pathogens viz., methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VREfs). Seven of the most active strains belonging to the phylum Firmicutes isolated from a brown seaweed (Phaeophyceae) Sargassum wightii exhibited spot-over-lawn assay guided inhibition zone of larger than 30 mm. Integrated phenotypic and genotypic studies have led to the characterization of the seaweed-associated bacteria particularly belonging to the phylum Firmicutes. The organic extracts of the studied bacteria exhibited promising antibacterial properties against MRSA and VREfs with minimum inhibitory concentration ranging between 6.25 and 12.50 µg/mL. Time-kill kinetic profiles of those bacteria displayed rapid bactericidal activity against both E. coli and MRSA, showing a ≥ 3log10 reduction in viable cell count than the initial. Among the studied bioactive Bacillus spp, B. tequilensis MTCC13043 and B. altiitudinis MTCC13046 were found to possess functional polyketide synthase (pks) gene (MW027664 and MW027660) that could be amplified. The outcome of amplified genes encrypting for polyketide synthase in conjunction with antibacterial activities unveiled the broad-spectrum antimicrobial activities of the marine heterotrophic Firmicutes, which could be further used against the emergent problem of antibiotic-resistant bacterial pathogens.

Application of Feature-Based Molecular Networking for Comparative Metabolomics and Targeted Isolation of Stereoisomers from Algicolous Fungi.

Seaweed endophytic (algicolous) fungi are talented producers of bioactive natural products. We have previously isolated two strains of the endophytic fungus, Pyrenochaetopsis sp. FVE-001 and FVE-087, from the thalli of the brown alga Fucus vesiculosus. Initial chemical studies yielded four new decalinoylspirotetramic acid derivatives with antimelanoma activity, namely pyrenosetins A-C (1-3) from Pyrenochaetopsis sp. strain FVE-001, and pyrenosetin D (4) from strain FVE-087. In this study, we applied a comparative metabolomics study employing HRMS/MS based feature-based molecular networking (FB MN) on both Pyrenochaetopsis strains. A higher chemical capacity in production of decalin derivatives was observed in Pyrenochaetopsis sp. FVE-087. Notably, several decalins showed different retention times despite the same MS data and MS/MS fragmentation pattern with the previously isolated pyrenosetins, indicating they may be their stereoisomers. FB MN-based targeted isolation studies coupled with antimelanoma activity testing on the strain FVE-087 afforded two new stereoisomers, pyrenosetins E (5) and F (6). Extensive NMR spectroscopy including DFT computational studies, HR-ESIMS, and Mosher's ester method were used in the structure elucidation of compounds 5 and 6. The 3'R,5'R stereochemistry determined for compound 6 was identical to that previously reported for pyrenosetin C (3), whose stereochemistry was revised as 3'S,5'R in this study. Pyrenosetin E (5) inhibited the growth of human malignant melanoma cells (A-375) with an IC50 value of 40.9 µM, while 6 was inactive. This study points out significant variations in the chemical repertoire of two closely related fungal strains and the versatility of FB MN in identification and targeted isolation of stereoisomers. It also confirms that the little-known fungal genus Pyrenochaetopsis is a prolific source of complex decalinoylspirotetramic acid derivatives.

Antibiotic-active heterotrophic Firmicutes sheltered in seaweeds: can they add new dimensions to future antimicrobial agents?

Appearance of drug-resistant microorganisms prompted researchers to unravel new environments for development of novel antimicrobial agents. Culture-supported analysis of heterotrophic bacteria associated with seaweeds yielded 152 strains, in that larger share of the isolates was embodied by Bacillus atrophaeus SHB2097 (54%), B. velezensis SHB2098 (24%), B. subtilis SHB2099 (12%), and B. amyloliquefaciens SHB20910 (10%). One of the most active strains characterized as B. atrophaeus SHB2097 (MW821482) with an inhibition zone more than 30 mm on spot-over-lawn experiment, was isolated from a seaweed Sargassum wightii, was selected for bioprospecting studies. Significant antibacterial potential was displayed by bacterial organic extract against vancomycin-resistant Enterococcus faecalis, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Klebsiella pneumonia with minimum inhibitory concentration 6.25 µg/mL and comparable to the antibiotics ampicillin and chloramphenicol. The genes of type 1 pks (MZ222383, 700 bp) and hybrid nrps/pks (MZ222389, 1000-1400 bp) of B. atrophaeus MW821482 could be amplified. The bacterium displayed susceptibility to the commercially available antibiotic agents, and was negative for the pore-forming non-hemolytic hemolysin BL (hbl) and enterotoxin (nhe) genes, and therefore, was not pathogenic. The bacterium was found to possess genes (1000-1400 bp) involved in the biosynthesis of siderophore-class of compounds (MZ222387 and MZ222388) that showed 99% of similarity in BLAST search, and showed production of siderophore. Noteworthy antibacterial activities against clinically important pathogenic bacteria in conjunction with occurrence of genes coding for antimicrobial metabolites inferred that the marine heterotrophic bacterium B. atrophaeus SHB2097 could be used for the development of antibacterial agents against the emerging antibiotic resistance.

Modelling Antifouling compounds of Macroalgal Holobionts in Current and Future pH Conditions.

Marine macroalgae are important ecosystem engineers in marine coastal habitats. Macroalgae can be negatively impacted through excessive colonization by harmful bacteria, fungi, microalgae, and macro-colonisers and thus employ a range of chemical compounds to minimize such colonization. Recent research suggests that environmental pH conditions potentially impact the functionality of such chemical compounds. Here we predict if and how naturally fluctuating pH conditions and future conditions caused by ocean acidification will affect macroalgal (antifouling) compounds and thereby potentially alter the chemical defence mediated by these compounds. We defined the relevant ecological pH range, analysed and scored the pH-sensitivity of compounds with antifouling functions based on their modelled chemical properties before assessing their distribution across the phylogenetic macroalgal groups, and the proportion of sensitive compounds for each investigated function. For some key compounds, we also predicted in detail how the associated ecological function may develop across the pH range. The majority of compounds were unaffected by pH, but compounds containing phenolic and amine groups were found to be particularly sensitive to pH. Future pH changes due to predicted average open ocean acidification pH were found to have little effect. Compounds from Rhodophyta were mainly pH-stable. However, key algal species amongst Phaeophyceae and Chlorophyta were found to rely on highly pH-sensitive compounds for their chemical defence against harmful bacteria, microalgae, fungi, and biofouling by macro-organisms. All quorum sensing disruptive compounds were found the be unaffected by pH, but the other ecological functions were all conveyed in part by pH-sensitive compounds. For some ecological keystone species, all of their compounds mediating defence functions were found to be pH-sensitive based on our calculations, which may not only affect the health and fitness of the host alga resulting in host breakdown but also alter the associated ecological interactions of the macroalgal holobiont with micro and macrocolonisers, eventually causing ecosystem restructuring and the functions (e.g. habitat provision) provided by macroalgal hosts. Our study investigates a question of fundamental importance because environments with fluctuating or changing pH are common and apply not only to coastal marine habitats and estuaries but also to freshwater environments or terrestrial systems that are subject to acid rain. Hence, whilst warranting experimental validation, this investigation with macroalgae as model organisms can serve as a basis for future investigations in other aquatic or even terrestrial systems.

An investigation of antibacterial activity of New Zealand seaweed-associated marine bacteria.

Aim: To explore seaweed-associated bacteria as prospective producers of bioactive material with antibacterial properties. Materials & methods: 143 bacterial species were isolated from the surface of 15 New Zealand marine macroalgae. Bacterial extracts obtained using dimethyl sulfoxide and ethyl acetate were screened for antagonistic activities against three antimicrobial susceptibility indicators: Kocuria rhizophila, Staphylococcus epidermidis and Escherichia coli, using well-diffusion method. For selected species, minimum inhibitory concentration was determined, followed by a phylogenetic identification based on 16S rRNA gene sequences. Results: Among all bacteria screened, seven that belonged to the genera Vibrio, Pseudoalteromonas, Psychromonas and Cobetia, showed antagonistic activity against all three indicators. Conclusion: Seaweed-associated bacteria produce bioactive compounds with antimicrobial potential and possible biomedical application in aquatic habitats.

Novel amylomacins from seaweed-associated Bacillus amyloliquefaciens as prospective antimicrobial leads attenuating resistant bacteria.

The rise in antibiotic-resistant bacterial strains prompting nosocomial infections drives the search for new bioactive substances of promising antibacterial properties. The surfaces of seaweeds are rich in heterotrophic bacteria with prospective antimicrobial substances. This study aimed to isolate antibacterial leads from a seaweed-associated bacterium. Heterotrophic Bacillus amyloliquefaciens MTCC 12716 associated with the seaweed Hypnea valentiae, was isolated and screened for antimicrobial properties against drug-resistant pathogens. The bacterial crude extract was purified and three novel amicoumacin-class of isocoumarin analogues, 11'-butyl acetate amicoumacin C (amylomacin A), 4'-hydroxy-11'-methoxyethyl carboxylate amicoumacin C (amylomacin B) and 11'-butyl amicoumacin C (amylomacin C) were isolated to homogeneity. The studied amylomacins possessed potential activities against Pseudomonas aeruginosa, vancomycin-resistant Enterococcus faecalis, Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, and Shigella flexneri with a range of minimum inhibitory concentration values from 0.78 to 3.12 µg/mL, although standard antibiotics ampicillin and chloramphenicol were active at 6.25-25 µg/mL. Noticeably, the amylomacin compound encompassing 4'-hydroxy-11'-methoxyethyl carboxylate amicoumacin C functionality (amylomacin B), displayed considerably greater antagonistic activities against methicillin-resistant S. aureus, vancomycin-resistant E. faecalis, Vibrio parahaemolyticus, Escherichia coli, and K. pneumoniae (minimum inhibitory concentration 0.78 µg/mL) compared to the positive controls and other amylomacin analogues. Antimicrobial properties of the amylomacins, coupled with the presence of polyketide synthase-I/non-ribosomal peptide synthetase hybrid gene attributed the bacterium as a promising source of antimicrobial compounds with pharmaceutical and biotechnological applications.