Targeting antimalarial metabolites from the actinomycetes associated with the Red Sea sponge Callyspongia siphonella using a metabolomic method.

Malaria is a persistent illness that is still a public health issue. On the other hand, marine organisms are considered a rich source of anti­infective drugs and other medically significant compounds. Herein, we reported the isolation of the actinomycete associated with the Red Sea sponge Callyspongia siphonella. Using "one strain many compounds" (OSMAC) approach, a suitable strain was identified and then sub-cultured in three different media (M1, ISP2 and OLIGO). The extracts were evaluated for their in-vitro antimalarial activity against Plasmodium falciparum strain and subsequently analyzed by Liquid chromatography coupled with high-resolution mass spectrometry (LC-HR-MS). In addition, MetaboAnalyst 5.0 was used to statistically analyze the LC-MS data. Finally, Molecular docking was carried out for the dereplicated metabolites against lysyl-tRNA synthetase (PfKRS1). The phylogenetic study of the 16S rRNA sequence of the actinomycete isolate revealed its affiliation to Streptomyces genus. Antimalarial screening revealed that ISP2 media is the most active against Plasmodium falciparum strain. Based on LC-HR-MS based metabolomics and multivariate analyses, the static cultures of the media, ISP2 (ISP2-S) and M1 (M1-S), are the optimal media for metabolites production. OPLS-DA suggested that quinone derivatives are abundant in the extracts with the highest antimalarial activity. Fifteen compounds were identified where eight of these metabolites were correlated to the observed antimalarial activity of the active extracts. According to molecular docking experiments, saframycin Y3 and juglomycin E showed the greatest binding energy scores (-6.2 and -5.13) to lysyl-tRNA synthetase (PfKRS1), respectively. Using metabolomics and molecular docking investigation, the quinones, saframycin Y3 (5) and juglomycin E (1) were identified as promising antimalarial therapeutic candidates. Our approach can be used as a first evaluation stage in natural product drug development, facilitating the separation of chosen metabolites, particularly biologically active ones.

pH-Responsive Chitosan-Doped ZnO Hybrid Hydrogels for the Encapsulation of Bioactive Compounds in Aquaculture.

In this study, we synthesized and characterized pH-responsive Chitosan-AgCl-doped ZnO hybrid hydrogels and evaluated their potential for loading aquaculture bioactive compounds, and assessed their antimicrobial properties against a threatening pathogen associated with disease across a broad spectrum of warm water fish and invertebrates. Hydrogel characterization consisted of assessing morphology via SEM, composition via EDS, hydrogels' network components interactions via FT-IR and pH response through swelling behavior determinations. The swelling characterization of the synthesized hydrogels demonstrated a pH-responsive behavior, showing that low pH values caused the hydrogel polymeric network to expand and capture more of the aqueous solution. These characteristics make the synthesized hydrogels suitable for the encapsulation and controlled release of drugs and bioactive compounds in aquaculture. Chitosan_ZnO hybrid hydrogels showed great antimicrobial activity against Vibrio harveyi, even better than that of loaded PB hydrogels. Here, we provide evidence for the potential capacity of Chitosan_ZnO hybrid hydrogels for the preventive and curative treatment of diseases that impact aquaculture animal health and prevent drug resistance by bacteria.

The endophytic Fusarium strains: a treasure trove of natural products.

The complexity and structural diversity of the secondary metabolites produced by endophytes make them an attractive source of natural products with novel structures that can help in treating life-changing diseases. The genus Fusarium is one of the most abundant endophytic fungal genera, comprising about 70 species characterized by extraordinary discrepancy in terms of genetics and ability to grow on a wide range of substrates, affecting not only their biology and interaction with their surrounding organisms, but also their secondary metabolism. Members of the genus Fusarium are a source of secondary metabolites with structural and chemical diversity and reported to exhibit diverse pharmacological activities. This comprehensive review focuses on the secondary metabolites isolated from different endophytic Fusarium species along with their various biological activities, reported in the period from April 1999 to April 2022.

Rare actinobacteria isolated from the hypersaline Ojo de Liebre Lagoon as a source of novel bioactive compounds with biotechnological potential.

The Ojo de Liebre Lagoon is a Marine Protected Area that lies within a UNESCO World Heritage Site and is a critical habitat for important migratory species such as the grey whale and bird species. Unique hypersaline environments, such as the Ojo de Liebre Lagoon, are underexplored in terms of their bacterial and chemical diversity, representing a potential source for new bioactive compounds with pharmacological properties. Actinobacteria are one of the most diverse and prolific taxonomic bacterial groups in terms of marine bioactive compounds. This study aimed to identify the culturable actinobacterial community inhabiting the Lagoon, as well as to test their potential as new sources of anticancer compounds with pharmacological potential. A selective isolation approach focused on spore-forming bacteria from 40 sediment samples generated a culture collection of 64 strains. The 16S rRNA gene analyses identified three phyla in this study, the Actinobacteria, Firmicutes and Proteobacteria, where the phylum Actinobacteria dominated (57%) the microbial community profiles. Within the Actinobacteria, nine different genera were isolated including the Actinomadura, Micromonospora, Nocardiopsis, Plantactinospora and Streptomyces sp. We observed seasonal differences on actinobacteria recovery. For instance, Micromonospora strains were recovered during the four sampling seasons, while Arthrobacter and Pseudokineococcus were only isolated in February 2018, and Blastococcus, Rhodococcus and Streptomyces were uniquely isolated in June 2018. Ethyl acetate crude extracts derived from actinobacterial cultures were generated and screened for cytotoxic activity against six cancer cell lines. Strains showed promising low percentages of viability on lung (H1299), cervical (SiHa), colon (Caco-2) and liver (HepG2) cancer lines. Molecular networking results suggest many of the metabolites produced by these strains are unknown and they might harbour novel chemistry. Our results showed the Ojo de Liebre Lagoon is a novel source for isolating diverse marine actinobacteria which produce promising bioactive compounds for potential biotechnological use as anticancer agents.

Six novel species of the obligate marine actinobacterium Salinispora, Salinispora cortesiana sp. nov., Salinispora fenicalii sp. nov., Salinispora goodfellowii sp. nov., Salinispora mooreana sp. nov., Salinispora oceanensis sp. nov. and Salinispora vitiensis sp. nov., and emended description of the genus Salinispora.

Ten representative actinobacterial strains isolated from marine sediments collected worldwide were studied to determine their taxonomic status. The strains were previously identified as members of the genus Salinispora and shared >99 % 16S rRNA gene sequence similarity to the three currently recognized Salinispora species. Comparative genomic analyses resulted in the delineation of six new species based on average nucleotide identity and digital DNA-DNA hybridization values below 95 and 70 %, respectively. The species status of the six new groups was supported by a core-genome phylogeny reconstructed from 2106 orthologs detected in 118 publicly available Salinispora genomes. Chemotaxonomic and physiological studies were used to complete the phenotypic characterization of the strains. The fatty acid profiles contained the major components iso-C16 : 0, C15 : 0, iso-17 : 0 and anteiso C17 : 0. Galactose and xylose were common in all whole-sugar patterns but differences were found between the six groups of strains. Polar lipid compositions were also unique for each species. Distinguishable physiological and biochemical characteristics were also recorded. The names proposed are Salinispora cortesiana sp. nov., CNY-202T (=DSM 108615T=CECT 9739T); Salinispora fenicalii sp. nov., CNT-569T (=DSM 108614T=CECT 9740T); Salinispora goodfellowii sp. nov., CNY-666T (=DSM 108616T=CECT 9738T); Salinispora mooreana sp. nov., CNT-150T (=DSM 45549T=CECT 9741T); Salinispora oceanensis sp. nov., CNT-138T (=DSM 45547T=CECT 9742T); and Salinispora vitiensis sp. nov., CNT-148T (=DSM 45548T=CECT 9743T).

Awakening ancient polar Actinobacteria: diversity, evolution and specialized metabolite potential.

Polar and subpolar ecosystems are highly vulnerable to global climate change with consequences for biodiversity and community composition. Bacteria are directly impacted by future environmental change and it is therefore essential to have a better understanding of microbial communities in fluctuating ecosystems. Exploration of Polar environments, specifically sediments, represents an exciting opportunity to uncover bacterial and chemical diversity and link this to ecosystem and evolutionary parameters. In terms of specialized metabolite production, the bacterial order Actinomycetales, within the phylum Actinobacteria are unsurpassed, producing 10 000 specialized metabolites accounting for over 45 % of all bioactive microbial metabolites. A selective isolation approach focused on spore-forming Actinobacteria of 12 sediment cores from the Antarctic and sub-Arctic generated a culture collection of 50 strains. This consisted of 39 strains belonging to rare Actinomycetales genera including Microbacterium, Rhodococcus and Pseudonocardia. This study used a combination of nanopore sequencing and molecular networking to explore the community composition, culturable bacterial diversity, evolutionary relatedness and specialized metabolite potential of these strains. Metagenomic analyses using MinION sequencing was able to detect the phylum Actinobacteria across polar sediment cores at an average of 13 % of the total bacterial reads. The resulting molecular network consisted of 1652 parent ions and the lack of known metabolite identification supports the argument that Polar bacteria are likely to produce previously unreported chemistry.

Targeted antibiotic discovery through biosynthesis-associated resistance determinants: target directed genome mining.

Intense competition between microbes in the environment has directed the evolution of antibiotic production in bacteria. Humans have harnessed these natural molecules for medicinal purposes, magnifying them from environmental concentrations to industrial scale. This increased exposure to antibiotics has amplified antibiotic resistance across bacteria, spurring a global antimicrobial crisis and a search for antibiotics with new modes of action. Genetic insights into these antibiotic-producing microbes reveal that they have evolved several resistance strategies to avoid self-toxicity, including product modification, substrate transport and binding, and target duplication or modification. Of these mechanisms, target duplication or modification will be highlighted in this review, as it uniquely links an antibiotic to its mode of action. We will further discuss and propose a strategy to mine microbial genomes for these genes and their associated biosynthetic gene clusters to discover novel antibiotics using target directed genome mining.

Nitrosopyridine Probe To Detect Polyketide Natural Products with Conjugated Alkenes: Discovery of Novodaryamide and Nocarditriene.

An optimized nitroso-based probe that facilitates the discovery of conjugated alkene-containing natural products in unprocessed extracts was developed. It chemoselectively reacts with conjugated olefins via a nitroso-Diels-Alder cyclization to yield derivatives with a distinct chromophore and an isotopically unique bromine atom that can be rapidly identified using liquid chromatography/mass spectrometry and a bioinformatics tool called MeHaloCoA (Marine Halogenated Compound Analysis). The probe is ideally employed when genome-mining techniques identify strains containing polyketide gene clusters with two or more repeating KS-AT-DH-KR-ACP domain sequences, which are required for the biosynthesis of conjugated alkenes. Comparing the reactivity and spectral properties of five brominated arylnitroso reagents with model compounds spiramycin, bufalin, rapamycin, and rifampicin led to the identification of 5-bromo-2-nitrosopyridine as the most suitable probe structure. The utility of the dienophile probe was then demonstrated in bacterial extracts. Tylactone, novodaryamide and daryamide A, piperazimycin A, and the saccharamonopyrones A and B were cleanly labeled in extracts from their respective bacterial producers, in high regioselectivity but with varying degrees of diastereoselectivity. Further application of the method led to the discovery of a new natural product called nocarditriene, containing an unprecedented epoxy-2,3,4,5-tetrahydropyridine structure, from marine-derived Nocardiopsis strain CNY-503.

Genomic insights into specialized metabolism in the marine actinomycete Salinispora.

Comparative genomics is providing new opportunities to address the diversity and distributions of genes encoding the biosynthesis of specialized metabolites. An analysis of 119 genome sequences representing three closely related species of the marine actinomycete genus Salinispora reveals extraordinary biosynthetic diversity in the form of 176 distinct biosynthetic gene clusters (BGCs) of which only 24 have been linked to their products. Remarkably, more than half of the BGCs were observed in only one or two strains, suggesting they were acquired relatively recently in the evolutionary history of the genus. These acquired gene clusters are concentrated in specific genomic islands, which represent hot spots for BGC acquisition. While most BGCs are stable in terms of their chromosomal position, others migrated to different locations or were exchanged with unrelated gene clusters suggesting a plug and play type model of evolution that provides a mechanism to test the relative fitness effects of specialized metabolites. Transcriptome analyses were used to address the relationships between BGC abundance, chromosomal position and product discovery. The results indicate that recently acquired BGCs can be functional and that complex evolutionary processes shape the micro-diversity of specialized metabolism observed in closely related environmental bacteria.

Phylogenomic Insight into Salinispora (Bacteria, Actinobacteria) Species Designations.

Bacteria represent the most genetically diverse kingdom of life. While great progress has been made in describing this diversity, it remains difficult to identify the phylogenetic and ecological characteristics that delineate groups of bacteria that possess species-like properties. One major challenge associated with species delineations is that not all shared genes have the same evolutionary history, and thus the choice of loci can have a major impact on phylogenetic reconstruction. Sequencing the genomes of large numbers of closely related strains provides new opportunities to distinguish ancestral from acquired alleles and assess the effects of recombination on phylogenetic inference. Here we analyzed the genomes of 119 strains of the marine actinomycete genus Salinispora, which is currently comprised of three named species that share 99% 16S rRNA gene sequence identity. While 63% of the core genome showed evidence of recombination, this had no effect on species-level phylogenomic resolution. Recombination did however blur intra-species relationships and biogeographic resolution. The genome-wide average nucleotide identity provided a new perspective on Salinispora diversity, revealing as many as seven new species. Patterns of orthologous group distributions reveal a genetic basis to delineation the candidate taxa and insight into the levels of genetic cohesion associated with bacterial species.

Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways by Target-directed Genome Mining.

Recent genome sequencing efforts have led to the rapid accumulation of uncharacterized or "orphaned" secondary metabolic biosynthesis gene clusters (BGCs) in public databases. This increase in DNA-sequenced big data has given rise to significant challenges in the applied field of natural product genome mining, including (i) how to prioritize the characterization of orphan BGCs and (ii) how to rapidly connect genes to biosynthesized small molecules. Here, we show that by correlating putative antibiotic resistance genes that encode target-modified proteins with orphan BGCs, we predict the biological function of pathway specific small molecules before they have been revealed in a process we call target-directed genome mining. By querying the pan-genome of 86 Salinispora bacterial genomes for duplicated house-keeping genes colocalized with natural product BGCs, we prioritized an orphan polyketide synthase-nonribosomal peptide synthetase hybrid BGC (tlm) with a putative fatty acid synthase resistance gene. We employed a new synthetic double-stranded DNA-mediated cloning strategy based on transformation-associated recombination to efficiently capture tlm and the related ttm BGCs directly from genomic DNA and to heterologously express them in Streptomyces hosts. We show the production of a group of unusual thiotetronic acid natural products, including the well-known fatty acid synthase inhibitor thiolactomycin that was first described over 30 years ago, yet never at the genetic level in regards to biosynthesis and autoresistance. This finding not only validates the target-directed genome mining strategy for the discovery of antibiotic producing gene clusters without a priori knowledge of the molecule synthesized but also paves the way for the investigation of novel enzymology involved in thiotetronic acid natural product biosynthesis.

CRISPR-Cas systems in the marine actinomycete Salinispora: linkages with phage defense, microdiversity and biogeography.

BACKGROUND: Prokaryotic CRISPR-Cas systems confer resistance to viral infection and thus mediate bacteria-phage interactions. However, the distribution and functional diversity of CRISPRs among environmental bacteria remains largely unknown. Here, comparative genomics of 75 Salinispora strains provided insight into the diversity and distribution of CRISPR-Cas systems in a cosmopolitan marine actinomycete genus. RESULTS: CRISPRs were found in all Salinispora strains, with the majority containing multiple loci and different Cas array subtypes. Of the six subtypes identified, three have not been previously described. A lower prophage frequency in S. arenicola was associated with a higher fraction of spacers matching Salinispora prophages compared to S. tropica, suggesting differing defensive capacities between Salinispora species. The occurrence of related prophages in strains from distant locations, as well as spacers matching those prophages inserted throughout spacer arrays, indicate recurring encounters with widely distributed phages over time. Linkages of CRISPR features with Salinispora microdiversity pointed to subclade-specific contacts with mobile genetic elements (MGEs). This included lineage-specific spacer deletions or insertions, which may reflect weak selective pressures to maintain immunity or distinct temporal interactions with MGEs, respectively. Biogeographic patterns in spacer and prophage distributions support the concept that Salinispora spp. encounter localized MGEs. Moreover, the presence of spacers matching housekeeping genes suggests that CRISPRs may have functions outside of viral defense. CONCLUSIONS: This study provides a comprehensive examination of CRISPR-Cas systems in a broadly distributed group of environmental bacteria. The ubiquity and diversity of CRISPRs in Salinispora suggests that CRISPR-mediated interactions with MGEs represent a major force in the ecology and evolution of this cosmopolitan marine actinomycete genus.

Diversity and evolution of secondary metabolism in the marine actinomycete genus Salinispora.

Access to genome sequence data has challenged traditional natural product discovery paradigms by revealing that the products of most bacterial biosynthetic pathways have yet to be discovered. Despite the insight afforded by this technology, little is known about the diversity and distributions of natural product biosynthetic pathways among bacteria and how they evolve to generate structural diversity. Here we analyze genome sequence data derived from 75 strains of the marine actinomycete genus Salinispora for pathways associated with polyketide and nonribosomal peptide biosynthesis, the products of which account for some of today's most important medicines. The results reveal high levels of diversity, with a total of 124 pathways identified and 229 predicted with continued sequencing. Recent horizontal gene transfer accounts for the majority of pathways, which occur in only one or two strains. Acquired pathways are incorporated into genomic islands and are commonly exchanged within and between species. Acquisition and transfer events largely involve complete pathways, which subsequently evolve by gene gain, loss, and duplication followed by divergence. The exchange of similar pathway types at the precise chromosomal locations in different strains suggests that the mechanisms of integration include pathway-level homologous recombination. Despite extensive horizontal gene transfer there is clear evidence of species-level vertical inheritance, supporting the concept that secondary metabolites represent functional traits that help define Salinispora species. The plasticity of the Salinispora secondary metabolome provides an effective mechanism to maximize population-level secondary metabolite diversity while limiting the number of pathways maintained within any individual genome.

Multilocus sequence typing reveals evidence of homologous recombination linked to antibiotic resistance in the genus Salinispora.

The three closely related species that currently comprise the genus Salinispora were analyzed using a multilocus sequence typing approach targeting 48 strains derived from four geographic locations. Phylogenetic congruence and a well-supported concatenated tree provide strong support for the delineation of the three species as currently described and the basal relationship of Salinispora arenicola to the more recently diverged sister taxa S. tropica and S. pacifica. The phylogeny of the initial region of the rpoB gene sequenced was atypical, placing the related genera Micromonospora and Verrucosispora within the Salinispora clade. This phylogenetic incongruence was subsequently ascribed to a homologous-recombination event in a portion of the gene associated with resistance to compounds in the rifamycin class, which target RpoB. All S. arenicola strains produced compounds in this class and possessed resistance-conferring amino acid changes in RpoB. The phylogeny of a region of the rpoB gene that is not associated with rifamycin resistance was congruent with the other housekeeping genes. The link between antibiotic resistance and homologous recombination suggests that incongruent phylogenies provide opportunities to identify the molecular targets of secondary metabolites, an observation with potential relevance for drug discovery efforts. Low ratios of interspecies recombination to mutation, even among cooccurring strains, coupled with high levels of within-species recombination suggest that the three species have been described in accordance with natural barriers to recombination.