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.

Nocardiopsis codii sp. nov., and Rhodococcus chondri sp. nov., two novel actinomycetal species isolated from macroalgae collected in the northern Portuguese coast.

Two novel actinomycetal strains, designated CC-R113T and CC-R104T, were isolated from the tissues of two macroalgae collected on the northern Portuguese coast. Phylogenetic analyses based on the 16S rRNA gene showed that strain CT-R113T belongs to the genus Nocardiopsis, being closely related to Nocardiopsis umidischolae 66/93T and Nocardiopsis tropica VKM Ac-1457T, with 98.65 and 98.39 % sequence similarity, respectively. The clade formed between the three type strains was confirmed by phylogenomic analysis. The genome of strain CT-R113T was 7.27 Mb in size with a G+C content of 71.3 mol %, with average nucleotide identity (ANI) values of 89.59 and 90.14 % with strains 66/93T and VKM Ac-1457T, respectively. The major cellular fatty acids were identified as C18 : 1 ω9c, iso-C16 : 0 and anteiso-C17 : 0. Menaquinone 10 (MK-10) was the major respiratory quinone. Comparative analysis of 16S rRNA gene sequences showed that strain CC-R104T belongs to the genus Rhodococcus and is most closely related to Rhodococcus pyridinivorans DSM 44555T, with 98.24 % sequence similarity. However, phylogenomic analysis revealed that strain CC-R104T establishes a clade with Rhodococcus artemisae DSM 45380T, being more distant from Rhodococcus pyridinivorans DSM 44555T. The genome of strain CC-R104T was 5.34 Mb in size with a G+C content of 67.01 mol%. The ANI value between strains CC-R104T and DSM 45380T was 81.2 % and between strains CC-R104T and DSM 44555T was 81.5 %. The major cellular fatty acids were identified as C18 : 1 ω9c, C16 : 0 and summed feature 3. Menaquinone 8 (MK-8) was the only respiratory quinone. For both CC-R113T and CC-R104T, optimum growth was observed at pH 7.0, 28 °C and 0-5 % NaCl and whole-cell hydrolysates contained meso-diaminopimelic acid as the cell-wall diamino acid. On the basis of phenotypic, molecular and chemotaxonomic characteristics, strains CT-R113T and CC-R104T are considered to represent novel species, for which the names Nocardiopsis codii sp. nov. (type strain CT-R113T=LMG33234T=UCCCB172T) and Rhodococcus chondri sp. nov. (type strain CC-R104T=LMG33233T=UCCCB171T) are proposed.

Cellulamides: A New Family of Marine-Sourced Linear Peptides from the Underexplored Cellulosimicrobium Genus.

Bioprospecting the secondary metabolism of underexplored Actinomycetota taxa is a prolific route to uncover novel chemistry. In this work, we report the isolation, structure elucidation, and bioactivity screening of cellulamides A and B (1 and 2), two novel linear peptides obtained from the culture of the macroalga-associated Cellulosimicrobium funkei CT-R177. The host of this microorganism, the Chlorophyta Codium tomentosum, was collected in the northern Portuguese coast and, in the scope of a bioprospecting study focused on its associated actinobacterial community, strain CT-R177 was isolated, taxonomically identified, and screened for the production of antimicrobial and anticancer compounds. Dereplication of a crude extract of this strain using LC-HRMS(/MS) analysis unveiled a putative novel natural product, cellulamide A (1), that was isolated following mass spectrometry-guided fractionation. An additional analog, cellulamide B (2) was obtained during the chromatographic process and chemically characterized. The chemical structures of the novel linear peptides, including their absolute configurations, were elucidated using a combination of HRMS, 1D/2D NMR spectroscopy, and Marfey's analysis. Cellulamide A (1) was subjected to a set of bioactivity screenings, but no significant biological activity was observed. The cellulamides represent the first family of natural products reported from the Actinomycetota genus Cellulosimicrobium, showcasing not only the potential of less-explored taxa but also of host-associated marine strains for novel chemistry discovery.

Decylprodigiosin: a new member of the prodigiosin family isolated from a seaweed-associated Streptomyces.

Bioprospecting actinobacterial secondary metabolism from untapped marine sources may lead to the discovery of biotechnologically-relevant compounds. While studying the diversity and bioactive potential of Actinomycetota associated with Codium tomentosum, a green seaweed collected in the northern Portuguese cost, strain CT-F61, identified as Streptomyces violaceoruber, was isolated. Its extracts displayed a strong anticancer activity on breast carcinoma T-47D and colorectal carcinoma HCT116 cells, being effective as well against a panel of human and fish pathogenic bacteria. Following a bioactivity-guided isolation pipeline, a new analogue of the red-pigmented family of the antibiotics prodigiosins, decylprodigiosin (1), was identified and chemically characterized. Despite this family of natural products being well-known for a long time, we report a new analogue and the first evidence for prodigiosins being produced by a seaweed-associated actinomycete.

Diversity and Bioactive Potential of Actinobacteria Isolated from a Coastal Marine Sediment in Northern Portugal.

Natural compounds have had increasing applications in the biotechnological sector, with a large fraction of these substances being channeled to the pharmaceutical industry due to their important pharmacological properties. The discovery of new bioactive molecules with novel mechanisms of action constitutes a promising solution for the design of alternative therapeutic solutions. Actinobacteria are a large group of morphologically and physiologically diverse bacteria well known for their production of biotechnologically relevant compounds. The Portuguese coast is scantly explored in terms of Actinobacteria diversity and respective bioactive potential, offering a good opportunity to find new Actinobacteria taxa and bioactive natural products. In this study, we investigated the Actinobacteria diversity associated with a sediment sample collected from the intertidal zone of a beach in northern Portugal, through a cultivation-dependent approach, and screened its antimicrobial and cytotoxic potential. A total of 52 Actinobacteria strains were recovered from the marine sediment, with the largest fraction of the isolates belonging to the genus Micromonospora. Bioactivity screening assays identified crude extracts of six Streptomyces strains active against C. albicans, exhibiting minimum inhibition concentration (MIC) values in the range of 3.90-125 µg mL-1. Twenty-five Actinobacteria crude extracts (obtained from strains of the genera Micromonospora, Streptomyces and Actinomadura) exhibited significant effects on the viability of at least one tested cancer cell line (breast ductal carcinoma T-47D and liver hepatocellular carcinoma HepG2). The Actinobacteria extracts demonstrating activity in the antimicrobial and/or cytotoxic assays were subjected to metabolomic analysis (Mass spectrometry (MS)-based dereplication and molecular networking analyses), indicating the presence of four clusters that may represent new natural products. The results obtained demonstrate the importance of bioprospecting underexplored environments, like the Portuguese coast, for enhancing the discovery of new natural products, and call attention to the relevance of preserving the natural genetic diversity of coastal environments.

Actinobacteria Isolated From Laminaria ochroleuca: A Source of New Bioactive Compounds.

Nature is the major reservoir of biologically active molecules. The urgent need of finding novel molecules for pharmaceutical application is prompting the research of underexplored environments, such as marine ecosystems. Here, we investigated cultivable actinobacteria associated with the macroalgae Laminaria ochroleuca and assessed their potential to produce compounds with antimicrobial or anticancer activities. A specimen of L. ochroleuca was collected in a rocky shore in northern Portugal, and fragments of tissues from different parts of the macroalgae (holdfast, stipe, and blades) were surface sterilized and plated in three culture media selective for actinobacteria. A total of 90 actinobacterial strains were isolated, most of which affiliated with the genus Streptomyces. Isolates associated with the genera Isoptericola, Rhodococcus, Nonomuraeae, Nocardiopsis, Microbispora, and Microbacterium were also obtained. Organic extracts from the isolates were tested for their antimicrobial activity using the agar-based disk diffusion method, followed by determination of minimum inhibitory concentration (MIC) values. Forty-five isolates inhibited the growth of Candida albicans and/or Staphylococcus aureus, with MIC values ranging from <0.5 to 1000 µg mL-1. The actinobacterial isolates were also tested for their anticancer potential on two human cancer cell lines. Twenty-eight extracts affected the viability of at least one human cancer cell line (breast carcinoma T-47D and neuroblastoma SH-SY5Y) and non-carcinogenic endothelial cell line (hCMEC/D3). Seven extracts affected the viability of cancer cells only. This study revealed that L. ochroleuca is a rich source of actinobacteria with promising antimicrobial and anticancer activities and suggests that macroalgae may be a valuable source of actinobacteria and, consequently, of new molecules with biotechnological importance.