Uncovering the biotechnological capacity of marine and brackish water Planctomycetota.

An appealing strategy for finding novel bioactive molecules in Nature consists in exploring underrepresented and -studied microorganisms. Here, we investigated the antimicrobial and tumoral anti-proliferative bioactivities of twenty-three marine and estuarine bacteria of the fascinating phylum Planctomycetota. This was achieved through extraction of compounds produced by the Planctomycetota cultured in oligotrophic medium followed by an antimicrobial screening against ten relevant human pathogens including Gram-positive and Gram-negative bacteria, and fungi. Cytotoxic effects of the extracts were also evaluated against five tumoral cell lines. Moderate to potent activities were obtained against Enterococcus faecalis, methicillin-sensitive and methicillin-resistant Staphylococcus aureus and vancomycin-sensitive and vancomycin-resistant Enterococcus faecium. Anti-fungal effects were observed against Trichophyton rubrum, Candida albicans and Aspergillus fumigatus. The highest cytotoxic effects were observed against human breast, pancreas and melanoma tumoral cell lines. Novipirellula caenicola and Rhodopirellula spp. strains displayed the widest spectrum of bioactivities while Rubinisphaera margarita ICM_H10T affected all Gram-positive bacteria tested. LC-HRMS analysis of the extracts did not reveal the presence of any known bioactive natural product, suggesting that the observed activities are most likely caused by novel molecules, that need identification. In summary, we expanded the scope of planctomycetal species investigated for bioactivities and demonstrated that various strains are promising sources of novel bioactive compounds, which reenforces the potential biotechnological prospects offered by Planctomycetota.

Aeoliella straminimaris sp. nov., a novel member of the phylum Planctomycetota with an unusual filamentous structure.

Organisms with distinctive biological features and cellular organization constitute the bacterial phylum Planctomycetota. In this study, we formally describe a novel isolate, strain ICT_H6.2T, isolated from sediment samples collected in the brackish environment of the Tagus River estuary (Portugal) using an iChip-based culturing technique. The 16S rRNA gene analysis placed this strain into the phylum Planctomycetota and family Lacipirellulaceae, with a similarity value of 98.0 % to its closest relative Aeoliella mucimassa Pan181T, the currently only known member of the genus. Strain ICT_H6.2T has a genome size of 7.8 Mbp and a DNA G+C content of 59.6 mol %. Strain ICT_H6.2T is heterotrophic, aerobic and capable of microaerobic growth. This strain grows from 10 to 37 °C and from pH 6.5 to 10.0, requires salt to grow, and can tolerate up to 4 % (w/v) NaCl. Diverse nitrogen and carbon sources are utilized for growth. Morphologically, strain ICT_H6.2T is white to beige pigmented, spherical to ovoid in shape and around 1.4×1.1 µm in size. The strain clusters mainly in aggregates and younger cells show motility. Ultrastructural studies showed a cell plan with cytoplasmatic membrane invaginations and unusual filamentous structures with hexagonal organization in transversal section. Morphological, physiological and genomic comparison between strain ICT_H6.2T and its closest relatives strongly suggests it represents a novel species within the genus Aeoliella, for which we propose the name Aeoliella straminimaris sp. nov., represented by strain ICT_H6.2T as the type strain (=CECT 30574T=DSM 114064T).

Streptomyces marispadix sp. nov., isolated from marine beach sediment.

A novel actinomycetal strain, designated M600PL45_2T, was isolated from marine sediments obtained from Ingleses beach, Porto, on the Northern Coast of Portugal and was subjected to a polyphasic taxonomic characterisation study. The here described Gram-reaction-positive strain is characterised by the production of a brown pigment in both solid and liquid medium and forms typical helical hyphae that differentiate into smooth spores. The results of a phylogenetic analysis based on the 16S rRNA gene sequence indicated that M600PL45_2T has a high similarity to two members of the genus Streptomyces, Streptomyces bathyalis ASO4wetT (98.51 %) and Streptomyces daqingensis NEAU ZJC8T (98.44 %). The genome of M600PL45_2T has a size of 6 695 159 bp, a DNA G+C content of 70.71 mol% and 5538 coding sequences. M600PL45_2T grows at 15-37 °C and with a maximal growth rate between 25 °C and 30 °C. Growth at pH 6.0 to 9.0 with the optimal range between 6.0 and 7.5 was observed. M600PL45_2T showed a high salinity tolerance, growing with 0-10 % (w/v) NaCl, with best growth with 1-3% (w/v) NaCl. Major cellular fatty acids are iso-C15:0 (25.03 %), anteiso-C15:0 (17.70) and iso-C16:0 (26.90 %). The novel isolate was able to grow in media containing a variety of nitrogen and carbon sources. An antimicrobial activity screening indicated that an extract of M600PL45_2T has inhibitory activity against Staphylococcus aureus. On the basis of the polyphasic data, M600PL45_2T (= CECT 30365T = DSM 114036T) is introduced as the type strain of a novel species, that we named Streptomyces marispadix sp. nov.

Stieleria tagensis sp. nov., a novel member of the phylum Planctomycetota isolated from Tagus River in Portugal.

A bacterial strain was isolated from a brackish water sample of Tagus river, Alcochete, Portugal and was designated TO1_6T. It forms light pink colonies on M13 medium supplemented with N-acetylglucosamine. Cells are pear-shaped to spherical, form rosettes and divide by budding. Strain TO1_6T presents a mesophilic and neutrophilic profile, with optimum growth at 20 to 25 °C and pH 7.0 to 7.5, and vitamin supplementation is not required to promote its growth. The genome of the novel isolate is 7.77 Mbp in size and has a DNA G + C content of 56.3%. Based on its 16S rRNA gene sequence, this strain is affiliated with the phylum Planctomycetota. Further taxonomic characterization using additional phylogenetic markers, namely rpoB gene sequence (encoding the ß-subunit of the DNA-dependent RNA polymerase), as well as Percentage of conserved proteins, average nucleotide identity and average amino acid identity, suggest the affiliation of strain TO1_6T to the genus Stieleria, a recently described taxon in the family Pirellulaceae, order Pirellulales and class Planctomycetia. Based on the genotypic, phylogenetic and physiological characterization, we here describe a new species represented by the type strain TO1_6T (= CECT 30432T, = LMG 32465T), for which the name Stieleria tagensis sp. nov. is proposed.

Rubinisphaera margarita sp. nov., a novel planctomycete isolated from marine sediments collected in the Portuguese north coast.

The phylum Planctomycetota is constituted by bacteria with unique features that are well adapted to a vast range of habitats. Here, we describe a novel planctomycete isolated from marine sediments collected on a beach in Matosinhos (Portugal) using an iChip-based culturing technique. Strain ICM_H10T forms beige-coloured colonies in modified M14 medium and its cells are spherical to ovoid in shape, stalked, rosette-forming and showing motility in a phase of the life cycle. Transmission electron microscopy observations showed a typical planctomycetal cell plan and cell division by budding. This strain requires salt for growth and grows in the range of 2.0-5.0 % (w/v) NaCl, from 20 to 37 °C, within a pH of 6.0-9.0 and is able to use diverse nitrogen and carbon sources. It is heterotrophic, aerobic and capable of microaerobic growth. This strain has a genome size of approximately 6.0 Mb and a G+C content of 58.1 mol%. A 16S rRNA gene-based phylogenetic analysis supports the association of strain ICM_H10T to the phylum Planctomycetota and the family Planctomycetaceae, as it shares only 96.8 and 96.4% similarity to its closest relatives Rubinisphaera italica Pan54T and Rubinisphaera brasiliensis IFAM 1448T, respectively. Other phylogenetic markers also support the separation of this strain into a novel species. Morphological, physiological and genomic comparisons between strain ICM_H10T and its closest relatives strongly suggest that ICM_H10T represents a new species of the genus Rubinisphaera, for which we propose the name Rubinisphaera margarita sp. nov., with ICM_H10T (=CECT 30326T=LMG 32234T) as type strain.

The Planctomycetia: an overview of the currently largest class within the phylum Planctomycetes.

The phylum Planctomycetes comprises bacteria with uncommon features among prokaryotes, such as cell division by budding, absence of the bacterial tubulin-homolog cell division protein FtsZ and complex cell plans with invaginations of the cytoplasmic membrane. Although planctomycetes are ubiquitous, the number of described species and isolated strains available as axenic cultures is still low compared to the diversity observed in metagenomes or environmental studies. An increasing interest in planctomycetes is reflected by the recent description of a large number of new species and their increasing accessibility in terms of pure cultures. In this review, data from all taxonomically described species belonging to Planctomycetia, the class with the currently highest number of characterized members within the phylum Planctomycetes, is summarized. Phylogeny, morphology, physiology, ecology and genomic traits of its members are discussed. This comprehensive overview will help to acknowledge several aspects of the biology of these fascinating bacteria.

Diketopiperazines and other bioactive compounds from bacterial symbionts of marine sponges.

Humanity faces great challenges, such as the rise of bacterial antibiotic resistance and cancer incidence. Thus, the discovery of novel therapeutics from underexplored environments, such as marine habitats, is fundamental. In this study, twelve strains from the phylum Firmicutes and thirty-four strains from the phylum Proteobacteria, isolated from marine sponges of the Erylus genus, collected in Portuguese waters, were tested for bioactivities and the secondary metabolites were characterised. Bioactivity screenings comprised antimicrobial, anti-fungal, anti-parasitic and anti-cancer assays. Selected bioactive extracts were further analysed for already described molecules through high performance liquid chromatography and mass spectrometry. Several bioactivities were observed against the fungus Aspergillusfumigatus, the bacteria (methicillin-resistant Staphylococcus aureus and Escherichia coli), the human liver cancer cell line HepG2 and the parasite Trypanosoma cruzi. Medium scale-up volume extracts confirmed anti-fungal activity by strains Proteus mirabilis #118_13 and Proteus sp. (JX006497) strain #118_20. Anti-parasitic activity was also confirmed in Enterococcus faecalis strain #118_3. Moreover, P. mirabilis #118_13 showed bioactivity in human melanoma cell line A2058 and the human hepatocellular carcinoma cell line HepG2. The dereplication of bioactive extracts showed the existence of a variety of secondary metabolites, with some unidentifiable molecules. This work shows that bacterial communities of sponges are indeed good candidates for drug discovery and, as far as we know, we describe anti-parasitic activity of a strain of E. faecalis and the presence of diketopiperazines in Proteus genus for the first time.

Isolation, diversity and antimicrobial activity of planctomycetes from the Tejo river estuary (Portugal).

The discovery of new bioactive compounds is an invaluable aid to the development of new drugs. Strategies for finding novel molecules can focus on the exploitation of less studied organisms and ecosystems such as planctomycetes and brackish habitats. The unique cell biology of the underexplored Planctomycetota mean it is of particular interest. In this study, we aimed to isolate planctomycetes from the estuary of the Tejo river (Portugal). To reach this goal, macroalgae, water and sediments were sampled and diverse media and isolation techniques applied. Sixty-nine planctomycetal strains were brought into pure culture. An analysis of the 16S rRNA genes found that the majority of the isolates were affiliated to the genus Rhodopirellula. Putative novel taxa belonging to genera Stieleria and Rhodopirellula were also isolated and characterized morphologically. Enterobacterial repetitive intergenic consensus fingerprinting analyses showed higher diversity and different genotypes within close strains. Relevant biosynthetic gene clusters were found in most isolates and acetone extracts from representative strains exhibited mild antimicrobial activities against Escherichia coli and Staphylococcus aureus. Our work has not only enlarged the number and diversity of cultured planctomycetes but has also shown the potential for the discovery of bioactive compounds from the novel taxa.

Rhodopirellula aestuarii sp. nov., a novel member of the genus Rhodopirellula isolated from brackish sediments collected in the Tagus River estuary, Portugal.

Bacteria within the phylum Planctomycetota are biologically relevant due to unique characteristics among prokaryotes. Members of the genus Rhodopirellula can be abundant in marine habitats, however, only six species are currently validly described. In this study, we expand the explored genus diversity by formally describing a novel species. The pink-coloured strain ICT_H3.1T was isolated from brackish sediments collected in the Tagus estuary (Portugal) and a 16S rRNA gene sequence-based analysis placed this strain into the genus Rhodopirellula (family Pirellulaceae). The closest type strain is Rhodopirellula rubra LF2T, suggested by a similarity of 98.4% of the 16S rRNA gene sequence. Strain ICT_H3.1T is heterotrophic, aerobic and able to grow under microaerobic conditions. The strain grows between 15 and 37 °C, over a range of pH 6.5 to 11.0 and from 1 to 8% (w/v) NaCl. Several nitrogen and carbon sources were utilized by the novel isolate. Cells have an elongated pear-shape with 2.0 ± 0.3 × 0.9 ± 0.2 µm in size. Cells of strain ICT_H3.1T cluster in rosettes through a holdfast structure and divide by budding. Younger cells are motile. Ultrathin cell sections show cytoplasmic membrane invaginations and polar fimbriae. The genome size is 9,072,081 base pairs with a DNA G + C content of 56.1 mol%. Genomic, physiological and morphological comparison of strain ICT_H3.1T with its relatives suggest that it belongs to a novel species within the genus Rhodopirellula. Hence, we propose the name Rhodopirellula aestuarii sp. nov., represented by ICT_H3.1T (=CECT30431T = LMG32464T) as the type strain of this novel species. 16S rRNA gene accession number: GenBank = OK001858. Genome accession number: The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAMQBK000000000. The version described in this paper is version JAMQBK010000000.

Stieleria sedimenti sp. nov., a Novel Member of the Family Pirellulaceae with Antimicrobial Activity Isolated in Portugal from Brackish Sediments.

The phylum Planctomycetota is known for having uncommon biological features. Recently, biotechnological applications of its members have started to be explored, namely in the genus Stieleria. Here, we formally describe a novel Stieleriaisolate designated as strain ICT_E10.1T, obtained from sediments collected in the Tagus estuary (Portugal). Strain ICT_E10.1T is pink-pigmented, spherical to ovoid in shape, and 1.7 µm ± 0.3 × 1.4 µm ± 0.3 in size. Cells cluster strongly in aggregates or small chains, divide by budding, and have prominent fimbriae. Strain ICT_E10.1T is heterotrophic and aerobic. Growth occurs from 20 to 30 °C, from 0.5 to 3% (w/v) NaCl, and from pH 6.5 to 11.0. The analysis of the 16S rRNA gene sequence placed strain ICT_E10.1T into the genus Stieleria with Stieleria neptunia Enr13T as the closest validly described relative. The genome size is 9,813,311 bp and the DNA G+C content is 58.8 mol%. Morphological, physiological, and genomic analyses support the separation of this strain into a novel species, for which we propose the name Stieleria sedimenti represented by strain ICT_E10.1T as the type of strain (=CECT 30514T= DSM 113784T). Furthermore, this isolate showed biotechnological potential by displaying relevant biosynthetic gene clusters and potent activity against Staphylococcus aureus.

Novel and Conventional Isolation Techniques to Obtain Planctomycetes from Marine Environments.

Bacteria from the distinctive Planctomycetes phylum are well spread around the globe; they are capable of colonizing many habitats, including marine, freshwater, terrestrial, and even extreme habitats such as hydrothermal vents and hot springs. They can also be found living in association with other organisms, such as macroalgae, plants, and invertebrates. While ubiquitous, only a small fraction of the known diversity includes axenic cultures. In this study, we aimed to apply conventional techniques to isolate, in diverse culture media, planctomycetes from two beaches of the Portuguese north-coast by using sediments, red, green, and brown macroalgae, the shell of the mussel Mytilus edulis, an anemone belonging to the species Actinia equina, and seawater as sources. With this approach, thirty-seven isolates closely related to seven species from the families Planctomycetaceae and Pirellulaceae (class Planctomycetia) were brought into pure culture. Moreover, we applied an iChip inspired in-situ culturing technique to successfully retrieve planctomycetes from marine sediments, which resulted in the isolation of three additional strains, two affiliated to the species Novipirellula caenicola and one to a putative novel Rubinisphaera. This work enlarges the number of isolated planctomycetal strains and shows the adequacy of a novel methodology for planctomycetes isolation.

From Ocean to Medicine: Pharmaceutical Applications of Metabolites from Marine Bacteria.

Oceans cover seventy percent of the planet's surface and besides being an immense reservoir of biological life, they serve as vital sources for human sustenance, tourism, transport and commerce. Yet, it is estimated by the National Oceanic and Atmospheric Administration (NOAA) that eighty percent of the oceans remain unexplored. The untapped biological resources present in oceans may be fundamental in solving several of the world's public health crises of the 21st century, which span from the rise of antibiotic resistance in bacteria, pathogenic fungi and parasites, to the rise of cancer incidence and viral infection outbreaks. In this review, health risks as well as how marine bacterial derived natural products may be tools to fight them will be discussed. Moreover, an overview will be made of the research pipeline of novel molecules, from identification of bioactive bacterial crude extracts to the isolation and chemical characterization of the molecules within the framework of the One Health approach. This review highlights information that has been published since 2014, showing the current relevance of marine bacteria for the discovery of novel natural products.

Alienimonas chondri sp. nov., a novel planctomycete isolated from the biofilm of the red alga Chondrus crispus.

The phylum Planctomycetes comprises bacteria with peculiar and very unique characteristics among prokaryotes. In marine environments, macroalgae biofilms are well known for harboring planctomycetal diversity. Here, we describe a novel isolate obtained from the biofilm of the red alga Chondrus crispus collected at a rocky beach in Porto, Portugal. The novel strain LzC2T is motile, rosette-forming with spherical- to ovoid-shaped cells. LzC2T forms magenta- to pinkish-colored colonies in M13 and M14 media. Transmission and scanning electron microscopy observations showed a division by polar and lateral budding. Mother cells are connected to the daughter cells by a tubular neck-like structure. The strain requires salt for growth. Vitamins are not required for growth. Optimal growth occurs from 15 to 30°C and within a pH range from 5.5 to 10.0. Major fatty acids are anteiso-C15:0 (54.2%) and iso-C15:0 (19.5%). Phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid represent the main lipids and menaquinone 6 (MK-6) is the only quinone present. 16S rRNA gene-based phylogenetic analysis supports the affiliation to the phylum Planctomycetes and family Planctomycetaceae, with Alienimonas as the closest relative. Strain LzC2T shares 97% 16S rRNA gene sequence similarity with Alienimonas californiensis. LzC2T has a genome size of 5.3 Mb and a G+C content of 68.3%. Genotypic and phenotypic comparison with the closest relatives strongly suggest that LzC2T (=CECT 30038T=LMG XXXT) is a new species of the genus Alienimonas, for which we propose the name Alienimonas chondri sp. nov., represented by LzC2T as type strain. 16S rRNA gene accession number: GenBank=MN757873.1. Genome accession number: GenBank=WTPX00000000.

Bioactivities and Extract Dereplication of Actinomycetales Isolated From Marine Sponges.

In the beginning of the twenty-first century, humanity faces great challenges regarding diseases and health-related quality of life. A drastic rise in bacterial antibiotic resistance, in the number of cancer patients, in the obesity epidemics and in chronic diseases due to life expectation extension are some of these challenges. The discovery of novel therapeutics is fundamental and it may come from underexplored environments, like marine habitats, and microbial origin. Actinobacteria are well-known as treasure chests for the discovery of novel natural compounds. In this study, eighteen Actinomycetales isolated from marine sponges of three Erylus genera collected in Portuguese waters were tested for bioactivities with the main goal of isolating and characterizing the responsible bioactive metabolites. The screening comprehended antimicrobial, anti-fungal, anti-parasitic, anti-cancer and anti-obesity properties. Fermentations of the selected strains were prepared using ten different culturing media. Several bioactivities against the fungus Aspergillus fumigatus, the bacteria Staphylococcus aureus methicillin-resistant (MRSA) and the human liver cancer cell line HepG2 were obtained in small volume cultures. Screening in higher volumes showed consistent anti-fungal activity by strain Dermacoccus sp. #91-17 and Micrococcus luteus Berg02-26. Gordonia sp. Berg02-22.2 showed anti-parasitic (Trypanosoma cruzi) and anti-cancer activity against several cell lines (melanoma A2058, liver HepG2, colon HT29, breast MCF7 and pancreatic MiaPaca). For the anti-obesity assay, Microbacterium foliorum #91-29 and #91-40 induced lipid reduction on the larvae of zebrafish (Danio rerio). Dereplication of the extracts from several bacteria showed the existence of a variety of secondary metabolites, with some undiscovered molecules. This work showed that Actinomycetales are indeed good candidates for drug discovery.