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

Abstract

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 also shown the potential for the discovery of bioactive compounds from the novel taxa.

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

“…Basal medium was adapted from the modified M14 medium [33] and prepared using, per litre: 880 ml natural sea water (previously filtered through a 0.22 µm pore filter), 0.1 % ammonium sulphate (w/v) and 50 ml of Tris-HCl buffer (0.1M, pH 7.5). After autoclaving, the basal medium was supplemented: 10 ml of a vitamin solution [32], 20 ml of Hutner’s solution [76] and 0.1 % (w/v) of each carbon source (glucose, xylose, N -acetylglucosamine (NAG), arabinose, carrageenan, dextran, starch, mannitol, lactose, fructose, galactose and cellobiose). For nitrogen sources, per litre, 880 ml natural sea water (previously filtered through a 0.22 µm pore filter) and 50 ml Tris-HCl buffer (0.1M, pH 7.5) were firstly autoclaved.…”

“…Genomic DNA was obtained with the EZNA Bacterial DNA Isolation Kit (Omega BIO-TEK Norcross) following the protocols of the manufacturer. The 16S rRNA gene was amplified by PCR using the universal primers 27F and 1492R following previously defined protocols [32]. Purification of the PCR product was done using the Illustra GFX PCR DNA and Gel Band Purification Kit following the company protocols and the sequencing was done at Eurofins Genomics (Germany).…”

“…During low tide, water-submerged sediments samples were collected into sterile falcon tubes and brought to the laboratory under refrigerated conditions (approximately 8 °C). The isolation methodology used in this study was an in situ iChip-based culturing technique [30] that was previously adapted for the isolation of Planctomycetota [31, 32]. In this study, the bacterial inoculum was prepared by adding 10 ml sterile sea water to 20 g retrieved sediment and shaking the mixture vigorously.…”

Streptomyces meridianus sp. nov. isolated from brackish water of the Tagus estuary in Alcochete, Portugal

“…For initial identification, the partial 16S rRNA gene sequence was amplified with the primers 27F and 1492R [18]. PCR was performed as described previously [19]. Subsequently, the PCR amplicon obtained was purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (Cytiva).…”

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

“…For the primary identification, the 16S rRNA gene was amplified with the primers 27F and 1492R [18]. PCR was performed as described by Vitorino and colleagues [19]. The DNA was sequenced using Sanger sequencing at Eurofins Genomics (Ebersberg, Bavaria, Germany).…”