Novel and Conventional Isolation Techniques to Obtain Planctomycetes from Marine Environments

Abstract

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.

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

“…S1). After plate incubation for 45 days at room temperature and in darkness, the content of each pit was re-inoculated into modified M13 medium plates [8,46] and incubated at 25 °C. A colony with beige pigmentation assigned as strain ICM_H10 T was obtained after 3 weeks incubation.…”

“…Water submerged sediments were collected in Memória beach (Matosinhos), Portugal (41° 13′ 50.35″ N 8° 43′ 16.61″ W) [46] (Fig. S1, available in the online version of this article) in October 2020 during a low tide and brought immediately into the laboratory in a cold chamber.…”

“…S1, available in the online version of this article) in October 2020 during a low tide and brought immediately into the laboratory in a cold chamber. The methodology used for the isolation of strain ICM_H10 T was based on an enrichment which used an iChip-inspired [48] culturing technique, which was for the first time applied for the targeted isolation of planctomycetes [46]. Briefly, an environmental bacterial inoculum was prepared by mixing 25 g of the collected sediments in 10 ml sterile sea water.…”

“…The current known diversity of the phylum Planctomycetota is thus still very incomplete. Several isolation efforts in recent years have led to a substantial number of strains to be brought into pure culture and the formal description of more than half of the currently known species [4,14,[32][33][34][35][36][37][38][39][40][41][42][43][44][45][46]. Taxonomic rearrangements within the phylum were also recently made [47].…”

“…Recently, we successfully applied an iChip-inspired [48] culturing technique for a novel targeted isolation methodology of planctomycetes from marine sediments, which resulted in the isolation of three strains, two affiliated to the species Novipirellula caenicola and one putative novel Rubinisphaera (based on a 16S rRNA gene analysis) [46]. In this study, we formally describe Rubinisphaera margarita sp.…”