The phylum Planctomycetes

The phylum Planctomycetes is a group of bacteria within the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum (Wagner and Horn 2006), which is presently also formed by Lentisphaerae and other sister phyla with Candidatus status (Lage et al. 2019). The first representative of this phylum was identified nearly one century ago and mistakenly classified as a floating fungus, which explains the name of the phylum (Gimesi 1924).

Taxonomically, the phylum Planctomycetes is presently divided into two classes, Planctomycetia (Krieg et al. 2010) and Phycisphaerae (Fukunaga et al. 2009). In addition to members of these two classes, Planctomycetes capable of anaerobic ammonium oxidation (anammox) (Strous et al. 1999) form the proposed Candidatus order Brocadiales (Jetten et al. 2010), a status that results from the current lack of an axenic culture for any described member of the order.

Planctomycetes and other PVC members such as the Chlamydiae are unique in many aspects, such as their cell biology (van Niftrik and Devos 2017). The planctomycetal cell plan is distinctive from other bacteria because of invaginations of the cytoplasmic membrane which give rise to an enlarged periplasmic space in several strains (Boedeker et al. 2017; Devos 2014; Lage et al. 2013; Santarella-Mellwig et al. 2013). Planctomycetes, as well as Chlamydiae, are solely capable of dividing without the otherwise universal division protein FtsZ, which is unique in prokaryotes (Ouellette et al. 2020; Rivas-Marin et al. 2016a). Key proteins involved in cell division are still unknown (Wiegand et al. 2020c). The phenomenon of phagocytosis-like cell engulfment (exclusively known until now in eukaryotes and therefore quite unique in prokaryotes) was also spotted recently in the putative planctomycete Candidatus ‘Uab amorphum’ (Shiratori et al. 2019).

Presently, Planctomycetes are still mysterious bacteria (Lage et al. 2019; Wiegand et al. 2018) and, although ubiquitous, only a small percentage of the known diversity is covered by axenic cultures (Wiegand et al. 2018). The description of novel taxa and the expansion of our knowledge on the existing ones are equally important to have a better understanding of the biology of this fascinating group of bacteria. In fact, recent efforts in the development of new isolation techniques and sampling in different habitats resulted in the isolation of numerous strains in the last years (Boersma et al. 2020; Dedysh et al. 2020c; Devos et al. 2020; Kaushik et al. 2020; Kulichevskaya et al. 2017a; Kulichevskaya et al. 2020a; Kulichevskaya et al. 2020b; Kumar et al. 2020a; Kumar et al. 2020b; Kumar et al. 2020c; Lage et al. 2017; Pradel et al. 2020; Vitorino et al. 2020; Vitorino et al. 2021b; Wiegand et al. 2020c). Wiegand and collaborators made a remarkable contribution to the current planctomycete collection by bringing into culture and characterizing 79 planctomycetal strains, most of which are new taxa (Wiegand et al. 2020c). Furthermore, recent re-arrangements in the planctomycetal taxonomy have allowed the introduction of new taxonomic groups (Dedysh et al. 2020c).

The main goal of this overview article is to gather current knowledge on Planctomycetia, which is currently the class within the phylum Planctomycetes with the highest number of characterized members. This review, which puts the emphasis on taxonomy, is based on data available on the currently and effectively or validly described representatives. All compiled data from hitherto published species descriptions is given in Supplementary Table 1a-d while an overview of the major features and differences between each family is presented in Table 1. Based on the dataset, the phenotypic and genomic features of the taxa are discussed. Additionally, information on other isolates and environmental 16S rRNA gene sequences, when available, was obtained by searching for hits within the species threshold of 99% similarity of the 16S rRNA gene in the National Center for Biotechnology Information (NCBI) database using BLAST search (data given in Supplementary Table 1a).

Table 1 Overview on the main features of the families of the class Planctomycetia. NDA- no data available
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The class Planctomycetia

Taxonomy

With a total of 108 formally described species, Planctomycetia is currently the best studied class within the phylum Planctomycetes (Supplementary Table 1a-d). By comparison, the current class Phycisphaerae consists of only 9 published species. More than half of the species described falling within the class Planctomycetia (70) were either published in 2019, 2020 or 2021, which reflects the increasing interest in an exploration of the diversity within this class. The class is currently subdivided into four orders, namely the type order Planctomycetales and the orders Pirellulales, Isosphaerales and Gemmatales (Dedysh et al. 2020c). The order Planctomycetales has only one family, Planctomycetaceae. The current order Pirellulales in turn is divided into three families, Pirellulaceae, Lacipirellulaceae and Thermoguttaceae. The orders Isosphaerales and Gemmatales harbour a single family, Isosphaeraceae and Gemmataceae, respectively. A partial 16S rRNA gene-sequence-based tree was computed based on all type strains to summarize the phylogeny of the families and orders within the class Planctomycetia (Fig. 1).

Fig. 1
figure 1

Phylogeny of the families and orders of class Planctomycetia. The number of described species with 16S rRNA gene available are given. The partial 16S rRNA gene-based tree was constructed with MEGA X (Kumar et al., 2018) using type strain sequences, which were retrieved from the NCBI database. The alignment of sequences was performed with CLUSTALW (Larkin et al., 2007) and the phylogeny was inferred by using the Maximum Likelihood method and General Time Reversible mode using the gamma substitution and estimation of proportion of invariable sites option. Members belonging to the same family were collapsed and the number of species within each one presented. The outgroup consists of 3 members from the phylum Verrucomicrobia

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To achieve a better insight on the phylogenetic diversity within the class, we additionally searched for the respective full-length 16S rRNA gene sequences in the SILVA SSU Ref NR database (release 138.1 from 27 August 2020) (Quast et al. 2013) that putatively belong to class Planctomycetia. We found 4871 non-redundant sequences in total, defined by a 99% identity threshold. However, a large part of the list of Operational taxonomic units (OTUs) corresponds to organisms that have not yet been cultivated, which underlines that only a small fraction of the known diversity of the class is presently covered by axenic cultures (approximately 2%) (Wiegand et al. 2018). In that regard, the class Planctomycetia is largely unexplored. Nevertheless, the current number of axenic cultures reflects the predicted abundance of this class within the phylum: in comparison, the number of OTUs for the phylum is 8657, of which more than half belong to the class Planctomycetia while only 2484 correspond to members of the class Phycisphaerae and the rest to other lineages. Based on the available information, Planctomycetia seems to be the most diverse class of the phylum Planctomycetes. However, this assumption is purely based on the present knowledge regarding the data available. As already discussed previously (Wiegand et al. 2018), the entire phylum Planctomycetes is still heavily underexplored and novel taxa and even lineages are most likely present in the environment but have not yet been discovered.

Genomic characteristics

Of all hitherto described species falling in the class Planctomycetia, the majority of the type strains have currently a genome sequence available in the NCBI database (as of October 2021), which has allowed in the recent years for a more extensive analysis of this group (Kallscheuer and Jogler 2021; Wiegand et al. 2020c) (Supplementary Table 1d). In total, in the NCBI database, 145 non-redundant genome assemblies belonging to cultured organisms taxonomically assigned to class Planctomycetacia are currently available (excluding Metagenome-assembled genomes (MAGs)). These genomes were used to compute a genomic multilocus-sequence analysis-based tree (MLST) (Alanjary et al. 2019) to visualize the phylogenetic organization of the current class Planctomycetia (Fig. 2). Furthermore, core information clustered in this study for all Planctomycetia species was additionally displayed in association to the tree (Fig. 2).

Fig. 2
figure 2

available at GenBank. Excluded species with currently no genome available are Rhodopirellula lusitana, Thermostilla marina, Novipirellula caenicola, N. rosea, Bremerella cremea, Thermogutta hypogea, "Singulispaera mucilagenosa", S. rosea, Tundrisphaera lichenicola, Planctomyces bekefii, P. stranskae and P. guttaeformis. The datasets containing the summarized information of Planctomycetia species were added to the tree using the Interactive Tree Of Life (iTOL) v5 online tool (Letunic and Bork, 2021)

Multi-locus- sequence analysis-based tree (MLST) showing the phylogenomics of class Planctomycetia and a summary of the core information clustered in this study. All genomes currently available in the NCBI database of organisms assigned to the class Planctomycetia were utilized to construct the MLS tree, which was computed using the autoMLST: Automated Multi-Locus Species Tree pipeline using default gene parameters (Alanjary et al., 2019). The outgroup consists of three members from phylum Actinobacteria (Streptomyces spp.). Name labels in blue correspond to type strains of the described species and names in black correspond to other strains with the genome

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The genome size is highly variable among species within this class, with the smallest size of 4.3 Mb for “Botrimarina hoheduenensis” Pla111T (Lacipirellulaceae) (Wiegand et al. 2020a) and the biggest size of 12.4 Mb belonging to Fimbriiglobus ruber SP5T (Gemmataceae) (Kulichevskaya et al. 2017a). Genome sizes are overall higher in planctomycetes from families such as Isosphaeraceae and Gemmataceae, which are almost exclusively present in terrestrial and freshwater habitats (Table 1). The DNA G + C content is also highly diverse, ranging from 45.1 mol% in Gimesia aquarii V144T (Planctomycetaceae) (Wiegand et al. 2020b) to 71.3 mol% in Urbifossiella limnaea ETA_A1T (Gemmataceae) (Kallscheuer et al. 2020d). The higher values are also correlated with planctomycetes that are inhabitants of terrestrial environments (Table 1). In general, more than 40% of the protein-coding genes are associated with unknown functions (Wiegand et al. 2020c).

Data on genome-encoded features in the central carbon metabolism was also gathered when available. All examined strains harbour genes coding for enzymes involved in glycolysis (Embden-Meyerhof-Parnas pathway or the alternative Entner-Doudoroff pathway), although, in some cases, not all genes could be identified. The same was noticed for the tricarboxylic acid cycle and gluconeogenesis. Most species had a fully functional pentose phosphate pathway but no planctomycetal genome analysed so far showed a complete glyoxylate shunt pathway. This pathway is normally required for anaplerosis during growth with fatty acids or acetate as sole carbon and energy source (Chew et al. 2019). The lack of genes for this pathway might support the hypothesis that members of class Planctomycetia prefer sugars over carboxylic acids as carbon and energy sources.

The bioactive potential of this class was also evidenced by the presence of related biosynthetic gene clusters in most available genomes (Wiegand et al. 2020c; Wiegand et al. 2018). These showed to be rich in different genes encoding large multimodular proteins such as Non-ribosomal peptide synthetases (NRPSs) and Polyketide synthases (PKSs), as well as in genes for production of terpenes, ectoines and antibiotics such as bacteriocins and lanthipeptides, among others (Kallscheuer and Jogler 2021; Wiegand et al. 2020c; Wiegand et al. 2018). The production of antimicrobial compounds empowers members of this class with the ability to compete against other fast-growing bacteria for space and food resources, rendering them competitive in challenging environments.

Morphology

Morphologically, members of the class Planctomycetia are mostly spherical to ovoid, elongated or pear-shaped and 0.5 to 2.5 µm in diameter or length and can form aggregates, rosettes and even chains (Supplementary Table 1b). Most species also have the capacity to produce extracellular materials (e.g. holdfast or mucus substances or fibrous materials) that allows them to live in an attached life style in biofilms. Most species also display crateriform pits, either distributed uniformly over the cell surface or only on the reproductive pole. Overall, members of the class Planctomycetia display what is considered a typical planctomycetal cell structure, including a complex pattern of cytoplasmic membrane invaginations (Boedeker et al. 2017; Lage et al. 2019; Wiegand et al. 2018). Colony colours within the class range from unpigmented/white/beige to pink/red or orange. The pigmentation is caused by the production of carotenoids (Kallscheuer et al. 2019), while their exact function remains to be elucidated.

Members of the class Planctomycetia divide by budding (Lage et al. 2019; Wiegand et al. 2020c). This is a striking difference to most other bacteria, which divide by binary fission, including members of the class Phycisphaerae (Fukunaga et al. 2009), as well as the anammox planctomycetes (van Niftrik and Jetten 2012). Budding in bacteria is a rare division mode shared only by a scarce number of taxa, such as Caulobacter spp. and Hyphomicrobium spp., and diverges from the budding normally associated to yeasts (Stackebrandt et al. 1988). Planctomycetal budding is characterized by the outgrowth of a bud from the mother cell, which is regularly on the polar side of the cell but can be formed laterally (Vitorino et al. 2020; Wiegand et al. 2020c). Furthermore, other unique division variations such as the formation of a tubular neck-like structure between the mother and daughter cells and consecutive budding were also spotted in different Planctomycetia strains (Boersma et al. 2020; Kohn et al. 2016; Lage 2013; Vitorino et al. 2020).

Ecology

Data on the ecology of the currently described members of the class was gathered (Supplementary Table 1a) and summarized in Fig. 3. Marine and brackish environments are well-known habitats for members of the class and material used for strain isolation included diverse sources such as water, sediments, marine organisms like algae, plants and invertebrates (i.e. sponges, shrimps), etc. (Fig. 3a). In particular, macroalgae are hotspot organisms that harbour planctomycetal diversity (Bengtsson and Ovreas 2010; Bondoso et al. 2014b, 2017; Lage and Bondoso 2011, 2014). In fact, environments rich in organic carbon sources such as macroalgal biofilms that are composed of Extracellular polymeric substances (EPS) seem to favour the presence of members of the class Planctomycetia. Furthermore, strains belonging to the families Pirellulaceae and Lacipirellulaceae were isolated from natural (wood pellets) or artificial (polystyrene particles or polyethylene) materials retrieved from marine/brackish environments (Fig. 3a). On the other hand, freshwater and terrestrial environments, seem to be promising spots for the isolation of members of the families Gemmataceae and Isosphaeraceae (Fig. 3b). Strains affiliated to all families were also isolated from ‘extreme’ environments (such as hot springs and hydrothermal vents) (Fig. 3b). The ranges regarding temperature preference points towards potential differences in the capacity of different taxa to adapt to such conditions. Also, deep-surface environments such as gold mines served as a source for the isolation of Isosphaeraceae and Thermoguttaceae (Fig. 3b).

Fig. 3
figure 3

Habitat distribution of the currently taxonomically described members of the families within the class Planctomycetia. In (a), the marine and brackish water ecosystems display a very distinctive planctomycetal distribution compared to freshwater/terrestrial/other environments (b). Data from type strains was obtained from species description studies and information on other isolates and environmental 16S rRNA gene sequences, when available, was obtained by searching for hits defined by a threshold of 99% similarity of the 16S rRNA gene, in the NCBI database using the BLAST search

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Although no strains were isolated from oil/petroleum/metal-contaminated environments so far, a variety of environmental 16S rRNA sequences likely belonging to the families Pirellulaceae, Planctomycetaceae and Gemmataceae were detected (Fig. 3b). The indication of the presence of strains belonging to this class in such environments demonstrates that Planctomycetia members are promising organisms to be studied in the context of bioremediation (either with hydrocarbons or metals). So far, only uncultured Planctomycetaceae, Gemmataceae, Lacipirellulaceae and Thermoguttaceae were detected in anaerobic reactors/activated sludge or wastewater treatment plants (Fig. 3b). Taken together, known members of this class, although mostly distributed in marine and freshwater environments, appear to adapt well to a variety of different habitats (Fig. 3).

In terms of the geographical distribution of known Planctomycetia (Fig. 4), sampling and isolation campaigns focused on a limited number of locations around the globe, in particular the European coast. The Mediterranean Sea around Italy, including the Aeolian Islands, turned out to be a promising location for the isolation of members of all families with exception of Gemmataceae. A variety of strains from the families Pirellulaceae, Lacipirellulaceae and Planctomycetaceae was also obtained from German coastlines of the North and Baltic Sea and the Spanish and French coastlines/islands (Wiegand et al. 2020c). The North Coast of Portugal in the Atlantic ocean is also inhabited by diverse Planctomycetaceae and Pirellulaceae (Bondoso et al. 2014b, 2017; Lage and Bondoso 2011, 2014), as well as the Norwegian coast (Bengtsson and Ovreas 2010). Additional coastlines from which Planctomycetaceae and Pirellulaceae were isolated are the Californian coast (Wiegand et al. 2020c) and the eastern Australian coast (Izumi et al. 2013). Valu Fa Ridge in the Southwest Pacific is also a location with abundant Pirellulaceae and Planctomycetaceae, as well as Lacipirellulaceae (Storesund et al. 2018). Numerous locations in Russia, namely peat bogs, subarctic lands and lakes, also serve as habitats for a variety of known Gemmataceae and Isosphaeraceae, as well as some Pirellulaceae and Lacipirellulaceae (Dedysh and Ivanova 2019; Ivanova et al. 2016; Kulichevskaya et al. 2017a, 2015, 2017b). An isolation campaign in India also revealed diversity of Planctomycetia (Gaurav et al. 2021; Kaushik et al. 2020; Kumar et al. 2020a; Kumar et al. 2020b; Kumar et al. 2021a; Kumar et al. 2020c; Kumar et al. 2021b; Kumar et al. 2021c). Based on NCBI data, additional locations with shown planctomycetal variety include territories in the United States and India but also other regions on the globe, including China, Mexico, Antarctica, Pacific Ocean, Philippines, Indonesia, Papua New Guinea, Japan and South Korea (Fig. 4), which makes them good candidates for future diversity and isolation studies. Taken together, known members of the class Planctomycetia seem to be well distributed around the globe and found when searched for (Fig. 4). Exploring different niches and locations is crucial to help bringing new taxa into pure culture and unveil the untapped planctomycetal diversity.

Fig. 4
figure 4

Geographical distribution of the currently taxonomically described members of the families within the class Planctomycetia. Data from type strains was obtained from species description studies and information on other isolates and environmental 16S rRNA gene sequences, when available, was obtained by searching for hits, defined by a threshold of 99% similarity of the 16S rRNA gene, in the NCBI database using the BLAST search

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Physiology and metabolism

Most of the currently described members of the class Planctomycetia are aerobic (Lage et al. 2019; Wiegand et al. 2020c) while members of the class Phycisphaerae are mostly facultatively or strictly anaerobic (Dedysh et al. 2020a; Wiegand et al. 2018). In general, Planctomycetia comprises heterotrophic, neutrophilic, and mesophilic strains (Supplementary Table 1c). Members of this class are rather slow-growing bacteria, with the shortest doubling time of around 5 h (Yadav et al. 2018) and the largest of up to 140 h (Salbreiter et al. 2020), both belonging to family Planctomycetaceae. A variety of species have a motile stage in a dimorphic lifecycle (Gade et al. 2005). Most Planctomycetia are resistant to several antibiotics (Supplementary Table 1c) (Cayrou et al. 2010; Godinho et al. 2019; Ivanova et al. 2021b), e.g. the beta-lactam ampicillin and the aminoglycoside streptomycin, which are the components of a common antibiotic mixture used in isolation medium for the selective enrichment of planctomycetes (Lage and Bondoso 2012; Wiegand et al. 2020c) and also glycopeptides (Godinho et al. 2019). Although most species do not require vitamins for growth, the use of vitamin B12 (cyanocobalamin) as supplement enhances their growth.

Analysis of carbohydrate utilization patterns of Planctomycetia assessed by traditional assays (Supplementary Table 1c) showed that most species are capable of using diverse sugars and other complex polysaccharides as carbon and energy source. Furthermore, most species can also utilize peptone, yeast extract, urea, nitrate, and ammonium as nitrogen sources. N-acetyl glucosamine can act both as a source of carbon and nitrogen (Schlesner 1994). Members of Gemmataceae and Isosphaeraceae and, more specifically, strains isolated from the microbial community inhabiting boreal Sphagnum peat bogs and lichen-dominated tundra wetlands, also show the ability to grow on compounds like xylan, pectin, starch, lichenan, cellulose, chitin and polysaccharides of microbial origin, which demonstrates their versatile hydrolytic capabilities and wide repertoires of carbohydrate-active enzymes (Dedysh and Ivanova 2019; Rakitin et al. 2021).

The fatty acid (FA) composition present in the various families is dominated by C16:0 with the exception of the family Gemmataceae, in which C18:0 and C16:1ω5c are the major fatty acids. Besides C16:0, the families Pirellulaceae, Lacipirellulaceae and Isosphaeraceae also have C18:1ω9c as another major FA and the representatives of Thermoguttaceae have a large content of C18:0. In general, the Planctomycetaceae possesses C16:1ω7c. Although a comprehensive analysis on all the described species is not available, FA profiles are, to a certain extent, taxonomically indicative. The main respiratory quinone found in this class is menaquinone 6 (MK-6).

In the following sections, data will be discussed at the family level, including the main distinctive features between the six families of class Planctomycetia which are summarized in Table 1. Furthermore, particular aspects of Planctomycetia species currently described will also be presented.

Family Planctomycetaceae

The type family Planctomycetaceae currently comprehends 14 genera and 29 species and is the second largest family in terms of isolated and studied members (Table 1). The type genus Planctomyces comprises three members, including the first planctomycete, Planctomyces bekefii (Gimesi 1924). The genus is constituted by as-yet-uncultivated planctomycetes with validly published names, namely the type species P. bekefii, P. stranskae and P. guttaeformis (Gimesi 1924; Starr and Schmidt 1984). Certain pieces of information are available for these taxa, however, it is crucial to bring them into pure culture to allow a full description, as already evidenced by Dedysh et al. (Dedysh et al. 2020b). The remaining described species are listed in Table 2, as well as their main features.

Table 2 Main features of the described species of family Planctomycetaceae
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Members of this family are mostly spherical to ovoid but can also be rice/pear-shaped. Thalassoglobus polymorphus Mal48T, as the name indicates (“polymorphus”: multiform, various shapes of the cells), has typically pear-shaped cells but other forms (such as coccoid and ovoid cells) are also observed (Rivas-Marin et al. 2020c). Members of Planctomycetaceae can be generally found in aggregates or rosettes, often harbour stalk-like structures and many members produce a holdfast structure. Morphologically, members of the genus Planctomyces are distinct: P. bekefii has a unique morphotype of rosettes formed by cells separated through stalk-like structures, which was the morphotype initially used for the distinction of this phylum (Dedysh et al. 2020b; Gimesi 1924). The other two Planctomyces species have also distinctive morphotypes: P. stranskae was described as a bulbiform bacterium with numerous multifibrillar appendages and bristles extending from the spherical end of the cell, while P. guttaeformis is described as a bulb-shaped bacterium with only one prominent multifibrillar appendage, like a spike (but not a stalk) (Starr and Schmidt 1984). Other species also possess distinct morphological features, such as Caulifigura coniformis Pan44T, which has an uncommon cell texture comprised of triangles or rectangles (resembling a pinecone) (Kallscheuer et al. 2020e), and Schlesneria paludicola MPL7T, which has short stalk-like structures which resemble twisted fibrils connecting the cells (Kulichevskaya et al. 2007). Thalossoglobus neptunius KOR42T differs from others by being the only family member capable of occasional aggregation in filaments (Kohn et al. 2020a). Calycomorphotria hydatis V22T possesses striking cell internal characteristics, such as putative filamentous cytoskeletal elements (Schubert et al. 2020). Although all known class members divide by budding, several members of this family have distinctive variations of the cell division process, such as polar and lateral budding in Alienimonas chondri LzC2T (Vitorino et al. 2020; Vitorino et al. 2021a), consecutive budding in A. californiensis CA12T (Boersma et al. 2020) and the formation of a tubular-like structure between mother and daughter cells in A. chondri LzC2T, Planctopirus hydrillae JC280T (Yadav et al. 2018) and Fuerstiella marisgermanici NH11T (Kohn et al. 2016; Kohn et al. 2019). Most members are white/cream to pink pigmented while species Maioricimonas rarisocia (type strain Mal4T) and Rubinisphaera brasiliensis (type strain IFAM 1448 T) are orange coloured (Kallscheuer et al. 2019; Rivas-Marin et al. 2020b; Schlesner 1989).

The family comprises, in general, heterotrophic, mesophilic, aerobic and neutrophilic organisms, although some members such as the strains isolated from the microbial communities inhabiting boreal tundra wetlands can be psychrotolerant and/or acid-tolerant (Supplementary Table 1c). Overall, the most common fatty acids found in the family are palmitic acid (C16:0), palmitoleic acid (C16:1ω7c) and C18:1ω7 but some members display a different lipid profile, such as F. marisgermanici NH11T, which is mainly constituted by fatty acids C16:1ω6c and C18:1ω7c (Kohn et al. 2016; Kohn et al. 2019), R. italica Pan54T with the uncommon fatty acid C15:0 iso2-O (Kallscheuer et al. 2020b) and A. chondri LzC2T which produces iso-C15:0 and anteiso-C15:0 in major amounts (Vitorino et al. 2020, 2021a). Additional uncommon fatty acids (either C14:0,3-OH or C16:1iso) are also part of the lipid composition of most known members of the genus Gimesia. The main polar lipids found in the family are phosphatidylglycerol, di-phosphatidylglycerol, phosphatidylcholine, phosphatidyl-dimethylethanolamine, phosphocholine and phosphatidyl-monomethylethanolamine besides a variety of non-identified lipids. Planctomicrobium piriforme P3T is different from other family members by having a specific polar lipid composition (diacylglycerol-O-(N,N,N-trimethyl)homoserine lipid plus phosphocholine (Kulichevskaya et al. 2015). Although menaquinone 6 is the main respiratory quinone found in the class, an additional one, menaquinone 7 (MK-7), was found in P. hydrillae JC280T (Yadav et al. 2018).

As referred previously, members of this family are geographically widely distributed (Fig. 4) and present in a variety of marine habitats, such as sediments, the water column and in association with macro- and microalgae, plants and animals (Fig. 3). Gimesia spp. are particularly widely spread in different marine locations and have also been isolated and detected in extreme environments, such as hot lakes and petroleum- and metal-contaminated sites (Supplementary Table 1a) which makes the members of the genus Gimesia promising organisms for future studies dealing with bioremediation of contaminated locations. Some members of the family such as Planctopirus spp., P. piriforme and S. paludicola are exclusively found in freshwater associated environments.

Members of family Planctomycetaceae turned out to be good candidates for future studies focusing on bioactive molecules: P. limnophila, P. hydrillae and R. brasiliensis strains showed antimicrobial activity (Graca et al. 2016; Jeske et al. 2016; Yadav et al. 2018). Additionally, the first detected phage capable of infecting a planctomycete was observed in P. ephydatiae spb1T and posteriorly isolated and characterized (Kohn et al. 2020b).

The average genome size in this family is approximately 6.9 ± 1.1 Mb and it ranges from 5.16 to 8.92 Mb. The mean DNA G + C content is 54.8 ± 6.6 mol% (the lowest of the class) and it ranges from 45.1 to 70.7 mol%. P. limnophila MU 290 T, the only family member harbouring a plasmid, is considered a model organism for planctomycetal studies and has been one of the few successfully genetically modified planctomycetes (Boedeker et al. 2017; Jeske et al. 2016; Rivas-Marin et al. 2016b; Rivas-Marin et al. 2020a).

During the analysis of the family, we encountered a taxonomic conflict regarding the genus Gimesia. The 16S rRNA gene comparison between G. benthica E7T and G. chilikensis JC646T indicated that these two species are very similar (99.9% similarity). Both species descriptions were published almost at the same time (May and June 2020) (Kumar et al. 2020a; Wang et al. 2020) and both names are currently valid. The resulting conflict required reassessment of the phylogeny of the two strains. In fact, Wiegand and collaborators already employed several phylogenetic markers (rpoB gene identity, Average nucleotide identity (ANI), Amino acid identity (AAI) and percentage of conserved proteins) to re-analyse phylogenetic positions and concluded that the two strains belong to the same species (in this case, G. chilikensis, which was published and validated first) (Wiegand et al. 2020b). By the comparative analysis of our data clustered for both strains (Supplementary Table 1) we corroborate that these strains are indeed highly similar in morphological and physiological traits. Strain E7 should therefore be re-classified as a strain of the species G. chilikensis.

Family Pirellulaceae

The family Pirellulaceae is the group with the highest number of studied members in the entire class Planctomycetia (Table 1) and comprises 15 described genera and 39 species. The type genus Pirellula is constituted by a single species, Pirellula staleyi (Schlesner and Hirsch 1987) that was the first Pirellulaceae isolated, although it was originally named Planctomyces staleyi (Staley 1973) and then Pirella staleyi (Schlesner and Hirsch 1984). All described species are listed in Table 3 and their main traits presented. Very recently, an additional member of this family, Candidatus "Laterigemmans baculatus" was described (Kumar et al. 2021a), which possesses the Candidatus status due to the loss of viability in the laboratory culture (Kumar et al. 2021a).

Table 3 Main features of the described species of family Pirellulaceae
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Members of the family Pirellulaceae are often ovoid/pear-shaped/elongated (“pirellula” = small pear) and rosettes are the most common form of aggregation, with exception of species Rhodopirellula solitaria CA85T that does not aggregate at all (Supplementary Table 1b). The recently described Candidatus "Laterigemmans baculatus" was the first reported rod-shaped planctomycete in the family (Kumar et al. 2021a). Stalk-like structures have never been observed in members of this family. Most species have a motile stage and produce a holdfast structure or fibrous materials. A strong glycocalyx or extracellular polymeric substance (which renders the cells adhesive) were additionally observed for some members of the genus Rubripirellula (R. obstinata LF1T and R. tenax Poly51T) (Bondoso et al. 2015; Kallscheuer et al. 2020c). The main colony colours in the family are white and pink while Stieleria varia Pla52T is the sole member of the family to form orange-pigmented colonies (Surup et al. 2020).

Current data on known Pirellulaceae shows that they are wide-spread around the globe (Fig. 4) and live mostly in a variety of marine habitats (Fig. 3). Members of the genus Rhodopirellula seem to be the most disseminated one in these environments (Supplementary Table 1a) and they are often strongly associated with different macroalgal species (Bondoso et al. 2017; Lage and Bondoso 2011, 2012; Schlesner et al. 2004; Winkelmann and Harder 2009). Strains from the genera Pirellula and “Anatilimnocola” were only found in freshwater environments. Pirellulaceae strains belonging to additional genera, such as Novipirellula and Rubripirellula, were isolated from non-natural and abiotic surfaces like polystyrene or polyethylene particles. One strain affiliated to the genus Bremerella was isolated from an anaerobic, sulfide- and sulfur-rich spring in Oklahoma (Elshahed et al. 2007) and an uncultured clone putatively from the same genus was detected in chromium-contaminated tannery sludge, which indicates that Bremerella spp. may be promising organisms with relevance in bioremediation studies.

In general (Supplementary Table 1c), Pirellulaceae are heterotrophs and aerobes, with few exceptions capable of microaerobic or anaerobic growth, such as Rhodopirellula rubra, Rhodopirellula lusitana and Blastopirellula marina. They all divide by polar budding, with the exception of the recently described Candidatus "Laterigemmans baculatus", which is also capable of lateral budding (Kumar et al. 2021a). Overall, Pirellulaceae are neutrophilic and mesophilic, with a few psychrotolerant species. Members of this family have the smallest generation times of the current class Planctomycetia, with a mean value of 15.3 ± 7.4 h, with exception of Aureliella helgolandensis Q31aT, which has a relatively high doubling time (41 h). Some members require vitamin B12 for growth. Phosphatidylcholine, diphosphatidylglycerol and phosphatidylglycerol are the main polar lipids found in the studied strains, however, a chemotaxonomic analysis was not presented in a considerable number of studies describing members of this family. Moreover, still unknown (phospho)lipids are also present in most species. Studies with Novipirellula rosea LHWP3T (Roh et al. 2013) showed that it produces an extra polar lipid rare in the family: phosphatidylethanolamine. The major fatty acids found in members of this family are oleic/elaidic acid (C18:1ω9) and palmitic acid (C16:0), while P. staleyi ATCC 27377 T also produces the long-chain unsaturated fatty acid C20:1ω11c and Mariniblastus fucicola FC18T the C14:0 myristic acid in major amounts, which are unique in the family. M. fucicola FC18T is also the only member in the class Planctomycetia to produce menaquinone 5 (MK-5) besides the standard menaquinone 6 (MK-6) found in planctomycetes.

As briefly discussed previously, Planctomycetia have the genomic potential to produce secondary metabolites with potential biotechnological applications (Kallscheuer and Jogler 2021). The natural function of such compounds is probably linked to the survival in complex and competitive habitats such as macroalgae biofilms (Graca et al. 2016). In fact, Pirellulaceae have been demonstrated to be promising organisms for bioprospection, in particular Bremerella members. Cell extracts of these bacteria have been linked to anti-cancer activity (Calisto et al. 2019), Roseimaritima ulvae UC8T showed antimicrobial activity (Graca et al. 2016) and diverse strains from the genus Rhodopirellula showed antimicrobial and anti-cancer activities (Calisto et al. 2019; Graca et al. 2016; Jeske et al. 2016). Members of the genus Stieleria were the source for the isolation of a new type of secondary metabolite with moderate antimicrobial activity named stieleriacine (Kallscheuer et al. 2020a; Sandargo et al. 2020). Moreover, R. rubra LF2T was studied for its adequacy as a supplementary food source for Daphnia magna (Marinho et al. 2019, 2018).

R. baltica SH1T was the first planctomycete with a sequenced genome (Glockner et al. 2003) and currently 64 genomes of strains belonging to this family are available. The average genome size in this family is approximately 7.7 ± 1.2 Mb with a minimum and maximum size of 6.1 and 11.0 Mb, respectively. The mean DNA G + C content is 56.5 ± 2.5 mol% and it ranges from 49.5 to 62.4 mol%. No current member of the family harbours a plasmid.

A phylogenetic analysis of the family led to a taxonomic conflict between the validly published genus Stieleria and the more recently and not yet validated name of the genus “Roseiconus” (Kumar et al. 2020c). This is possibly related to the fact that the genus description of Stieleria was not published in a taxonomy journal, but in the context of the analysis of identified stieleriacines. Based on the 16S rRNA gene comparison between the five taxa (2 species of “Roseiconus” and 3 species of Stieleria), we found that “Roseiconus lacunae” JC635T is more similar to other Stieleria than to “Roseiconus nitratireducens”, with a 16S rRNA gene sequence similarity of 99.36% between “R. lacunae” JC635T and its closest relative S. neptunia Enr13T. On the other hand, “R. nitratireducens” JC645T shares 96.9% 16S rRNA gene similarity with its closest relative S. maiorica Mal15T. Following the rules of priority, these analysis suggest that “Roseiconus lacunae” JC635T is a strain that belongs to species S. neptunia and that “R. nitratireducens” JC645T is probably a new species within the genus Stieleria (following the well-established thresholds for delineation of new species of 98.7% and new genera of < 94.5% (Yarza et al. 2014)). However, the use of the 16S rRNA gene as sole phylogenetic marker is not always reliable for phylogenetic inference in the phylum Planctomycetes (Kallscheuer et al. 2020a; Kohn et al. 2020b; Wiegand et al. 2020b). Re-analysis of the phylogenetic position of both “Roseiconus” species using other phylogenetic markers is thus envisaged, ideally before the names of the genus “Roseiconus” and the proposed species belonging to the genus are validly published.

Family Lacipirellulaceae

The current family Lacipirellulaceae comprises 8 genera and 13 species (Table 1 and 4). Bythopirellula goksoeyrii Pr1dT (initially described as “Bythopirellula goksoyri”), was the first identified member of this family (Storesund and Ovreas 2013, 2021), however, the type genus is Lacipirellula and the type species L. parvula (Dedysh et al. 2020c).

Table 4 Main features of the described species of family Lacipirellulaceae
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Most known members of this family were isolated in marine habitats, either on natural (algae, wood, sediments) or artificial (polyethylene) surfaces (Fig. 3). Dedysh and collaborators (Dedysh et al. 2020c) also demonstrated that several members are preferably found in low-oxygen aquatic habitats, which is corroborated by the isolation and detection of strains in sites such as wastewater treatment plants, hydrothermal vents and the gut microbiome of some aquatic invertebrates (Supplementary Table 1a). The genus Lacipirellula is the sole genus of the family to be exclusively found in freshwater habitats.

Members of this family are overall pear-shaped/ovoid/ellipsoidal and cells are mostly observed in aggregates (Supplementary Table 1b), although rosettes were additionally found in L. parvula PX69T and Posidoniimonas corsicana KOR34T (Dedysh et al. 2020c; Wiegand et al. 2020a). No stalks were observed. Most members produce fibrous extracellular materials or a holdfast structure which helps in cell aggregation. Aeoliella mucimassa Pan181T was described as forming very fibrous materials and slime (Wiegand et al. 2020a) and Bythopirellula polymerisocia Pla144T was found to be able to attach to polymeric material (Wiegand et al. 2020a). Pirellulimonas nuda Pla175T differs from its relatives by an uncommon absence of matrix or fibers (Wiegand et al. 2020a). Members from this family are either unpigmented/white or hot pink/red pigmented, with currently no orange-pigmented species.

In general, Lacipirellulaceae are aerobic, mesophilic, neutrophilic and heterotrophic, with the exception of L. parvula PX69T which is microaerobic and facultatively anaerobic (Supplementary Table 1c). This family has the highest mean doubling time (43.0 h ± 26.6 h) of the class Planctomycetia with the lowest doubling time of 17 h of Pseudobythopirellula maris Mal64T and the highest of B. polymerisocia Pla144T(94 h). The fatty acids C16:1ω9c, palmitic acid (C16:0), oleic/elaidic acid (C18:1ω9), palmitoleic acid (C16:1ω7c) and stearic acid (C18:0) are the major ones found in this family, although a chemotaxonomic analysis was not presented for most members. The same is true for the polar lipid content: the only compound detected until now was dimethylphosphatidylethanolamine in L. parvula PX69T (Dedysh et al. 2020c).

Although the family Lacipirellulaceae was only recently introduced, the biotechnological potential of its members is already starting to be unveiled: antimicrobial activity was recently detected in L. parvula PX69T (Belova et al. 2020). Similar to Pirellulaceae and Planctomycetaceae, Lacipirellulaceae are present in complex habitats rich in microbial diversity, and we can hypothesize that production of antimicrobial metabolites by these organisms can give them advantage to survive in such environments.

The average genome size in this family is 6.1 ± 0.8 Mb and it ranges from 4.30 to 6.83 Mb. The average DNA G + C content is 62.1 ± 5.3 mol% and it ranges from 52.8 to 66.7 mol% (Supplementary Table 1d). The presence of a plasmid was only reported in one member, L. parvula PX69T (Dedysh et al. 2020c).

Family Thermoguttaceae

The family Thermoguttaceae is underrepresented in terms of isolated strains and is constituted by only 2 genera and 3 species (Table 1 and 5). The genus Thermogutta includes two species, T. terrifontis and T. hypogea (Slobodkina et al. 2015). An additional strain of this genus that was initially designated “Thermopirellula anaerolimosa” VM20-7 was isolated from an anaerobic sludge blanket and described as an obligate anaerobic hydrogen-producing thermophilic bacterium (Liu et al. 2012). However, the genus ‘Thermopirellula’ was not formally described and thus the strain was added as a member of the species T. terrifontis (Slobodkina et al. 2015). The genus Thermostilla has only one species, T. marina (Slobodkina et al. 2016).

Table 5 Main features of the described species of family Thermoguttaceae
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Members of this family are coccoid to ellipsoidal, form aggregates and are motile. No pink or orange pigmentation was observed in this family, as all members are white/cream pigmented (Supplementary Table 1b).

They can be found in extreme environments (such as hot springs, hydrothermal vents, high-temperature horizons) and deep-subsurface sites (such as gold mines) and were also detected in anaerobic reactors (Fig. 3b and Supplementary Table 1a).

All current members are thermophilic, facultatively anaerobic and capable of microaerobic growth, neutrophilic, halotolerant and chemoorganotrophic (Supplementary Table 1c). Palmitic acid (C16:0), stearic acid (C18:0) and eicosanoid acid (C20:0) are the major fatty acids in the family and T. marina produces an additional fatty acid (11-methyl C18:0) (Slobodkina et al. 2016). Data on polar lipid and respiratory quinone content is currently not available.

Only one member (Thermogutta terrifontis R1T) has a genome sequence available for further analyses: with 4.8 Mb, the genome is relatively small and no plasmids are present (Supplementary Table 1d). The average DNA G + C content between the three current members is 60.8 ± 0.8 mol% and the range is from 57.3 to 66.6 mol%.

Family Isosphaeraceae

The family Isosphaeraceae is constituted by 6 genera and 13 published species (Table 6).

Table 6 Main features of the described species of family Isosphaeraceae
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The type genus Isosphaera was the first described genus of the family. The type species is Isosphaera pallida (Giovannoni et al. 1987).

Members of this family (Supplementary Table 1b) have spherical cells which are found singly, in pairs, aggregates or even chains (filaments). Cells connected by chains were observed in Paludisphaera borealis PX4T, Tundrisphaera lichenicola P12T and Isosphaera pallida 1S1BT (Giovannoni et al. 1987; Kulichevskaya et al. 2017b, 2016). I. pallida 1S1BT is the only planctomycete that forms long filaments with often more than one hundred cells (Giovannoni et al. 1987). No stalk-like structures were observed in members of this family. Cells are non-motile and colonies pink pigmented while Singulisphaera acidiphila MOB10T and “Singulisphaera mucilagenosa” Z0071T are the sole unpigmented/milky-yellow-pigmented members of the family. Distinctive characteristics are observed in some species, such as phototactic gliding motility in I. pallida 1S1BT (Giovannoni et al. 1987) and the tendency to attach strongly to plastic surfaces for Tautonia plasticadhaerens ElPT, a characteristic that justifies its name (Jogler et al. 2020).

Members of this family are primarily found in freshwater and terrestrial environments (Fig. 3). The majority of the isolated strains were retrieved from boreal regions in Russia and acidic wetlands (Fig. 4), while other species such as I. pallida and T. sociabilis were isolated from extreme environments such as hot springs (Giovannoni et al. 1987) and a deep subsurface environment (gold mine) (Kovaleva et al. 2019), respectively. The genus Tautonia is the only genus of the family with isolates from marine environments, in this case macroalgae biofilms and sediments (Jogler et al. 2020).

Members of this family are heterotrophic, neutrophilic, mesophilic and aerobic, although some members such as P. borealis and Singulisphaera spp. are also capable of microaerobic growth (Supplementary Table 1c). As most members were isolated from cold regions and acidic wetlands, most species are also acid-tolerant and psychrotolerant, while others isolated from extreme environments are thermotolerant, which is the case for I. pallida 1S1BT and T. sociabilis (Giovannoni et al. 1987; Kovaleva et al. 2019). Almost all known members are salt-sensitive, with the exception of the strains isolated from marine environments belonging to the genus Tautonia (Gaurav et al. 2021; Jogler et al. 2020). The average doubling time in the family is 29.9 ± 8.0 h and Aquisphaera giovannonii OJF2T has the highest doubling time (48 h) of the family, although this data is not available for most of the other family members. The major fatty acids in the family are oleic/elaidic acid (C18:1ω9), palmitic acid (C16:0) and stearic acid (C18:0). Members of the genus Singulisphaera produce an additional rare fatty acid: C18:2ω6c,12c. The main polar lipids are phosphatidylcholine, phosphatidylglycerol, trimethylornithine and phosphocholine.

The average genome size in this family is 8.3 ± 1.7 Mb, the average DNA G + C content is 64.1 ± 6.0 mol% (the highest in the class) and almost all known members possess more than one plasmid, which is very distinctive in comparison to the other families. The I. pallida type strain IS1BT has the smallest genome of the family (5.4 Mb) and A. giovannonii OJF2T the largest one (10.4 Mb).

Family Gemmataceae

The family Gemmataceae contains 9 genera and 11 species, which are listed in Table 7. Gemmata was the first identified genus with the description of G. obscuriglobus UQM 2246 T (Franzmann and Skerman 1984).

Table 7 Main features of the described species of family Gemmataceae
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Gemmataceae are mostly spherical and can be found singly and more often in shapeless aggregates, although some species can assemble in rosettes or in short chains, such as the cells of Frigoriglobus tundricola PL17T (Kulichevskaya et al. 2020a). Stalk-like structures are observed in some species, namely Limnoglobus roseus PX52T, Telmatocola sphagniphila SP2T and Zavarzinella formosa A10T. All known members are pink/red-pigmented. The cytoplasmic membrane invaginations (a characteristic cell biological features of planctomycetes) can be re-arranged in an even more complex way in some Gemmataceae (Sagulenko et al. 2014), which is the case of G. obscuriglobus, which shows a very complex cytoplasmic membrane invagination system (Santarella-Mellwig et al. 2013). In fact, several studies regarding cell structure, division and genetic manipulation have focused on this species (Boedeker et al. 2017; Jeske et al. 2015; Rivas-Marin et al. 2016b; Sagulenko et al. 2014; Sagulenko et al. 2017). A tubulo-vesicular network was also reported in G. obscuriglobus, which is unique among prokaryotes and reveals similarities with the endocytosis that is exclusively associated with eukaryotes (Acehan et al. 2014). Taken together, these studies demonstrated how complex and unique it is the Planctomycetes cell biology. Other distinctive morphotypes are also seen in this family, such as the cells from T. sphagniphila SP2T which cluster in unique dendriform-like structures (Kulichevskaya et al. 2012b). Z. formosa A10T is the only taxon of the family forming a holdfast structure, which supports aggregation in rosettes besides the formation of abnormally thick stalk-like structures (Kulichevskaya et al. 2009).

Known members of the family are found in freshwater and terrestrial environments (Fig. 3). “G. massiliana” IIL30T was initially isolated from a water network in a hospital (Aghnatios et al. 2015) and consequently Gemmata-like organisms have been hypothesized as possible opportunistic human pathogens (Aghnatios and Drancourt 2016), however, this still remains to be clarified (Wiegand et al. 2018).

Thermogemmata fonticola 2918 T is the only Gemmataceae member isolated from a terrestrial hot spring (Elcheninov et al. 2020). Furthermore, uncultured strains belonging to this species were detected in other hot springs (including the radioactive Paralana hot spring in Australia), in sediments from an U.S. Department of Energy contaminated site (Abulencia et al. 2006) and associated with the marine invertebrate Pocillopora meandrina found in the Pacific Ocean.

The family comprises, in general, heterotrophic, mesophilic, neutrophilic and aerobic organisms, however, a small number of members are also psychrotolerant (F. tundricola and T. sphagniphila) or thermophilic (T. fonticola) or acid tolerant (e.g. Frigoriglobus ruber SP5T and T. sphagniphila) (Supplementary Table 1c). All the isolated strains are halophobic. The average doubling time in the family is 21.8 ± 14.5 h, however, this data is not available for all members. Strain MBLW1T of the species Tuwongella immobilis has a low doubling time (6 h) in comparison to the other family members (Seeger et al. 2017). The main fatty acids in the family are stearic acid (C18:0), C18:1ω5c and C16:1ω5c. In T. fonticola 2918 T, C20:0 fatty acids were also detected. The fatty acid composition in F. ruber SP5T differs from the other family members as it includes C20:1ω9c, C16:1ω9c and C16:0 as the major fatty acids. In this family, the fatty acid C18:1ω7c is produced in major amounts by only L. roseus PX52T and the uncommon fatty acid (βOH-C16:1) by F. tundricola PL17T. The main polar lipid is trimethylornithine, although others such as phosphatidylglycerol, dimethylphosphatidylethanolamine, monomethylornithine and dimethylornithine can be present.

The average genome size in this family is approximately 9.1 ± 2.2 Mb (among the highest in the class). The largest genome of the class Planctomycetia (and even of the entire phylum) belongs to F. ruber SP5T (12.4 Mb), while T. fonticola 2918 T has a small genome (4.81 Mb) compared to the average size in the family (9.1 Mb). The average DNA G + C content is 63.5 ± 4.6 mol% and Urbifossiella limnaea ETA_A1T has the highest DNA G + C content of the class Planctomycetia (71.3 mol%). F. tundricola PL17T is the only member of the family that possesses a plasmid.

Conclusions

In this overview article, the clustered data obtained from species description studies dealing with members of the class Planctomycetia, allowed for a more holistic view and comparison of phylogeny, ecology, morphology, physiology, and genomic traits of the cultured taxa in this group. Shared as well as distinctive (or even unique) features of the different families are presented and discussed. This overview showed the importance of a complete characterization of the novel taxonomic groups, namely genomic information, chemotaxonomy and physiology of the strains, to allow a better discrimination between taxa.

The last decades were a great momentum for the discovery of planctomycetes and in particular of the class Planctomycetia. However, the planctomycetal scientific community faces the great challenge of bringing into culture much of the biodiversity that still stays beyond our capacity of isolation. The bias in the number of isolated strains towards the class Planctomycetia and specifically towards mesophilic, neutrophilic and aerobic members may be due to several factors such as the uniform isolation techniques and growth conditions, the media formulation used, the use of selective factors like N-acetylglucosamine as the only source of carbon and nitrogen and also our unawareness of the nutritional and metabolic requirements of so far non-culturable planctomycetes. In fact, the media developed for isolation are based on the knowledge gathered on the nutritional needs of the isolated strains which are mainly from the class Planctomycetia. As the majority of the isolated planctomycetes are resistant to betalactam antibiotics and streptomycin, a mixture of these antibiotics is currently used to achieve an enrichment of planctomycetes, making it impossible to isolate non-resistant planctomycetes. Furthermore, most planctomycetes seem to be resistant to most classes of antibiotics, however, the mechanisms behind these resistances are still unknown. It is hypothesized that many of the antimicrobial resistance genes in pathogens are obtain via horizontal gene transfer events from environmental bacteria, which is why it is also important to gather knowledge on the resistome-mobilome of highly resistant bacteria such as the planctomycetes.

To overcome the isolation difficulties here discussed, new strategies are needed to bring the untapped diversity of uncultured clades of planctomycetes into culture. This can possibly by achieved by the exploitation of less investigated ecological niches and by focusing on different culture conditions (such as anaerobic growth) and testing new medium formulations (e.g. by incorporating nutrients from the isolation environments or the use of other selective antibiotics). The development and refinement of novel isolation techniques is also essential, such as the use of in-situ methodologies, which recently proved to be useful to isolate novel taxa. As many strains have long doubling times, it is always needed to wait for a long period to allow slow growing planctomycetes to appear in culture. Another approach should be the dilution of the initial inoculum or its cell sorting to obtain single cell cultures per well in order to avoid completion from fast growing bacteria.

Thanks to the isolation and description of many species that lead to a taxonomic revolution in the phylum in the last years, scientists became aware of several aspects of the cell biology of this group. The availability of many planctomycetal strains and genomes will facilitate the investigation of the secrets in their cell biology. In the light of more than 40% of the genome-encoded proteins with an unknown function, their genomes are another enigmatic feature of their divergent morphology and physiology. Future studies with members of the current family Planctomycetia will contribute to understand their division mode, their resistance to antibiotics, the function behind many genes, their complex structure, the role played in many ecosystems and the biotechnological potential that starts to be discovered.