Maioricimonas rarisocia gen. nov., sp. nov., a novel planctomycete isolated from marine sediments close to Mallorca Island

Planctomycetes are ubiquitous bacteria with environmental and biotechnological relevance. Axenic cultures of planctomycetal strains are the basis to analyse their unusual biology and largely uncharacterised metabolism in more detail. Here, we describe strain Mal4T isolated from marine sediments close to Palma de Mallorca, Spain. Strain Mal4T displays common planctomycetal features, such as division by polar budding and the presence of fimbriae and crateriform structures on the cell surface. Cell growth was observed at ranges of 10–39 °C (optimum at 31 °C) and pH 6.5–9.0 (optimum at 7.5). The novel strain shows as pear-shaped cells of 2.0 ± 0.2 × 1.4 ± 0.1 µm and is one of the rare examples of orange colony-forming Planctomycetes. Its genome has a size of 7.7 Mb with a G+C content of 63.4%. Phylogenetically, we conclude that strain Mal4T (= DSM 100296T = LMG 29133T) is the type strain representing the type species of a novel genus, for which we propose the name Maioricimonas rarisocia gen. nov., sp. nov.


Introduction
Planctomycetes are bacteria that belong to the PVC superphylum (Wagner and Horn 2006), which includes the phyla Planctomycetes, Verrucomicrobia, Chlamydiae, Lentisphaerae and Kirimatiellaeota as well as some uncultured candidate phyla, such as Candidatus Omnitrophica. The PVC superphylum has environmental, medical and biotechnological relevance (Devos and Ward 2014).
Planctomycetes have been shown to be present in several environments, in which they play important roles in biogeochemical cycles, such as the carbon and nitrogen cycle (Wiegand et al. 2018). One example are Planctomycetes of the class Candidatus Brocadiae, which perform unique reactions during anaerobic ammonium oxidation (anammox) (Strous et al. 1999;Peeters and van Niftrik 2019). Members of the phylum Planctomycetes, in particular of the class Planctomycetia, colonise a variety of environments from terrestrial to aquatic, being able to dwell on various marine algal surfaces (Bengtsson et al. 2012;Bondoso et al. 2014Bondoso et al. , 2015Bondoso et al. , 2017Lage and Bondoso 2014;Vollmers et al. 2017). They form biofilms on biotic surfaces (Bengtsson and Øvreås 2010), on which they metabolise complex carbon substrates (Lachnit et al. 2013;Jeske et al. 2013). Unique pili-forming crateriform structures and an enlarged periplasm are probably required for uptake and also cleavage of large polysaccharides obtained from the environment .
Planctomycetes possess large genomes with sizes of up to 12.4 Mb (Ravin et al. 2018), in which the presence of giant genes has been reported (Jeske et al. 2013;Guo et al. 2014;Kohn et al. 2016;Faria et al. 2018). These genome sizes are in line with their assumed capacity for secondary metabolite production (Graça et al. 2016;Jeske et al. 2016;Yadav et al. 2018). Furthermore, several members of the phylum Planctomycetes produce carotenoids, which could be associated with an increased tolerance against UV radiation or oxidative stress (Kallscheuer et al. 2019b).
Planctomycetes were considered exceptional due to several presumptively eukaryotic features, such as the lack of a peptidoglycan (König et al. 1984), a compartmentalised cell plan (Lindsay et al. 1997), a nucleus-like structure (Fuerst and Webb 1991) and the endocytosis-like uptake of macromolecules for an intracellular degradation (Lonhienne et al. 2010). However, with advances of microscopy techniques and the development of genetic tools (Jogler et al. 2011;Rivas-Marín et al. 2016b;Boedeker et al. 2017), many of these traits have been refuted or reinterpreted.
In recent years, the presence of peptidoglycan has been reported in several members of the Planctomycetes (Jeske et al. 2015;van Teeseling et al. 2015) and also in the sister phyla Verrucomicrobia  and Chlamydiae (Pilhofer et al. 2013;Liechti et al. 2014Liechti et al. , 2016. With the exception of anammox-performing Planctomycetes (Jogler 2014;Neumann et al. 2014), the proposed cell plan has been found to feature large invaginations of the cytoplasmic membrane instead of closed compartments (Santarella-Mellwig et al. 2013;Acehan et al. 2014;Boedeker et al. 2017). These discoveries contributed to the reinterpretation of Planctomycetes as bacteria with a cell envelope architecture resembling that of Gram-negative bacteria, but with some variations (Devos 2014a, b;Boedeker et al. 2017). Nevertheless, Planctomycetes remain exceptional in other ways, e.g. they lack the protein FtsZ normally essential for bacterial division as well as other division proteins (Pilhofer et al. 2008;Jogler et al. 2012;Rivas-Marín et al. 2016a). Beyond that, phylum members divide by binary fission, budding or intermediate mechanisms (Wiegand et al. 2018. Presence and essentiality of sterols in the membranes of one of its members was recently reported (Pearson et al. 2003;Rivas-Marin et al. 2019).
The unusual cell biology of Planctomycetes prompted us to explore the uncharacterised planctomycetal diversity. In the present study, we describe the novel strain Mal4 T isolated from marine sediments in Palma de Mallorca (Spain) in terms of physiological, microscopic as well as genomic properties. Supported by phylogenetic analyses, we conclude that strain Mal4 T represents a novel species of a novel genus within the family Planctomycetaceae.

Cultivation conditions and isolation
Strain Mal4 T was isolated from marine sediments at the coast of S'Arenal close to Palma de Mallorca (Spain) on the 23th of September 2014 (sampling location: 39.5126 N 2.7470 E) as previously described . For strain isolation and cultivation M1H NAG ASW medium was used. Medium preparation was previously described (Kallscheuer et al. 2019a). Cultures were incubated in baffled flasks at 28°C with constant agitation at 110 rpm. Plates were cultivated at 28°C for 2-3 weeks and isolated colonies were then streaked on fresh M1H NAG ASW plates. Initial amplification and sequencing of the 16S rRNA gene, intended to check whether isolated strains are members of the phylum Planctomycetes, was performed as previously described ).

Genome analysis
The genome of strain Mal4 T was previously published . The genome (accession number CP036275) and 16S rRNA gene sequence (accession number MK559979) are available from the GenBank database. The primary metabolism was analysed by examining locally computed InterProScan (Mitchell et al. 2019) results cross-referenced with information from the UniProt database (UniProt 2019) and BlastP results of 'typical' protein sequences. Numbers of carbohydrate-active enzymes were determined by employing dbCAN2 (Zhang et al. 2018), which automatically mines the CAZy database (Lombard et al. 2014).

Light microscopy and scanning electron microscopy
Phase contrast microscopy and scanning electron microscopy were performed according to protocols published earlier (Kallscheuer et al. 2019a).

Phylogenetic analyses
16S rRNA gene sequence-based phylogeny was computed for strain Mal4 T , the type strains of all described planctomycetal species (assessed in January 2020) and all isolates recently published (Kohn et al. 2016(Kohn et al. , 2020aBoersma et al. 2019;Kallscheuer et al. 2019a;Dedysh et al. 2020;Wiegand et al. 2020). An alignment of 16S rRNA gene sequences was performed with SINA (Pruesse et al. 2012). The phylogenetic analysis was conducted employing a maximum likelihood approach with 1000 bootstraps, the nucleotide substitution model GTR, gamma distribution and estimation of proportion of invariable sites (GTRGAMMAI option) (Stamatakis 2014). Three 16S rRNA genes of bacterial strains from the PVC superphylum, but outside of the phylum Planctomycetes, were used as outgroup. The rpoB nucleotide sequences (encoding the RNA polymerase bsubunit) were taken from publicly available genome annotations and the sequence identities were determined as described previously (Bondoso et al. 2013) using Clustal Omega (Sievers et al. 2011). Alignment and matrix calculation were done after extracting only those parts of the sequence that would have been sequenced with the described primer set. The average nucleotide identity (ANI) was calculated using OrthoANI (Lee et al. 2016). The average amino acid identity (AAI) was obtained with the aai.rb script of the enveomics collection (Rodriguez-R and Konstantinidis 2016). The percentage of conserved proteins (POCP) was calculated as described before (Qin et al. 2014). The unique single-copy core genome of all analysed genomes for the multi-locus sequence analysis (MLSA) was determined with proteinortho5 (Lechner et al. 2011) ('selfblast' option enabled). The sequences of the obtained orthologous groups were aligned using MUSCLE v.3.8.31 (Edgar 2004). After clipping, partially aligned C-and N-terminal regions and poorly aligned internal regions were filtered using Gblocks (Castresana 2000). The final alignment was concatenated and clustered using the maximum likelihood method implemented by RaxML (Stamatakis 2014) with the 'rapid bootstrap' method and 500 bootstrap replicates.

Phylogenetic inference
In the phylogenetic trees obtained after analysis of 16S rRNA gene sequences, as well as MLSA (Fig. 1), strain Mal4 T clusters stably with members of two genera of the family Planctomycetaceae, namely Planctomicrobium and Thalassoglobus. 16S rRNA gene sequence identity between strain Mal4 T and the two genera is between 91.4% and 91.9% (Fig. 2). These values are below the proposed genus threshold of 94.5%, but above the threshold for separate families of 86.5% (Yarza et al. 2014), indicating that strain Mal4 T represents an distinct genus in the family Planctomycetaceae. Coherently, average nucleotide identities (ANI) below 95% confirm that strain Mal4 T is a distinct species. Phylogenetic assumptions on the genus level can also be obtained by analysing the rpoB gene sequence identities, AAI and POCP. For delineation of genera, the proposed threshold values for the above-mentioned markers are 75.5-78% (Kallscheuer Bootstrap values from 1000 re-samplings (500 re-samplings for MLSA) are given at the nodes (in %) et al. 2019c), 45-65% (Konstantinidis et al. 2017) and 50% (Qin et al. 2014), respectively. The rpoB identity value and the AAI between strain Mal4 T and the members of the genus Thalassoglobus, which comprises Thalassoglobus neptunius KOR42 T (Kohn et al. 2020a) and Thalassoglobus polymorphus Mal48 T (Rivas-Marin et al. 2020), are below the given thresholds. POCP was found to be slightly above the threshold (51.2%), this, however, does not significantly influence the overall conclusion that strain Mal4 T belongs to a separate genus. Minimal comparative values of strain Mal4 T and the genus Rubinisphaera, another closely related genus; featuring Rubinisphaera italica (Kallscheuer et al. 2019a) and Rubinisphaera brasiliensis (Scheuner et al. 2014), are below these thresholds for all three phylogenetic markers (Fig. 2). Analogously, POCP between strain Mal4 T and Planctomicrobium piriforme P3 T (Kulichevskaya et al. 2015) was also found to fall below the proposed threshold (Fig. 2), whilst the AAI value (56%) was in a 'grey zone' (45-65%), but well below the upper limit. Although the rpoB gene sequence identity of 78.6% is slightly above the proposed threshold, this sole deviance should not overrule the distinctiveness of the other values. In summary, the majority of analysed phylogenetic markers suggests that strain Mal4 T belongs to a novel genus.

Morphological and physiological analyses
Light microscopy and scanning electron microscopy ( Fig. 3) were applied to analyse the morphological characteristic of Mal4 T cells harvested during the exponential growth phase. Detailed information on morphology and cell division is summarised in comparison to the current closest relatives (Table 1). Mal4 T cells are pear-shaped (2.0 ± 0.2 lm 9 1.4 ± 0.1 lm) (Fig. 3a-c), occur as single cells and in rare cases form aggregates (Fig. 3d). The cell surface appears rough, evenly covered with crateriform structures and short fimbriae (Fig. 3d, e). A holdfast structure was not observed during electron microscopy. As shown for all described members of the family Planctomycetaceae, cell division takes place by polar budding with the daughter cell displaying a round shape. Optimal temperature and pH for growth were shown to be 31°C and pH 7.5, respectively, however, Mal4 T cells are able to grow over a range of 10-39°C and pH 6.5-9.0 (Fig. 4). These values are comparable to the two Thalassoglobus species, but differ from P. piriforme P3 T , which did not grow at temperatures exceeding 30°C and favours moderate acidic conditions. The maximal observed growth rate of strain Mal4 T in M1H NAG ASW medium was determined to be 0.041 h -1 , corresponding to a generation time of approximately 17 h. Strain Mal4 T is amongst the rare examples of planctomycetal strains forming orange colonies and might thus be an interesting strain for further analysis of carotenoid production and their function in Planctomycetes. Since most of the planctomycetal strains characterised so far are either pink to red or lack pigmentation (white), the pigmentation of the novel strain is an important phenotypic feature separating it from its current close phylogenetic neighbours. Strain Mal4 T is an aerobic heterotroph.

Genomic characteristics
The genomic characteristics of strain Mal4 T in comparison to T. polymorphus Mal48 T , T. neptunius KOR42 T and P. piriforme P3 T are summarised in Table 1. Its genome is 7.7 Mb in size, which is around 1 Mb larger compared to the other three strains. The G?C content is also the highest of the four strains. Automated gene prediction and annotation identified 5829 putative protein-encoding genes, of which 39% (2257 genes) are annotated as hypothetical proteins. These values correspond to 753 protein-coding genes per Mb and a coding density of 85.9%. Although the genome size of strain Mal4 T is larger, the coding density is in the same range in the other three species. Similar to its relatives, the strain lacks plasmids. Numbers of 41-55 tRNA genes are similar, except for T. neptunius KOR42 T , which has a slightly higher number of 70 tRNA genes. As for T. polymorphus Mal48 T , strain Mal4 T harbours two copies of the 16S rRNA gene, whereas the gene occurs in single copy in the other two strains. pH range (optimum) 6.5-9.0 (7.5) 6.5-8.0 (7.5) 5.5-8.5 (7.0-7.5) 4.2-7.1 (6.0-6.5) Aggregates Yes  (Table 2). Genes coding for enzymes participating in glycolytic pathways, gluconeogenesis, the tricarboxylic acid (TCA) cycle and anaplerotic reactions, such as pyruvate or phosphoenolpyruvate carboxylation and the glyoxylate shunt, were included. The resulting data suggest that strain Mal4 T is able to metabolise carbohydrates using at least two glycolytic pathways, the Embden-Meyerhof-Parnas pathway (the most common glycolytic pathway) and the pentose phosphate pathway. Additionally, its genome bears genes coding for putative 2-dehydro-3-deoxyphosphogluconate aldolase and phosphogluconate dehydratase, both involved in the alternative Entner-Doudoroff pathway. All four strains harbour a complete gene set required for a functional TCA cycle, which suggests that the central carbon metabolism of the strains is similar to canonical heterotrophic bacteria. With regard to gluconeogenesis, a minimal gene set required for this pathway has been identified, suggesting that the three strains are capable of de novo sugar biosynthesis. All four strains lack the glyoxylate shunt, which is typically required during growth either with acetate or with compounds that are degraded to acetate or acetyl-CoA units. The lack of the glyoxylate shunt suggests that the strains are not able to use such compounds as the exclusive energy and carbon source. Alternatively, they may follow a different pathway with a similar function.
Based on the physiological, morphological and phylogenetic analyses of strain Mal4 T , we conclude that the characterised strain represents a novel species within the novel genus Maioricimonas. Thus, we propose the name Maioricimonas rarisocia gen. nov., sp. nov., represented by the type strain Mal4 T .
Cells have a Gram-negative cell envelope architecture and divide by polar budding. Cells are mesophilic, neutrophilic, aerobic and heterotrophic and present crateriform structures and matrix or fimbriae. The genus is part of the family Planctomycetaceae, order Planctomycetales, class Planctomycetia, phylum Planctomycetes. The type species of the genus is Maioricimonas rarisocia.
In addition to the features described for the genus, cells are pear-shaped (2.0 9 1.4 lm), form orange colonies and mostly occur as single cells. Temperature and pH optimum of the type strain are 31°C and 7.5, respectively, however growth is observed over a range  Author contributions E.R.M. wrote the manuscript, analysed the data and prepared the figures, S.W. and M.J. performed the genomic and phylogenetic analysis, A.H. isolated the strain and performed the initial strain cultivation and deposition, S.H.P. and C.B. performed the light microscopic analysis, N.K. and M.S.M.J. contributed to text preparation and revised the manuscript, M.R. performed the electron microscopic analysis, C.J. took the samples, supervised A.H. and the study. All authors read and approved the final version of the manuscript.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical statement This article does not contain any studies with animals performed by any of the authors.
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