Rubinisphaera italica sp. nov. isolated from a hydrothermal area in the Tyrrhenian Sea close to the volcanic island Panarea

Planctomycetes is a fascinating phylum of mostly aquatic bacteria, not only due to the environmental importance in global carbon and nitrogen cycles, but also because of a unique cell biology. Their lifestyle and metabolic capabilities are not well explored, which motivated us to study the role of Planctomycetes in biofilms on marine biotic surfaces. Here, we describe the novel strain Pan54T which was isolated from algae in a hydrothermal area close to the volcanic island Panarea in the Tyrrhenian Sea, north of Sicily in Italy. The strain grew best at pH 9.0 and 26 °C and showed typical characteristics of planctomycetal bacteria, e.g. division by polar budding, formation of aggregates and presence of stalks and crateriform structures. Phylogenetically, the strain belongs to the genus Rubinisphaera. Our analysis suggests that Pan54T represents a novel species of this genus, for which we propose the name Rubinisphaera italica sp. nov. We suggest Pan54T (= DSM 29369 = LMG 29789) as the type strain of the novel species.


Introduction
Together with Verrucomicrobia, Lentisphaerae, Kirimatiellaeota and Chlamydiae, Planctomycetes form the medically and biotechnologically relevant PVC superphylum (Spring et al. 2016;Wagner and Horn 2006;Devos and Ward 2014). In the past, Planctomycetes were postulated as the missing link between bacteria and eukaryotes (Devos and Reynaud 2010) beyond the bacterial cell plan (Devos et al. 2013;Fuerst and Sagulenko 2011). This finding was based on proposed exceptional planctomycetal features, such as, lack of peptidoglycan (König et al. 1984), a compartmentalised cell plan (Lindsay et al. 1997), a nucleus-like structure (Fuerst and Webb 1991) and performance of endocytosis (Lonhienne et al. 2010). Further investigation of the planctomycetal physiology and morphology based on the advent of novel techniques changed this picture (Jogler et al. 2011;Jogler and Jogler 2013;Rivas-Marin et al. 2016). In particular, presence of peptidoglycan in some Planctomycetes was confirmed (Jeske et al. 2015;van Teeseling et al. 2015) and thus the cell plan of Planctomycetes was reinterpreted to be Gram-negative Devos 2014a, b). But still, Planctomycetes remain exceptional and enigmatic in comparison to well-characterised canonical bacteria. They e.g. divide unusually, either by budding, binary fission or even a combination of both (Wiegand et al. 2019) and lack canonical divisome proteins including the otherwise universal FtsZ (Jogler et al. 2012;Pilhofer et al. 2008).
Many Planctomycetes survive in oligotrophic environments, such as seawater, by utilising a range of highmolecular-weight sugars derived from algae (Jeske et al. 2013;Lachnit et al. 2013) after attaching to these nutrient-rich surfaces (Bengtsson et al. 2012;Bondoso et al. 2015Bondoso et al. , 2014Bondoso et al. , 2017Lage and Bondoso 2014;Vollmers et al. 2017). In this context, a specialised morphology including the unique pili-forming crateriform structures and an extremely enlarged periplasm might be involved in the uptake and cleavage of such polymeric compounds .
It is frequently observed that Planctomycetes are highly abundant in biofilms on nutrient-rich marine surfaces (Bengtsson and Øvreås 2010;Kohn et al. 2019b). This is astonishing when considering their moderate growth rates compared to faster-growing competitors in this ecological niche (Frank et al. 2014;Wiegand et al. 2018). It is thus likely that Planctomycetes are 'talented' producers of secondary metabolites, which could mediate (symbiotic) interactions with algae or act as antibiotics (Jeske et al. 2013). Gene clusters involved in small molecule production were predicted in planctomycetal genomes, which substantiates Planctomycetes as a promising source of such bioactive compounds (Graca et al. 2016;Wiegand et al. 2019).
Taken together, Planctomycetes are amongst the most maverick of all bacteria known thus far (Wiegand et al. 2018). We are steadily aiming to expand the collection of known Planctomycetes and recently presented 79 novel strains in an overview article (Wiegand et al. 2019). Here, we introduce and validly describe Pan54 T , a novel planctomycetal strain that was isolated close to the volcanic island Panarea, and describe its morphology, physiology and phylogeny. The sampling location Panarea is located in the Tyrrhenian Sea, has an area of 3.3 km 2 and is the second smallest of the Aeolian islands. The island itself is only a small part of a sub-marine edifice in form of a truncated cone with an eastern protrusion with its base being 1500 m below sea level (Gabbianelli et al. 1990). The entire cone has a diameter of 23 km and an area of 460 km 2 and several thermal springs are located in proximity to the island. In the surroundings of Panarea areas with increased temperatures, higher levels of nutrients including nitrogen and sulfur sources are present, which motivated us to choose this geographical location as a valuable source of so far unknown species of prokaryotes (Gugliandolo et al. 2015).
Strain Pan54 T was isolated on the 10th of September 2013 from an algal surface in hydrothermal area 26 (sampling site 38.6392 N, 15.1051 E) close to the island Panarea in the north of Sicily, Italy. Algae leaves were initially washed with 0.5 9 artificial sea water (ASW) and placed on M1H ASW solid medium (lacking NAG) containing 8 g/L gelrite additionally supplemented with 20 mg/L cycloheximide, 1000 mg/ L streptomycin and 200 mg/L ampicillin. Two different pH values (6.5 and 8.0) were tested and plates were cultivated at 20°C for 2-3 weeks. Isolated colonies were then streaked on a new plate and maintained in liquid M1H NAG ASW medium. Initial amplification and sequencing of the 16S rRNA gene was performed as previously described ).

Deposition of genomic data
Genome data (acc. no. SJPG00000000) and the 16S rRNA gene sequence (acc. no. MK554545) were deposited in the GenBank database.

Light microscopy
Phase contrast (Phaco) analyses were performed employing a Nikon Eclipse Ti inverted microscope with a Nikon DS-Ri2 camera (blue LED). Specimens were immobilised in MatTek glass bottom dishes (35 mm, No. 1.5) employing a 1% agarose cushion . Images were analysed using the Nikon NIS-Elements software (version 4.3). To determine the cell size, at least 100 representative cells were counted manually (Annotations and Measurements, NIS-Elements) or by using the NIS-Elements semi-automated object count tool (smooth: 49, clean: 49, fill holes: on, separate: 49).

Electron microscopy
For field emission scanning electron microscopy (FESEM) bacteria were fixed in 1% (v/v) formaldehyde in HEPES buffer (3 mM HEPES, 0.3 mM CaCl 2 , 0.3 mM MgCl 2 , 2.7 mM sucrose, pH 6.9) for 1 h on ice and washed once employing the same buffer . Cover slips with a diameter of 12 mm were coated with a poly-L-lysine solution (Sigma-Aldrich) for 10 min, washed in distilled water and airdried. 50 lL of the fixed bacteria solution was placed on a cover slip and allowed to settle for 10 min. Cover slips were then fixed in 1% glutaraldehyde in TE buffer (20 mM TRIS, 1 mM EDTA, pH 6.9) for 5 min at room temperature and subsequently washed twice with TE buffer before dehydrating in a graded series of acetone (10, 30, 50, 70, 90, 100%) on ice for 10 min at each concentration. Samples from the 100% acetone step were brought to room temperature before placing them in fresh 100% acetone. Samples were then subjected to critical-point drying with liquid CO 2 (CPD 300, Leica). Dried samples were covered with a gold/palladium (80/20) film by sputter coating (SCD 500, Bal-Tec) before examination in a field emission scanning electron microscope (Zeiss Merlin) using the Everhart Thornley HESE2 detector and the inlens SE detector in a 25:75 ratio at an acceleration voltage of 5 kV. Transmission electron microscopy (TEM) was performed as described before (Kohn et al. 2016).

Physiological analyses
For determination of the pH optimum 100 mM HEPES was used for cultivations at pH 7.0, 7.5 and 8.0. For cultivation at pH 5.0 and 6.0 HEPES was replaced by 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), whereas 100 mM N-cyclohexyl-2aminoethanesulfonic acid (CHES) served as a buffering agent at pH 9.0 and 10.0. Cultivations for determination of the pH optimum were performed at 28°C. For determination of the temperature optimum Pan54 T was cultivated in M1H NAG ASW medium at pH 7.5 at different temperatures ranging from 10 to 40°C. Fatty acid composition of Pan54 T was analysed based on a protocol described previously (Kohn et al. 2016).

Phylogenetic analyses
The genome of strain Pan54 T was published previously (Wiegand et al. 2019) and is available from GenBank under acc. no. SJPG00000000. The Gen-Bank acc. no. of the 16S rRNA gene is MK554545. 16S rRNA gene phylogeny was computed for strain Pan54 T , the type strains of all described planctomycetal species (May 2019) and all isolates recently published (Wiegand et al. 2019). The 16S rRNA gene sequences were aligned with SINA (Pruesse et al. 2012). The phylogenetic analysis was done employing a maximum likelihood (ML) approach with 1,000 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 served as outgroup. The rpoB nucleotide sequences (encoding the RNA polymerase b-subunit) were taken from publicly available genome annotations and the sequence identities were determined as described previously (Bondoso et al. 2013) with Clustal Omega (Sievers et al. 2011) alignment and matrix calculation upon 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 gained 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).

Phylogenetic analysis
In our phylogenetic analysis Pan54 T appears in a monophyletic clade with its closest relative Rubinisphaera brasiliensis DSM 5305 T (Fig. 1). R. brasiliensis DSM 5305 T was originally isolated from a water sample of Lagoa Vermelha, a salt pit near Rio de Janeiro, Brasil, initially described as ''Planctomyces brasiliensis'' in 1989 and was later reclassified (Scheuner et al. 2014;Schlesner 1989). R. brasiliensis is currently the only validly described species within the genus Rubinisphaera. Pan54 T and R. brasiliensis DSM 5305 T share a 16S rRNA gene sequence identity of 96.2%. This value is below the threshold of 98.7% for a novel species (Stackebrandt and Ebers 2006), but above the threshold for a novel planctomycetal genus of 94.5% (Yarza et al. 2014). Both strains share 76.5% rpoB gene identity, which is below the 96.3% cutoff proposed to distinguish between different planctomycetal species and above the threshold for novel genera of 72% sequence identity (Bondoso et al. 2013). Average nucleotide identity (ANI) between both strains is 69.7% and distinctly below the species threshold of 95 -96% for ANIb (ANI calculated with the BLAST algorithm) (Kim et al. 2014). The average amino acid identity (AAI) between Pan54 T and its relative R. brasiliensis DSM 5305 T is 58.1% and thus fits in the range for species belonging to the same bacterial genus (Rodriguez et al. 2018). Finally, the percentage of conserved proteins (POCP) of 62.3% between both strains also indicates that they belong to the same genus, as a prokaryotic genus is proposed to be formed by a group of species with pairwise POCP values higher than 50% (Qin et al. 2014). Close relatives apart from R. brasiliensis DSM 5305 T include Planctomicrobium piriforme P3 T (Kulichevskaya et al. 2015), Gimesia maris DSM 8797 T (Scheuner et al. 2014), Fuerstiella marisgermanici NH11 T (formerly designated ''Fuerstia marisgermanicae'') (Kohn et al. 2019a(Kohn et al. , 2016 and species of the Planctopirus genus (Fig. 1).

Morphological, physiological and biochemical analyses
For a morphological characterization, Pan54 T cells were harvested during the exponential growth phase. Pan54 T cells were found to be pear-shaped (1.6 ± 0.2 lm 9 0.8 ± 0.1 lm) (Fig. 2a-c) and form strong aggregates and biofilms. Cells have a textured surface and contain evenly distributed crateriform structures (Fig. 2d, e). Pan54 T divides by polar budding (Fig. 2a). Daughter cells have the same shape as mother cells. Thin sections of Pan54 T cells show a condensed nucleoid and cytoplasmic invaginations (Fig. 2f, g). Colonies are white indicating a lack of carotenoids as pigmenting compounds. Detailed information on morphology, locomotion and cell division is summarised in Table 1. Pan54 T has a very similar morphology as R. brasiliensis. In the direct comparison Pan54 T has a slightly elongated shape and crateriform structures appear to be more pronounced. Similar to R. brasiliensis rosette formation was observed rather than formation of larger aggregates (Scheuner et al. 2014). The colony colour of non-pigmented Pan54 T differs from the yellow to orange pigmentation of R. brasiliensis.
In physiological experiments, Pan54 T grew at a temperature range of 14-27°C (Fig. 3a) and a pH range of 6.0-10.0 (Fig. 3b), but failed to grow at 30°C or above (Fig. 3a). The optimal conditions turned out to be pH 9.0 and 26°C. The observed temperature optimum of Pan54 T is lower compared to R. brasiliensis (30-33°C) (Schlesner 1989). R. brasiliensis even grows at temperatures of 37°C (Schlesner 1989), while Pan54 T failed to grow at 30°C or higher. Taken together, growth of Pan54 T is mesophilic and slightly alkaliphilic. A maximal growth rate of 0.039 h -1 was observed under the given conditions corresponding to a generation time of 18 h (Fig. 3). This value is in the range of 0.01-0.09 h -1 (generation times of 8-70 h), which we typically observed for planctomycetal strains isolated and characterised in our lab so far.

Genomic characteristics
The genome of Pan54 T has a size of 6.7 Mb and is slightly larger compared to R. brasiliensis (6.0 Mb), whereas the GC content is lower (48.8% for Pan54 T , 56.4% for R. brasiliensis). Relevant genome characteristics are summarised in Table 1. Automated gene The ''Thermophilic clade'' comprises the genera Thermostilla, Thermogutta and Thermopirellula prediction and annotation identified 5275 putative protein-encoding genes, of which 42% (2229 genes) are annotated as hypothetical proteins. The calculated values correspond to 787 protein-coding genes per Mb and a coding density of 85%. Except for differences in GC content no striking differences between genome features of Pan54 T and R. brasiliensis DSM 5305 T were observed (Table 1). The number of 16S rRNA in both strains is identical and number of tRNAs is very similar.

Conclusion
Based on the data collected during strain characterisation, Pan54 T represents a novel species within the genus Rubinisphaera. We propose the name Rubinisphaera italica sp. nov. and present Pan54 T as the type strain of the species.
Emended description of the genus Rubinisphaera Scheuner et al. (2014) The description of the genus Rubinisphaera given previously (Scheuner et al. 2014), with the following modification: The GC content is between 48 and 57%.
Description of Rubinisphaera italica sp. nov.
Rubinisphaera italica (i.ta'li.ca. L. fem. adj. italica of Italy; corresponding to the isolation of the strain from Italy). Cells are pear-shaped (length: 1.6 ± 0.2 lm, Fig. 2 Microscopy images and cell size plot of Pan54 T . Pictures from light microscopy (LM) (a, b), scanning electron microscopy (SEM) (d, e) and transmission electron microscopy (TEM) (f, g) are shown. The scale bars are 1 lm. For determination of the cell size (c) at least 100 representative cells were counted manually or by using a semi-automated object count tool during scanning electron microscopy width: 0.8 ± 0.1 lm), form aggregates and divide by polar budding. Cells grow at ranges of 14-27°C (optimum 26°C) and pH 6.0-10.0 (optimum 9.0).
Colonies are white. The genome (acc. no. SJPG00000000) and 16S rDNA sequence (acc. no. MK554545) are available from the GenBank database. The genome has a GC content of 48.8% and is 6.70 Mb in length. The proposed type strain is Pan54 T (DSM 29369 = LMG 29789) isolated from an algal surface at a hydrothermal area close to Panarea, Italy.