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Blastopirellula retiformator sp. nov. isolated from the shallow-sea hydrothermal vent system close to Panarea Island

Aquatic bacteria belonging to the deep-branching phylum Planctomycetes play a major role in global carbon and nitrogen cycles. However, their uncommon morphology and physiology, and their roles and survival on biotic surfaces in marine environments, are only partially understood. Access to axenic cultures of different planctomycetal genera is key to study their complex lifestyles, uncommon cell biology and primary and secondary metabolism in more detail. Here, we describe the characterisation of strain Enr8T isolated from a marine biotic surface in the seawater close to the shallow-sea hydrothermal vent system off Panarea Island, an area with high temperature and pH gradients, and high availability of different sulphur and nitrogen sources resulting in a great microbial diversity. Strain Enr8T showed typical planctomycetal traits such as division by polar budding, aggregate formation and presence of fimbriae and crateriform structures. Growth was observed at ranges of 15–33 °C (optimum 30 °C), pH 6.0–8.0 (optimum 7.0) and at NaCl concentrations from 100 to 1200 mM (optimum 350–700 mM). Strain Enr8T forms white colonies on solid medium and white flakes in liquid culture. Its genome has a size of 6.20 Mb and a G + C content of 59.2%. Phylogenetically, the strain belongs to the genus Blastopirellula. We propose the name Blastopirellula retiformator sp. nov. for the novel species, represented by the type strain Enr8T (DSM 100415T = LMG 29081T).


Introduction
Members of the family Planctomycetaceae, which belong to the environmentally, medically and biotechnologically relevant PVC superphylum (Planctomycetes-Verrucomicrobia-Chlamydiae, and others) (Spring et al. 2016;Wagner and Horn 2006), are ubiquitous microorganisms dwelling mostly in aquatic environments, in which they play key roles in cycling of carbon and nitrogen. However, the mechanisms as to how these microorganisms gain nutrients from biotic material in the oceans (in particular carbon and nitrogen for biomass and energy formation) are only partially understood. Species belonging to the family Planctomycetaceae are typically found to be attached to marine biotic surfaces, such as algae or kelp, and probably form biomass by using algal compounds as carbon and energy sources (Bondoso et al. 2015;Wiegand et al. 2019). Such nutrient-rich environments-in contrast to the oligotrophic seawaterattract also faster-growing competing microorganisms, e.g. Roseobacter sp. (Frank et al. 2014;Wiegand et al. 2018), but the latter fail to outcompete the slower-growing species of the family Planctomycetaceae. Two theories provide plausible explanations for this counter-intuitive observation: (I) Planctomycetaceae harbour a specialised machinery for uptake and degradation of complex polysaccharides released by algae or (II) they produce small molecules as a metabolic defence strategy. Both theories are supported by large planctomycetal genomes and the high number of predicted enzymes involved in the catabolism of complex sugars (Naumoff et al. 2014;Reisky et al. 2018;Wegner et al. 2013) (supporting theory I) or for production of secondary metabolites (supporting theory II) (Graça et al. 2016;Jeske et al. 2016;Wiegand et al. 2018Wiegand et al. , 2019. Uptake and catabolism of polymeric sugars might be facilitated by the unique pili-forming crateriform structures and an enlarged periplasm (Boedeker et al. 2017), while the mostly unexplored secondary metabolism could be a promising source for novel compounds, including those with health-promoting activities in humans (Graça et al. 2016).
Not only from a metabolic perspective, but also structurally, the phylum Planctomycetes is an interesting research topic. Eukaryotic-like morphological traits of Planctomycetes led to the conclusion that they might be beyond the bacterial cell plan (Fuerst and Sagulenko 2011;König et al. 1984;Lonhienne et al. 2010). In recent years, this picture changed with the advent of novel microscopic techniques and detailed physiological analyses of Planctomycetes (Jeske et al. 2015;Jogler et al. 2011;Jogler and Jogler 2013;Rivas-Marin et al. 2016;van Teeseling et al. 2015). The cell envelope of Planctomycetes was ultimately found to resemble that of Gram-negative bacteria (Boedeker et al. 2017;Devos 2014). But still, Planctomycetes remain exceptional. They divide by budding, binary fission or even a combination of both and lack proteins of the canonical divisome ). Many strains have been shown to be resistant to several antibiotics (Cayrou et al. 2010;Godinho et al. 2019), either because of their degradation, or intrinsic resistance due to the lack of targets (Jogler et al. 2012;Pilhofer et al. 2008).
In conclusion, Planctomycetes have some characteristic traits (Wiegand et al. 2018, which encourages more detailed research and motivates us to steadily expand the collection of axenic cultures. In this study, we took samples in the surroundings of Panarea Island in the Tyrrhenian Sea off the southwestern coast of Italy. This region includes a shallowsea hydrothermal vent system and consists of areas with increased temperatures, steep temperature gradients and high levels of different nitrogen and sulphur sources (Maugeri et al. 2010). Due to the expected microbial diversity (Manini et al. 2008), we considered this location a valuable source of so far unknown species of the family Planctomycetaceae and here describe the characterization of the novel strain Enr8 T isolated from a hydrothermal area close to Panarea Island.

Isolation and cultivation conditions
Strain Enr8 T was isolated on the 10th of September 2013 from a marine biotic surface (Fig. 1) in a hydrothermal area in the Tyrrhenian Sea (sampling site 38.6387 N 15.1068 E) 2.5 km east of the port of Fig. 1 Representative picture of the sampling location. Strain Enr8 T was isolated from the surface of the marine plant shown on the right side of the photograph Panarea Island. The strain was isolated as described previously ) and subsequently cultivated in M1 medium with HEPES as buffering agent and supplemented with N-acetyl glucosamine (NAG) and artificial seawater (ASW) (medium designation M1H NAG ASW) (Kallscheuer et al. 2019a).

Physiological analyses
The temperature optimum of strain Enr8 T was determined in M1H NAG ASW medium by cultivation at temperatures of 10,15,20,22,24,27,30,33,36 and 40°C in a shaking incubator at 110 rpm with an initial pH of 8.0. For the determination of the pH optimum 100 mM HEPES was used as buffering agent for cultivations at pH 7.0, 7.5 and 8.0. For cultivations at pH 5.0 and 6.0 HEPES was replaced by 100 mM 2-(Nmorpholino)ethanesulfonic acid (MES) and at pH 9.0 and 10.0 100 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES) was used as buffering agent. Cultivations for determination of the pH optimum were performed at 28°C in a shaking incubator at 110 rpm. For analysis of the salt tolerance, strain Enr8 T was cultivated in M1H NAG ASW medium with different concentrations of NaCl. The tested concentrations were 0, 100, 200 (standard NaCl concentration in ASW), 350, 500, 700, 900, 1200 and 1600 mM NaCl. Due to formation of white flakes by aggregation of cells in all experiments, growth was assessed by visual inspection of the culture taking the number and size of white flakes into account. Measurement of the optical density at 600 nm (OD 600 ) was not possible.

Light microscopy and electron microscopy
Microscopic analyses were performed according to a previously published protocol (Kallscheuer et al. 2019a).

Genome information and analysis of genomeencoded features
The genome (accession no. SJPF00000000) and 16S rRNA gene sequence (accession no. MK554541) of strain Enr8 T are available from GenBank ). The primary metabolism was analysed by examining locally computed InterProScan (Mitchell et al., 2019) results cross-referenced with information from the UniProt database and BLASTp results of 'typical' protein sequences.
Phylogenetic analysis 16S rRNA gene phylogeny was computed for strain Enr8 T , the type strains of all described planctomycetal species (as available in May 2019) and all isolates recently published ), including the strains described recently (Boersma et al. 2019;Kallscheuer et al. 2019a, b, c, d;Kohn et al. 2019). The 16S rRNA gene sequences were aligned with SINA (Pruesse et al. 2012). The phylogenetic analysis was performed with RAxML (Stamatakis 2014) employing a maximum likelihood approach with 1000 bootstraps, the nucleotide substitution model GTR, gamma distributed rate variation and estimation of proportion of invariable sites (GTRGAMMAI option). Three 16S rRNA genes of bacterial strains from the PVC superphylum were used as outgroup. The average nucleotide identity (ANI) was calculated using OrthoANI (Lee et al. 2016) and the average amino acid identity (AAI) was calculated using the aai.rb script of the enveomics collection (Rodriguez-R and Konstantinidis 2016). The percentage of conserved proteins (POCP) was calculated as described (Qin et al. 2014).

Phylogenetic analysis
According to maximum likelihood-based 16S rRNA gene sequence analysis shown in Fig. 2, the current closest relative of strain Enr8 T is Blastopirellula marina, which is also the type species of the genus (Schlesner et al. 2004). The type strain of B. marina, DSM 3645 T , was originally isolated from brackish water in the Baltic Sea (Schlesner 1986) and together with Blastopirellula cremea (Lee et al. 2013) forms the current genus Blastopirellula. B. marina DSM 3645 T and strain Enr8 T share a 16S rRNA similarity of 98.9%. This value is slightly above the proposed species threshold of 98.7% (Stackebrandt and Ebers 2006), which could indicate that strain Enr8 T belongs to the species B. marina. However, it was shown before that 16S rRNA genes are not in all cases a reliable marker for Planctomycetes as some strains have high 16S rRNA gene sequence identities, but nevertheless belong to different species (Bondoso et al. 2013;Kohn et al. 2019). Thus, other phylogenetic markers need to be considered in order to achieve a reliable classification of strain Enr8 T . For example, the similarity of the analysed partial rpoB sequence is 88.4%, which is clearly below the proposed threshold of 95.5% for strains belonging to the same species Gimesia clade includes species of the genera Gimesia, Planctopirus, Fuerstiella, Schlesneria, Rubinisphaera and Planctomicrobium, while the thermophilic clade includes species of the genera Thermostilla, Thermogutta and Thermopirellula (Bondoso et al. 2013). The same is true for ANI values of 78.3% when applying the species threshold of 95-96% (Kim et al. 2014). Taken together, rpoB similarity and ANI values clearly reinforce the conclusion that strain Enr8 T belongs to a separate species and does not represent a strain of the species B. marina. Thus, it appears likely that 16S rRNA gene identity comparisons in this clade do not follow the proposed species threshold of 98.7% (Stackebrandt and Ebers 2006). Accordingly, strain Enr8 T and B. marina have an AAI of 75.8% and a POCP of 77.6%. Both values are above the respective genus threshold ranges (AAI: 60-80% and POCP: 50%) (Luo et at. 2014;Qin et al. 2014), confirming that both strains belong to the same genus. In order to show that strain Enr8 T and B. marina DSM 3645 T do not belong to the recently proposed genus 'Bremerella' (Rensink et al. 2019), minimal 16S rRNA gene sequence identities, AAI values, rpoB similarities and POCP values of strain Enr8 T and 'Bremerella vulcanica' Pan97 T were compared. Comparison yields a 16S rRNA gene identity of 94.3%, an AAI value of 54.7% and a partial rpoB sequence of 74.7%, which are below the genus thresholds of 94.5%, 60-80% and 75.5-78%, respectively (Kallscheuer et al. 2019d;Luo et al. 2014;Yarza et al. 2014). The POCP value of 60.4% is only slightly above the genus threshold of 50% (Qin et al. 2014).

Morphological and physiological analyses
Strain Enr8 T was cultivated in M1H NAG ASW medium and exponentially growing cells were used for morphological characterisation using phase contrast microscopy and scanning electron microscopy (Fig. 3). Detailed information on morphology, locomotion and cell division in comparison to the closely related species B. marina is summarised in Table 1. Cells of strain Enr8 T appear ovoid to pear-shaped (length: 1.3 ± 0.2 lm, width: 0.7 ± 0.1 lm) (Fig. 3a), form strong aggregates and contain crateriform structures at the cell poles (Fig. 3b-e). Polar budding was observed as mode of cell division with the shape of the bud being similar to that of the mother cell (Fig. 3a). Fibres together with an extracellular matrix facilitate attachment to other cells or surfaces, thereby causing white flake formation visible with the naked eye in broth cultures. Colonies of strain Enr8 T  (Table 1).
In cultivation experiments, strain Enr8 T was found to grow at a pH range of 6.0-8.0 and at temperatures from 15 to 33°C (Fig. 4a, b). The optimal conditions were determined to be pH 7.0 and 30°C. During cultivation, formation of strong aggregates was observed, which became visible as white flakes settling down in the cultivation tubes, for example as shown for the determination of the pH optimum (Fig. 4a). This rendered measurement of cell densities using OD 600 impossible. Instead, optimal conditions (pH, temperature and NaCl concentration) were determined by visual inspection taking the observed differences in the number and size of flakes into account. The temperature optimum at 30°C is the same as for B. marina and both failed to grow at 36-38°C (Schlesner 1986). Due to aggregate formation, a maximal growth rate could not be calculated for strain Enr8 T . In the salt tolerance experiments, the strain showed growth in the presence of 0.6-7.0% (w/ v) NaCl (100-1200 mM), but failed to grow without NaCl or with 9.3% (w/v) NaCl (1600 mM) (Fig. 4c). Optimal growth was observed in the range of 2.1-4.1% (w/v) NaCl (350-700 mM), which is in the range of natural seawater in the Mediterranean Sea with a total salt concentration of around 3.8% (w/v). The high salt tolerance of strain Enr8 T is a distinctive difference compared to B. marina. While concentrations of 1% NaCl were lethal for B. marina (Schlesner 1986), Enr8 T showed optimal growth at 2-4% NaCl and could even grow in the presence of 7% NaCl. Genome-based analysis of the central carbon metabolism Based on the genome sequences of Enr8 T and B. marina DSM 3645 T , we analysed the presence of key metabolic enzymes of the central carbon metabolism. The analysis included glycolytic pathways, gluconeogenesis, the tricarboxylic acid (TCA) cycle and anaplerotic reactions (Table 2). Both strains harbour the genes coding for enzymes involved in glycolytic reactions either using the Embden-Meyerhof-Parnas pathway (referred to as glycolysis) or the alternative Entner-Doudoroff pathway. However, we failed to identify the gene coding for phosphoglycerate mutase (Pgm), which catalyses the reversible isomerisation of 3-phosphoglycerate to 2-phosphoglycerate and is required for the activity of both pathways. As the degradation of glucose by both strains suggests that the glycolysis is active, the two strains either bypass the reaction, e.g. by using 2,3-bisphosphoglycerate as intermediate or the protein sequence of Pgm differs from canonical sequences and escaped our analysis. The pentose phosphate pathway and the TCA cycle appear to be fully functional in both strains since we were able to assign genes to all participating enzymes (Table 2). Both strains also encode phosphoenolpyruvate carboxykinase (ATP) and fructose-1,6-bisphosphatase, the key enzymes required for de novo   (Table 2), which is required e.g. during growth with acetate or fatty acids as sole carbon and energy source. This would imply that both strains are unable to use acetate or fatty acids as sole carbon and energy source, which may support the hypothesis that species of the family Planctomycetaceae consume complex sugars derived from phototrophs rather than short-or long-chain carboxylic acids.
Putative gene clusters involved in secondary metabolite production To gain a first insight into the potential of strain Enr8 T as a source of secondary metabolites an AntiSMASH analysis based on its genome sequence was conducted (Blin et al. 2019). It appears reasonable to assume that the complex lifestyle and growth in competitive environments such as marine surfaces is related to the production of secondary metabolites allowing strain Enr8 T to cope with abiotic and biotic stresses. Of major interest in this case are multi-domain protein complexes of the families of polyketide synthases (PKSs) or non-ribosomal peptide synthetases (NRPSs) as these serve as molecular assembly lines for production of secondary metabolites starting from metabolites of the primary carbon metabolism (e.g. malonyl-CoA or amino acids) (Park et al. 2019). Strain Enr8 T harbours three genes or clusters involved in the production of terpenoids. These are probably not involved in the production of carotenoids (a major class of terpenoids) as the strain is white and thus lacks pigmentation. In addition, Enr8 T harbours genes coding for a putative type I PKS, a putative NRPS, a putative mixed NRPS-type I PKS and a protein related to bacteriocin biosynthesis. The same set of clusters is also encoded in the genome of B. marina DSM 3645 T except for the putative NRPS gene, which was not identified in B. marina. The exact products formed by the identified PKS and NRPS enzymes should be identified in future studies to gain insights into their biological significance. Taken together, our phylogenetic analysis and the results of the morphological and physiological characterisation support the conclusion that strain Enr8 T represents a novel species of the genus Blastopirellula, for which we propose the name Blastopirellula retiformator sp. nov.
Colonies are white. Cells are pear-shaped (length: 1.3 ± 0.2 lm, width: 0.7 ± 0.1 lm) and form large aggregates which become visible as white flakes in liquid culture. Cells divide by polar budding and grow at ranges of 15-33°C (optimum 30°C), pH 6.0-8.0 (optimum 7.0) and NaCl concentrations from 100 to 1200 mM (optimum 350-700 mM). The genome (accession no. SJPF00000000) and 16S rRNA gene sequence (accession no. MK554541) of the type strain are available from GenBank. The type strain genome has a size of 6.20 Mb and a G ? C content of 59.2%. The type strain is Enr8 T (DSM 100415 T = LMG 29081 T , also designated Enrichment 8), isolated from a hydrothermal area close to the island Panarea, Italy.