Planctomycetes bacterium strain Mal48 16S ribosomal RNA gene, partial sequence

Abstract

Cultivation conditions and isolation Strain Mal48T was isolated on the 23th of September 2014 from phytoplankton collected at the coast of S’Arenal close to Palma de Mallorca (Spain) (sampling location: 39.5126 N 2.7470 E). After centrifugation of the sampling material, the pellet was resuspended in 100 µL sterile artificial seawater (ASW) and streaked on a plate containing M1H medium with N-acetylglucosamine (NAG) and ASW (designated M1H NAG ASW) (Kallscheuer et al. 2019a) solidified with 15 g/L agar and additionally supplemented with 200 mg/L ampicillin, 500 mg/L streptomycin and 20 mg/L cycloheximide. Plates were incubated at 28 °C for 3–4 weeks and isolated colonies were then streaked on fresh M1H NAG ASW plates. Initial amplification and sequencing of the 16S rRNA gene was performed as previously described (Rast et al. 2017). This step was included to ensure that the isolated strain is indeed a member of the phylum Planctomycetes.

Physiological analyses For temperature and pH optima determination M1H NAG ASW medium was used. The strain was cultivated at pH 8 at different temperatures ranging from 10 to 40 °C. For pH optimum identification 100 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.0–6.5), HEPES (pH 7.0–8.0), HEPPS (pH 8.5) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES, pH 9.0–9.5) were used as buffering agents. Cultivations for determination of the pH optimum were performed at 28 °C. Growth was determined from optical density measurements at 600 nm (OD600) of triplicate cultures.

Genome analysis The genome of strain Mal48T is available from NCBI under GenBank accession number CP036267 and the 16S rRNA gene sequence under accession number MK625061. Sequencing of the genome is described in a previous study (Wiegand et al. 2020). 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.

Light microscopy and scanning electron microscopy Phase contrast light microscopy and scanning electron microscopy were performed as previously described (Kallscheuer et al. 2019a).

Phylogenetic analyses The 16S rRNA gene-based phylogenetic analysis of strain Mal48T was computed along with sequences of all described planctomycetal species (assessed in January 2020), including recently published isolates (Kohn et al. 2016, 2020a, b; Kulichevskaya et al. 2015; Boersma et al. 2019; Kallscheuer et al. 2019a; Dedysh et al. 2020). SINA was used to perform the 16S rRNA gene sequence alignment (Pruesse et al. 2012). The phylogenetic inference was performed 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 from members of the PVC superphylum, but outside of the phylum Planctomycetes, were used as outgroup (Opitutus terrae, acc. No. AJ229235; Kiritimatiella glycovorans, acc. no. NR_146840 and Lentisphaera araneosa, acc. no. NR_027571). The average nucleotide identity (ANI) was calculated using OrthoANI (Lee et al. 2016) and the average amino acid identity (AAI) was gained 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). The rpoB gene sequences were extracted from the genome annotations and the sequence identities were determined as described (Bondoso et al. 2013) with Clustal Omega (Sievers et al. 2011). Alignment and matrix calculation were performed by extracting only those parts of the sequence that would have been sequenced with the described primer set. 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 of 709 ubiquitous genes with a combined length of 356,576 conserved amino acid residues was concatenated and clustered using the maximum likelihood method implemented by RaxML (Stamatakis 2014) (‘rapid bootstrap’ method and 500 bootstrap replicates).