Planctomycetes bacterium strain Pla52n 16S ribosomal RNA gene, partial sequence

Abstract

Isolation of strain Pla52nT and cultivation Strain Pla52nT was isolated from wood particles placed in an incubator and stored for two weeks (August–September 2014) at a depth of 2 m in the Baltic Sea, below a landing stage at Heiligendamm (‘Seebrücke Heiligendamm’, 54.146 N 11.843 E) (Oberbeckmann et al. 2018). In the laboratory, biofilms formed on the wood particles were removed by incubation with β-galactosidase (2 mg/mL, 30 °C, pH 4.7) for 30 min and subsequent sonication for 10 min at 30 °C. M1 medium buffered with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and supplemented with N-acetyl glucosamine (NAG) and artificial seawater (ASW) (designated M1H NAG ASW medium) (Boersma et al. 2019) was used for the cultivation. The medium was solidified with 8 g/L gellan gum and additionally supplemented with 500 mg/L streptomycin, 100 mg/L ampicillin and 20 mg/L cycloheximide. The cell suspension obtained after sonication was streaked on an M1H NAG ASW plate, incubated at 20 °C for six weeks and regularly checked for the presence of colonies. Colonies obtained were then subjected to 16S rRNA gene amplification and sequencing according to a previously published protocol (Rast et al. 2017). This step was included to check whether strains are members of the phylum Planctomycetes. Colonies of strains confirmed as members of the phylum Planctomycetes were re-streaked on M1H NAG ASW plates, which then served as a source for the inoculation of liquid cultures in M1H NAG ASW medium. After several days of cultivation, exponentially growing cells were used for subsequent cultivation experiments. Determination of the pH optimum for growth was performed by cultivation of strain Pla52nT in M1H NAG ASW at 28 °C with 100 mM of the following buffers: 2-(N-morpholino)ethanesulfonic acid (MES) for pH 5.0-6.5, HEPES for pH 7.0-8.0, 3-(4-(2-hydroxyethyl)piperazin-1-yl)propane-1-sulfonic acid (HEPPS) for pH 8.0 and N-cyclohexyl-2-aminoethanesulfonic acid (CHES) for pH 9.0-10.0. Cultivations for determination of the temperature optimum for growth were performed in M1H NAG ASW medium at pH 7.5. Growth of the strain was measured as optical density at 600 nm (OD600). Maximal growth rates µmax were obtained by determination of the slope in the plot of the natural logarithmic function of average OD600 values from biological triplicates against the cultivation time. The slope from at least five data points in the exponential growth phase was used as growth rate µmax (in h−1). The generation time td (in h) was calculated using the equation td = ln(2)/µmax. Microscopy Microscopic analyses included phase contrast light microscopy and field emission scanning electron microscopy (SEM) and were performed as previously described (Boersma et al. 2019). Genome information and antiSMASH analysis Sequencing of the genome of strain Pla52nT was conducted as part of a previous study (Wiegand et al. 2020). Genome and 16S rRNA gene sequence of strain Pla52nT are available from GenBank under accession numbers GCA_007860045 and MK554582, respectively. Analysis of secondary metabolite-associated gene clusters was performed using antiSMASH bacterial version 5.1.2 with relaxed strictness and the following extra features enabled: KnownClusterBlast, ActiveSiteFinder and SubClusterBlast (Blin et al. 2019)