Abstract
Isolation and cultivation of the strain
Strain Pla85_3_4T was isolated from submerged wood pellets suspended near the discharge of a wastewater treatment plant in the estuary of Warnow river next to the city Rostock located in Germany (exact location 54.106 N 12.096 E). This location is close to the Baltic Sea and has brackish water. Isolation was performed as described previously (Oberbeckmann et al. 2018; Wiegand et al. 2020). For further investigation, the strain was grown in M1H medium supplemented with N-acetyl glucosamine (NAG) and artificial seawater (ASW) (M1H NAG ASW medium) as described previously (Wiegand et al. 2020) and was cultivated at 28 °C under constant agitation at 110 rpm.
Light microscopy and electron microscopy
Phase contrast microscopy was performed with a Nikon Eclipse Ti inverted microscope with a Nikon DS-Ri2 camera. Cells were immobilised in MatTek glass bottom dishes (35 mm, No. 1.5) using a 1% (w/v) agarose cushion (Boedeker et al. 2017). ImageJ (Rueden et al. 2017) was used to examine cell size by sequentially applying an Otsu threshold, then the watershed function, and finally the count particles function, excluding particles smaller than 0.05 µm.
Field emission scanning electron microscopy was performed as described previously (Boersma et al. 2019). The bacteria were fixed in formaldehyde, washed and placed on poly-L-lysine-coated cover slips. Samples were then fixed in 1% (v/v) glutaraldehyde and washed twice before dehydrating in graded series of acetone [10, 30, 50, 70, 90, 100% (v/v)] on ice. Samples from the last acetone treatment step were brought to room temperature before placing them in fresh 100% acetone. Samples were then subjected to critical-point drying with liquid CO2 (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 an Everhart–Thornley HESE2 detector and an inlens SE detector in a 25:75 ratio with an acceleration voltage of 5 kV.
Physiological and biochemical analyses
The pH optimum was determined at 28 °C, with buffering agents 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) at pH 5 and 6, 100 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES at pH 7, 7.5 and 8, and 100 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at pH 9 and 10. Temperature optimum determination was performed at pH 7.5 and temperatures ranging from 10 to 40 °C in steps of 5 °C. Cell densities were inferred from optical densities (λ = 600 nm).
Genome information and analysis of genome-encoded features
The genome and 16S rRNA gene sequences of strain Pla85_3_4T are available from GenBank (accession numbers MK559988 and CP036433, respectively). Numbers of carbohydrate-active enzymes were obtained from the CAZY database (Lombard et al. 2014). Gene clusters potentially involved in the production of secondary metabolites were determined using antiSMASH 4.0 (Blin et al. 2017).
