Poriferisphaera corsica strain KS4 16S ribosomal RNA gene, partial sequence

Abstract

Cultivation-Independent Bacterial Community Analysis The calcareous sponge C. clathrus was sampled in September 2015 near the coast of the island Corsica (exact location: 42.579° N 8.725° E; Figure 1A). One individual was sampled, and three pieces were used for DNA extraction and pooled for further processing. The sample for DNA extraction from the sponge surface was obtained by scraping off the sponge-associated biofilm from the three pieces using single-use scalpels and resuspension of the pooled material into sterile natural seawater. The surrounding seawater was sampled at three different locations in the immediate vicinity of the sponge and further processed within 8 h. In the laboratory, the triplicate water samples (approximately 300 ml each) were homogenized by gentle stirring and subsequently filtered through a single polycarbonate membrane filter (Ø 47 mm, Isopore, Merck) with a pore size of 0.22 μm to collect planktonic bacteria. Filters were frozen and stored at −20°C until DNA extraction. Isolation of DNA and all subsequent steps for amplification of DNA and amplicon sequencing were performed as previously described (Kohn et al., 2020a). The analysis of operational taxonomic units present in the samples was performed using the SILVAngs pipeline (Quast et al., 2012). The analysis was based on 22,364 sequences of the variable V3 region of 16S rRNA genes with an average length of 143 bp. Classification of the sequences was performed using SILVA reference version 132 and BLASTn version 2.2.30+ with a sequence similarity of 93%.

Isolation of Strain KS4T and Physiological Characterization For the isolation of strain KS4T, a small piece of C. clathrus was washed with sterile-filtered seawater, additionally supplemented with 100 mg/L cycloheximide to prevent fungal growth. Afterward, the material was swabbed over three M1H NAG ASW plates solidified with 8 g/L gellan gum and supplemented with 1,000 mg/L streptomycin, 200 mg/L ampicillin, and 20 mg/L cycloheximide (Wiegand et al., 2020). Plates were subsequently incubated at 20°C for 8 weeks and regularly inspected for the presence of colonies. Slow-growing colonies with a white or pink color were subjected to colony-PCR to amplify the 16S rRNA gene, which was subsequently sequenced. Strains identified as members of the phylum Planctomycetes, but with a 16S rRNA gene similarity below 98% to already chracterized strains, were further cultivated. Strain KS4T, isolated using the above-mentioned strategy, showed good growth also in liquid M1H NAG ASW medium (Boersma et al., 2019) and was characterized in detail. Unless otherwise stated, growth experiments were performed at pH 7.5, and 27°C. For determination of the pH optimum for growth, different buffering compounds were used: 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) for pH 5–6, 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) for pH 7-8, or 100 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES) for pH 9–10. In the growth experiments without either NaCl (0% w/v NaCl) or artificial seawater (ASW) (0% ASW), the medium still contained all chemical elements required for growth (see medium recipe; Wiegand et al., 2020). Concentrated ASW contained 46.94 g/L NaCl, 7.84 g/L Na2SO4, 21.28 g/L MgCl2 x 6 H2O, 2.86 g/L CaCl2 x 2 H2O, 0.384 g/L NaHCO3, 1.384 g/L KCl, 0.192 g/L KBr, 0.052 g/L H3BO3, 0.08 g/L SrCl2 x 6 H2O, and 0.006 g/L NaF. 250 ml of concentrated ASW were used for the preparation of 1 L of M1H NAG ASW medium and was considered 100% ASW. A range of 0–200% ASW was tested in the cultivation experiments.

Morphological Characterization Phase contrast and field emission scanning electron microscopy were performed as previously described (Kohn et al., 2016).

Genome Information and Analysis of Genome-Encoded Features The genome sequence of strain KS4T is available from GenBank under the accession number CP036425. The 16S rRNA gene sequence can be found under accession number MK559978. DNA isolation and genome sequencing are part of a previously published study (Wiegand et al., 2020). The number of carbohydrate-active enzymes was obtained from the CAZy database (Terrapon et al., 2017). Biosynthetic gene clusters associated to secondary metabolite production were determined using antiSMASH version 4.0 (Blin et al., 2017).

Phylogenetic Analysis The 16S rRNA gene sequence-based phylogeny was computed for strain KS4T, the type strains of all described planctomycetal species (assessed in January 2020) and all isolates recently published and described (Boersma et al., 2019; Kallscheuer et al., 2019a,b,c, 2020; Dedysh et al., 2020; Kohn et al., 2020a; Peeters et al., 2020). The 16S rRNA gene sequences were aligned with SINA (Pruesse et al., 2012), and the phylogenetic inference was calculated with RAxML (Stamatakis, 2014) using a maximum likelihood approach with 1,000 bootstraps, nucleotide substitution model GTR, gamma distributed rate variation, and estimation of proportion of invariable sites (GTRGAMMAI option). The 16S rRNA genes of the following bacterial strains from the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum outside of the phylum Planctomycetes were used as the outgroup: Opitutus terrae (NCBI accession number: AJ229235), Kiritimatiella glycovorans (NCBI accession number: NR_146840), and Lentisphaera araneosa (NCBI accession number: NR_027571). For the multi-locus sequence analysis (MLSA), the unique single-copy core genome of the analyzed genomes was determined with proteinortho5 (Lechner et al., 2011) with the “selfblast” option enabled. The protein sequences of the resulting 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 was concatenated and clustered using the maximum likelihood method implemented by RAxML (Stamatakis, 2014) with the “rapid bootstrap” method and 500 bootstrap replicates. Two strains from the family Isosphaeraceae (Singulisphaera acidiphila, accession number CP003364.1, and Isosphaera pallida, accession number CP002353.1) were used as outgroup in the MLSA-based phylogenetic tree. Average nucleotide identities (ANI) were calculated using OrthoANI (Lee et al., 2016). The average amino acid identity (AAI) was calculated using the aai.rb script of the enveomics collection (Rodriguez-R and Konstantinidis, 2016) and the percentage of conserved proteins (POCP) was calculated as described (Qin et al., 2014).