ALBERT

Description: The present study aimed at a first characterization of the enigmatic JTB255 marine benthic group in deep-sea sediments, by: i) confirming the abundance and ubiquitous distribution of JTB255 in deep-sea sediments globally, ii) refining the phylogenetic positioning of the JTB255 clade within the \u03b3-Proteobacteria, iii) distinguishing potential ecotypes within the JTB255 clade, iv) providing first insights into the metabolic potential of deep-sea representatives of this clade. Therefore, two single cell genomes from Arctic HAUSGARTEN deep-se surface sediments were obtained and CARD-FISH counts of total cells, y-Proteobacteria and the JTB255 marine benthic group performed.

Description: Samples in this dataset were collected at the long-term ecological research (LTER) site HAUSGARTEN in Fram Strait, and the central Arctic Ocean. On board, the samples were fixed with formalin in a final concentration of 2% for 10 – 12 hours, then filtered onto 0.2 µm polycarbonate Nucleopore Track-Etched filters, and stored at -20°C for further analysis. Cell abundances of the groups Alteromonas, Bacteroidia, Polaribacter, Gammaproteobacteria and the SAR11 clade were asses using CAtalyzed reporter deposition Fluorescence In Situ Hybridization (CARD-FISH) following the protocol established by (Pernthaler et al., 2002). The filters were evaluated microscopically under an automated microscope (Zeder et al., 2011). Cell enumeration was performed with the software Automated Cell Measuring and Enumeration Tool (ACMETool3, 2018-11-09; Zeder et al., 2011). Cells were counted as objects according to manually defined parameters separately for the DAPI and FISH channels.

Description: We aimed to explore the community composition and turnover of eukaryotic and bacterial microorganisms associated with ice-associated and sinking algal aggregates, as well as their similarity to potential source communities of sea ice, water and deep-sea sediments using Illumina tag sequencing. We sampled algae aggregates growing in melt ponds on sea ice, deposited algae aggregates at the seafloor in more than 4000 m water depth, sea ice, upper water column, sediment surface and the gut content of holothurians feeding on the deposits. For Illumina sequencing, the Amplicon libraries of the bacterial V4-V6 region of the 16S rRNA gene and the eukaryotic V4 region of the 18S rRNA gene were generated according to the protocol recommended by Illumina (16S Metagenomic Sequencing Library Preparation, Part # 15044223, Rev. B). For Bacteria we selected the S-D-Bact-0564-a-S-15 and S-*Univ-1100-a-A-15 primer pair based on a primer evaluation by Klindworth et al. (2013, doi:10.1093/nar/gks808) and for Eukaryota the TAReukFWD1 and TAReukREV3 primers (Stoeck et al., 2010; doi:10.1111/j.1365-294X.2009.04480.x). Libraries were sequenced on an Illumina MiSeq platform in 2x300 cycles paired end runs. For Sequence data cleaning & processin, we used cutadapt (v. 1.8.1; Martin, 2011; doi:10.14806/ej.17.1.200) for the removal of primer sequences and a custom awk script to ensure the correct orientation of reads prior to merging. For merging forward and reverse reads we used pear (v. 0.9.5; Zhang et al., 2014; doi:10.1093/bioinformatics/btt593) and trimmed and quality filtered all sequences using trimmomatic (v. 0.32; Bolger et al., 2014; doi:10.1093/bioinformatics/btu170). We reassured correct formatting of the fastq files with bbmap (v. 34.00; B. Bushnell - sourceforge.net/projects/bbmap) before clustering the reads into OTUs by applying a local clustering threshold of d=1 and the fastidious option in swarm (v. 2.1.1; Mahé et al., 2015; doi:10.7717/peerj.1420). Subsequently, we used the SINA aligner (v. 1.2.10; Pruesse et al., 2012; doi:10.1093/bioinformatics/bts252) to align and classify the seed sequence of each OTU with the SILVA SSU database release 123 (Quast et al., 2013; doi:10.1093/nar/gks1219). OTUs that were classified as chloroplasts, mitochondria, archaea, or those that could not be classified at domain level were removed from further analysis. OTUs that were classified as bacteria within the eukaryotic dataset and vice versa, were removed as well. Furthermore, we removed all absolute singletons, OTUs that were only represented by a single sequence across the complete dataset. Filtering and removal of absolute singletons resulted in a final number of 8,869 bacterial and 7,627 eukaryotic OTUs. All further analyses were performed on these processed OTU abundance tables.

Description: We fixed 0.5 g aliquots of sediment with a 4% formaldehyde solution for 2-4 h, washed the fixed sediments three times with 1x phosphate-buffered saline (PBS), before storing them in 50% ethanol/PBS at -20°C. For water samples, we fixed 10 ml (for samples from the deep chlorophyll maximum and 100 m water depth) and 30 ml (for meso- and bathypelagic samples) with formaldehyde to a final concentration of 2-4% for 2-4 h, then filtered over a 0.22 µm polycarbonate filter, and stored samples at -20°C. We performed total cell counts as described by Schauer et al. (2011, doi:10.1111/j.1462-2920.2011.02530.x) using the nucleic acid dye 4'-6-diamidino-2-phenylindole (DAPI). A minimum of 1,000 cells in 20 independent grids were counted using a Zeiss Axio Imager M1 epifluorescence microscope equipped with a 100x/1.25 oil plan-apochromat objective. We used Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) according to Ishii et al. (2004) to count Gammaproteobacteria and JTB255 cells. We used the GAM42a oligonucleotide probe and the BET42a competitor probe to target members of the Gammaproteobacteria (Manz et al., 1992, doi:10.1016/S0723-2020(11)80121-9). We designed the JTB819a and JTB897 probes to target 16S rRNA gene sequences assigned to JTB255 in SILVA release 128 and the cJTB897 competitor probe to target all non-JTB255 16S sequences in SILVA release 128 that have a single mismatch to JTB819a and JTB897 probes. We obtained total gammaproteobacterial and JTB255 cell counts from duplicate filters derived from each sampling site.

Description: Hypersaline aqueous environments at subzero temperatures are known to be inhabited by microorganisms, yet information on community structure in subzero brines is very limited. Near Utqiaġvik, Alaska, we sampled subzero brines (–6°C, 115–140 ppt) from cryopegs, i.e. unfrozen sediments within permafrost that contain relic (late Pleistocene) seawater brine, as well as nearby sea-ice brines to examine microbial community composition and diversity using 16S rRNA gene amplicon sequencing. We also quantified the communities microscopically and assessed environmental parameters as possible determinants of community structure. The cryopeg brines harbored surprisingly dense bacterial communities (up to 108 cells mL–1) and millimolar levels of dissolved and particulate organic matter, extracellular polysaccharides and ammonia. Community composition and diversity differed between the two brine environments by alpha- and beta-diversity indices, with cryopeg brine communities appearing less diverse and dominated by one strain of the genus Marinobacter, also detected in other cold, hypersaline environments, including sea ice. The higher density and trend toward lower diversity in the cryopeg communities suggest that long-term stability and other features of a subzero brine are more important selective forces than in situ temperature or salinity, even when the latter are extreme.