Sampling, bacterial isolation, and colony screening
Samples were collected from thermally active sediments as evidenced by temperature and/or emission of steam in November 2010 and 2012 from Harry’s Dream (HD2 and HD3), Warren Cave (WC1, WC2, WC3, WC4, WC7, and WC8), Haggis Hole, (HH), Mammoth Cave (MC), Hut Cave (Hut), and Heroine Cave (HC) (fumarolic ice caves on Mt. Erebus volcano [21]). The samples were divided into four types: Mainly weathered basaltic/phonolitic sand (HD2, HD3, WC3, WC4, WC8 and Hut); pebbles and rock fragments (WC7, HH, and MC); black porous glassy materials that appeared to be solidified lava (WC1, WC2, and HC); and ash sediment (WC10) with little to no organic material (Table S1). The sediments were collected aseptically using sterile 50 mm conical tubes and immediately sealed. Additional information and environmental parameters on the sampling locations are provided in Table S1 and in Tebo et al. [21].
To isolate bacteria found in these oligotrophic environments, we employed a culture strategy of long-term incubation in a nutrient-poor medium and screening of slow-growing colonies by direct PCR identification. Reasoner’s 2 A gellan gum medium (10% R2AG) [23] and FS1VG medium [24] were used for bacterial isolation. The 10% R2AG is a 10-fold diluted R2A broth (Nihon Seiyaku, Tokyo, Japan), solidified with 15 g/L gellan gum (Kanto chemical, Tokyo, Japan) with 2 g/L CaCl2. The 10% R2AG and FS1VG media were adjusted to a pH of 4.5 or 6.0, with or without 30 mg/L sodium azide. Samples used for isolation were selected from Warren Cave and Harry’s Dream, where the presence of relatively large numbers of bacteria (>107/g) was confirmed in a previous report [21], and a total of nine samples, WD1, 2, 3, 4, 7, 8, and 10 and HD2 and 3, were used without any other pretreatment such as drying or dilution. For the sandy and ash samples with fine particles (WC3, 4, 8, 10, and HD2, 3), approximately 50 mg of sample were spread directly on plates. Glassy materials (WC1 and WC2) were embedded directly in plates using 2-3 pieces (approx. 200 mg). Pebbles and rock fragments (WC7) were crushed and approximately 50 mg of debris were spread directly onto the plates. All plates were incubated at 15 °C, 30 °C or 37 °C under dark conditions. New colonies were marked as they appeared and selection of the isolates was performed by picking only colonies that appeared after four weeks of incubation. We selected colonies around the sediment or in the cracks created during spreading using a magnifying glass.
For identification, colonies were picked with a sterile toothpick, re-streaked, stabbed on fresh medium, and subsequently suspended in 20 μL sterilized 0.05 M NaOH. Suspensions were heated at 100 °C for 15 min, and supernatants were used as template DNAs for PCR. Partial 16 S rRNA gene sequences were amplified by PCR using commonly used bacterial primer set 27 F (5′-AGATTTGATCCTGGCTCAG-3′) and 1492 R (5′-GGTTACCTTGTTACGACTT-3′) or 536 R (5′-GTA TTA CCG CGG CTG CTG-3′) with TaKaRa ExTaq DNA polymerase (Takara Bio, Shiga, Japan) as previously described [23]. Sequencing was performed at Eurofins Genomics (Louisville, KY, USA), using a 3730xl DNA analyzer (Applied Biosystems, CA, USA). Sequence similarities with closest species were calculated using EZbiocloud’s Identify Service (https://www.ezbiocloud.net/identify). Subsequently, cells in the stab identified as “Ca. Eremiobacterota” by the direct PCR identification were serially diluted and stabbed onto new plates until multiple pure cultures of Eremiobacterota were obtained. The isolate was designated as strain WC8-2.
Whole genome sequencing and annotation
Genomic DNA was extracted from WC8-2 cells grown in 10% R2A broth (pH6.0) with air/CO2 (90:10, v/v) at 30 °C for 30 days under a 12/12 h light/dark regime with incandescent light (250 μmol m–2s–1), using a Puregene Yeast/Bact. Kit B (Qiagen, Germantown, MD, USA) [25]. Sequencing was performed by Macrogen Japan Corp., on a NovaSeq 6000 (Illumina, Inc., San Diego, CA, USA) and PacBio RSII (Pacific Biosciences of California, Inc. Menlo Park, CA, USA). The gap-free complete genome was assembled de novo using the Unicycler version 0.4.8 hybrid assembly pipeline with default settings [26]. Completeness and contamination levels were estimated using CheckM [27]. The genome was annotated using the DDBJ Fast Annotation and Submission Tool (DFAST) [28] and the BlastKOALA web server version 2.2, and was visualized using CGView Server [29] (http://cgview.ca/).
Phylogenetic analysis of “Ca. Eremiobacterota”
Identification of strain WC8-2 was performed using the Genome Taxonomy Database Toolkit (GTDB-Tk) (ver. 2.1.0), which produces standardized taxonomic labels that are based on those used in the Genome Taxonomy Database [30]. Terrabacterial genomes including “Ca. Eremiobacterota” MAGs and related genomes were retrieved from the Genome Taxonomy Database (GTDB) (July 2022) and the NCBI RefSeq database (July 2022). Full-length 16 S rRNA gene sequences were retrieved from the WC8-2 genome (WPS_r00030) and the NCBI database (Table S2). Multiple sequences were aligned using SINA (version 1.2.11) [31]. IQ-TREE version 1.6.12 [32] was used to build the phylogeny. ModelFinder [33] was used to determine the optimal evolutionary model for phylogeny building, which selected the TNe+I + G4 model. Branch support was calculated using 1000 ultrafast bootstraps [34]. The pairwise 16 S rRNA gene sequence similarities were determined using SDT software. Phylogenomic analysis based on 400 marker proteins was carried out using PhyloPhlAn v3.0 [35]. Diamond v5.2.32 [36], MAFFT v7.453 [37], and TrimAI were utilized for orthologs searching, multiple sequence alignment within the superphylum Terrabacteria, and gap-trimming, respectively. Gappy sites and sequences with >50% gaps were deleted from the alignments. IQ-TREE version 1.6.12 [32] was used to build the phylogenomic tree. ModelFinder [33] was used to determine the optimal evolutionary model for phylogeny building, which selected the LG + F + R9 model. These analyses were conducted using the “AOBA-B” super- computer (NEC, Tokyo, Japan) with 2CPUs (EPYC7702, AMD, CA, US) and 256GB of RAM. The related similarity of genomes between strain WC8-2 and relatives was estimated using average nucleotide identity (ANI) values, which were calculated using OrthoANI calculator in the EzBio-Cloud web service [38]. The related similarity between strain WC8-2 and its sister phyla with one representative from each class was assessed by pairwise Average Amino acid Identity (AAI) values using the online tools at the Kostas Konstantinidis Lab website Environmental Microbial Genomics Laboratory (http://enve-omics.ce.gatech.edu/aai/). The MAGs used in the tree are listed in Table S3.
Phylogeny of photosynthesis- and “atmospheric chemosynthesis”- associated genes
We retrieved phototrophy- and “atmospheric chemosynthesis”-related protein sequences from the WC8-2 genome, “Ca. Eremiobacterota” MAGs, and known phototrophs genomes (Table S3) using the local BLAST server (SequenceServer 1.0.14 [39]) with reference sequences (Table S3) or annotated sequences as queries. Sequences were aligned using MUSCLE and poorly aligned regions were removed using Gblocks version 0.91b [40] or by manual inspection. The alignment sequences were concatenated into a single sequence. The ML tree was constructed using IQ-TREE with the best-hit evolutionary rate model: LG + I + G4 for HhyL, CbbL, BchXYZ and CbbL, LG + F + I + G4 for BchLNB, and LG + F + G4 for PufML, BchI, and BchD. All trees were visualized using iTOL (version 5.0) [41]. All sequences used in the trees are listed in Table S3.
Analysis of bacterial communities in the fumarolic ice caves
Environmental DNA was extracted from the samples (0.1 g) (WC7, WC8, HD3, MC, Hut, HH, HC) using a DNeasy PowerSoil Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The V4 region of the 16 S rRNA gene was PCR amplified using primers with adapter sequences (V3-V4f_MIX) [42]. The PCR cycling was carried out using the following parameters: 94 °C for 2 min followed by 25 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min with a final extension at 72 °C for 2 min. Library construction and sequencing were performed at the Bioengineering Lab (Kanagawa, Japan) using MiSeq (Illumina). Briefly, adaptor and primer regions were trimmed using the FASTX-Toolkit v0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit). Read sequences of ≤40 bp with ambiguous bases and low-quality sequences (quality score, ≤Q20), together with their paired-end reads, were filtered out using Sickle v1.33 (https://github.com/najoshi/sickle). High-quality paired-end reads were merged using PEAR v0.9.10 with default settings [43]. Merged sequences of ≤245 and ≥260 bp were discarded using SeqKit v0.8.0 [44]. Operational taxonomic units (OTUs) were classified using QIIME v1.9.1 and the SILVA database (release 132) with 97% identity. To study the phylogeny of the OTUs assigned to the “Ca. Eremiobacterota”, a neighbor-joining (NJ) tree of the OTUs was constructed [45] as described above.
Microscopic observation
Electron microscopic observations of the cells were performed at Tokai Electron Microscopy (Nagoya, Japan). Briefly, the cells grown in 10% R2A broth (pH6.0) with air/CO2 (90:10, v/v) at 30 °C for 15 days under a 12/12 h light/dark regime with incandescent light (250 μmol m–2s–1) were fixed with 4% paraformaldehyde (PFA) and 4% glutaraldehyde (GA) in 0.1 M phosphate buffer (PB) at pH7.4, and postfixed with 2% OsO4 in 0.1 M PB. Cells were then dehydrated using graded ethanol solutions. The dehydrated cells were polymerized with resin, ultrathin sectioned, stained with 2% uranyl acetate, then secondary-stained with Lead stain solution. A transmission electron microscope (TEM) (JEM-1400Pus; JEOL, Tokyo, Japan) was used to observe the ultrathin sectioned cells at 100 kV acceleration voltage. To observe the negative-stained cells, PFA- and GA-fixed cells were adsorbed on formvar film-coated copper grids and stained with 2% phosphotungstic acid solution (pH 7.0) and observed using a TEM at 100 kV. DAPI (4,6-diamidino2-phenylindole) and Nile-Red staining was performed by incubating 0.1 mL cell suspension with a 1 mL staining solution (1 mg/L DAPI and 1 mg/L Nile Red in PBS buffer) for 10 min. The stained cells were observed under a fluorescence microscope (Olympus AX80T; Olympus Optical; Tokyo, Japan).
Growth assay
Unless otherwise noted, all cultures were grown in 100 mL butyl stopper- and screw-cap-sealed glass vials containing 50 mL liquid medium (pH6.0) at 30 °C. Growth was monitored by optical density at 600 nm (OD600) using a spectrophotometer (BioSpectrometer Basic; Eppendorf; Tokyo, Japan). Initial cell density was adjusted to 0.005 (OD600). Specific culture conditions are described below. All anaerobic growth tests were conducted with 100% N2 gas in the headspaces and supplemented with a reducing agent (0.3 g/L cysteine-HCl) and a redox indicator (1 mg/L resazurin).
Optimal culture conditions
Cell growth in different media was examined using 1, 10, 20, 100% (a full strength) R2A broth and Basal_YE with air/CO2 (90:10, v/v) under a 12/12 h light/dark regime with incandescent light (250 μmol m–2s–1) for 25 days. Basal_YE contained (l−1) 0.44 g KH2PO4, 0.1 g (NH4)2SO4, 0.1 g MgSO4.7H2O, 0.3 g yeast extract, and 1 ml trace element SL-8 [refer to DSMZ745]. Cell growth at different temperatures (10, 13, 20, 25, 30, 33 and 37 °C; pH6.0), pH (3.7, 4.5, 6.0, 7.0, 8.0, and 9.0), and NaCl (0, 1, 10, 20, and 30 g [l−1]; pH6.0) was examined using Basal_YE with air/CO2 (90:10, v/v) for 25 days under a 12/12 h light/dark regime with incandescent light (250 μmol m−2s−1). The following pH buffer solutions were used: acetic acid/sodium acetate for pH 4–6, K2HPO4/KH2PO4 for pH 6–8, sodium bicarbonate/sodium carbonate for pH 9–10. To examine colony formation on solid media, WC8-2 was streaked or stabbed on 10% R2AG medium (pH 6.0) and incubated at 30 °C for 30 days under a 100% air atmosphere.
Optimal oxygen and carbon dioxide conditions
To determine the preferred O2 concentration, WC8-2 was grown in Basal_YE (pH 6.0, 30 °C, no NaCl) in butyl stopper-sealed glass bottles with the atmosphere in the headspace adjusted to different N2/O2/CO2 ratios (70:20:10%, 80:10:10%, 89:1:10%, 90:0:10% v/v) after removing oxygen with 100% N2 gas. Cultures were incubated for 25 days under a 12/12 h light/dark regime with incandescent light (250 μmol m–2s–1). To determine the CO2 preference, the gas phase was adjusted to different N2/O2/CO2 ratios (70:20:10%,75:20:5%, 80:20:0% v/v) in sealed bottles or 100% air (plugged with a BIO-SILICO N-38 sponge plug; Shin-Etsu Polymer Co., Ltd, Tokyo, Japan; breathable culture-plug) under the same conditions as the O2 preference test.
Photoorganoheterotrophic (or photoorganoautotrophic) and chemoorganoheterotrophic (or chemoorganoautotrophic) growth
The utilization of organic compounds as carbon sources/organic electron donors was tested in Basal medium (Basal_YE without yeast extract, pH 6.0) supplemented with one of the following sources (l-1): 0.3 ml of glycerol, or 0.3 g of sucrose, d-glucose, d-ribose, maltose, l-leucine, l-isoleucine, l-valine, l-serine, l-lysine, taurine, yeast extract or gellan gum, 1 ml of vitamin B12 solution (2 mg/L). Utilization was assessed by measuring growth of the cultures during a 25-day incubation at 30 °C in continuous light (250 μmol m–2s–1) for photoorganoheterotrophy, or continuous dark for chemoorganoheterotrophy under aerobic (air/CO2 [90:10, v/v]) and anaerobic (N2/CO2 [90:10, v/v]) conditions.
Photolithoautotrophic and chemolithoautotrophic growth
Cells were inoculated into amended PSB2 [46] as described below or Basal medium with 5 mM Na2S or Na2S2O3 or 1% H2 (v/v; in the gas phase) as electron donors, and cultivated in continuous light (250 μmol m–2s–1) for photolithoautotrophic growth, or continuous dark for chemolithoautotrophic growth under aerobic (air/CO2 [90:10, v/v]) and anaerobic (N2/CO2 [90:10, v/v]) conditions for 60 days at 30 °C. The amended PBS2 contained (L-1): 0.5 g NH4Cl, 1.0 g KH2PO4, 0.2 g NaCl, 0.4 g MgSO4.7H2O, 0.05 g CaCl2.2H2O, 4.2 g NaHCO3, 1 ml trace element SL-8 [refer to DSMZ745], and 1 ml vitamin B12 solution (2 mg/L), pH6.0.
Fermentative or anaerobic growth
Anaerobic growth was examined in continuous dark in 20% R2A broth (pH 6.0) with N2/CO2 (90:10, v/v) supplemented with 5 mM Na2SO4, NaNO3, or dimethyl sulfoxide (DMSO) as electron acceptors for 60 days at 30 °C.
Pigment assays
Cells grown in Basal_YE (pH6.0) with air/CO2 (90:10, v/v) at 30 °C for 14 days (exponential growth phase) under continuous light (250 μmol m–2 s–1) and continuous dark were used for the pigment assays. The absorption spectrum was determined in a cell suspension in 60% (w/v) sucrose and in a 100% methanol extract using a spectrophotometer (V-630; JASCO, Tokyo, Japan) at 350–1100 nm. The BChl a concentration was determined spectroscopically in 100% methanol [47]. Dry cell weight was measured after harvested cells were washed twice with Milli-Q water and dried at 80 °C for 3 days. The extract was also analyzed by HPLC (NEXERA X2; Shimadzu; Kyoto, Japan) equipped with a 4.6 × 250 mm COSMOSIL 5C18-AR (Nakarai Taque; Tokyo, Japan) with isocratic elution of 92.5% (v/v) methanol in water at a flow rate of 1.0 mL/min. BChl a was monitored at 766 nm using a diode-array spectrophotometer detector (SPD-M20A; Shimadzu; Kyoto, Japan).
Observation of taxis
To study phototaxis in WC8-2, cells were grown in 20% R2A broth with air/CO2 (90:10, v/v) for 14 days under a 12/12 h light/dark regimen. Cultures were transferred to tissue culture flasks (175 cm2, canted neck, Iwaki, Shizuoka, Japan). Light sources were ultraviolet (UV) at 395 nm (Linkman, Fukui, Japan), blue at 470 nm (CREE, Durham, NC, USA), green at 502 nm (Linkman), red at 653 nm (LENOO, Shinpei, Taiwan), and near-infrared (NIR) at 880 nm (LENOO). The light-emitting device was constructed by assembling LEDs on a breadboard with a power supply. Cultivations were illuminated with each wavelength using a light-emitting device from the underside and incubated at 20 °C for 18 h. Cells aggregating toward a light source was taken to indicate phototaxis. As a control, culture vessels were wrapped in aluminum foil to block light. Images and time-lapse video were captured using an iPhone 6 S camera.
Stable carbon isotope ratio mass spectrometry (IRMS)
The WC8-2 cells were grown in Basal_YE (pH6.0) with air/13CO2 [90:10, v/v] and air/unlabeled CO2 [90:10, v/v] under continuous light (250 μmol m–2s–1) for photoheterotroph and continuous dark for chemoheterotroph at 30 °C for 14 days (exponential growth phase). Approximately 10 mg culture biomass was collected, washed in HCl overnight, rinsed three times with deionized water, and placed into tin capsules. Stable carbon isotope ratios (δ13C) were analyzed at Shoko Science (Saitama, Japan) using a Delta V Advantage (EA-IRMS; Thermo Fisher Scientific, Bremen, Germany). The standard for C isotope ratio analysis was Vienna PeeDee Belemnite (VPDB). The δ13C values of 13CO2-cultivated cells exceeded the optimum calibration range of the instrument, but were used in this study to provide conclusive evidence that inorganic carbon was incorporated into the biomass.
Quantitative reverse transcription PCR (qRT-PCR)
Total RNA extraction and cDNA synthesis
Total RNA was extracted from cells grown in Basal_YE (pH6.0) with air/CO2 [90:10, v/v] in continuous light (250 μmol m–2s–1) for photoheterotrophic conditions and in continuous dark for chemoheterotrophic conditions at 30 °C for 14 days (exponential growth phase) using the Total RNA Purification Kit (Norgen, Biotek Corp, Ontario, Canada). DNA was removed from the extracted nucleic acids using an RNase-Free DNase I Kit (Norgen, Biotek Corp) according to the manufacturer’s protocol. The absence of DNA in the RNA samples was confirmed by PCR without reverse transcriptase. cDNA was generated from 500 ng total RNA using a TaKaRa PrimeScript™ 1st strand cDNA Synthesis Kit (TaKaRa Bio) with random hexamers according to the manufacturer’s protocol.
Primer design, specificity and efficiency
The following three photosynthesis- and CO2-fixation-related genes in the WC8-2 genome were selected for qRT-PCR: bchM encoding an enzyme involved in BChl synthesis, pufL encoding the anoxygenic Type II photochemical reaction centers L-subunit, and cbbL encoding the large subunit of type IE RuBisCO. The RNA polymerase subunit beta (rpoB) was used as a housekeeping reference gene. Primers for qRT-PCR were designed with Primer3 (v. 0.4.0) [48] with the following criteria: product size ranging from 80 to 150 bp, optimum Tm of 60 °C and GC content about 50 to 55%. Standard RT-PCR confirmed that each primer set amplified only a single product with expected size (data not shown), and the product was also sequenced using Sanger sequencing at Macrogen Japan Corp to confirm the candidate products. Primer efficiency was calculated for qRT-PCR using the slope of the calibration curve based on a 20-, 40-, 80-, 160-fold dilution series of cDNA samples [49]. In addition, the specificity of the primers was determined by the confirmation of a single peak in the melting curve. All information about the primers is shown in Table S4.
qRT-PCR
qRT-PCR was performed using a MiniOpticon Real-Time PCR System (Bio-Rad, Marnes la Coquette, France). The reaction mixture contained 10 μL TB Green Premix Ex Taq II (Tli RNaseH Plus, Takara Bio), 0.8 μL 10 mM primer, 2 μL of a 20-, 40-, 80-, 160-fold dilution series of cDNA, and 6.4 μL water. qRT-PCR was performed using the following protocol: denaturation at 95 °C for 30 s; denaturation and amplification at 95 °C for 5 s and 60 °C for 30 s, respectively (40 cycles). Fluorescence was measured at the end of the amplification step, and amplified products were examined by melting curve analysis from 60 to 95 °C. Each reaction was performed in three independent cultivations. Relative gene expression fold change was calculated using the comparative Ct method (2−ΔΔCt) [49]. Normalized expressions were used for reaction in dark. The 2−ΔΔCt values ≤0.5 were defined as downregulated and values ≥2.0 as upregulated, with P < 0.01 by the Student’s t-test between reactions in light and dark for each gene.
Chemotaxonomic analysis and other tests
Gram staining was conducted using a modified Hucker method [50]. To study the chemotaxonomic characteristics of WC8-2, we used the cells grown in 20% R2A (pH6.0) with air/CO2 (90:10, v/v) at 30 °C for 18 days under a 12/12 h light/dark regimen. Major menaquinone, polar lipids, and cellular fatty acids were determined following established protocols [4]. Briefly, the major menaquinone was isolated by thin-layer chromatography (TLC) [46] and identified using liquid chromatography. Polar lipids were identified using 2D TLC sprayed with detection reagents [51]. The fatty acid composition was identified using the Sherlock Microbial Identification System (v 6.0; MIDI) with the TSBA6 database. These chemotaxonomic analyses were conducted at Techno Suruga Laboratory Co., Ltd. (Japan).
Source: Ecology - nature.com
