Isolation and cultivation
We considered that culturing highly diverse bacteria is indeed possible when their ecological niche is sufficiently well mimicked. We then designed the “medium S” for cultures. The basal marine medium was designed in this study to match the salinity of seawater, which contained 80 % natural seawater, 20 % artificial seawater (consisting of 3.5 % NaCl, 0.32 % MgSO4, 0.22 % MgCl2, 0.12 % CaCl2, 0.07 % KCl, 0.02 % NaHCO3, and 1.0 % Na2S2O3), and 100 g l− 1 fresh sediment. After filtering with gauze, filtrates were obtained. For carbon and nitrogen sources, the initial cultivation medium for marine bacterial strains was supplemented with 0.33 g L− 1 sodium pyruvate, 0.5 g L− 1 peptone (Hopebio), and 0.1 g L− 1 yeast extract (Hopebio). The pH of the medium was adjusted to 7.5 with NaOH. All medium components were sterilised by autoclaving, except for NaHCO3, which was filtered through a 0.22 µM pore polyethersulfone filter.
Sediment samples were collected from Xiaoshi Island (37°31′ 36″ N, 122°00′ 58″ E), Weihai, PR China, which is a national marine nature reserve. The sediment sample was collected from approximately 10 cm depth in the intertidal zone after high tide, in November 2018. The sample was serially diluted to 10− 3 with sterilised seawater and 0.1 mL aliquots of each dilution were spread onto 1/10-strength marine medium using 2216 (MA; BD) plates as the basal medium. The plates were incubated in an aerobic environment for 30 d at 25 ℃. After incubation, a tiny pink colony was selected from the plate. After repeated streaking, the strain was routinely cultivated on MA at 30 ℃ for 10–15 d. The strain was stored in sterile 15 % (v/v) glycerol supplemented with 1 % (w/v) NaCl suspensions at -80 ℃. The novel bacterium, designated T37T, was isolated and selected for detailed taxonomic analyses, and strain T37T was cultivated on MA at 37 ℃ unless otherwise mentioned.
Phylogeny-based on 16S rRNA gene sequences
To classify this bacterium, the 16S rRNA gene was amplified using primers 27F and 1492R (Lane 1991). Purified PCR products were sequenced by BGI Co., Ltd. (Qingdao, China), and a mostly complete 16S rRNA gene sequence was obtained. The mostly complete 16S rRNA gene sequence of strain T37T was preliminarily identified by searching for matches in the EzBioCloud (http://www.ezbiocloud.net) and GenBank databases (https://www.ncbi.nlm.nih.gov) for further phylogenetic analysis. Comparative sequence analyses of the mostly complete 16S rRNA gene sequences were used to determine the phylogenetic position of strain T37T to related strains.
To determine its exact taxonomic status, we performed further phylogenetic analyses. The 16S rRNA gene sequences of the novel strains, all the published verrucomicrobial strains and the uncultured strains, which the 16S rRNA gene sequence similarities were higher than the 86.5 % family threshold identity, were collected. Additionally, 16S rRNA gene sequences from genome sequences were extracted using RNAmmer version 1.2 (Karin et al. 2007) and compared with the 16S rRNA gene reference database from phylogenetic trees through the analysis of BLAST sequence alignment to ensure authenticity. Next, according to similarity and completeness, duplicate 16S rRNA genome sequences were discarded from the study. Finally, the 16S rRNA sequences of 125 strains were obtained. Using the MUSCLE program (Edgar 2004), trimAl (Capella-Gutierrez et al. 2009) was used to automatically remove heterotypic sequences and differential regions according to the length and number of ambiguous bases. A comprehensive sequence alignment was generated using sequences extracted from genomic sequences or 16S rRNA gene sequences previously obtained from type strains. Based on the 16S rRNA gene sequence, the phylogenetic tree analysis was reconstructed using the FastTree 2 program (Price et al. 2010) and RAxML version 8 (Alexandros 2014) and visualised using MEGA version 7.0 (Sudhir et al. 2016). Bootstrap analysis was performed with 1,000 replications to provide confidence estimates for tree topologies (Felsenstein and Joseph 1985).
Genome sequencing, annotation, and analysis
The biomass of strain T37T was collected, and genomic DNA was extracted according to the instructions of the genome extraction kit (TAKARA). The extracted DNA was detected by agarose gel electrophoresis for degradation and contamination, and a microspectrophotometer (Nanodrop, Thermo Fisher Science and Technology) was used to determine the extracted genomic DNA content, to ensure the content of at least 12 µg. Using the extracted genomic DNA as a template for the PCR, the 16S rRNA gene sequence was amplified and sequenced, and the sequencing results were verified by BLAST comparison. After the bacterial strain information was confirmed, the extracted genomic DNA was sent to Tianjin Novozhiyuan Bio-Information Technology Co., Ltd. for genome sequencing. The PacBio platform and Illumina platform were used for library construction and inspection. After qualified library inspection, PacBio Sequel and Illumina Novaseq PE150 were used for sequencing and genome assembly was completed using SMRT Link Version5.0.1 software (Simon et al. 2018). Protein-encoding regions were identified using the Cluster of Orthologous Group of Proteins (COG) (Tatusov et al. 2003). Gene content was annotated using the NCBI Prokaryotic Genome Annotation Pipeline, and the genes involved in metabolic pathways were analysed in detail using information from the KEGG database (Minoru et al. 2016). The secondary metabolic gene clusters were predicted by antiSMASH (Medema et al. 2011), active carbohydrate enzymes were predicted by Cazy (Carbohydrate-Active enZYmes Database) (Cantarel et al. 100AD), resistance genes were predicted by Comprehensive Antibiotic Research Database (CARD) (Alcock et al. 2019), and membrane transporters were predicted by Transporter Classification Database (TCDB) (Saier et al. 2006).
From the EzBioCloud (https://www.ezbiocloud.net) and NCBI databases (https://www.ncbi.nlm.nih.gov) all the genome of published verrucomicrobial strains were downloaded. Then, the bacterial core gene set coverage was used to examine genome integrity, determine whether the genome is polluted compared to the ContEst16S (Lee et al. 2017), and retain genomes that have fewer than 500 contigs, completeness of 95.0 % or greater, and no contamination. For the genomic phylogenetic tree, UBCG (Na et al. 2018) was used to analyse the phylogenetic relationships. Based on 49 genomes, FastTree 2 (Price et al. 2010) using GTR + CAT parameters and IQtree (Jana et al. 2016) using the GTR + F + I + G4 model was used to reconstruct the phylogenetic tree, and 1000 bootstrap replicates were used for analysis. Phylogenetic analysis was performed using MEGA version 7.0 (Sudhir et al. 2016).
The genome sequences of the strains of the order Verrucomicrobiales were obtained from the NCBI database. Calculations of genomic G + C content values and genomic size were also consistent with the taxonomic mill (García-López et al. 2019). The genomic sequences of 22 strains were analysed for their related species, including the average amino acid identity (AAI) (Rodriguez and Konstantinidis 2014), percentage of conserved proteins (POCP) (Qin et al. 2014), average nucleotide identity (ANI) (http://jspecies.ribohost.com/jspeciesws/) (Yoon et al. 2017), and the digital DNA-DNA hybridisation (dDDH) (http://ggdc.dsmz.de/distcalc2.php) (Goris et al. 2007) were determined. However, we focused on the genetic relationships among different genera; therefore, we focused on analysing the AAI values between the genomes. The AAI calculator estimates, which were calculated using Enve-omics (http://enve-omics.ce.gatech.edu/aai/index), using both best hits (one-way AAI) and reciprocal best hits (two-way AAI) between two genomic datasets of proteins. The AAI values were set at a threshold of 60.0 % as the boundary of the genus (Rodriguez and Konstantinidis 2014).
Cell morphological analysis
The determination of the cell characteristics of strain T37T was performed using cells grown on MB for 3 d at 37 ℃. Cell division, morphological analysis, and size were examined by light microscopy (Carl Zeiss Axioscope A1), scanning electron microscopy (model Nova NanoSEM450; FEI), and transmission electron microscopy (JEM1200, Japan).
The cells were collected and diluted 1000 times (OD600 = 1.0), washed twice with 0.1 mol/L phosphoric acid buffer (PBS) at pH 7.0, and resuspended in 200 µL staining solution FM4-64/water = 1:19; the final concentration of FM4-64 was 3 µg/mL, and the mixture was incubated at room temperature for 10 min. The cells were resuspended in 200 µL staining solution DAPI:water (1:25), the final concentration of DAPI was 1 µg/mL, and the mixture was incubated at room temperature for 10 min. The cells were washed twice with PBS and centrifuged at 2,500 × g for 1.5 min. Finally, the cells were resuspended in 200 µL PBS. During the entire process, the cells were observed and photographed using a fluorescence microscope. The FM4-64 dye stains cell membrane lipids and emits red fluorescence (maximum excitation/emission wavelength is approximately 515/640 nm) (Amerik and Hochstrasser 2006), and DAPI is a fluorescent dye that strongly binds to DNA (Piotr et al. 2001). According to the intensity of fluorescence, the amount of DNA can be determined by the amount of blue light emitted (maximum excitation/emission wavelength is approximately 340 nm/488 nm).
Transmission electron microscopy of ultrathin sections of strain T37T was performed as described by Pimentel-Elardo et al. (Pimentel-Elardo et al. 2003). For transmission electron microscopy, cells were grown in liquid culture and fixed with 2.5 % glutaraldehyde for 24 h at 4 ℃, washed three times with PBS (0.1 M), pH 7.0. In the fume hood, the sample was fixed with 1 % osmium acid solution for 1–2 h, the osmium acid waste solution was carefully removed, and the sample was washed three times with 0.1 mol/L PBS buffer at pH 7.0, 15 min each time. The samples were dehydrated with increasing concentrations of ethanol (from 30 to 100 %) for 15 min at each concentration and then treated with 100 % ethanol for 20 min. A drop of culture was incubated on a copper grid, the liquid was removed, and the cells were stained with a drop of 0.5 % uranyl acetate for 5 min. The cells were examined using transmission electron microscopy (JEM1200, Japan).
For scanning electron microscopy, colonies with surrounding material were fixed in 2.5 % glutaraldehyde for 4 h at 4 ℃, washed three times with 0.1 M PBS, pH 7.0, and dehydrated with increasing concentrations of ethanol (from 30 to 100 %) for 10 min. After critical-point drying and platinum coating of the dried material, the colonies were examined using scanning electron microscopy (model Nova NanoSEM450; FEI). Before photographing, it was important to select the field of view where the cells did not overlap and block each other. Additionally, it was necessary to select a relatively high magnification to view single cells.
The temperature range for growth was determined on MA at 4–45 ℃. The NaCl concentrations for growth were determined by incubating the bacteria in modified marine broth 2216 made with 0.5 % peptone, 0.1 % yeast extract, and artificial seawater (0.32 % MgSO4, 0.22 % MgCl2, 0.12 % CaCl2, 0.07 % KCl, 0.02 % NaHCO3, w/v) in the presence of 0.0–10.0 % (w/v) NaCl at intervals of 0.5 %. The effects of pH were determined by adding the appropriate buffers (Sangon), including MES (pH 5.5 and 6.0), PIPES (pH 6.5 and 7.0), HEPES (pH 7.5 and 8.0), Tricine (pH 8.5), and CAPSO (pH 9.0–9.5) to MB at a concentration of 20 mM, and the pH of the control groups was checked after autoclaving. The OD600 values of the cultures were measured after incubation for 3 d at 37 ℃. Cells of strain T37T were obtained from cultures grown on MA at 37 ℃ for 3 d, and the following phenotypic tests were performed as described in previous studies (Feng et al. 2020). Motility was determined by the hanging-drop method, and gliding motility was tested by inoculating the bacteria on 0.5 % agar. The Gram reaction was determined using the bioMérieux Gram-stain kit according to the manufacturer’s instructions. Growth under anaerobic conditions (10 % H2, 5 % CO2, and 85 % N2) was determined after incubation for 14 d in an anaerobic chamber with or without 0.1 % (w/v) KNO3. Susceptibility to antibiotics was investigated on MA using the disc diffusion method, and according to procedures outlined by the Clinical and Laboratory Standards Institute (Cockerill et al. 2011). Catalase activity was determined by observing bubble formation in a 3 % H2O2 solution. Oxidase activity was examined using an oxidase reagent kit (bioMérieux) according to the manufacturer’s instructions. Tests were performed for the hydrolysis of starch, casein, alginate, carboxymethylcellulose, and Tweens 20, 40, 60, and 80. All experiments were performed in triplicates. Biochemical tests were performed using API 50CHB (bioMérieux) kits (http://www.biomerieux-diagnostics.com/apir-id-strip-range) and API 20E (bioMérieux) kits were performed according to the manufacturer’s instructions, using the biomass of strain T37T grown on MA at 37 ℃ for 3 d. Production of other enzymes was assessed using API ZYM kits (bioMérieux). Carbon source oxidation was checked using BIOLOG GEN III microplates (http://www.biolog.com/). All API tests were performed according to the manufacturer’s instructions, except that the salinity was adjusted to 1.5 %. All API and BIOLOG tests were performed with two replicates.
Chemotaxonomic characterizations analysis
Biomass for the study of chemotaxonomic features of strain T37T was obtained from cultures grown in MB for 3 d (late logarithmic phase). Polar lipids were determined using 2D thin-layer chromatography (TLC) (Minnikin et al. 1982). Four separate TLC plates (EMD Millipore, 1.16487.0001) were prepared for each sample and individually stained using phosphomolybdic acid solution (total lipids), molybdenum blue solution (phosphates), α-naphthol sulfuric solution (carbohydrates), and ninhydrin (amines); all reagents were from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Isoprenoid quinones of strain T37T were analysed using reverse-phase HPLC (Kroppenstedt and Reiner 1982). The preparation and extraction of fatty acid methyl esters from biomass and their subsequent separation and identification by gas chromatography were performed as previously described (Athalye et al. 2010). The fatty acids were extracted according to the standard protocol of MIDI (Sherlock Microbial Identification System, version 6.1), methylated, and analysed using an Agilent 6890N gas chromatograph. Cellular fatty acids were identified using the TSBA40 database of the microbial identification system.