Genomic DNA extraction, whole genome sequencing, and genomic analysis of YS11
Genomic DNA of Lysinibacillus boronitolerans YS11 was extracted using Wizard Genomic DNA purification Kit (Promega, USA). Whole-genome sequencing was performed using Pac Bio RSII Single Molecule Real Time (SMRT) sequencing method (Pacific Biosciences, USA) with a SMRTbell template library in Chunlab (Seoul, South Korea) according to manufacturers’ instructions. The complete genome was achieved using CLgenomics program provided by Chunlab. Gene annotation and assembly were conducted with NCBI Prokaryotic Genomes Automatic Annotation Pipeline. CLgenomics program provided by Chunlab and kegg pathway searching were used for genomic analysis of YS11.
L. boronitolerans YS11 culture conditions
For growth test of strain YS11, a seed culture was incubated in Luria-Bertani (LB) medium, which is composed of 1% tryptone (Bioshop), 1% sodium chloride (Sigma-Aldrich), .5% yeast extract (Bioshop), at 30 °C overnight with shaking at 220 rpm. Next, 1 ml of the seed culture was transferred to a 1.7-ml microtube and washed twice with phosphate buffered saline (PBS), which is composed of .08% NaCl, .02% KCl, .144% Na2HPO4, .024% KH2PO4. Next, strain YS11 at density of 1 × 106 CFU/ml was transferred into 25 ml of calcium acetate (CaAc) medium (15.8 mM calcium acetate, .4% yeast extract, .5% glucose) or sodium acetate (NaAc) medium (15.8 mM sodium acetate, .4% yeast extract, .5% glucose) in a 50-ml flask to observe the growth on Ca-rich or Ca-poor condition. Growth curves were generated based on colony-forming unit (CFU). The culture was serially diluted from 100 to 10−8 using PBS for dilution. Then, 200 µl of the diluted bacteria was streaked into pre-heated LB plates. The colonies were counted after 12 h incubation at 30 °C.
RNA isolation and transcriptomic analysis by RNA-seq
Total RNA was obtained from mid-exponentially grown YS11 cells (6 h) from NaAc and CaAc media using a RNeasy kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The extracted total RNA was then sent to Chunlab (Seoul, South Korea) for RNA sequencing and alignment. Ribo‐Zero rRNA removal kit (Epicentre, Medison, WI, USA) was used for ribosomal RNA depletion according to the manufacturer’s instructions. Libraries for Illumina sequencing were constructed with TruSeq Stranded mRNA sample prep kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. RNA sequencing was conducted on Illumina HiSeq 2500 platform using single-end 50 bp sequencing. Sequence data for the reference genome (Lysinibacillus boronitolerans YS11) were retrieved from the NCBI database. Quality-filtered reads were aligned to the reference‐genome sequence using Bowtie2. The abundance of relative transcript was shown by reads per kilobase of the exon sequence per million mapped sequence reads (RPKM) defined as total exon reads/(mapped reads in millions × exon length in kilobases). Metabolic pathways were analyzed based on KEGG pathway analysis and BLAST alignment with proteins using CLRNASeq program provided by Chunlab.
Isolation procedure for strain AK13
To attain bacteria native to the environment of L. boronitolerans YS11, soil sample was amassed from the rhizosphere of Miscanthus sacchariflorus near Seongbukcheon in South Korea (37° 34′ 44.9″ N 127° 01′ 28.8″ E) (Lee et al. 2017). A 1 g of soil sample was prepared and washed with PBS (pH 7.5). It was then spread onto pH 13 adjusted LB agar. Plates were cultured overnight at 30 °C. Colonies that appeared on the agar plate were isolated and single colonies were roughly cocultured with YS11 in LB medium with three replicates to observe any difference in optical density (OD) compared to YS11 single culture. Cells were grown in PVC 96-well microtiter plates (BD Biosciences) at 30 °C and measured optical density at 595 nm (OD595) using biophotometer (Eppendorf, Germany). An isolate that showed a bump that had more than 1% increase in OD565 value in the growth curve during coculture was observed and named as AK13.
Phylogenetic analysis of 16S rRNA gene sequence
The 16S rRNA gene of newly isolated alkaliphilic strain AK13 was amplified using universal bacterial 16S rRNA gene primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). Polymerase chain reaction (PCR) was conducted with the following cycling conditions: 94 °C for 90 s followed up by 25 cycles of 94 °C for 45 s, 58 °C for 45 s, and 72 °C for 45 s, and finally an extension step at 72 °C for 5 min. Sequence similarity with other bacteria was determined with EzTaxon program. A neighbor-joining tree was constructed using a distance matrix calculation method in MEGA 7. Bootstrapping was conducted with 1000 iterations. The fasta file of 16S rRNA gene sequence of strain AK13 was deposited at GenBank database (MK517564).
Cocultivation for L. boronitolerans YS11 and Bacillus sp. AK13
Strain YS11 was incubated overnight in pH 6.8 LB broth while strain AK13 was cultured in pH 8 LB broth at 30 °C overnight. Cells were then washed twice with PBS. For coculture of YS11 plus AK13, 1 × 106 CFU/ml of overnight isolates were inoculated into YL medium composed of .4% yeast extract and .34% calcium-acetate at 1:1 ratio. For single culture of YS11 and AK13 each, 2x106 CFU/ml of overnight isolates were inoculated into YL medium. They were incubated at 30 °C with shaking at 220 rpm.
The growth rate of coculture and single culture was measured by counting colony-forming unit (CFU). Normal neutral LB agar was used as a selective medium for strain YS11 while LB agar with pH 12 was used as a selective medium for alkaliphilic strain AK13. Alteration of pH and unbound calcium ion concentration were measured using a pH electrode (Thermo Fisher Scientific, USA) and a calcium-ion selective electrode (ISE) (Thermo Fisher Scientific, USA), respectively. The 7 ml volume of sub-samples were removed from the master volume, over time for 24 h. For these sub samples, organic matters were removed by centrifugation at 4000 rpm for 10 min prior to measurements. These experiments were conducted in triplicates.
For coculture experiment in agar plates, two types of modified B4 medium, CaAc medium and NaAc medium, were used. Then 20 µl of each YS11 and AK13 overnight cultures (1 × 106 CFU/ml) were spotted on the edge and opposite sides of the plate and then cultured at 30 °C for 24 h.
FE-SEM and FTIR for morphological analysis of CaCO3
Mineral from YS11 single culture or YS11 plus AK13 coculture in YL medium was dried in an oven drier at 100 °C overnight prior to FE-SEM analysis. The sample was placed onto a carbon tape for 1 h to be dried. After blowing unattached particles with nitrogen gas, samples were Pt-coated and analyzed with a Quanta 250 FEG FE-SEM (FEI, USA). For FTIR analysis, an oven dried sample was resuspended in distilled water and analyzed using ATR method in FTIR (Agilent).
pH measurement in pH increasing condition
To determine pH increasing mechanisms in various conditions, 106 CFU/ml of overnight YS11 was inoculated into media consisting of either .8% yeast extract or .8% nutrient broth or MSB basal medium each supplemented with .1% pyruvate, .1% l-serine, or .1% pyruvate plus .1% l-serine. Total of 50 ml volume culture was incubated in 100 ml volume of Erlenmeyer flasks. OD600 and pH were measured in 24-h interval during their incubation at 30 °C with agitation at 220 rpm. 7 ml volume of sub samples were taken out from three replicates and measured right away. For the optical density measurement, 10−1 dilutions were done for each sample.
Ammonia measurement assay
Ammonia concentration during incubation of YS11 in YL medium was measured using ammonia assay kit (Sigma-Aldrich). Briefly, supernatant (50 µl) of YS11 cultured in YL medium was mixed with ammonia assay reagent (500 µl) containing α-ketoglutaric acid and NADPH. Then, 5 µl of l-glutamate dehydrogenase was added to each sample and incubated for 5 min at 30 °C. The absorbance of each solution was measured at wavelength of 340 nm.
CLSM analysis to examine biofilm and calcium carbonate development
Biofilms from YS11 and YS11 plus AK13 were stained for 30 min with FilmTracer™ SYPRO® Ruby biofilm matrix dye at room temperature and visualized using CLSM (LSM700; Carl Zeiss, Jena, Germany). FilmTracer™ SYPRO® Ruby biofilm matrix stained biofilm cells were used to obtain confocal images under red fluorescent light (excitation wavelength: 450 nm, emission wavelength: 610 nm). Calcium carbonate was visualized under blue fluorescent light by reflection signal of excitation between 483 and 493 nm (Bai et al. 2017). Both biofilms and precipitated calcium carbonate were evaluated for height and density of morphology (C-Apochromat 40×/1.20 W Korr M27; Carl Zeiss).
Biofilm formation assay
Overnight cultures of YS11 and AK13, with the same condition mentioned in cocultivation section, were washed with room-temperature PBS twice at room-temperature. Overnight cells were then inoculated into 1 ml volume of YL medium to contain 2 × 106 CFU/ml of strain YS11 or mixture of YS11 and AK13 at cell density of 1 × 106 CFU/ml each in 48-well microtiter plates. The volume of the cells inoculated was measured by converting OD600 value of the overnight cells and converting them to the standard CFU/ml graph. Cell inoculated microtiter plates were then incubated at 30 °C with different time intervals (24 h, 48 h, 72 h) in static condition. Biofilms were stained with crystal violet dye using crystal violet staining assay (O’Toole 2011). Crystal violet attached to biofilms was dissolved in 95% ethanol solution. The optical density at 595 nm (OD595) was then measured for biofilm quantification using a multi-detection microplate photometer (HIDEX Sense, Finland).
FAME analysis
Fatty acids from YS11 cultured in NaAc and CaAc media were extracted. Fatty acids were then saponified and methylated to be transformed into fatty acid methyl esters (FAMEs). At first, the bacterial cells washed twice with PBS, and were harvested with centrifugation. The cells were resuspended in 1 ml of saponification reagent into 15 ml falcon tube. This was vortexed for 30 s and heated at 100 °C for 5 min. The cells were once again vortexed for 10 s and heated at 100 °C for 25 min. Then, they were heated at room-temperature for 1 min and 2 ml of methylation reagent were added. This was vortexed for 10 s and heated at 80 °C for 10 min. The solution was cooled at room-temperature for 1 min. 1.25 ml of extraction solvent was added and vortexed for 10 min where the solution divides into two layers. The upper later was transferred into new 15 ml falcon tube. 3 ml of base wash solution was added and vortexed for 5 min. The upper layer of the solution was finally transferred into GC vials. These extracted FAMEs were analyzed using an Agilent 7890 GC system with a flow ionization detector and an HP-Ultra-2 capillary column (crosslinked 2.5% phenylmethyl silicone, 25 m, 200 mm i.d., film thickness: .33 mm). MIDI-2000 calibration standard was used to calibrate FAME values. FAMEs were identified and qualified using the Sherlock 6.0B MIDI software according to their equivalent chain value.
FE-SEM/EDX analysis for CaCO3 nanoparticle formation
YS11 was incubated in CaAc medium for 6 and 12 h at 30 °C prior to FE-SEM and EDS analyses. Cells were fixed first with low-strength Karnovsky’s solution (2% paraformaldehyde, 2.5% glutaraldehyde, and .1 M phosphate buffer, final pH 7.2) for 2 h. Secondary fixation was done using 2% osmium tetroxide solution for 2 h. These fixed samples were gradually dehydrated with ethanol (30%, 50%, 70%, 100%) for 10 min each and placed onto aluminum stub for 4 days to be dried at room-temperature. These samples were then coated with platinum and analyzed using field-emission scanning electron microscope Quanta 250 FEG (FEI, USA) and energy dispersive X-ray spectrometer (EDS).
Bacterial strains culture deposition number
A strain YS11 culture was stored in Agricultural Culture Collection (KACC) under number KACC 81048BP. A strain AK13 culture was stored in KACC with a deposition number of KACC 81070BP.
Nucleotide and SRA sequence accession number
The complete genome sequence of Lysinibacillus boronitolerans YS11 has been deposited in NCBI under the GenBank accession number CP026007.1. The RNA-seq of Lysinibacillus boronitolerans YS11 has been deposited in NCBI under the SRA number SRR8083459.