Sampling and isolation
An electronic waste associated bioaerosol sample collected from Guiyu, Guangdong province of China (23.32N, 116.36E) in November 2019, was used to isolate bacterial strains (Pan et al. 2021). Bioaerosols were collected by six-stage Andersen Cascade Impactors with 10 × diluted nutrient agar (NA) followed by aerobic incubation at 37 ℃ for 48 h. Single colonies were separated and purified by repeated streaking on fresh 2 × diluted NA plates. Only one light yellow isolate GA224T, a unique alkalophilic microorganism compared to other potentially novel bacteria, was selected for further study (Kim et al. 2014). The isolate was routinely cultured on 2 × diluted NA at 37 °C and preserved as a suspension in 2 × diluted nutrient broth (NB) with glycerol (15%, w/v) at − 80 °C. Strain GA224T has been deposited to the Japan Collection of Microorganisms (JCM) and Guangdong Microbial Culture Collection Center (GDMCC). For the comparative study, the reference strain Yonghaparkia alkaliphile KCTC 19126T was obtained from GDMCC and tested using the same laboratory conditions as for strain GA224T.
Morphological, physiological and biochemical characteristics
The morphological, physiological and biochemical characteristics of strain GA224T were characterized after 48 h of incubation at 37 ℃ on 2 × diluted NA or 2 × diluted NB with the pH adjusted to 8.0. Cell morphology, size, shape, flagellation and Gram staining were examined by transmission electron microscope (Harris 2015) and light microscopy, using freshly grown cells. Growth at various temperatures (4, 15, 25, 30, 37, and 40 °C) and numerous pH conditions (pH 4.5−10.5, 0.5 pH unit interval) were evaluated in 2 × diluted NB after 48 h of incubation at 37 °C. Two different buffers, 1M HCl (for pH 4.5–6.5), and 1M NaOH (for pH 7.0–10.5) were used for pH test. Salinity tolerance was evaluated in 2 × diluted trypticase soy broth supplemented with 0, 0.5 and 1.0−5.0% (w/v), at intervals of 1.0% after 48 h of incubation at 37 °C. Growth was determined by checking the optical density at 600 nm. Growth under anaerobic conditions was determined after incubation in an anaerobic incubator on 2 × diluted NA (pH 8.0). Catalase, and oxidase activities were determined using 3% (v/v) H2O2 and 1% (v/v) dimethyl-p-phenylenediamine hydrochloride reagent, respectively (Harrigan and McCance 1966). Enzyme activities and utilization of carbon sources were further determined using the commercial kits API ZYM (Biomerieux) and GEN III MicroPlate system (BIOLOG), according to the manufacturer’s instructions. Antibiotic sensitivity was tested by broth microdilution method (Balouiri et al. 2016) with 2 × diluted NB containing the following antibiotics: streptomycin sulphate, gentamycin solution, kanamycin sulfate, vancomycin hydrochloride, tetracycline hydrochloride, ciprofloxacin hydrochloride, cephalosporin, penicillin G, imipenem, and ampicillin.
For cellular fatty acid analysis, 40 mg of cells were harvested from 2 × diluted NA plates after incubation for 48 h at 37 ºC. Fatty acids were extracted, methylated, and saponified according to the manufacturer’s protocol of Microbial Identification System and identified by gas chromatography (Sasser 1990; Sun et al. 2018). The peaks obtained were then labeled, and the equivalent chain length values were computed by the Sherlock software. Menaquinones were extracted as described by Collins (Collins et al. 1977) and analyzed by reverse phase high-performance liquid chromatography (Ranmadugala et al. 2018). The polar lipids profile was analyzed by extracting cells with methanol-chloroform-saline (2:1:0.8, v/v/v) from 1 g of freeze-dried bacterial cells. Separation of lipids was performed by two-dimensional chromatography on a silica gel thin-layer chromatography plate using chloroform-methanol-water (65:25:4, v/v/v) in the first dimension and chloroform-methanol-acetic acid-water (80:12:18:5, v/v/v/v) in the second dimension (Park et al. 2018). The total polar lipids profile was detected using several color developing agents. The dodecamolybdophosphoric acid was used for detecting total lipid, molybdenum blue for phosphate, ninhydrin for free amino group, Dragendorff reagent for quaternary nitrogen and methyl naphthol for glycolipids as described by Tindall (Tindall 1990). The diamino acid of the cell wall was determined using thin-layer chromatography (Liu et al. 2017).
Phylogeny and genome analysis
Genomic DNA was extracted using the phenol-chloroform method (Wagner et al. 2015). The 16S rRNA gene sequence was amplified by using a PCR with bacterial universal primers (27F, 1492R) (Baker et al. 2003). The 16S rRNA gene sequence was determined by Sanger DNA sequencing. The sequence is available from the National Centre for Biotechnology Information (NCBI, https ://www.ncbi.nlm.nih. gov/) under the accession number MT214126.1. The BLASTN program was used to compare 16S rRNA gene sequence of strain GA224T with deposited reference sequences in the GenBank and Ezbiocloud database. The multiple alignments were accomplished using the CLUSTAL W program. The phylogenetic trees were constructed using both the neighbor-joining method (Saitou and Nei 1987) and the maximum-likelihood method (Felsenstein 1981) in the MEGA 7.0 program (Kumar et al. 2016), with all branches having a bootstrapping value of at least 50% based on 1000 bootstrap replicates (Felsenstein et al. 1985).
The draft genome sequence of strain GA224T was performed with Illumina HiSeq sequencing system with paired-end DNA libraries that were prepared at Magigene Company (Shenzhen, China). The reads of each data set were filtered, and de novo assemblies were performed by using the software SPAdes version 3.5.0 (Bankevich et al. 2012). The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (Hyatt et al. 2010). The sequence is available from the NCBI under the accession number JABANT000000000. To estimate the degree of pairwise relatedness between GA224T, and close relatives, average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) was calculated. ChunLab's online Average Nucleotide Identity (ANI) calculator (https://www.ezbiocloud.net/tools/ani), Average Nucleotide Identity calculator (http://enve-omics.ce.gatech.edu/ani/) and Genome-to-Genome Distance Calculator (http://ggdc.dsmz.de/ggdc.php#) were used for the calculation of Ortho ANIu, ANIb and in silico DDH (is DDH) respectively.