Bacteria isolate and culture conditions
Strain LY-0111T was isolated using a settling dish of trypticase soy agar (TSA, BD) medium. Generally, the dish was placed in a ventilated environment of the manufacturing shop for a standing time of 24 h. The dish was then covered and incubated at 28°C for 7 days. Single colonies were selected and cultivated on TSA plates. The strain LY-0111T was maintained on TSA medium and stored as aqueous glycerol suspensions (20%, v/v) at -80°C. N. flava CCTCC AB 207010T (Luo et al. 2008) and N. haasae Hz 6–5T (Wang et al. 2022; Wang et al. 2021) were used as reference strains for morphological, physiological, biochemical and chemotaxonomic analyses under the same experimental conditions as strain LY-0111T.
Morphology, physiology, and biochemical analysis
Gram staining was performed using the Gram Stain kit (G1060, Solarbio, China) according to the manufacturer’s instructions. The temperature range for growth were tested at 4, 10, 20, 28, 35, 37, 42, 45 and 50℃ on TSA plates. Cell morphology was examined using optical microscopy (Axio Vert A1, Zeiss), and scanning electron microscopy (SEM, HITACHI HT7800) after 2-day-incubation on TSA medium at 35oC with. Growth conditions were tested at 35°C for 3 days on R2A, tryptic soy agar (TSA), marine agar 2216 (MA), and Luria–Bertani agar (LB). The salt tolerance range were tested at different NaCl concentrations (0%, 0.5%, 1%, 2%, 3%, 5%, 7%, 10%, 12%, and 15%, w/v) and pH tolerance range were determined at different pH (pH 4.0–11.0, at intervals of 1.0 unit) in TSA medium. Motility was determined by observing growth of cells in test tubes containing semisolid TSA medium with 0.5% agar after 3 days of incubation at 28 ℃. Oxidase activity was determined by the oxidation of tetramethyl-p-phenylenediamine. Catalase activity was determined by 3% H2O2, and gas production was identified as a positive reaction. Nitrate reduction, production of H2S, and hydrolysis of cellulose, gelatin, starch, Tweens 20, 60 and 80, and urea were determined by using standard procedures (Goodfellow 1971). Other physiological and biochemical characterization were performed using the BIOLOG GEN III microtest system (BIOLOG, USA) according to the manufacturer’s instructions.
Phylogenetic and genome sequence analysis
Genomic DNA was extracted from strain LY-0111T by using the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, China) according to the manufacturer’s instructions. The 16S rRNA gene was amplified using the following primers: PA (5’-CAG AGT TTG ATC CTG GCT-3’) as the forward primer and PB (5’-AGG AGG TGA TCC AGC CGC A-3’) as the reverse primer (Weisburg et al. 1991). PCR amplification was performed under the following conditions: 4 min at 95 ºC; 30 cycles of 1 min at 94 ºC, 1 min at 56 ºC, and 1.5 min at 72 ºC, plus an additional 10 min cycle at 72 ºC. And then, amplicon was determined by Sangon Biotech (Shanghai, P.R. China) Co., Ltd. The analysis of the 16S rRNA gene sequences of the strains was performed on the EzBioCloud server (https://www.ezbiocloud.net/) (Yoon et al. 2017). After multiple sequence alignment, the neighbor-joining (NJ) (Saitou and Nei 1987), Minimum Evolution (ME) (Rzhetsky and Nei 1992) and maximum-parsimony (MP) (M. 1971) methods were used to construct phylogenetic tree in the MEGA X software package (Kumar et al. 2018). Kimura’s two parameter model (Kimura 1980) was used to calculate evolutionary distance matrices of the phylogenetic trees. A bootstrap analysis with 1,000 replicates was also performed (Felsenstein 1985).
Genomic DNA was sheared randomly to construct libraries with lengths ~ 500 bp by a Bioruptor ultrasonicator (Diagenode, Denville, NJ, USA) and physico-chemical methods. The paired-end fragment libraries were sequenced according to the Illumina MiSeq system’s protocol. Raw reads of low quality from paired-end sequencing (those with consecutive bases covered by fewer than five reads) were discarded. Genome assembly and gap filling were done using SPAdes version 3.13.0 (Nurk et al. 2013) in UGENE software package (Okonechnikov et al. 2012). The average nucleotide identity (ANI) and average amino acid identity (AAI) were calculated on the ANI/AAI-Matrix server (http://enve-omics.ce.gatech.edu/aai/) (Qin et al. 2014). Digital DNA–DNA hybridization (dDDH) estimates between the type strains was calculated based on the genome sequences using the Genome-to-Genome Distance Calculator (GGDC) web server version 3.0 (https://ggdc.dsmz.de) (Meier-Kolthoff et al. 2013). The whole genome sequences of the strains were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (Tatusova et al. 2016) and eggNOG online server (https://eggnog-mapper.embl.de/) (Huerta-Cepas et al. 2017). DNA G + C content was determined according to the genome sequences.
In order to construct a robust phylogeny, a well-characterized core gene sets, bac120 (120 genes in the domain Bacteria) (Parks et al. 2017), was used to construct genome-based maximum-likelihood (ML) phylogenetic trees. Hidden Markov models (HMMs) model of each gene was retrieved from the Pfam-A (www.pfam.org/) and NCBI (https://ftp.ncbi.nih.gov/hmm/) HMM databases for homologous gene calling with the program hmmsearch from HMMER software package (http://hmmer.org/). Clustal Omega (Sievers et al. 2011) was employed for multiple sequence alignments, trimAl (Capella-Gutierrez et al. 2009) for trimming and conserved region selection, and FastTree (Price et al. 2009) for phylogeny inference. The workflow was implemented automatically using a Perl (https://www.perl.org/) script named as EasyCGTree (https://github.com/zdf1987/EasyCGTree4) (Xue et al. 2022).
Chemotaxonomic characterization
Cells of strain LY-0111T and the reference type strains were harvested from cultures grown on TSA liquid medium for 2 days at 35 ℃. Cellular fatty acids were extracted and analyzed according to the MIDI standard protocol (Sherlock Microbial Identification System, version 6.0) and identified by using the TSBA6.0 database of the Microbial Identification System as described previously (Sasser 1990). Respiratory quinone was purified (Hiraishi et al. 1996) and analyzed by HPLC (Tamaoka 1986). Polar lipids were separated by two dimensional thin-layer chromatography and identified according to the previously described method (Collins and Jones 1981; Minnikin et al. 1984). A purified cell-wall peptidoglycan was prepared for amino acid analysis by HPLC (AccQ.Tag Ultra Derivatization kit, Waters) after derivatization with 6-aminoquinolyl-N-hydro-sysuccinimidylcarbamate as described previously (Schleifer 1985).