2. MATERIALS AND METHODS

2.1 Chemicals, bacterial strains and growth conditions

DBP,DMP,DEP,DEHP were purchased from Yonghua Chemical Co., Ltd. (Jiangsu, China), with a purity greater than 99.5%. Methanol was high-performance liquid chromatography (HPLC) grade and was purchased from Tianjin Siyou Fine Chemicals Co., Ltd. All other reagents were pure analytical grade.
Arthrobacter sp. ZJUTW, isolated from sludge of river of Hangzhou City, China had been deposited with the China Center of Typical Culture Collection (CCTCCM2012246).
Luria-Bertani (LB) medium was applied for bacterial enrichment. A modified basic inorganic salt medium (BSM) used for the degradation tests consisted of: K2HPO4·3H2O 1.0 g,NaCl 1.0 g,(NH4)2SO4 0.5 g,MgSO4·7H2O 0.4 g,CaCl2 0.0755 g,and FeCl3 0.0143 g.

2.2 DBP degradation by Arthrobactersp. ZJUTW

Arthrobacter sp. ZJUTW strain was inoculated into LB medium, cultured at 30 °C for 12 h at 180 rpm. Bacterial cells were harvested by centrifuging at 4000 rpm for 10 min after the OD600 reached about 0.6. Pellets were washed three times with 0.1 mM phosphate buffered saline (PBS, pH = 7.2). The re-suspended cells in the inorganic saline liquid medium were inoculated into BSM containing different initial DBP concentrations (50 mg/L,125 mg/L, 250 mg/L, 500 mg/L, and 1000 mg/L, respectively), and then incubated under the optimum conditions (pH = 7.0-8.0, 30 °C, and 180 rpm). All experiments were performed in triplicate.
The supernatant was collected by centrifugation at 12000 rpm for 10 min after 48 h of incubation, then mixed with the equal volume of dichloromethane with violently oscillation. The aqueous phase was transferred to a rotary evaporator for drying. The dried substances were dissolved in 2 ml methanol and after filtering through a 0.22 μm membrane filter, the DBP concentration in methanol was measured using an Agilent 1260 HPLC (USA) under the following conditions: Diamonsil C18 column, 250 mm × 4.6 mm, 5 μm; the UV wavelength 235 nm; the mobile phase contained 90% (vol) methanol and 10% (vol) water; the flow rate,1.0 ml/min.

2.3 Genomic sequencing

After Arthrobacter sp. ZJUTW was cultured to the mid-exponential phase in LB medium, cells were harvested by centrifugation (10000 rpm, 10 min, 4 °C). Genomic DNA was extracted using an EasyPure Bacteria Genomic DNA Kit (TransGenBiotech, Beijing, China). The whole-genome sequencing of the strain ZJUTW was performed using PacBio SMRT. The raw reads were polished by using the SMRT Analysis workflow from Pacific Biosciences to obtain clean reads. The genome of the strain ZJUTW was re-sequenced by Illumina Hiseq to correct the PacBio results. The complete genome was obtained by sequence assembly using software canu, SPAdes (Bankevich et al., 2012). If there was a certain length of overlap at both ends of the final assembly sequence, the sequence was looped and one of the overlap sequences was truncated. Finally, the complete chromosome and plasmid sequences were obtained.

2.4 Genome annotations

After sequencing, gene prediction of the chromosome was carried out using Glimmer 3.02 software (http://ccb.jhu.edu/software/glimmer/index.shtml) and the GeneMarkS (http://opal.biology.gatech.edu/GeneMark/ ) was used for gene prediction of the plasmid. The predicted gene sequences were performed by sequence alignment with NR (Non-Redundant Protein Database), Swiss-Prot (https://web.expasy.org/docs/swiss-prot_guideline.html ), GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genome databases) to obtain functional annotation information. CGview software was used to implement circular genome visualization (Stothard & Wishart, 2005). The rRNA and tRNA contained in the genome were predicted using Barrnap 0.4.2 and tRNAscan-SE v1.3.1 software. Tandem Repeats Finder was used to predict tandem repeats. Gene Island (GI) was directed using soft IslandViewer (Bertelli et al., 2017). PHAST (Zhou, Liang, Lynch, Dennis, & Wishart, 2011) and Minced (Bland et al., 2007) were used to find phages and CRISPR-Cas.

2.5 Comparative genomic analysis

A total of 25 Arthrobacter strains chromosome sequences have been fully sequenced and were downloaded from NCBI. The strain ZJUTW was used as a reference genome and its genome’s circular comparison with 25 strains were performed and visualized using BLAST Ring Image Generator (BRIG, version 0.95) (Alikhan, Petty, Ben Zakour, & Beatson, 2011). The specific fragments of the strain ZJUTW were identified based on the alignment results.
The phylogenetic position of ZJUTW among the 26 Arthrobacter strains was validated by using CVTree3 (tlife.fudan.edu.cn/cvtree). According to the phylogenetic tree of 26 strains of the genome, four strains with close evolutionary relationship were obtained. Their orthologous gene alignment analysis was based on protein sequence alignment. The BLAST software was used for comparison and extraction (E- value < 10-5) of homologous gene pairs. Finally, the orthologous genes and the specific genes in the ZJUTW strain were displayed using a Venn diagram.

2.6 Mining of genes and gene clusters involved in PAE metabolism

Ester hydrolase and dioxygenase play a key role in PAE degradation. Ester hydrolase is responsible for removing the side chain of PAEs and the ring-cleavage process of aromatic compounds results from the action of dioxygenase. Based on the genome sequence obtained and the annotation results, all dioxygenase coding sequences and hydrolase coding sequences were retrieved and manually screened to identify dioxygenases involved in the catabolism of aromatic compounds. The genes of interest (dioxygenase and hydrolase genes) were located by the Basic Local Alignment Search Tools (BLAST) of National Center for Biotechnology Information (NCBI). In addition, the upstream and downstream sequences of the target genes were analyzed to identify possible gene clusters. Moreover, to obtain identified genes and gene clusters related to PAE degradation, we reviewed a large body of relevant literature. The proposed genes and gene clusters were also further analyzed by blast-2.4.0+ to show their similarity with reported gene clusters.

2.7 Total RNA isolation and sequencing

For transcriptomic analysis, Arthrobacter sp. ZJUTW was inoculated in BSM, supplemented with 0.1% glucose and 400 mg/L (w/v) DBP, respectively. After incubation at 30 °C and 180 rpm until the broth OD600 reached 0.4, the cells were harvested by centrifugation (10000 rpm, 10 min, 4 °C ) and immediately mixed with RNA isolater from Vazyme Biotech stored at 80 °C for RNA extraction. The quality of extracted RNA was determined by the ND-5000 spectrophotometer (USA) and gel electrophoresis. The extracted RNA was sent to Beijing Nuohe Zhiyuan Technology Co., Ltd for RNA sequencing. The transcriptome sequencing of Arthrobacter sp. ZJUTW was performed based on the Illumina sequencing platform. The transcriptomic data was subjected to parametric analysis usingArthrobacter sp. ZJUTW as a reference genome. The clean reads were compared to the reference genome and reference gene sequences using the HISAT2 software and the Bowtie2 software, respectively. Quantitative analysis using RSEM (RNASeq by Expectation Maximization) software based on the alignment results and calculates the expression level of the gene. The analysis of differential expression focuses on finding genes that are differentially expressed between samples and conducting an in-depth analysis of these genes. The differentially expressed genes were screened for GO and KEGG enrichment by hypergeometric analysis.