Whole Genome Sequence of Sphingobacterium sp. G1-14, a Strain With Effective Paichongding Biodegradation

In this study, we obtained a Paichongding (RR/SS-IPP) degrading Sphingobacterium sp. G1-14 by UV irradiation of the original strain G1-13. This new mutant strain showed excellent RR/SS-IPP degradation performance, and the degradation of ratio was up to 30 per cent after 7 days. Subsequently, we determined the mutant strain G 1-14 as Sphingobacterium based on the phylogenomic analyses. The circular chromosome of Sphingobacterium sp. G1-14 was presented in this paper by Illumina Hiseq platform combined with a third-generation sequencing platform. 5583 protein-coding sequences of the complete genome sequence were obtained, which is beneficial to deduced genes related to RR/SS-IPP degradation.

At present, genes and enzymes related to the degradation of RR/SS-IPP have not been reported. Based on the newly selected RR/SS-IPP efficient degrading bacteria, this study provides insights into the degradation mechanism of RR/SS-IPP from a genetic perspective through the whole-genome sequencing and bioinformatics analysis combined with liquid chromatography tandem high-resolution mass spectrometry (LC-MS/MS).

Chemicals and Microorganism Strain
Paichongding, (IPP, chemical purity 98.3%; Formula Weight, FW 366. Fig. 1) was obtained from Jiangsu Kesheng Company Ltd., HPLC grade methanol and acetonitrile were purchased from Burdick & Jackson (MI, USA). All other reagents and common chemicals were analytical grade and purchased from Sinopharm Chemical Reagent Company (Shanghai, China).
Sphingobacterium sp. G1-14 (GenBank accession numbers: KP657689, CGMCC No. 10454) was irradiated from the original strain G1-13, which was isolated from soils in the south of Changzhou, Jiangsu province, China. It had good IPP-degrading activity and could use IPP as a sole carbon and energy source. Sphingobacterium sp. G1-14 was cultivated aerobically at 30 o C on Luria-Bertani (LB) broth. Stock cultures were stored in 20% glycerol at -80 o C.

Biodegradation Assays
To illustrate the biodegradation pathway of IPP in aquatic system, 250 mL glass flasks were -1 ) was added into replicates. After culture for 72 h, cells of Sphingobacterium sp. G1-14 were harvested by centrifugation (21,000×g, 4 o C, 10min) and washed twice with 0.85% (w/w) sodium chloride solution.

Extraction and Pretreatment
The cultivation was collected by centrifugation (21,000×g, 4 o C, 10min). The supernatant was extracted with dichloromethane three times. The recovery of IPP ranged from 91.22% to 107.29, the relative standard deviation (RSD) was less than 7.58%. The results showed that the extraction method is feasible and satisfactory for the analysis of IPP residues. The dichloromethane extracts were combined and concentrated through vacuum rotary evaporator at 40 o C to almost dry and then the extracts were dissolved in 1 ml of methanol. IPP and its biodegradation intermediates were monitored and analyzed.

Genomic DNA Extract and Sequence
The commercial kits (PureLink™ Microbiome DNA Purification Kit, ThermoFisher Scientific) were used for extracting and purifying of genomic DNA from Sphingobacterium sp. G1-14. The genomic DNA was sequenced using Illumina Hiseq 2500 at BGI company (Shenzhen, China).

Results
As a chiral insecticide, IPP can be divided into four stereoisomers according to two asymmetrically substituted C atoms (Fig. 1). Compared with enantiomers RS-IPP and SR-IPP, enantiomers RR-IPP and SS-IPP exhibit higher stablility and difficulty in degradation (Fu, Wang, et al., 2013;J. Wang et al., 2016). In this study, we found a gram-negative mutant strain G1-14 from G1-13 through UV mutagenesis. As is shown in Fig 2, G1-14 in culture plates has higher growth rate and larger single colonies than G1-13. After seven days of fermentation, the IPP degradation rate was about 30%, which was superior to original strain significantly. The phylogenetic tree based on 16S rRNA gene sequence was constructed using MEGA (v5.1) (Fig. 3). It could reveal the evolutionary status of Sphingobacterium sp. G1-14. Our group attempted to speculate the conceivable metabolic pathway of RR/SS-IPP by Sphingobacterium sp. G1-14 though the liquid chromatography tandem high-resolution mass spectrometry (LC-MS/MS). Owing to the complicated mechanism of RR/SS-IPP, the whole genome sequence was added to provide insights about the degradation mechanism of RR/SS-IPP from the genetic point of view. The Sphingobacterium sp. G1-14 was incubated at 30°C, 180 revolutions min −1 in 100 mL of LB medium. The genomic DNA was extracted using the Takara DNA kit and DNA concentration was measured using Qubit3.0 to ensure DNA quality for the subsequent experiments. Illumina HiSeq 2500 system combined with Pacfic Biosciences SMRT (PacBio RSII) have been used to complete genome sequence. The Illumina PE library and PacBio library generated 2021 Mb data (14,532,302 reads with 335 bp average coverage) and 625 Mb data (83839 subreads with 7,453 bp average length) respectively. We assembled the genome into one contig based on the reads obtained from Illumina PE library and PacBio library by SOAPdenovo (v2.04) and Celera Assembler (v8.0). GapCloser (v1.12) was used to complete the gap closure and confirm a 6,325,678 bp chromosome with circular topology (Koren et al., 2012) . The function annotations of protein-coding genes (predicted by Glimmer 3.0) were obtained from BLASTP with COG, KEGG and Swiss-Prot databases (Delcher, Bratke, Powers, & Salzberg, 2007). Finally, we indentified the rRNA and tRNA contained in the genome using RNAmmer (v1.2) and tRNAscan-SE (v1.3.1). The assembly result of Sphingobacterium sp. G1-14 genome revealed

Discussion
In combination with deduced metabolic pathways (J. Wang et al., 2016), we found that: (i) IPP and imidacloprid are structurally consistent in Phase Ⅰ (Fig 5). KEGG pathway analysis suggested that CYP450 enzymes were not exist in the genome sequence. CYP450s were the only proven enzymes that involved in imidazole ring hydroxylation in imidacloprid with stable chloropyridine ring (Casida, 2011;Fu, Zhang, et al., 2013;Schulz-Jander, Leimkuehler, & Casida, 2002) . Our results deduced that the hydroxylation of IPP occurs in the tetrahydropyridine ring based on six hydroxylases in the genome. (ii) According to the KEGG database, MqnB was found in the chromosome of G1-14 which could hydrolyze the C-N bond attached to the imidazole (Hiratsuka et al., 2008). The formation of I1 and I2 were obtained from the removal of C-N bonds between 2-chloro-5-ethylpyridine and 8-amino octahydroimidazo [1,2-alpha] -pyridin-7-ol. Based on these observations,we hypothesized that the break of this C-N bond was mainly due to the candidate enzyme MqnB.

Conclusion
In this study, we obtained the whole-genome sequence of the Sphingobacterium sp. G1-14. Through bioinformatics analysis, hydroxylation of RR/SS-IPP may occur on relatively active tetrahydropyridine rings. From the metabolic point of view, the candidate enzyme MqnB might be responsible for the removal of the chloropyridine ring.