Characterization of the diethyl phthalate-degrading bacterium Sphingobium yanoikuyae SHJ

A newly isolated bacterial strain SHJ was found to be capable of degrading diethyl phthalate (DEP) very efficiently. Its growth characteristics and 16S rDNA gene sequence were analyzed. Its whole genome was also sequenced. Strain SHJ was identified as Sphingobium yanoikuyae SHJ.


Subject area
Biology More specific subject area Microbial characterization, identification and phylogenetic analysis Type of data

Value of the data
The whole genome sequence data of strain SHJ is available by its accession number. Characterization and identification of the newly isolated Sphingobium yanoikuyae SHJ. Biodiversity with capability of bio-remediating phthalate esters-contaminated aquifer.

Data
A new bacterium strain SHJ was isolated from the shallow aquifer sediment of Jianghan plain, Hubei, China. It grew on NB agar plate containing 400 mg L À 1 DEP as sole carbon source and appeared to be yellow colony (Fig. 1a), and it was observed to be short rod under a scanning electron microscope (Fig. 1b). It was found to be capable of degrading DEP very efficiently under simulated shallow aquifer (SSA) conditions which are dark, oxygen-limited, at pH 7 and 18°C [1]. However, the most well-known DEP-degrading bacterial isolates that are purely aerobic are listed in Table 1. Classification and general features of the strain SHJ were listed in Table 2. Its 16S rDNA gene sequence (GenBank accession number JFFT01000000) showed the highest similarity with Sphingobium yanoikuyae ATCC 51230 (Fig. 2). Therefore, strain SHJ was classified as Sphingobium yanoikuyae SHJ. The Whole Genome Shotgun project of S. yanoikuyae SHJ has been deposited at DDBJ/EMBL/GenBank under the accession JFFT00000000 and the release date of its GenBank Data is February 28, 2017.

Chemicals and reagents
DEP was purchased from Tianjin Hengxing Chemical Reagent Co., Ltd., China. DEP standard solutions were prepared at various concentrations in methanol and kept in dark at 4°C.

DEP-degrading bacterial strain
The DEP-degrading strain SHJ was isolated from the sediments collected from the quaternary shallow aquifer from a depth of 2.2 m in Jianghan Plain, Hubei, China, with a precise GPS location of 30°28 0 19″N, 113°59 0 13″E (longitude, latitude). The strain SHJ was grown using the method described previously [18]. It was pre-grown for 24 h at pH 7.2 and 30°C in nutrient broth (NB), which contained peptone 5 g L À 1 , beef extract 3 g L À 1 , NaCl 5 g L À 1 . Nutrient agar plates were prepared using NB supplemented with agar (1.5%). NB-DEP agar plate was prepared by diffusing 400 mg L À 1 DEP solution into the nutrient agar medium. All media were sterilized for 20 min at 121°C before inoculation. Detection and identification of DEP degradation intermediates ethyl methyl phthalate (EMP), monoethyl phthalate (MEP), monomethyl phthalate (MMP) and phthalic acid (PA) was carried out as described previously [1].

Identification of strain SHJ
Colonies of the strain SHJ on NB agar plate were picked for Gram staining, and the morphology of the strain was observed using an optical microscope.
Microbial identification and phylogenetic analysis of strain SHJ were performed by 16S rDNA gene sequencing. One ml overnight culture of bacterium grown in NB media in a rotary shaker (150 rpm) at 30°C was centrifuged at 6000 Â g for 10 min. The cells obtained were washed three times using sterile water and re-suspended in sterile water. Genome DNA was extracted from the isolate using UltraClean s Microbial DNA Isolation Kit (MoBio, USA) according to the manufacturer's protocol. 16S rDNA gene of the strain SHJ was amplified from its genomic DNA by using PCR procedures [18]. The bacterial universal primers F27 and R1492 were used for amplifying the full length of 16S rRNA gene fragments. The Shanghai Personal Biotechnology Co., Ltd performed the sequencing and assembly of strain SHJ using Illumina MiSeq sequencing platform, and gene prediction and annotation were completed using National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP, https://www.ncbi.nlm.nih. gov/genome/annotation_prok/) [1]. The 16S rDNA gene sequence of strain SHJ was searched against Gen-Bank database under the accession JFFT00000000 using BLASTn at the website of NCBI (http://www.ncbi. nlm.nih.gov/BLAST/). Based on the 16S rDNA gene sequences obtained, phylogenetic analysis of strain SHJ was performed by molecular evolutionary genetics analysis (MEGA 6, https://www.megasoftware.net/ index.php) after all sequences alignment by using Clustal W (https://www.ebi.ac.uk/Tools/msa/clustalw2/).   , not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project.