Draft genome sequence of furan-degrading Rhodococcus erythropolis strain FUR100

ABSTRACT

Rhodococcus erythropolis FUR100 was isolated from a mixture of soil and activated sludge. It can use furan as a sole source of carbon and energy. Its draft genome sequence may provide insight into the genetics of furan catabolism.

ANNOUNCEMENT

Furan is an aromatic five-membered heterocyclic compound containing one oxygen atom. It is a precursor used in the synthesis of fine chemicals (1). In rat and human liver, furan is transformed by P450 monooxygenases to the carcinogenic cis-2-butene-1,4-dial, possibly via the unstable intermediate 2,3-epoxy-2,3-dihydrofuran (2–4). Bacterial aerobic metabolism of furan derivatives furfural, 2-furoic acid, 5-hydroxy-methylfurfural, and dibenzo[b,d]furan is well researched (5–7) but not of the heterocycle itself. Rhodococcus erythropolis FUR100 can use furan as a sole source of carbon and energy. The strain was obtained by enrichment in 100 mL liquid mineral salt medium (8), inoculated with a mixture of 10 g soil sample and 10 mL activated sludge from a wastewater treatment plant (Stuttgart-Büsnau, Germany, 48.75191 N 9.08968 E), supplied with 5 mM furan, and incubated at 30°C, 250 rpm, in a baffled flask (500 mL), sealed gas tight with a polytetrafluoroethylene septum. After 7 days, 1 mL of suspension was transferred to fresh medium supplied with furan and incubated until a visible increase in turbidity was observed. This step was repeated a second time. A dilution of a 1 mL sample was plated on mineral salts medium plates and incubated at 30°C in a desiccator supplied with a 0.001% furan atmosphere. Single colonies were picked and streaked onto fresh plates to obtain pure cultures. These pure cultures were tested again for growth in liquid culture to exclude the use of agar as a carbon source. One of the isolated strains, FUR100, was further examined.

DNA was obtained from a fresh liquid culture (same conditions as isolation procedure). Cells were harvested after 4 days, washed twice with PBS (4°C, 10,000 × g, 10 min), and resuspended in 5 mL PBS. Cell pellets were created by pipetting droplets (~100 µL) directly into liquid nitrogen and disrupted using a Mixer Mill MM 200 (Retsch). DNA was isolated with the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. The sequencing library was prepared from 50 ng of input material using the Nextera DNA Library Preparation Kit (Illumina) according to the manufacturer’s protocol and sequenced on an Illumina HiSeq2500 platform in paired-end mode for 150 cycles. A sequencing depth of 5,866,912 reads was generated. Sample integrity and sequencing library quality were ensured by using a Fragment Analyzer (Agilent). Raw sequencing reads were demultiplexed with the bcl2fastq software (Illumina, 2015). Default parameters were used for all software unless otherwise specified. Adapter trimming and low-quality read removal were performed using BBDuk (BBMap v34.41 http://sourceforge.net/projects/bbmap/) with the parameters: trimpolyg = 10 hdist = 1 mink = 12 maxns = 10 minlen = 50 trimq = 20 qtrim = t ktrim = r k = 28. The quality of the sequencing was controlled with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The genome was de novo assembled using ABySS 2.0.2 (9), followed by an evaluation with Quast v5.0.2 (10). The genome was annotated by NCBI using PGAP (11). It has a size of 7,667,056 bp with a GC content of 62.5% and a coverage of 215.0×. It is split into 105 contigs with an N₅₀ of 145,273 bp. There are 7,186 predicted genes of which 6,992 are protein coding. The taxonomy of strain FUR100 was determined using the TYGS webserver (v389), closest match being Rhodococcus erythropolis JCM 3201^T (dDDH d₄ 88.5%, confidence interval 86.0%–90.5%) (12, 13).

DATA AVAILABILITY

The draft genome sequence of Rhodococcus erythropolis FUR100 has been deposited in GenBank under BioProject accession PRJNA680546, BioSample accession number SAMN16882220, assembly accession GCA_016019085.1, and SRA accession SRR26192813.