Identification of essential β-oxidation genes and corresponding metabolites for estrogen degradation by actinobacteria¶

Introduction¶

This narrative was used for a comparative transcriptomic analysis of an actinobacterium Rhodococcus sp. strain B50 incubated under three different steroid conditions. Via this analysis, the candidate genes and gene clusters of aerobic estrogen degradation in Strain B50 could be revealed.

RNA sample collection, purification and sequencing¶

RNA Sample Collection and purification¶

Strain B50 was incubated in the chemically defined mineral medium supplemented with 1 mM of estrone, testosterone, or cholesterol as the sole carbon source. Bacterial cells was harvested when substrate was consumed. Cell pellet was resuspended in 200 μL lysozyme buffer contained 20 mM Tris-HCl, 2 mM EDTA, 1% TritonX-100 and 0.8 mg lysozyme. After incubation at 37°C for 1 hour, 600 μL of TRI reagent^® (Sigma-Aldrich, St. Louis, MO, USA) was added into the sample and kept in -80°C until further RNA extraction. Total RNA was extracted using Direct-zol^TM RNA MiniPrep kit (Zymo Research, Irvine, CA, USA), and residual DNA was removed with the TURBO DNA-free^TM Kit (Thermo Fisher Scientific, Waltham, MA, USA). The quality and quantity of the resulting RNA samples were determined using BioAnalyzer 2100 (Agilent, Santa Clara, CA, USA) and Qubit RNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively, while the DNA-removing efficacy was evaluated using Qubit 1x dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

RNA Sequencing¶

The QC-passed RNA samples were depleted for rRNA using the Ribo-Zero™ Magnetic Kit for Bacteria (EPICENTRE Biotechnologies, Madison, WI, USA). Further library construction, including cDNA synthesis, adaptor ligation, and enrichment was performed following the instruction of TruSeq Stranded mRNA Library Prep (Illumina, San Diego, CA, USA). The constructed libraries were sequenced as pair-end reads (with a 301-bp read length) on the Illumina MiSeq system (Illumina, San Diego, CA, USA). Finally, we obtained 19,493,886, 20,592,482 and 19,110,700 raw reads from estrone, testosterone and cholesterol treatments, respectively.

Data Import, Trimming, Alignment and Assembling Transcripts¶

The Strain B50 genome was downloaded in the GenBank format from the NCBI. Later, it was imported using default parameters through the Import GenBank File as Genome from Staging Area.
Three different pairs of transcriptomic raw reads were imported using default parameters through the Import FASTQ/SRA File as Reads from Staging Area.
All these transcriptomic raw reads were combined into a sample set through the Create RNA-seq Sample set.
After quality check using Assess Read Quality with FastQC, the RNA-seq sample set was trimmed using default parameters through the Trim Reads with Trimmomatic.
The trimmed RNA-seq reads were aligned to Strain B50 genome using default parameters through the Align Reads using HISAT2.
Finally, each gene expression strength under different growth conditions was evaulated using default parameters through the Assemble Transcripts using StringTie.

Created Object Name	Type	Description
B50_trimmed_RNA_sampleset_trimm_paired	ReadsSet	Trimmed Reads
B50_trimmed_RNA_sampleset_trimm_paired_SampleSet	RNASeqSampleSet	Trimmed Reads
B50_trimmed_RNA_sampleset_trimm_unpaired_fwd	ReadsSet	Trimmed Unpaired Forward Reads
B50_trimmed_RNA_sampleset_trimm_unpaired_fwd_SampleSet	RNASeqSampleSet	Trimmed Unpaired Forward Reads
B50_trimmed_RNA_sampleset_trimm_unpaired_rev	ReadsSet	Trimmed Unpaired Reverse Reads
B50_trimmed_RNA_sampleset_trimm_unpaired_rev_SampleSet	RNASeqSampleSet	Trimmed Unpaired Reverse Reads

Created Object Name	Type	Description
Estrone_trimm_paired_alignment	RNASeqAlignment	Reads 89341/102/1;89341/16/4 aligned to Genome 89341/100/1
Cholesterol_trimm_paired_alignment	RNASeqAlignment	Reads 89341/102/1;89341/19/4 aligned to Genome 89341/100/1
Testosterone_trimm_paired_alignment	RNASeqAlignment	Reads 89341/102/1;89341/13/4 aligned to Genome 89341/100/1
B50_trimmed_RNA_sampleset_trimm_paired_alignment_set	ReadsAlignmentSet	Set of all new alignments