2.1 Chemicals, reagents, and culture media
Analytical HPLC-grade nicosulfuron (NS; CAS 111991-09-4; 1-(4,6-dimethoxypyrimidin-2-yl)-3-(3-dimethylcarbamoyl-2-pyridylsulfonyl)urea), chlorimuron-ethyl (CAS 90982-32-4; ethyl 2-(4-chloro-6-methoxy-2-pyrimidinylcarbamoylsulfamoyl)benzoate), and cinosulfuron (CAS 94593-91-6; 1-(4,6-dimethoxy-1,3,5-triazin-2-yl)-3-(2-(2-methoxyethoxy)phenylsulfonyl)urea) were purchased from the Aladdin Industrial Shanghai Co., Ltd. (China). Chlorimuron-ethyl is a selective post-emergence herbicide for controlling actively growing weeds in peanut, soybeans, and non-crop areas. Cinosulfuron is a broad spectrum triazinylsulfonylurea herbicide used for post-emergence control of many weeds, including European water plantain, annual sedge, aquatic ferns, and pod weeds. These herbicides are highly water soluble and can leach from soil to groundwater and to surface water.
The Luria Bertani (LB) medium containing (in g/L) 10.0 peptone and 5.0 yeast extract (both obtained from Beijing Aoboxing Bio-tech Ltd), 10.0 NaCl and pH 7.0 was used for culturing strain LAM1902. The sulfonylurea herbicide degradation studies using strain LAM1902 were carried out according to Li et al. 33 using glucose supplemented medium (GSM) containing (in g/L) 1.0 NH4Cl, 1.0 NaH2PO4·12H2O, 0.5 KH2PO4, 0.2 MgSO4·12H2O, and 20 µL trace element solution, pH 7.0), 5 g glucose /L, and 50 mg/L of NS, chlorimuron-ethyl or cinosulfuron in distilled water 24. The trace element solution (g/L; pH 7.0) contained: 5.5 CaCl2, 50.0 EDTA, 1.1 (NH4)6MoO2 4H2O, 5.0 FeSO4 7H2O, 2.2 ZnSO4, 5.1 MnCl2 4H2O, 1.6 CuSO4 5H2O, and 1.6 CoCl 6H2O. All the chemicals and reagents (at least analytical grade) were obtained from commercial sources, as listed in Supplementary Information (Table S1).
2.2 Optimization of the nicosulfuron degradation conditions for P. nicosulfuronedens LAM1902
The degradation of NS by strain LAM1902 was determined after 6 d incubation, according to Li et al. 33. Different incubation conditions included: different sources of carbon (1.0 g/L: sodium acetate, glycerol, glucose, sodium succinate, peptone, yeast extract, sucrose, and starch) and nitrogen (1.0 g/L: NH4Cl, (NH4)2SO4, NH4H2PO4, yeast extract, and peptone), increasing from pH 5 to pH 9, increasing temperatures from 15 to 45°C, and using different volumes (0, 1, 3, 5, 7, and 10%, v/v). Different initial NS concentrations (10, 25, 50, 100, 200, or 500 mg/L GSM) were used at 30°C on a rotary shaker at 150 rpm. Based on these NS degradation optimization studies, the strain LAM1902 was incubated in 50 mg NS/L in GSM at pH 6, at 30°C, and with an incubation volume of 5%, unless otherwise specified. The residual NS concentrations (determined using HPLC analysis) were determined daily. All treatments were performed in triplicate.
2.3 Sulfonylurea herbicide degradation studies using P. nicosulfuronedens LAM1902
Chemical analysis of NS concentrations was determined according to others 28, 33 using a high-performance liquid chromatography (HPLC) system (Agilent 1200, Waldbronn city, Germany) with a 10 µL injection volume, a C18 column (50 mm ⊆ 2.1 mm), a mobile phase of acetonitrile/water/acetic acid (30/68/2, v/v/v), and a flow rate of 1.0 mL/min. The photodiode array detector had a wavelength of 245 nm and the column temperature was 30°C. Peak identification was based on our earlier paper and the HPLC peak retention time (Rt of NS was 5.6 min) of the authentic NS sample 33. The photodiode array detector had a wavelength of 210 nm and the column temperature was 30°C. The NS concentrations were determined by the standard curve. The NS degradation efficiency (%) of strain LAM1902 was calculated using Eq. 1.
where, [NS]i is the initial (i) nicosulfuron (NS) concentration (mg/L) at time (t) = 0
(d), [NS]f is the final (f) NS concentration at t = 6 d. This equation was used for the chlorimuron-ethyl and cinosulfuron degradation studies (described below).
The degradation of NS by strain LAM1902 was compared with chlorimuron-ethyl and cinosulfuron (50 mg/L) after 6 d incubation at pH 6, 30°C, using a 5% incubation volume. The negative control group was incubated with autoclaved sterilized LAM1902 cells (abiotic control). The residual concentrations (determined using HPLC analysis) and cell growth (OD600) were determined daily for six days. The chlorimuron-ethyl and cinosulfuron concentrations were determined HPLC using an injection volume of 10 µL and a C18 column (50 mm ⊆ 2.1 mm). For chlorimuron analysis, the mobile phase was mixture of methanol/water/acetic acid (67/32/1, v/v/v, flow rate of 1.0 mL/min) and a photodiode array detector set at 236 nm, and a column temperature of 30°C. For the cinosulfuron studies, the HPLC used a photodiode array detector at 240 nm, with a column temperature at 28°C; the mobile phase was a mixture of acetonitrile/methanol/water/acetic acid (45/15/40/0.1, v/v/v, flow rate of 1.0 mL/min). The respective Rt of chlorimuron-ethyl and cinosulfuron were respectively 10 min and 6 min, using authentic reference samples. The % efficiency of strain LAM1902 to degrade chlorimuron-ethyl and cinosulfuron was calculated using Eq. 1.
2.4 Identification of oxalic acid production and the degradation effect on nicosulfuron.
The oxalic acid produced by strain LAM1902 in GSM medium were identified by HPLC with an injection volume of 10 µL. The mobile phase was mixture of ammonium dihydrogen phosphate (0.02 mM, pH 2, adjusted with H3PO4) and methanol (85:15 by volume, flow rate of 1.0 mL/min). The photodiode array detector had a wavelength of 210 nm and the column temperature was 30°C. And the degradation effect on nicosulfuron oxalic acid (200 mg/L) were compared with strain LAM1902. All treatments were performed in triplicate.
2.5 Total RNA isolation and sequencing
P. nicosulfuronedens LAM1902 was incubated in the GSM medium at 3% (v/v) (i.e., 3 mL strain LAM1902 plus 97 mL GSM) in presence of 50 mg NS/L, which served as the experimental group (YG). In parallel, the culture was grown at absence of NS and was considered as a control group (NG) 42. After culturing for 3 d (the logarithmic growth phase of strain LAM1902) at 30°C and at 160 rpm in the dark, the samples were centrifuged at 8000 rpm (Sigma, Germany) for 10 min. The pellet was washed three times with 130 mM phosphate buffer (pH 7.2), then quickly frozen in liquid nitrogen, and stored at -80°C. All experiments were conducted in triplicate.
Total RNA was extracted using commercial kits following the manufacturer’s instructions (Ambion, Foster City, CA). RNA degradation and contamination were monitored on 1% agarose gels. RNA quantity was measured using Qubit 2.0 (Thermo Fisher Scientific, MA, USA) and Nanodrop One (Thermo Fisher Scientific, MA, USA). RNA integrity was detected using the Agilent 2100 system (Agilent Technologies, Waldbronn, Germany).
Whole mRNAseq libraries were generated by Guangdong Magigene Biotechnology (Guangzhou, China) using the NEB Next Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, MA, USA) following the manufacturer’s recommendations. Briefly, the 16S ribosomal RNA (rRNA) transcripts in total RNA samples were reduced by the Ribo-Zero rRNA removal kit. Fragmentation was carried out using the NEBNext RNA First Strand Synthesis reaction buffer. The first strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). For synthesizing the second strand of cDNA, a chain-specific library was constructed by replacing dTTP with dUTP to improve the accuracy of results. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with its hairpin loop structures were ligated in preparation for hybridization. To choose cDNA fragments of preferentially 150 ~ 200 bp in length, the fragments were selected using AMPure XP beads (Beckman Coulter, Beverly, USA). PCR was then performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer (Premier Biosoft International, Palo Alto, USA). PCR products were purified with AMPure XP beads and the library insert size was assessed on the Agilent 2100 system (Agilent Technologies, Waldbronn, Germany). The clustering of the index-coded samples was performed on a cBot Cluster Generation System. The sequencing library was operated and sequenced on the Illumina Hiseq Xten platform by Guangdong Magigene Biotechnology (Guangzhou, China). Libraries for transcriptome analysis were established by using RNA collected from the YG and NG groups.
2.6 Analysis of RNA-Seq data
The fastq format raw data were treated with Trimmomatic (v.0.36) to obtain the clean reads 43, with mapping to NCBI Rfam databases for removing the rRNA sequences by Bowtie2 (v2.33). The residual mRNA sequences were compared with the reference genome using Hisat2 (2.1.0) 44. The read count and function of each gene was obtained using HTSeq-count (v0.9.1). The reads per kilobase per million for each gene was used to compare the expression level of genes among the experimental and control groups. The read count of each gene (acquired from HTSeq-count) was mainly used for analyzing the differential expression. The DEGs between two groups were identified using the edgeR (v3.16.5) 45–47. The GO and KEGG enrichment analyses of DEGs were performed by the cluster Profiler software (v3.4.4) to analyze their potential biological pathways 48.
2.7 Statistical analyses
Data was analyzed using one-way analysis of variance (ANOVA) and SPSS software (ver.18.0, SPSS Inc., Chicago, IL, United States). The resulting p-value was adjusted for judging the false discovery rate. Genes with the false discovery rate correction, p ≤ 0.05, and |log2 (fold change)| ≥ 1 were taken as the candidates of DEGs. The GO terms and KEGG pathways with p ≤ 0.05 were identified as significantly enriched by DEGs.
2.8 Data availability
All the RNA-seq data have been deposited in the NCBI Sequence Read Archive (SRA) database (accession number: PRJNA785098).