Citrobacter sp. Y3 Harboring a Novel Gene HBCD-hd-1 Mineralizes Hexabromocyclododecane (HBCD) with New Metabolic Pathways Based on Multi-omics Characterization

doi:10.21203/rs.3.rs-1782615/v1

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Citrobacter sp. Y3 Harboring a Novel Gene HBCD-hd-1 Mineralizes Hexabromocyclododecane (HBCD) with New Metabolic Pathways Based on Multi-omics Characterization

https://doi.org/10.21203/rs.3.rs-1782615/v1

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Background: Hexabromocyclododecane (HBCD) is a typical persistent organic pollutant that is widely detected in the environment. Until now, a microorganism resource that can degrade and even completely mineralizing HBCD is lacking.

Results: By stable isotope analysis, we found that the Citrobacter sp. strain Y3 can use ¹³C-HBCD as its sole carbon source and degrade or even mineralize it to ¹³CO₂with a maximum conversion rate of 100% in approximately 14 days. Genomics, proteomics, and metabolomics were used to study the catabolic pathway of HBCD biodegradation by strain Y3. Several enzymes reportedly involved in the degradation of HBCD were identified in the strain Y3. A new functional gene named HBCD-hd-1 encoding a haloacid dehalogenase was cloned and heterologously expressed in Escherichia coli. The recombinant E. coli transformed the typical intermediate 4-bromobutyric acid (4-BBA) to 4-hydroxybutanoic acid (4-HDBA) and showed excellent degradation performance on HBCD with a degradation rate of 100% in only 2 days. Meanwhile, HBCD was well debrominated by the recombinant E. coli, because nearly 100% bromine ions were detected by ion chromatograph (IC) in vitro.

Conclusion: This is the first report on the degradation function of haloacid dehalogenase in HBCD treatment. Strain Y3 can potentially degrade brominated flame retardants such as HBCD especially in a low-nutrient environment.

Hexabromocyclododecane

Mineralization

Multi-omics

Catabolic pathway

Identification of functional gene

Hexabromocyclododecane (HBCD), as a typical brominated flame retardant, is easily released to the environment during its manufacture, storage, and use. HBCD production is over 31,000 metric tons in China, Europe, and United States, according to the latest estimation on its global production [1]. Considering its persistent, long-distance migration ability, bioaccumulation effect, and toxicity in wildlife and human bodies [2, 3], HBCD has been listed by the Stockholm Convention as persistent organic pollutant (POP) in 2013 and can only be used under strict regulatory rules [4]. It has been frequently and widely detected in air, water, sediment, soil, and organisms [5]. HBCD residues have attracted much public attention, and its remediation in the environment has become urgent and necessary. Biodegradation plays an important role in HBCD removal in soil, wastewater treatment plants, and sediment [6]. Acquiring enough microbial resource to treat HBCD and understanding its biodegradation mechanism has become essential.

Previous studies in this area reported the removal efficiency and degradation pathway of HBCD in activated sludge and soil [6, 7]. In addition, several pure cultures that could degrade HBCD have been isolated from the environment [6], including aerobic bacteria Bacillus sp. HBCD-sjtu [8], Pseudomonas sp. GJY [9], and Pseudomonas aeruginosa HS9 [10] and anaerobic bacteria Sphingobium chinhatense IP26 [11] and Achromobacter sp. HBCD-1 [12]. All these pure cultures could partly convert HBCD to typical intermediates, including pentabromocyclododecanols (PBCDOHs), tetrabromocyclododecadiols (TBCDDOHs), and 1,5,9-cyclododecatriene (CDT), by debromination. Some cultures can convert the intermediates by hydrolyzation. However, most HBCD-degrading bacteria isolated to date cannot use HBCD as a sole carbon source and therefore cannot degrade it into basic biomass and CO₂. In addition, studies about HBCD mineralization by pure cultures have not been performed. If not completely mineralized, then the intermediates may lead to secondary pollution during the degradation process.

Known enzymes involved in HBCD conversion, including gamma-hexachlorocyclohexane dehydrochlorinase (LinA), dehydrochlorinase, haloalkane dehalogenase (LinB), and haloalkane dehalogenase (DhaA), were verified in vitro and studied in detail [13–15]. LinA converts HBCDs to pentabromocyclododecene (PBCDE) [16]. It has several variants, such as LinA1 and LinA2, which share about 90% similarity in their amino acid sequences [17]. Both of these variants originated from Sphingobium indicum B90A. LinB, which can be isolated from S. chinhatense IP26, can initially catalyze HBCD to become PBCDOHs; it can further catalyze it to become TBCDDOHs [18]. Although a LinB homology of enzyme DehHZ1 extracted from B. sp. HBCD-sjtu transformed HBCD, different products of CDT were determined. Despite that HBCD-degrading enzymes of dehydrochlorinase and DhaA have been identified, other clades of enzymes for HBCD removal are still lacking. In particular, enzymes responsible for the later mineralization after CDT degradation are completely unknown.

Previously, we have confirmed that Citrobacter sp. Y3 could transform HBCD using sodium citrate as additional carbon source [19]. In the present work, with the stable isotope labelling ¹³C-HBCD (α-, β-, and γ-HBCD), the degradation efficiency and mineralization rate of α-, β-, and γ-HBCD by strain Y3 were studied (Fig. 1). The detailed genomic and proteomic characterization of strain Y3 was obtained by high-throughput sequencing technique. The degradation pathway of HBCD was rebuilt by identifying intermediates through ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-Q-TOF-MS), the annotation of functional genes, and protein expression by omics. A hypothetical gene HBCD-hd-1 that encodes a new dehalogenase was cloned and identified. It possessed high performance on HBCD and its intermediates of 4- bromobutyric acid (4-BBA).

Mineralization of HBCD by strain Y3

The strain Y3 could effectively bio-transform HBCD, including its isomers, i.e., α-, β-, and γ-HBCD [20]. It could utilize ¹³C-HBCD (α-, β-, and γ-HBCD) as sole carbon source and degrade it to ¹³CO₂to various extents within a certain timeframe (Fig. 2). The degradation rate of β-¹³C-HBCD was approximately 100% in 12 days, whereas those of α-¹³C-HBCD and γ-¹³C-HBCD were approximately 80% and 95%, respectively. The removal rates of ¹³C-HBCD diastereomers were in the following order: β- > γ- > α-¹³C-HBCD. This order was consistent with that obtained by Heeb et al [11]. This was different from results of another study, in which the degradation rates of α- and β-HBCD were faster than that of γ-HBCD [9]. The produced ¹³CO₂ by β-¹³C-HBCD mineralization reached approximately 8.2 ppm within 18 days, which indicated that the mineralization rate reached 100%. The concentrations of ¹³CO₂ produced by γ- and α-¹³C-HBCD were 7.5 and 6.7 ppm, respectively. The conversion to ¹³CO₂was 91.5% and 81.7%, respectively. The HBCD mineralization rates of the three diastereomers were in the following order: β- > γ- > α-HBCD. The α-HBCD, which had the lowest degradation, was the most stable, because of its physico-chemical properties, such as water solubility and octanol-water partition coefficient [20, 21]. It is the predominant HBCD diastereoisomer in biological tissues and can be converted from β- and γ-HBCD [22].

Genomic characterization

DNA was extracted from Citrobacter sp. Y3 and subjected to whole-genome sequencing. The genome of Citrobacter sp. Y3 consisted of one circular chromosome (5 246 113 bp) and two circular plasmids (34 341 and 89 705 bp). The GC content of strain Citrobacter sp. Y3 was 54.19%. It contained 5240 protein-coding genes, as shown in Table S2. The assembly of strain Y3 genome yielded one contig with 76 241 reads and with a mean read length of 12 685 bp and an N50 contig length of 4 542 216 bp. The chromosome and plasmid of strain Y3 contained 5240 ORFs, of which 5092 (97%) were putative coding sequences (CDS). There were 22 RNA genes (5s, 16s, and 23s rRNA genes), 84 tRNA genes, and 59 ncRNA genes (Fig. S1).

Overview of proteomics

Proteomic analysis identified a total of 5006 proteins, which were equivalent to the predicted 95.5% of CDS. Of these, 492 proteins were upregulated and 670 were downregulated in the presence of HBCD (Fig. 3a). The number of proteins quantified and differentially abundant by less than 2-fold [−1.0 ≤ log₂ (fold abundance) ≤1.0, p ≤ 0.05] in response to HBCD are shown in Fig. 3b. The shaded areas represent proteins with more than 2-fold differential abundance [−1.0 ≥ log₂ (fold abundance) or log₂ (fold abundance) ≥ 1.0, p ≤ 0.05]. All identified proteins were divided into three categories in Fig. 3c, namely, biological processes (BP), cellular components (CC), and metabolic functions (MF). The presence of HBCD led to the overexpression of proteins related to ionic glutamate receptor active protein, arginine biosynthesis process protein, and ribosome. Strain Y3 can completely biodegrade HBCD. Thus, the metabolism process may involve three metabolic pathways, namely, carbon metabolism (CM), energy metabolism (EM), and xenobiotics metabolism (XM). According to further analysis based on the functional annotation of KEGG annotated genes, 45 proteins related to CM (Table S3), EM (Table S4), and XM (Table S5) were screened out from the 492 upregulated proteins. As shown in Fig. 3d, 26 upregulated proteins were related to CM. The abundance of 11 kinds of upregulated proteins closely related to the process of EM more than tripled. XM proteins were most likely involved with HBCD degradation. Thus, particular research focus was given to the study of XM. Among them, 11 kinds of upregulated proteins were found. The proteins involved in dehalogenation mainly included haloacid dehalogenase (DhaG) [23], DhaA [24], LinA [25], and LinB [14]. Some proteins, such as glutathione S-transferase (GST) [26] and cytochrome P450 (CYP450) [27], were found in other metabolism pathways; they probably participated in the debromination of HBCD.

Reconstruction of HBCD catabolic pathway using genomic, proteomic, and metabolic analyses

Seventeen biodegradation intermediates were identified by UHPLC-MS/MS (Table S6 and Fig. S2). According to the analysis of genomic, proteomic, and metabolic intermediates, HBCD degradation pathways by strain Y3 were proposed, as shown in Fig. 4. Obviously, HBCD debrominated via two pathways. The first pathway involved the detaching of bromine (Br) atoms from the compound during the process. Some typical products generated by gradual debromination include PBCDE (7.13 min, m/z of 573), tetrabromocyclododecane (TBCD) (9.49 min, m/z of 494), tribromocyclodedecane (TriBCD) (6.42 min, m/z of 413), dibromocyclododecane (DBCD) (1.76 min, m/z of 334), and CDT (4.41 min, m/z of 174). Similar intermediates were found in the degradation of HBCD by other strains of B. sp. HBCD-sjtu, Dehalococcoides mccartyi 195, and P. aeruginosa HS9 [8, 28]. LinA catalyzed dehalogenation processes, including debromination [16, 18, 29]. The expression volume of LinA was upregulated with 1- to 2-fold abundance increase (log₂ of 8.0 to 12.3) after HBCD treatment.

The CDT could be further transformed to intermediates, such as (4Z,8Z)-13-oxabicyclo [10.1.0] trideca-4,8-diene (ECDD) (1.02 min, m/z of 190), cyclododecane (CDDA) (3.76 min, m/z of 180), (1Z,5Z)-cyclododeca-1,5-diene (CDDE-diene) (5.93 min, m/z of 176), (Z)-cyclododecene (CDDE) (7.97 min, m/z of 178), 2-dodecene (6.61 min, m/z of 180), and formaldehyde (2.82 min, m/z of 31), which can be detected in the downstream products. Table S6 and Fig. S2 present the specific mass spectrometric information. Three intermediates (D-gluconate, 3-hydroxypropanoic, and formaldehyde) were proposed to enter the TCA cycle by forming acetyl-SCoA and succiny-CoA, because the functional enzymes of alcohol dehydrogenase (YiaY), salicylate hydroxylase (Slhe), and galactonate dehydratase (GanD) were detected during the degradation process [30-32]. Considering that 3-hydroxypropanoic and formaldehyde were identified, it could be deduced that they were converted to 2-oxoglutaratc and acetaldehyde when the reactions were catalyzed by Slhe and YiaY, respectively. Similar to other reports, 2-oxoglutaratc was converted by alpha-oxoglutarate dehydrogenase (SucA) to succinyl-SCoA, which is a precursor to TCA cycle, and then to CO₂ and H₂O [33].

The other pathway was the substitution of bromine atoms by hydroxylation of HBCD to form 9-boraneyl-2,5,6,10-tetrabromocyclododecan-1-ol (PBCD-ols) (5.18 min, m/z of 591), 6-boraneyl-5,9,10-tribromocyclododecane-1,2-diol (TetrBCD-diols) (6.67 min, m/z of 528), 2,6,10-tribromocyclododecane-1,5,9-triol (TriBCD-triols) (6.25 min, m/z of 465), and others. HBCD-degrading strains IP26 [11], HS9 [34], and GJY [9] have similar pathways. These strains’ debromination capability on HBCD and some bromo products via hydroxyl substitution was due to the action of functional proteins, such as LinB [14], DhaA [24], GST [35], and CYP450 [27]. During the debromination pathway in this study, three enzymes (DhaA, LinB, and GST) were upregulated by 1.4-, 1.6-, and 2.0-fold, respectively. CYP450 was downregulated and may not be fully involved in the debromination of HBCD. This finding was partly different from the previous description of GST and CYP450, which reportedly participate in the debromination reaction during the degradation of HBCD [35].

The typical product of TriBCD-triols might undergo ring-cleavage to transform 4-bromobutan-1-ol (BMBTL) through oxygenases, such as quercetin 2,3-dioxygenase (QDGE) and monooxygenase (MOGE) [36, 37]. These oxygenases may be responsible for the conversion of TetrBCD-diols to 5-bromohexane-1,4-diol (BHEDL) [38, 39]. The key chemical reactions catalyzed by these oxidative enzymes with a broad range of substrates are hydroxylation and epoxidation [40]. 4-BBA and other intermediate products decomposed to form succinyl-SCoA and oxaloacetate into TCA cycle by alka monooxygenase alpha chain (LuxA), YiaY, and DhaG, which were upregulated by 3.2-, 1.2-, and 2.0-fold, respectively. The abundance of DhaG increased by more than 2-fold in the process of HBCD degradation. Surprisingly, the gene dhaG that encoded DhaG was not found in genome sequences, but DhaG was found in 11 upregulated proteins (Fig. 3d). A new gene encoding DhaG–hypothetical gene (HBCD-hd-1) may exist in strain Y3. Its function will be verified in a subsequent section.

Comparative genomic analysis and gene arrangement

By phylogenic analysis, strain Y3 was most closely related to Acinetobacter venetianus JKSF02 based on 16S rRNA genes (Fig. S3), whereas it was most closely related to S. indicum B90A with a similarity of 81.65% based on the whole genome, as previously reported for HBCD-degrading bacteria (Fig. 5a and Table S2). Comparative genomic mapping showed a complete synteny conservation between strains Y3 and B90A (Table S2 and Fig. 5b). However, the average amino acid identity (AAI) between the two strains was only 42.39% (Fig. S4). According to the species classification criteria of AAI (95%–96%) [41], strains F2 and IP 26 may be the same species, whereas strain Y3 was distantly related to other HBCD-degrading strains in the phylogenic analysis (Fig. S3).

The degrading enzymes were encoded by functional gene clusters, as shown in the Fig. 5c. Three key dehalogenation genes (linA, linB, and dhaA) [42-44] and other major potential biodegradation genes (gst, cyp450, yiaY, frmA, and catB) [26, 27, 31, 45, 46] were found in the gene cluster of strain Citrobacter sp. Y3. The expressions of all enzymes encoded by these genes were upregulated to different extents in HBCD-treatment groups. The other eight strains contained only some of the key dehalogenation genes. Strains HBCD-sjtu contained only dhaG. Strains F2 and A. venetianus JKSF02 contained only genes dhaA, linA, and dhaG. Notably, strain Y3 contained multiple dehalogenation genes, thereby indicating its more powerful potential for the efficient degradation of HBCD.

Most of the reported HBCD-degrading bacteria contained linA, whereas only strains Y3 and B90A harbored linB. A coordinated gene linC was found in the upstream of linA in four strains (B90A, P25, F2, and TKS). The linC, also named P-450lin, was a member of the cyp450 gene family [47]. Enzyme LinC encoded by linC was responsible for electron transfer and substrate binding, and it cooperated with LinA to replace bromine with hydroxy [48]. However, the genes of strain Y3 coordinated with linA responsible for debromination was different with other strains. The upstream of linA was a single operon composed of purH and purD [49]. Based on the Paul W. Sternberg operon hypothesis, the demand for gene expression machines for gene expression can be minimized by operons [50]. It may contribute to a higher expression of LinA, because findings were consistent with the nearly 2-fold increase of LinA expression in protein analysis. The same situation was found for the catB, pcag, and frma genes in strain Y3. Although yiaY, pobA, and catB were found in the downstream of the gst in four strain genomes (B90A, F2, and TKS), their gene sequences and arrangement were totally different. Genes trspE (coding transposase) and resA (coding resolvase), which were closely related to dhaA, were also found in four strains, namely, TKS, F2, IP26, and B90A. The genes were almost always adjacent to each other on the chromosome and formed defined secondary metabolite gene clusters [51]. However, trspE and resA were not found in the upstream and downstream of dhaA in strain Y3. Instead, ygfk and ygfM were found, and these encoded molybdate-containing enzymes and polypeptides carrying the FAD domain, respectively. They can mediate electron transfer in a wide variety of metabolic reactions, which may be more conducive to the participation of dhaA in the dehalogenation process in the degradation of HBCD [51]. By genomic annotation, the dhaG gene was not found in strain Y3, but enzyme DhaG was sharply upregulated after HBCD treatment. By genome mapping, DhaG corresponded to a hypothesis gene, which indicated that the gene (named HBCD-hd-1) might be a new gene encoding DhaG. To further explore the homology of HBCD-hd-1 and dhaG, dhaG sequences from 44 bacteria were selected from NCBI and compared with the HBCD-hd-1 using NJ phylogenetic tree analysis (Fig. S5). The HBCD-hd-1 had low homology with the reported dhaG sequences, including those from B90A and P25. These results were consistent with the results of gene cluster analysis.

Verification of HBCD and its intermediate degradation by key enzyme encoded by HBCD-hd-1

The HBCD-hd-1 gene with a size of 2550 bp was successfully cloned and heterologously expressed in E. coli (Fig. 6a). The results of first-generation sequencing of E. coli are presented in Table S7. The recombinant E. coli could sharply transform HBCD with 100% removal rate within 3 days of treatment (Figs. 6b and c). The removal efficiency by recombinant E. coli was much faster than that by strain Y3, indicating that a higher concentration of functional enzyme showed stronger degradation performance. Meanwhile, about 7.1 mg/L of bromine ion was generated, and the production reached a plateau after 3 days. Theoretically, six Br^- was produced when one HBCD was completely debrominated. The ratio of the detected Br^-concentration divided by the theoretically transformed Br^-concentration could serve as a marker of debromination. In the present study, about 98.61% HBCD were completely debrominated. Like other haloacid dehalogenases, the enzyme encoded by HBCD-hd-1 gene could debrominated a typical mother compound of 4-BBA (Figs. 6e and f) with the peak appearing at a retention time of 3.1 mins with an iron at m/z 167 in ESI negative mode. After 4 d of treatment, the concentration of 4-BBA obviously decreased with the gradual increase in the concentration of 4-hydroxybutanoic acid (4-HDBA) (m/z of 104) (Figs. 6g and h). A similar functional description was presented previously [23, 52]. The results verified for the first time that haloacid dehalogenase encoded by a new functional gene HBCD-hd-1 not only transforms 4-BBA to 4-HDBA but also efficiently degrades HBCD (including α-, β-, and γ-HBCD) in just 2 days. The performance was sharply faster than the performance obtained by using other strains.

Several isolates from the genus of Citrobacter have been reported to degrade environmental pollutants, such as 2,4,6-trinitrotoluene [53], polyethylene, plastic mixtures [54], textile dye [55], and radioactive pollutants [56]. They have the degradation potential to treat a wide range of environmental pollutants. They can reproduce and develop in a barren environment. They can efficiently degrade HBCD and use it as sole carbon source. Their use can eventually lead to the complete mineralization of HBCD without secondary contamination. They deserve more attention because of their good potential for use in treating HBCD and other compounds with similar structure in the natural environment, especially in low-nutrient soils and water. Six key genes and their corresponding encoded proteins that were responsible for dehalogenation were obviously upregulated. The strain Y3 contained more abundant and effective genes for debromination. Furthermore, a new functional gene HBCD-hd-1 that encoded DhaG was verified to degrade HBCD (including α-, β-, and γ-HBCD) for the first time. Results confirmed that this was a new dehalogenation gene. The identification of this enzyme may provide new perspectives and ideas for researchers focusing on the removal of HBCD in the environment.

Chemicals, reagents, and bacterial strain. HBCD (99% in purity) and its three diastereomers with ¹³C-HBCD (α-, β-, and γ-HBCD, 99% in purity) were used as target compounds and purchased from Sigma Chemical, Co. (St Louis, MO, USA). It was dissolved in acetone to yield a stock solution (1000 mg/L). After filtration via a 0.22 µm nylon membrane, the stock solution was added into the mineral media to obtain the desired concentrations. 4-BBA (98%) was obtained from Aladdin Biochemical Technology, Co. (Shanghai, China). All solvents and reagents used in this study were of HPLC grade and purchased from Merck Company (Darmstadt, Germany). Citrobacter sp. Y3 was previously isolated from activated sludge obtained from a wastewater treatment plant in an electronic industrial park [19]. DNA maker, clone vector pEASY-T1, vector pCold-Ⅲ, endonuclease NdeI and XbaI, Competent Cell DH5α, and Escherichia coli BL21 (DE3) were bought from Beijing TransGen Biotech, Co. (Beijing, China). PCR primers were obtained from Sangon Biotech, Co. (Shanghai, China).

Batch biodegradation experiments. Experiments for product analysis were conducted, in which three diastereomers of ¹³C-HBCD served as the sole carbon sources (10 mg/L). The sample was collected at many different reactive times and then centrifuged at 7,000 rpm for 5 min to obtain the supernatant. To obtain better product analysis results, the sample was extracted and concentrated; the details of the specific method are presented in the Supporting Information [57]. To detect ¹³CO₂, the experimental conditions and settings used were completely consistent with the batch experiment, and the vacuum sampling bag was used to collect the gas (60 ml) produced by the reaction system daily from 1 d to 6 d. All experiments were performed in triplicate, and the average value with standard deviation (error bar) was obtained.

Chemical analytical. The HBCD concentration was analyzed by LC-MS/MS (Trip Quad 5500, SCIEX, Singapore). The gradient elution method was adopted, and the mobile phase consisted of water (10%) and acetonitrile (90%). The flow rate was 0.3 mL/min. The injection volume was 5 µL and the total elution time was 6 min for each sample. The collected extract was injected into UPLC-Q-TOF-MS (Agilent 1290, Palo Alto, CA, United States; Bruker, Germany) using electrospray ionization (ESI) in both positive and negative modes to identify the major degradation products of HBCD and 4-BBA. The ¹³CO₂ produced by ¹³C-HBCD (α-, β-, and γ- HBCD) mineralization was detected by high-precision laser isotope analyzer (Picarro L2130i, USA). The detection mode was set to ¹³CO₂, ¹²CO₂-dry, and H₂O. Before testing, the tested gas and N₂ were mixed at a ratio of 9:1 in the vacuum gas sampling bag. Bromide ions were detected by ion chromatography (Metrohm model 733, Herisau, Switzerland).

DNA and protein extraction. DNA was extracted by using a DNA extraction kit (TIANGEN, China), and the concentration and purity of DNA samples were detected by using a spectrophotometer (Thermo, Nanodropone, USA). The protein was extracted by centrifugation to collect bacteria and rinsed quickly with precooled PBS 2–3 times to avoid bacterial contamination. After each cleaning, the supernatant was completely discarded by 5000 g centrifugal 5 min at 4 ℃, and the bacteria were collected in 1.5 mL centrifuge tube (frozen tube). The volume of bacteria was recorded, and they were stored in refrigerator at -80 ℃ after quick freezing with liquid nitrogen.

Genome sequencing, assembly, and analysis. The whole genome sequence of strain Citrobacter sp. Y3 was prepared and completed. The library was sequenced using PacBio RS to obtain clean data and reads that can be used for analysis. These reads were assembled using SMTR portal (v2.0). Open reading frames (ORFs) were predicted using Prodigal (v2.60) [58]. The completeness and contamination were estimated by CheckM [59]. The whole genome of Citrobacter sp. Y3 was uploaded to GenBank with the accession number GCA011602505.1.

Proteomic analyses. The mass spectrometry analysis of label-free quantitative proteome was completed by Thermo fusion mass spectrometry. The raw data files were processed with MaxQuant v.1.6.1.0 to search the database and for quality control [60]. The positive and negative mixed library search strategy was adopted. The false positive rate of the peptide was estimated to be 0.01, and the protein was quantitatively analyzed by label-free quantitative analysis. Proteins were considered differentially expressed when they exhibited at least 1.5-fold change in abundance between treatment and reference proteomes. Proteins were mapped into metabolic pathways using Kyoto Encyclopedia of Genes and Genomes (KEGG) [61]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with the dataset identifiers (PXD033344).

Fuctional verification of gene HBCD-hd-1. The HBCD-hd-1 gene was expressed and purified in E. coli, and the degradation of 4-BBA was carried out. After enrichment and culture of strain Y3, the DNA was extracted by DNA extraction kit, and the DNA fragment of HBCD-hd-1 was amplified with primers 2448-F and 2448-R (Table S1). Then, the HBCD-hd-1 gene was cloned and ligated into the cloning vector pEASY-Simple. The plasmid was transformed and expressed in DH5α competent cells and extracted by plasmid extraction kit. The plasmids and pCold-Ⅲ vectors were digested with NdeI and XbaI for 1 h at 37 ℃, respectively. After recovery and purification, the target gene HBCD-hd-1 was ligated with an empty vector with the T4 ligase (16 ℃, 12 h). E. coli harboring HBCD-hd-1 was grown in LB culture at 37°C to an OD₆₀₀ of 0.6–0.8 and subsequently induced for 12 h by adding 0.1 mM isopropyl-beta-D-thiogalactopyranoside at 16°C. Electrophoresis and sequencing were performed to detect the gene sequence of HBCD-hd-1 and to ensure that no mutation occurred. After expanded culture, batch experiments were carried out with 4-BBA to verify the debromination function of HBCD-hd-1. Functional genes HBCD-hd-1 was uploaded to NCBI with the accession number ON155918.

Analytical methods. On the basis of genomic and proteomic analysis, the expressions of genes involved in the degradation process were detected and screened out as differentially expressed genes (DEGs). Enrichment analysis of DEGs from the three groups was performed to illustrate their distribution on the KEGG pathways. This helped elucidate their possible functions in the biological process and pathway. The phylogenetic tree of the whole genome was drawn by GTDB-Tk [62]. The data of average amino acid identity (AAI) and 16S rRNA gene identity were analyzed by CompareM [63]. The gene cluster arrangements were based on the length of functional genes. The phylogenetic tree of functional genes was generated by Neighbor Joining method [64]. Evolutionary distance bootstrap values were determined using the bootstrap method on MEGA-X [65]. To analyze the specific differences among several kinds of strain, genetic sequence alignment was made by using the sequence comparison tool, DNAMAN, and visualized through Circos [66].

Author contributions

Xingxing Peng: Original draft writing, Experiment conducting, Data analysis, Software, Visualization, Research design, Supervision. Tianyu Li: Experiment conducting, Data analysis, Visualization, Revision. Qihang Zheng: Original draft writing, Experiment conducting, Data analysis, Software, Visualization. Yingyuan Lu: Experiment conducting, Data analysis. Yuzhe He: Experiment conducting, Data analysis. Yetao Tang: Research design, Supervision. Rongliang Qiu: Review & editing.

Funding

This work was financially supported by the National Natural Science Foundation of China (no. 42077128), the Pearl River Nova Program of Guangzhou (no. 201806010100).

Availability of data materials

The whole genome of Citrobacter sp. Y3 was uploaded to GenBank with the accession number GCA011602505.1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with the dataset identifiers (PXD033344). Functional genes HBCD-hd-1 was uploaded to NCBI with the accession number ON155918.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author details

¹School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510006, Guangdong, China.²Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, Sun Yat-sen University, Guangzhou 510275, China.³Guangdong Provincial Key Laboratory of Agricultural & Rural Pollution Abatement and Environmental Safety, College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, China.⁴Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou 510642, China

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Citrobacter sp. Y3 Harboring a Novel Gene HBCD-hd-1 Mineralizes Hexabromocyclododecane (HBCD) with New Metabolic Pathways Based on Multi-omics Characterization

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