Mineralization of HBCD by strain Y3
The strain Y3 could effectively bio-transform HBCD, including its isomers, i.e., α-, β-, and γ-HBCD . It could utilize 13C-HBCD (α-, β-, and γ-HBCD) as sole carbon source and degrade it to 13CO2 to 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 . 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 . 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 13CO2 was 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 .
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) , DhaA , LinA , and LinB . Some proteins, such as glutathione S-transferase (GST)  and cytochrome P450 (CYP450) , 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 .
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 , HS9 , and GJY  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 , DhaA , GST , and CYP450 . 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 .
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 . 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%) , 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 . Enzyme LinC encoded by linC was responsible for electron transfer and substrate binding, and it cooperated with LinA to replace bromine with hydroxy . 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 . Based on the Paul W. Sternberg operon hypothesis, the demand for gene expression machines for gene expression can be minimized by operons . 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 . 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 . 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.