Cupriavidus metallidurans CH34 Possesses Aromatic Catabolic Versatility and Degrades Benzene in the Presence of Mercury and Cadmium

Heavy metal co-contamination in crude oil-polluted environments may inhibit microbial bioremediation of hydrocarbons. The model heavy metal-resistant bacterium Cupriavidus metallidurans CH34 possesses cadmium and mercury resistance, as well as genes related to the catabolism of hazardous BTEX aromatic hydrocarbons. The aims of this study were to analyze the aromatic catabolic potential of C. metallidurans CH34 and to determine the functionality of the predicted benzene catabolic pathway and the influence of cadmium and mercury on benzene degradation. Three chromosome-encoded bacterial multicomponent monooxygenases (BMMs) are involved in benzene catabolic pathways. Growth assessment, intermediates identification, and gene expression analysis indicate the functionality of the benzene catabolic pathway. Strain CH34 degraded benzene via phenol and 2-hydroxymuconic semialdehyde. Transcriptional analyses revealed a transition from the expression of catechol 2,3-dioxygenase (tomB) in the early exponential phase to catechol 1,2-dioxygenase (catA1 and catA2) in the late exponential phase. The minimum inhibitory concentration to Hg (II) and Cd (II) was significantly lower in the presence of benzene, demonstrating the effect of co-contamination on bacterial growth. Notably, this study showed that C. metallidurans CH34 degraded benzene in the presence of Hg (II) or Cd (II).


Introduction
Benzene, toluene, ethylbenzene, and xylene isomers, commonly known as BTEX, are volatile monoaromatic hydrocarbons often present in crude oil spilled sites and polluted industrial areas [1,2]. These compounds can easily spread to distant locations from their original locations, polluting natural water bodies, groundwater, and atmospheric air [3]. BTEX are hazardous to living organisms; their toxicity, mutagenicity, and carcinogenic effects increase during bioaccumulation in animal and human tissues. Benzene is the most unsafe and toxic component of this group, causing cancer and leukemia in humans [4]. BTEX are also toxic for microorganisms, disturbing the cell membranes, disrupting the electron transport systems, and provoking oxidative stress damage [2,5].
In polluted sites, bacteria are often exposed to a complex scenario wherein co-contamination of hydrocarbons and heavy metal ions may reduce the degradation potential of bacterial strains and effectiveness of the bioremediation process [6][7][8][9][10][11][12]. Aerobic bacterial degradation of hydrocarbons may be inhibited by heavy metals such as mercury and cadmium [6,[13][14][15][16]. Cadmium is a by-product of non-ferrous metal ore processing, a component of battery manufacturing, and a plastic stabilizer [17,18]. Mercury has been extensively used in gold extraction by amalgam and is released from heavy metal mining (e.g., copper, silver, and gold) and industrial activities [11,[19][20][21]. BTEX biodegradation in the presence of cadmium and mercury has barely been studied. Cd (II) was found to cause inhibition of toluene, ethylbenzene, and o-xylene degradation by Bacillus sp. [22], while limited studies have assessed the role of Hg on BTEX bacterial catabolism. Cadmium and mercury are toxic for microorganisms and may affect their bioremediation capabilities, mainly through the binding to sulfhydryl groups (cysteine and methionine) and substitutive metal binding to metalloproteins [8,[22][23][24][25].
Genomic analyses of Cupriavidus metallidurans strain CH34 revealed three bacterial multicomponent monooxygenases (BMM), which are a rare trait and may lead to new mixed or hybrid BMM with novel substrate specificities [48]. The aims of this study were to analyze the aromatic catabolic potential of C. metallidurans CH34 and to determine the functionality of the predicted benzene catabolic pathway and the influence of cadmium and mercury on benzene degradation.

Growth of C. metallidurans CH34 on Benzene and Other Aromatic Compounds
For benzene and aromatic compound growth experiments, CH34 cells grown in LPTMS medium on succinate (overnight culture), washed twice with NaCl 0.85% p/v and suspended in saline solution NaCl 0.85% p/v, were used as inoculum. Growth was followed by measuring the turbidity at 600 nm.
Strain CH34 was cultured in LPTMS medium using benzene (5 mM) as the sole carbon and energy source in 250 mL flasks with PTFE/silicone-screw caps at 150 rpm. These cultures were carried out after an adaptation protocol consisting in successive passages in LPTMS medium using 0.5 mM and 2.0 mM benzene using succinate-grown cells as initial inoculum (1% v/v, initial turbidity 600nm ≈ 0.006). Strain CH34 growth on benzene was determined by measuring turbidity at 600 nm. Values were calculated as the mean ± SD of the results of at least three independent experiments.
During growth on some aromatics, bacterial aggregation was identified by naked-eye observation. For BTEX compounds and p-cymene, growth by CH34 cells was also assessed in LPTMS agar plates containing sterile-filter papers soaked with each compound placed on the inside cover plate to supply the hydrocarbon via vapor-phase. Values were calculated as the mean ± SD of the results of at least three independent experiments.

Aromatic Catabolism Reconstruction of C. metallidurans Strain CH34
C. metallidurans CH34 genome was retrieved from NCBI (Accession Nº GCA_000196015.1). A curated dataset of catabolic enzymes (ring-hydroxylating oxygenases, multicomponent monooxygenases, extradiol, and intradiol dioxygenases) involved in aromatic compound catabolism reported in UniProtKB-Swissprot, EAWAG, KEGG, and scientific literature were analyzed in CH34 genome using a best-bidirecctional hit (BBH) approach with the BLASTP and TBLASTN tools of BLAST 2.7.1. software [50]. An identity ≥ 30%, coverage ≥ 70%, and e-value cut-off ≤ 1 × 10 −10 were used as a filter for gene identification. The genomic context of each gene identified was analyzed to identify a complete aromatic catabolic pathway. Missing enzymes of an incomplete pathway were manually searched on the CH34 genome using the same BBH approach and filter parameters.

RNA Isolation and Gene Expression Analysis
Total RNA was isolated from C. metallidurans CH34 cells grown until early exponential phase (turbidity 600nm = 0.2-0.3) and late exponential phase (turbidity 600nm = 0.5-0.6) on benzene (5 mM) or succinate (6.2 mM) using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. TURBO DNAfree set (LifeTechnologies, Carlsbad, CA, USA) was used to degrade residual DNA. A final qPCR test with the gyrB gene primers designed by Primer 3.0 (Table S2) was performed to confirm genomic DNA absence. The RNA concentration was quantified using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) and a Nanodrop spectrophotometer (Thermo Scientific, Carlsbad, CA, USA). RNA integrity was observed by agarose (1%) gel electrophoresis.
Reverse transcription was carried out using 200 ng of RNA and a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline was used as standard protocol [60]. Real-time PCR was performed using 20 ng of cDNA on a StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific, Carlsbad, CA, USA) and 0.3 µM of each primer. cDNA was initially denatured at 95 • C for 5 min. A 40 cycle amplification and quantification protocol (95 • C for 15 s, 55 • C for 15 s, and 60 • C for 15 s) with a single fluorescence measurement per cycle followed by a melting-curve program (95 • C for 15 s, 25 • C for 1 s, 50 • C for 15 s, and 95 • C for 1 s) were used according to the manufacturer's recommendations. PCR melting curves confirmed the amplification of a single product for each primer pair. Primers yielded products between 200 and 250 bp. The gyrB gene (RMET_RS00030) was amplified as a reference gene, yielding an amplicon of 233 bp. A standard curve was made in triplicate using serial dilutions (10-fold) for each amplicon in a linear range (10 ng-0.1 pg) of genomic DNA. qPCR efficiencies were calculated from the slopes of the log-linear portion of calibration curves by using the equation E = 10(1/slope). The reference gyrB gene was stably expressed according to the algorithms of BestKeeper [61]. Relative gene expression ratios were determined as outlined by Pfaffl (2001) [62], thereby normalizing gene expression levels of CH34 cells grown on benzene versus cells grown on succinate.

Determination of Mercury and Cadmium Minimum Inhibitory Concentrations
The resistance to mercury and cadmium for strains CH34 and F1 was determined by the value of minimum inhibitory concentration (MIC) of metals that prevent growth [35,37]. In order to assess MIC of C. metallidurans CH34 and P. putida F1 using succinate as sole carbon and energy source, we grew the cells until the late exponential phase in LPTMS medium using succinate (6.2 mM) as a carbon source and inoculated them in fresh LPTMS medium at the same succinate concentration at initial turbidity 600 nm = 0.06. On the other hand, for MIC determination using benzene (5 mM) as the sole carbon source, the cells were subjected to a previous adaptation protocol to induce the expression of the catabolic machinery prior to heavy metal exposure. This adaptation consisted of benzene exposure to succinate-grown cells, as previously described, inoculating the cells in fresh LPTMS with benzene (1 mM) and 12 h incubation under the same culture conditions. Benzene-adapted cells were subsequently washed twice using NaCl (0.85% p/v) and Tween20 (0.05% v/v) in order to eliminate remaining benzene and inoculated in fresh LPTMS medium using benzene (5 mM) as the sole carbon and energy source to reach an initial turbidity 600 nm = 0.006 in glass tubes closed using PTFE/silicone-screw caps. Succinate and benzene-grown cells were exposed to increasing concentrations of Hg (II) or Cd (II) from stock solutions of HgCl 2 and CdCl 2 . Data were collected after 48 h at 30 • C. Values were determined using at least three independent biological replicates.

Benzene Degradation Assays
Benzene degradation assays were performed using borosilicate glass tubes (25 mL) with PTFE/silicone-screw caps and arranged in horizontal position. Glass tubes were equilibrated with 1.25 mL of resting cell buffer (Tris-Cl 50 mM (pH 7.0), Tween20 (0.05% v/v)) supplemented with benzene (10 mM) for 12 h under culture conditions at 30 • C. Tween20 (0.05% v/v) was added to increase bioavailability in buffer solution and mitigate volatilization. C. metallidurans CH34 and P. putida F1 cells were subjected to the benzene adaptation protocol described in Section 2.7. Washed cells were concentrated to obtain a cell suspension at turbidity 600nm = 10 in the resting cell buffer. Aliquots of 1.25 mL of cell suspensions were added to the benzene-equilibrated glass tubes, resulting in a 2.5 mL resting cell suspension at turbidity 600nm = 5.0 and benzene (5 mM). Final concentrations of Hg (II) and Cd (II) were obtained after the addition of aliquots from HgCl 2 and CdCl 2 100 × stock solutions. Samples (100 µL) were taken at 3 h intervals during 24 h of incubation at culture conditions. Cells were removed by centrifugation (19,000× g for 1 min) for subsequent analyses of benzene and their metabolites by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) and spectrophotometry. A boiled cell suspension was used as a dead cell control to determine abiotic loss of benzene. Cells were killed by boiling for 10 min in a water bath. Resting cells assays were performed in triplicate, and values were calculated as the mean ± SD of the results of at least three independent experiments.
HPLC-DAD analysis was carried out using a Jasco HPLC model LC-2000 equipped with a diode array detector (DAD) Jasco model MD-2015 using a RP 18e/Chromolith column of 100-4.6 mm and a pore size of 13 nm (Merck, Darmstadt, Germany). The solvents used for sample elution were 0.1% formic acid in water (A) and 100% acetonitrile (B). The flow rate was 1.0 mL/min, and the elution profile was 70% A/30% B for 4 min, then changed linearly to 0% A/100% B over a 1 min period and kept at this ratio for 3 min and finally changed linearly to 30% A/70% B over 1 min and kept at this ratio for 2 min. Benzene, phenol, and catechol concentrations were monitored using calibration curves with authentic standards. The injection volume was 10 µL. Benzene, phenol, and catechol determinations were performed at 200, 270, and 276 nm wavelengths, respectively.
The tmoX (RMET_RS06590) gene, part of the CMGI-2, encodes a TbuX/FadL-type outer membrane transport protein that possesses a 43% identity with the transporter TmoX of Pseudomonas mendocina KR1, which is part of the toluene X superfamily of mono-aromatic outer membrane transport proteins [64].

Benzene Catabolism by C. metallidurans CH34
The functionality of the benzene catabolic pathway of C. metallidurans CH34 was evaluated. Benzene was selected as a model BTEX compound due to its environmental relevance, high toxicity, and presence in hydrocarbon-polluted sites. During C. metallidurans CH34 growth on benzene, at early exponential phase (22 h), a yellow color formation was observed. The absorbance spectrum of the yellow-colored culture by spectrophotometry indicated the formation of 2-hydroxymuconic semialdehyde (2-HMS) during the metacleavage pathway by the catechol 2,3-dioxygenase (C23O) TomB ( Figure 3B).
To identify other metabolic intermediates of the benzene catabolism, we analyzed culture supernatants of CH34 cells grown on benzene (5 mM) by HPLC-DAD. Benzene concentration decreased during the incubation, while the formation of phenol was detected after 15-26 h ( Figure 3B). However, catechol was not detected by HPLC-DAD.

Transcriptional Analysis during Benzene Degradation
The expression of genes encoding the T2MO (tomA3), TMO (tmoA), PH (phyC), catechol 1,2-dioxygenase (C12O; catA1 and catA2), C23O (tomB), semialdehyde-2-hydroxymuconate dehydrogenase (tomC), and semialdehyde-2-hydroxymuconate hydrolase (tomD) were quantified by RT-qPCR analysis. Transcriptional analysis of CH34 cells grown on benzene as the sole carbon source showed a simultaneous expression of genes encoding monooxygenases in both early and late exponential phase (Figure 4). The tomB, catA1 and catA2 genes displayed differential transcriptional expression. The tomB gene encoding C23O was induced at the early and late exponential phases, whereas the catA1 and catA2 genes showed a higher induction at the late exponential phase. The tomC and tomD genes were expressed at the early and late exponential phases, suggesting that the hydrolytic and the 4-oxalocrotonate branches of the meta-cleavage pathways are active. In addition, the transcription of the sigma-38 factor rpoS gene, as well as the LysR-type (catM and benM) and the XylR/NtrC-type (tomR and poxR) transcriptional regulators, were studied. The tomR gene encoding a XylR/NtrC-type transcriptional regulator was expressed at early and late exponential phase. Conversely, the sigma-38 factor rpoS gene, and the benM, catM, and poxR genes encoding for transcriptional regulators were expressed only during the late exponential phase (Figure 4). On the basis of these results, we propose that benzene is degraded in C. metallidurans CH34 by an upper pathway that involves successive monooxygenation reactions catalyzed by the BMMs: PH, T2MO, and TMO, producing phenol and catechol as metabolic intermediates ( Figure 5). The central pathway starts with a metaor ortho-cleavage by catechol dioxygenases TomB in order to form 2-HMS, or by CatA1 and CatA2 in order to form cis-cis muconate, respectively. Transcriptional data and the metabolite formation suggest a predominant meta-cleavage pathway via C23O, while an induction of the catA1 and catA2 genes was observed at a late exponential phase. The induction of the tomC and tomD genes at early exponential phase suggest that both branches of the meta-cleavage pathway are active during benzene degradation ( Figure 5).

C. metallidurans CH34 and P. putida F1 growth on Benzene in the Presence of Mercury and Cadmium
To evaluate the effects of mercury and cadmium during growth on benzene, we determined the MICs of Hg (II) and Cd (II) in C. metallidurans CH34, and the model benzene-degrader P. putida F1. P. putida F1 was incorporated as a model proteobacterium for aerobic benzene degradation [52,65].
The MICs of Hg (II) and Cd (II) were significantly lower when the strains were previously grown on benzene (5 mM) compared to succinate-grown cells ( Table 2). Strain CH34 showed higher MICs for Hg (II) than strain F1, independently of the carbon source. For strain F1, the impact of benzene growth on MIC was higher in the presence of Cd (II) than in presence of Hg (II), reaching only 10% of the MIC observed during growth on succinate ( Table 2).

The Effects of Mercury or Cadmium on Benzene Degradation by C. metallidurans CH34 and P. putida F1
The effects of mercury (32.5 µM) or cadmium (200 µM) on benzene degradation by C. metallidurans CH34 was studied and compared with the degradation by benzenedegrader P. putida F1. The metal ion concentrations were selected after preliminary experiments to determine which concentration caused an inhibitory effect in bacterial catabolism. Benzene degradation is achieved in strain CH34 via BMMs, while strain F1 activates the aromatic ring using a benzene/toluene dioxygenase [66]. The benzene, 2-HMS, phenol, and catechol concentrations were monitored in resting cell assays (Figures 6, 7, and S1). The resting cell assay considered an initial concentration of 5 mM of benzene and a cellular turbidity 600nm = 5.0.
In the absence of heavy metals, C. metallidurans CH34 showed a lower benzene degradation rate in comparison with P. putida F1, which showed no residual benzene with the concomitant highest 2-HMS concentration after 3 h (Figure 6). A decrease in benzene was observed for dead cells for both strains, indicating abiotic benzene loss from the aqueous phase, probably by volatilization and biosorption. For the resting cell assay of benzene by strain CH34, phenol was detected at 3 h ( Figure S1), while 2-HMS was detected at 6 h ( Figures 6A and 7A), supporting the proposed benzene degradation pathway in C. metallidurans CH34.    Table S2. The fold-change in gene expression was calculated relative to CH34 cells grown on succinate. p value = 0.1%.  The presence of mercury or cadmium had no detrimental effects on benzene degradation by C. metallidurans CH34 (Figures 6 and 7, Table S5). Of note, despite a decay in phenol production by both heavy metals ( Figure S1), mercury exposure slightly stimulated the benzene degradation, supported by an increase of 2-HMS production at 6 h incubation ( Figure 6A, Table S5). After longer incubation, CH34 2-HMS production rate was lower during Hg (II) exposure compared to the control condition. In contrast, cadmium slightly inhibited CH34 production of 2-HMS at shorter incubations (6 and 9 h), but not after 12 h ( Figure 7A, Table S5). In P. putida F1, the presence of mercury strongly inhibited benzene degradation, showing almost negligible 2-HMS production ( Figure 6B, Table S5). Cadmium showed no effect on strain F1 benzene oxidation; however, a slight inhibition in 2-HMS degradation was observed ( Figure 7B, Table S5).

Aromatic Catabolic Reconstruction of C. metallidurans CH34
In this study, we reported a wide variety of aromatic compounds that can be used as carbon source by C. metallidurans CH34, and the aromatic catabolic pathways including the routes to degrade BTEX, chlorobenzoates, and lignin-derived compounds were inferred from genomic studies. We also reported new metabolic insights to describe the benzene catabolic pathways observing intermediates at different growth stages and by functional expression of key genes. Finally, we studied the effects of the presence of heavy metals on benzene degradation since the capabilities of strain CH34 to degrade aromatic compounds such as benzene in the presence or absence of toxic heavy metals have been scarcely reported in the wild-type strain CH34 [44]. Springael et al. transferred the plasmid pSS50, which is involved in polychlorobiphenyl (PCB) degradation, into strain CH34 to study the effect of nickel and zinc ions on PCB degradation by the transconjugant strain AE707, overlooking the innate catabolic potential of strain CH34 [26,45]. Aromatic degradation by Cupriavidus strains has been described, but only three genomes have been sequenced and analyzed to prospect their overall aromatic degradation capabilities: the chloroaromatic degrader C. pinatubonensis JMP134, the lignin degrader C. basilensis B-8, and the 3-CBA degrader C. necator NH9 [27,67,68]. Of note are the harboring of C1 and C2 genes by C. metallidurans CH34, which confers a similar catabolic versatility to strain JMP134, a well-known aromatic degrader model [69], including multiple central pathways for aromatic degradation (the protocatechuate and the catechol β-ketoadipate pathways; the meta-cleavage catechol pathway; the HQ pathway; the homogentisate pathway; the 3-hydroxyanthranilate pathway; and the hybrid aerobic pathways 2-aminobenzoyl-CoA, phenylacetyl-CoA, and benzoyl-CoA), three BMMs, and genes encoding benzoate and lignin-derived degradation enzymes. Morever, these results showed the ability of strain CH34 to grow on 3-CBA and 3,5-CBA despite the absence of canonical chlorocatechol pathways (tfd and clc genes). Following these results, previous studies reported that C12O of strain CH34 possesses a unique spectrum to cleave substituted-catechols in ortho position, including tetrachlorocatechol, 4-fluorocatechol, 4-methylcatechol, and 3-methylcatechol [70,71]. Even though specific pathways for chloro-substituted aromatics were not identified in the CH34 genome, the benzoate 1,2-dioxygenase BenABCD may perform the dioxygenase activation with a lower affinity to convert 3-CBA and 3,5-CBA into chlorocatechols, which has been reported during degradation of 3-CBA by C. pinatubonensis JMP134 [72]. Transcriptome profiling of C. necator NH9 cells grown on 3-CBA compared to benzoate supports the hypothesis that 3-CBA and benzoate are degraded by a common pathway [68].
In the present study, we also observed that C. metallidurans CH34 grew on benzene, toluene, ethylbenzene, and xylenes. In addition, CH34 cells are also able to grow on pcymene, BA, 3-CBA, 3,5-CBA, salicylate, 3-HBA, 4-HBA, pABA, PA, 3-HPA, 4-HPA, vanillin, and cinnamate. These results support the corresponding catabolic pathways identified by genomic analyses (Table 1). Although strain CH34 grew on salicylate, p-cymene, 3-HBA, 3-HPA, 4-HPA, benzamide, pABA, and cinnamate, ortholog genes for their previously described catabolic pathways were not found in the CH34 genome, suggesting the presence of still undescribed catabolic pathways in the Cupriavidus genus. The absence of degradative canonical pathways of phenylpropanoids (cinnamate, phenylpropionates) and 4-HPA in Cupriavidus strains B-8, NH9, and JMP134, despite their growth on these compounds [27,31,67], indicate that members of the Cupriavidus genus may degrade them via novel catabolic pathways. Cytochrome P450 OhpA is a self-sufficient cytochrome involved in the transformation of 2-HPA into homogentisate, which was described recently in C. pinatubonensis JMP134 [73], being also present in the genome of strain CH34 (RMET_RS25305), evidencing the need for further characterization of novel enzymatic activities in the Cupriavidus genus.

BMMs in C. metallidurans CH34
The presence of more than one BMM observed in C. metallidurans strain CH34 is rare in bacteria, with its co-expression potentially leading to the formation of a complex modularity, generating new substrate specificity and providing optimized metabolic capabilities [40,48,74]. The first BMM of strain CH34 was constituted by T2MO (encoded by the tomA012345 gene cluster), which possesses a high similarity in amino acid sequence and gene organization to the B. vietnamiensis G4 T2MO subunits (Figure 2 and Table S1), suggesting a regiospecific hydroxylation of toluene into o-cresol and, subsequently, an oxidation into 3-methylcatechol [75][76][77]. In addition, T2MO catalyzes the oxidation of dichloroethylenes, chloroform, 1,4-dioxane, aliphatic ethers, and diethyl sulphide [76,78] and enables the formation of epoxides from a variety of alkene substrates [79]. The second CH34 BMM is the TMO encoded by the tmoABCDEF gene cluster, which belongs to the group II BMMs. The products of these genes are similar to the corresponding subunits of the BMMs TbcABCDEF (C. pinatubonensis JMP134 and B. cepacia JS150) and TmoABCEDF (P. mendocina KR1) ( Figure 2). All these BMMs have different substrate specificities, enabling the oxidation of toluene and other BTEX compounds [54,80]. For example, T4MO of strain KR1 oxidizes toluene into 4-methylcatechol and catalyzes the formation of epoxides [79], as well as the oxidation of phenols and methylphenols into catechol [75]. An outstanding broad substrate group II BMM is the toluene o-xylene monooxygenase (ToMO) of P. stutzeri OX1, which oxidizes o-xylene, m-xylene, p-xylene, toluene, benzene, ethylbenzene, styrene, naphthalene, and tetrachloroethylene [74]. The characterization of the hydroxylation capabilities of CH34 TMO should be performed due to their potential broad substrate specificity and the classification of the CH34 TmoE subunit as a singleton in the phylogenetic analysis of γ-hydroxylase subunit of the group II BMMs. C. metallidurans CH34 genome encodes a third BMM, a PH that is encoded by the phyZABCDE gene cluster. This enzyme is also present in C. pinatubonensis JMP134 and transforms benzene into phenol, and then into catechol ( Figure 5). In accordance, the phyZABCDE gene cluster is located upstream of the catA1 gene, which encodes a catechol 1,2-dioxygenase (C12O) (Figure 2, Table S3) [39]. The phy-catA1 gene cluster might be controlled by PoxR, a phenol-sensitive XylR/NtrC-like transcriptional activator (Table S3). Furthermore, an additional C12O is encoded on the C2 (RMET_RS25045) and belongs to the benzoate catabolic ben-cat gene cluster [28]. Multiple BMMs were encountered in other Burkholderiales degraders (B. cenocepacia JS150 and C. pinatubonensis JMP134), which could account for their exceptionally versatile catabolic capabilities [28].

Functionality of CH34 BMMs Associated with the Degradation of Benzene
In this study, the functionality of CH34 BMMs associated with the degradation of benzene was reported for the first time. Transcriptional analyses and kinetics of the metabolic intermediates indicated that CH34 BMM activities depend on the growth phase. Phenol accumulation during CH34 growth on benzene was observed ( Figure 3B). The results of this study suggest that one or more BMMs could be activated simultaneously during the process, thereby indicating the occurrence of a complex degrading module to generate a catabolic strategy. Interestingly, activation of diverse BMMs has been proposed in C. pinatubonensis JMP134, R. pickettii PKO1, and B. cenocepacia JS150 [48,54,80]. RT-qPCR revealed the successive use of two transcription profiles according to the growth phase. At early exponential phase, two BMM-encoding gene clusters (tomA012345 and tmoABCDEF), and tomB, tomC, and tomD genes forming the C23O pathway that all belong to the the CMGI-2 catabolic gene cluster ( Figure 4) were induced. In contrast, at late exponential phase, the phyZABCDE gene cluster encoding the PH, and the catA1 and catA2 genes that compose the C12O pathway were upregulated. This transcription profile associated with the late exponential growth phase during benzene catabolism is in agreement with the downstream location of a rpoS promoter and the reported inhibition of C12O activity in the presence of phenol [54]. Therefore, these results suggest an induction of the C12O pathway only at late growth phase on benzene at ultimately lower phenol concentrations (Figure 7). Transcription analyses and metabolic intermediate detection suggest that meta-cleavage of catechol is catalyzed by a C23O (TomB) at early stages of growth, whereas ortho-cleavage, catalyzed by a C12O (CatA1, CatA2) at late stages of growth, is driven by starvation and the general stress transcriptional factor RpoS. Although versatile predominance between the C12O or C23O degradation pathways has been previously reported as dependent of substrate and concentration [81,82], more analyses should be performed to determine to which extent each CH34 catabolic pathway is involved in benzene degradation.

Effect of Benzene on Bacterial Cadmium MICs
Cadmium MICs of C. metallidurans CH34 and P. putida F1 grown on benzene were significantly lower compared to MICs of succinate-growing bacteria. However, cadmium showed a higher impact on strain F1. These results are in agreement with the heavy metal resistance niche specialization of the CH34 strain, reflexed on its horizontal acquisition and duplication of genes that encode cadmium efflux systems [39,83,84], some of which are represented as homologous genes in the genome of strain F1, although to a significant lower extent [85].
Previously, we have reported in strain CH34 that Cd (II) induces genes encoding the cadmium efflux system CadA and the mercury reductase MerA that are involved in heavy metal response [41]. In addition, cadmium decreases the second messenger c-di-GMP, induces a metal regulated phosphodiesterase, and inhibits biofilm formation [41]. Aromatic hydrocarbons may increase membrane fluidity, causing non-specific permeabilization and membrane protein disruption [86,87], which may affect the cation uptake and explain the reduced MIC values. The occurrence of futile cycles during cadmium detoxification, but not in mercury detoxification systems, has been reported due to the volatile nature of the mercury detoxification product [88]. Therefore, a faulty efflux system for Cd (II) may originate from benzene exposure in both CH34 and F1 strains. Cd (II) induces oxidative stress in bacteria [88]. An increase in reactive oxygen species by cadmium can lead to protein carbonylation, lipoperoxidation, and oxidative damage to DNA, which may act in synergy with benzene-induced membrane disruption, strongly reducing cell viability [89].

Effects of Mercury and Cadmium on Benzene Degradation
In the present study, the effects of mercury and cadmium on benzene degradation by C. metallidurans CH34 and the model benzene/toluene-degrader P. putida F1 were studied and compared. In the absence of heavy metals, P. putida strain F1 showed a higher benzene degradation rate than C. metallidurans CH34. Benzene transformation into catechol via dioxygenases is achieved using a single oxygen molecule, whereas monooxygenase-mediated activation converts benzene to catechol in two hydroxylation steps, with the accumulation of the toxic metabolic intermediate phenol [54]. While mercury and cadmium concentrations used in the benzene degradation assays by a resting cell approach were not equitoxic in comparison with respective MICs of C. metallidurans CH34 or P. putida F1, these concentrations corresponded to minimal heavy metal concentrations that showed observable effects on benzene degradation in both strains. Heavy metal effect on benzene catabolism in both strains could also be attributed to a general stress response, affecting other enzymatic systems that could indirectly influence the degradation rate of each compound. Nevertheless, the concentrations evaluated and their effects are in accordance with the differences within both cytoplasmic detoxification systems aforementioned in Section 4.4.
In the presence of mercury, C. metallidurans CH34 showed higher MICs during growth on benzene and on succinate than P. putida strain F1. Strain CH34 possesses a moderate mercury resistance, whereas P. putida F1 is a mercury-sensitive strain [35,40,41,90], which is in accordance with the effect of mercury exposure on benzene degradation assays observed. Notably, C. metallidurans CH34 showed a slightly higher degradation rate of benzene in the presence of mercury (32.5 µM; Figure 6A) than in the absence of this heavy metal; in contrast, P. putida F1 was unable to degrade benzene in the presence of mercury. The increased CH34 benzene degradation in the presence of mercury is noteworthy since the reported detrimental effect of mercury (36,180, and 360 µM) on the catabolism of aromatic compounds in soil microbiota [14]. Although the modest increase in the benzene degradation rate was observed by strain CH34, 2-HMS production rate was lower than the unexposed condition after the 6 h, suggesting dissimilar effects for the upper or lower benzene catabolic pathway. Interestingly, the induction by arsenic of the m-xylene degradation genes in P. putida mt2 has been described [91]; therefore, the induction of some catabolic genes by some metals might not be discarded. Future studies should investigate the transcriptional regulation and possible co-activation of the benzene degradation pathway and the mer genes in C. metallidurans CH34. Strains CH34 and F1 were able to degrade benzene in the presence of cadmium. Cd (II) (200 µM) slightly decreased the benzene degradation by C. metallidurans CH34 during the first 6 h ( Figure 7A), but no inhibition on degradation was observed after longer incubation (Figures 7 and S1). The impairment of phenol production during the short incubation time may have been caused by the destabilization of T2MO and TMO (2Fe-2S) clusters by cadmium, which has been reported for copper and zinc [92][93][94]. The partial decrease in 2-HMS production caused by cadmium could be explained by the mixed nature of C. metallidurans CH34 catabolic pathway, in which the cytoplasmic presence of Cd (II) may favor C12O activity instead of C23O. The location of the catA1 gene downstream of a RpoS promoter is in accordance with an induction of its expression under cadmium stress [95][96][97]. In addition, cadmium impairs C23O activity in Stenotrophomonas maltophilia KB2 and Variovorax sp. 12S while increasing in vitro C12O activity by up to 270% [98,99]. It has been reported that cadmium binds to C23O extradiol aromatic ring cleaving dioxygenases, replacing Fe (II) in its catalytic binding site [100], but not to the C12O dioxygenase active site, which contains Fe (III) [71].
On the other hand, P. putida strain F1 achieved a similar benzene degradation rate in the presence or absence of cadmium, suggesting that the dioxygenase-mediated catabolism is not affected by cadmium. In accordance, the toluene dioxygenase active site containing Fe (III) showed non-susceptibility to divalent cation ligand substitution [101].

Conclusions
This study showed that C. metallidurans CH34 possesses a wide aromatic catabolic versatility and is able to grow on a broad range of monoaromatic compounds including BTEX compounds, chlorobenzoates, and lignin-derived aromatics. CH34 benzene degradation occurs by the activation of the aromatic ring through three BMMs and a central mixed route composed by C12O and C23O catechol dioxygenases that are differentially regulated at early and late exponential growth phases. Notably, strain CH34 degraded benzene in the presence of the toxic heavy metals mercury and cadmium. The presence of mercury (32.5 µM) slightly favored benzene degradation in C. metallidurans CH34; however, it inhibited benzene degradation in P. putida F1. Cadmium (200 µM) decreased benzene degradation by strain CH34 during short incubation (6 h) but not after longer time periods, whereas P. putida F1 degradation was not affected. These results indicate that C. metallidurans CH34 is an attractive biocatalyst for the bioremediation of BTEX in sites polluted with aromatic compounds and heavy metals such as mercury and cadmium.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/microorganisms10020484/s1, Figure S1: Effects of cadmium and mercury on phenol production in C. metallidurans CH34 during resting cell assays. Table S1: Bacterial multicomponent monooxygenase operon information of Cupriavidus metallidurans strain CH34 and other Proteobacteria. Table S2: Primer sets designed and used in this study. Table S3: Predicted aromatic central pathways in C. metallidurans CH34. Table S4: Predicted aromatic peripheral pathways in C. metallidurans CH34. Table S5: Statistic analysis of mercury and cadmium effects on benzene degradation and intermediate production by C. metallidurans CH34 and P. putida F1.