The Cytochrome bd Complex Is Essential for Chromate and Sulfide Resistance and Is Regulated by a GbsR-Type Regulator, CydE, in Alishewanella Sp. WH16-1

Sulfate-reducing bacteria are a group of microorganisms that use sulfate as an electron acceptor. These bacteria are useful in the bioremediation of heavy metal pollution since they can reduce/precipitate metals. Previously, we identified the Alishewanella strain WH16-1 from soil of a copper and iron mine and determined that it can reduce sulfate and chromate and that it was tolerant to many heavy metals. In this study, we investigated the chromate reduction mechanism of strain WH16-1 through Tn5 transposon mutagenesis. A cytochrome bd (cytbd) Tn5 mutant was generated (Δcytbd), and a detail analysis showed that the following: (1) gene cydE (coding for a GbsR-type regulator) was co-transcribed with the two subunits coding genes of the Cytochrome bd complex (Cytbd), namely, cydA and cydB, based on RT-PCR analysis, and similar gene arrangements were also found in other Alteromonadaceae family strains; (2) the chromate resistance level was dramatically decreased and chromate reduction efficiency also decreased in strain Δcytbd compared to the wild-type and a complemented strain (Δcytbd-C); (3) Cytbd could catalyze the decomposition of H2O2 according to the analyses of H2O2 decomposition ability, cellular H2O2 contents, H2O2 inhibition zone, and H2O2 sensitivity tests; (4) surprisingly, chromate was not an inducer of the expression of Cytbd, but sulfate induced expression of Cytbd, and sulfate/sulfide resistance levels were also decreased in the Δcytbd strain; (5) the addition of sulfate enhanced the chromate resistance level and reduction efficiency; (6) Cytbd expression was repressed by CydE and derepressed by sulfate based on an in vivo bacterial one hybrid system and in vitro EMSA tests; and (7) DNA footprinting and short-fragment EMSA tests revealed two binding sites of CydE in its promoter region. All these results showed that Cytbd is negatively regulated by CydE and derepressed by sulfate. In addition, Cytbd contributes to the resistance of sulfate and sulfide, and sulfide could be used as a reductant to reduce chromate. Moreover, Cytbd is essential to decompose H2O2 to decrease cellular oxidative stress. Thus, the regulation and function of Cytbd may explain why sulfate could enhance chromate reduction.


INTRODUCTION
Sulfate-reducing bacteria (SRB) are a diverse group of prokaryotes that use sulfate as the terminal electron acceptor and produce H 2 S (Muyzer and Stams, 2008). They are widely distributed and play a key role in the environment (Muyzer and Stams, 2008; Barton and Fauque, 2009). Various SRB have exhibited great potential for environmental bioremediation applications, such as participating in the precipitation of heavy metals to produce metal sulfides Zhou et al., 2016), reduction of toxic metals (Barton and Fauque, 2009), degradation of azo dyes (Pandey et al., 2007), and nitroaromatic compound respiration (Barton and Fauque, 2009).
Chromate is chemically analogous to sulfate and enters cells mediated by the sulfate ABC transporter CysPUWA in various bacteria (Viti et al., 2014). Some SRB are also chromate-reducing bacteria (CRB) (Viti et al., 2014;Xia et al., 2016). Accordingly, Cr(VI) induces the expression of the sulfate transporter and competes with sulfate in some bacteria (Viti et al., 2014). Moreover, the products of sulfur assimilation are involved in Cr(VI) detoxification. H 2 S, cysteine, and glutathione (GSH) are capable of directly reducing Cr(VI) (Cheung and Gu, 2007;Thatoi et al., 2014;Joutey et al., 2015). In addition, GSH also plays an important role in maintaining cellular sulfhydryl groups in their reduced form when exposed to oxidative stress induced by Cr(VI) (Presnell et al., 2013;Viti et al., 2014). However, many details concerning the effects of sulfur metabolism on Cr(VI) detoxification remain unclear.
Cytbd is a terminal respiratory oxidase found in many prokaryotes and is composed of two subunits, CydA and CydB (Giuffre et al., 2014). CydC and CydD are also needed for the assembly of Cytbd in Escherichia coli (Borisov et al., 2011), while CydX is also essential for the activity of Cytbd in some bacteria (Sun et al., 2012;VanOrsdel et al., 2013;Chen H. et al., 2015). Cytbd is involved in energy supply, bacterial virulence, and resistance to oxidative and nitrosative stresses (Borisov et al., 2013;Giuffre et al., 2014;Roop et al., 2015). Recently, Cytbd was also found to be associated with sulfide resistance in E. coli, since sulfide could inactivate heme-copper family respiratory oxygen reductases (cytochrome bo 3 ) but not the copper-free Cytbd (Forte et al., 2016;Korshunov et al., 2016). In addition, the expression of Cytbd is regulated by the transcriptional regulators Arc, Fnr, and CydR, depending on environmental oxygen concentrations in E. coli and Azotobacter vinelandii (Wu et al., 1997;Borisov et al., 2011). Potential regulator genes containing helix-turn-helix (HTH) conserved domain sequences were observed adjacent to the cytbd operon in some bacteria (Degli Esposti et al., 2015). However, these potential regulators, such as the GbsR type (Nau-Wagner et al., 2012;Lee et al., 2013), have not been reported to regulate the expression of Cytbd. Furthermore, no study concerning the relevance between Cytbd and chromate resistance has been reported thus far.
Alishewanella sp. WH16-1 (=CCTCC M201507) was isolated from soil of a copper and iron mine. It possesses great potential in metal bioremediation since it reduces sulfate and chromate or generates CdS/PbS precipitation (Xia et al., , 2018Zhou et al., 2016). Strain WH16-1 also showed a high tolerance to Cr(VI) (MIC of 45 mM) . However, its Cr(VI) resistance and reduction mechanisms remain to be explored. A first step of this study was to investigate the Cr(VI) detoxification mechanism of strain WH16-1 through Tn5 transposon mutagenesis. Later, we found that Cytbd was relevant to chromate, sulfate, and sulfide resistance. Interestingly, the transcription of cytbd was repressed by a GbsR-type regulator (named CydE) and depressed by sulfate.

Transposon Mutagenesis and Construction of a Complemented Strain
To identify the molecular mechanism of Cr(VI) detoxification of strain WH16-1, Tn5 transposon mutagenesis was used for screening Cr(VI) resistance genes. Transposon insertion mutants were generated with a suicide plasmid pRL27 (Larsen et al., 2002) transferred from the donor strain E. coli S17-1 to the recipient strain WH16-1 using the filter mating method (Smith and Guild, 1980). After conjugation, the Tn5 (Km r ) transposon was randomly inserted into the chromosome DNA of strain WH16-1, generating a library of insertion mutants. Selection was done on LB plates with Rif (50 µg/mL) and Km (50 µg/mL) to obtain strains in which transposition had occurred. The transconjugants were then plated on two LB plates with or without 20 mM K 2 CrO 4 , and the colonies that were unable to grow in the presence of K 2 CrO 4 were reserved and subjected to further analyses. Cloning of genes neighboring the Tn5 transposon was performed according to the plasmid rescue method described before (Chen F. et al., 2015). The resulting neighboring sequences were searched against the whole genome of strain WH16-1  using the NCBI BLAST server.
To identify the function of Cytbd, a complemented strain was constructed. The whole cytbd operon was cloned into the pCT-Zori plasmid using SacI and HindIII restriction enzyme sites. The generated plasmid was transferred into the mutant strain cytbd by conjugation from E. coli S17-1 to obtain a complemented strain, cytbd-C.
For co-transcription analysis, strain WH16-1 was incubated to an OD 600 of approximately 0.3 in 100 mL LB broth, followed by incubation with 1 mM K 2 CrO 4 for 3 h. Total RNA was extracted by Trizol reagent (Invitrogen), and DNA was removed by digestion with DNase I (Takara). Reverse transcription was conducted with a RevertAid First Strand cDNA Synthesis Kit (Thermo) with 300 ng total RNA for each sample. The resulting cDNA was used as a template to amplify the fragments between genes in the cytbd operon. Genomic DNA was used as a positive control. The total RNAs of strain WH16-1 and ddH 2 O were used as negative controls. Primers are shown in Supplementary  Table S2.

Reporter Gene Construction
The putative promoter and promoter-cydE regions (Supplementary Figure S1A) were each PCR amplified from genomic DNA of strain WH16-1. Each DNA fragment was then cloned into plasmid pLSP-kt2lacZ using EcoRI-BamHI restriction enzyme sites. The resulting constructs were designated as pLSP-promoter-lacZ (Supplementary Figure S1B) and pLSPpromoter-cydE-lacZ (Supplementary Figure S1C). E. coli DH5α containing pLSP-promoter-lacZ or pLSP-promoter-cydE-lacZ was incubated in LB medium. Overnight cultures were diluted 100 times with fresh medium and incubated for approximately 4 h (OD 600 approximately 0.3). Next, Na 2 SO 4 (0, 5, 25, and 50 mM) and K 2 CrO 4 (0, 1, and 5 mM) were added to the cultures. The cultures were then distributed into tubes after a 6-h incubation. β-Galactosidase enzymatic assays were performed using the method described by .

Chromate/Sulfate/Sulfide Sensitivity and Chromate Reduction Assay
Strains WH16-1, cytbd, and cytbd-C were each inoculated into 5 mL LB and incubated at 37 • C with shaking at 150 rpm. When the OD 600 reached approximately 0.8-1.0, the strains were each inoculated into 100 mL LB with the presence of 500 mM Na 2 SO 4 , 200 µM Na 2 S, 3 mM K 2 CrO 4 , or no addition. Na 2 SO 4 powder was added to LB medium before sterilization, while K 2 CrO 4 and Na 2 S were added from stock solutions. In addition, LB plates with 0 or 3 mM K 2 CrO 4 were used for Cr(VI) sensitivity analysis. For observing Cr(VI) reduction, strains were incubated in LB broth with 1 mM K 2 CrO 4 . To maintain consistent growth conditions, K 2 CrO 4 was added when OD 600 reached 0.6. At designated times, culture samples were taken for measuring OD 600 and chromate amounts by spectrophotometry (DU800, Beckman) and atomic absorption spectrometry (AAS; 986A, Beijing Puxi General Instrument Co., Beijing, China), respectively.

Effects of Cytbd on Cellular Oxidative Stress
To analyze the effects of Cytbd on oxidative stress, membrane proteins of strains WH16-1, cytbd, and cytbd-C were extracted to react with H 2 O 2 and chromate. The membrane protein extraction was performed as described previously by Das et al. (2005), and the protein concentration was determined by the Lowry method (Lowry et al., 1951). Then, 10 mg/L of membrane proteins was reacted with 10 mM hydroquinone and 10 mM H 2 O 2 or K 2 CrO 4 in Tris-HCl (pH 8.5) buffer for 30 min under anoxic conditions in a N 2 chamber. The residual H 2 O 2 was measured by the Amplex red/horseradish peroxidase assay (Mishin et al., 2010), and chromate concentrations were determined as mentioned above.
To gain more insight into the effects of Cytbd on cellular oxidative stress, cellular H 2 O 2 contents of the WH16-1, cytbd, and cytbd-C strains were determined. The strains were incubated to an OD 600 of approximately 0.3 in 100 mL LB. K 2 CrO 4 was then added to the cultures until the final concentrations reached 1 mM. Cells were centrifuged and washed twice with potassium phosphate buffer (50 mM, pH 7.7). The pellets were lysed via sonication on ice for 3 min and centrifuged for 5 min at 12,000 rpm to remove particulate materials. H 2 O 2 amounts were measured as mentioned above.
Moreover, inhibition zone and H 2 O 2 sensitivity tests were performed. For the inhibition zone test, cultures of each strain (OD 600 approximately 0.8-1.0) were added to LB agar medium, and 200 µL of 3% H 2 O 2 was added to the Oxford cup . For the H 2 O 2 sensitivity assay, 5 µL overnight cultures of WH16-1, cytbd, and cytbd-C (OD 600 0.8-1.0) were dropwise added onto LB agar media containing various amounts of H 2 O 2 (0, 0.05, 0.1, 0.5, and 1 mM).

Effects of Sulfate on Chromate Reduction and Resistance
Chemically defined medium (CDM) was selected to test the effects of sulfate on Cr(VI) reduction and resistance in strain WH16-1. The components of the CDM medium were the same as previously described (Weeger et al., 1999), except for replacing sodium lactate, magnesium sulfate and sodium sulfate with maltose, magnesium, and sodium chloride, respectively. This medium contained 0.12 mM SO −2 4 and no other forms of sulfur. Strain WH16-1 was incubated with or without 100 µM Cr(VI) and additional 0, 5, or 10 mM sulfate in CDM medium. The remaining Cr(VI) in the medium was determined as mentioned above.

Bacterial One-Hybrid System Assay
The DNA binding activity of CydE was tested in vivo with a bacterial one-hybrid system (Guo et al., 2009). The cydE coding sequence was amplified and cloned into the pTRG vector using BamHI-EcoRI restriction enzyme sites to obtain a plasmid pTRG-cydE. The promoter sequence of the cydE (Supplementary Figure S1A) was amplified and inserted directly into XcmI site of pBXcmT, yielding the pBX-promoter plasmid. The next steps were followed as previously described (Guo et al., 2009;Shi et al., 2017). The pTRG-cydE and pBX-promoter plasmids were cotransformed into E. coli XL1-Blue and grew on selective screening medium plates (Guo et al., 2009). In addition, E. coli XL1-Blue containing the pBX-MthspXp and pTRG-Rv3133c plasmids served as the positive controls, while E. coli XL1-Blue containing the empty vectors pBXcmT or pTRG was used as negative controls (Guo et al., 2009;Shi et al., 2017).

Cloning, Expression, and Purification of CydE
The CydE coding sequence was also amplified from DNA of strain WH16-1 using specific primers (Supplementary Table S2) that were designed to contain the restriction sites for BamHI and HindIII. The PCR product was digested with these enzymes and cloned into pET28a generating plasmid pET28a-cydE. After DNA sequencing confirmation, the plasmid was introduced into E. coli BL21 (DE3) cells. CydE was overexpressed by adding 0.1 mM IPTG to cells at an OD 600 of 0.3-0.4 that were further cultured for 4 h at 28 • C. The cells were then harvested by centrifugation (8,000 rpm for 10 min at 4 • C). After washing twice with 50 mM Tris-HCl (pH 8.0), the pellets were lysed via French Press at 120 MPa. Next, the soluble supernatant was mixed with 1 mL preequilibrated Ni-NTA His Bind Resin (7sea Biotech) and gently agitated at 4 • C for 1 h. The resin was transferred into a 10-mL gravity-flow column and washed with 4 mL Tris-HCl with 200 mM imidazole to elute the miscellaneous proteins. The Histagged CydE protein was eluted in 1 mL Tris-HCl with 500 mM imidazole, and the eluted fractions were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The quality and quantity of the proteins were assessed with spectrophotometry (NanoDrop 2000, Thermo) and SDS-PAGE.

DNA Footprinting
One hundred nanograms of FAM-labeled DNA probe was incubated with 0, 0.2, and 0.4 µg CydE, respectively (the reaction system was the same as in EMSA), then digested by 6 × 10 −4 U/µL DNase I (New England Biolabs) for 10 min at room temperature. Next, the reaction was stopped by addition of 50 mM EDTA and incubation in a water bath at 65 • C for 10 min. The digested DNA fragments were purified with a PCR clean-up Gel extraction kit (Macherey-Nagel). Samples were analyzed in a 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, United States), and the electropherograms were aligned with GeneMapper v3.5 (Applied Biosystems, Foster City, CA, United States). For verification of the binding sites, a short-fragment EMSA test was used. The DNA sequences of the two binding sites identified by DNA footprinting were synthesized by Tsingke (Biological Technology Company, Beijing, China). The process of short-fragment EMSA was performed as described above. The final gel was stained by ethidium bromide.

Characterization of the Chromate-Sensitive Mutants by Transposon Mutagenesis
Random mutants were generated by mobilization of the suicide plasmid pRL27 from the donor strain E. coli S17-1 into the recipient strain WH16-1. Approximately 8,000 Kmand Rif-resistant clones were randomly chosen and initially tested for their ability to grow on LB plates containing 20 mM K 2 CrO 4 . After 48 h of incubation, 40 Cr(VI)-sensitive mutants were obtained. Mutation sites were identified in 13 mutants with decreased Cr(VI) resistance. These mutant genes encoded CydB, ChrB, ferredoxin, iron transporter, DNA repair proteins (UvrC, UvrD, RecA, RecB, RecC, and YebC), and three other proteins (ComEC, ScpA, and a hypothetical protein). The cydB mutant ( cytbd) was selected for this study since it may reveal potentially novel Cr(VI) detoxification mechanisms. The BLAST results showed that the Tn5 was inserted in the middle of the cydB (AAY72_09260) gene.
The cytbd Operon in Strain WH16-1 The cytbd operon sequence is conserved in the Alteromonadaceae strains based on phylogenetic analysis ( Figure 1A). Moreover, the gene arrangement is also similar in these strains ( Figure 1A). The genes coding for CydE (AAY72_09270) and CydA (AAY72_09265) were identified adjacent to cydB. The operon was located in contig 1 of the genome sequence. To gain more insight, RT-PCR was carried out. The forward and inverse primers used for RT-PCR were designed to overlap each two adjacent genes. The results of RT-PCR showed FIGURE 1 | Analysis of cytbd gene operon, cydB mutation, and complementation. (A) The amino acid-based phylogenetic relationship of the cytbd gene cluster and its adjacent gene arrangement in Alteromonadaceae family members. (B) cydE, cydA, and cydB were co-transcribed. (C) Physical evidence of cydB mutation and complementation. Lanes 1-3 are the PCR-amplified products of wild-type, cytbd, and cytbd-C by primers cydBF/cydBR, respectively. Lane 4 is a negative control. The Tn5 insert fragment is also approximately 1.8 kb. Consequently, the PCR product of the mutant strain is approximately 1.8 kb longer than PCR products of the wild-type and complemented strains.
Frontiers in Microbiology | www.frontiersin.org FIGURE 2 | Cytbd is induced by sulfate and contributes to sulfate and sulfide resistance. (A) Sulfate effects on Cytbd expression. β-Galactosidase activities were increased after adding sulfate or removing cydE behind the promoter. The growth of WH16-1 (wild type), cytbd (mutant strain), and cytbd-C (complemented strain) with 0 (B), 500 mM Na 2 SO 4 (C), or 200 µM Na 2 S (D) in LB medium. The values represent averages and standard deviations of three replicates.
that DNA fragments between the three genes (cydE/cydA and cydA/cydB) were amplified with DNA and cDNA templates. It implied that cydE, cydA, and cydB were cotranscribed in an operon ( Figure 1B). To verify the function of Cytbd, a complementation experiment was carried out. The complete cytbd operon including cydE, cydA, and cydB was introduced into the mutant strain cytbd and confirmed by PCR using primers cydBF/cydBR ( Figure 1C) and DNA sequencing. This generated the complemented strain cytbd-C.

The Expression of Cytbd Was Induced by Sulfate
Escherichia coli DH5α-pLSP-promoter-lacZ and E. coli DH5α-pLSP-promoter-cydE-lacZ were constructed (Supplementary Figure S1) to test the expression of Cytbd protein. When cells were incubated in LB medium for 4 h without sulfate, the β-galactosidase activity was higher without CydE, indicating that CydE repressed the activity of the cydE promoter (Figure 2A). Furthermore, the β-galactosidase activity of E. coli DH5α-pLSP-promoter-cydE-lacZ was upregulated when sulfate was added (Figure 2A). However, Cytbd was constitutively expressed when chromate or sulfide were added (data not shown).

Cytbd Contributes to Sulfide and Sulfate Resistance
The wild-type, mutant, and complemented strains were used in sulfate-and sulfide-sensitivity tests. Cultures containing corresponding strains without sulfate and sulfide were used as controls (Figure 2B). The results showed that with the addition of 500 mM Na 2 SO 4 or 200 µM Na 2 S, the wild-type strain grew almost as well as the ones without the addition of Na 2 SO 4 or Na 2 S (Figures 2C vs. B). However, the growth of strain cytbd was partially inhibited with the addition of sulfate ( Figure 2C) and completely inhibited with the addition of sulfide (Figure 2D), and the complemented strains were partially recovered to the wild-type levels. These results indicated that Cytbd was weakly associated with sulfate resistance, but it was very essential for sulfide resistance in strain WH16-1. Sulfate appeared not to be very toxic to strain WH16-1 since the addition of 500 mM sulfate had almost no effect on its growth.

Cytbd Contributes to Chromate Resistance and Reduction
A chromate sensitivity test was also performed. The chromate sensitivity test was performed on LB plates and in LB medium. The results showed that the chromate resistance of the mutant strain was noticeably weaker than in the wild-type and the complemented strain (Figures 3A,B). The chromate minimal inhibition concentration (MIC) of the mutant strain was 3 mM, while for the wild type, it was 45 mM . In addition, the Cr(VI) reduction ability of the mutant strain was also somewhat weaker than the wild-type and complemented strain ( Figure 3D) under similar growth conditions ( Figure 3C).

Cytbd Protects Against Cellular Oxidative Stress
To achieve a better understanding of how Cytbd contributes to chromate resistance, a series of experiments were carried out. First, the membrane protein of the wild-type, mutant, and complemented strain was extracted and reacted with H 2 O 2 and chromate. The H 2 O 2 decomposition activity of the cytbd membrane protein was noticeably lower than the wild type and cytbd-C (Supplementary Figure S2A), while chromate reduction showed no significant difference (data not shown). Furthermore, the cytoplasmic H 2 O 2 contents were measured to reflect the cellular oxidative stress. Without K 2 CrO 4 , there were no significant differences in H 2 O 2 content among strains WH16-1, cytbd, and cytbd-C (Supplementary Figure S2B). When exposed to 1 mM K 2 CrO 4 , the H 2 O 2 contents of all three strains were increased (Supplementary Figure S2B). However, the H 2 O 2 content in the mutant strain was higher than that in the wild-type and the complemented strain with K 2 CrO 4 (Supplementary Figure S2B). A similar result was obtained based on the inhibition zone test for H 2 O 2 sensitivity. The diameter of The growth curve of WH16-1, cytbd, and cytbd-C with 3 mM K 2 CrO 4 in LB medium. K 2 CrO 4 was added at the beginning (A and B). The growth (C) and K 2 CrO 4 reduction (D) curves of WH16-1, cytbd, and cytbd-C with 1 mM K 2 CrO 4 in LB medium. To maintain similar growth conditions, K 2 CrO 4 was added until OD 600 reached 0.6 (C and D). Every sample was prepared in triplicate, and the results are presented as the mean values. the inhibition zone of the mutant strain was visibly larger than for the other two strains (Supplementary Figure  S2C). This finding means that the mutant strain was more sensitive to H 2 O 2 . In addition, the MIC to H 2 O 2 of the mutant strain was 0.5 mM, which was lower than that of the wild-type and the complemented strain (Supplementary Figure S2D). Interestingly, the mutant strain lost partial capability for H 2 O 2 decomposition. Hence, it was more sensitive to H 2 O 2 compared to the wild-type and complemented strain. As a result, we inferred that the Cytbd catalyzes the reduction of cytoplasmic oxidative stress to enhance Cr(VI) resistance in strain WH16-1, but it does not directly catalyze chromate reduction.

Sulfate Enhances Chromate Resistance Level and Reduction Efficiency
The effects of sulfate on chromate resistance and reduction were examined since sulfur metabolism is relevant to chromate metabolism in many bacteria (Viti et al., 2014), and the above results showed that sulfate and chromate resistance are both associated with Cytbd. The growth of strain WH16-1 showed no significant difference with or without additional Na 2 SO 4 in the absence of added K 2 CrO 4 (data not shown) but was affected after adding K 2 CrO 4 ( Figure 4A). However, growth was much better with the addition of Na 2 SO 4 ( Figure 4A). Additionally, the Cr(VI) reduction ability of strain WH16-1 increased with increasing concentrations of Na 2 SO 4 ( Figure 4B).

Interaction Between Regulator CydE and the Promoter Region of cytbd Operon
CydE is homologous to the GbsR-type regulator based on the results of BLASTP in NCBI. It shares 18.8% and 19.4% similarities with GbsR and OpcR, respectively. Next, we aligned CydE with the two reported GbsR regulators ( Figure 5A). The results showed that CydE harbored the same conserved amino acids as the GbsR-type regulators. These conserved amino acid residues may be involved in DNA binding. GbsR-type regulators usually act as repressors of gene expression (Nau-Wagner et al., 2012;Lee et al., 2013).
To examine the regulation function of CydE, we first used a bacterial one-hybrid system to test the protein-DNA interaction based on the transcriptional activation of HIS3 (imidazoleglycerol-phosphate dehydratase gene involved in histidine biosynthesis) and aadA (streptomycin resistance gene) (Guo et al., 2009). The promoter of cydE (Supplementary Figure S1A) was cloned into upstream of HIS3-aadA in the reporter vector pBXcmT, while the CydE coding region (Supplementary Figure S1A) was introduced into the pTRG vector. Both constructed vectors were then transferred into a histidine synthesis defective and streptomycin (Str) sensitive strain. The generated strain and positive control strain grew well on the screening plate (Guo et al., 2009) containing 3-amino-1,2,4-triazole (3-AT) and Str, while the negative control strain did not grow. The results demonstrated that CydE could interact with the promoter of the cytbd operon in vivo ( Figure 5B).
Next, the purified His-tag CydE (Supplementary Figure  S3) and cydE promoter DNA (Supplementary Figure S1A) were used to test the interaction in vitro using EMSA. With increasing amounts of CydE, the free DNA substrates gradually disappeared, while the intensity of the shifted DNA band increased ( Figure 5C). Moreover, the unlabeled DNA substrate could competitively inhibit the binding of CydE to the labeled DNA substrate (Figure 5C).
To identify the binding site of CydE, DNA footprinting was carried out. With increasing amounts of CydE, decreases in two sites of the peaks were observed (Figure 6A), indicating that there are two binding sites. This is consistent with the EMSA results. The sequences of the binding sites were TATTTCAGAAATTTCTGAAAGTTCA and GGGATGCGCATATGCAAAT ( Figure 6B). The two binding site sequences were synthesized and then incubated with CydE. The EMSA result showed that both binding sites could interact with CydE ( Figure 6C).

CydE Is a Repressor and Can Be Derepressed by Sulfate
To investigate the repression ability of CydE, an EMSA derepression experiment was performed. The results showed that the free DNA increased when more sulfate was added ( Figure 6D). However, the phenomenon was not observed when chromate was added ( Figure 6D). These results are coincided with those of the lacZ reporter assay (Figure 2A).
All these results demonstrated that CydE can repress the expression of the cytbd operon and sulfate addition results in derepression.

DISCUSSION
Alishewanella sp. WH16-1 is a sulfate-and chromate-reducing bacterium. According to our previous study, this strain can FIGURE 5 | Sequence alignment of CydE and its interaction with the cydE promoter region. (A) Multiple sequence alignment was performed using ClustalW and Espript 3.0. The identical and elevated level of similarity residues are shown in red backgrounds and red boxes, respectively. The amino acid sequences of Bacillus subtilis GbsR (P71015) and Bacillus subtilis_OpcR (O34709) were selected from the UniProt database. (B) Bacterial one-hybrid assay. Co-transformants containing pBX-Mt2031p/pTRG-Rv3133c (Rv3133c protein can integrate with Mt2031p promoter) and empty vector pBXcmT/pTRG (without protein and promoter DNA) were used as positive and negative controls, respectively. Cells of positive and negative controls and the reporter strain containing plasmids pBX-promoter (promoter sequence of cydE) and pTRG-cydE (coding for CydE protein) were grown to an OD 600 of 1.0, and 2 µL of each was spotted onto His-selective medium (+3AT, +Str r ) and LB plates (-3AT, -Str r ). (C) EMSA assay. Lanes 1-4, band shifts were enhanced by increased CydE. Lanes 6-8, FAM-labeled DNA was competed by unlabeled DNA. The result showed two migration bands. It indicated that there are two binding sites for the CydE in the cydE promoter region. produce H 2 S during cultivation and has complete sulfate assimilation reduction pathway genes (cysCDNHIJ) Zhou et al., 2016). It possesses high chromate resistance and reduction ability (Xia et al., , 2018. In this study, we found that Cytbd was involved in sulfide and chromate resistance in Alishewanella sp. WH16-1. The function of Cytbd in sulfide resistance was previously reported in E. coli (Forte et al., 2016;Korshunov et al., 2016), where sulfide can inactivate heme-copper family cytochrome oxidase but not Cytbd (Forte et al., 2016;Korshunov et al., 2016). Under sulfide stress, Cytbd may play a key role in cell respiration. To our knowledge, this is the first report showing that Cytbd is associated with chromate resistance and reduction.
In strain WH16-1, Cytbd is induced by sulfate and is essential for decomposing H 2 O 2 to reduce cellular oxidative stress. In addition, Cytbd contributed to sulfide resistance, and sulfide can be used as a reductant to reduce chromate. These findings explain why Cytbd is important in coupling with chromate stress and the chromate resistance mechanism of strain WH16-1 appears to be indirect (Figure 7). Cytbd also plays an important role in resistance to other environmental stresses such as low oxygen, nitrosative, and oxidative stresses, since Cytbd can use O 2 , NO, and H 2 O 2 as electron acceptors (Borisov et al., 2013;Giuffre et al., 2014;Roop et al., 2015). The electron transformation models between Cytbd and O 2 /NO have been clarified (Giuffre et al., 2014). Under low oxygen conditions, Cytbd is regulated by Arc, Fnr, or CydR in E. coli (Borisov et al., 2011) and A. vinelandii (Wu et al., 1997).
Another important achievement of this study is the finding of a novel regulation mechanism of Cytbd transcription. Previously, a GbsR-family protein was reported as an intracellular choline sensor (Nau-Wagner et al., 2012;Lee et al., 2013). In this study, we identified a GbsR-family protein, CydE, which is Cytbd's repressor and it is inactivated by high sulfate concentration. In this way, high amount of sulfate can stimulate Cytbd transcription. Thus, sulfate could enhance chromate resistance in Alishewanella sp. WH16-1 (Figure 7). We speculate that numerous factors including the following may cause such enhancement. (i) Sulfate assimilation products such as S 2− , Cys, and GSH can directly reduce chromate (Thatoi et al., 2014;Joutey et al., 2015;Qian et al., 2016). (ii) S 2− can be used for Fe-S cluster synthesis. A potential Cr(VI) reductase (4Fe-4S ferredoxin, AAY72_06850) was also identified by the Tn5 transposon mutagenesis in this study. Previously, ferredoxin and hydrogenase, which contain the Fe-S cluster as the active group, were reported to be associated with chromate reduction (Chardin et al., 2003). In addition, proteins associated with Fe-S cluster FIGURE 7 | The proposed mechanism by which Cytbd contributes to sulfide and chromate resistance in Alishewanella sp. WH16-1. CydE, GbsR family regulator; CydA, Cytochrome bd complex subunit A; CydB, Cytochrome bd complex subunit B; ChrA, chromate efflux protein; CysPUWA, sulfate/chromate transporter; CysND, sulfate adenylyltransferases; CysC, adenylylsulphate kinase; CysH phosphoadenylylsulphate reductase; CysIJ, sulfite reductase. biogenesis, such as IscRs, are involved in multiple stress responses (Liu et al., 2015;Romsang et al., 2015). (iii) Sulfate induces the expression of Cytbd, and Cytbd is essential for chromate resistance and reduction.
On the other hand, sulfate was reported to have no effect on Cr(VI) reduction in some bacteria (Shen and Wang, 1994;Campos et al., 1995;Liu et al., 2006) or even inhibited Cr(VI) reduction in some cases (Wang, 2000;Çetin et al., 2008). The different phenomena reflect various Cr(VI) reduction mechanisms of bacteria. Some bacteria cannot reduce sulfate to produce H 2 S and do not use sulfate and chromate as terminal electron acceptors (Liu et al., 2006). Accordingly, sulfate has no noticeable effect on chromate reduction in these bacteria. Other bacteria use chromate as a terminal electron acceptor under anaerobic conditions (Wang, 2000;Liu et al., 2006). In some cases, sulfate could inhibit the activity of chromate reductase competitively (Park et al., 2000) and consequently inhibit chromate reduction in these microorganisms. These reports and our results suggest that the chromate detoxification mechanisms of selected bacteria are quite varied.

CONCLUSION
We showed that Cytbd contributes to chromate resistance, which can be explained by the ability of Cytbd to catalyze the decomposition of H 2 O 2 to protect against H 2 O 2 -related oxidative stresses. Furthermore, Cytbd contributes to the resistance of sulfide, and sulfide could act as a reductant to reduce chromate. In addition, Cytbd's expression is negatively regulated by the GbsR family regulator CydE and derepressed by sulfate. Hence, sulfate could enhance chromate resistance and reduction in Alishewanella sp. WH16-1.

AUTHOR CONTRIBUTIONS
XX designed, analyzed and interpreted the experiments, and prepared the manuscript. SW participated in the lacZ report gene and sulfate-/sulfide-sensitive experiments. LL and BX participated in the Tn5 transposon test. GW designed the study and revised the draft manuscript.