ChrASO, the chromate efflux pump of Shewanella oneidensis, improves chromate survival and reduction

The chromate efflux pump encoding gene chrASO was identified on the chromosome of Shewanella oneidensis MR1. Although chrASO is expressed without chromate, its expression level increases when Cr(VI) is added. When deleted, the resulting mutant ΔchrASO exhibits a chromate sensitive phenotype compared to that of the wild-type strain. Interestingly, heterologous expression of chrASO in E. coli confers resistance to high chromate concentration. Moreover, expression of chrASO in S. oneidensis and E. coli significantly improves Cr(VI) reduction. This effect could result either from extracytoplasmic chromate reduction or from a better cell survival leading to enhanced Cr(VI) reduction.


Introduction
The transition metal chromium has different oxidation states, but only two of them are stable, namely the trivalent Cr(III) and the hexavalent Cr(VI) forms. The Cr(III) form which is relatively insoluble is considered to be poorly toxic since it can hardly penetrate the cells. The Cr(VI) form (chromate and dichromate) is soluble and can be conveyed through cell membranes by different transporters, making it a very toxic compound for both eukaryotic and prokaryotic cells [1].
Chromate, which is chemically and structurally similar to sulfate, is thought to enter the bacterial cells mainly via sulfate ABC transporters. Once inside the cells, Cr(VI) can be reduced, either enzymatically or not, giving rise to intermediates which are responsible for various damages to DNA and cellular components. Some of these alterations are due to the generation of reactive oxygen species (ROS). When present inside the cells, Cr(III), the final product of Cr(VI) reduction, causes toxic effects on both DNA and proteins by binding respectively to phosphate, and to carboxyl and thiol groups [2].
Bacteria have developed different strategies to resist chromate [2,3]. One strategy is to limit its cell entry by reducing Cr(VI) to Cr(III) extracellularly. Reduction can be achieved anaerobically by membrane bound reductases like flavin reductases, cytochromes and

Strains, medium and growth conditions
The E. coli strains CC118 λpir and 1047/pRK2013 used for conjugation, and MC1061 derivatives used for chromate resistance and reduction assays were routinely grown in Lysogeny Broth (LB) medium at 37˚C, or 30˚C when specified [23,24]. All S. oneidensis strains used in this study are derivatives of strain MR1-R (referred as wildtype and used instead of MR-1 to allow counter-selection by rifampicin in the conjugation experiments) and were routinely grown at 28˚C in LB under aerobic (agitation) or anaerobic (static) conditions. In the latter case, LB medium was supplemented with 20 mM trimethylamine oxide (TMAO) as electron acceptor. When growth was performed in the presence of chromate, disposable tubes were used and incubated either statically or under agitation. The dissolved oxygen (DO) of the latter condition measured by a Clark electrode was 10%. This condition was then referred as semi-aerobic. Chromate challenge was carried out by supplementing LB medium at the required final concentration with a filter-sterilized stock solution of potassium chromate (K 2 CrO 4 , Sigma-Aldrich).
If required, media were solidified by adding 17 g.L -1 agar. When needed, chloramphenicol (Cm) was used at 25 μg.mL -1 . Growth was determined spectrophotometrically by monitoring changes in optical density at 600 nm compared to the same medium without bacterium (OD 600nm ).
Chromate resistance assays in S. oneidensis S. oneidensis pre-cultures grown overnight on LB plates were suspended in LB medium and used to inoculate fresh LB medium to an initial OD 600nm = 0.2. Cells were then grown until an OD 600nm = 0.5 and submitted to two different assays.
For the spot assay, 10-fold serial dilutions of cell cultures were spotted on LB plates supplemented or not with 0.5 mM of chromate and incubated at 28˚C. Plates were scanned after 4 days of incubation.
For the viability assay, chromate was added to the cell cultures (OD 600nm = 0.5) to a final concentration of 0.2 mM. After 5 hours, cells were appropriately diluted in LB and spread onto LB agar, and incubated at 28˚C. The total number of viable cells was estimated based on the number of colony-forming unit (CFU). Results were expressed as the percentage of viable counts measured in these conditions compared to that of the same culture grown in the absence of chromate.

Cr(VI)-reduction assays in S. oneidensis
In vivo Cr(VI) measurements were carried out on both semi-aerobic and anaerobic cultures. When fresh cultures reached an OD 600nm = 0.5, chromate was added to a final concentration of 1 mM. After 2h of incubation at 28˚C, cell cultures were centrifuged at 8,000 g for 5 min and residual Cr(VI) concentration present in the supernatant was determined using the Sdiphenylcarbazide (DPC) method [25] slightly modified. Briefly, 10, 20, and 50 μL supernatant samples were added in a 2 mL tube containing 1 mL of H 3 PO 4 0.5% and the volume of water necessary to obtain a 1960 μL final volume. Forty μL of DPC reagent (5 mg.mL -1 in 95% acetone and stored in dark at room temperature) were added, gently mixed and kept at room temperature for 5-10 min. Absorbance was measured at 540 nm. Cr(VI) concentration in the samples was calculated from a standard curve. These experiments were repeated three times.
In vitro measurements of chromate reductase activity were performed on crude extracts as described previously [26] with slight modifications. Briefly, wild-type and chrA SO deleted mutant strains were grown overnight in LB medium at 28˚C under agitation. The cells were then harvested, washed with sodium phosphate buffer (pH7) and resuspended in the same buffer before disruption by French press. The cell lysates were then centrifuged and the supernatants (crude extracts) recovered. Protein concentrations were measured using Protein Assay Dye Reagent (Bio-Rad), and bovine serum albumin as standard. Reaction mixtures (1 mL) containing 100 μM of K 2 CrO 4 [Cr(VI)] and 1.5 mg of proteins (crude extracts) were incubated at 30˚C for 30 min. The residual Cr(VI) concentration in the reaction mixture was estimated using the DPC method as described above.

Construction of plasmid pchrA SO
To evaluate the impact of ChrA SO on chromate resistance and reduction in E. coli, the chrA SO open reading frame (so0986) was PCR-amplified from MR1-R chromosomal DNA, from upstream of the ATG start codon (with an optimized Shine Dalgarno) to the TAA stop codon, and cloned between the SacI and the XbaI restriction sites of the pBAD33 vector, under the control of the inducible P ara promoter [27]. The resulting plasmid, called pchrA SO , was introduced into strain MC1061 and controlled by DNA-sequencing (S1 Table).

Chromate resistance and reduction assays in E. coli
Pre-cultures of MC1061 derivatives containing either the plasmid pchrA SO or the pBAD33 vector were used to inoculate fresh LB medium to an initial OD 600nm = 0.05. Cells were grown until an OD 600nm = 0.2 prior to the addition of chromate at various final concentrations (0, 0.2, 0.4, 0.8 and 1.2 mM), then the cells were incubated at 30˚C. The cultures were regularly sampled to measure the OD 600nm and to quantify the chromate using the DPC method.
In vitro chromate reductase activities were measured on crude extracts from E. coli cells containing either the pBAD33 vector or the plasmid pchrA SO as described above for the measurements on S. oneidensis crude extracts.As ChrA SO is most probably inserted inside the cytoplasmic membrane, its overexpression is toxic for the host. Preliminary growth assays of the E. coli strain harboring pchrA SO were carried out at different temperatures (30˚C or 37˚C) in the presence of different concentrations of arabinose (0, 0.005 or 0.01%). We observed that a temperature of 30˚C and the absence of inducer allowed a better growth of the strain, thus these parameters were used in further experiments.

Construction of a deletion mutant
The chrA SO -deleted strain was constructed as described previously [28]. Briefly, upstream and downstream regions flanking the gene were amplified by PCR from a MR1-R strain (by using primers SO_0986 D2 to D5, S1 Table) and cloned into the suicide vector pKNG101 at the restriction sites BamHI and SpeI. The ligation product was transformed into the E. coli CC118 λpir strain. The resulting plasmid was introduced into MR1-R strain by conjugation using the E. coli helper strain 1047/pRK2013 [29]. The plasmid was integrated in the MR1-R chromosome by a first recombination event and removed by a second recombination event in the presence of 6% sucrose. Deletion of chrA SO gene was confirmed by PCR.

Reporter gene assay
A putative promoter region was searched upstream of the start codon of chrA SO using the BPROM Softberry online software (http://www.softberry.com/berry.phtml?topic= bprom&group=programs&subgroup=gfindb). A DNA fragment corresponding to the 400 bp upstream from the ATG was amplified by PCR using S. oneidensis MR1-R chromosomal DNA, digested with XbaI and SpeI, and ligated upstream of the lacZ gene of vector pACYC184-lacZ, which was previously constructed by cloning the β-galactosidase-encoding gene lacZ from pGE593 into the vector pACYC184 (S1 Table) [30,31]. The ligation product was transformed into E. coli strain MC1061. The resulting plasmid called pchrA SO ::lacZ was introduced into S. oneidensis MR1-R by conjugation. Plasmid construction was checked by DNA sequencing. As a control, another fusion, named pmxd 450 ::lacZ, was similarly constructed using a DNA fragment corresponding to the 450 bp upstream from the ATG of mxdA [32]. S. oneidensis MR1-R strain containing pchrA SO ::lacZ or pmxd 450 ::lacZ was grown overnight on LB-agar plate. The overnight culture was diluted to an OD 600nm = 0.1 in fresh liquid LB medium containing various concentrations of chromate (0, 0.05, 0.1 and 0.2 mM) and incubated in semi-aerobiosis at 28˚C for 16 hours prior to β-galactosidase activity quantification. The β-galactosidase activity was determined by a Miller assay adapted for use with plate reader [33]. Briefly, cells were transferred into a microtiter plate and OD 600nm was measured using a Tecan Spark 10M microplate reader. Cells were then lysed using lysozyme and PopCulture reagent (Sigma-Aldrich) prior to incubation with Z buffer (62 mM Na 2 HPO 4 , 45 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , 50 mM β-mercaptoethanol). O-nitrophenyl-ß-D-galactopyranoside (ONPG) was added to the mix before kinetic quantifications of OD 420nm . Slope of the obtained curves were calculated and βgalactosidase activity (arbitrary units) was determined by the following equation: ð1000 Â slopeÞ=ðOD 600nm Â volumeof reactionðmLÞÞ:

Bioinformatics analysis
The amino acid sequence of ChrA SO was retrieved from the MicroScope Microbial Genome Annotation and Analysis Platform (https://www.genoscope.cns.fr/agc/microscope/home/). Prediction of transmembrane helices was carried out using the TMHMM online software (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Proteins sharing homologies with ChrA SO were searched in the Bacteria kingdom using the BlastP software on the NCBI server (https://blast.ncbi.nlm.nih.gov). Representative sequences of different phyla were subsequently selected, as well as the sequences of four plasmid-encoded ChrA proteins that were previously characterized. For the phylogenetic analysis, we used the "Phylogeny.fr" software in the "one-click" mode, i.e. with the default parameters optimized by the authors (http://www.phylogeny.fr/). For the tree rendering step, we used the software "Fig-Tree" version 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/) in which we entered the result in Netwick format obtained with "Phylogeny". The tree rooting was performed with the midpoint option.

S. oneidensis chromosome contains a chromate efflux pump encoding gene
The S. oneidensis MR-1 genome contains only one gene (so0986) whose product is annotated as a putative chromate efflux pump. This gene is not located on the megaplasmid but on the chromosome. Accordingly, a Blastp search performed on S. oneidensis using well-known chromate ion transporters, such as the ChrA proteins of P. aeruginosa and C. metallidurans, only identified the SO0986 protein as a ChrA homolog. In contrast, S. oneidensis does not contain homologs of the chrB and chrF genes which sometimes co-occur with chrA.
For clarity, we will refer to the SO0986 protein as ChrA SO . This protein of 383 amino acids with a molecular mass of 40.9 kDa contains two chromate transporter regions (PF02417) and therefore belongs to the LCHR subfamily of the chromate ion transporter (CHR) superfamily. The TMHMM program predicted that ChrA SO contains 10 transmembrane helices, which is usual for the proteins of the LCHR family. The ChrA SO protein is most likely inserted in the cytoplasmic membrane with its N-terminus oriented towards the periplasm, as schematized in Fig 1A. A Blastp search was performed using ChrA SO as query. Several hundreds of homologous proteins were identified in bacteria belonging for the vast majority of them to the Proteobacteria group, but also for some of them to the Terrabacteria group. As expected, the highest percentages of identities were found between ChrA SO and proteins from other Shewanella species. ChrA SO is also highly homologous to proteins belonging in particular to Vibrio and Pseudoalteromonas species. A phylogenetic tree was constructed using a subset of proteins taken from species representative of the different taxonomic families (Fig 1B). Not surprisingly, ChrA SO clusters with ChrA proteins of the γ-proteobacterial class but more particularly with those of the Aeromonadales, Alteromonadales and Vibrionales orders. Strinkigly, ChrA SO shows the highest percentage of homology with the chromosome-encoded ChrA proteins from Shewanella sp. ANA-3, P. aeruginosa and C. metallidurans compared with their plasmid-encoded ChrA proteins, that were functionally characterized [12,13,34]. As an example, ChrA SO displays 89% sequence identity with the chromosome-encoded ChrA and only 26% with the plasmid-encoded ChrA proteins of Shewanella ANA-3. Moreover S. oneidensis megaplasmid does not harbor chrA gene.

ChrA SO is involved in chromate resistance in S. oneidensis
Since ChrA SO clearly belongs to the chromate ion transporter family, we wondered if it could be involved in chromate resistance in S. oneidensis. We therefore constructed a strain deleted of the chrA SO gene and compared its capability to survive in the presence of chromate to that of the wild-type strain.
Wild-type and mutant cells were first allowed to grow at 28˚C until an OD 600 of 0.5. These pre-cultures were then submitted to two chromate resistance tests. In the first one, cells were allowed to grow for 5 hours after addition of 0.2 mM chromate in the pre-cultures. Afterwards, bacterial cells were diluted and plated on LB-agar to determine the percentage of survival after "colony forming unit" counting. The same experiment was carried out in the absence of chromate as a control. As observed on Fig 2A, the survival percentage is about 40% in the wild-type strain while it is only about 25% in the chrA SO deleted strain. In the second test, serial 10-fold dilutions of the bacterial pre-cultures were performed and then spotted on plates containing either no chromate or 0.5 mM chromate. The plates were incubated at 28˚C to allow bacterial growth. In the absence of chromate, no growth difference is observed between the wild-type and the chrA SO mutant strains (Fig 2B). The presence of chromate impairs the growth of both strains, but the effect is clearly more pronounced for the chrA SO mutant strain (Fig 2B). These results strongly suggest that ChrA SO is involved in chromate resistance in S. oneidensis.

Chromate reduction is impaired in a chrA SO deleted strain
The impact of ChrA SO on the capability of S. oneidensis to reduce Cr(VI) was evaluated by comparing the percentage of Cr(VI)-reduction obtained when the wild-type, the chrA SO mutant and the chrA SO mutant containing the plasmid pchrA SO were exposed to 1 mM chromate either in semi-aerobic (as defined in the Materials and methods section) or anaerobic conditions during 2 hours. The concentration of Cr(VI) was evaluated by the DPC method. As shown in Fig 3, in both conditions (semi-aerobiosis and anaerobiosis), the deletion of chrA SO has a dramatic impact on Cr(VI)-reduction. Indeed, the amount of Cr(VI) reduced by the chrA SO mutant is about one third of that reduced by the wild-type strain. As expected, the presence of pchrA SO in the chrA SO mutant restores a reduction efficiency comparable to that of the wild-type strain.  To decipher whether ChrA SO possesses a chromate reductase activity, we performed in vitro chromate reductase activity on crude extracts from the wild-type and the chrA SO mutant strains and the results show that the specific activity of chromate reductase is similar for both crude extracts (0.78 ± 0.1 and 0.88 ± 0.05 nmol reduced-Cr(VI).min -1 .mg -1 , respectively), indicating that ChrA SO is not directly involved in Cr(VI)-reduction. The chrA SO expression is induced by chromate Since chrA SO is involved in chromate resistance, we wondered if its expression could be regulated by chromate. We thus constructed a transcriptional fusion using the lacZ gene as a reporter. The sequence upstream of the start codon of chrA SO was first analyzed with Bprom software (Softberry) to search for a putative promoter. Sequences corresponding to putative -10 (CATAAT) and -35 (TTTTCA) boxes separated by 20-bp were identified respectively 33 and 59-bp upstream from the start codon. The 400-bp upstream from the start codon and containing these boxes were amplified and cloned upstream of the promoterless lacZ gene. The plasmid bearing this transcriptional fusion was introduced in S. oneidensis. β-galactosidase activities were then measured on the resulting strain grown with various concentrations of chromate. As shown in Fig 4, a significant level of β-galactosidase is observed when the strain is grown in the absence of chromate, meaning that the putative promoter of chrA SO is functional in this condition. Interestingly, β-galactosidase activity increases with rising concentrations of chromate. Indeed expression from the fusion is nearly 2-fold higher in the presence of 0.2 mM chromate than in its absence. As controls, β-galactosidase activities were measured on E. coli containing the chrA SO ::lacZ fusion and on S. oneidensis containing another transcriptional fusion (mxdA::lacZ). In both cases, the activities were similar whatever the growth conditions. These results indicate that chrA SO expression is induced by chromate in S. oneidensis.

Heterologous expression of chrA SO gene confers higher chromate resistance and reduction to E. coli
We demonstrated that chrA SO plays a key role in chromate resistance and reduction in S. oneidensis and we wondered if it could play a similar role when heterologously expressed. For this purpose, E. coli strains containing either the plasmid pchrA SO or the pBAD33 vector were grown in the presence of various concentrations of chromate (0, 0.2, 0.4, 0.8 and 1.2 mM) and the time course of growth was followed by measuring the OD 600 of the different cultures. To avoid toxicity due to ChrA SO overproduction, cultures were performed at 30˚C without the inducer arabinose (for details see the Materials and methods section). As shown in Fig 5A, when the initial concentration of chromate is 0.2 mM, growth of both strains is similar during the first 8 hours. Then, cells containing the pchrA SO continue to grow whereas cells containing only the vector stop growing. With higher initial concentrations of chromate, the growth remains possible when the pchrA SO is present, although it slows down as concentration in chromate increases. In contrast, higher concentrations of chromate have a dramatic impact on cells harboring only the vector. The residual concentration of Cr(VI) was evaluated after 2 and 7 hours of growth. As shown on Fig 5B, the percentage of Cr(VI)-reduction is quickly improved when the cells contain the pchrA SO compared to the ones with the vector. Reduction seems to be inefficient after 7 hours of incubation when the vector is present, whereas it is clearly improved in cells harboring the pchrA SO . These results suggest that ChrA SO has also a positive effect on the reduction efficiency of Cr(VI) in E. coli.
We performed in vitro chromate reductase activity assays on crude extracts from E. coli strains containing either the pBAD33 vector or the pchrA SO plasmid and the results show that the specific activity of chromate reductase is similar for both crude extracts (1.1 ± 0.12 and 1.05 ± 0.06 nmol reduced-Cr(VI).min -1 .mg -1 , respectively), confirming that ChrA SO is not directly involved in Cr(VI)-reduction.

Discussion
A bioinformatics analysis revealed that ChrA SO (SO0986) belongs to the large family of chromate ion transporters. In agreement with this prediction, a S. oneidensis strain deleted of the chrA SO gene is less resistant to chromate than the wild-type strain. Strikingly, expression of chrA SO in E. coli makes the strain capable of growing in the presence of high chromate concentrations, as also observed for the plasmid-encoded ChrA of Shewanella ANA-3 and for several ChrA of Burkholderia xenovorans LB400 [34,35]. Therefore ChrA SO most probably functions as a chromate efflux pump and expulses chromate ions from the cytoplasm, as demonstrated for some members of the CHR family [13,34].
It is noteworthy that, in the presence of ChrA SO , the percentage of Cr(VI) reduction is higher than in its absence in both S. oneidensis and E. coli. A direct role of ChrA SO in Cr(VI) reduction was ruled out, since the specific activity of chromate reductase measured in vitro on crude extracts from both S. oneidensis and E. coli strains is similar in the absence and the presence of ChrA SO . One hypothesis to explain this phenomenon is that ChrA SO promotes Cr(VI) reduction by expulsing chromate outside the cells where it can then be reduced. In S. oneidensis, this scenario is worth considering as two outer membrane decaheme cytochromes, MtrC and OmcA, are known to be involved in extracellular chromate reduction [21]. Another hypothesis could be that the presence of ChrA SO improves cell survival by lowering intracellular Cr(VI) concentration and consequently Cr-induced damages inside the cells, which allows the cells to grow and reduce Cr(VI) over an extended period of time. This could be true for both S. oneidensis and E. coli.
This study also shows that, although chrA SO is expressed without chromate, the level of expression is higher in its presence. Two previous studies performed on S. oneidensis using transcriptomic approaches missed this regulation, probably because the applied cut-offs were quite stringent [19,36]. Indeed, these studies considered only the genes showing at least a 2-fold or a 3-fold change in expression, respectively. Moreover the experimental conditions used in both studies were different from ours, which can also explain this discrepancy. The fact that chrA SO expression is chromate-induced is reminiscent of what was observed for the expression of several chr genes in other bacteria. For example, in C. metallidurans, transcription of the plasmidic chrA1 gene was shown to be up-regulated by chromate [12]. In B. xenovorans LB400, which possesses six genes encoding chromate ion transporters, only the chrA2 gene present on a megaplasmid is subject to chromate-induced expression [35]. Interestingly, it was recently demonstrated that ChrA2 is the major determinant of chromate resistance in this organism [37]. The chrBACF operon of Ochrobactrum tritici 5bvl1, which is located in a chromosomally integrated transposon, proved also to be chromate-induced [38]. It was subsequently shown that the ChrB protein is a transcriptional regulator that binds to the chrBACF promoter region to regulate its expression [39]. As mentioned in the result section, there is no ChrB homolog in S. oneidensis, meaning that the regulation of chrA SO expression probably depends on a regulatory protein belonging to a family different from that of ChrB. It will be interesting to identify this protein, since no chromate-sensing regulator except from ChrB was previously described.
In conclusion, although ChrA SO plays a key role in chromate resistance in S. oneidensis, other transporters could be involved in this process. Of interest, genes encoding the three components of a putative heavy metal efflux pump (SO0518 to SO0520, CzcCBA) were observed to be up-regulated by chromate [19]. This suggests a role in chromate resistance, although this pump does not belong to the CHR family.
Supporting information S1 Table. Plasmids and primers used in this study. (TIF)