Proteomic analysis to unravel the biochemical mechanisms triggered by Bacillus toyonensis SFC 500-1E under chromium(VI) and phenol stress

Bacillus toyonensis SFC 500-1E is a member of the consortium SFC 500-1 able to remove Cr(VI) and simultaneously tolerate high phenol concentrations. In order to elucidate mechanisms utilized by this strain during the bioremediation process, the differential expression pattern of proteins was analyzed when it grew with or without Cr(VI) (10 mg/L) and Cr(VI) + phenol (10 and 300 mg/L), through two complementary proteomic approaches: gel-based (Gel-LC) and gel-free (shotgun) nanoUHPLC-ESI–MS/MS. A total of 400 differentially expressed proteins were identified, out of which 152 proteins were down-regulated under Cr(VI) and 205 up-regulated in the presence of Cr(VI) + phenol, suggesting the extra effort made by the strain to adapt itself and keep growing when phenol was also added. The major metabolic pathways affected include carbohydrate and energetic metabolism, followed by lipid and amino acid metabolism. Particularly interesting were also ABC transporters and the iron-siderophore transporter as well as transcriptional regulators that can bind metals. Stress-associated global response involving the expression of thioredoxins, SOS response, and chaperones appears to be crucial for the survival of this strain under treatment with both contaminants. This research not only provided a deeper understanding of B. toyonensis SFC 500-1E metabolic role in Cr(VI) and phenol bioremediation process but also allowed us to complete an overview of the consortium SFC 500-1 behavior. This may contribute to an improvement in its use as a bioremediation strategy and also provides a baseline for further research.


Introduction
Environmental pollution by heavy metals and aromatic compounds has increased dramatically in recent years (Dong et al. 2021).Cr(VI) accompanied by phenols have been widely found in wastewater discharged by tanneries, textile-dyeing industries, electroplating, printing, and wood preservation (Cheng et al. 2015;Deghles and Kurt 2016;Yang et al. 2021).Both contaminants are designated as a priority by the United States Environmental Protection Agency (USEPA) due to their carcinogenic and teratogenic effects (Wang et al. 2016).Despite their toxicity, some microorganisms can reduce or oxidize them, and such mechanisms are utilized in the bioremediation process (Malla et al. 2018).In this context, the consortium SFC 500-1, including two strains identified as Acinetobacter guillouiae SFC 500-1A and Bacillus toyonensis SFC 500-1E, was previously isolated from tannery sediments polluted with Cr(VI) and phenol (Ontañon et al. 2015b).This consortium was characterized and selected by its remarkable ability to bioremediate Cr(VI) and phenol from diverse matrix (Fernandez et al. 2019a;Ontañon et al. 2017Ontañon et al. , 2015b;;Pereira et al. 2021).However, complete biochemical mechanisms related to its survival and persistence under Cr(VI) and phenol simultaneous exposure have not been deeply explored yet.In previous works, our research group has demonstrated that A. guillouiae SFC 500-1A induces the β-ketoadipate pathway for phenol oxidation and assimilates the degradation products through TCA cycle and glyoxylate shunt (Ontañon et al. 2015a(Ontañon et al. , 2018b)).Besides, phenol exposure increased the abundance of proteins associated with energetic processes and ATP synthesis, but it also triggered cellular stress in this bacterium.Membrane lipids remodeling seemed to be a bacterial response to Cr(VI) and phenol exposure (Fernandez et al. 2019b).The involvement of flavoproteins in Cr(VI) reduction to Cr(III) was also proposed, as well as the important role of antioxidant response, SOS-induced proteins and chaperones in the ability of the bacterium to response the damage generated by phenol and Cr(VI) (Ontañon et al. 2018b).On the other hand, we have demonstrated that B. toyonensis SFC 500-1E is able to efficiently remove Cr(VI) from synthetic media and simultaneously tolerate high phenol concentrations despite its inability to metabolize this aromatic compound (Ontañon et al. 2018a).The ability to reduce Cr(VI) and grow in the presence of other contaminants such as phenol makes this strain interesting for application in bioremediation.The main mechanism involved in the Cr(VI) remediation would be the reduction through cytosolic NADH-dependent chromate reductases (Ontañon et al. 2018a).However, there are still many questions to be answered in relation to other molecular networks associated with the adaptation of B. toyonensis SFC 500-1E to stress conditions, such as those produced by Cr(VI) and Cr(VI) + phenol treatment.In this sense, various authors reported that most of the proteins involved in normal Gram(+) cell functioning were found to be downregulated under chromium stress (Shah andDamare 2019, 2020).Besides, Matilda et al. (2019) described that Bacillus cereus VITSH1 combats metal stress probably by restricting the entry of metal inside the cell either by possessing a modified outer membrane or by moving away from the toxicant.In addition, the induction of the thiol-specific oxidative stress response seems to be a detoxifying and highly conserved mechanism among Gram(+) bacteria under phenol stress (Antelmann et al. 2008).For all of the above, our present interest is to study the behavior of B. toyonensis SFC 500-1E under two stressful situations in order to deepen our understanding related to its metabolic strategies to cope with Cr(VI) bioremediation process.
Proteins are involved in almost all biochemical reactions that occur in a cell (Heyer et al. 2017).Accordingly, the development of proteomics technologies to study all proteins in a species has opened new opportunities for elucidating the bacterial molecular mechanisms in response to specific stress (Chen et al. 2020;Guo et al. 2021).In particular, comparative proteomics is a useful approach that allows the analysis of proteins expressed and repressed differently under normal conditions with respect to stressful conditions (Gao et al. 2020).Furthermore, it provides clues of proteins with biotechnological potential such as pollution biomarkers (López-Pedrouso et al. 2020;Shiny Matilda et al. 2020).Among the available technologies, gel-based and gel-free are widely used (Wöhlbrand et al. 2013).The combination of gel-based (Gel-LC) with shotgun technologies (gelfree) improves the resolution of the proteins' dynamic range, positively impacting the number of proteins identified, the sequence percentage coverage, and the quality of the results during the mass spectrometry analysis (Mastroleo et al. 2009;Piersma et al. 2013;Heyer et al. 2015;Aebersold and Mann 2016;Nilo-Poyanco et al. 2021).Current, both technologies have used as complementary approaches given the limitations and advantages that present each one (Bonilla et al. 2020;Monoyios et al. 2018).There has been considerable proteomic research devoted to understanding the mechanisms adopted by microorganisms to remove a particular contaminant such as heavy metals (Dekker et al. 2016;Zhai et al. 2017;Izrael-Živkovic et al. 2018) or multiple metals (Yung et al. 2014;Bonilla et al. 2016;Shah and Damare 2020;Völkel et al. 2020) even under Cr(VI) or phenol exposure separately (Koçberber et al. 2010;Ceylan et al. 2011;Gu et al. 2017;Gang et al. 2019).However, most of these studies have focused on Gram(−) bacteria and only a few on the effect of Cr(VI) or phenol on the proteome of Gram(+) bacteria (Li et al. 2016;Shah and Damare 2019).To our knowledge there are no studies on the proteomic profile of a Gram(+) strain growing with Cr(VI) and phenol simultaneously, being able to remove this heavy metal and to tolerate this aromatic compound.
The main goal of the present work was to evaluate the changes in the proteomic profile of B. toyonensis SFC 500-1E growing with Cr(VI) and Cr(VI) plus phenol through Gel-LC in combination with shotgun proteomics.Data from this research widened the knowledge on mechanisms triggered by this strain, which also would help to deepen our understanding of key metabolic pathways involved in the bioremediation process of Cr(VI) and phenol by the consortium SFC 500-1.

Bacterial strain and maintenance
Bacillus toyonensis SFC 500-1E used in this study is a member of the consortium SFC 500-1, isolated from sediment contaminated with tannery effluents (Ontañon et al. 2015b).The strain was deposited in the CChRGM Collection (WDCM 1067, accession number RGM 2898).The maintenance of the strain was developed at 28 °C on a solid TY medium supplemented with Cr(VI) 10 mg/L and phenol 300 mg/L (Ontañon et al. 2015b).
Vol:. ( 1234567890) Culture conditions and physico-chemical analysis Initially, cells were grown at 150 rpm and 28 ± 2 °C overnight in 20 mL TY medium (Beringer 1974).Culture was centrifuged at 15,000×g for 10 min and the biomass obtained was washed with 0.85% NaCl solution and further re-suspended in the same solution up to an OD(600 nm) = 1, using Beckman DU640 UV-vis spectrophotometer.This bacterial suspension was employed to inoculate (1% v/v) Erlenmeyer flasks containing MMYE medium 400 mL (Na 2 HPO 4 , 2.0 g/L; KH 2 PO 4 , 9.0 g/L; NaCl, 2.5 g/L; NH 4 Cl, 1.0 g/L; 0.3% yeast extract, pH 7) with and without pollutants and incubated for 16 h at 150 rpm and 28 ± 2 °C.Three treatments were tested by triplicate: medium MMYE (control), medium MMYE supplemented with Cr(VI) 10 mg/L (Cr(VI) treatment) and with 10 mg/L Cr(VI) plus 300 mg/L phenol (Cr(VI) + phenol treatment).These conditions were determined in concordance with our previous studies (Ontañon et al. 2018a, b).The cells grew for 16 h in each condition and then were harvested by centrifugation at 15,000×g for 10 min at 4 °C.Pellets were twice washed with phosphate buffer saline-PBS-at pH 7.4 (NaCl 137 mM, KCl 2.7 mM, Na 2 HPO 4 24.2 mM, KH 2 PO 4 5.2 mM) and kept at − 20 °C until proteins extraction.
At the beginning and end of the experiment, the cell viability (CFU/mL) was determined using the microdroplet method (Somasegaran and Hoben 1994), while residual Cr(VI) and phenol concentrations were measured, according to APHA-AWWA (1989) and Wagner and Nicell (2002), respectively.Other parameters were also monitored such as pH and conductivity using a Waterproof Tester (models HI98130), while Chemical Oxygen Demand (COD) was quantified using a commercial HACH kit (HR, Cat.2125915), following the method suggested by US EPA 410.4.

Protein extraction and quantification
Protein extraction was performed according to the methodology described by Malherbe et al. (2019), with modifications.For that, pellets were re-suspended in 2 mL PBS and newly re-suspended in 8 mL of spheroplast buffer on ice (0.1 M Tris pH 8.0, 500 mM sucrose, 0.5 mM EDTA pH 8.0), incubated for 5 min and centrifuged (20,817×g, 4 °C, 3 min).The obtained pellets were then re-suspended in 3.2 mL of hypotonic solution (1 mM MgCl 2 ) and the samples were incubated for 15 s on ice before adding 160 μL MgSO 4 (20 mM).After centrifugation, the subsequent supernatants were carefully discarded and the pellets were re-suspended in 7 mL of spheroplast buffer and strongly mixed.Then, the suspensions were diluted with 7 mL of double-distilled water and incubated for 5 min on ice.After centrifugation, pellets were re-suspended in 3 mL Tris buffer pH 8.0 (10 mM).For cell lysis stage, we employed three cycles of sonication on ice (40 W, pulse 6 s, during 2 min), followed by the freezing in liquid nitrogen for 2 min and thawing in a 37 °C water bath for 3 min.The lysed cells were incubated with 50 μL 1 M MgSO 4 for 10 min, prior to centrifugation for 25 min at 20,817×g.The resulting supernatant was considered as the cytoplasmic fraction, which was split into two aliquots.The first one was stored at − 80 °C and the second one was lyophilized for the following gelbased and gel-free proteomics assays, respectively.
Protein concentration was measured using Bradford method, using bovine serum albumin as a standard (Bradford 1976).

Protein separation and trypsin digestion
Protein aliquots stored at − 80 °C were used to evaluate the protein profile by one dimensional electrophoresis in polyacrylamide gel (PAGE) using 12% of polyacrylamide and sodium dodecyl sulfate as denaturing agent (SDS) (Laemmli 1970).For that, twentyfive micrograms of proteins from each condition tested were dissolved in sample buffer (100 mM Tris pH 6.8, 2% SDS, 0.05% bromophenol blue, 5% betamercaptoethanol and 15% glycerol) and incubated at 100 °C for 5 min.The electrophoretic run was performed at 80 V for 20 min and then at 120 V for 2 h, using MINIPROTEAN IV (Bio-Rad).The obtained gel was subjected to staining with Coomassie R-250 0.25% w/v (Meyer and Lamberts 1965).Afterward, each line in the gel was cut in different regions following two criteria: molecular weight regions or specific regions with high abundance proteins to resolve possible issues such as dynamic range (Gel-LC analysis) (McConnell et al. 2011;Paez and Callegari 2022).The gel regions were bleached with Water/ Ethanol/Acetic Acid (50:40:10), destained with 50 mM Ammonium Bicarbonate/50% Acetonitrile, reduced with Dithiothreitol (DTT, Sigma-Aldrich, Saint Louis, MO) at room temperature for 30 min, followed by alkylated with Iodoacetamide (Sigma-Aldrich, Saint Louis, MO) at room temperature in dark condition for 30 min and then in gel digestion with trypsin sequencing grade (Promega, Madison, WI) (Callegari 2016).Finally, the digested peptides were extracted from the gel twice using Formic Acid/ Acetonitrile/Water (1:2:97) and Water/Acetonitrile (50:50), respectively.
As a complementary approach to 1D electrophoresis analysis and to improve the relative abundance analysis, shotgun proteomics from proteins in solution was performed.For that, lyophilized proteins corresponding to each condition were solubilized in 100 mM ammonium bicarbonate/5% of acetonitrile pH 8, up to obtain a final protein concentration of 1 μg/μL.The solubilized proteins were reduced and alkylated using DTT and Iodoacetamide, respectively, and finally digested with Trypsin at 37 °C overnight, as it was mentioned previously.The tryptic digested peptides were concentrated in a centrifuge concentrator (SpeedVac, Thermo Savant).

Mass spectrometry analysis
Peptide analysis was performed using nanoUPLC-ESI-MS/MS according to highly standardized protocols by Proteomics Core Facility in the Biomedical Research Infrastructure Network (BRIN)-Sanford School of Medicine, University of South Dakota (Callegari 2016;Paez and Callegari 2022).
Briefly, the tryptic digested peptides coming either from the gel or in solution digestion were inline desalted, concentrated and separated using an Easy nLC UHPLC 1200 in nanoflow configuration (Thermo Scientific, Bremen, Germany) for 110 min (see supplementary information X1).The eluted ions were analyzed through a QExactive Plus (Thermo Scientific, Bremen, Germany) Quadrupole Orbitrap through a nano-electrospray ion source using Full MS followed by ddMS2 (DDA) mode for 110 min.Parent mass (MS) and fragment mass (MS/MS) peak ranges were 300-1800 Da (resolution 70,000) and 65-2000 Da (resolution 17,500), respectively (Kelstrup et al. 2012;Sun et al. 2013;Liu et al. 2020).Mascot Distiller v2.6.2.0 in-house licensed (www.matri xscie nce.com) and Proteome Discoverer v2.1 (Thermo Scientific) were used to generate the peak list at the mascot generic format (mgf) to identify + 1 or multiple charged precursor ions from the mass spectrometry data file.
For the gel-free proteomic analyses, biological triplicates were used in three independent experiments.Also, a technical duplicate of each replicate previously mentioned was used.Instead, for gel-based proteomic assays, 9 areas per gel line were cut, where each line corresponds to one triplicate.A series of blanks and wash cycles were running between replicates to avoid carryover in the replicates (see supplementary information X1).

Data processing
Each peak list with + 1 or multiple charged precursor ions will be submitted to the Mascot server v2.7.0.1 and Daemon toolbox v2.5 (www.matri xscie nce.com) with a local license to search against the B. toyonensis SFC 500-1E protein database 20210430 (from Assembly Genome GCA_014236635.1, BioProject PRJNA612184, NCBI database, 5573 sequences, 1,561,288 residues).The following search parameters were set: carbamidomethyl on cysteine was set as fixed modification, whereas methionine oxidation and asparagine and glutamine deamidation were set as variable modifications.Only one missed cleavage was considered; monoisotopic masses were counted; the precursor peptide mass tolerance and fragment mass tolerance were set at 15 ppm and 0.02 Da, respectively; the ion score or expected cut-off was set at 5. The MS/MS spectra were searched with MAS-COT using a 95% confidence interval (C.I. %) threshold (p < 0.05), with which a minimum score of 26 was used for peptide identification.When the peptides identified match equally well to multiple protein ID, only those proteins that appeared in at least two or more replicates were considered to be included in the list (Bonilla et al. 2021).
The comparison of treatments expression patterns and relative quantification by "label free" technology through the spectral counting was performed using ProteoIQ v2.8 software (www.premi erbio soft.com) (local license) and only peptides that passed a false discovery rate (FDR) ≤ 1% (FDR-adjusted p < 0.01) were considered for analysis, which reduced the data set from 1623 to 918 proteins.The program also performs data normalization considering the relationship between protein molecular weight to number of peptides and fragments generated after the digestion and mass spectrometry analysis to avoid bias related with the relative abundance quantification.
After the data were normalized, a comparison between the proteins relative expression (log2) in the presence and absence of contaminants was made to ascertain whether the proteins were up-or downregulated, where log2 > 1 indicates up-expression, while log2 < − 1 indicates down-regulation, and t-test p-values lower than 0.05 (corresponding to values greater than 1.3 on the y axis of the graph, Fig. 3a) were considered as statistically significant.BlastKO-ALA (version 2.2) was used to assign Kyoto Encyclopedia for Genes and Genomes (KEGG) metabolic pathways to differentially expressed proteins (Kanehisa et al. 2016).This was carried out against a database by Bacillus toyonensis (taxonomy ID: 155322).Clusters of Orthologous Groups (COGs) of proteins were also assigned manually from NCBI (Tatusov et al. 2001).Furthermore, proteins were categorized families-based according to their function using KEGG BRITE identifier (ko) assigned by KEGG classification.

Growth and removal of Cr(VI) by B. toyonensis SFC 500-1E
Bacillus toyonensis SFC 500-1E was able to grow in the presence of Cr(VI) and Cr(VI) plus phenol.However, it was only capable of removing the heavy metal from the culture medium.Its inability to remove phenol despite being a hypertolerant bacterium had been previously described (Ontañon et al. 2018a).Table 1 shows how bacterial growth was negatively affected in presence of Cr(VI) plus phenol, but not in presence of Cr(VI) alone, which suggests certain toxicity given by phenol.The decrease in the organic nutrients removal indirectly measured as COD levels also suggested growth reduction in the treatment with both contaminants (3%).After 16 h, Cr(VI) removal above 55% was detected in Cr(VI) and Cr(VI) plus phenol treatments.These results were in agreement with previous studies that indicated the capability of B. toyonensis SFC 500-1E to remove Cr(VI) through cytosolic NADH-dependent chromate reductases, even in the presence of phenol (Ontañon et al. 2018a).

Overall protein identification results
From the analysis with FDR ≤ 1, a total of 918 unique proteins were identified, representing 16% of the B. toyonensis SFC 500-1E proteome.Figure 1 represents a Venn diagram of the proteomic analysis using gel-based and gel-free combined methodology.A number of 886 proteins were identified as overlapping under all treatments.Also, 1, 5 and 3 proteins were expressed exclusively at blue, red and green groups respectively.In this sense, ATPase containing AAA domain was only found in the presence of Cr(VI), while five proteins, namely hemL, BacA, metN, QueC, and a glycosyltransferase were observed exclusively when grown in presence of Cr(VI) + phenol.The hemL enzyme is implicated in the heme and siroheme biosynthesis (Table S1, Supplementary material).The siroheme is a prosthetic group bound to nitrite reductases, among other enzymes and both this like so flavin and iron sulfur center play essential roles in electron transfer and consequently is likely to be involved in chromate reduction (Viamajala et al. 2002;Bhattacharya et al. 2015).Moreover, sulfate transport is widely known as a common bacterial regulation mechanism to deal with chromate.Chromate is chemically analogous to sulfate and in a variety of bacterial species, it enters the cells by means of sulfate ABC transporter (Monsieurs et al. 2011;Viti et al. 2014).On the other hand, metN is described as a methionine ABC transporter.In this sense, Decorosi (2010) hypothesized that when methionine is available in the medium, the sulfate transport by bacterium is possible, allowing at the same time the uptake of chromate.In a control condition, the unique proteins identified were those belonging to aldehyde dehydrogenase family protein, two-component system response regulator and a hypothetical protein (Peptidase U61).
A comparative analysis of the relative protein abundances based on the normalized spectral counts (SpCs) was performed and a total of 11, 7 and 5 proteins only were found among the following treatments: control-Cr(VI), control-Cr(VI) + phenol and Cr(VI)-Cr(VI) + phenol, respectively (Figs. 1, 2).Functional category of this set of proteins based on the clusters of orthologous groups (COG, Fig. 2) and other classifiers were also included (Table S2, Supplementary material).In this analysis, only four proteins showed significant differences in their relative abundances, where one was down-regulated in the presence of the metal, while two were downregulated and one up-expressed in the presence of the Cr(V) + phenol (significant log2 value is indicated on the right of each bar, Fig. 2a, b).The hom protein was the only one up-regulated in the presence of the Cr(V) + phenol (and shared with control treatment) and it is involved in amino acid metabolism as an oxidoreductase (E).The proteins down-regulated in the presence of Cr(VI) + phenol were related to coenzyme transport and metabolism (LYS5, acpT; H) and nitrogen metabolism such as an oxidoreductase (ncd2, npd; Table S2, Supplementary material), while YhfH family protein (MBC2686979.1),downregulated in the presence Cr(VI), remains functionally uncharacterized.
It is important to highlight that among the 11 proteins only shared in cells grown in the presence of Cr(VI) and control conditions, two proteins were detected (MBC2686289.1 and MBC2686495.1) as transcriptional regulators with conserved helix-turnhelix motif (MarR and TetR/AcrR family, respectively) and another (PPOX, hemY) was again related with heme cofactor biosynthesis (Fig. 2a).The ubiquitous MarR and TetR/AcrR families include proteins that regulate a wide range of cellular activities, including osmotic stress, homeostasis, antibiotic resistance, metal-tolerance, efflux pumps, among others (Deng et al. 2013;Baksh and Zamble 2020).The most frequently encountered function of TetR family members was regulation of efflux pumps and transporters involved in antibiotic resistance and tolerance to toxic chemical compounds (Deng et al. 2013).Similarly, CarD, which is also known as a stress-responsive transcriptional regulator (Zhu et al. 2019) was found in Cr(VI) + phenol and control treatments (Fig. 2b).Therefore, these results could suggest that the MarR, TetR/AcrR and CarD family transcriptional regulator would have potential to serve as biomarkers for selecting new bacteria able to cope with these contaminants.

Functional characterization of differentially expressed proteins
Up to 886 proteins were identified in common under all treatments, of which 400 proteins were differentially expressed in some of them (Tables S5 and S6, Supplementary material).As shown in Fig. 3a, volcano plot highlighted the drastic down-regulation (152) and up-regulation (205) of proteins under Cr(VI) and Cr(VI) + phenol treatment respectively, suggesting there is a major shift in cellular metabolism under these conditions.The KEGG pathway enrichment results indicated that these metabolic responses were mainly associated with (i) Carbohydrate metabolism, chiefly including glycolysis, citrate cycle and pyruvate metabolism; (ii) Energy metabolism through carbon fixation pathways; (iii) Amino acid metabolism; (iv) Translation, being important the aminoacyl-tRNA biosynthesis and (v) Membrane transport, getting relevance the ABC transporters (Fig. 3c and Table S3 Supplementary material).Unclassified proteins contributed on average almost 20% of the total identified proteins, which indicates the incomplete annotation/lack and poor incorporation of new sequenced genomes in databases for further functional descriptions.On the contrary, 64 upregulated proteins in Cr(VI) and 62 down-regulated proteins in Cr(VI) + phenol were also mainly associated in Carbohydrate metabolism and Amino acid metabolism (Fig. 3b, c).A similar observation was made with COG-based analysis (Fig. S1, Supplementary material).
To gain a better understanding of the protein functions, they were classified into families by KEGG (Fig. 4 and Table S4, Supplementary material).There were a large number of enzymes (oxidoreductases, transferases, ligases and aminoacyl-tRNA synthetases) down-regulated and up-regulated under Cr(VI) and Cr(VI) + phenol, respectively.Another most striking result in these conditions was the increase in the number of ABC-2 type transporters and others involved in amino acid and metallic ion transport (Fig. 4 and Table S4, Supplementary material).Proteins related to genetic information processing were mostly associated with Helix-turn-helix motif transcription factors (GntR, ArsR, LacI, AraC, DeoR, XRE families); the biogenesis of ribosome, transfer RNA and mitochondria as well as heat shock  S1 (Supplementary material) and chromosome partitioning proteins (ko03110 and ko03036, respectively).Other family-based observations (Fig. 4 and Table S4, Supplementary material) showed proteins related to DNA repair and recombination such as those responsible of translesion DNA synthesis, single and double strand breaks repair (SSBR and DSBR) which followed the same pattern of expression under Cr(VI) (-down) and Cr(VI) + phenol (-up) treatment.
Results from the KEGG analysis give the overall impression that B. toyonensis SFC 500-1E induces a generalized metabolism reduction as a strategy to cope Cr(VI), whereas after phenol addition (Cr(VI) + phenol) it would be forced to assimilate carbon from MMYE medium and obtain energy in the form of ATP, which is subsequently used for ATPdependent chaperones/transporters and other energydependent stress responses.

Proteome responses of B. toyonensis SFC 500-1E
to Cr(VI) and Cr(VI) + phenol stress Carbohydrates, amino acids and nucleotides metabolism Differentially expressed proteins under the three treatments were summarized in Table 2. Most of the proteins were involved in glucose oxidation, for instance, in the glycolytic pathway (FBA and GAPDH; ko00010), in the oxidative decarboxylation of pyruvate (PDHA/B; Table 2, ko00010), in the citrate cycle as well as in the oxidative phosphorylation (ACO, sdhA/B/C, sucB/ C/D, mdh and ndh; Table 2, ko00020 and ko00190) and they were up-regulated when B. toyonensis SFC 500-1E grew with Cr(VI) and phenol.Probably, these changes would generate ATP for the production of proteins involved in counteracting the Cr(VI) + phenol effects.Similarly, acetate kinase (ackA) which facilitates the production of acetyl-CoA was also upregulated under this condition.Interestingly, we noted that the majority of these enzymes related to carbon assimilation, energetic processes and lipid metabolism decreased in presence of Cr(VI) alone.It has been previously reported that Cr(VI) exposure decreased the abundance of bacterial proteins involved in pyruvate metabolism (Bhat et al. 2015;Wang et al. 2014).Besides, under Cd stress the phdA/B/C/D downregulation in some bacteria has been reported, and it would switch TCA cycle to a branched or noncyclic anaerobic form, that would be regarded as an energy conservation strategy (Pagès et al. 2007;Zhai et al. 2017).Furthermore, oxidative phosphorylation was described as another important pathway inhibited under Cr(VI) stress (Yung et al. 2014;Gang et al. 2019).The decreased flux through the electron transport chain may be a mechanism of reducing reactive oxygen species (ROS), which could protect B. toyonensis SFC 500-1E against Cr-induced toxicity.
On the other hand, important enzymes for the biosynthesis of fatty acids (Table 2, ko00620) such as acetyl-CoA carboxylase (accA/C/D) and malic   enzyme (ME2) that provide the malonyl-CoA substrate and the reducing power, respectively; were upregulated in the studied strain grown in presence of both contaminants.This could be related to the derivation of part of generated Acetyl-CoA under these conditions.It is also known that the reducing power provided by malic enzyme has a role in the Cr(VI) reduction as well as in the repair of cellular damage resulting from oxidative stress (Decorosi et al. 2011;Viti et al. 2014), which could explain the increased expression of this enzyme detected in our study.Moreover, these changes in the abundance of proteins associated with fatty acid biosynthesis were highly expected and possibly they were needed to reorganize cellular membranes, due to toxic compounds that can change the fluidity of bacterial membranes (Murínová and Dercová 2014).In fact, a decrease in B. toyonensis SFC 500-1E membrane fluidity or a greater rigidity caused by Cr(VI) + phenol exposure was detected in a previous study (Fernandez et al. 2020).The studied experimental conditions, proteins involved in pyrimidine metabolism (pyrG, upp, thyA, ymdB) were in higher abundance under Cr(VI) + phenol treatment, whereas in lower abundance with Cr(VI) (Table 2, ko00240).A similar behavior was observed in Lactobacillus plantarum and Brevibacterium casei exposed to Cd and Cr(VI) plus arsenic, respectively (Zhai et al. 2017;Shah and Damare 2020).Protein expression levels related to amino acid metabolism instead (Table 2, ko00250 and ko00260) were variable in this study, i.e. there were proteins with enhanced and inhibited activities in both conditions (Fig. 3b and for more detail Table S3, Supplementary material), suggesting that Cr(VI) + phenol, simultaneously applied, have an important impact on the protein-building machinery of the cells.For example, synthases of glutamate, asparagine, argininosuccinate and threonine were down-regulated in one and other conditions or both.However, the majority of the rest of the protein were related to alanine, aspartate and glutamate (gudB, purB, purB) and they were found once again to be down-regulated and up-regulated in the presence of Cr(VI) and Cr(VI) + phenol, respectively.Proteomic studies of Brevibacterium casei and Arthrobacter sp.FB24 in response to Cr(VI) indicated that the majority of the proteins involved in amino acid metabolism showed down-regulation (Henne et al. 2009;Shah and Damare 2020).
By up-regulating carbon and energy pathways as well as lipid, amino acid and nucleotide metabolism, B. toyonensis SFC 500-1E was able to adapt to the Cr(VI) + phenol induced stress.Energy and reducing power were needed to counteract the toxic effects of Cr(VI) + phenol exposure through reducing activities, ATP-dependent transport mechanism and repair.Surprisingly, these processes may be an important target when this metal was used alone, by inducing the opposite response on the majority of these enzymes.
Cr(VI) metabolism and stress-associated global response It has been previously demonstrated that B. toyonensis SFC 500-1E is able to reduce Cr(VI) to Cr(III) through NADH dependent chromate reductases localized in cytosolic and membrane fraction (Ontañon et al. 2018a).In addition, a possible chromate efflux mechanism involving the chrA transporter protein was also described (Ontañon et al. 2018a).However, such proteins were not detected in this study, which might be due to the limitations of proteomic analysis itself or due to lack once again of annotation in the repositories.Our proteomic study revealed that exposure to Cr(VI) caused a significant increase in cellular concentration of oxidoreductases enzymes in B. toyonensis SFC 500-1E, such as nitroreductase (MBC2687515.1),nitrite/sulfite reductase (nirA) and NAD(P)H-dependent oxidoreductase (azr) (Table 2, ko01000).The combined treatment (Cr(VI) + phenol) induced expression of other enzymes involved in redox reactions namely (ubi)quinone reductases (ndh, qor), dihydrolipoamide dehydrogenase (DLD), FMN-dependent NADHazoreductase (acpD/azoR) and other nitroreductase (nfrA1).These enzymes, with different metabolic functions, are not obligate chromate reductases, but they have also been reported to catalyze Cr(VI) reduction in bacteria (Bae et al. 2005;Gonzalez et al. 2005;Thompson et al. 2007;Thatoi et al. 2014;Ma et al. 2019;Jiang et al. 2021).
Intracellular reduction of chromium through physiological reducing agents (i.e.cysteine, glutathione, and ascorbate) and by transiently formed intermediates, such as Cr(V) by DLD activity for example, leads to the generation of reactive oxygen species (ROS).This could cause oxidative stress and consequently the inactivation of essential enzymes, DNA and lipid damage may occur (Ahemad 2014;Bellenberg et al. 2019).To counteract the oxidative stress, the studied bacterial strain showed higher expression of oxidoreductases and thioredoxins such as homogentisate 1,2-dioxygenase and proteins of BrxA/BrxB family under Cr(VI) treatment, whereas dehydrogenases (sdhA/frdA, mdh) and ferre/thiodoxin type protein (korB, DsbA family) in presence of Cr(VI) + phenol.Interestingly, the induction of the thiol-specific oxidative stress response under aromatic compounds treatment is highly conserved among Gram(+) bacteria (Antelmann et al. 2008).Likewise, both oxidoreductases and thioredoxins have been proposed to be responsible for maintaining the cytosol reduced environment under chromium stress of various species belonging to Staphylococcus genera and Shewanella oneidensis MR-1 (Gang et al. 2019;Pereira et al. 2018;Shah & Damare 2019).Furthermore, in a previous study performed by our group, it was possible to detect Cr(VI) associated to B. toyonensis SFC 500-1E biomass.Besides, interaction between Cr(III) and the bacterial biomass was detected, either surface sorption and accumulation within cells, constituting another mechanism to counteract the Cr(VI) effect (Ontañon et al. 2018a).
In addition, the results revealed induction of global stress response associated with DNA repair and recombination proteins (Table 2, ko03400).In this regard, an enhanced activity of MutT protein was found exclusively under Cr(VI) exposure, whereas proteins RecA recombinase and UvrA excinuclease were only up-expressed in presence of Cr(VI) + phenol.The MutT protein, the prototype for Nudix superfamily, has been identified as responsible for preventing incorporation of the highly mutagenic 8-oxo-dGTP nucleotide into DNA (Makarova et al. 2001).Meanwhile, the activation of the SOS system that involves RecA recombinase for DNA repairing has been mentioned among the possible mechanisms displayed by diverse bacteria to alleviate the damage by redox intermediate Cr(VI) (Ontañon et al. 2018b;Zhai et al. 2017).By contrast, other proteins were up-expressed under Cr(VI) + phenol treatment, although down-expressed in presence of Cr(VI) alone, such as umuC DNA polymerase also associated with SOS response and gyrB DNA topoisomerase, that catalyze transient single-or double-strand breaks.This reflects the need of maintaining the chromosome in a topological state according to the particular replicative and transcriptional needs (Brown et al. 2006;Viti et al. 2014).Noticeably, DNA helicases (uvrD, addA) which catalyze the unwinding of the stable duplex DNA in the recently mentioned processes were overexpressed under both treatments with the contaminants, suggesting that would play a pivotal role in all aspects of DNA metabolism and repair.
Chaperones, with a key role in the repair of misfolded proteins and in the prevention of their aggregation, are also part of global stress response (Kim et al. 2021).In this sense, it is important to highlight that several chaperones such as those ATP dependent heat shock-type (clpC, clpB, clpX, hslU, groES and dnaJ) and degP membrane protease as well as the protein secretion machinery (secA/Y; Table 2, 02024) were exclusively up-regulated when B. toyonensis SFC 500-1E grew with Cr(VI) plus phenol (Table 2,  ko03110), which might be related with the toxicity produced by phenol.The induction of heat-shock proteins by phenol was also detected in Bacillus subtilis and seems to be required for stabilization of macromolecular structures (Tam et al. 2006;Antelmann et al. 2008).It is well known that chaotrope solutes such as phenol cause water stress in some bacteria and, thus, the induction of heat-shock proteins seems to be required for stabilization of macromolecular structures (Tam et al. 2006).Clp, a protease that functions in preventing cytoplasmic proteins aggregation and maintaining their quality (Santos et al. 2019), was also found in microorganisms such as Lactobacillus reuteri, Listeria monocytogenes and Lactobacillus plantarum after environmental stresses (Wall et al. 2007;Zhai et al. 2017;Santos et al. 2019).Moreover, aromatic compounds can induce chaperone expression (Ceylan et al. 2011) like the dnaK protein under phenol exposure (Ontañon et al. 2018b;Roma-Rodrigues et al. 2010) and HslU protein under toluene exposure (Wijte et al. 2011), reflecting the occurrence of protein misfolding in the cytoplasm and the consequent need to refold them.Furthermore, DegP protease is also associated with different stresses (thermal, osmotic and oxidative) and is known to play a central role in the protein quality control network in the periplasm (Gunasekera et al. 2008;Kim et al. 2021).In contrast with our results, the induction of chaperones by Cr(VI) exposure (groES and hslU, for example) was reported in different bacterial strains (Bonilla et al. 2020;Chourey et al. 2006).
Taken together, the presented data provide clear evidence that the presence of phenol [Cr(VI) + phenol Vol.: ( 0123456789) treatment] would trigger a more complete global stress response to deal with DNA and protein damage.We have previously demonstrate that B. toyonensis SFC 500-1E is unable to metabolize phenol (Fernandez et al. 2020;Ontañon et al. 2015a), and the present proteomic study did not reveal enzymes involved in the aerobic degradation pathways of this compound.Thus, it could result toxic for the cell and would explain the special effort made by the strain to cope the damage.
Transcription and translation processes Most of the proteins involved in ribosomal structure/ biogenesis and translation process (Table 2, ko03011, ko03009, ko03016 and ko03029) were increased due to stress caused by Cr(VI) + phenol, whereas they were down-regulated in presence of Cr(VI) alone.These findings were in accordance with the corresponding changes in global protein level under both conditions; i.e. a greater number of proteins up-regulated under Cr(VI) + phenol respect to Cr(VI) (Figs. 3a, S4).There is no consensus regarding to the expression levels of ribosomal proteins and others involved in the transcription and replication processes under stress conditions.In Bacillus cereus VITSH1 and Streptomyces sp.MC1 exposed to Cr(VI) these proteins were up-expressed (Bonilla et al. 2020;Matilda et al. 2019), while in Brevibacterium casei and A. guillouiae SFC 500-1A cells grown on arsenic/ chromium and phenol, respectively the results were variable (Ontañon et al. 2018b;Shah and Damare 2020).A central point in Cr(VI) stress response previously reported (Malaviya and Singh 2014;Joutey et al. 2015) is that the reduction to Cr(III), a cationic species that can remain uncomplexed inside the cell, binds with the negatively charged phosphate of DNA which could explain the stop in transcription and translation processes.
As expected, the majority of the factors that regulate the transcriptional process (Table 2, ko03000) were also negatively regulated under Cr(VI) treatment and positively regulated under Cr(VI) + phenol exposure, except two ArsR family proteins such as hlyU and smtB that were up-regulated under both treatments.The ArsR/SmtB family members sense a wide variety of metal ions like As, Sb, Bi, Zn, Cd, Pb, Co, Ni, Cu, Ag (Ma et al. 2009).They are released from their operator site due to metal binding and they not only modulate the expression of genes associated with metal homeostasis (uptake, storage and efflux), but can also alter metabolism to decrease the cellular demand for metals, allowing these organisms to survive in challenging environments (Osman and Cavet 2010;Saha et al. 2017).There is currently no data indicating the existence of ArsR family transcriptional regulators specific for Cr(VI).However, regulators can bind various metals indicating their potential to also transport Cr(VI).Additionally, CadC and CodY that were up-regulated under Cr(VI) + phenol in B. toyonensis SFC 500-1E, have been reported in Gram(+) bacteria as important to confer full resistance to several heavy metals and phenol, respectively (Tam et al. 2006;Liu et al. 2021).
Interestingly, the only transcriptional regulator upexpressed under Cr(VI) was Fur, which is involved in iron homeostasis in response to changes in intracellular levels of Fe 2+ , this last is an indirect chemical Cr(VI) reductants under aerobic environmental conditions (Troxell and Hassan 2013;Bansal et al. 2019;Bellenberg et al. 2019).Noticeably, Fur was originally described as a repressor of genes involved in iron uptake, storage and metabolism (Berg et al. 2020).Recent studies however, have indicated that Fur may also function as an activator (Butcher et al. 2012).This finding could suggest an important role Fur in Cr(VI) metabolism.More studies will be needed to clarify its role as regulator in B. toyonensis SFC 500-1E and its relation with iron intracellular levels and Cr(VI) reduction.Besides, considering the evidence that metalloregulators can bind to different metals, it is also possible to think that Fur binds Cr(VI) and that the increase in the expression of this regulator may be a tolerance mechanism of the bacteria to prevent further entry of the heavy metal.
The results could suggest that these proteins are important to face the stress caused by Cr(VI) presence, even when phenol was simultaneously present (Cr(VI) + phenol treatment) and help the microorganism to counteract the toxicity of these compounds.
Transporters Eighteen transporters were identified up-regulated in Cr(VI) + phenol treatment, whereas only five were up-regulated in presence of Cr(VI) and thirteen down-regulated.Among them, the ironsiderophore transporter (yclQ/ceuA) and a member of NitT/TauT family involved in the mineral and organic ion transport were the only two transporters up-regulated in both treatments (Table 2, ko02000).It is well known that siderophore biosynthesis by bacteria is regulated by the amount of iron present in the environment and the amount of intracellular iron (Schalk et al. 2011).For example, iron deprivation activates the expression of components of the siderophore-mediated iron acquisition systems (yclNOPQ) in Bacillus species (Zawadzka et al. 2009).In B. toyonensis SFC 500-1E, the yclQ/ceuA up-regulation occurred when Cr(VI) was present whereas the other iron-siderophore transporters (fhuD/ftsB/siuD and sirA/fecB/cbrA) were up-regulated in presence of both contaminants.This might be related to sensing low levels of intracellular Fe 2+ (Fur-mediated or not) as a consequence of its participation act as electron donor in the Cr(VI) reduction previously proposed or, why not, by simply sensing Cr(VI).In this sense, there is increasing evidence that metals other than iron, especially heavy metals, can activate the production of siderophores by bacteria (Schalk et al. 2011).However, their mechanisms of action are still unclear.In Bacillus megaterium, for example, siderophore production slowed the passive transport of aluminum as a protecting mechanism against metal toxicity (Hu and Boyer 1996).All these data suggest the implication of siderophores in Cr(VI) tolerance, but would be useful to deepen the study related to their exact role.
Table 2 shows the substrates related to each transporter.In presence of both contaminants, proteins involved in the transport also included those transporting sugars (treB/treP and nagE), phosphate and amino acids (fliY/tcyA and metN), oligopeptides (oppD) as well as a great amount of ABC-2 type transporters (sufC, ftsX/E, ABC.CD.A, ecsA, ABC-2.A, acrB).This was consistent with the increased metabolism of carbohydrate, energy, lipid and amino acids in this condition, as shown in Fig. 3b.In this sense, it is clear that ABC transporters play an important role in bacteria, importing various nutrients required for survival in different niches and exporting toxic substances such as metal ions to the cell (Garmory and Titball 2004; Mandal et al. 2019).
When B. toyonensis SFC 500-1E grew with Cr(VI) alone, the phosphocarrier protein (HPr) was up-regulated.This protein is an essential component of the sugar-transporting phosphotransferase system (PTS) in Gram(+) bacteria and plays a prominent role as global regulator to adjust catabolic capacities to ensure preferential use of readily metabolizable carbon sources (Huynh et al. 2000;Rodionova et al. 2017).This could suggest the key implication of carbon and energy metabolism in B. toyonensis SFC 500-1E to cope Cr(VI) toxicity, which can be observed in Fig. 3b.The rest of the identified transporters were generally down-regulated under this condition.For example, most subunits (OppF, OppC, OppD) belonging to oligopeptide Opp transport system involved in peptides transport used as carbon and nitrogen sources, were down-regulated under Cr(VI) alone as well as the majority of amino acid transporters and ABC-type transporters.Similarly, Lactobacillus plantarum needs to shut down carbohydrate transporters to survive under Cd stress (Zhai et al. 2017).These changes might indicate that in order to survive under Cr stress, the studied strain needs to shut down the majority of the transporters although optimizing iron-siderophore transporter, whereas the need to different adapt to both contaminants requires an increase in the proportion of numerous transporter systems related to carbon, energy and amino acids metabolism.
Proteins probably involved in other adaptive strategies to Cr(VI) plus phenol stress Bacterial sporulation is considered the last resort response that bacilli use to cope and survive under extreme, adverse conditions (Rodriguez Ayala et al. 2020).In our study, the spo0A protein (Table 2, 02020) involved in the sporulation process was identified to be up-regulated under Cr(VI) + phenol growth, while it was down-regulated in presence of Cr(VI) alone.The role of Spo0A in the induction of biofilm formation was also widely demonstrated (Dawson et al. 2012;Haggett et al. 2018).In this sense, the exopolysaccharide (EPS) matrix as a constituent part provides the scaffold bonding together spores and vegetative cells, as well as protecting to vegetative cells against different stresses (Dawson et al. 2012).A previous study performed with B. toyonensis SFC 500-1E shown a significantly increase in the EPS content and in the biofilm formation when cells were exposed to Cr(VI) + phenol (Fernandez et al. 2020), which could be related to the increased spo0A levels found in this work.

Final considerations
The label-free shotgun proteomic in combination with gel-based approach described here provides a full overview about molecular data and also on the probable metabolic pathways involved in the response of B. toyonensis SFC 500-1E against Cr(VI) and Cr(VI) + phenol (Fig. 5).The complete genome sequence of this strain provides the starting point for a detailed analysis of the triggered mechanisms.However, the available functional enrichment software's have limitations to recently sequenced species, something which should be enhanced upon for more precise results.
Results from the KEGG analysis give the overall impression that B. toyonensis SFC 500-1E induces a generalized reduction of carbon, lipid and energetic metabolism as a strategy to cope Cr(VI).Contrary, when phenol was also present (Cr(VI) + phenol) we may assume that this strain would be forced to obtain energy via carbon assimilation and reducing power through malic enzyme (Table S3).The energy stored as ATP will be subsequently used by ATP-dependent mechanisms such as transport, DNA repair and protein folding, whereas the reducing power could be useful for the Cr(VI) reduction mainly mediated by NAD(P) H-dependent reductases.Our findings have highlighted that the changes in the abundance of malic enzyme together with acetyl-CoA carboxylase (accA/C/D) also associated to fatty acids biosynthesis were possibly crucial for the B. toyonensis SFC 500-1E membrane reorganize under Cr(VI) + phenol and the restoration of its state fluidity.Concerning the Cr(VI) reduction process, this leads to the generation of ROS which causes oxidative stress and consequently enzymes, DNA and lipid damages.The B. toyonensis SFC 500-1E effort to mitigate the oxidative stress and regulate the intracellular redox state mainly through thioredoxins and also oxidoreductases, dehydrogenases was more evident during the Cr(VI) + phenol co-remediation process.In this condition, the strain also responded to DNA and protein damage by expression of DNA repair/ recombination proteins and chaperones.Altogether, these responses may contribute to B. toyonensis SFC 500-1E adaptation during growth with both contaminants.Particularly, this strain lacks the enzymatic machinery for phenol degradation.In fact, phenol inhibited cell growth given its toxicity, which could explain the special effort made by this strain to cope with the damage.It is likely for the ArsR/SmtB family metalloregulators and the iron-siderophore transporter (yclQ/ceuA) highly expressed in Cr(VI) + phenol exposed cells and even Fur regulator up-regulated in the presence of Cr(VI) alone, to play some role in Cr(VI) metabolism.The implication of these proteins in Cr(VI) reduction as well as their relation with iron intracellular levels remain to be clarified.
Taken together with previous studies, we can suggest that the increased spo0A levels found in this work could be related to the increase in the EPS content and in the biofilm formation previously described for B. toyonensis SFC 500-1E cells exposed to Cr(VI) + phenol (Fernandez et al. 2020).Moreover, Cr(VI) and Cr(III) biosorption, either surface sorption and accumulation within cells, have been other adaptive responses previously reported for this strain (Ontañon et al. 2018a).Thus, they were also added into the schematic representation as hypothetical mechanisms triggered by the strain under study (Fig. 5).The results obtained in this work further shed light on the molecular mechanisms, at the proteomic level, triggered by B. toyonensis SFC 500-1E when grown with Cr(VI) and phenol and highlight the extra effort made by the strain to adapt itself and keep growing when phenol was also present.Moreover, this work could help us to deeply study certain key proteins found as well as providing a starting point to prioritize new specific targets.
Our findings plus the previous proteomic analysis performed in the other member of consortium SFC 500-1 (Ontañon et al. 2018b), allowed us to complete an overview of the Cr(VI) + phenol remediate pathway in this consortium for the first time.Understanding the role of each member within a consortium will facilitate and optimize its application as a bioremediation strategy and can help us tackle the biodegradation of more complex environments.

Fig. 3
Fig. 3 Volcano plot of relative expression of proteins (a), number of down-regulated and up-regulated proteins (b) and KEEG-based enrichment analysis of these differentially expressed proteins (c) during B. toyonensis SFC 500-1E growth under Cr(VI), Cr(VI) + phenol and control condition.log2 > 1 indicates up-regulation, while log2 < − 1 indicates down-regulation, and a p-value below 0.05 (above 1.3 on the

Table 1
Bacterial growth, physico-chemical characteristics of culture medium and contaminant removal Vol.: (0123456789)

Table 2
Differentially expressed proteins in the three treatments Classification of differently expressed proteins was based on pathways and protein families annotated in the database of KEGG Vol:. (1234567890) d e Relative expression (log2); log2 > 1 indicates up-expression (bold), while log2 < − 1 indicates down-regulation (italics).Differentially expressed proteins (-down or -up) only in the control treatment were not included.For more detailed information (protein length and weight, F/P-Statistic, p value, MASCOT score, peptides number, % seq.coverage and normalized spectral count) see TableS5(Supplementary material).Proteins also belonging to the classification of (*) Amino acid related enzymes, (**) Mitochondrial biogenesis, (PM) Pyruvate metabolism, (CF) Carbon fixation, (GCT) Glycine, serine and threonine metabolism; (TCS) Two-component system; (QS) Quorum sensing Vol.: (0123456789)