Cellular response of Brevibacterium casei #NIOSBA88 to arsenic and chromium—a proteomic approach

Abstract

Cellular response against different heavy metal stress differs with the metal. Arsenic and chromium are heavy metals and toxic to living systems. The concentration of these metals in seawater is very low. However, due to their solubility in nature, they actively enter cells via various transport mechanisms and cause damage to the cells. Brevibacterium casei #NIOSBA88, a marine-derived, gram-positive isolate was multi-metal tolerant. Proteomic analysis of this isolate in response to arsenic and chromium resulted in the identification of total 2549 proteins, out of which 880 proteins were found to be commonly expressed at 750 mgL⁻¹ arsenic and 100 mgL⁻¹ chromium and in absence of both the metals. In contrast, 533, 212, and 270 proteins were found to be unique in the absence of any metal, 750 mgL⁻¹ of arsenic and 100 mgL⁻¹ of chromium respectively. Proteins such as antibiotic biosynthesis monooxygenase, ArsR family transcriptional regulator, cytochrome C oxidase subunit II, and thioredoxin reductase were exclusively expressed only in response to arsenic and chromium. Other proteins like superoxide dismutase, lipid hydroperoxide reductase, and thioredoxin-disulfide reductase were found to be upregulated in response to both the metals. Most of the proteins involved in the normal cell functioning were found to be downregulated. Major metabolic functions affected include amino acid metabolism, carbohydrate metabolism, translation, and energy metabolism. Peptide mass fingerprinting of Brevibacterium casei #NIOSBA88 exposed to arsenic and chromium respectively revealed the deleterious effect of these metals on the bacterium and its strategy to overcome the stress.

Electronic supplementary material

The online version of this article (10.1007/s42770-020-00353-7) contains supplementary material, which is available to authorized users.

Introduction

Heavy metals are the elements that have density > 5 gcm⁻³, higher atomic weight, and molecular mass [1]. They are a group of essential elements (Mn, Fe, Zn, Co, Cu) which are necessary in trace amounts for vital biological processes as well as non-essential elements (Cr, As, Cd, Pb, Hg) that could be toxic even at low concentrations [2]. These metals are non-biodegradable and recalcitrant [3].

Arsenic is existing in the environment in various oxidation states (arsenate (+ 5), arsenite (+ 3), arsenic (0), and arsine gas (− 3)) [4, 5]. Arsenic concentration is in trace amounts and released from the volcanic activities, effluent discharges, and agricultural run-offs [6–8]. The trivalent (arsenite) and pentavalent (arsenate) are the most abundant forms of arsenic [9]. Arsenic is one of the most prevalent toxins in the environment [10]. Arsenic is toxic and carcinogenic in nature affecting the global health [11, 12]. It is known to cause cancer in kidney, bladder, lungs, and skin, along with cardiovascular and neurological effects [13, 14]. However, there is no known arsenic-specific uptake system reported in any organism. Hence, arsenic uptake occurs via other non-specific transporters [15].

Arsenate is a structural analogue to phosphate and interferes with the phosphate metabolism in organisms [16]. Arsenite enters the cells through aquaglyceroporins [17]. Although arsenic is toxic, a large number of arsenic-tolerant bacteria have been reported [16]. Different mechanisms adopted by bacteria for arsenic transformations include arsenite oxidation, arsenite methylation, and arsenate reduction. [18, 19]. Different arsenic-specific gene clusters or operons in different organisms responsible for tolerance and detoxification have been reported so far [9, 20, 21]. Similarly, there are a few reports with respect to active gene products using transcriptomic and proteomic approach [22–25].

Chromium exists in two major oxidation states in the environment, trivalent (+ 3) and hexavalent (+ 6) chromium, both stable and water-soluble [26]. Various sources of chromium include textile-dyeing industries, tanneries, electroplating, wood preservation, and petroleum refining [27, 28]. Due to its soluble nature, chromium can actively enter cells and damage their constituents [29]. Chromium (Cr (VI)) is a toxic and carcinogenic-causing genotoxicity, mutagenicity, and lung cancer in humans and can enter body through inhalation, skin absorption, and ingestion [12]. Several bacteria have been reported for their chromate tolerance and detoxification [30–32]. Various chromium tolerance mechanisms exhibited by bacteria include intracellular accumulation, biotransformation, periplasmic bio-sorption, production of exopolysaccharides, and efflux pumps [33].Genomics has been a widely used tool to study cellular responses towards different stresses. However, proteomics is a better tool to study the differentially expressed proteins since the expression of the genome under various conditions is demonstrated through metabolic activity carried out by the expressed proteins [34, 35]. Several proteomic studies reporting arsenic and chromium tolerance in different bacteria have been published so far. Peptide mass fingerprinting using liquid chromatography coupled with mass spectrometry (LCMS) is one of the reproducible and extremely sensitive methods to assess proteomic response of any organism, but this has a limitation that it depends on the database used for identification of the proteins; the well-annotated the database, the better is the protein data for analysis. Proteomic response to arsenic in Pseudomonas [36], Thiomonas [37], and Staphylococcus sp. [19, 25] have been reported. Proteomic response to chromium in Pseudomonas aeruginosa [38], Pycnoporus sanguineus [39], Staphylococcus sp. NIOMR8 [40, 41], Pannonibacter phragmitetus [42], and Staphylococcus cohnii #NIOSBK35 [32] has been reported. However, few studies have shown multi-metal tolerance in bacteria. Bitton and Freihofold [43] have reported cellular response in Klebsiella aerogenes to cadmium and copper. Afzal et al. [44] have reported the effect of nickel and copper, and the biosorptive potential of Klebsiella variicola. Similarly, Figueroa et al. [45] have reported several bacteria tolerant to multiple metals (Te, Se, Au, Ag) belonging to different genera such as Enterobacter, Staphylococcus, Acinetobacter, and Exiguobacterium.

It is quite interesting to know that a single bacterium can be tolerant of multiple metals. This adaptation of bacteria can be employed for various processes such as bioremediation and bioleaching of metals. A lot of information is available about the bacterial response to different metals. This study focuses on the effect of arsenic and chromium on the marine-derived Brevibacterium casei #NIOSBA88, and its proteomic response to these metals analyzed using peptide mass fingerprinting.

Materials and methods

Growth characteristics of Brevibacterium casei #NIOSBA88 in the presence of arsenic and chromium

Brevibacterium casei #NIOSBA88 was isolated on Zobell marine agar from water sampled from a depth of 200 m from the Arabian Sea (9° 0′ 0.4068″ N, 67° 59′ 59.6436″ E). Luria-Bertani broth (HiMedia, India) containing 750 mgL⁻¹ arsenic (NaAsO₂) (v/v) and 100 mgL⁻¹ chromium (K₂Cr₂O₇) (v/v), discretely, were inoculated with a synchronous culture of Brevibacterium casei #NIOSBA88. A control flask (without metal) inoculated with culture was maintained. Similarly, a media blank with metal and no culture was also maintained for each metal. The flasks were incubated at 28 °C at 120 rpm for about 96 h. Growth of the organism was checked by measuring absorbance at 600 nm using a UV visible spectrophotometer (Genesys 20 Visible Spectrophotometer, Thermo Scientific, USA) after every 3 h. Biological replicates (triplicates) were maintained, and the standard deviation was calculated.

Proteome identification and characterization in Brevibacterium casei #NIOSBA88

For the whole-cell protein extraction, cell pellets at 13 different time points (6 h, 9 h, 12 h, 18 h, 24 h, 27 h, 33 h, 42 h, 48 h, 54 h, 57 h, 72 h, and 96 h) were obtained from the experimental flasks. The cell pellets from the triplicates were pooled together for protein extraction. These time points were chosen so as to cover different bacterial growth phases. The cell pellets were washed with 1× phosphate buffer and then resuspended in lysis buffer [25].

Protein extraction was performed using urea-thiourea buffer [25]. Extracted proteins were subjected to methanol precipitation and quantified using Folin-Lowry method for protein estimation [46]. The quality of extracted proteins was analyzed by performing SDS-PAGE. For protein profiling of Brevibacterium casei #NIOSBA88, a gel-free bottom-up proteomic approach was used. Proteins were resuspended in 6 M urea, followed by reduction using 200 mM dithiothrietol (DTT) and alkylation by 200 mM idoacetamide (IAA) and trypsin digestion [25].

Peptide mass fingerprinting of Brevibacterium casei #NIOSBA88

The tryptic-digested peptides were loaded on to Prot ID chip 150 II 300A C18 150 mm column followed by ionization with an HPLC Chip with a source voltage of 1900 V (6538 UHD accurate mass QToF LCMS, Agilent Technologies, USA) [25]. A method was created using an offline method editor, and the spectral data was acquired using the Mass-Hunter Data Acquisition software B.06.00 (Agilent Technologies, USA). The flow rate for sample loading was maintained at 2.0 μL min⁻¹, and separation was done at 0.40 μL min⁻¹ throughout the run. Samples were run as technical replicates of four. Peptides were separated with a non-linear gradient from 3 to 97% for 120 min with formic acid (0.1%) as adduct in a water-acetonitrile solvent system. The separation was performed for 115 min in positive polarity for data acquisition and the remaining 5 min in negative polarity for cleaning of the residual ions. For generating the mass spectra, the instrument was set for the following parameters: The scan range for MS1 was set to 200–2000 m/z with a scan rate of 2 spectra/s. For MS2, the scan range was set to 100–3000 m/z with a scan rate of 1 spectra/s maximum precursors to be selected per cycle was set to 14 with intensity over 1000 and purity stringency of 100% and purity cutoff of 30%. The dynamic exclusion duration of fragmented precursor ions was set to 60 s. Collision-induced dissociation (CID) was used as fragmentation mode with collision energy offset of 5, slope as 3.2, and charge preference as 2, 3, > 3, and 1, in that order. Here, quadrupole was the mass analyzer for MS2.

The mass spectra (MS/MS spectra) were matched against species-specific NCBI protein database (Brevibacterium casei, Txid-33889 with 17,147 entries as on November 12, 2017) using Spectrum Mill MS Proteomic Workbench ver. B.06.00.201 (Agilent Technologies, USA). The precursor and product mass tolerances were set to 50 and 100 ppm respectively and charge state range of 2 to 8. A maximum of 2 missed trypsin cleavages was allowed, while carbamidomethylation was chosen as fixed modification. Auto-validation was performed on all the sample replicates with a charge state range of 2 to 8 and 1.2% false discovery rate (FDR). Peptide and protein summary files were generated with a score of more than one and percent SPI of more than 10. The protein summary files were exported containing score, spectral intensity, start AA position, sequence, retention time, precursor m/z, protein molecular weight, species name, accession number, and protein name. The mass spectrometry data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) [47] via the PRIDE partner repository [48] with the dataset identifier PXD016588.

Identified proteins were analyzed for their expression pattern in Mass Profiler Professional ver. 14.9.1 (GeneSpring GX, Agilent Technologies, USA). The sample replicates were first grouped as control (absence of metal) and experiment (presence of metal). After grouping, interpretation for the grouped set was created categorically based on the metal (arsenic or chromium) and its concentration followed by the time point at which the cells were harvested for protein extraction. A comparison between the proteins expressed in the presence and absence of metal was made to ascertain whether the proteins were up- or downregulated. For calculation of fold change (cutoff 2.0), first, the dataset was log2 normalized, and then the expression pattern was analyzed. Expressed proteins based on their functions were categorized into the metabolic pathways using the Kyoto Encyclopedia for Genes and Genomes (KEGG) [49] pathway ver. 37 database.

Results

Proteome identification in Brevibacterium casei #NIOSBA88

Global proteomic response of Brevibacterium casei #NIOSBA88 resulted in the identification of 2549 proteins across all the conditions. Discretely, 1946, 1450, and 1571 proteins at 0 mgL⁻¹ and 750 mgL⁻¹ of arsenic and 100 mgL⁻¹ of chromium respectively. Of all these identified proteins, it was found that 880 proteins were common across all conditions, while 533, 212, and 270 proteins were unique to 0 mgL⁻¹ and 750 mgL⁻¹ of arsenic and 100 mgL⁻¹ of chromium respectively (Fig. 1).

Fig. 1.

Venn diagram representing total identified proteins in Brevibacterium casei #NIOSBA88 at (a) 0 mgL⁻¹ and (b) 750 mgL⁻¹ of arsenic, and (c) 100 mgL⁻¹ of chromium respectively

Proteomic response of Brevibacterium casei #NIOSBA88 to metals

All the proteins identified in the presence of arsenic and chromium were further analyzed for their regulation based on the metal concentration alone as well as concentration and time points at which cells were harvested during the growth. A fold change cutoff of 2.0 with for 750 mgL⁻¹ arsenic against control and 100 mgL⁻¹ of chromium against that of control respectively was maintained. It was found that 205 proteins were upregulated and 390 proteins downregulated at 750 mgL⁻¹ arsenic against that of control, while 290 proteins were found to be upregulated and 401 proteins downregulated at 100 mgL⁻¹ of chromium against that of control. Protein expression studies across different time points were carried out to see the effect of the arsenic and chromium at different growth stages of the bacterium. A comparison of the proteins expressed in the presence of arsenic and chromium was made, which showed that 392 proteins were common to both the conditions.

Proteins such as 3-carboxy-cis, cis-muconate cycloisomerase, ABC transporter permease, alcohol dehydrogenase, aspartate aminotransferase family protein, ATP synthase subunit beta, glycine dehydrogenase, NAD-dependent aldehyde dehydrogenase, ubiquinol-cytochrome C reductase, and UTP-glucose-1-phosphate uridylyl transferase were found to be upregulated in the presence of arsenic, while 30S ribosomal protein S2, 3-hydroxyisobutyrate dehydrogenase, 50S ribosomal proteins (L18, L9), acetate-CoA ligase, adenylosuccinate synthetase, chlorite dismutase, enterochelin ABC transporter periplasmic protein, Fe-S cluster assembly protein SufB, glycerol dehydrogenase, and phosphoenolpyruvate carboxykinase were some of the downregulated proteins in the presence of arsenic. The up- and downregulated proteins with their fold change values are given in Table 1 respectively.

Table 1.

Protein regulation in the presence of 750 mgL⁻¹ arsenic and their corresponding metabolic pathways

Protein name	Fold change	Functional pathway	Metabolic pathway (KEGG)	Accession ID (NCBI)
3-carboxy-cis, cis-muconate cycloisomerase	1.482	Benzoate degradation	Xenobiotics degradation and metabolism	1017131651
ABC transporter permease	1.313	ABC transporters	Membrane transport	1017129676
Alcohol dehydrogenase	1.411	Glycolysis	Carbohydrate metabolism	1017132330
Aspartate aminotransferase family protein	1.503	Alanine, aspartate and glutamate metabolism	Amino acid metabolism	1238047174
ATP synthase subunit beta	1.436	Oxidative phosphorylation	Energy metabolism	1017132306
Glycine dehydrogenase	1.115	Glycine, serine and threonine metabolism	Amino acid metabolism	425480421
NAD-dependent aldehyde dehydrogenase	2.520	Glycolysis	Carbohydrate metabolism	425479298
Ubiquinol-cytochrome C reductase	1.414	Oxidative phosphorylation	Energy metabolism	1017127611
UTP-glucose-1-phosphate uridylyltransferase	2.901	Pentose and glucouronate interconversions	Carbohydrate metabolism	1017134956
30S ribosomal protein S2	− 1.296	Ribosome	Translation	1017134332
3-Hydroxyisobutyrate dehydrogenase	− 1.347	Synthesis and degradation of ketone bodies	Lipid metabolism	1238047929
50S ribosomal protein L18	− 1.971	Ribosome	Translation	1017134113
50S ribosomal protein L9	− 1.275	Ribosome	Translation	1017129105
Acetate-CoA ligase	− 2.466	Pyruvate metabolism	Carbohydrate metabolism	1238044129
Adenylosuccinate synthetase	− 2.037	Purine metabolism	Nucleotide metabolism	1017134709
Chlorite dismutase	− 1.615	Stress protein	Stress proteins	1017130501
Enterochelin ABC transporter periplasmic protein	− 1.247	ABC transporters	Membrane transport	425480436
Fe-S cluster assembly protein SufB	− 1.082	Thiamine metabolism	Amino acid metabolism	1017129675
Glycerol dehydrogenase	− 2.513	Glycerolipid metabolism	Lipid metabolism	1238047435
Phosphoenolpyruvate carboxykinase	− 1.184	Glycolysis	Carbohydrate metabolism	1017126007

Positive values indicate upregulation, while negative values indicate downregulation of proteins

Similarly, 30S ribosomal protein S5, 3-hydroxy-2-methylbutyryl-CoA dehydrogenase, 50S ribosomal protein L13, branched-chain amino acid aminotransferase, DNA polymerase III subunit beta, ferritin, GCN5 family acetyltransferase, NAD(P)-dependent oxidoreductase, polyphosphate glucokinase, and thioredoxin are some of the upregulated proteins in response to chromium. Likewise, 30S ribosomal protein S1, 50S ribosomal protein L17, acyl-CoA dehydrogenase, adenosine deaminase, aldehyde dehydrogenase, argininosuccinate lyase, ATP synthase F0F1 subunit delta, betaine aldehyde dehydrogenase GbsA, chorismate synthase, enoyl-ACP reductase, glutamine-fructose-6-phosphate aminotransferase, iron ABC transporter ATP-binding protein, methylmalonyl-CoA carboxyltransferase, serine/threonine protein kinase, and type I glyceraldehyde-3-phosphate dehydrogenase were some of the downregulated proteins in the presence of chromium. Table 2 represents the proteins expressed in response to chromium.

Table 2.

Protein regulation in the presence of 100 mgL⁻¹ chromium and their corresponding metabolic pathways

Protein name	Fold change	Functional pathway	Metabolic pathway (KEGG)	Accession ID (NCBI)
30S ribosomal protein S5	1.679	Ribosome	Translation	1017134112
3-Hydroxy-2-methylbutyryl-CoA dehydrogenase	1.404	Valine, leucine and isoleucine degradation	Amino acid metabolism	1017133159
50S ribosomal protein L13	2.577	Ribosome	Translation	1017133731
Branched-chain amino acid aminotransferase	2.123	Cysteine and methionine metabolism	Amino acid metabolism	1238046825
DNA polymerase III subunit beta	1.519	DNA replication	Replication and repair	1238045853
Ferritin	2.186	Porphyrin and chlorophyll metabolism	Metabolism of cofactors and vitamins	1017127630
GCN5 family acetyltransferase	1.880	Stress protein	Stress proteins	1017131990
NAD(P)-dependent oxidoreductase	2.181	Stress protein	Stress proteins	1017129873
Polyphosphate glucokinase	1.349	Glycolysis	Carbohydrate metabolism	1017127586
Thioredoxin	1.620	Selenocompound metabolism	Metabolism of other amino acids	425481290
30S ribosomal protein S1	− 1.218	Ribosome	Translation	1017129649
50S ribosomal protein L17	− 1.324	Ribosome	Translation	1017134096
Acyl-CoA dehydrogenase	− 2.372	Fatty acid degradation	Lipid metabolism	1017133638
Adenosine deaminase	− 1.081	Purine metabolism	Nucleotide metabolism	1017134505
Aldehyde dehydrogenase	− 2.120	Glycolysis	Carbohydrate metabolism	1238735675
Argininosuccinate lyase	− 1.547	Arginine biosynthesis	Amino acid metabolism	1017131190
ATP synthase F0F1 subunit delta	− 1.492	Oxidative phosphorylation	Energy metabolism	1017132303
Betaine aldehyde dehydrogenase GbsA	− 1.103	Glycine, serine, and threonine metabolism	Amino acid metabolism	425478780
Chorismate synthase	− 1.695	phenylalanine, tyrosine and tryptophan biosynthesis	Amino acid metabolism	1017129606
Enoyl-ACP reductase	− 2.075	fatty acid biosynthesis	Lipid metabolism	1017129687
Glutamine- fructose-6-phosphate aminotransferase	− 2.688	Alanine, aspartate and glutamate metabolism	Amino acid metabolism	1017133720
Iron ABC transporter ATP-binding protein	− 1.146	ABC transporters	Membrane transport	1017129691
Methylmalonyl-CoA carboxyltransferase	− 2.515	Propanoate metabolism	Carbohydrate metabolism	1017133648
Serine/threonine protein kinase	− 1.831	Glycine, serine, and threonine metabolism	Amino acid metabolism	1017123174
Type I glyceraldehyde-3-phosphate dehydrogenase	− 1.096	Glycolysis	Carbohydrate metabolism	1238046172

Positive values indicate upregulation, while negative values indicate downregulation of proteins

Apart from the proteins that were up- and downregulated in response to metal, there were several proteins expressed only in response of arsenic and chromium respectively while absent in control (Table 3). It was observed that some of the expressed proteins were commonly up- and downregulated in the presence of arsenic as well as chromium (Table 4).

Table 3.

Proteins expressed exclusively in response to 750 mgL⁻¹ arsenic and 100 mgL⁻¹ chromium (absent in control) and their corresponding metabolic pathways

Protein name	Functional pathway	Metabolic pathway (KEGG)	Accession ID (NCBI)
30S ribosomal protein S16	Ribosome	Translation	1017134345
Antibiotic biosynthesis monooxygenase	Biosynthesis of type II polyketide products	Metabolism of terpenoids and polyketides	1017133906
ArsR family transcriptional regulator	Arsenical resistance proteins	Arsenical resistance proteins	1017127542
Aspartate kinase	Glycine, serine and threonine metabolism	Amino acid metabolism	1017123517
ATPase AAA	Oxidative phosphorylation	Energy metabolism	425478730
ATP-binding protein	ABC transporters	Membrane transport	1017132259
Cystathionine beta-lyase	Cysteine and methionine metabolism	Amino acid metabolism	1017127612
Cysteine synthase	Cysteine and methionine metabolism	Amino acid metabolism	1017134445
Cytochrome C oxidase subunit II	Oxidative phosphorylation	Energy metabolism	1017127607
Disulfide bond formation protein DsbA	Naphthalene degradation	Xenobiotics degradation and metabolism	1017132328
Lysophospholipase	Glycerophospholipid metabolism	Lipid metabolism	1017132281
N-Acetylneuraminate synthase	Amino sugar and nucleotide sugar metabolism	Carbohydrate metabolism	1017124789
Peptidase C56	Stress protein	Stress proteins	1238046526
Peptidase M24	Stress protein	Stress proteins	1017128614
Polynucleotide phosphorylase/ polyadenylase	Purine metabolism	Nucleotide metabolism	425479831
Thioredoxin reductase	Selenocompound metabolism	Metabolism of other amino acids	1017129116

Table 4.

Protein regulation in the response of 750 mgL⁻¹ arsenic and 100 mgL⁻¹ chromium respectively and their corresponding metabolic pathways

Protein name	Regulation	Functional pathway	Metabolic pathway (KEGG)	Accession ID (NCBI)
30S ribosomal protein S7	+	Ribosome	Translation	1017134137
50S ribosomal protein L25/general stress protein Ctc	+	Ribosome	Translation	1017129853
50S ribosomal protein L7/L12	+	Ribosome	Translation	1017134151
ATP synthase subunit alpha	+	Oxidative phosphorylation	Energy metabolism	1017132304
Choline oxidase	+	Glycine, serine and threonine metabolism	Amino acid metabolism	917388870
Dihydrolipoamide acyltransferase	+	Glycolysis	Carbohydrate metabolism	425482522
Elongation factor Tu	+	Ribosome	Translation	1017134135
Lactoyl-glutathione lyase	+	Pyruvate metabolism	Carbohydrate metabolism	1017133195
Lipid hydroperoxide peroxidase	+	Stress protein	Stress proteins	1017121252
Lipoyl synthase	+	Lipoic acid metabolism	Metabolism of cofactors and vitamins	1017127597
Succinate-CoA ligase subunit alpha	+	Citrate cycle	Carbohydrate metabolism	1238046916
Superoxide dismutase	+	Stress protein	Stress proteins	1017134298
Thioredoxin-disulfide reductase	+	Selenocompound metabolism	Metabolism of other amino acids	749994889
2,3,4,5-Tetrahydropyridine-2,6-dicarboxylate N-Succinyltransferase	–	Lysine biosynthesis	Amino acid metabolism	1017121203
2,5-didehydrogluconate reductase	–	Pentose and glucuronate interconversions	Carbohydrate metabolism	425479034
2-Oxoisovalerate dehydrogenase	–	Valine, leucine, and isoleucine degradation	Amino acid metabolism	1017133021
4-Carboxymuconolactone decarboxylase	–	Benzoate degradation	Xenobiotics degradation and metabolism	1238046634
50S ribosomal protein L5	–	Ribosome	Translation	1017134116
ATP-dependent RNA helicase	–	Spliceosome	Transcription	1238047530
Beta-ketoadipyl CoA thiolase	–	Fatty acid elongation	Lipid metabolism	1238048479
Cell division protein DivIVA	–	Cell division	Cell division	749996074
Glucose-6-phosphate isomerase	–	Glycolysis	Carbohydrate metabolism	1017129665
Glutamate-1-semialdehyde aminotransferase	–	Porphyrin and chlorophyll metabolism	Metabolism of cofactors and vitamins	1017133877
Glyceraldehyde-3-phosphate dehydrogenase	–	Glycerophospholipid metabolism	Lipid metabolism	1017120073
GMP synthase	–	Purine metabolism	Nucleotide metabolism	1017133742
Hypoxanthine phosphoribosyltransferase	–	Purine metabolism	Nucleotide metabolism	1017133928
LuxR family transcriptional regulator	–	Transcriptional factors- prokaryotic type	Transcription	1017133168
Molybdenum cofactor biosynthesis protein	–	Folate biosynthesis	Metabolism of cofactors and vitamins	1017135002
Phenylacetic acid degradation protein	–	Phenylalanine metabolism	Amino acid metabolism	1017136161

+Indicates upregulation

−Indicates downregulation of proteins

Metabolic classification of all the expressed proteins

All the expressed proteins were classified into their respective KEGG metabolic pathways using COG functional categories and KEGG pathway database. It was found that the majority of these proteins were categorized under a group of hypothetical proteins (223 proteins). This was followed by proteins involved in amino acid metabolism (111), carbohydrate metabolism (81), stress proteins (71), translation (57), lipid metabolism (44), membrane transport (44), metabolism of cofactors and vitamins (41), replication and repair, energy metabolism (31), and so on (Fig. 2).

Fig. 2.

Pie chart representing the metabolic classification of all the proteins expressed by Brevibacterium casei #NIOSBA88 under all conditions into their respective pathways

Discussion

Arsenic and chromium, both heavy metals, belong to different groups of elements in the periodic table. Bacteria can have one to many metal tolerance mechanisms. In this study, we focused on the differential protein expression of Brevibacterium casei #NIOSBA88 in response to arsenic and chromium stress, probable tolerance mechanisms, and the metabolic pathways affected due to these metals. Brevibacterium casei #NIOSBA88 isolated from the Arabian Sea showed multi-metal tolerance and at high concentrations. Bottom-up shotgun proteomics approach enabled us to understand the probable mechanisms for the bacterial tolerance against arsenic and chromium, respectively. Here, the expressed proteins were assessed for their functions and their pathways based on their expression patterns (Tables 1, 2, 3, and 4).

Genes conferring arsenic tolerance and involved in arsenic metabolism are known to be present in both gram-positive and gram-negative bacteria [50, 51]. Proteins such as ATP synthase subunit beta, ubiquinol-cytochrome C reductase involved in energy metabolism, alcohol dehydrogenase, and NAD-dependent aldehyde dehydrogenase involved in carbohydrate metabolism were found to be upregulated in response to arsenic indicating that these proteins could be aiding in the survival of the organism under arsenic stress. Similar results have also been observed in other studies. Cliess-Arnold et al. [52] in their transcriptomic analysis have reported induction and expression of genes that are involved in energy metabolism and transport and cellular processes under arsenic stress. Also, Srivastava et al. [19] and Zhang et al. [22] have reported overexpression of proteins involved in carbohydrate metabolism and carbon metabolism pathways in response to arsenic. Patel et al. [36] have reported the presence and expression of arsenic-specific proteins in Pseudomonas sp. as a response to arsenic in their study, which is contrasting in our study.

Majority of the house-keeping proteins such as ribosomal proteins, proteins involved in lipid metabolism, were found to be downregulated, indicating metal stress on bacterial cells. Downregulation of stress proteins in response to arsenic observed in our study has previously been reported by Shah and Damare [25] in Staphylococcus cohnii NIOSBK35, Jones et al. [53] in Arabidopsis thaliana in root hairs.

Brevibacterium casei #NIOSBA88’s response to chromium showed that majority of the house-keeping proteins such as ribosomal proteins (30S ribosomal protein S1, 50S ribosomal protein L17), proteins involved in amino acid metabolism (argininosuccinate lyase, betaine aldehyde dehydrogenase GbsA, glutamine- fructose-6-phosphate aminotransferase, serine/threonine protein kinase), and lipid metabolism (acyl-CoA dehydrogenase, enoyl-ACP reductase) were found to be downregulated in the presence of chromium. Similar results have been reported in Arthrobacter sp. strain FB24, where the majority of the proteins involved in amino acid metabolism and energy metabolism showed altered expression [54]. Some stress proteins were upregulated in response to chromium alone like GCN5 family acetyltransferase, NAD(P)-dependent oxidoreductase, and thioredoxin which have also been reported in Staphylococcus cohnii NIOSBK35 [32], Pycnoporus sanguineus [39], and Staphylococcus sp. NIOMR8 [41] to be expressed under chromium stress.

Some proteins were found to be expressed in the presence of arsenic and chromium and absent in control. These could be the significant proteins that would be responsible for bacterial tolerance to heavy metals. These proteins include antibiotic biosynthesis monooxygenase, ArsR family transcriptional regulator, cytochrome C oxidase subunit II, Peptidase C56 and M24, cystathionine beta-lyase, ATPase AAA, ATP-binding protein, and thioredoxin reductase. Peptidase M28, a stress protein, was upregulated in the presence of chromium in Staphylococcus cohnii NIOSBK35 [32]. Similarly, ArsR family transcriptional regulator was upregulated in the presence of arsenic and chromium [25, 32]. Bacteria are known to tolerate arsenite and chromate due to the presence of efflux mechanisms [55, 56]. However, no such efflux-specific proteins were detected in this study.

Ribosomal proteins such as 30S ribosomal protein S7, 50S ribosomal protein L25/general stress protein Ctc, 50S ribosomal protein L7/L12, and elongation factor Tu were found to be upregulated. Similarly, ATP synthase subunit alpha (energy metabolism), dihydeolipoamide acyltransferase and succinate-CoA ligase subunit alpha (carbohydrate metabolism), lipid hydroperoxide peroxidase and superoxide dismutase (stress proteins) were also found to be upregulated indicating a cellular response to metals and its mechanism for survival against the metal stress. Similar results of overexpression of stress proteins against heavy metal stress have been reported in many organisms such as Staphylococcus cohnii NIOSBK35 [25, 32], Staphylococcus sp. NIOMR8 [41], Enterobacteriaceae strain LSJC7 [22], and Pannonibacter phragmitetus BB [42]. Proteins that play a significant role in the usual cellular processes such as cell division protein DivIVA (cell division), glucose-6-phosphate isomerase and 2,5-didehydrogluconate reductase (carbohydrate metabolism), GMP synthase, and hypoxanthine phosphoribosyl transferase (nucleotide metabolism) were found to be downregulated indicating the cellular tactics to cope metal stress and its survival.

Hypothetical proteins contributed almost 25% of the total identified proteins which indicates that the databases are not well-curated and needs further annotation. Majority of the expressed proteins were found to be involved in amino acid metabolism, suggesting that arsenic and chromium have a considerable impact on the protein-building machinery of the cells. This was followed by the proteins involved in carbohydrate metabolism, stress proteins, translation, lipid metabolism, membrane transport, etc. (Fig. 2). These are major metabolic processes that regulate cellular functions. Alteration in the functioning of any of the proteins involved in these metabolic pathways may prove fatal to the cells. Exposure of Brevibacterium casei #NIOSBA88 to arsenic and chromium may have altered one to many of these proteins; however, a different cellular repair mechanism helped the organism to overcome the metal stress and retain its metabolic functions.

Conclusion

Brevibacterium casei #NIOSBA88, a marine-derived isolate, is multi-metal-tolerant bacterium showing tolerance to arsenic and chromium. Peptide mass fingerprinting of Brevibacterium casei #NIOSBA88 revealed a unique set of proteins that are involved in bacterial tolerance to arsenic and chromium. Expression of a set of proteins exclusively in the presence of metal indicated the vital role played by these proteins in bacterial survival against metal stress. No chromium-specific proteins were detected in this study, indicating a parallel or similar mechanism for the organism to be tolerant to both the metals.

Authors’ contributions

The work was conceptualized by the corresponding author and carried out by the first author. The manuscript was written by both the authors.

Funding information

The authors received funding support from the project BSC0111 and OLP1901 by the CSIR, India. The first author received a fellowship from the Council of Scientific and Industrial Research (CSIR) under RF84179 (CSIR-SRF 31/26(0330)/2019-EMR-I).

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Conflict of interest

The authors declare that they have no conflict of interest.

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