New insights into aniline toxicity: Aniline exposure triggers envelope stress and extracellular polymeric substance formation in Rubrivivax benzoatilyticus JA2

Journal Pre-proof New insights into aniline toxicity: Aniline exposure triggers envelope stress and extracellular polymeric substance formation in Rubrivivax benzoatilyticus JA2 Mujahid Mohammed, Lakshmi Prasuna Mekala, Sasikala Chintalapati, Venkata Ramana Chintalapati

PII:

S0304-3894(19)31525-0

DOI:

https://doi.org/10.1016/j.jhazmat.2019.121571

Reference:

HAZMAT 121571

To appear in:

Journal of Hazardous Materials

Received Date:

12 June 2019

Revised Date:

12 September 2019

Accepted Date:

29 October 2019

Please cite this article as: Mohammed M, Prasuna Mekala L, Chintalapati S, Ramana Chintalapati V, New insights into aniline toxicity: Aniline exposure triggers envelope stress and extracellular polymeric substance formation in Rubrivivax benzoatilyticus JA2, Journal of Hazardous Materials (2019), doi: https://doi.org/10.1016/j.jhazmat.2019.121571

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New insights into aniline toxicity: Aniline exposure triggers envelope stress and extracellular polymeric substance formation in Rubrivivax benzoatilyticus JA2

Mujahid Mohammeda,c, Lakshmi Prasuna Mekalaa,d, Sasikala Chintalapatib , Venkata Ramana Chintalapati*a a

Department of Plant Sciences, School of Life Sciences, University of Hyderabad, P.O.

Central University, Hyderabad 500 046, India. Bacterial Discovery Laboratory, Center for Environment, IST, JNT University Hyderabad,

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b

Kukatpally, Hyderabad -500 085, India. c

Present address: Department of Botany, Bharathidasan Government College for Women,

d

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Puducherry U.T-605003, India

Present Address: Department of Plant Sciences, Avvaiyar Government College for Women,

author. Ch.V. Ramana, e-mail: Mailing address: Department of Plant

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*Corresponding

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Karaikal, Puducherry U.T-609602.

Sciences, School of Life Sciences, University of Hyderabad, P.O. Central University,

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Gachibowli, Hyderabad-500 046, India. Phone: +9140 23134502.

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E-mail:[email protected];

AUTHOR INFORMATION

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Corresponding Author * E-mail: [email protected]. Phone: +91-040-23134502. Fax: +91-040-23010120. ORCID Venkata Ramana Chintalapati: 0000-0002-0146-1672

Graphical abstract

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Highlights

Aniline induces extracellular polymeric substance and aggregates formation in strain

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

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JA2

Proteomic data reveal the up-regulation of envelope stress response

proteins

indicating that aniline exposure causes acute envelope stress Our study revealed the induction of putative CpxAR, two component envelope stress

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

response pathway

The overall study suggested that strain JA2

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activates envelope stress response

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mechanism to counter the aniline stress 

Formation of EPS to aniline is possibly regulated by envelope stress response

signaling

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ABSTRACT Aniline is a major environmental pollutant of serious concern due to its toxicity. Although microbial metabolism of aniline is well-studied, its toxic effects and physiological responses of microorganisms to aniline are largely unexplored. Rubrivivax benzoatilyticus JA2, an aniline non-degrading bacterium, tolerates high concentrations of aniline and produces extracellular polymeric substances(EPS). Surprisingly, strain JA2 forms EPS only when exposed to aniline and other toxic compounds like organic solvents and heavy metals indicating that EPS formation is coupled to cell toxicity. Further, extensive reanalysis of the previous proteomic data of aniline exposed cells revealed up-regulation of envelope stress response(ESR) proteins such as periplasmic protein folding, envelope integrity,

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transmembrane complex, and cell-wall remodelling proteins. In silico analysis and molecular modeling of three highly up-regulated proteins revealed that these proteins were homologous to CpxARP proteins of ESR signalling pathway. Furthermore, EPS formation to known ESR activators(Triton-X-100, EDTA) suggests that envelope stress possibly regulating the EPS

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production. The present study suggests that aniline triggers envelope stress; to counter this strain JA2 activates ESR pathway and EPS production. Our study revealed the hitherto

unknown toxic effects of aniline as an acute envelope stressor thus toxicity of aniline may be

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more profound to life-forms than previously thought.

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ABBREVIATIONS: EPS, extracellular polymeric substance; ESR, Envelope stress response; TIRTC, Tetramethylrhodamine isothiocyanate; TCR, two component

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transcriptional regulator;

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KEY WORDS

Extracellular polymeric substance, Envelope stress response, Aniline toxicity, Rubrivivax benzoatilyticus, CpxAR-signalling

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1. INTRODUCTION Aromatic amines (AAs) are one of the major classes of anthropogenic environmental pollutants widely distributed in the environment. Most of the AAs are cytotoxic or genotoxic to life forms [1, 2] and they account for 12% of the synthetic chemicals known as potential carcinogens [2, 3]. Aniline is one such important class of aromatic amine pollutant of serious environmental concern [4-6]. Aniline and its derivatives are widely used in the preparation of pesticides, herbicides, paints, pharmaceuticals, and dyes [3, 4, 7]. Owing to their continuous use, aniline and its derivatives are constantly released and accumulated in the environment

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through industrial effluents, accidental spills and their direct application to the soil[4, 8, 9].

Aniline is mainly accumulated and contaminated the soil and aquatic ecosystems [10, 11]. Two major accidental aniline spills in China released large amounts of aniline into the

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environment and contaminated aquatic ecosystems and posed a severe health hazard to

human as well as other life forms [7-9] . Aniline is highly recalcitrant organic pollutant [12]

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and due to its toxic and persistent nature, it is in priority pollutants list of EPA (USA, EEC, 1976, Federal Register, 1979). Aniline may remain as a major environmental threat as global aniline use is on the rise and its contamination would pose a severe environmental risk [9,

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13]. Though, few studies were reported on toxic effects of aniline in animal models[14, 15], eukaryotic organisms [16] or in vitro cell cultures [17], toxic implications of aniline to life forms are not yet completely understood particularly in microorganisms. Though microbial

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metabolism of aniline has gained considerable attention, very few studies were carried out to understand the toxicity of the aniline and its derivatives in microorganisms [8, 18] [19].

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Aniline or its metabolites induce free radical formation in Yeast thereby leading to the oxidative stress and cytotoxicity[16]. Aniline or its derivatives inhibited that methanogenic

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activity in acetoclastic methanogenic bacteria[20]. Quantitative structure-activity relationship (QSAR) studies on algae and Vibrio fischeri, (luminescent bacterium) suggest that toxicity of aniline to microorganisms depends on the chemical characteristic and hydrophobicity[19]. Study on short-time exposure of soil microbial communities to aniline revealed that aniline significantly reduces the soil microbial respiratory activity and soil enzyme activities (βglucosidase, urease, acid-phosphatase, and dehydrogenase) and thereby adversely affecting the soil microbial activity[8]. However, studies could not reveal the molecular mechanism of aniline toxicity mainly, due to an incomplete understanding of molecular targets of aniline.. 4

Although aniline can be a potential nutritional source for a few bacteria, it is an acute physiological stressor to both degraders and non-degraders and thus may affect microbial communities [12]. Thus, understanding the molecular mechanisms of aniline toxicity and cellular responses to aniline is significant to access its impact on microbial communities and also in developing effective bioremediation methods. A previous study by our group revealed the systemic responses of a photosynthetic bacterium, Rubrivivax benzoatilyticus JA2 to high (25 mM) concentration of aniline using an omics approach [12]. Integrated omics study on strain JA2 revealed that strain employs multilayered tolerance mechanisms and displayed extensive metabolic rearrangements to aniline stress [12]. The study also uncovered the extracellular polymeric substance formation and

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associated biochemical changes to aniline stress [12]. Considering strain JA2’s ability to survive in the presence of a high concentration of aniline and leads from the previous study

we carried out further study to gain insights into the mode of aniline toxicity and the interplay between toxicity and stress response mechanisms of strain JA2. In the present

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communication, our study revealed aniline exposure causes severe envelope stress and

activated the envelope stress response mechanism to counter the envelope assault in strain

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JA2. Our study also suggests that EPS formation in strain JA2 is plausibly triggered by envelope stress and formation of EPS may be a protective response to counter the envelope

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stress caused by toxicants.

2. MATERIAL AND METHODS

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2.1 Organism and growth conditions

Rubrivivax benzoatilyticus JA2 (ATCCBAA-35) is a metabolically versatile anoxygenic photosynthetic purple bacterium [21, 22] capable of surviving under high

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concentration of aniline (25 mM) and its derivatives without using them as a nutrient source [5, 6]. Rubrivivax benzoatilyticus JA2 was grown photoheterotrophically (anaerobic, 30

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±1ºC; light 2,400 lux) in a mineral medium, malate (20 mM) as carbon source and ammonium chloride (7 mM) as nitrogen source in fully filled screw cap test tubes (10x100 mm)/reagent bottles (250 ml) at pH 6.8. For induction of EPS, culture was grown in malate media under photoheterotrophic conditions and mid-log phase culture (0.4 OD at 660 nm) was exposed to aniline (20 mM, sub-lethal ) or other solvents (10 mM), incubated at 30 ºC for desired period (maximum for 56 h). To monitor the effect of the solvents on growth, strain JA2 was grown in mineral media amended with different concentrations of organic solvents under photoheterotrophic conditions. Growth of strain JA2 was measured 5

turbidometrically at 660 nm. To test the EPS production under different conditions, mid-log phase (OD660 nm 0.4) culture was exposed to salt stress (1 and 2% NaCl), pH 4.5 or 8.5 (adjusted using buffers) heavy metals (Cobalt chloride, Zinc sulfate, Nickle chloride 0.5 mM). For nutritional stress, mid-log phase culture was centrifuged under sterile conditions and cell pellet suspended in carbon or nitrogen free mineral media and allowed grow under photoheterotrophic conditions. For envelope stress, mid-log phase culture of strain JA2 (OD660 nm 0.4) was exposed to different concentrations of Triton-X-100 (0.1%), SDS (0.1%) and EDTA (2 and 5 mM) then the culture was incubated photoheterotrophically. 2.2 Scanning electron microscopic and confocal microscopy analysis Scanning electron microscopic studies were carried out according to [23] using

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control and toxic compounds exposed cultures. EPS were stained with calcofluor white (β polysaccharides), Nile red (lipids), Acridine orange (nucleic acids), rhodamine isothiocyanate (proteins, amino groups). 5 μl of each dye (1 mg.ml-1) was added to EPS, after 20 min

incubation in the dark, excess of the dye was washed with sterile Mill-Q water. For viability,

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cell aggregates were stained with vital dyes, fluorescein diacetate (FDA), resazurin and

polysaccharides were stained using calcofluor white and aniline blue in a similar way as

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mentioned for EPS. Stained samples were mounted on to glass slide and visualized by confocal laser microscopy. Confocal laser microscopic analysis was performed on Zeiss Observer LSM 710 confocal microscope equipped with blue, green and red lasers. Images

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were taken with 10x and 40x objectives. Probes were visualized at their respective excitation and emission wavelengths. Calcofluor white (Ex400/Em480 nm), Nile red (Ex552/Em636 nm), Acridine orange (Ex504/Em526 nm), tetramethylrhodamine isothiocyanate

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(Ex547/Em572 nm), FDA (Ex490/Em514 nm) and resazurin (Ex 570/Em590 nm). Five random places were visualized for each sample.

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2.3 Proteomic data analysis

The proteomic data published as supporting information in our previous publication[12] was

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reanalyzed to identify the altered envelope stress response and other related proteins. Statistical parameters considered for data analysis were as described by Mujahid et al 2015. briefly, proteins identified with protscore> 2, two unique peptides, false discovery rate <1, p-value < 0.05 and error factor <2 were subjected to further analysis. Protein with 1.5 fold change and p-value <0.05 was considered as statically significant to categorize up and down regulated proteins. Functional classification of proteins was done according to the KEGG database. 3.4 Bioinformatics analysis and homology modeling 6

All the protein sequences were retrieved from NCBI genome project of strain JA2, functional domain search was performed against the Pfam and NCBI conserved domain databases. Homology models of hypothetical protein RBXJA2-18423(Accession No EGJ12324) and histidine kinase RBXJA2-18413(Accession No EGJ12322) were generated using the SWISS-MODEL homology modelling server in the fully automated mode. CpxP (PDB ID 3qzc-A; 25% identity) and CpxA (4biz-C; 30% identity) were used as template for modelling of hypothetical, histidine kinase proteins of strain JA2, respectively. The model quality was evaluated using the PROCHECK by Z-score of ProSA-Web validation method and structural homology search was done using the DALI server. Subcellular localization of proteins was predicted using CELLO2GO server and interacting proteins, functional protein network analysis was done using the STRING program. A dimeric model of hypothetical

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protein was generated by submitting the two identical monomer models of hypothetical

protein to the ClusPro server. Electrostatic surface potentials were calculated using the APBS program through the Poisson-Boltzmann equation and PyMOL was used for molecular visualization.

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3. RESULTS

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3.1 Extracellular polymeric substances (EPS) and aggregates formation by strain JA2 Aniline exposed culture of strain JA2 was viscous compared to control (observation)

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and SEM analysis revealed no EPS formation in control cells (aniline unexposed) (Fig.1A) while aniline exposed cells showed deposition of EPS on the cell surface (Fig.1B). Further, time course analysis revealed the formation of EPS after 12 h of aniline exposure, and

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thereafter EPS steadily accumulated (Fig. 1C). In addition to EPS production, SEM analysis revealed aggregates formation (EPS encased cells) in aniline exposed cells of strain JA2 (Fig. 2A, B) and such aggregates were not found in control cells indicating the aniline induced

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EPS/aggregate formation.

Further, aggregates were stained with Calcofluor white, a EPS specific component

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fluorescent dye revealed the presence of polysaccharides in aggregates (Fig. S1A,B). Vital (metabolism specific) dyes such as fluorescein diacetate (FDA) (Fig. S1C) and resazurin staining (Fig. S1D) revealed presence of metabolically active cells in the aggregates. Staining patterns of FDA and resazurin were similar (Fig. S1C, D) indicating the metabolic activity and redox status of the cells. Different fluorescent specific probes were used to detect the components of EPS and their distribution in the aggregates (Fig. 2C). Confocal microscopic studies revealed the presence of polysaccharides (Fig. 2D; calcofluor white), nucleic acids

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(Fig. 2E; Acridine orange), proteins (Fig. 2F; TRITC) and lipids (Fig. 2G; nile red) in aggregates. Remarkably, similar staining pattern of polysaccharides, proteins, nucleic acids and lipids in aggregates (Fig. 2H) indicate their co-localization and heterogenous nature of the EPS. 3.2 Formation of EPS to the toxic compounds and other environmental stressors To know whether the EPS formation in strain JA2 is specific to aniline exposure or other stressors, we exposed strain JA2 to physiological stressors such as temperature (15 and 40º C), pH (5.0, 8.5), NaCl (1, 1.5%), aerobic growth (150 rpm), nutritional stress (carbon and nitrogen) and heavy metal stress (Zn, Co, Cu). EPS and aggregates formation was not observed when strain JA2 exposed to physiological stressors, while heavy metal stress

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induced EPS as well as aggregates formation under the tested conditions in strain JA2 (Fig.

S1). When strain JA2 was exposed to different organic solvents such as aromatic (Toluene, acetophenone, aniline, benzene, xylene, nitrobenzene) and aliphatic solvents (ethanol,

methanol, dimethyl sulfoxide, isopropanol, octanol, cyclohexane), surprisingly, all the

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organic solvents tested, induced EPS formation (Fig. 3A). EPS production was significantly high in presence of cyclohexane (43±4 µg mg dry wt.) followed by aniline(28.25±6 µg mg

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dry wt.), octanol (27.5±3 µg mg dry wt.), nitrobenzene (23±5 µg mg dry wt.), acetophenone (22.16±2 µg mg dry wt.), xylene (21±1 µg mg dry wt.) (Fig. 2A). Organic solvents with logP

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(octanol/water partition coefficient) value <1 (ethanol, methanol, DMSO, isopropanol) induced less EPS production compared to solvents with logP value >1 (acetophenone, benzene, nitrobenzene, toluene, xylene). Interestingly, aniline with the logP value 1, also

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induced higher EPS production (Fig. 2A). Some of the tested toxic compounds are known to disrupt the cell membranes, we hypothesise that envelope stress may be signal for EPS

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formation.

3.3 Proteome reveals molecular signatures of envelope stress caused by aniline

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To understand the possible envelope stress caused by aniline, we mined the proteomic

data of aniline exposed cells for molecular signatures of the envelope stress response (ESR). Out of the 756 proteins that were detected in aniline exposed cells, 16% (120) were related to envelope associated proteins and 4% were (30) related to folding sorting and degradation (cytoplasmic protein quality control machinery) (Fig. 3B). Of the 120 envelope associated proteins, 33 proteins showed differential regulation wherein 18 proteins were up-regulated and 15 were downregulated (Fig. 3C). Among proteins related to folding sorting and degradation, 15 proteins were up-regulated and 1 was downregulated (Fig. 3C). Further, up8

regulation of proteins related to periplasmic protein quality control such as thiol-disulfide interchange protein (DsbA), peptidylprolyl cis-trans isomerase, membrane protease, and aminopeptidase was observed in aniline exposed cells (Fig. 4). Proteins related to transmembrane protein targeting (TolB, SedD, YidC, HlyD, OmpA, porin) (Fig. 4) and envelope integrity maintaining proteins (peptidoglycan-associated lipoprotein (PAL), membrane lipoproteins) were upregulated to aniline exposure (Fig. 4). Further, cell wall remodeling proteins (lytic transglycosylase and penicillin-binding protein) were also upregulated to aniline exposure (Fig. 4). Among, up-regulated proteins, hypothetical protein18423, histidine kinase, and two component transcriptional regulator (TCR) proteins were highly up-regulated (Fig. 4).

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3.4 In silico characterization and homology modelling revealed presence of putative CpxARP proteins

To gain the functional insights of up-regulated proteins, in silico analysis of three

highly up-regulated proteins was carried out. The highly up-regulated hypothetical protein

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RBXJA2T_18423 (Accession No EGJ12324) consisted of 182 amino acids with a theoretical molecular weight of 19.7 kDa. Conserve motifs analysis of the hypothetical protein revealed

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the presence of LTXXQ motif (PF07813) and showed 42% identity at an amino acid level to the CpxP protein (GeneBank protein Accession No EHR70557.1) of Burkholderiales

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bacterium JOSHI_001. Further, the in silico subcellular localization analysis revealed that the hypothetical protein-18423 is a periplasmic protein (cello2go score 2.2) and the presence of periplasmic signal peptide and plausible cleavage site (at 30-31th amino acid residues) was

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predicted by the signalP.3 server.

A 3D model of hypothetical protein was generated using CpxP of E.coli as template PDB ID 3QZC and predicted (monomeric) model has four alpha helices, typically

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found in CpxP related proteins[24] (Fig. 5A). Model quality was validated using PROCHEK, ProSA and Ramachandran plot revealed 89.2% residues were in the most favoured region

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followed by 8.8% residues in additionally allowed areas indicating the good stereo-chemical quality of the model (Fig. S2A). ProSA, Z-score (-3.7) of the predicted model was similar to the Z-score (-3.67) of template (3QZC) indicating the structural homology between experimentally determine the structure of the template and predicted model (Fig. S2B). Further, a dimer model of hypothetical protein was generated by docking the two identical monomers using ClusPro and predicted dimeric model has cap-shaped structure (Fig. 5B), as observed in case of crystal structure resolved CpxP protein[24]. When 3D dimer model of hypothetical protein was searched against DALI server for similar folding protein, DALI 9

server identified the CpxP protein (PDB ID:3qzc, z-score 10.6, rmsd-0.2 in 105 amino acids) as the closest structural homolog. The second best structural homolog of DALI search was also a CpxP protein (PDB ID: 3itf, z-score 8.5, rmsd 1.9) followed by Spy protein (PDB ID: 3o39, z-score 7.7, rmsd 1.6) of E.coli and best 10 hits were also either CpxP or Spy proteins. Electrostatic surface potential analysis of predicted dimer model of the hypothetical protein revealed the highly positive concave and mixed (positive and negative) as well as hydrophobic convex surface a characteristic feature of CpxP /Spy proteins[24] (Fig. S3A). STRING analysis was carried out to identify the interacting partners (interactome) of highly up-regulated hypothetical protein and STRING identified the interacting partners with high confidence (0.<7). On the basis of the neighbourhood (green color), hypothetical protein interacted with the uncharacterized histidine kinase, TCR and putative lipoprotein

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(Fig. S3). Interestingly, histidine kinase and TCR identified as interacting partners of the hypothetical proteins were also highly up-regulated to aniline stress (Fig. 4). The up-

regulated histidine kinase protein (Accession No EGJ12322) of strain JA2 consisted of 470 amino acids with a theoretical molecular mass of 50.5 kDa. The histidine kinase of strain

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JA2 showed 43% identity to the CpxA protein (GeneBank accession KWT94346.1 ) of the

Variovorax sp. WDL1 and subcellular localization studies revealed that histidine kinase as an

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inner membrane/cytoplasmic protein (cello2go score 2.6/3.7). PDB search showed the structural similarity (33%) to the CpxA (PDB ID: 4CB0).

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A 3D model of up-regulated histidine kinase was generated (Fig. 5C) using the CpxA (PDB ID:4CB0, 33% identity) of E.coli as template. The model was validated using PROCHECK, Ramachandran plot revealed the 91% of residues were in the most favoured

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region and 7% in the additionally allowed area indicating the good stereo-chemical quality of the model (Fig. S3C). Overall model quality analysis with ProSA revealed Z-score -7.3(Fig. S3D) which is similar to the Z-score of the template (4CB0-7.8) indicating the structural

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similarity between predicted model and template. Predicted 3D model of histidine kinase was searched for similar folding proteins in protein databank using DALI server and first six hits

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were different chains of CpxA of E.coli (PDB ID:4BIZ). Dali identified the CpxA protein (PDB ID: 4BIZ-A, Z score-20.4, and 1.8 in 217 amino acids) and PDB ID: 4BIV-A (Z score22.5, and 1.9 in 217 amino acids) as the closest structural homologs. The second interacting partner of hypothetical protein, a two-component transcriptional protein (Accession No EGJ12323) which was highly up-regulated to aniline exposure has 234 and theoretical mass of 26.1 kDa. Protein consisted of response regulator receiver domain (REC; PF00072) and TCRy domain (helix turn helix motif; PF00486). The up-regulated TCR showed 84% identity to the CpxR (KNZ32387.1) of the Methylibium sp. NZG and subcellular localization studies 10

indicated TCR as a cytoplasmic protein (cello2go score 6.1). The TCR of strain JA2 showed 47%, and 39% identity to the crystal structure of CpxR receiver domain (4HUJ), DNA binding domain (4UHT) of E.coli, respectively.

4. DISCUSSION Aniline and its derivatives are one of the important class of pollutants of serious environmental concern and its growing global demand and usage would further escalate the environmental threat [9]. Thus understanding the toxicity of aniline to various life forms and its mechanism of toxicity is crucial in assessing its environmental impact. In the present study, we uncovered the mechanism of aniline toxicity and its role in EPS formation in an anoxygenic photosynthetic bacterium, by integrating the physiology, proteomic and

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bioinformatics.

4.1 Aniline and other envelope damaging toxic compounds induces EPS/aggregates formation

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Our recent study revealed multi-layered tolerance mechanisms employed by strain JA2 to aniline and detected the EPS formation [12]. Formation of EPS by aniline exposed

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cells but not in control suggest aniline stress induces EPS formation in strain JA2. EPS is a major component of biofilm or cell aggregates and its production is prerequisite for biofilm

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or aggregates formation. Strain JA2 forms cell aggregates (surface free) in response to aniline (Fig. 2A, B) and other toxic substances such as solvents (data not shown), heavy metal zinc ( Fig. S1F). Aggregates consist of EPS encased cells [25] and bacteria form aggregates or

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flocks in response to various toxic substances [25-27]. Similarly, aggregates of strain JA2 also composed of viable cells (Fig. S1C, D) encased in EPS evident from specific

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fluorescence probes staining (Fig. S1E; Fig. 2). 4.2 Reanalysis of proteomic data of aniline exposed cells reveals molecular signatures of

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envelope stress

Formation of EPS to aniline and other toxic compounds suggests that it may be a

specific response to toxic compound stress and membrane damage may be a molecular signal for EPS formation as many of the toxic compounds (organic solvents) are known to disorganize membranes by interacting with lipids or proteins [28-30]. In support of this proteome data on aniline exposed cells revealed differential regulation of a large number of envelope-associated proteins and proteins related to cytoplasmic protein quality control (Fig. 3B, C). Taken together, these results suggest that aniline exposure plausibly causing protein 11

denaturation thereby the membrane damage. This is further evident from up-regulation of proteins related to envelope biogenesis, periplasmic proteins quality control in aniline exposed cells (Fig. 4). Proteins related to envelope homeostasis and periplasmic protein quality control are known to up-regulate in bacteria when exposed to envelope damaging compounds [30-32]. Bacteria senses envelope assault by a dedicated two-component system consisting of a histidine kinase sensor and response regulator which modulates expression of genes related to envelope repair [29, 32, 33]. Different ESR pathways are identified in bacteria and CpxARP is one among them which senses envelope assault [29]. Interestingly, in our study we found a highly upregulated hypothetical protein showing homology with CpxP related proteins with respect to

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having LTXXQ motif predicted periplasmic localization and cap-shaped 3D structure (Fig. 5A) characteristic of CpxP/Spy proteins involve in ESR[24]. Based on this, we speculate that the up-regulated hypothetical protein is likely to be a putative-CpxP/Spy protein of strain JA2 and this needs functional validation. CpxP and Spy proteins are structurally and functionally

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similar [24, 34], CpxP, a periplasmic protein interacts with CpxA and regulates CpxR and

CpxARP system is a widely found ESR pathway in gram-negative bacteria [24, 32]. CpxP or

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Spy proteins are known to express to envelope stress and assist in maintaining periplasmic protein quality by their chaperone activity in gram-negative bacteria [24, 35, 36]. Similarly, up-regulation of putative CpxP/Spy to aniline exposure in strain JA2 suggests that aniline

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possibly causes envelope stress.

Moreover, identification of putative CpxAR proteins( involved in ESR signalling)

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in strain JA2 based on their interaction with CpxP, co-expression, homology, subcellular localization and structural similarity to the characterized CpxAR proteins further supports the aniline induced envelope stress. For the first time, we report possible putative CpxARP

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proteins in strain JA2. Co-expression of putative CpxARP proteins along with other envelope homeostasis proteins to aniline (Fig.4) strongly suggests that aniline causes envelope stress.

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Though five different ESR signalling pathways are known in bacteria[37], genome mining of strain JA2 revealed only genes coding for the putative CpxARP proteins suggesting that it may be a principle ESR signalling pathway in strain JA2. The functional validation of proteins of putative CpxARP in strain JA2 is highly warranted however this is beyond the scope of the current manuscript. Many toxic compounds cause envelope damage and to combat this cell activates envelope reinforcing mechanisms [30, 32, 37] and envelope stress also regulates biofilm[38, 12

39]/EPS formation in bacteria [40, 41]. Similarly, up-regulation of ESR specific proteins and EPS formation in aniline exposed cells indicates that envelope damage may be triggering the EPS/aggregates formation in strain JA2. This is further confirmed by EPS formation in strain JA2 when exposed to well-characterized envelope stressors Tritan-X-100, and EDTA (Fig. 5D) Recent reports revealed envelope stress sensing CpxAR system role in biofilm formation and activation of biofilm matrix (EPS) producing genes in bacteria [38, 40].

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accordance with this, our study suggests that aniline causes envelope stress leading to the activation of the putative CpxAR pathway and thereby possibly activating the EPS formation in strain JA2 (Fig. 6). CpxAR system mainly responds to denatured or misfolded periplasmic

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proteins and activates ESR in bacteria [42, 43]. Similarly aniline possibly denaturing the periplasmic proteins leading to activation of CpxAR driven stress response in strain JA2. Based on the findings from the current study we propose a model depicting the interplay between aniline toxicity and cellular response in terms of EPS formation (Fig. 6).

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5. CONCLUSIONS

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Previous studies revealed that aniline causes oxidative stress and DNA damage [14, 16, 17, 44] however, for the first time, our study revealed that aniline is an acute envelope stressor. Further, activation of periplasmic and cytoplasmic protein quality control systems

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strongly suggests that proteins are one of the molecular targets of aniline. As membranes are universal to all living systems and vital for cell survival, membrane assault by stressor like aniline potentially affects cell survival. Demonstration of aniline as an acute envelope

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stressor in the current study and previously reported toxic effects of aniline all together suggests that aniline is of more serious environmental concern than previously thought.

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However, extant of aniline toxicity and tolerance to aniline would possibly depend on intrinsic nature of the species. Our study indicates aniline plausibly interacts directly or

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indirectly with proteins of the membrane thereby cause envelope stress. Future investigations on functional validation of putative CpxARP envelope stress response system in strain JA2 and understanding how aniline damages proteins/lipids would provide more insights of aniline toxicity as envelope stressor. Our study also highlighted how omic approaches would assist in deciphering the molecular mechanisms of toxicity. The presents study expanded the current knowledge on aniline toxicity and provided new insights into the mechanism of aniline toxicity.

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SUPPLEMENTARY MATERIAL The Supplementary material is available free of charge on the ACS Publications website Figure S1; Aggregates formation by strain JA2 to aniline and heavy metal stress, Figure S2: Surface charge distribution of putative CpxP model and STRING based protsin network analysis Figure S3: PROCHECK and ProSA based CpxP and CpxA model validation

DECLARATION OF INTEREST: Authors declare no competing financial interest ACKNOWLEDGEMENTS VRC thank the Department of Biotechnology, Government of India for the award of Tata

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FIST (SR/FST/LSII-030/2013(G)) are acknowledged.

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Innovative Fellowship (BT/HRD/35/01/02/2015). Infrastructural facilities funded by DST-

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REFERENCES [1] P.L. Skipper, M.Y. Kim, H.L. Sun, G.N. Wogan, S.R. Tannenbaum, Monocyclic aromatic amines as potential human carcinogens: old is new again, Carcinogenesis, 31 (2010) 50-58. [2] D. Kim, F.P. Guengerich, Cytochrome P450 activation of arylamines and heterocyclic amines, Annu Rev Pharmacol Toxicol, 45 (2005) 27-49. [3] M. Martins, F. Rodrigues-Lima, J. Dairou, A. Lamouri, F. Malagnac, P. Silar, J.M. Dupret, An acetyltransferase conferring tolerance to toxic aromatic amine chemicals:

ro of

molecular and functional studies, J Biol Chem, 284 (2009) 18726-18733. [4] Q. Liang, M. Takeo, M. Chen, W. Zhang, Y. Xu, M. Lin, Chromosome-encoded gene

cluster for the metabolic pathway that converts aniline to TCA-cycle intermediates in Delftia tsuruhatensis AD9, Microbiology, 151 (2005) 3435-3446.

-p

[5] M. Mujahid, C. Sasikala, V. Ramana Ch, Aniline is an inducer, and not a precursor, for indole derivatives in Rubrivivax benzoatilyticus JA2, PLoS One, 9 (2014) e87503. [6] M. Mujahid, C. Sasikala, V. Ramana Ch, Aniline-induced tryptophan production and

re

identification of indole derivatives from three purple bacteria, Curr Microbiol, 61 (2010) 285-290.

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[7] S.Y. Lee, G.H. Kim, S.H. Yun, C.W. Choi, Y.S. Yi, J. Kim, Y.H. Chung, E.C. Park, S.I. Kim, Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24,

na

PLoS One, 11 (2016) e0154233.

[8] H. Chen, R. Zhuang, J. Yao, F. Wang, Y. Qian, K. Masakorala, M. Cai, H. Liu, Short-

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term effect of aniline on soil microbial activity: a combined study by isothermal microcalorimetry, glucose analysis, and enzyme assay techniques, Environ Sci Pollut Res Int, 21 (2014) 674-683.

Jo

[9] W. Sun, Y. Li, L.R. McGuinness, S. Luo, W. Huang, L.J. Kerkhof, E.E. Mack, M.M. Haggblom, D.E. Fennell, Identification of Anaerobic Aniline-Degrading Bacteria at a Contaminated Industrial Site, Environ Sci Technol, 49 (2015) 11079-11088.

[10] A.S. Vangnai, W. Petchkroh, Biodegradation of 4-chloroaniline by bacteria enriched from soil, FEMS Microbiol Lett, 268 (2007) 209-216.

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[11] H.Y. Kahng, J.J. Kukor, K.H. Oh, Characterization of strain HY99, a novel microorganism capable of aerobic and anaerobic degradation of aniline, FEMS Microbiol Lett, 190 (2000) 215-221. [12] M. Mujahid, M.L. Prasuna, C. Sasikala, V. Ramana Ch, Integrated Metabolomic and Proteomic Analysis Reveals Systemic Responses of Rubrivivax benzoatilyticus JA2 to Aniline Stress, J Proteome Res, 14 (2015) 711-727. [13] Y. Jiang, K. Yang, Y. Shang, H. Zhang, L. Wei, H. Wang, Response and recovery of aerobic granular sludge to pH shock for simultaneous removal of aniline and nitrogen, Chemosphere, 221 (2019) 366-374. [14] X. Fan, J. Wang, K.V. Soman, G.A. Ansari, M.F. Khan, Aniline-induced nitrosative stress in rat spleen: proteomic identification of nitrated proteins, Toxicol Appl

ro of

Pharmacol, 255 (2011) 103-112.

[15] Y. Okazaki, K. Yamashita, M. Sudo, M. Tsuchitani, I. Narama, R. Yamaguchi, S.

Tateyama, Neurotoxicity induced by a single oral dose of aniline in rats, J Vet Med Sci, 63 (2001) 539-546.

-p

[16] R.J. Brennan, R.H. Schiestl, Aniline and its metabolites generate free radicals in yeast, Mutagenesis, 12 (1997) 215-220.

re

[17] Y. Wang, H. Gao, X.L. Na, S.Y. Dong, H.W. Dong, J. Yu, L. Jia, Y.H. Wu, Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes, Int J Environ

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Res Public Health, 13 (2016).

[18] C.E. Cerniglia, J.P. Freeman, C. Van Baalen, Biotransformation and toxicity of aniline and aniline derivatives of cyanobacteria, Arch Microbiol, 130 (1981) 272-275.

na

[19] V. Aruoja, M. Sihtmae, H.C. Dubourguier, A. Kahru, Toxicity of 58 substituted anilines and phenols to algae Pseudokirchneriella subcapitata and bacteria Vibrio fischeri: comparison with published data and QSARs, Chemosphere, 84 (2011) 1310-1320.

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[20] B.A. Donlon, E. Razo-Flores, J.A. Field, G. Lettinga, Toxicity of N-substituted aromatics to acetoclastic methanogenic activity in granular sludge, Appl Environ

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Microbiol, 61 (1995) 3889-3893.

[21] M. Mohammed, A. Isukapatla, L.P. Mekala, R.P. Eedara Veera Venkata, S. Chintalapati, V.R. Chintalapati, Genome sequence of the phototrophic betaproteobacterium Rubrivivax benzoatilyticus strain JA2T, J Bacteriol, 193 (2011) 2898-2899.

[22] L.P. Mekala, M. Mohammed, S. Chintalapati, V.R. Chintalapati, Stable Isotope-Assisted Metabolic Profiling Reveals Growth Mode Dependent Differential Metabolism and Multiple Catabolic Pathways of l-Phenylalanine in Rubrivivax benzoatilyticus JA2, J Proteome Res, 17 (2018) 189-202. 16

[23] J.H. Priester, A.M. Horst, L.C. Van de Werfhorst, J.L. Saleta, L.A. Mertes, P.A. Holden, Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy, J Microbiol Methods, 68 (2007) 577-587. [24] G.L. Thede, D.C. Arthur, R.A. Edwards, D.R. Buelow, J.L. Wong, T.L. Raivio, J.N. Glover, Structure of the periplasmic stress response protein CpxP, J Bacteriol, 193 (2011) 2149-2157. [25] G.P. Sheng, H.Q. Yu, X.Y. Li, Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review, Biotechnol Adv, 28 (2010) 882-894. [26] M.Y. Chen, D.J. Lee, J.H. Tay, K.Y. Show, Staining of extracellular polymeric substances and cells in bioaggregates, Appl Microbiol Biotechnol, 75 (2007) 467-474.

ro of

[27] G.P. Sheng, H.Q. Yu, Z.B. Yue, Production of extracellular polymeric substances from Rhodopseudomonas acidophila in the presence of toxic substances, Appl Microbiol Biotechnol, 69 (2005) 216-222.

[28] J.L. Ramos, E. Duque, M.T. Gallegos, P. Godoy, M.I. Ramos-Gonzalez, A. Rojas, W.

-p

Teran, A. Segura, Mechanisms of solvent tolerance in gram-negative bacteria, Annu Rev Microbiol, 56 (2002) 743-768.

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[29] S. Jordan, M.I. Hutchings, T. Mascher, Cell envelope stress response in Gram-positive bacteria, FEMS Microbiol Rev, 32 (2008) 107-146.

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[30] R. Mazzoli, P. Fattori, C. Lamberti, M.G. Giuffrida, M. Zapponi, C. Giunta, E. Pessione, High isoelectric point sub-proteome analysis of Acinetobacter radioresistens S13 reveals envelope stress responses induced by aromatic compounds, Mol Biosyst, 7

na

(2011) 598-607.

[31] M. Merdanovic, T. Clausen, M. Kaiser, R. Huber, M. Ehrmann, Protein quality control in the bacterial periplasm, Annu Rev Microbiol, 65 (2011) 149-168.

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[32] S.L. Vogt, T.L. Raivio, Just scratching the surface: an expanding view of the Cpx envelope stress response, FEMS Microbiol Lett, 326 (2012) 2-11.

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[33] M. Grabowicz, T.J. Silhavy, Envelope Stress Responses: An Interconnected Safety Net, Trends Biochem Sci, 42 (2017) 232-242.

[34] E. Kwon, D.Y. Kim, C.A. Gross, J.D. Gross, K.K. Kim, The crystal structure Escherichia coli Spy, Protein Sci, 19 (2010) 2252-2259.

[35] X. Zhou, R. Keller, R. Volkmer, N. Krauss, P. Scheerer, S. Hunke, Structural basis for two-component system inhibition and pilus sensing by the auxiliary CpxP protein, J Biol Chem, 286 (2011) 9805-9814.

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[36] I. Kouidmi, L. Alvarez, J.F. Collet, F. Cava, C. Paradis-Bleau, The Chaperone Activities of DsbG and Spy Restore Peptidoglycan Biosynthesis in the elyC Mutant by Preventing Envelope Protein Aggregation, J Bacteriol, 200 (2018). [37] G. Rowley, M. Spector, J. Kormanec, M. Roberts, Pushing the envelope: extracytoplasmic stress responses in bacterial pathogens, Nat Rev Microbiol, 4 (2006) 383-394. [38] H. Li, F. Liu, W. Peng, K. Yan, H. Zhao, T. Liu, H. Cheng, P. Chang, F. Yuan, H. Chen, W. Bei, The CpxA/CpxR Two-Component System Affects Biofilm Formation and Virulence in Actinobacillus pleuropneumoniae, Front Cell Infect Microbiol, 8 (2018) 72. [39] C. Dorel, O. Vidal, C. Prigent-Combaret, I. Vallet, P. Lejeune, Involvement of the Cpx

(1999) 169-175.

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signal transduction pathway of E. coli in biofilm formation, FEMS Microbiol Lett, 178

[40] J. Schmid, V. Sieber, B. Rehm, Bacterial exopolysaccharides: biosynthesis pathways and engineering strategies, Front Microbiol, 6 (2015) 496.

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[41] T.L. Raivio, Everything old is new again: an update on current research on the Cpx envelope stress response, Biochim Biophys Acta, 1843 (2014) 1529-1541.

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[42] A. Delhaye, J.F. Collet, G. Laloux, Fine-Tuning of the Cpx Envelope Stress Response Is Required for Cell Wall Homeostasis in Escherichia coli, MBio, 7 (2016) e00047-

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00016.

[43] S. Hunke, R. Keller, V.S. Muller, Signal integration by the Cpx-envelope stress system, FEMS Microbiol Lett, 326 (2012) 12-22.

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[44] P. Makhdoumi, H. Hossini, G.M. Ashraf, M. Limoee, Molecular Mechanism of Aniline Induced Spleen Toxicity and Neuron Toxicity in Experimental Rat Exposure: A

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Review, Curr Neuropharmacol, 17 (2019) 201-213.

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FIGURE LEGENDS

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Figure. 1 EPS formation by aniline exposed cells of strain JA2. Scanning electron micrograph of control (A) and aniline exposed (B) cells of Strain JA2.

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EPS production by aniline exposed culture of strain JA2 (C).

Time course of

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Figure. 2: Aggregates formation by aniline exposed to culture and CLSM images of the aggregates. SEM micrograph of the aggregates of strain JA2 (A) and magnified aggregates showing EPS encased cells (B). CLSM phase image of aggregates (C), exopolysaccharides staining-calcofluor white(D), nucleic acids staining by acridine orange (E), proteins staining

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by rhodamine esters(F), Lipids staining by Nile red (G) and merged image (H).

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Figure. 3: EPS formation by strain JA2 to different toxic compounds (A). Pie chart showing

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envelope associated and folding sorting and degradation related proteins detected in aniline exposed cells (B). Bar-graph showing the differentially expressed proteins related to folding

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sorting and enveloped associated proteins (C).

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Figure. 4: Bar graph of upregulated envelope associated proteins to aniline exposure in strain JA2. Data mean ± standard deviation of three independent experiments. The dotted line

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represents 1.5 fold thresh hold of up-regulated proteins.

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Figure. 5: Homology models of up-regulated proteins and EPS formation to envelope

stressors. Superimposed monomeric structure of CpxP of E.coli in blue and model CpxP of

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strain JA2 in red (A), Dimeric model of putative CpxP of strain JA2 generated by

ClusPro(B), superimposed monomeric structure of CpxA of E.coli in blue and model of CpxAof strain JA2 in red (C). Formation of EPS by strain JA2 to known envelope stressors

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(D). Data mean ± standard deviation of three independent experiments.

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Figure. 6: Proposed model depicting the aniline exposure leading to envelope stress and

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triggering the EPS formation in strain JA2. The shaded region represents the putative

CpxARP signaling pathway sensing envelope assault. ? represents unknown signaling events

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and the metabolic process involved in EPS formation to aniline exposure.

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