HosA, a MarR Family Transcriptional Regulator ... - ACS Publications

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HosA, a MarR Family Transcriptional Regulator, Represses Nonoxidative Hydroxyarylic Acid Decarboxylase Operon and Is Modulated by 4‑Hydroxybenzoic Acid Ajit Roy†,‡ and Akash Ranjan*,† †

Computational and Functional Genomics Group, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana 500001, India ‡ Graduate studies, Manipal University, Manipal 576104, India S Supporting Information *

ABSTRACT: Members of the Multiple antibiotic resistance Regulator (MarR) family of DNA binding proteins regulate transcription of a wide array of genes required for virulence and pathogenicity of bacteria. The present study reports the molecular characterization of HosA (Homologue of SlyA), a MarR protein, with respect to its target gene, DNA recognition motif, and nature of its ligand. Through a comparative genomics approach, we demonstrate that hosA is in synteny with nonoxidative hydroxyarylic acid decarboxylase (HAD) operon and is present exclusively within the mutS-rpoS polymorphic region in nine different genera of Enterobacteriaceae family. Using molecular biology and biochemical approach, we demonstrate that HosA binds to a palindromic sequence downstream to the transcription start site of divergently transcribed nonoxidative HAD operon and represses its expression. Furthermore, in silico analysis showed that the recognition motif for HosA is highly conserved in the upstream region of divergently transcribed operon in different genera of Enterobacteriaceae family. A systematic chemical search for the physiological ligand revealed that 4-hydroxybenzoic acid (4-HBA) interacts with HosA and derepresses HosA mediated repression of the nonoxidative HAD operon. Based on our study, we propose a model for molecular mechanism underlying the regulation of nonoxidative HAD operon by HosA in Enterobacteriaceae family.

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INTRODUCTION Environmental cues are sensed by bacteria directly or indirectly through proteins, mainly by regulators, to adapt and survive in a particular condition. Different regulators act in highly coordinated ways that enable the bacteria to better fit in an ever challenging environment. MarR, an important family of regulatory proteins in prokaryotes has been shown to regulate diverse cellular and physiological processes. MarR, the eponymous member of this family acts as a repressor of marRAB operon, which confers resistance to antibiotics, household disinfectants, and organic solvents in E. coli.1−5 Different members of MarR family have also been shown to control bacterial response to oxidative stress,6−11 virulence determinants expression,12−17 multidrug exposure,18−24 transition metal transport,25 and catabolism of aromatic xenobiotics.26−32 Members of the MarR family generally act as repressors, although some proteins of this family operate as activators. Ligand-responsive transcriptional repressors of MarR family are modulated by small molecule ligands, usually of aromatic nature. Ligands cause the derepression through conformational change in the regulator, induced either by direct association with a small molecule ligand33,34 or by signal molecules produced on exposure of ligands that in-turn decreases the affinity of the regulator with its cognate binding sequence.35 In © XXXX American Chemical Society

a subset of regulators within the MarR family, attenuation of DNA binding occur through the formation of disulfide bond in the regulator with response to different oxidative stresses.6−11 Different aromatic molecules present in the environment show structural resemblance to ligands of MarR family.36 Hydroxyarylic acids like ferulic acid, gallic acid, syringic acid, vanillic acid, and 4-hydroxy benzoic acid are the common plant derived molecules present in the environment that have been shown to act as natural antimicrobials.37−39 Hydroxyarylic acid decarboxylases (HADs) are broadly categorized as either oxidative or nonoxidative type. Compared to the oxidative, nonoxidative HADs are less studied in prokaryotes, but are required to negate the toxic effect of aromatic acids and their derivatives in anoxic environment.40−43 Phylogenetic analysis and enzymatic assays suggest a nonoxidative HAD gene cluster is present in different members of Enterobacteriaceae like Salmonella, Shigella, Escherichia, and Klebsiella. In pathogenic strains of E. coli, a gene cluster containing three genes: ecdB, ecdC, and ecdD (renamed for pad1, yclC, and yclD, respectively) present in the mutS-rpoS polymorphic region have been shown hydroxyarylic acid Received: October 26, 2015 Revised: January 14, 2016

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DOI: 10.1021/acs.biochem.5b01163 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry Table 1. Bacterial Strains and Plasmids strain or plasmid Escherichia coli K12 DH5α BL21(DE3) MC4100 MC41AA MC41AB MC41AR E. coli strain UMN026 plasmids pET21b (+) pEThosA pHYD3025 pHYDhosA pETslyA pCL1920 pMU575 pMUPecdB pMUPslyA pCLPhosA pCLPslyA pMUPecdBM pMUPecd(M1-M6)

relevant genotype or phenotype F− endA1 gln V44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ (lacZYA-argF) U169, hsdR17 (rK− mK+), λ− F− OmpT gal dcm lon hsdSB (rB− mB−) λ (DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) F± araD319 D (argF-lac) U169 relA1¯bB5301 deoC1 ptsF25 rbsR MC4100 derivative (ΔaaeA), Kmr MC4100 derivative (ΔaaeB), Kmr MC4100 derivative (ΔaaeR), Kmr Escherichia coli O17:K52:H18 UMN026 (ExPEC) (ATCCRBAA1161)

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6x-histidine tag protein expression vector hosA gene cloned in pET21b (+) vector derivative of the expression vector pTrc99A hosA gene cloned in pHYD3025 vector slyA gene cloned in pET21b (+) vector low copy number vector for regulated low-level expression of cloned genes promoterless low copy number reporter vector PecdB (216 bp) containing the intergenic region between the genes hosA and ecdB cloned in pMU575 vector Upstream region of slyA (PslyA, 302 bp) cloned in pMU575 vector hosA gene along with its upstream region (166 bp) cloned in pCL1920 vector slyA gene along with its upstream region (250 bp) cloned in pCL1920 PecdB (216 bp) containing the mutations in −10 region cloned in pMU575 PecdB (216 bp) containing different HosA binding site mutants (M1-M6) cloned in pMU575

Novagen this study ref 90 This study This study ref 58 ref 59 this study this study this study this study this study this study

decarboxylase activity, when expressed in heterologous host.42 Despite having different enzymatic studies on nonoxidative HADs in Enterobacteriaceae, a detailed understanding of its conditional expression and regulation is still lacking. The present study reports the regulation of nonoxidative HAD gene cluster by a divergently transcribed MarR family protein; HosA in uropathogenic E. coli strain UMN026. HosA was previously renamed as “Homologue of SlyA” and has been shown as a regulator of temperature dependent motility in enteropathogenic E. coli strain E2348/69,44,45 but an understanding of its DNA recognition site and ligand modulating its activity is still lacking. By utilizing comparative genomics, electrophoretic mobility shift assay (EMSA), site-directed mutagenesis, and transcription assays, the regulatory mechanism of HosA and its modulation by 4-hydroxybenzoic acid (4HBA) was elucidated. This study explicates the importance of the constant 2.9 kb sequence (often referred to as “segment O”),46 present within the mutS-rpoS polymorphic region in the physiology of different strains of bacteria in Enterobacteriaceae family.

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and ethanol were used as nonaromatic ligand molecules. The different aromatic and nonaromatic molecules either dissolved in ethanol or water were used at varying range of concentrations (50 μM to 10 mM) in media or in reaction mixture. DNA Manipulations, Protein Expression, and Purification. Standard methods for DNA manipulation like genomic and plasmid DNA isolation, digestion with restriction enzymes, ligation with T4 DNA ligase, and transformation were carried out as described elsewhere.47 DNA fragments were either purified by PCR purification kit (Invitrogen) or by nucleotide purification kit (Qiagen) based on their length. Commercially synthesized oligonucleotides were procured from Europhins Genomics (Bangalore, India). Nucleotide sequences of different clones were verified by dideoxy chain termination method.48 Site-directed mutagenesis of PecdB were performed by overlap extension PCR49 using different palindrome or promoter specific primers. 4-HBA exporter knockouts of E. coli strain MC4100 were generated by P1 transduction as described elsewhere.47 Different primers used in this study are listed in Supporting Information Table 3. Primers used for generating different constructs that are used in β-Galactosidase assay are given in Supporting Information Table 2. The coding region of hosA (ECUMN_3063) and slyA (ECUMN_1933) were amplified by PCR using primer pairs: hosA FP, hosA RP and slyA FP, slyA RP, respectively, using genomic DNA of E. coli strain UMN026 as a template. Amplified PCR fragments were ligated between the NdeI and XhoI restriction sites in pET21b (+) expression vector (Novagen) to generate vectors pEThosA and pETslyA, respectively. The clones were verified by sequencing and were used for overexpression in E. coli strain BL21 (DE3). The different proteins were purified from cultures after induction with 1 mM IPTG at 37 °C, using Ni-NTA chromatography. Eluted fractions enriched with HosA were pooled and were dialyzed against Tris-Cl buffer (10 mM, pH 8.0). The proteins

MATERIALS AND METHODS

Bacterial Strains, Plasmids, and Growth Conditions. The bacterial strains and plasmids used in this study are listed in Table 1. Different E. coli strains were grown with continuous stirring at 37 °C either in Luria−Bertani (LB) or glucose minimal A media,47 depending on the experimental procedures. The antibiotics used in this study were at following concentrations: 100 μg/mL ampicillin, 60 μg/mL trimethoprim and 50 μg/mL spectinomycin. Different aromatic molecules that were tested as putative ligands of HosA in this study, that is, 4-hydroxybenzoic acid (4-HBA), ferulic acid, cinnamic acid, salicylic acid, acetyl salicylate, methyl 4-hydroxybenzoic acid (4MHBA), vanilic acid, 2,5-dihyroxybenzoic acid (2,5-DHBA), 3hydroxybenzoic acid (3-HBA), 3,4-dihydroxybenzoic acid (3,4DHBA), and phenol, were purchased from Sigma. Acetic acid B


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Biochemistry

and exposed to phosphorimager screen, and radioactivity was detected using image scanner FLA9000 (Fujifilm). DNase I Footprinting. An amount of 20 ng of uniquely radiolabeled PecdB was incubated with increasing molar concentrations of HosA in binding buffer (100 mM HEPES pH 8.0, 200 mM KCl, 15 mM MgCl2, 0.1 M dithiothreitol, 10% NP40, and 50% glycerol) for 30 min. The reaction mix was then treated with 0.1 μg of DNase I solution (Roche) for 45 s, and the reaction was terminated using stop solution (1% SDS, 200 mM NaCl and 20 mM EDTA). The reaction mix was phenol-chloroform extracted, ethanol precipitated, and airdried. The samples were dissolved in formamide dye mix, before being electrophoresed in 6% denaturing urea polyacrylamide gel. A dideoxy sequencing ladder corresponding to the coding strand was also run along with the samples. Primer Extension. Primer extension analysis was performed to determine the transcription start site of nonoxidative HAD operon. RNA was isolated using RNeasy Mini kit (Qiagen) from mid log phase grown cultures of E. coli strains UMN026 and MC4100, after treatment with 4-HBA (5 mM), according to the manufacturer’s instructions. AMV reverse transcriptase was used to transcribe cDNA from RNA according to the manufacturer’s instructions (Promega) using radiolabeled primer PEecdB. Primer extension products were electrophoresed in 6% denaturing urea polyacrylamide gel along with sequencing ladder generated from vector pMUPecdB by Sanger’s chain termination method using the radiolabeled primer PEecdB. Semiquantitative PCR. RNA was isolated from uropathogenic E. coli strain UMN026 grown in LB medium that was treated either with 4-HBA (5 mM, dissolved in ethanol) or with equal volume of ethanol using RNeasy mini kit (Qiagen). Equal amounts of RNA from both the samples were used as starting material to transcribe cDNAs by primers IG1RP, IG2RP, HosARTRP, and 16SRP using SuperScript III reverse transcriptase, according to the manufacturer’s instructions (Life Technologies). Equal amounts of cDNAs thus formed were utilized as templates for PCR amplification using the following primers: IG1FP and IG1RP for IG1 (200 bp), IG2FP and IG2RP for IG2 (200 bp), HosARTFP and HosARTRP for internal region of hosA (178 bp), and 16SFP and 16SRP for 16S rRNA internal region (542 bp), for 24 cycles in thermocycler (Bio-Rad) and were analyzed using 1% agarose gel. Bioinformatics Analysis. Protein sequence of HosA (accession no. YP_002413753.1) was used as a query in SyntTax web server51 for synteny analysis within the Enterobacteriaceae family, as per the instructions given by the author. Sequences with highest identity to HosA and located within the mutS-rpoS polymorphic region were analyzed for the presence of a palindromic sequence in their upstream region. Intergenic regions between hosA and nonoxidative HAD operon in different bacterial strains were extracted manually (Supporting Information Table 1) and were used for motif identification using Gibbs Motif Sampler.52 The predicted palindromic sites were utilized in WebLogo program53 to generate sequence logo for possible consensus site for HosA binding. Espirit web server (http://espript.ibcp.fr/ESPript/ESPript/) was used for alignment of upstream sequences of nonoxidative HAD operon in the selected members of Enterobacteriaceae family.

were concentrated using 10 kDa cutoff concentrator (Amicon) and were analyzed for purity through 12% SDS-PAGE. The concentration of the proteins was measured using Bradford assay.50 Gel Exclusion Chromatography. The native molecular weight of HosA was determined by gel exclusion chromatography using Sephacryl S-200 resin. The column containing Sephacryl S-200 resin was equilibrated with Tris-Cl buffer (10 mM, pH 8.0) at a flow rate of 1 mL/min. The protein was eluted from the column as a single peak at the same flow rate. To determine the molecular weight of HosA in solution, the column was calibrated with the following molecular weight standards (GE Healthcare Life Science): Ribonuclease (13.7 kDa), Carbonic anhydrase (29 kDa), Ovalbumin (44 kDa) and Conalbumin (75 kDa). Blue dextran 2000 was used to determine the void volume of the column. The molecular weight of HosA was calculated from the plot between Kav (calculated using the formula: Kav = (Ve − V0/Vc − V0), where Ve is the elution volume of a protein, V0 is the column void volume, and Vc is the column geometric volume) and molecular weight of different standards. β-Galactosidase Assay. β-Galactosidase assay was performed by using strains carrying various plasmids in E. coli strain MC4100 background, and the activity was expressed in Miller units. Overnight grown cultures of the transformed strains were diluted to 1/20 in glucose minimal A media supplemented with different antibiotics and when required different aromatic compounds were added to the cultures. Different mid log phase grown cultures were used to determine β-Galactosidase activity as described before.47 MacConkey agar containing 2% lactose was used for qualitative β-Galactosidase assay. All the experiments were replicated three times, and Student’s t-test was performed to analyze the statistical significance for the data showing relative expression of promoter activity. P < 0.05 was considered to be significant. Electrophoretic Mobility Shift Assay (EMSA). EMSA was carried out to demonstrate DNA−protein interaction. The complementary DNA fragments of PecdB (F1−F7), Fc, Fc (A), Fc (B), U, and mutated palindromic fragments (M1−M7) (as given in Supporting Information Table 2) were synthesized commercially. Then 50 pmol of single strand of each fragment was radiolabeled at the 5′-end with γP32 dATP (3000 Ci mmol−1) (BRIT, India) using T4 PNK enzyme, as per the manufacturer’s instructions (NEB), and was purified using a nucleotide purification kit (Qiagen). The double stranded DNA of each radiolabeled fragment was formed by addition of 50fold molar excess of unlabeled complementary fragment to the labeled mix, heated at 95 °C for 10 min, followed by gradual cooling to room temperature. The DNA fragment PecdB (216 bp) was amplified by primers PecdBFP and PecdBRP, using vector pMUPecdB as a template. The amplified fragment was radiolabeled and purified through gel extraction. Then 10 ng (3.5 nM) of radiolabeled double stranded DNA fragments was incubated with different molar concentrations of purified Histagged HosA in binding buffer (100 mM HEPES pH 8.0, 200 mM KCl, 15 mM MgCl2, 0.1 M dithiothreitol, 10% NP40 and 50% glycerol). For in vitro effect of 4-HBA on HosA-DNA interaction, different molar concentration of 4-HBA was added to the binding buffer containg radiolabeled DNA fragment and HosA. Salmon sperm DNA (10 μg/mL) was used as nonspecific competitor. Samples were electrophoresed in 6% native polyacrylamide gel at constant 10 V/cm for 2−3 h, dried, C


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Biochemistry

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RESULTS Comparative Genomics Analysis Revealed a Syntenic Relationship between hosA and Nonoxidative HAD Gene Cluster. The region between the gene mutS and rpoS is highly polymorphic in Enterobacteriaceae family. Previous studies have shown the presence of hosA, encoding a MarR family protein, in the mutS-rpoS polymorphic region. The presence of hosA was shown to vary within the clinical isolates of Escherichia and Salmonella and was reported in very few sequenced members of Enterobacteriaceae.46,54−57 To determine its presence and location, hosA was searched using BLAST in all the sequenced bacteria included in the Enterobacteriaceae family. Interestingly, hosA was found to be present along with a nonoxidative HAD gene cluster in the sequenced genomes of nine genera of Enterobacteriaceae family. The relative co-occurrence of hosA and nonoxidative HAD gene cluster was analyzed. It was observed that despite having a difference in relative co-occurrence in each genus, the presence of both was conserved in more than half of the total strains analyzed (Figure 1B). To quantify the co-occurrence, synteny analysis of hosA was performed for all the members of Enterobacteriaceae using “SyntTax” web server.51 SyntTax identified that the gene cluster coding for the nonoxidative HAD gene cluster is in synteny with hosA in all the sequenced strains of Enterobacteriaceae retaining hosA (Figure 1A). HosA Acts as a Repressor of Nonoxidative HAD Gene Cluster. The syntenic relationship of hosA and nonoxidative HAD gene cluster prompted us to check the possible regulation of the gene cluster by HosA. Due to difficulty in manipulating the pathogenic E. coli strain UMN026, we have performed all the in vivo experiments in E. coli strain MC4100. In order to determine the in vivo effect of HosA on nonoxidative HAD gene cluster, β-Galactosidase assay was performed. For this, two compatible replicative vectors, one pCLPhosA, a derivative of low copy number protein expression vector pCL192058 containing hosA gene under the control of its natural promoter and the other, pMUPecdB, a derivative of promoterless reporter vector pMU57559 with cloned 216 base pair (bp) DNA fragment containing the intergenic region between the gene hosA and ecdB (named as “ PecdB” and will be referred as the promoter of nonoxidative HAD gene cluster throughout this manuscript) upstream to β-Galactosidase gene, were used. Two plasmids pCL1920 and pMU575, containing either their respective cloned fragments or plasmid alone were cotransformed in different combinations in E. coli strain MC4100. The transformed cells were grown on glucose minimal A medium, before being analyzed for β-Galactosidase activity. A cotransformed strain containing reporter vector pMUPecdB showed nearly 30-fold decrease in β-Galactosidase activity in the presence of vector pCLPhosA, when compared to the presence pCL1920 vector (Figure 1C). However, neither the closest homologue of HosA-SlyA-had any effect on PecdB activity, nor did HosA have any effect on the promoter of slyAPslyA (Figures 1D and E). Thus, it was concluded that HosA and not SlyA represses the promoter activity of nonoxidative HAD operon in heterologous strain of E. coli. HosA Binds to PecdB in Vitro. The hosA gene was amplified using genomic DNA of E. coli strain UMN026 as a template, cloned downstream to T7 promoter in expression vector and overexpressed. The His6-tagged HosA was purified to apparent homogeneity (Figure 2A). Since most members of MarR family exist as dimers, size exclusion chromatography was

Figure 1. HosA exist in syntenic relationship with nonoxidative HAD operon and negatively regulates its expression. (A) Schematic representation showing synteny of hosA and nonoxidative HAD gene cluster (within rectangular box) from nine genera within Enterobacteriaceae family. (B) Relative co-occurrence of the gene hosA and nonoxidative HAD gene cluster within nine genera in Enterobacteriaceae family (where gray and white bars represent the total number of sequenced genome and number of genome in which the hosA and nonoxidative HAD gene cluster co-occur in a particular genera, respectively). (C) β-Galactosidase assay showing the promoter activity of PecdB in the presence and absence of HosA and (D) nonspecific protein SlyA in heterologous E. coli strain MC4100. ***P < 0.0001 between promoter activities in glucose minimal A media. (E) β-Galactosidase assay showing the promoter activity of PslyA in presence and absence of HosA.

used to check the oligomeric status of purified HosA protein. The molecular weight of HosA calculated from the plot between Kav and molecular weight of different standards (Figure 2B) was found to be 33.6 kDa, a value close to theoretical molecular weight of the HosA dimer (34.6 kDa) including the histidine tag, suggesting that HosA in solution exists as dimer. Since HosA was shown to repress the promoter activity of PecdB in vivo, the direct interaction of HosA was checked with the radiolabeled PecdB through EMSA. A band shift of radiolabeled PecdB was observed with increasing concentrations D


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Biochemistry

Figure 2. Purification of HosA and its interaction with PecdB. (A) SDS-PAGE analysis of purified His-tagged HosA. Lane 1: Protein molecular weight marker (14.3−97.4 kDa). Lane 2: Purified His-tagged HosA protein (∼17 kDa). (B) Gel filtration chromatographic analysis of purified HosA. The standard curve was plotted between Kav versus molecular weight of different standards (gray squares). Kav of HosA is shown as a dark circle. (C) Genomic organization of ecdB, ecdC, and ecdD of nonoxidative HAD operon and hosA (in upper case italic letters). Promoter of nonoxidative HAD gene cluster; PecdB (216 bp, shown in thick black line along with its genomic locus). Seven overlapping fragments (F1−F7) of PecdB are shown by straight lines. The rectangular box (in gray) showing 25 bp overlapping sequence between F2 and F3 fragments. (D) In vitro binding of HosA to radiolabeled PecdB (PecdB(R)) through EMSA. Lane 1: PecdB(R) with no protein. Lanes 2−11: PecdB(R) incubated with increasing concentrations of HosA (10−100 nM). Lane 12: PecdB(R) incubated with HosA (100 nM) along with either 50-fold molar specific cold probe or (Lane 13) with nonspecific cold probe (PslyA(C)). Lane 14: Nonspecific protein SlyA incubated with PecdB(R). Lane 15: Nonspecific radiolabeled probe PslyA (PslyA(R)) incubated with HosA (100 nM). (E) EMSA of overlapping fragments of PecdB incubated with HosA. Lane1: Radiolabeled free probe (F1). Lanes 2−9: HosA incubated with radiolabeled fragments of PecdB (F1−F7). Free probe (FP) and DNA−protein binary complex (BC) are shown by arrows.

HosA Recognizes a Perfect Palindromic Sequence within PecdB. The β-Galactosidase assay followed by EMSA demonstrated that HosA negatively regulates nonoxidative HAD gene cluster by directly interacting with its promoter region. Therefore, the exact nature of binding site recognized by HosA within PecdB was elucidated at the molecular level. Seven DNA fragments (F1−F7) of PecdB containing 25 bp overlapping region were generated, radiolabeled, and used to perform EMSAs. The position of different fragments within

of HosA from 10 to 100 nM. The specificity of binding of HosA with PecdB was confirmed by addition of 50-fold molar excess of specific cold probe, which abolished the binary complex of protein and DNA while, a 50-fold molar excess of nonspecific cold probe (PslyA) had no effect. Neither a band shift was observed by incubating HosA with nonspecific radiolabeled fragment (PslyA), nor could a negative control, SlyA, retard the mobility of radiolabeled PecdB (Figure 2D). E


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Biochemistry PecdB is depicted in Figure 2C. Together, they cover the nucleotide sequence from 3 169 666 to 3 169 811 in the genome of E. coli strain UMN026. The purified HosA showed binding only to fragments F2 and F3, but no band shift was observed with fragments F1, F4, F5, F6, and F7, suggesting a DNA recognition site for HosA is present within the overlapping fragments F2 and F3 (Figure 2E). The putative binding site of HosA was limited to 25 bp overlapping sequence of fragments F2 and F3 covering the DNA sequence from 3 169 725 to 3 169 749 in the genome of E. coli strain UMN026 as observed by means of previous experiment. A 14 bp palindrome was identified within the 25 bp overlapping fragment using the program EMBOSS Einverted v1.5.60 Further, to validate the palindrome as the true binding site of HosA, three different fragments (Fc, Fc (A), and Fc (B)) were generated from the overlapping 25 bp region with and without the intact palindrome (Figure 3A). Two fragments Fc and Fc (A) containing the intact palindrome showed band shift, while the fragment Fc (B) lacking the intact palindrome did not show any band shift, when incubated with HosA (Figure 3B). In addition, DNase I footprinting assay of uniquely radiolabeled PecdB demonstrated a clear protected region covering the DNA sequence from 3 169 735 to 3 169 748 within PecdB with increasing concentrations of HosA (Figure 3C), which was in accordance with the palindromic site obtained through EMSA. The above experiments showed a perfect palindrome: 5′GTTCGTATACGAAC-3′ present within the PecdB as the recognition site for HosA. To elucidate the importance of each base within the palindrome recognized by HosA, 25 bp probes containing specific point mutations were generated in the respective sites on each half of the palindrome (Figure 4A) and were utilized in binding studies. EMSA of radiolabeled probes incubated with HosA showed a band-shift in the following probes: Unaltered (U), M2, M3, M5, and M7, while probes M1 and M4 showed no binding and the probe M6 showed partial binding. This indicated that the specific bases mutated in M1, M4, and M6 are critical for HosA binding (Figure 4B). To validate the role of specific bases (mutated in probes M1, M4 and M6) on binding of HosA under in vivo condition, individual point mutations were introduced in PecdB and cloned in vector pMU575. The effect of HosA on palindrome mutant derivatives of PecdB was checked in cotransformed strains of E. coli containing vector pCLPhosA along with different palindrome mutant derivatives of vector pMUPecdB (pMUPecd(M1-M7)) through β-galactosidase assay. Strains containing the following palindrome specific mutant derivatives of vector pMUPecdB: pMUPecdM2, pMUPecdM3, pMUPecdM5, and pMUPecdM7 did not show any changes in promoter activity (Supporting Information Figure 1B), while a nearly 20-fold higher expression of promoter activity was observed in palindrome mutant derivatives pMUPecdM1, pMUPecdM4, and pMUPecdM6 in the presence of HosA, when compared to native promoter (Figure 4C). This indicated binding of HosA with palindrome specific mutants (M1, M4, and M6) is defective under in vivo condition, which is in support to the EMSA studies demonstrating that the specific bases within the palindrome are critical for interaction of HosA to PecdB. Thus, it was concluded that the HosA mediated repression of PecdB is through the direct interaction of HosA with a palindromic sequence within the promoter of nonoxidative HAD operon.

Figure 3. Identification of HosA recognition site. (A) Three fragments (Fc, Fc (A), and Fc (B)) were generated from the overlapping region of fragments F2 and F3 (marked in rectangular box). Fragments Fc and Fc (A) contains the intact palindromic region (marked as horizontal arrows), while the fragment Fc (B) has incomplete palindrome. (B) EMSA of fragments (Fc, Fc (A), and Fc (B)) incubated with constant concentration of HosA, free probe (FP), and DNA−protein binary complex (BC) are shown by arrows. (Gel picture is given in exponential scale.) (C) DNase I footprinting assay of radiolabeled PecdB in the presence of HosA. Lanes G, A, T, and C: The sequencing ladder generated using dideoxy nucleotides ddG, ddA, ddT, and ddC. Lane 1: DNase I treated uniquely radiolabeled PecdB. Lanes 2−4: DNase I treated radiolabeled PecdB that was incubated with increasing molar concentrations of HosA (25−100 nM). DNase I protected region within PecdB is shown by arrows within rectangular box. Arrows outside the rectangular box corresponds to flanking sequences on both side of the palindrome. (Gel picture is given in exponential scale.)

Identification of Binding Site Consensus for HosA. In vivo and in vitro results identified a palindrome of 14 bp length within PecdB to be crucial for HosA binding. As previously mentioned, organization of nonoxidative HAD gene cluster is conserved in nine different genera of Enterobacteriaceae family therefore, intergenic regions of hosA and nonoxidative HAD gene cluster were aligned. It was observed that despite having F


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Biochemistry

HosA Binds Downstream to Transcription Start Site of Nonoxidative HAD Operon. In order to understand the mechanism of repression mediated by HosA, the transcription origin of nonoxidative HAD gene cluster was mapped. Primer extension assay was performed using RNA isolated from 4-HBA (a molecule identified as inducer of HAD operon in this work and reported in the following section) treated culture of pathogenic E. coli strain UMN026 (as repeated attempts for obtaining the transcript signal were unsuccessful using RNA from the culture without 4-HBA treatment). Primer extension analysis suggested a transcription start site originating 33 bp upstream to translation start site (Figure 6A and B). Six bases further upstream, a potential −10 site (TATTTT), separated by 17 bp from a putative −35 site (TTGCCG) was observed. To validate the transcription start site observed through primer extension assay, two specific point mutations were introduced in the conserved positions in the −10 site (i.e., wild type −10 site (TATTTT) changed to mutant (TCTTTC)) within PecdB and cloned upstream to β-galactosidase gene in vector pMU575 to obtain vector pMUPecdBM. E. coli strain MC4100 transformed with pMUPecdBM showed a drastic decrease in promoter activity as compared to strain transformed with pMUPecdB, as observed qualitatively in MacConkey agar plate (Figure 6C) and quantitatively through β-Galactosidase assay (Figure 6D). The HosA binding site as delineated previously through DNase I footprinting analysis was mapped two bases downstream to the transcription start site of nonoxidative HAD gene cluster (Figure 6A). 4-HBA Acts As a Physiological Ligand for HosA. Phenolic small molecules have been shown to bind to MarR proteins and modulate its activity.34 In a quest to identify the physiological ligand of HosA, different aromatic and nonaromatic compounds, including the prototypical modulator sodium salicylate were tested under in vivo condition. One mM concentrations of each aromatic and nonaromatic molecules were added separately to the cultures cotransformed with vectors pMUPecdB and pCLPhosA and their ability to modulate HosA mediated repressions were tested through βGalactosidase assay. Out of the different molecules tested, the culture treated with 4-HBA was observed to derepress HosA mediated repression of PecdB in heterologous E. coli strain MC4100. More than 6-fold increase in promoter activity of PecdB was observed in treated, when compared to the untreated culture transformed with both the vectors. On the contrary, promoter activity of PecdB did not show any significant change in 4-HBA treated cultures which was cotransformed with vectors pMUPecdB and pCL1920 (Figure 7A and B), indicating 4-HBA mediated induction of PecdB activity is through the derepression of HosA mediated repression. When tested with increasing concentration, derepression of HosA mediated repression of PecdB was shown to be elevated. A gradual increase in β-Galactosidase activity values from 2- to 8-fold was observed, on increasing the 4-HBA concentrations from 50 μM to 10 mM (Figure 7C) in a strain cotransformed with plasmids pMUPecdB and pCLPhosA. To further verify that intracellular increase in 4-HBA concentration modulates the repression caused by HosA, plasmids pMUPecdB and pCLPhosA were cotransformed in different strains of E. coli (MC41AA, MC41AB, and MC41AR) lacking genes (aaeA, aaeB, and aaeR) that are involved in 4HBA export.61 Addition of 1 mM of 4-HBA to different cultures with specific exporter gene knockout that were

Figure 4. Identification of critical residues within palindrome required for HosA binding. (A) Schematic representation of probes (25 bp) containing the unaltered palindrome (probe U, shown within the rectangular box) and mutated palindromes (M1−M7) containing point mutations (purine changed to pyrimidine) at specific position within the palindrome (shown within the square boxes). (B) EMSA of radiolabeled probes [unaltered (U) and mutated (M1−M7)] that were incubated with HosA. Free probe (FP) and DNA−protein binary complex (BC) are shown by arrows. (C) β-Galactosidase assay showing the effect of palindromic mutations within PecdB on binding of HosA.

differences, conservation in a locus corresponding to palindromic recognition site of HosA is present in all intergenic regions (Figure 5A). To find a consensus for binding site of HosA, possible palindromic sites were searched among the intergenic regions of hosA and nonoxidative HAD gene cluster in all the bacteria showing the synteny for both (Supporting Information Table 1), using Gibbs motif sampler.52 A single palindrome, similar to the site obtained from DNase I footprinting assay of PecdB was identified within different intergenic regions by Gibbs motif sampler. Different variant of HosA binding site, both predicted through Gibbs motif sampler and obtained from in vitro mutagenesis experiment were used to generate a possible consensus binding sequence for HosA using WebLogo program (Figure 5B).53 G


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Figure 5. In silico binding site analysis for HosA. (A) Multiple sequence alignment of the intergenic regions between hosA and first gene of the nonoxidative HAD operon (given by flanking gene names) from selected members of Enterobacteriaceae. The conserved palindromic region that corresponds to HosA recognition site is shown within rectangular box. (B) Binding site consensus of HosA generated through WebLogo program. The relative frequencies of bases at different position are shown by the height of the nucleotides.

cotransformed with both the vectors showed an additional 1.5to 2-fold increase in derepression, when compared to parent E. coli strain MC4100, indicating a rise in intracellular 4-HBA concentration in exporter knockout strains indeed caused the higher derepression of HosA mediated repression (Figure 7D). To validate the direct effect of 4-HBA on interaction of HosA with probe (U), EMSA was performed. As expected, a decrease in protein−DNA binary complex was observed on increasing the concentration of 4-HBA from 0.5 mM to 5 mM, which indicates that 4-HBA inhibits the interaction of HosA with its cognate site in a concentration dependent manner (Figure 8C).

In vivo and in vitro studies identified 4-HBA interacts with HosA and disrupts its binding to PecdB, and thus causing the derepression. These results emboldened us to determine the effect of 4-HBA on the transcript level of nonoxidative HAD gene cluster. The addition of 4-HBA to glucose minimal A grown culture of pathogenic E. coli strain UMN026 resulted in elevated transcript levels of intergenic regions IG1 (region between the gene ecdB and ecdC) and IG2 (region between the gene ecdC and ecdD), when compared to the untreated culture, as shown by semiquantitative PCR (Figure 8A and B). Increase in transcript level of nonoxidative HAD operon upon exposure H


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Figure 6. Identification of transcription origin of nonoxidative HAD operon. (A) Nucleotide sequence of PecdB containing intergenic region (marked in upper case) and genic region (marked in lower case) of the genes ecdB and hosA, respectively. The −10 and −35 regions are underlined. Transcription start site (+1) and HosA binding site (in italic) are shown by an arrow and within rectangular box, respectively. (B) Primer extension analysis of nonoxidative HAD operon. Lanes G, A, T, and C: Sequencing ladder that was generated using dideoxy nucleotides ddG, ddA, ddT, and ddC, respectively. Lane 1: Primer extension product generated using RNA either from logarithmic phase of pathogenic E. coli strain UMN026, or (lane 2) MC4100, treated with 4-HBA (5 mM). (C) MacConkey agar (containing 2% lactose) grown cultures of E. coli strain MC4100 transformed with vectors pMUPecdB (shown by pink colored growth) and pMUPecdBM (without colored growth). (D) Quantitative βGalactosidase assay showing the activity of cloned wild type (pMUPecdB) and mutated (pMUPecdBM) promoter transformed in E. coli strain MC4100.

Figure 7. Ligand identification of HosA. (A) β-Galactosidase assay showing the ability of different aromatic and nonaromatic compounds to cause the derepression of HosA mediated repression of PecdB. (B) β-Galactosidase assay showing the effect of 4-HBA on the promoter activity of PecdB, with and without the presence of HosA. (C) βGalactosidase assay showing the effect of varying concentration of 4HBA (50 μM-10 mM) on derepression of HosA mediated repression of PecdB. (D) Effect of 4-HBA (1 mM) on HosA mediated repression of PecdB in different 4-HBA exporter knockout strains as shown through β-Galactosidase assay (T, cultures with 4-HBA treatment; U/ UT, cultures without 4-HBA treatment). ***P < 0.0001, **P < 0.001, and *P < 0.01 between promoter activities of 4-HBA treated and untreated cells in glucose minimal A media.

HosA belongs to MarR family of transcription factors. The members of this family are associated with the regulation of genes that are involved in antibiotic resistance and detoxification of noxious compounds. The close proximity of hosA with HAD operon and the regulation of this operon by HosA demonstrates a prototypical regulatory functions shown by MarR family proteins. Despite relative abundance of HosA in the pathogenic strains of Enterobacteriaceae and its characterization as a different regulatory protein than SlyA, a detailed study of HosA regulon is still lacking. The first two genes of the HAD operon, ecdB and ecdC, show high similarity with the paralogous isofunctonal genes ubiX and ubiD of E. coli and PAD1 and FDC of Saccharomyces cerevisiae.63 However, the regulation of either ubiX and ubiD of E. coli or the homologous genes (PAD1 and FDC) of S.cerevisiae is not well-studied. This is the first study that ascribes HosA as a transcriptional regulator of HAD operon. Although, the role of HAD operon in antibiotic resistance has not been previously explored, however its role in decarboxylation and detoxification of antimicrobial benzoic acids has been substantiated from different organisms.42 For instance, in S. cerevisiae, the product of the orthologus genes of ecdB and ecdC, that is, PAD1 and FDC (yclC) have been

to 4-HBA was in accordance with increase in derepression of HosA mediated repression on exposure to 4-HBA in heterologous E. coli strain MC4100 and thereby confirmed 4HBA as a physiological ligand of HosA.

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DISCUSSION In the Enterobacteriaceae family, the region between mutS and rpoS genes has been observed to be highly polymorphic with a size that ranges from 88 bp in Yersinia to >12 000 bp in Salmonella.57 In E. coli, this region varies in different pathogenic strains. Through comparative genomics analysis a syntenic relationship between a nonoxidative HAD operon and hosA in the mutS-rpoS region was observed in nine different genera of the sequenced members of Enterobacteriaceae family. This locus has been suggested to be acquired through lateral gene transfer as a single transcriptional unit and protends to follow selfish operon model of gene clustering.46 Presence of HosA within this locus explicates the requirement of the regulators often associated with operons which are acquired through lateral gene transfer.62 I


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EMSA and DNase I footprinting established the binding of HosA dimer to hitherto unknown palindromic site: 5′GTTCGTATACGAAC-3′ that is present upstream of HAD operon. The fact that HosA recognizes and binds a complete palindrome was unexpected as the proteins of the MarR family mostly recognize an incomplete palindrome. Also, as opposed to other members of MarR family which bind to multiple sites within the promoter region2,17,26,29 only single binding site was observed for HosA within PecdB. This conservation of highly similar and single binding site of HosA in different bacterial genera explains the intactness of binding site throughout the process of evolution. Identification of cognate sequence recognized by HosA revealed its dissimilarity from the sites recognized by its closest homologue SlyA17 and indicates that a different set of genes might be regulated by both of these transcription factors. However, the binding site of HosA resembles to that recognized by phylogenetically closer regulator; MepR(having consensus half site “GTTAGAT”) of Staphylococcus aureus21 (Supporting Information Figure 4). The detailed characterization of HosA binding site in the present study will further help to identify HosA-regulon across the genome and understand its role in the bacterial physiology. Autoregulation has been observed in number of MarR family proteins.17 HosA has been previously shown to positively regulate its own expression,44 although it was speculated that it might not be directly involved in its own expression. This is in agreement with our present study on HosA autoregulation, as the overexpression of HosA only transiently autorepressed its expression (Supporting Information Figure 2). MarR proteins act as repressors through blocking the transcript formation by occlusion of RNA polymerase binding site. For example, SlyA represses its own expression through preventing the open complex formation of RNA polymerase by occupying the region between the −10 and −35 site of the promoter.17 In the present study, HosA binding site was determined to be centered around +10 position with respect to transcription origin and suggests a possible road block mechanism of repression by the steric hindrance. The aromatic ligands have been shown to interact and modulates the DNA binding activity of MarR family of transcriptional factors. This is the first study that has demonstrated the interaction of 4-HBA with HosA within micromolar to milimolar concentrations using in vivo and in vitro techniques. Higher derepression of HosA mediated repression at a milimolar concentration of 4-HBA might be due to the fact that the uncharged form of 4-HBA was present in a significantly lower concentration at neutral pH of culture media, which is the active form transported inside E. coli.61 The absence of complete derepression of HosA mediated repression even at higher concentrations of 4-HBA indicated that, it is either metabolized or exported from the cell through other exporters.71,72 Moreover, phylogenetic analysis suggested evolutionary relatedness of HosA to other MarR proteins which are regulated by the ligands that are structurally similar to 4-HBA, that is, 3-HBA (MobR) and 3,4-DHBA (PcaV) (Supporting Information Figure 4).26,73 These proteins acts as the regulators of different aromatic acid metabolism pathways and indicates the evolutionary conserveness of their ligandbinding domains with HosA. The optimum intracellular concentration of 4-HBA is critical for ubiquinone biosynthesis and is required for a number of pertinent processes like motility,74 osmotic tolerance,75 antibiotic and thiol resistance,76,77 aerobic growth and number of

Figure 8. Effect of 4-HBA on expression level of HAD operon and HosA−DNA interaction. (A) Schematic diagram of nonoxidative HAD operon along with the primer pairs (shown by arrows) used for amplifying intergenic regions IG1 and IG2. (B) Semiquantitative PCR of intergenic regions IG1 and IG2 of nonoxidative HAD operon. Lane UT: PCR amplified cDNA template that was transcribed from RNA of cultures without 4-HBA treatment and (Lane T) with 4-HBA treatment. Amplified internal sequence of 16S rRNA transcript was taken as internal control. (C) Effect of 4-HBA on interaction of HosA with probe (U) through EMSA. Lane 1: Radiolabeled free probe (U). Lane 2: Radiolabeled probe (U) incubated with HosA. Lanes 3−8: Different molar concentrations of 4-HBA (0.5−5 mM) incubated with probe and HosA.

previously demonstrated to detoxify the antimicrobial benzoic acids through decarboxylation to its less toxic derivative.64−66 Benzoic acids and its derivatives have been demonstrated to passively diffuse through the cell membrane and cause internal acidification of cytoplasm, which result in disruption of membrane potential, denaturation of vital proteins, membrane damage, generation of reactive oxygen species (ROS), and leakage of ions from the bacteria.67 Therefore, the presence of nonoxidative HAD operon in pathogenic E. coli might provide a selective advantage in overcoming the toxicity of 4-HBA and its widely used chemical analogue; parabens which has been described to be metabolized inside body to produce 4HBA.68,69 The proteins of MarR family of transcriptional factors have been shown to exists as dimer under the physiological condition.17 The dimeric state of a regulator necessitates its binding to palindromic or near palindromic DNA sequences.70 In the present study, HosA was demonstrated to exist as homodimer using gel exclusion chromatography. Furthermore, J


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Figure 9. Proposed model for regulation of nonoxidative HAD operon by HosA. Genes of the nonoxidative HAD operon (ecdB, ecdC, and ecdD) and hosA are denoted by large arrows. Two parallel vertical lines represent the cell membrane. Elliptical aligned and nonaligned symbols (in black) represents the HosA unbound and bound to 4-HBA, respectively. An inverted vertical dotted arrow below HosA represents autoregulation. Different exporters of 4-HBA are shown by elliptical symbols (in gray color above the parallel lines). Arrows above the exporters represent the transport of negatively charged 4-HBA from cytoplasm to external environment. Arrow above the cell membrane represent diffusion of uncharged form of 4-HBA from external environment to cytoplasm. The green and red color arrows represent the transcription progression and blockage of nonoxidative HAD operon, respectively. The rectangular box represents possible metabolic channeling of phenol by enzymes of different pathway.

of UbiX/PAD1 as a flavin prenyltransferase which modifies the isoalloxazine ring of FMN80,81 and provides a novel cofactor required for decarboxylation activity of the enzyme UbiD/ FDC.82 Unlike the paralogous gene pair, present study have demonstrated the HAD gene cluster in pathogenic E. coli to exists as an operon through semiquantitative PCR. This supports the fact that product of all the genes in HAD operon, rather than any single gene product are invovled in decarboxylation of 4-HBA or related compounds, as reported previously.42 Therefore, similar to paralogous proteins, it would be interesting to determine the nature of the cofactor and the

energy dependent pathways.78 In E. coli, the product of the isofunctional genes ubiX and ubiD are required for a functional ubiquinone biosynthetic pathway. Strikingly, the gene PAD1 of S. cerevisiae has been shown to complement ubiX null strain of E. coli.79 It is intriguing that 4-HBA, the physiological ligand of HosA, is also the starting compound of ubiquinone synthesis; therefore, it is plausible that HAD operon might be involved in the ubiquinone biosynthetic pathway. Recent reports have unraveled the function of paralogous isofunctional proteins UbiX and UbiD that are required to decarboxylate an intermediate in ubiquinone biosynthesis: 3octaprenyl-4-hydroxybenzoate. It demarcates a unique function K


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by induction of HAD operon. The present model illustrates the direct binding of 4-HBA to HosA which disrupts its interactions with DNA, probably by inducing a conformational change and results in transcription progression of HAD operon. Structural studies of HosA in complex with 4-HBA in future will further enhance the understanding of the mechanistic basis of ligandmediated modulation of HosA function.

mechanism of decarboxylation used by the proteins of the HAD operon to carry out its function. MarR regulators have been shown to act as an oxidation sensing molecule that are generated in response to the exposoure of different chemicals in the form of reactive oxygen species (ROS) or mediated by metal ions.6−11,35 To examine the possibility of HosA as an oxidation sensing molecule, the in vivo effect of oxidizing agent (H2O2) and different ions released by membrane damage were tested for their ability to cause derepression of HosA mediated repression. It was observed that the DNA binding ability of HosA was not altered by the oxidizing agent and ions that are released by the membrane damage. This can be attributed to the fact that HosA does not contain cysteine residue (Supporting Information Figure 1B). Benzoic acid decarboxylases through metabolic engineering have been utilized for biological synthesis of industrially important chemicals. For example, PAD1 and FDC genes from S. cerevisiae have been engineered in E. coli for commercial production of styrene and higher alcohols using glucose as a nutrient source.83,84 Similarly, hydroxystyrene,85 a monomer used for synthesis of many industrially important polymers and the plant growth hormone Indole Acetic Acid (IAA) was produced by the engineered strains of E. coli that contained phenolic acid decarboxylases (PAD1 and yclC) from Bacillus amyloliquefaciens.86 E. coli 4-hydroxybenzoic acid decarboxylase has shown to be utilized for commercial production of phenol.87 The molecular mechanism of regulation and function of HAD operon will therefore provide a novel avenue for its technological implications in biological synthesis of commercially important chemicals. Proposed Model for HosA Mediated Regulation of Nonoxidative HAD Operon. Taken together, we propose a model for HosA mediated regulation of HAD operon as shown in Figure 9. Similar to most of the proteins of MarR family, HosA regulates its contiguous operon. HosA as compared to divergently transcribed nonoxidative HAD operon is expressed in a definite amount under physiological condition in different strains.44 The same has been observed in E. coli strain UMN026 in this study (Supporting Informationy Figure 4). We assume that the minimal amount of HosA produced in normal physiological condition is sufficient to repress HAD operon by binding downstream to the transcription start site (Figure 6A). The presence of 4-HBA at very low concentration under physiological condition inside the cell, as an intermediate in ubiquinone biosynthesis pathway will be insufficient to induce the nonoxidative HAD operon. On external exposure, the concentration of 4-HBA increases inside the cell due to diffusion of its uncharged form. The dissociation of 4-HBA to its negatively charged derivative inside the cell lowers the cytoplasmic pH, which can cause impairment of cell physiology. This stress caused by intracellular drop in pH is relieved by the cell through the export of negatively charged 4-HBA by different membrane exporters.61,71 We propose another putative pathway of 4-HBA assimilation through its decarboxylation by proteins of HAD operon. Although, the product of the decarboxylation of 4-HBA produces a toxic analogue phenol, but its conversion to metabolic intermediates by the enzymes (i.e., aromatic ring hydroxylases and extradiol dioxygenases) of other pathways, for example: 4-hydroxy phenyl acetate and 3-hydroxyphenyl propionic acid degradation pathways88,89 cannot be ruled out which will require further investigation. Our study has indicated a probable way of assimilation of excessive 4-HBA through metabolic channeling

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b01163. Effect of different membrane assciated ions and oxidizing agent on HosA mediated repression and in vivo effect of HosA binding on cognate site mutants, Supporting Figure 1. Supporting Figures 2−4 describe the autoregulation, stage specific expression and phylogenetic analysis of HosA, respectively. Supporting Tables provide the list of contiguous genes the intergenic regions of which are used for consensus HosA binding site determination (Supporting Table 1), primer pairs used for making constructs used in β-Galactosidase assay (Supplementary Table 2), primer sequences used for PCR amplification of the target sequences (Supporting Table 3), and MarR family proteins (along with their accession numbers) used for phylogenetic analysis (Supporting Table 4). (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +91-40-2474 9395. Fax: +91-40-2474 9448. E-mail: [email protected]. Funding

This work is supported by a core grant from CDFD, Hyderabad under Department of Biotechnology, Government of India. A.R. is the recipient of a doctoral research fellowship from Council of Scientific and Industrial Research (CSIR), Government of India and is registered under the academic program (PhD) of Manipal University. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the assistance of Rohan Misra, Bhavik Sawhney, and Suhail Yousuf for constructive criticism of the manuscript and Mr. Shaffiqu T. S. for the technical input.

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ABBREVIATIONS EMSA, electrophoretic mobility shift assay; HAD, hydroxyarylic acid decarboxylase; MarR, multiple antibiotic resistance Regulator; 4-HBA, 4-hydroxybenzoic acid

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REFERENCES

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