The diphtheria toxin repressor, DtxR, of Corynebacterium diphtheriae has been shown to be an iron-activated transcription regulator that controls not only the expression of diphtheria toxin but also of iron uptake genes. This study aims to identify putative binding sites and operons controlled by DtxR to understand the role of DtxR in patho-physiology of Corynebacterium diphtheriae.
Result
Positional Shannon relative entropy method was used to build the DtxR-binding site recognition profile and the later was used to identify putative regulatory sites of DtxR within C. diphtheriae genome. In addition, DtxR-regulated operons were also identified taking into account the predicted DtxR regulatory sites and genome annotation. Few of the predicted motifs were experimentally validated by electrophoretic mobility shift assay. The analysis identifies motifs upstream to the novel iron-regulated genes that code for Formamidopyrimidine-DNA glycosylase (FpG), an enzyme involved in DNA-repair and starvation inducible DNA-binding protein (Dps) which is involved in iron storage and oxidative stress defense. In addition, we have found the DtxR motifs upstream to the genes that code for sortase which catalyzes anchoring of host-interacting proteins to the cell wall of pathogenic bacteria and the proteins of secretory system which could be involved in translocation of various iron-regulated virulence factors including diphtheria toxin.
Conclusions
We have used an in silico approach to identify the putative binding sites and genes controlled by DtxR in Corynebacterium diphtheriae. Our analysis shows that DtxR could provide a molecular link between Fe+2-induced Fenton's reaction and protection of DNA from oxidative damage. DtxR-regulated Dps prevents lethal combination of Fe+2 and H2O2 and also protects DNA by nonspecific DNA-binding. In addition DtxR could play an important role in host interaction and virulence by regulating the levels of sortase, a potential vaccine candidate and proteins of secretory system.
Background
Iron is an important inorganic component of a cell. Iron is required as co-factor for various essential enzymes and proteins some of which are involved in electron transport (Cytochromes), redox reactions (oxidoreductases) and regulation of gene expression (fumarate-nitrate reduction regulatory protein, iron-binding protein) [1]. However a higher level of intracellular iron can catalyze formation of hydroxyl radicals and reactive oxygen species through Fenton's reaction which could be lethal to the cell [2]. Hence, a careful regulation of iron-requiring enzymes/proteins and iron uptake proteins/enzymes is required for the survival of bacteria.
Inorganic iron is also known to influence virulence in many pathogenic bacteria such as Corynebacterium diphtheriae, Escherichia coli, and Bordetella bronchiseptica [3–5]. The diphtheria toxin repressor DtxR is known as an iron-activated global transcription regulator that represses the transcription of various iron-dependent genes in C. diphtheriae [6, 7]. Eight DtxR-binding sites in upstream sequences of operons/genes named as tox, hmuO, irp1, irp2, irp3, irp4, irp5 and irp6 have been identified by DNA footprinting methods [6]. The product of tox gene is diphtheria toxin which catalyzes the NAD-dependent ADP ribosylation of eukaryotic aminoacyl-transferase-II, thereby causing inhibition of protein synthesis and subsequent death of the host. The hmuO gene, which encodes a haem oxygenase, oxidizes the haem to release free iron. The operons irp1 and irp6 encode the products with homology to ABC-type ferric-siderophore transport systems. The gene irp3 encodes a homologue of AraC-type transcriptional activators. The products of irp2, irp4 and irp5 do not show any homology to the other known proteins. In addition, C. diphtheriae with inactive DtxR has been shown to be sensitive to killing by exposure to high iron conditions or hydrogen peroxide than the wild type [8].
This work uses an in silico method to identify additional DtxR-binding sites and target genes to understand the role of DtxR in virulence and patho-physiology of C. diphtheriae.
Results
In silico identification of putative DtxR-binding sites
Experimentally characterized DtxR-binding motifs were collected from the literature (Table 1). These binding sites were used to identify additional putative DtxR-binding sites along with associated operons in C. diphtheriae NCTC13129 genome (see materials and methods). Table 2 shows the predicted DtxR-binding sites with score 3.7438 or more. We could identify five (tox, irp4, irp5, irp6 and hmuO) of the eight known DtxR-binding sites, in sequenced C. diphtheriae NCTC13129 genome. We could not find irp1 and irp2 motifs as the corresponding genes (irp1, irp2) are not present in the sequenced strain NCTC13129 [9]. The regulator binding sites of irp3, irp4 and irp6 genes in the strain NCTC13129 shows one base change from the binding sites reported in strain C7 [6]. Binding site of irp3 gene (TTAGGTGAGACGCACCCAT) although exists in strain NCTC13129, but not there in the predicted sites, because it is located within the coding region of irp3 ORF. The predicted ORF of irp3 in the sequenced strain NCTC13129 has different start position and is larger than what was previously reported in strain C7 [9, 10].
Table 1
Known DtxR-binding sites from C. diptheriae
Binding site
Gene
Product
Reference
TTAGGATAGCTTTACCTAA
tox
Diphtheria toxin
[25]
TTAGGTTAGCCAAACCTTT
Irp1
Periplasmic protein of siderophore transport system
[26]
GCAGGGTAGCCTAACCTAA
Irp2
Hypothetical protein
[26]
TTAGGTGAGACGCACCCAT
Irp3
AraC-type transcription regulator
[10]
ATTACTAACGCTAACCTAA
Irp4
Hypothetical protein
[10]
CTAGGATTGCCTACACTTA
Irp5
Hypothetical protein
[10]
TTTCCTTTGCCTAGCCTAA
Irp6
Periplasmic protein of siderophore transport system
[6]
TGAGGGGAACCTAACCTAA
hmuO
Haem oxygenase
[27]
Table 2
Predicted DtxR-binding sites in C. diphtheriae
Score
Position
Site
Gene
Synonym
Product
4.45904
-80
TGAGGGGAACCTAACCTAA
hmuO
DIP1669**
heme oxygenase
4.39003
-52
TTAGGATAGCTTTACCTAA
Tox
DIP0222**
Diphtheria toxin precursor
4.25877
-60
ATAGGCTACACTTACCTAA
-
DIP0624
Putative membrane protein
4.21068
-168
TTGGATTAGCCTACCCTAA
-
DIP2162**
ABC-type peptide transport system periplasmic component
4.2033
-21
TTAGGGTAGCTTCGCCTAA
iucA
DIP0586
Putative siderophore biosynthesis related protein
4.17632
-78
ATAGGCATGCCTAACCTCA
-
DIP2330
Putative membrane protein
4.07921
-130
TTAGGTCAGGGTACCCTAA
-
DIP0370
Putative succinate dehydrogenease cytochrome B subunit
4.03559
-30
TTAGCTTAACCTTGCCTAT
arsR
DIP0415
Putative ArsR family regulatory protein
4.01967
-239
TTAGGGTAGGCTAATCCAA
sidA*
DIP2161
nonribosomal peptide synthase
3.99985
-74
TTTTCTTTGCCTAGCCTAA
irp6A
DIP0108**
Ferrisiderophore receptor Irp6A
3.99195
-241
TTAGGCACCCCTAACCTAG
-
DIP0539
Putative sugar ABC transport syste ATP-binding protein
3.98554
-72
TTAGCTTAGCCCTAGCTAA
-
DIP0169
Putative secreted protein
3.9296
-26
CTAGGATTGCCTACACTTA
Irp5
DIP0894**
Conserved hypothetical protein
3.9073
-93
GTTGGGTTGCCCAACCTAC
-
DIP2106
Putative ABC transport system, ATP-binding subunit
3.89763
-86
ATAGGTTAGGTTAACCTTG
chtA*
DIP1520
Putative membrane protein
3.89676
-130
TTGTGTTAGCCTAGGCTAA
secA
DIP0699
Translocase protein
3.89169
-26
TTGGGGTGGCCTATCCTTA
-
DIP2304
Putative DNA-repair glycosylase
3.88042
-172
TTAGGTAAGTGTAGCCTAT
htaA*
DIP0625
Putative membrane protein
3.86534
-69
ATTACTAATGCTAACCTAA
Irp4
DIP2356**
Putative conserved membrane protein
3.85539
-173
TTAGGGTGGGCTAACCTGC
deoR*
DIP1296
Putative DNA-binding protein
3.84889
-75
TTAGGGAACTCTTGCCTTA
piuB*
DIP0124
Putative membrane protein
3.83816
-121
TTAGCTAGGGCTAAGCTAA
-
DIP0168
Putative glycosyl transferase
3.83576
-219
GTAACAAAGGCAAGCCTAA
xerD
DIP1510
Putative integrase/recombinase
3.8224
-216
ATAGGCAAGGTTAAGCTAA
-
DIP0417
Putative membrane protein
3.81905
-47
GTTGGACAGGTTACCCTAA
frgA*
DIP1061
Putative iron-siderophore uptake system permease
3.8148
-37
TGTGGGCACACCAACCTAA
-
DIP2272
possible sortase-like protein
3.76235
-136
TTGGGGTTGCCCTTCCTAA
-
DIP0142
Hypothetical protein
3.76233
-268
CTAGGTTAGGGGTGCCTAA
secY*
DIP0540
preprotein translocase SecY subunit
3.74673
-110
TAAACATAGCCAAACCAAA
nrdF1
DIP1865
ribonucleotide reductase beta-chain 1
3.7438
-81
TAAGGATAGGCCACCCCAA
Dps
DIP2303
Starvation inducible DNA-binding protein
Note: **Indicate the gene synonym with experimentally identified binding site in C. diphtheriae [6]. * Indicates the genes known to be regulated by DtxR [7]. The binding sites in Italics were verified by EMSA. The gene pairs, DIP0624-DIP0625, DIP2161-DIP2162, DIP0168-DIP0169, DIP0539-DIP0540 and DIP2303-DIP2304 are divergently transcribed and contain common regulatory regions.
In addition, we have identified binding sites in upstream sequences of eight genes recently reported to be regulated by DtxR [7]. However, our prediction differs from the previous report for five (secY, deoR, chtA, frgA, sidA) of the seven sites which were identified by BLAST search (Table 2). Our prediction agreed with the previous report that the genes such as recA (DIP1450) and ywjA (DIP1735) are not under a direct DtxR regulation as we could not detect any motif upstream to these gene with scores above the cutoff value [7].
Experimental validation of predicted binding sites
Since our approach to identify DtxR-regulated genes is purely computational in nature, we decided to test the validity of our predictions. A sample of predicted regulator binding motifs (Table 2) (upstream to ORFs: DIP2161, DIP0699, DIP0586, DIP2304, DIP2272) were experimentally verified by EMSA using IdeR, an orthologue of DtxR from M. tuberculosis. DtxR and IdeR are iron-dependent regulators. A pair wise sequence comparison of the two proteins shows a high (58%) overall sequence identity (similarity 72%) which increases further to 92% identity and 100% similarity in DNA recognition domain. In addition, the structural comparison of two regulators also shows a very similar 3D organization, suggesting that the IdeR regulator would be able to recognize the DtxR motif [11].
Synthetic double stranded oligonucleotides corresponding to DNA-binding sites were labeled with 32P and mixed with purified IdeR in presence of manganese ions and was assayed for the formation of DNA-protein complex using EMSA. Manganese was used as the divalent metal in the binding reactions on account of its redox stability compared with ferrous ion. Electrophoretic mobility of all five double stranded oligonucleotides tested was retarded by IdeR (Figure 1). However a synthetic motif (TTTTCATGACGTCTTCTAA) used as a negative control did not show any complex formation. These results indicate that the predicted DtxR-binding sites can indeed bind to DtxR.
Figure 1
IdeR binds the predicted DtxR-binding DNA fragments. 30 pmoles of IdeR was added to 32P-labelled DNA probes in the presence of 200 μM Mn2+, and complexes were resolved on a 7% Tris-borate polyacrylamide gel containing 150 μM Mn2+. Lane 1: Control gel retardation using Radiolabeled DNA motif without DtxR-binding site. Lane 2: Radiolabeled DIP2161 motif without IdeR. Lane 3: Radiolabeled DIP2161 motif with IdeR. Lane 4: Radiolabeled DIP0699 motif with IdeR. Lane 5: Radiolabeled DIP0586 motif with IdeR. Lane 6: Radiolabeled DIP2304 motif with IdeR. Lane 7: Radiolabeled DIP2272 motif with IdeR.
Identification and annotation of DtxR-regulated genes C. diphtheriae genome
In addition to the binding site prediction, we have also identified co-regulated genes (operons) downstream to the predicted DtxR-binding site (Table 3). Function of the proteins encoded by the putative genes in Table 2 and Table 3 was predicted by RPS-BLAST search against conserved domain database [12].
Table 3
Predicted DtxR-regulated operons in C. diphtheriae
Synonym
Gene
Orthologue
Product
DIP2158
COG1131
ABC-type transport system permease and ATPase component
DIP2159
COG1131
ABC-type transport system permease and ATPase component
DIP2160
-
COG3321
Polyketide synthase modules and related proteins
DIP2161*
-
COG1020
Non-ribosomal peptide synthetase modules and related proteins
DIP0586
iucA
Pfam04183
Catalyse discrete steps in biosynthesis of the siderophore aerobactin
DIP0587
-
-
Putative membrane protein
DIP0588
-
-
Putative membrane protein
DIP1059
fepC
COG1120
ABC-type cobalamin/Fe3+-siderophores transport systems
DIP1060
fepG
COG4779
ABC-type enterobactin transport system
DIP1061*
fepD
COG0609
ABC-type Fe3+-siderophore transport system
DIP2162
ddpA
COG0747
ABC-type peptide transport system periplasmic component
DIP2163
ddpB
COG0601
ABC-type peptide/nickel transport systems permease components
DIP2164
ddpC
COG1173
ABC-type peptide/nickel transport systems permease components
DIP2165
dpdD
COG0444
ABC-type peptide/nickel transport systems ATPase component
DIP0169
lraI
COG0803
ABC-type metal ion transport system, periplasmic component
DIP0170
znuC
COG1121
ABC-type Mn/Zn transport systems, ATPase component
DIP0171
znuB
COG1108
ABC-type Mn2+/Zn2+ transport systems, permease components
DIP0172
znuB
COG1108
ABC-type Mn2+/Zn2+ transport systems, permease components
DIP0173
lraI
COG0803
ABC-type metal ion transport system, periplasmic component
DIP2106
mdlB
COG1131
ABC-type multidrug transport system, ATPase and permease component
DIP2107
mdlB
COG1131
ABC-type multidrug transport system, ATPase and permease component
DIP0625
htaa
Pfam04213
Haemin transporter associated protein
DIP0626
hmuT
COG4558
ABC-type haemin transport system
DIP0627
hmuU
COG0609
ABC-type Fe3+-siderophore transport system
DIP0628
hmuV
COG4559
ABC-type haemin transport system
DIP0629*
htaa
Pfam04213
Haemin transporter associated protein
DIP1519*
htaa
pfam04213
Haemin transporter associated protein
DIP1520*
htaa
pfam04213
Haemin transporter associated protein
DIP2303
dps
COG0783
Starvation inducible DNA-binding protein
DIP2304
-
COG0266
Formamidopyrimidine-DNA glycosylase
DIP2305
-
COG0063
Predicted sugar kinase
DIP1510
xerD
COG4974
Site-specific recombinase
DIP1288
-
-
Conserved hypothetical protein
DIP1289
uup
COG0488
ATPase components of ABC transporters with duplicated ATPase domains
ABC-type transport system involved in Fe-S cluster assembly
DIP1294
-
COG0719
ABC-type transport system involved in Fe-S cluster assembly
DIP1295
sufB
COG0719
ABC-type transport system involved in Fe-S cluster assembly
DIP1296*
deoR
COG2345
DeoR family transcriptional regulator
DIP0370
-
-
Putative succinate dehydrogenease (cytochrome b)
DIP0371
-
COG1053
Succinate dehydrogenase/fumarate reductase
DIP0372
-
COG0479
Succinate dehydrogenase/fumarate reductase
DIP0373
-
-
Putative membrane protein
DIP0374
-
-
Putative membrane protein
DIP0375
-
-
Putative membrane protein
DIP0376
-
-
Putative membrane protein
DIP0377
-
-
Putative membrane protein
DIP1864
ctaD
COG0843
Heme/copper-type cytochrome/quinol oxidases
DIP1865
nrdF1
COG0208
Ribonucleotide reductase
DIP2330
-
-
Putative membrane protein
DIP2331
-
COG1012
NAD-dependent aldehyde dehydrogenases
DIP0124*
-
Pfam03929
Uncharacterized iron-regulated membrane protein (DUF337)
DIP0622
-
-
Putative membrane protein
DIP0623
metA
COG2021
Homoserine acetyltransferase
DIP0624
-
-
Putative membrane protein
DIP0415
-
Pfam01022
Bacterial regulatory protein
DIP0539
-
COG3839
ABC-type sugar transport systems
DIP0168
-
-
Putative glycosyl transferase
DIP0417
-
-
Putative membrane protein
DIP0142
-
-
Hypothetical protein
DIP0143
-
-
-
DIP0144
tra8
COG2826
Transposase and inactivated derivatives
DIP2271
-
-
Putative membrane protein
DIP2272
-
COG3764
Sortase (surface protein transpeptidase)
DIP0699
secA
COG0653
Preprotein translocase subunit SecA (ATPase
DIP0700
-
-
Hypothetical protein
DIP0540*
secY
Pfam00344
Eubacterial secY protein
DIP0541
Adk
COG0563
Adenylate kinase and related kinases
DIP0542
mapA
Methionine aminopeptidase
DIP0543
-
-
Sialidases or neuraminidases;
DIP0544
erfK
Pfam03734
This family of proteins contains a conserved histidine and cysteine
DIP0545
infA
COG0361
Translation initiation factor 1 (IF-1)
DIP0546
rpsM
COG0099
Ribosomal protein S13
DIP0547
rpsK
COG0100
Ribosomal protein S11
DIP0548
rpsD
COG0522
Ribosomal protein S4 and related proteins
DIP0549
rpoA
COG0202
DNA-directed RNA polymerase
DIP0550
rplQ
COG0203
Ribosomal protein L17
DIP0551
truA
COG0101
Pseudouridylate synthase
Note: * Indicate the genes reported be regulated by DtxR. Genes listed together belongs to same operon.
Discussion
Our analysis identified putative DtxR motifs upstream to various operons/genes which could be involved in siderophore biosynthesis, ABC-type transport systems, iron storage, oxidative stress defense and iron-sulfur cluster biosynthesis. In addition, we have also identified the motifs upstream of operons that could be involved in anchoring of host-interacting proteins to the cell wall and secretion of various virulence factors. Important functions of some of these DtxR-regulated genes and their role in C. diphtheriae physiology are discussed here.
Regulation of siderophore biosynthesis and ABC-type transport systems
Predicted member of the DtxR regulon, the gene DIP0586, codes for the IucA/IucC family of enzymes that catalyze discrete step in the biosynthesis of the aerobactin [13]. In addition to known DtxR-regulated siderophore transport genes (irp1, irp6), DtxR could also regulate other ABC-type transport systems similar to Manganese/Zinc, peptide/Nickel and multidrug subfamilies of ABC transporters. The peptide/nickel transport system (DIP2162-DIP2165) has been suggested to be recently acquired by pathogenic C. diphtheriae [9].
Regulation of iron storage and oxidative stress defense
We predict that DtxR could regulate divergently transcribed genes DIP2303 and DIP2304 whose products are similar to starvation inducible DNA-binding protein (Dps) and Formamidopyrimidine-DNA glycosylase (Fpg), respectively. Dps in Escherichia coli is induced in response to oxidative or nutritional stress and protects DNA from oxidative stress damage by nonspecific binding [14]. Dps also catalyzes oxidation of ferrous iron to ferric iron by hydrogen peroxide (2Fe2+ + H2O2 + 2H2O → 2Fe+3OOH(core) + 4H+) which in turn prevents hydroxyl radical formation by Fenton's reaction (Fe2+ + H2O2 → Fe+3 + HO- + HO.) and thereby prevents subsequent DNA damage [15]. The enzyme, formamidopyrimidine-DNA glycosylase is a primary participant in the repair of 8-oxoguanine, an abundant oxidative DNA lesion [16]. The gene DIP1510 which codes for the site-specific recombinase XerD could also be regulated by DtxR. The xerD gene in E. coli belongs to the oxidative stress regulon [17].
Regulation of proteins involved in iron-sulfur cluster biosynthesis and iron-sulfur cluster containing proteins
We predict that the operon DIP1288-DIP1296, which is similar to the suf operon of E. coli, could be regulated by DtxR. The suf operon in bacteria encodes the genes for Fe-S cluster assembly machinery [18]. In addition, genes encoding the iron-sulfur containing proteins such as succinate dehydrogenase (Sdh), cytochrome oxidase (CtaD) and Ribonucleotide reductase (NrdF1) in C. diphtheriae also show DtxR motif in their upstream sequences.
Regulation of sortases
We predict that DtxR could regulate the recently acquired pathogenic island DIP2271-DIP2272, encoding the sortase srtA and hypothetical protein, respectively [9]. Sortases are membrane-bound trans-peptidases that catalyze the anchoring of surface proteins to the cell wall peptidoglycan [9]. Such systems are often used by gram-positive pathogens to anchor host-interacting proteins to the bacterial surface [19].
Regulation of protein translation and translocation system
DtxR could regulate two operons that contain genes DIP0699 (secA) and DIP0540 (secY) that code for the protein translocation system. The sec Y-containing operon, which is similar to the streptomycine operon spc from B. subtilis and other bacteria, involves the genes required for protein translation and translocation [20]. The operon contains additional sialidase gene (DIP0543) in comparison to non pathogenic Corynebacterium species. Activity of sialidase has been linked to virulence in several other microbial pathogens and may enhance fimbriae mediated adhesion in Corynebacterium diphtheriae by unmasking receptors on mammalian cells [9].
The Sec system can both translocate proteins across the cytoplasmic membrane and insert integral membrane proteins into it. The former proteins but not the latter possess N-terminal, cleavable, targeting signal sequences that are required to direct the proteins to the Sec system. Some of the DtxR-regulated genes including diphtheria toxin (Table 4) show predicted signal sequences by SignalP 3.0 [21] and hence they may play an important role in host interaction and virulence of Corynebacterium diphtheriae [9].
Table 4
DtxR-regulated genes containing the potential signal sequence
Gene
Product
DIP0222
Diphtheria toxin
DIP0109
IRP6B
DIP2356
IRP4
DIP2162
ABC-type peptide transport system periplasmic component
DIP0172
Putative membrane protein
DIP2107
Putative integral membrane transport protein
DIP0625
Haemin transporter associated protein
DIP0626
ABC-type haemin transport system
DIP0627
ABC-type haemin transport system
DIP1519
Haemin transporter associated protein
DIP0629
Haemin transporter associated protein
DIP1520
Haemin transporter associated protein
DIP2330
Putative membrane protein
DIP0543
Sialidases or neuraminidases
Conclusions
The bioinformatics method used to predict the targets of DtxR in C. diphtheriae NCTC13129 genome is promising, as some of the predicted targets were experimentally verified. The approach identified novel DtxR-regulated genes, which could play an important role in physiology of C. diphtheriae NCTC13129. DtxR, generally known as a repressor of diphtheriae toxin and iron siderophore/transport genes, can also regulate other metal ion transport genes, iron storage, oxidative stress, DNA-repair, biosynthesis of iron-sulfur cluster, Fe-S-cluster containing proteins, and even protein sortase and translocation systems.
Methods
Source of genome sequence
The complete genome sequence of C. diphtheriae was downloaded from NCBI ftp site [22], and the DtxR-binding sites identified by experimental methods were collected from literature [6, 10, 25–27].
Prediction of DtxR-binding sites
DtxR-binding site recognition profile was calculated by positional Shannon relative entropy method [23, 24]. The positional relative entropy Qiat position i in a binding site is defined as
where b refers to each of the possible base (A, T, G, C), fb,iis observed frequency of each base at position i and qbis the frequency of base b in the genome sequence. The contribution of each base to the positional Shannon's relative entropy is calculated by multiplying positional frequency of each base with positional relative entropy. The binding site profile thus generated was used to scan upstream sequences of all the genes of the Corynebacterium diphtheriae genome. The score of each site is calculated as the sum of the respective positional Shannon relative entropy of each of the four possible bases. A maximally scoring site is selected from the upstream sequence of each gene. The lowest score among the input binding sites is considered as cut-off score. The sites scoring higher than the cut-off value are reported as potential binding sites conforming to the consensus sequence.
Prediction of operons
Co-directionally transcribed genes, downstream to the predicted binding site were selected as potential co-regulated genes (operons) according to one of the following criteria (a) Co-directionally transcribed orthologous gene pairs, conserved in at least 4 genomes; (b) genes belong to the same cluster of orthologous gene function category and the intergenic distance is less than 200 base pairs; (c) the first three letters in gene names are identical (gene names for putative genes were assigned from COG database); (d) intergenic distance is less than 90 base pairs [24].
Functional assignment of genes
The function of predicted genes was inferred using the RPS-BLAST search against conserved domain database [12]. These genes were further classified according to their function.
Expression and purification of IdeR
The iron-dependent regulator IdeR from M. tuberculosis was expressed from a recombinant pRSET vector containing the IdeR gene fused to a six His affinity tag (P. Chakhiyar unpublished). The expressed protein was first purified using Ni-NTA Metal Chelate Affinity chromatography; later it was desalted and concentrated using Centricon Ultra filtration device. The concentration of the recombinant protein was estimated using Bradford method.
Electrophoretic mobility shift assay
Double-stranded oligonucleotides containing the predicted binding motif (19 bp long) were end labeled with T4 polynucleotide kinase and [γ32P]-ATP and were incubated with the recombinant purified IdeR protein in a binding reaction mixture. The binding reaction mixture (20-μl total volume) contain the DNA-binding buffer (20 mM Tris-HCl [pH 8.0], 2 mM DTT, 50 mM NaCl, 5 mM MgCl2, 50% glycerol, 5 μg of bovine serum albumin per ml), 10 μg of poly(dI-dC) per ml (for nonspecific binding) and 200 μM MnCl2. The reaction mixture was incubated at room temperature for 30 min. Approximately 2 μl of the tracking dye (50% sucrose, 0.6% bromophenol blue) was added to the reaction mixture at the end of incubation and was loaded onto 7% polyacrylamide gel containing 150 μM MnCl2 in 1 × Tris-borate-EDTA buffer. The gel was electrophoresed at 200 V for 2 hours. Subsequently the gel was dried and exposed to Fuji Storage Phosphor Image Plates for 16 hours. The image plates were subsequently scanned in Fuji Storage Phosphor Imaging workstation.
List of abbreviations
DtxR:
Diphtheria toxin repressor
IdeR:
Iron-dependent regulator
Dps:
DNA-binding protein from starved cells
RPS-BLAST:
Reversed Position Specific – Basic Local Alignment Search Tool
EMSA:
Electrophoretic Mobility Shift Assay
Declarations
Acknowledgements
This work is partially supported by CSIR NMITLI Grant to AR. SR is supported by CSIR NMITLI Grant. YS and PC is supported by CSIR Research Fellowships.
Authors’ Affiliations
(1)
Computational and Functional Genomics Group, Centre for DNA Fingerprinting and Diagnostics
(2)
Laboratory of Cellular and Molecular Biology, Centre for DNA Fingerprinting and Diagnostics
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