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. 2007 Aug 23;8:289. doi: 10.1186/1471-2164-8-289

GntR family of regulators in Mycobacterium smegmatis: a sequence and structure based characterization

Vaibhav Vindal 1, Katta Suma 1, Akash Ranjan 1,✉
PMCID: PMC2018728  PMID: 17714599

Abstract

Background

Mycobacterium smegmatis is fast growing non-pathogenic mycobacteria. This organism has been widely used as a model organism to study the biology of other virulent and extremely slow growing species like Mycobacterium tuberculosis. Based on the homology of the N-terminal DNA binding domain, the recently sequenced genome of M. smegmatis has been shown to possess several putative GntR regulators. A striking characteristic feature of this family of regulators is that they possess a conserved N-terminal DNA binding domain and a diverse C-terminal domain involved in the effector binding and/or oligomerization. Since the physiological role of these regulators is critically dependent upon effector binding and operator sites, we have analysed and classified these regulators into their specific subfamilies and identified their potential binding sites.

Results

The sequence analysis of M. smegmatis putative GntRs has revealed that FadR, HutC, MocR and the YtrA-like regulators are encoded by 45, 8, 8 and 1 genes respectively. Further out of 45 FadR-like regulators, 19 were classified into the FadR group and 26 into the VanR group. All these proteins showed similar secondary structural elements specific to their respective subfamilies except MSMEG_3959, which showed additional secondary structural elements. Using the reciprocal BLAST searches, we further identified the orthologs of these regulators in Bacillus subtilis and other mycobacteria. Since the expression of many regulators is auto-regulatory, we have identified potential operator sites for a number of these GntR regulators by analyzing the upstream sequences.

Conclusion

This study helps in extending the annotation of M. smegmatis GntR proteins. It identifies the GntR regulators of M. smegmatis that could serve as a model for studying orthologous regulators from virulent as well as other saprophytic mycobacteria. This study also sheds some light on the nucleotide preferences in the target-motifs of GntRs thus providing important leads for initiating the experimental characterization of these proteins, construction of the gene regulatory network for these regulators and an understanding of the influence of these proteins on the physiology of the mycobacteria.

Background

Being a fast growing, non-pathogenic mycobacteria, Mycobacterium smegmatis has been widely used as a model organism to study the biology of other virulent and extremely slow growing species like M. tuberculosis. The genome of M. smegmatis, as listed at the TIGR site, contains a large number of putative GntR-like regulators. These regulators play an important role in the cellular physiology. Many such regulators are involved in regulation of gene expression in response to various oxidized substrates related to either amino acid metabolism or at the branch points of various other metabolic pathways.

The GntR family of bacterial regulators is named after the Bacillus subtilis transcription regulator- GntR- a repressor of the gluconate operon [1]. Regulators of this family possess a conserved N-terminal domain that is involved in the DNA binding. Based on this conservation, these proteins can easily be recognized by a Conserved Domain Database (CDD) search [2]. However, the C-terminal domain, which is involved in the effector binding and/or oligomerization (E-b/O), is quite diverse and heterogeneous. As a consequence of this heterogeneity, the GntR regulators have been further classified into six subfamilies (FadR, HutC, MocR, YtrA, AraR and PlmA) [3,4]. The members of subfamilies possess conserved secondary structural features specific to their subfamily and interact with a limited number of molecules [5]. Considering these conserved secondary structural features in sequence analysis, GntR regulators are defined as a part of specific subfamily [6]. Earlier, we have characterized GntR regulators from M. tuberculosis [7]. In present study putative GntR regulators from M. smegmatis are classified into their specific subfamilies. Further, suitable orthologs of the M. smegmatis GntRs were also identified using reciprocal BLAST searches in M. tuberculosis, M. avium, M. bovis, M. ulcerans, M. sp. KMS, M. sp. MCS, M. vanbaalenii PYR-1 and B. subtilis. To identify the DNA targets of these regulators, we utilized the information about the nucleotide preferences for regulators of a given subfamily. All the upstream DNA sequences of the GntR coding genes were scanned to locate potential palindromes that matched the nucleotide preference criteria [5].

Results and discussion

Classification of the putative M. smegmatis GntRs into subfamilies

Unrooted tree of the M. smegmatis GntRs was constructed with the classified representatives of all subfamilies (Table 1) [5]. Among all putative M. smegmatis GntRs two proteins (MSMEG_1043 and MSMEG_2323) were found to be identical in sequence, hence only one of them MSMEG_1043 was taken for the classification. Each branch of the constructed tree represents a subfamily. Bootstrapping, involving 1000 replicates, shows all subfamily branches clustered with high bootstrap values. FadR subfamily is divided into two groups, FadR and VanR (Figure 1).

Table 1.

Details of GntR regulators used as representative from all subfamilies

Subfamily Organism Protein ID Amino acid Swiss Prot ID
FadR (FadR Group) Escherichia coli O157:H7 FadR 238 P0A8V8
FadR (VanR Group) Rhizobium leguminosarum MatR 222 Q9JP74
MocR Rhizobium meliloti MocR 493 P49309
HutC Pseudomonas putida HutC 248 P22773
YtrA Bacillus halodurans BH0651 123 Q9KF35
Bacillus halodurans BH2647 123 Q9K9J9
Staphylococcus aureus SAV1934 126 Q99SV4
Bacillus subtilis YhcF 121 P54590
Bacillus subtilis YtrA 130 O34712
AraR Bacillus subtilis P96711 362 P96711
Bacillus halodurans Q9KBQ0 375 Q9KBQ0
Bacillus stearothermophilus Q9S470 364 Q9S470
PlmA Synechocystis sp. strain PCC 6803 sll1961 388 P73804
Anabaena sp. strain PCC 7120 Q8YXY0 328 Q8YXY0
Synechococcus elongatus Q8DH43 367 Q8DH43
Trichodesmium erythraeum IMS101 Q3HFX5 327 Q3HFX5
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Figure 1.

Figure 1

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Unrooted tree of the proteins of GntR family regulators of M. smegmatis including representatives of all subfamily regulators from different Bacterial Genomes with 1000 bootstrap replicates. All the GntR regulators are clustered into six subfamilies. FadR subfamily is branched again into two groups (FadR and VanR). (Abbreviations are as indicated in Table 1 and Table 2).

FadR-like proteins of M. smegmatis

Of all the putative GntRs, 45 proteins were classified as the FadR-like regulators. These subfamily members are further classified into two groups FadR and VanR where the C-terminal effector binding and/or oligomerization domain length is about 170 and 150 amino acid residues respectively comprising all α-helices [5]. Among all FadR-like regulators, 19 regulators were clustered as members of the FadR group while 26 for the VanR group (Table 2). To study secondary structural features both the group members were dealt with separately. C-terminal domain of all the members of FadR group were predicted with seven α-helices except MSMEG_2599. All the regulators showed distinguishable predicted secondary structural features specific to this subfamily (Figure 2 and Figure 3) [5]. Secondary structural patterns of the regulator MSMEG_3959 revealed an extra secondary structural element, which could be significant in studying protein family evolution. FadR-like regulators are known to be involved in the regulation of gene expression in response to oxidized substrates related to either amino acid metabolism or at the branch point in various metabolic pathways such as glycolate [8], pyruvate [9], lactate [10], malonate [11] or gluconate [12]. One of FadR-like classified transcriptional regulator MSMEG_6700 is known to be involved in the regulation of piperidine and pyrrolidine metabolism [13]. These results provide a starting point for a detailed biochemical and genetic characterization of M. smegmatis FadR-like regulators.

Table 2.

List of Classified M. smegmatis GntR regulators

Gene Subfamily Amino acid Gene Subfamily Amino acid
MSMEG_0124 FadR 227 MSMEG_2546 FadR 239
MSMEG_0130 FadR 230 MSMEG_2599 FadR 224
MSMEG_0166 FadR 242 MSMEG_2605 FadR 255
MSMEG_0179 FadR 223 MSMEG_2682 FadR 262
MSMEG_0268 HutC 292 MSMEG_2794 FadR 225
MSMEG_0286 HutC 228 MSMEG_2910 FadR 235
MSMEG_0426 MocR 469 MSMEG_3345 FadR 258
MSMEG_0454 FadR 245 MSMEG_3822 FadR 267
MSMEG_0480 FadR 219 MSMEG_3527 FadR 240
MSMEG_0535 FadR 212 MSMEG_3959 FadR 290
MSMEG_0596 FadR 228 MSMEG_3980 FadR 214
MSMEG_0650 HutC 244 MSMEG_4042 FadR 252
MSMEG_0778 HutC 246 MSMEG_4057 FadR 221
MSMEG_0874 FadR 234 MSMEG_4121 FadR 229
MSMEG_0895 FadR 247 MSMEG_4140 MocR 508
MSMEG_2323 MocR 534 MSMEG_4659 HutC 245
MSMEG_1117 FadR 239 MSMEG_5174 YtrA 121
MSMEG_1227 HutC 274 MSMEG_5201 FadR 254
MSMEG_1317 FadR 229 MSMEG_5375 FadR 230
MSMEG_1572 MocR 470 MSMEG_5630 HutC 245
MSMEG_1995 FadR 241 MSMEG_5731 FadR 240
MSMEG_2009 FadR 226 MSMEG_5760 MocR 463
MSMEG_2104 MocR 449 MSMEG_6300 FadR 224
MSMEG_2164 FadR 262 MSMEG_6371 MocR 488
MSMEG_2173 FadR 230 MSMEG_6639 FadR 222
MSMEG_2209 FadR 222 MSMEG_6700 FadR 245
MSMEG_1043 MocR 534 MSMEG_6738 FadR 227
MSMEG_2453 FadR 244 MSMEG_6745 HutC 247
MSMEG_2480 FadR 246 MSMEG_6789 FadR 246
MSMEG_2489 FadR 240 MSMEG_6881 FadR 209
MSMEG_2531 FadR 253 MSMEG_6908 FadR 221
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Figure 2.

Figure 2

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Structure based sequence analysis of M. smegmatis GntR-like regulators by the multiple sequence alignment of the C-terminal domains of GntR regulators belonging to FadR Subfamily (FadR group). Abbreviations are as indicated in Table 1. Consensus sequence from the multiple sequence alignment has been drawn. High and low consensus levels were fixed arbitrarily at 80% and 40% of identity and are represented respectively by the capital and lowercase letters. Consensus symbol ! used for anyone of IV; $ is anyone of LM; % is anyone of FY; # is anyone of NDQEBZ. In graphical representation α-helix region and β-sheet regions are highlighted with light and dark gray background.

Figure 3.

Figure 3

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Structure based sequence analysis of M. smegmatis GntR-like regulators by the multiple sequence alignment of C-terminal domains of GntR regulators belonging to FadR Subfamily (VanR group). Abbreviations are as indicated in Table 1. Consensus sequence from the multiple sequence alignment has been drawn. High and low consensus levels were fixed arbitrarily at 80% and 40% of identity and are represented respectively by the capital and lowercase letters. Consensus symbol ! used for anyone of IV; $ is anyone of LM; % is anyone of FY; # is anyone of NDQEBZ. In graphical representation α-helix region and β-sheet regions are highlighted with light and dark gray background.

HutC-like proteins of M. smegmatis

Contrary to the FadR-like regulators, the regulators of this subfamily consist of both α-helices and β-sheet structures in the C-terminal domain. We identified eight GntRs as members of this subfamily (Table 2). All these members showed distinguishable predicted secondary structural features specific to this subfamily (Figure 4) [5]. These regulators are known to acquire the same protein fold as Escherichia coli UbiC; hence it is also named as UbiC transcription regulator-associated (UTRA) domain [14]. This effector-binding domain responds to various ligands like histidine (HutC) [15], long chain fatty acids [16], trehalose 6-phosphate [17] or alkylphosphonate [18]. A range of known ligands, specific to many HutC-like regulators, will help in characterizing the classified M. smegmatis regulators.

Figure 4.

Figure 4

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Structure based sequence analysis of M. smegmatis GntR-like regulators by the multiple sequence alignment of C-terminal domains of GntR regulators belonging to the HutC Subfamily. Abbreviations are as indicated in Table 1. Consensus sequence from the multiple sequence alignment has been drawn. High and low consensus levels were fixed arbitrarily at 80% and 40% of identity and are represented respectively by the capital and lowercase letters. Consensus symbol ! used for anyone of IV; $ is anyone of LM; % is anyone of FY; # is anyone of NDQEBZ. In graphical representation α-helix region and β-sheet regions are highlighted with light and dark gray background.

MocR-like protein of M. smegmatis

Among all the putative GntR regulators, eight were classified as members of the MocR subfamily (Table 2). All the eight regulators showed distinguishable predicted secondary structural features specific to this subfamily (Figure 5) [5]. MocR-like regulators show homology to the class I aminotransferase proteins [19], which requires pyridoxal 5'-phosphate (PLP) as a co-factor. All MocR-like regulators exhibit a PLP attachment site with a conserved lysine residue, which is also evident in the classified MocR-like regulators (Figure 5). It would thus be interesting to study the role of pyridoxal phosphate regulation in the classified regulators. [20].

Figure 5.

Figure 5

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Structure based sequence analysis of M. smegmatis GntR-like regulators by the multiple sequence alignment of C-terminal domains of GntR regulators belonging to the MocR Subfamily. Abbreviations are as indicated in Table 1. Consensus sequence from the multiple sequence alignment has been drawn. High and low consensus levels were fixed arbitrarily at 80% and 40% of identity and are represented respectively by the capital and lowercase letters. Consensus symbol ! used for anyone of IV; $ is anyone of LM; % is anyone of FY; # is anyone of NDQEBZ. In graphical representation α-helix region and β-sheet regions are highlighted with light and dark gray background.

YtrA-like protein of M. smegmatis

The YtrA subfamily is the least represented GntR-like regulator in the bacterial genomes. Among all M. smegmatis GntR regulators, only one regulator MSMEG_5174, showed the signatures of the YtrA subfamily member (Table 2, Figure 6). YtrA possesses a reduced C-terminal domain with only two α-helices. The average length of the putative effector binding and/or oligomerization domain is about 50 amino acids [5]. YtrA from B. subtilis is an experimentally explored regulator, which is part of a large self-regulated operon. This operon consists of genes encoding the ATP binding cassette (ABC) transport systems in addition to the YtrA [21]. It would be interesting to study further, whether MSMEG_5174 has any role in modulating such an operon.

Figure 6.

Figure 6

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Structure based sequence analysis of M. smegmatis GntR-like regulators by the multiple sequence alignment of the C-terminal domains of GntR regulators belonging to YtrA Subfamily. Abbreviations are as indicated in Table 1. Consensus sequence from the multiple sequence alignment has been drawn. High and low consensus levels were fixed arbitrarily at 80% and 40% of identity and are represented respectively by the capital and lowercase letters. Consensus symbol ! used for anyone of IV; $ is anyone of LM; % is anyone of FY; # is anyone of NDQEBZ. In graphical representation α-helix region and β-sheet regions are highlighted with light and dark gray background.

Operator/binding site analysis

We have tabulated a list of potential operator sites near the perfect palindrome sequence with conserved residues, which are found to be specific for most of the subfamily members (Table 3) [5]. We did not find an operator sequence in the upstream sequences of all the remaining regulators. All the predicted sites were found to be in the upstream region from the translation start site except MSMEG_2599. Identification of these sites is an important step to understand the GntR associated regulon or the gene regulatory network in the genome [22-25].

Table 3.

List of predicted potential operator sites

Subfamily Regulator Potential Operator sequence
FadR MSMEG_0124 --CCACTGTTCAACGAGCG---
MSMEG_0179 -AAGATCGTCCGACAATT----
MSMEG_0454 --CAATCGTCATACGATTG---
MSMEG_0596 --GTGTGGTCAGACCACAC---
MSMEG_0895 -----TCGTGGGACGA------
MSMEG_2164 -----CCGTTGAACGG------
MSMEG_2480 ---ACCGGTGGCACCAGGGT--
MSMEG_2599 ----ACCGTGGGACGGT-----
MSMEG_2682 -----TGGCAAGACCA------
MSMEG_2910 CCTTGATGTCCCACAACG----
MSMEG_3527 -----TGGTAAGACCA------
MSMEG_3822 -----TTGTTACTCAA------
MSMEG_3959 --TTGCCGCGCGACAA------
MSMEG_3980 -----TGGTGATACACCA----
MSMEG_4057 ----TTCGTGTCACAAGTCGAA
MSMEG_6789 ----TTTGTGTCACAAA-----
HutC MSMEG_0268 -----ACCGTCTACATCGT---
MSMEG_0650 ------TGGTCTATACCA----
YtrA MSMEG_5174 ---GCCATCATGTAGTGC----
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Preferred nucleotides in potential operator sites are printed in bold

Ortholog prediction

We have found a number of M. smegmatis GntR regulators that are orthologs of proteins from the other species of mycobacteria and B. subtilis (Table 4). As orthologs typically share the same function, these regulators could serve as a model to study homologues from the other species of mycobacteria. These characterized orthologs may provide clues for initiating detailed biochemical characterization of M. smegmatis proteins. Many putative orthologs were experimentally known like Rv0165c that is involved in regulation of mce1 operon [6]; GntR, a transcriptional repressor of gluconate operon [12]; YcbG, involved in utilization of D-glucarate and D-galactarate [26]; YcnF, involved in utilization of gamma-aminobutyrate [27]. However, we did not find the orthologs for all M. smegmatis GntRs in other pathogenic species.

Table 4.

Orthologs of M. smegmatis GntR-like regulators in other bacterial species

M.smeg M.tub M.aviump M.bov M.van M.spMCS M.spKMS M.ulc B.sub
MSMEG_0130 Rv0165c MAP3599c Mb0170c Mvan_0130 Mmcs_0114 Mkms_0123 MUL_1058 -
MSMEG_0179 - - - - - - MUL_1833 -
MSMEG_0268 - - - Mvan_5574 Mmcs_0189 Mkms_0198 - -
MSMEG_0286 - - - Mvan_0056 - - - -
MSMEG_0454 - - - Mvan_5910 - Mkms_5416 - -
MSMEG_0535 - - - - - - - GntR
MSMEG_0596 - - - - - Mkms_4471 - -
MSMEG_1043 - - - Mvan_2084 - Mkms_1901 - -
MSMEG_1227 - MAP1105 - - - - - -
MSMEG_1317 - - - Mvan_3051 - - - -
MSMEG_2104 - MAP1267 - - - - MUL_1552 -
MSMEG_2173 - - - Mvan_0294 - - - YcbG
MSMEG_2209 - MAP2404c - Mvan_1978 - Mkms_1807 MUL_3894 -
MSMEG_2599 - - - Mvan_2282 - Mkms_2107 - -
MSMEG_2794 - - - Mvan_0952 - Mkms_0349 MUL_1381 -
MSMEG_3527 Rv0586 - Mb0601 Mvan_2942 - Mkms_2771 MUL_4564 -
MSMEG_3822 - - - Mvan_0606 - Mkms_0519 - -
MSMEG_4057 - - - - - - - YdhC
MSMEG_4140 - - - - - - - YcnF
MSMEG_4659 Rv0792c MAP0628c Mb0816c Mvan_4015 - - MUL_0525 YvoA
MSMEG_5174 Rv1152 MAP2632c Mb1183 Mvan_4569 - - MUL_0993 YtrA
MSMEG_5201 Rv3060c MAP2347 Mb3086c Mvan_4590 - Mkms_4157 MUL_3832 -
MSMEG_5630 - MAP3505c - Mvan_4965 - Mkms_4496 MUL_4818 -
MSMEG_5731 - - - Mvan_0931 - Mkms_4957 - -
MSMEG_6371 - - - Mvan_5625 - Mkms_5086 - YhdI
MSMEG_6700 - - - Mvan_1846 - - - -
MSMEG_6908 Rv0043c MAP0053c Mb0044c Mvan_6046 - Mkms_5471 MUL_0061 -
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'-' Represents, corresponding orthologs are not present in the genome. M.smeg – M. smegmatis; M.tub – M. tuberculosis; M.aviump. – M. avium para.; M.bov – M. bovis; M.van – M. vanbaalenii PYR-1; M.spMCS – M. sp. MCS;M.spKMS – M. sp. KMS; M.ulc – M. ulcerans; B.sub – B. subtilis.

Our results help in extending the annotation of GntRs encoded in the M. smegmatis genome. We have classified putative M. smegmatis GntRs into four subfamilies. Though in the present study, we have made an attempt to explore M. smegmatis GntR regulators, this approach could also be effectively employed to extend the GntR family classification in other bacterial species as well.

Conclusion

This analysis has shown that M. smegmatis is equipped with large number of GntR-like regulators, belonging to four subfamilies. It further suggests that the GntR regulatory repertoires of M. smegmatis are far more complex than in M. tuberculosis. Indeed, additional GntR regulators possibly control a subset of genes required for adapting to a range of environmental conditions. One of the FadR-like regulators shows additional secondary structural elements, suggesting a possible origin of a new group within the FadR subfamily. Identified orthologs from M. smegmatis could serve as a model to decipher molecular regulation events taking place in the pathogenic mycobacteria. Potential operator sites were also identified based on the nucleotide recognition preferences of GntR-like regulators.

Methods

Selection of GntR-like Members

The sequences of M. smegmatis MC2 were downloaded from the Institute for Genomic Research Comprehensive Microbial Resource [28]. Apart from classified GntR regulators or proteins annotated as GntR-like regulator, other putative GntRs from M. smegmatis proteome were selected using GntR Pfam profile [29]. Among all predicted GntRs one protein (MSMEG_3400) was discarded for this study because of its unusual size (741 amino acid) and its annotation as glutamyl-tRNA(Gln) amidotransferase subunit A. Rest of the GntR regulators were retrieved from the SWISS-PROT/TrEMBL sequence database as per their Swiss-Prot ID (Table 1). Additionally published and annotated genome sequences of M. tuberculosis, M. avium subsp. paratuberculosis, M. bovis, M. ulcerans, M. sp KMS, M. sp. MCS, M. vanbaalenii PYR-1 and Bacillus subtilis were downloaded from the NCBI ftp site [30].

Secondary structure prediction

The secondary structural features of all bacterial GntR regulators including the M. smegmatis GntRs were analyzed (Table 1 and Table 2). Secondary structure predictions were made using Jpred [31], SsPro [32] and 3DPSSM [33]. A consensus of all the secondary structure predictions was considered for a better validity.

Multiple sequence alignments and Phylogenetic tree construction

Multiple sequence alignment was generated with MULTIALIN [34]. Distances between aligned proteins were computed with the PROTDIST program using the Dayhoff PAM matrix [35]. The FITCH program estimated phylogenies from distances in the matrix data using the Fitch-Margoliash algorithm [36]. The phylogenetic tree was drawn using the TREEVIEW program with incorporation of bootstrap values that were obtained involving 1000 replicates [37]. PROTDIST and FITCH programs are included in the PHYLIP package developed by Felsenstein [38].

Operator site analysis

To study the upstream region of GntR-like regulators, we considered sequences from 400 bases upstream to 50 bases downstream from the translation start site. As many GntR regulators are reported to recognize palindromes and also exhibit nucleotide recognition preferences among the same subfamily [5], we utilised these clues to scan the upstream sequences.

Reciprocal BLAST

Reciprocal BLAST hits are frequently utilized to identify the orthologs in two species [39,40]. In this method we searched for the best reciprocal BLAST hit for M. smegmatis GntR proteins with M. tuberculosis, M. avium, M. bovis, Mycobacterium ulcerans, Mycobacterium sp KMS, Mycobacterium sp. MCS, Mycobacterium vanbaalenii PYR-1 and B. subtilis.

Abbreviations

M. tuberculosis – Mycobacterium tuberculosis

M. bovis – Mycobacterium bovis

M. avium para. – Mycobacterium avium subsp. paratuberculosis

M. smegmatis – Mycobacterium smegmatis

M. ulcerans – Mycobacterium ulcerans

M. sp KMS – Mycobacterium sp. KMS

M. sp. MCS – Mycobacterium sp. MCS

M. vanbaalenii PYR-1 – Mycobacterium vanbaalenii PYR-1.

Authors' contributions

VV carried out the operator site prediction, subfamily data analysis, ortholog search and drafted the manuscript. KS participated in the multiple sequence alignment and structure based manual adjustment. AR participated in the study design and coordination. All authors read and approved the final manuscript.

Acknowledgments

Acknowledgements

We thank TIGR for making M. smegmatis mc2-155 sequences available through their web portal. Research in AR's laboratory is supported by the grants from Department of Biotechnology, Department of Science and Technology, Indian Council of Medical Research (ICMR), Council of Scientific & Industrial Research (CSIR) NMITLI. VV is supported by the UGC fellowship. KS is supported by the CSIR NMITLI Grant. Authors acknowledge the assistance of Ms. P. Umadevi, SRF and Mr. Rohan Misra, JRF for proof reading the manuscript and making useful suggestions.

Contributor Information

Vaibhav Vindal, Email: vaibhav@cdfd.org.in.

Katta Suma, Email: suma@cdfd.org.in.

Akash Ranjan, Email: akash@cdfd.org.in.

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