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

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.

Figure 1

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).

Figure 2

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

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.

Figure 4

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.

Figure 5

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.

Figure 6

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.