Characterisation of a putative AraC transcriptional regulator from Mycobacterium smegmatis

Dimitrios Evangelopoulos,Antima Gupta,Nathan A Lack,Arundhati Maitra,Annemieke M.C Ten Bokum,Sharon Kendall,Edith Sim,Sanjib Bhakta

doi:10.1016/j.tube.2014.08.007

Abstract

SummaryMSMEG_0307 is annotated as a transcriptional regulator belonging to the AraC protein family and is located adjacent to the arylamine N-acetyltransferase (nat) gene in Mycobacterium smegmatis, in a gene cluster, conserved in most environmental mycobacterial species. In order to elucidate the function of the AraC protein from the nat operon in M. smegmatis, two conserved palindromic DNA motifs were identified using bioinformatics and tested for protein binding using electrophoretic mobility shift assays with a recombinant form of the AraC protein. We identified the formation of a DNA:AraC protein complex with one of the motifs as well as the presence of this motif in 20 loci across the whole genome of M. smegmatis, supporting the existence of an AraC controlled regulon. To characterise the effects of AraC in the regulation of the nat operon genes, as well as to gain further insight into its function, we generated a ΔaraC mutant strain where the araC gene was replaced by a hygromycin resistance marker. The level of expression of the nat and MSMEG_0308 genes was down-regulated in the ΔaraC strain when compared to the wild type strain indicating an activator effect of the AraC protein on the expression of the nat operon genes.

Highlights

Transcriptional factors modulate gene expression through binding to a specific DNA sequence usually found upstream of the gene or the genomic area that they control
We describe the role of a transcriptional regulator from the AraC/XylS family in M. smegmatis, a fast growing, environmental species of Mycobacterium
We have identified that the AraC-family transcriptional regulator MSMEG_0307 protein recognises and binds to a palindromic motif upstream of the nat operon in M. smegmatis and that it is involved in the regulation of the expression of the nat and the MSMEG_0308 genes of the same operon

Summary

Introduction

Transcriptional factors modulate gene expression through binding to a specific DNA sequence usually found upstream of the gene or the genomic area that they control. They are important proteins that can help cells acclimatise to challenging environments based on the changing external stimuli. Common characteristics of the AraC proteins is the presence of a conserved region of 100 residues in the C-terminal region of the protein that form a helix-turn-helix structure responsible for DNA binding, a second region in the N-terminal region of the protein contains a ligand binding domain and a peptide-linker region connecting the two functional domains. The proteins that belong to the AraC/XylS family usually recognise palindromic DNA sequences and bind to them by forming dimers using the helix-turn-helix domain [2].

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Conclusion