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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.

SUBMITTER: Vindal V

PROVIDER: S-EPMC2018728 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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

Vindal Vaibhav V Suma Katta K Ranjan Akash A

BMC genomics 20070823

<h4>Background</h4>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 ...[more]

PMID: 17714599

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Project description:Mycobacterium smegmatis SigF is a group III sigma factor. Its ortholog in M. tuberculosis is reported to have role in regulation and function of cell wall components. In present study we have created an M. smegmatis M-NM-^TsigF mutant by allele exchange method. M. smegmatis sigF mutant shows non pigmented phenotype and is more sensitive to hydrogen peroxide generated oxidative stress. DNA microarray analysis of M. smegmatis wild type and M-NM-^TsigF mutant suggests that SigF in this species controls the expression of several energy and central intermediary metabolism genes along with regulation of carotenoid biosynthesis. Gene expression patterns of M.smegmatis wild type and M-NM-^TsigF mutant strains were compared at two growth stages i.e. log (OD600 ~1.4) and stationary (OD600 ~3.0). M. smegmatis strains were grown in DifcoTM MiddleBrook 7H9 broth base (BD Biosciences, Sparks, MD, USA) with 0.2% glycerol (v/v) and 0.05% Tween-80 (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% albumen dextrose catalase (BD Biosciences) (v/v) at 37 0C with continuous shaking. Total RNA was isolated using Trizole (Invitrogen) method and labelled with cyanine 3 (Cy3) as per Agilent 1-color labelling protocol (Version 5.5, February 2007). Six hundred nanograms of each Cy3 labelled samples were fragmented and hybridized. Fragmentation of labeled cRNA and hybridization were performed using Gene Expression Hybridization kit (Agilent Technologies). Hybridization was carried out in AgilentM-bM-^@M-^Ys Surehyb Chambers at 65 0C for 16 h. The hybridized slides were washed using Agilent Gene Expression wash buffers and scanned using the Agilent Microarray Scanner G Model G2565BA at 5 micron resolution. Feature extracted data was analysed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation. Set measurements less than 0.01 to 0.01 per chip, normalize to 50th percentile per gene, and normalize to specific samples.

Project description:AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two ?-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer.Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal ?-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains.

Dataset Information

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

Publications

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

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