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Expression, Functional Characterization and X-ray Analysis of HosA, A Member of MarR Family of Transcription Regulator from Uropathogenic Escherichia coli

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Abstract

Regulators belonging to multiple antibiotic resistance regulator (MarR) family are widespread in prokaryotes and are involved in regulation of genes that are responsible for virulence and pathogenicity in most of the clinically important pathogens. Here we report the transcriptional, biophysical, and X-ray analyses of homologue of SlyA (HosA), a member of MarR family that is predominantly present in the pathogenic strains of Enterobacteriaceae family. The initiation of hosA transcription was observed to occur at two independent start sites and subsequent binding study has revealed that the purified HosA interacts with its upstream region suggesting a probable autoregulation. The secondary structure analysis through circular dichroism spectroscopy demonstrated that HosA is predominantly composed of the alpha helix with higher thermal stability. To further understand the three-dimensional structure, HosA was crystallized and the crystals were diffracted to maximum of 2.9 Ǻ on exposure to X-rays. Analysis of the X-ray crystallographic data suggested a primitive space group (P 6 ? 2 2), with unit cell parameters a = b = 64.19 Å and c = 244.25 Å. The solvent content and Matthews coefficient were 41 % and 2.11 Å3 Da−1, respectively, which indicated the existence of two molecules of HosA in the asymmetric unit of crystal.

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Abbreviations

MALDI:

Matrix assisted laser desorption/ionization

EMSA:

Electrophoretic mobility shift assay

IEF:

Isoelectric focusing

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

TCEP:

Tris (2-carboxyethyl) phosphine

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Acknowledgments

This work is supported by the core grant from CDFD, Hyderabad, Department of Biotechnology, Government of India. Ajit Roy is the recipient of a doctoral research fellowship from Council of Scientific and Industrial Research (CSIR), Government of India and is registered under the academic program (Ph.D) of Manipal University. The authors would like to acknowledge the members of X-ray Crystallography facility at CSIR-CCMB, Hyderabad and Protein Crystallography beamline (PX-BL21), Indus 2 at RRCAT, Indore, India for their assistance during protein diffraction trials. This work has neither been published nor been simultaneously submitted for publication elsewhere. All authors agree to the submission to the journal.

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Authors and Affiliations

  1. Computational and Functional Genomics Group, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, 500001, India

    Ajit Roy, Bhavik Sawhney, Debasish Kumar Ghosh & Akash Ranjan

  2. Graduate Studies, Manipal University, Manipal, 576104, India

    Ajit Roy, Bhavik Sawhney & Debasish Kumar Ghosh

  3. Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad, Telangana, 500007, India

    Ravikumar Reddi & Anthony Addlagatta

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  1. Ajit Roy
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  2. Ravikumar Reddi
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  4. Debasish Kumar Ghosh
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Correspondence to Akash Ranjan.

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Fig. 1

Data analysis of gel exclusion chromatography. (A) Tabulated comparison of Kav of various molecular weight standards that were utilized for calibration in gel exclusion chromatography. The molecular weight of HosA was calculated from the plot between Kav and molecular weight of different standards and is highlighted in bold and italic. (B) Plot between the Kav and molecular weight (in kDa) of different protein standards. (TIFF 531 kb)

Fig. 2

Secondary structure analysis of different MarR proteins. Tabulated comparison of secondary structure content of different proteins from MarR family. (TIFF 480 kb)

Fig. 3

Representative diffraction image of poorly diffracted HosA crystal. (A) The diffraction image of HosA crystal that was obtained in sitting drop condition. (B) The diffraction image of HosA crystal that was obtained under oil in sitting drop condition. (TIFF 0 kb)

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Roy, A., Reddi, R., Sawhney, B. et al. Expression, Functional Characterization and X-ray Analysis of HosA, A Member of MarR Family of Transcription Regulator from Uropathogenic Escherichia coli . Protein J 35, 269–282 (2016). https://doi.org/10.1007/s10930-016-9670-1

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