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Overlapping protective roles for glutathione transferase gene family members in chemical and oxidative stress response in Agrobacterium tumefaciens

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Abstract

In the present work, we describe the characterisation of the glutathione transferase (GST) gene family from Agrobacterium tumefaciens C58. A genome survey revealed the presence of eight GST-like proteins in A. tumefaciens (AtuGSTs). Comparison by multiple sequence alignment generated a dendrogram revealing the phylogenetic relationships of AtuGSTs-like proteins. The beta and theta classes identified in other bacterial species are represented by five members in A. tumefaciens C58. In addition, there are three “orphan” sequences that do not fit into any previously recognised GST classes. The eight GST-like genes were cloned, expressed in Escherichia coli and their substrate specificity was determined towards 17 different substrates. The results showed that AtuGSTs catalyse a broad range of reactions, with different members of the family exhibiting quite varied substrate specificity. The 3D structures of AtuGSTs were predicted using molecular modelling. The use of comparative sequence and structural analysis of the AtuGST isoenzymes allowed us to identify local sequence and structural characteristics between different GST isoenzymes and classes. Gene expression profiling was conducted under normal culture conditions as well as under abiotic stress conditions (addition of xenobiotics, osmotic stress and cold and heat shock) to induce and monitor early stress-response mechanisms. The results reveal the constitutive expression of GSTs in A. tumefaciens and a modulation of GST activity after treatments, indicating that AtuGSTs presumably participate in a wide range of functions, many of which are important in counteracting stress conditions. These functions may be relevant to maintaining cellular homeostasis as well as in the direct detoxification of toxic compounds.

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Abbreviations

AtuGST:

Agrobacterium tumefaciens glutathione transferase

BCNB:

1-Bromo-2,4-dinitrobenzene

CDNB:

1-Chloro-2,4-dinitrobenzene

CuOOH:

Cumene hydroperoxide

Fluorodifen:

4-Nitrophenyl 2-nitro-4-trifluoromethylphenyl ether

FDNB:

1-Fluoro-2,4-dinitrobenzene

G-site:

Glutathione binding site

GSH:

Glutathione

GST:

Glutathione transferase

H-site:

Hydrophobic binding site

IDNB:

1-Iodo-2,4-dinitrobenzene

OaGST:

Ochrobactrum anthropi glutathione transferase

ORF:

Open reading frame

PmGST:

Proteus mirabilis glutathione transferase

pNBC:

p-Nitrobenzyl chloride

Nb-GSH:

S-(p-nitrobenzyl)-glutathione

pNPA:

p-Nitrophenyl acetate

RMSD:

Root mean square deviation

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Acknowledgements

This work was partially supported by the Hellenic General Secretariat for Research and Technology. ACP thanks the Academy of Finland for financial support (grant number 121278) and CSC-IT Center for Science Ltd. (Espoo, Finland) for access to computing resources.

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Authors and Affiliations

  1. Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, 11855, Athens, Greece

    Katholiki Skopelitou & Nikolaos E. Labrou

  2. Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, 20521, Finland

    Abdi W. Muleta & Anastassios C. Papageorgiou

  3. Laboratory of Plant Breeding and Biometry, Department of Crop Science, Agricultural University of Athens, Athens, Greece

    Ourania Pavli & Georgios N. Skaracis

  4. Laboratory of Molecular Biology, Department of Agricultural Biotechnology, Agricultural University of Athens, Athens, Greece

    Emmanouil Flemetakis

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  1. Katholiki Skopelitou
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  2. Abdi W. Muleta
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  3. Ourania Pavli
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  4. Georgios N. Skaracis
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  6. Anastassios C. Papageorgiou
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Correspondence to Nikolaos E. Labrou.

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Supplementary Table 1

Primers used for gene amplification and cloning. (DOCX 14 kb)

Supplementary Table 2

Primers used for real-time RT-qPCR. (DOCX 15 kb)

Supplementary Table 3

The crystal structures used as templates in homology modeling. (DOCX 17 kb)

Supplementary Table 4

RMSD (in Å) between the homology models. (DOCX 14 kb)

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Skopelitou, K., Muleta, A.W., Pavli, O. et al. Overlapping protective roles for glutathione transferase gene family members in chemical and oxidative stress response in Agrobacterium tumefaciens . Funct Integr Genomics 12, 157–172 (2012). https://doi.org/10.1007/s10142-011-0248-x

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