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Journal of Bacteriology, October 2009, p. 6292-6299, Vol. 191, No. 20
0021-9193/09/$08.00+0     doi:10.1128/JB.00824-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Organophosphate Hydrolase in Brevundimonas diminuta Is Targeted to the Periplasmic Face of the Inner Membrane by the Twin Arginine Translocation Pathway{triangledown}

Purushotham Gorla,1 Jay Prakash Pandey,1 Sunil Parthasarathy,1 Mike Merrick,2 and Dayananda Siddavattam1*

Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India,1 Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom2

Received 24 June 2009/ Accepted 10 August 2009

A twin arginine translocation (Tat) motif, involved in transport of folded proteins across the inner membrane, was identified in the signal peptide of the membrane-associated organophosphate hydrolase (OPH) of Brevundimonas diminuta. Expression of the precursor form of OPH carrying a C-terminal His tag in an opd-negative background and subsequent immunoblotting with anti-His antibodies showed that only the mature form of OPH associated with the membrane and that the precursor form of OPH was entirely found in the cytoplasm. When OPH was expressed without the signal peptide, most of it remained in the cytoplasm, where it was apparently correctly folded and showed activity comparable to that of the membrane-associated OPH encoded by the wild-type opd gene. Amino acid substitutions in the invariant arginine residues of the Tat signal peptide affected both the processing and localization of OPH, confirming a critical role for the Tat system in membrane targeting of OPH in B. diminuta. The localization of OPH to the periplasmic face of the inner membrane in B. diminuta was demonstrated by proteinase K treatment of spheroplasts and also by fluorescence-activated cell sorting analysis of cells expressing OPH-green fluorescent protein fusions with and without an SsrA tag that targets cytoplasmic proteins to the ClpXP protease.

* Corresponding author. Mailing address: Dept. of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India. Phone: 91 40 23134578. Fax: 91 40 23010120. E-mail: sdsl{at}uohyd.ernet.in

{triangledown} Published ahead of print on 21 August 2009.

Journal of Bacteriology, October 2009, p. 6292-6299, Vol. 191, No. 20
0021-9193/09/$08.00+0     doi:10.1128/JB.00824-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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