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Journal of Bacteriology, October 2008, p. 6428-6438, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00749-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of Functional Tat Signal Sequences in Mycobacterium tuberculosis Proteins ^,

Justin A. McDonough ,^, Jessica R. McCann, Erin McElvania Tekippe, Jason S. Silverman,^¶ Nathan W. Rigel, and Miriam Braunstein^*

Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7290

Received 26 May 2008/ Accepted 16 July 2008

The twin-arginine translocation (Tat) pathway is a system used by some bacteria to export proteins out from the cytosol to the cell surface or extracellular environment. A functional Tat pathway exists in the important human pathogen Mycobacterium tuberculosis. Identification of the substrates exported by the Tat pathway can help define the role that this pathway plays in the physiology and pathogenesis of M. tuberculosis. Here we used a reporter of Tat export, a truncated β-lactamase, 'BlaC, to experimentally identify M. tuberculosis proteins with functional Tat signal sequences. Of the 13 proteins identified, one lacks the hallmark of a Tat-exported substrate, the twin-arginine dipeptide, and another is not predicted by in silico analysis of the annotated M. tuberculosis genome. Full-length versions of a subset of these proteins were tested to determine if the native proteins are Tat exported. For three proteins, expression in a tat mutant of Mycobacterium smegmatis revealed a defect in precursor processing compared to expression in the wild type, indicating Tat export of the full-length proteins. Conversely, two proteins showed no obvious Tat export in M. smegmatis. One of this latter group of proteins was the M. tuberculosis virulence factor phospholipase C (PlcB). Importantly, when tested in M. tuberculosis a different result was obtained and PlcB was exported in a twin-arginine-dependent manner. This suggests the existence of an M. tuberculosis-specific factor(s) for Tat export of a proven virulence protein. It also emphasizes the importance of domains beyond the Tat signal sequence and bacterium-specific factors in determining if a given protein is Tat exported.

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* Corresponding author. Mailing address: Department of Microbiology and Immunology, CB#7290, 804 Mary Ellen Jones, University of North Carolina, Chapel Hill, NC 27599-7290. Phone: (919) 966-5051. Fax: (919) 962-8103. E-mail: Miriam_Braunstein{at}med.unc.edu

Published ahead of print on 25 July 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

J.A.M. and J.R.M. contributed equally to this work.

Present address: Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536.

^¶ Present address: Department of Medical Microbiology and Immunology, University of Wisconsin—Madison School of Medicine and Public Health, Madison, WI.

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Journal of Bacteriology, October 2008, p. 6428-6438, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00749-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.