Identification of functional Tat signal sequences in Mycobacterium tuberculosis proteins.
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 beta-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 Deltatat 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.
McDonough
Justin A
JA
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599-7290, USA.
McCann
Jessica R
JR
Tekippe
Erin McElvania
EM
Silverman
Jason S
JS
Rigel
Nathan W
NW
Braunstein
Miriam
M
D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015151:Immunoblotting; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D008957:Models, Genetic; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism / Q000502:physiology
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D001120:Arginine; D010738:Type C Phospholipases; C101029:MpcB protein, Mycobacterium tuberculosis