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Journal of Bacteriology, August 2002, p. 4500-4509, Vol. 184, No. 16
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.16.4500-4509.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Proteolytic Cleavage of the FlhB Homologue YscU of Yersinia pseudotuberculosis Is Essential for Bacterial Survival but Not for Type III Secretion

Moa Lavander,¹ Lena Sundberg,^1,2 Petra J. Edqvist,² Scott A. Lloyd,^2, Hans Wolf-Watz,² and Åke Forsberg^1,2*

Department of Medical Countermeasures, Division of NBC-Defense, Swedish Defense Research Agency, S-901 82 Umeå,¹ Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden²

Received 13 March 2002/ Accepted 17 May 2002

Pathogenic Yersinia species employ a type III secretion system (TTSS) to target antihost factors, Yop proteins, into eukaryotic cells. The secretion machinery is constituted of ca. 20 Ysc proteins, nine of which show significant homology to components of the flagellar TTSS. A key event in flagellar assembly is the switch from secreting-assembling hook substrates to filament substrates, a switch regulated by FlhB and FliK. The focus of this study is the FlhB homologue YscU, a bacterial inner membrane protein with a large cytoplasmic C-terminal domain. Our results demonstrate that low levels of YscU were required for functional Yop secretion, whereas higher levels of YscU lowered both Yop secretion and expression. Like FlhB, YscU was cleaved into a 30-kDa N-terminal and a 10-kDa C-terminal part. Expression of the latter in a wild-type strain resulted in elevated Yop secretion. The site of cleavage was at a proline residue, within the strictly conserved amino acid sequence NPTH. A YscU protein with an in-frame deletion of NPTH was cleaved at a different position and was nonfunctional with respect to Yop secretion. Variants of YscU with single substitutions in the conserved NPTH sequence—i.e., N263A, P264A, or T265A—were not cleaved but retained function in Yop secretion. Elevated expression of these YscU variants did, however, result in severe growth inhibition. From this we conclude that YscU cleavage is not a prerequisite for Yop secretion but is rather required to maintain a nontoxic fold.

Present address: Department of Pediatrics, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201.

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