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Journal of Bacteriology, December 2004, p. 8000-8009, Vol. 186, No. 23
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.23.8000-8009.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Two Outer Membrane Proteins Are Required for Maximal Type I Secretion of the Caulobacter crescentus S-Layer Protein

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada

Received 25 May 2004/ Accepted 26 August 2004

Transport of RsaA, the crystalline S-layer subunit protein of Caulobacter crescentus, is mediated by a type I secretion mechanism. Two proteins have been identified that play the role of the outer membrane protein (OMP) component in the RsaA secretion machinery. The genes rsaF_a and rsaF_b were identified by similarity to the Escherichia coli hemolysin secretion OMP TolC by using the C. crescentus genome sequence. The rsaF_a gene is located several kilobases downstream of the other transporter genes, while rsaF_b is completely unlinked. An rsaF_a knockout had 56% secretion compared to wild-type levels, while the rsaF_b knockout reduced secretion levels to 79%. When expression of both proteins was eliminated, there was no RsaA secretion, but a residual level of 9% remained inside the cell, suggesting posttranslational autoregulation. Complementation with either of the individual rsaF genes by use of a multicopy vector, which resulted in 8- to 10-fold overexpression of the proteins, did not restore RsaA secretion to wild-type levels, indicating that both rsaF genes were required for full-level secretion. However, overexpression of rsaF_a (with normal rsaF_b levels) in concert with overexpression of rsaA resulted in a 28% increase in RsaA secretion, indicating a potential for significantly increasing expression levels of an already highly expressing type I secretion system. This is the only known example of type I secretion requiring two OMPs to assemble a fully functional system.

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