| Summary: | The transport of RsaA, the S-layer subunit protein of Caulobacter crescentus, is mediated by ABC transporter (type I) secretion. The ABC transporter and membrane fusion protein (MFP) components were previously reported as downstream of rsaA, however the two outer membrane proteins (OMP) were not described. This study aims to elucidate the transcriptional regulation of the rsaADE genes as well as the role of two putative OMP genes, rsaFa and rsaFb, in the secretion of RsaA. The rsaADE genes were previously thought to be transcribed as an operon in a similar fashion to that of the Escherichia coli alpha-hemolysin (HlyA) system. Here I show that contrary to previous hypotheses, the rsaD and rsaE genes appear to be transcribed together using a promoter found between rsaA and rsaD suggesting that they are transcribed irrespective of rsaA. The outer membrane proteins of this system had been suggested, but not characterized. Two candidates for the OMP, rsaFa and rsaFb, were identified by similarity to the E. coli HlyA secretion OMP TolC, using the available C. crescentus genome sequence and were modeled using the solved TolC structure. rsaFa was found several Kb downstream of the other transporter genes, while rsaFb is in an apparently random location. The rsaF genes were disrupted to determine if they were involved in RsaA secretion. Knockout of rsaFa reduced secretion to ~54% of wild type levels while the rsaFb knockout reduced secretion levels to ~76%. When expression of both proteins was eliminated there was no RsaA secretion, but a residual level of ~9% remained intact inside the cell, suggesting posttransiational down regulation. Complementation with either of the individual rsaF genes using a multi-copy vector (and demonstration of overexpression) did not restore RsaA secretion to wild type levels indicating both rsaFa and rsaFb are required for normal levels of S-layer secretion. However, overexpression of rsaFa (with normal rsaFb levels) in concert with multi-copy expression of rsaA resulted in a 28% increase in RsaA secretion, indicating a potential for significantly increasing expression levels of an already high level type I secretion system. This is the only known example of type I secretion requiring two outer membrane proteins to assemble a fully functional system. It appears that production of RsaA is self-regulated, such that a build up of any amount of RsaA inside the cell due to blockage of transport limits RsaA production and severely impedes cell growth showing no signs of RsaA degradation. Secretion of RsaA appears to be a function of the number and type of outer membrane proteins as well as the amount of RsaA produced. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate
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