Analysis of high-level S-layer protein secretion in Caulobacter crescentus

Canadian Journal of Microbiology
Janny Ho Yu LauJ Smit

Abstract

Caulobacter crescentus exhibits a hexagonally arranged protein layer on its outermost surface. RsaA, the sole protein of this "S-layer", is secreted by a type I (ABC) transporter. Few type I transporters show high-level secretion, and few bacterial S-layers have been carefully examined for the amount of protein synthesis capacity needed to maintain cell coverage. Here we determined RsaA levels by quantitative immunoblotting methods, learned that very stable mRNA is a key factor in high-level secretion, and found that the transporter was capable of still higher secretion. A propensity for RsaA to aggregate was a barrier to quantitation, but with the use of S-layer shedding mutants and methods to keep RsaA soluble, we learned that approximately 31% of cell protein is RsaA. When multiple copies of rsaA were introduced, the level increased to approximately 51% of cell protein, a higher level than we are aware of for any protein in any bacterium. Unexpectedly, in comparing normal and S-layer shedding strains, an assembled S-layer was not a significant barrier to elevated secretion. The rsaA mRNA half-life was determined by real-time PCR to be 36 min, ranking with the most stable known in bacteria. A modification of the 5' region res...Continue Reading

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Citations

Sep 8, 2011·Antimicrobial Agents and Chemotherapy·Mark DuvalLisa A Cavacini
May 29, 2012·Applied and Environmental Microbiology·César de la Fuente-NúñezRobert E W Hancock
Sep 14, 2010·Journal of Bacteriology·Fernando AmatMark Horowitz
Sep 8, 2016·Acta Crystallographica. Section F, Structural Biology Communications·Michael D JonesJohn Smit
Dec 19, 2019·Proceedings of the National Academy of Sciences of the United States of America·Jonathan HerrmannSoichi Wakatsuki
May 13, 2017·Biophysical Journal·Jonathan HerrmannSoichi Wakatsuki

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