Analysis of high-level S-layer protein secretion inCaulobacter crescentus

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 ~31% of cell protein is RsaA. When multiple copies of rsaA were introduced, the level increased to ~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 resulted in a shorter half-life and a reduction in maximum protein synthesis levels. If secretion was prevented by knockout of type I transporter genes, RsaA levels dropped to 10% or less of normal, but with no significant reduction in rsaA mRNA. Overall, normal levels of RsaA were unexpectedly high, and still higher levels were not limited by transporter capability, the presence of an assembled S-layer, or the capacity of the cell’s physiology to produce large amounts of one protein. The normal upper limit of RsaA production appears to be controlled only by the level of an unusually stable message. Significant down-regulation is possible and is accomplished posttranscriptionally.