PNNL

Abstract

Caulobacter crescentus, a ubiquitous aquatic bacterium, holds great promise as a bioremediation agent due to its ability to facilitate uranium (U) biomineralization. However, the mechanism by which C. crescentus tolerates U is still not well understood. It was recently reported that two outer membrane proteins, RsaFa and RsaFb, are important for U tolerance. These proteins are homologous to E. coli TolC—part of a multidrug efflux pump—and are involved in the S-­-layer translocation pathway in C. crescentus. Here we provide evidence that, unlike TolC, RsaFa and RsaFb are not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFa and RsaFb are required to prevent intracellular accumulation and aggregation of the S-­-layer protein RsaA; deletion of RsaFa and RsaFb led to a general survival defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and RNA-­-seq, we show that loss of both RsaFa and RsaFb led to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFa and RsaFb in cellular fitness and tolerance to antimicrobial agents and increase our understanding of the S-­-layer export mechanism on both the transcriptional and translational levels in C. crescentus.