Shotgun Proteomic Analysis Unveils Survival and Detoxification Strategies byCaulobacter crescentusduring Exposure to Uranium, Chromium, and Cadmium

Abstract

The ubiquitous bacterium Caulobacter crescentus holds promise to be used in bioremediation applications due to its ability to mineralize U(VI) under aerobic conditions. Here, cell free extracts of C. crescentus grown in the presence of uranyl nitrate [U(VI)], potassium chromate [Cr(VI)], or cadmium sulfate [Cd(II)] were used for label-free proteomic analysis. Proteins involved in two-component signaling and amino acid metabolism were up-regulated in response to all three metals, and proteins involved in aerobic oxidative phosphorylation and chemotaxis were down-regulated under these conditions. Clustering analysis of proteomic enrichment revealed that the three metals also induce distinct patterns of up- or down-regulated expression among different functional classes of proteins. Under U(VI) exposure, a phytase enzyme and an ABC transporter were up-regulated. Heat shock and outer membrane responses were found associated with Cr(VI), while efflux pumps and oxidative stress proteins were up-regulated with Cd(II). Experimental validations were performed on select proteins. We found that a phytase plays a role in U(VI) and Cr(VI) resistance and detoxification and that a Cd(II)-specific transporter confers Cd(II) resistance. Interestingly, analysis of promoter regions in genes associated with differentially expressed proteins suggests that U(VI) exposure affects cell cycle progression.

Transposon Mutagenesis Paired with Deep Sequencing of Caulobacter crescentus under Uranium Stress Reveals Genes Essential for Detoxification and Stress Tolerance

“…Given that similar numbers of unique insertions are present in our data sets compared to those of other Tn-seq studies, we believe that the low fitness values are indicative of the fact that C. crescentus has a broad and relatively graded response to U toxicity. Consistent with this notion, previous proteomic and transcriptomic studies (23,24) also indicate that a small group of specialized genes cannot account for U resistance. Instead, the response occurs on a global level, involving many genes from different pathways serving functions that may act redundantly to mitigate U toxicity.…”

“…In summary, our Tn-seq screen for genes involved in detoxification and stress regulation during U exposure by C. crescentus has yielded several novel targets whose identity and function had previously remained elusive through other types of omics analyses (23,24). The results highlight that genes necessary for longer-term growth and survival under U stress do not significantly overlap those that are upregulated in response to acute U stress.…”

“…Proteins upregulated in response to U include the periplasmic protein UrcA, a phytase enzyme, two-component signaling factors, and an ABC transporter. However, individual deletion of these upregulated genes did not increase U susceptibility (23,24), indicating that these genes are not required for U tolerance. These results further suggest that the cellular response to U is complex and includes more than a direct response to acute toxicity.…”

“…C. crescentus is known to tolerate high levels of U(VI) and is able to facilitate U biomineralization through the formation of uranium phosphate precipitates (4). Transcriptomic and proteomic studies during U exposure in C. crescentus revealed dozens of genes with significant changes in expression in response to U; these genes do not appear to overlap substantially with those that are variably expressed in response to other heavy metal stresses, such as cadmium (Cd) and chromium, suggesting a divergent cellular response to U(VI) (23,24). This response manifests, in part, as a temporary arrest in cell cycle progression and DNA replication, along with some cell filamentation (a possible consequence of U-induced DNA damage) observed during growth recovery following U detoxification (25).…”

Modulation of Medium pH by Caulobacter crescentus Facilitates Recovery from Uranium-Induced Growth Arrest

“…C. crescentus exposed to high levels of U(VI) arrested cell cycle progression; the stalked-cell transition and the initiation of DNA replication were blocked in swarmer cells, while cell division was inhibited in stalked and predivisional cells. Recent proteomic analysis indicated that the levels of two master cell cycle regulators, CtrA and DnaA, were significantly reduced during U(VI) exposure (41). During the C. crescentus cell cycle, the abundance of these regulators is tightly controlled in order to precisely regulate the timing of DNA replication.…”