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

UNLABELLED: The ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in the C. crescentus genome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters (rsaFa and rsaFb), a stress-responsive transcription factor (cztR), or a ppGpp synthetase/hydrolase (spoT) exhibited a significantly lower survival rate under U stress. RsaFa and RsaFb, which are homologues of TolC in Escherichia coli, have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation of rsaFa and rsaFb by 4- and 10-fold, respectively, in the presence of U. We additionally show that rsaFa mutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaFa and RsaFb in U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance in C. crescentus. IMPORTANCE: Caulobacter crescentus is an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance in C. crescentus. The genes that we identified have previously remained elusive using other omics approaches and thus provide significant insight into the mechanisms of U resistance by C. crescentus. In particular, we show that outer membrane transporters RsaFa and RsaFb, previously known as part of the S-layer export machinery, may confer U resistance by U efflux and/or by maintaining membrane integrity during U stress.

Inhibition of herpes simplex virus type 1 with the modified green tea polyphenol palmitoyl-epigallocatechin gallate.

Green tea polyphenol epigallocatechin gallate (EGCG) is a strong antioxidant that has previously been shown to reduce the number of plaques in HIV-infected cultured cells. Modified EGCG, palmitoyl-EGCG (p-EGCG), is of interest as a topical antiviral agent for herpes simplex virus (HSV-1) infections. This study evaluated the effect of p-EGCG on HSV-infected Vero cells. Results of cell viability and cell proliferation assays indicate that p-EGCG is not toxic to cultured Vero cells and show that modification of the green tea polyphenol epigallocatechin gallate (EGCG) with palmitate increases the effectiveness of EGCG as an antiviral agent. Furthermore, p-EGCG is a more potent inhibitor of herpes simplex virus 1 (HSV-1) than EGCG and can be topically applied to skin, one of the primary tissues infected by HSV. Viral binding assay, plaque forming assay, PCR, real-time PCR, and fluorescence microscopy were used to demonstrate that p-EGCG concentrations of 50 µM and higher block the production of infectious HSV-1 particles. p-EGCG was found to inhibit HSV-1 adsorption to Vero cells. Thus, p-EGCG may provide a novel treatment for HSV-1 infections.