title: Transposon Mutagenesis Paired with Deep Sequencing of Caulobacter crescentus under Uranium Stress Reveals Genes Essential for Detoxification and Stress Tolerance.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Biosciences and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA.
authors.forename: Mimi C
authors.initials: MC
authors.lastname: Yung
authors.affiliation: Biosciences and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA.
authors.forename: Dan M
authors.initials: DM
authors.lastname: Park
authors.affiliation: Biosciences and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA.
authors.forename: K Wesley
authors.initials: KW
authors.lastname: Overton
authors.affiliation: DOE Joint Genome Institute, Walnut Creek, California, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Blow
authors.affiliation: DOE Joint Genome Institute, Walnut Creek, California, USA.
authors.forename: Cindi A
authors.initials: CA
authors.lastname: Hoover
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: John
authors.initials: J
authors.lastname: Smit
authors.affiliation: Department of Biology, California State University Northridge, Northridge, California, USA.
authors.forename: Sean R
authors.initials: SR
authors.lastname: Murray
authors.affiliation: Department of Developmental Biology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Dante P
authors.initials: DP
authors.lastname: Ricci
authors.affiliation: Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland.
authors.forename: Beat
authors.initials: B
authors.lastname: Christen
authors.affiliation: Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, USA.
authors.forename: Grant R
authors.initials: GR
authors.lastname: Bowman
authors.affiliation: Biosciences and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA jiao1@llnl.gov.
authors.forename: Yongqin
authors.initials: Y
authors.lastname: Jiao
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016935:Caulobacter crescentus / Q000235:genetics / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D016680:Genome, Bacterial; D016296:Mutagenesis; D013312:Stress, Physiological / Q000187:drug effects*; D059467:Transcriptome; D014501:Uranium / Q000633:toxicity*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D014501:Uranium