title: Identification of in vivo regulators of the Vibrio cholerae xds gene using a high-throughput genetic selection.
abstract: Vibrio cholerae, the causative agent of cholera, remains a threat to public health in areas with inadequate sanitation. As a waterborne pathogen, V. cholerae moves between two dissimilar environments, aquatic reservoirs and the intestinal tract of humans. Accordingly, this pathogen undergoes adaptive shifts in gene expression throughout the different stages of its lifecycle. One particular gene, xds, encodes a secreted exonuclease that was previously identified as being induced during infection. Here we sought to identify regulators responsible for the in vivo-specific induction of xds. A transcriptional fusion of xds to two consecutive antibiotic resistance genes was used to select transposon mutants that had inserted within or adjacent to regulatory genes and thereby caused increased expression of the xds fusion under non-inducing conditions. Large pools of selected insertion sites were sequenced in a high throughput manner using Tn-seq to identify potential mechanisms of xds regulation. Our selection identified the two-component system PhoB/R as the dominant activator of xds expression. In vitro validation confirmed that PhoB, a protein which is only active during phosphate limitation, was responsible for xds activation. Using xds expression as a biosensor of the extracellular phosphate level, we observed that the mouse small intestine is a phosphate-limited environment.
authors.affiliation: Howard Hughes Medical Institute and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, 02111, USA.
authors.forename: Emilykate
authors.initials: E
authors.lastname: McDonough
authors.forename: David W
authors.initials: DW
authors.lastname: Lazinski
authors.forename: Andrew
authors.initials: A
authors.lastname: Camilli
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000378:metabolism*; D015374:Biosensing Techniques; D004251:DNA Transposable Elements; D024881:Drug Resistance, Bacterial; D005092:Exonucleases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D057166:High-Throughput Screening Assays; D051379:Mice; D016254:Mutagenesis, Insertional; D010710:Phosphates / Q000032:analysis; D012641:Selection, Genetic; D015533:Transcriptional Activation; D014734:Vibrio cholerae / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D010710:Phosphates; C061278:PhoB protein, Bacteria; D005092:Exonucleases