title: Construction and application of a luxABCDE reporter system for real-time monitoring of Enterococcus faecalis gene expression and growth.
abstract: The present work describes the construction of a novel molecular tool for luciferase-based bioluminescence (BL) tagging of Enterococcus faecalis. To this end, a vector (pSL101) and its derivatives conferring a genetically encoded bioluminescent phenotype on all tested strains of E. faecalis were constructed. pSL101 harbors the luxABCDE operon from pPL2lux and the pREG696 broad-host-range replicon and axe-txe toxin-antitoxin cassette, providing segregational stability for long-term plasmid persistence in the absence of antibiotic selection. The bioluminescent signals obtained from three highly expressed promoters correlated linearly (R(2) > 0.98) with the viable-cell count. We employed lux-tagged E. faecalis strains to monitor growth in real time in milk and urine in vitro. Furthermore, bioluminescence imaging (BLI) was used to visualize the magnitude of the bacterial burden during infection in the Galleria mellonella model system. To our knowledge, pSL101 is the first substrate addition-independent reporter system developed for BLI of E. faecalis and an efficient tool for spatiotemporal tracking of bacterial growth and quantitative determination of promoter activity in real time, noninvasively, in infection model systems.
authors: Department of Chemistry, Biotechnology and Food Science, Laboratory of Microbial Gene Technology and Food Microbiology, Norwegian University of Life Sciences, Ås, Norway. sabina.leantilarosa@umb.no
authors: Sabina Leanti
authors: SL
authors: La Rosa
authors: Dzung B
authors: DB
authors: Diep
authors: Ingolf F
authors: IF
authors: Nes
authors: Dag Anders
authors: DA
authors: Brede
mesh_terms: D000818:Animals; D058491:Bacterial Load; D013293:Enterococcus faecalis / Q000235:genetics* / Q000254:growth & development*; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors; D007915:Lepidoptera / Q000382:microbiology; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism*; D008163:Luminescent Measurements; D050296:Microbial Viability; D008892:Milk / Q000382:microbiology; D009876:Operon; D010957:Plasmids; D014556:Urine / Q000382:microbiology
chemical_list: D008156:Luciferases