Biological sensor for sucrose availability: Relative sensitivities of various reporter genes

A set of three sucrose-regulated transcriptional fusions was constructed. F usions p61PYTIR, p61RYlac, and p61RYice contain the scrR sucrose repressor gene and the promoterless gfp, lacZ, and inaZ reporter genes, respectively, fused to the scrY promoter from Salmonella enterica serovar Typhimurium. C ells of Erwinia herbicola containing these fusions are induced only in medi a amended with sucrose, fructose, or sorbose, While a large variation in su crose-dependent reporter gene activity was observed in cells harboring all gene fusions, fusions to the inaZ reporter gene yielded a much wider range of activity and were responsive to lower levels of sucrose than either lacZ or gfp. The lacZ reporter gene was found to be more efficient than gfp, re quiring approximately 300-fold fewer cells for a detectable response over a ll concentrations of sucrose, Similarly, inaZ was found to be more efficien t than lacZ, requiring 30-fold fewer cells at 1.45 muM sucrose and 6,100-fo ld fewer cells at 29 mM sucrose for a quantifiable response. The fluorescen ce of individual cells containing p61RYTIR was quantified following epifluo rescence microscopy in order to relate the fluorescence exhibited by popula tions of cells in batch cultures with that of individual cells in such cult ures, While the mean fluorescence intensity of a population of individual c ells increased with increasing concentrations of sucrose, a wide range of f luorescence intensity was seen among individual cells. For most cultures th e distribution of fluorescence intensity among individual cells was log-nor mally distributed, but cells grown in intermediate concentrations of sucros e exhibited two distinct populations of cells, one having relatively low fl uorescence and another with much higher fluorescence, When cells were inocu lated onto bean leaves, whole-cell ice nucleation and gfp-based biological sensors for sucrose each indicated that the average concentration of sucros e on moist leaf surfaces was about 20 muM. Importantly, the variation in gr een fluorescent protein fluorescence of biosensor cells on leaves suggested that large spatial variations in sugar availability occur on leaves.