Biological sensor for sucrose availability: relative sensitivities of various reporter genes.
A set of three sucrose-regulated transcriptional fusions was constructed. Fusions p61RYTIR, 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. Cells of Erwinia herbicola containing these fusions are induced only in media amended with sucrose, fructose, or sorbose. While a large variation in sucrose-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, requiring approximately 300-fold fewer cells for a detectable response over all concentrations of sucrose. Similarly, inaZ was found to be more efficient than lacZ, requiring 30-fold fewer cells at 1.45 microM sucrose and 6,100-fold fewer cells at 29 mM sucrose for a quantifiable response. The fluorescence of individual cells containing p61RYTIR was quantified following epifluorescence microscopy in order to relate the fluorescence exhibited by populations of cells in batch cultures with that of individual cells in such cultures. While the mean fluorescence intensity of a population of individual cells increased with increasing concentrations of sucrose, a wide range of fluorescence intensity was seen among individual cells. For most cultures the distribution of fluorescence intensity among individual cells was log-normally distributed, but cells grown in intermediate concentrations of sucrose exhibited two distinct populations of cells, one having relatively low fluorescence and another with much higher fluorescence. When cells were inoculated onto bean leaves, whole-cell ice nucleation and gfp-based biological sensors for sucrose each indicated that the average concentration of sucrose on moist leaf surfaces was about 20 microM. Importantly, the variation in green fluorescent protein fluorescence of biosensor cells on leaves suggested that large spatial variations in sugar availability occur on leaves.
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA.
W G
WG
Miller
M T
MT
Brandl
B
B
Quiñones
S E
SE
Lindow
D001426:Bacterial Proteins; D015374:Biosensing Techniques; D004885:Erwinia / Q000235:genetics / Q000378:metabolism; D007887:Fabaceae / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007763:Lac Operon / Q000235:genetics / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010946:Plants, Medicinal; D010957:Plasmids / Q000235:genetics; D018272:Porins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D018272:Porins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C079242:ScrY protein, bacteria; C083535:scrR protein, bacteria; D049452:Green Fluorescent Proteins; D013395:Sucrose