Affiliation:
1. Department of Plant and Microbial Biology, University of California, Berkeley, California 94720
Abstract
ABSTRACT
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 μM 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 μM. Importantly, the variation in green fluorescent protein fluorescence of biosensor cells on leaves suggested that large spatial variations in sugar availability occur on leaves.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
80 articles.
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