Single live-bacterial cell assay of promoter activity and regulation.
Laboratory cultures of a single species of bacteria harboring the same genetic background include heterogeneous cell populations, each differing in apparent morphology and physiology, as found in natural environments. To get insights into difference in the genome expression between individual cells, we constructed various types of the cell chip for monitoring the growth and fate of individual bacterial cells. Immobilization of portions of Escherichia coli culture within these cell chips was established after raising the local temperature in the presence of poly-(N-isopropylacrylamide) (PNIPAAm). The newly developed cell-chip system allows the investigation of activity and regulation of green fluorescent protein (GFP)-fused promoter in single live-bacterial cells for prolonged time under controlled culture conditions. Using this single-cell observation system, we succeeded, for the first time, the real-time single-cell assay of promoter activity of the E. coli gcl gene encoding glyoxylate carboligase as a model system, and the kinetics of gcl induction by an effector glyoxylate. Marked heterogeneity was found in the expression level of the gcl promoter. The heterogeneity in gcl promoter activity was, however, confirmed by Flow cytometry of suspension cultures. Our success provides an experimental system for the increased demand of single-cell biology in bacterial studies.
Teramoto
Jun
J
Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan.
Yamanishi
Yoko
Y
Magdy
El-Shimy H
el-SH
Hasegawa
Akiko
A
Kori
Ayako
A
Nakajima
Masahiro
M
Arai
Fumihito
F
Fukuda
Toshio
T
Ishihama
Akira
A
D000178:Acrylamides / Q000737:chemistry; D001431:Bacteriological Techniques; D001681:Biological Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics
D000178:Acrylamides; D029968:Escherichia coli Proteins; C447153:GclR protein, E coli; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; C067295:N-isopropylacrylamide