Single live‐bacterial cell assay of promoter activity and regulation

Abstract

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 singlecell 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.

Molecular processes underlying synergistic linuron mineralization in a triple‐species bacterial consortium biofilm revealed by differential transcriptomics

“…Only a clear overexpression of the glycerate biosynthesis gcl operon was observed in WDL7 during consortium growth. This operon was shown to be induced by glyoxylate in Escherichia coli (Teramoto et al., ), suggesting that WDL7 senses a higher concentration of glyoxylate when grown in consortium conditions and thus that glyoxylate is a candidate metabolite that WDL7 could receive during consortium growth. As WDL7 does not show a strong change in regulation of metabolic pathways between growth conditions, 3,4‐DCA seems to remain the main carbon and energy source of WDL7 both in monoculture and consortium biofilm conditions.…”

Retropath: Automated Pipeline for Embedded Metabolic Circuits

“…32 When reacting with water and dioxygen, urate is transformed in (S)-(+)-allantoin, which is a regulator of the E. coli transcriptional activator AllS and repressor AllR, which participate in the regulation of more than 10 genes that are involved in the allantoin catabolism pathway 33 (see Figure 4). Teramoto et al 34 achieved a vector construct containing a green fluorescent protein (GFP) coding sequence next to the promoter of the E. coli gcl gene which is repressed by AllR. Such construct could be used to detect diabetes metillus since the presence of allantoin would repress the gcl gene promoter and thus the production of GFP, a detectable output signal.…”