Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multi-species context

Miao, Huang; Ratnasingam, Shalini; Pu, Chui San; Desai, Malan Manish; Sze, Chun Chau

doi:10.1016/j.mimet.2008.09.015

Cited by 40 publications

(33 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Physiological heterogeneity of E. coli populations was also detected on the basis of respiratory activity and membrane potential (Rezaeinejad & Ivanov 2010). Transcriptional heterogeneity within the E. coli population is influenced by partner species in mixed cultures with other bacterial species (Miao et al. 2009).…”

Section: Discussionmentioning

confidence: 99%

Single live‐bacterial cell assay of promoter activity and regulation

Teramoto¹,

Yamanishi²,

Magdy³

et al. 2010

Genes to Cells

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Single live‐bacterial cell assay of promoter activity and regulation

Teramoto¹,

Yamanishi²,

Magdy³

et al. 2010

Genes to Cells

View full text Add to dashboard Cite

show abstract

“…Two E. coli K-12 strains were used: MG1655 (F -λ -ilvG -rfb-50 rph-1); and SCC1 (MG1655 P A1/04/03 -gfpmut3*) which constitutively expresses green fluorescent protein (GFP; Miao et al, 2009). A single colony was picked from an agar plate and used to inoculate 20 mL of 2×LB (20 g/L tryptone, 10 g/L yeast extract (both Difco) and 10 g/L NaCl (Sigma)), which was grown overnight in a 250 mL…”

Section: Bacterial Strains and Methodsmentioning

confidence: 99%

The effects of orange juice clarification on the physiology of Escherichia coli; growth-based and flow cytometric analysis

Anvarian

Smith

Overton

2016

International Journal of Food Microbiology

View full text Add to dashboard Cite

Orange juice (OJ) is a food product available in various forms which can be processed to a greater or lesser extent. Minimally-processed OJ has a high consumer perception but presents a potential microbiological risk due to acid-tolerant bacteria. Clarification of OJ (such as removal of cloud) is a common processing step in many OJ products. However, many of the antimicrobial components of OJ such as essential oils are present in the cloud fraction. Here, the effect of clarification by filtration on the viability and physiology of Escherichia coli K-12 was tested using total viable count (TVC) and flow cytometric (FCM) analysis. The latter technique was also used to monitor intracellular pH during incubation in OJ. Removal of the OJ cloud fraction was shown to have dramatic effects on bacterial viability and physiology during storage at a range of incubation temperatures. For instance, at 4 °C, a significantly lower number of healthy cells and a significantly higher number of injured cells were observed in 0.22 μm-filtered OJ at 24 hours post-inoculation, compared to filtered OJ samples containing particles between 0.22 µm and 11 µm in size. Similarly, there was a significant difference between the number of healthy bacteria in the 0.7 μm-filtered OJ and both 0.22 μm-filtered and 1.2 μm-filtered OJ after 24 hours incubation at 22.5 °C. This indicated that OJ cloud between 0.7 μm and 0.22 μm in size might have an adverse effect on the viability of E. coli K-12. Furthermore, FCM allowed the rapid analysis of bacterial physiology without the requirement for growth on agar A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

show abstract

“…However, H 2 O 2 , lactic acid, and acetic acid are also known to induce the VBNC state in E. coli (Li, Ahn, & Mustapha, ; Oliver, ). E. coli K‐12 strain SSC1 used here constitutively expresses green fluorescent protein (GFP) (Miao, Ratnasingam, Pu, Desai, & Sze, ); It has previously been demonstrated that decreases in intracellular pH (pH i ) result in GFP deactivation (Kneen, Farinas, Li, & Verkman, ). The primary aim of this study was to use flow cytometry (FCM) to investigate the effects of washing E. coli K‐12 SCC1 with the sanitizers AC, H 2 O 2 , and organic acids on the viability, physiological state, and pH i of the cells as well as their culturability as measured using plate counts, before and after their inoculation in OJ.…”

Section: Introductionmentioning

confidence: 99%

Flow cytometry and growth‐based analysis of the effects of fruit sanitation on the physiology of Escherichia coli in orange juice

Anvarian

Smith

Overton

2019

Food Science & Nutrition

View full text Add to dashboard Cite

Chlorine‐based solutions are commonly used to sanitize orange fruits prior to juice extraction. We used flow cytometry ( FCM ) to investigate the physiology of Escherichia coli following its subjection to chlorine‐based solutions and alternative sanitizing agents (H 2 O 2 and organic acids). Green fluorescent protein ( GFP )‐generating E. coli K‐12 were washed with 50–200 ppm available chlorine ( AC ), 1%–5% H 2 O 2 , 2%–4% citric acid, 4% acetic acid, or 4% lactic acid, after which they were added to 1.2 μm‐filtered orange juice ( OJ ). Cell physiology was investigated with FCM during storage at 4°C, and culturability was determined using plate counting. Analysis of GFP fluorescence allowed estimation of intracellular pH ( pH i ). FCM results demonstrated an inverse relationship between the concentration of AC or H 2 O 2 and cellular health in OJ . Higher concentrations of sanitizer also resulted in a significantly greater number of viable but nonculturable ( VBNC ) cells. Real‐time FCM showed that supplementation of AC with 2% citric acid, but not with 100 ppm of Tween‐80, led to a significant reduction in pH i of the cells incubated in OJ , and that the majority of the reduction in pH i occurred during the first 2 min of incubation in OJ . Organic acids were found to be more effective than both AC and H 2 O 2 in reducing the pH i , viability, and culturability of the cells in OJ . The results confirmed the hypothesis that consecutive subjection of E. coli to maximum legally permitted concentrations of sanitizers and OJ induces the VBNC state. Furthermore, we demonstrate successful application of FCM for monitoring the efficacy of washing procedures.

show abstract

Dual fluorescence system for flow cytometric analysis of Escherichia coli transcriptional response in multi-species context

Cited by 40 publications

References 49 publications

Single live‐bacterial cell assay of promoter activity and regulation

Single live‐bacterial cell assay of promoter activity and regulation

The effects of orange juice clarification on the physiology of Escherichia coli; growth-based and flow cytometric analysis

Flow cytometry and growth‐based analysis of the effects of fruit sanitation on the physiology of Escherichia coli in orange juice

Contact Info

Product

Resources

About