Live-Cell Imaging Tool Optimization To Study Gene Expression Levels and Dynamics in Single Cells of Bacillus cereus

ABSTRACTSingle-cell methods are a powerful application in microbial research to study the molecular mechanism underlying phenotypic heterogeneity and cell-to-cell variability. Here, we describe the optimization and application of single-cell time-lapse fluorescence microscopy for the food spoilage bacteriumBacillus cereusspecifically. This technique is useful to study cellular development and adaptation, gene expression, protein localization, protein mobility, and cell-to-cell communication over time at the single-cell level. By adjusting existing protocols, we have enabled the visualization of growth and development of singleB. cereuscells within a microcolony over time. Additionally, several different fluorescent reporter proteins were tested in order to select the most suitable green fluorescent protein (GFP) and red fluorescent protein (RFP) candidates for visualization of growth stage- and cell compartment-specific gene expression inB. cereus. With a case study concerningcotDexpression during sporulation, we demonstrate the applicability of time-lapse fluorescence microscopy. It enables the assessment of gene expression levels, dynamics, and heterogeneity at the single-cell level. We show thatcotDis not heterogeneously expressed among cells of a subpopulation. Furthermore, we discourage using plasmid-based reporter fusions for such studies, due to an introduced heterogeneity through copy number differences. This stresses the importance of using single-copy integrated reporter fusions for single-cell studies.

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Measuring bacterial adaptation dynamics at the single-cell level using a microfluidic chemostat and time-lapse fluorescence microscopy

The Analyst ◽

10.1039/c4an00877d ◽

2014 ◽

Vol 139 (20) ◽

pp. 5254-5262 ◽

Cited By ~ 11

Author(s):

Zhicheng Long ◽

Anne Olliver ◽

Elisa Brambilla ◽

Bianca Sclavi ◽

Marco Cosentino Lagomarsino ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Single Cell ◽

Time Lapse ◽

Single Cell Level ◽

Cell Level ◽

Gfp Expression ◽

Bacterial Adaptation ◽

E Coli ◽

Cell Dimensions

We grewE. coliin a microfluidic chemostat and monitored the dynamics of cell dimensions and reporter GFP expression in individual cells during nutritional upshift or downshift.

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Generation of genetic tools for gauging multiple gene expression at single cell level

Applied and Environmental Microbiology ◽

10.1128/aem.02956-20 ◽

2021 ◽

Author(s):

Marta Mellini ◽

Massimiliano Lucidi ◽

Francesco Imperi ◽

Paolo Visca ◽

Livia Leoni ◽

...

Keyword(s):

Gene Expression ◽

Image Processing ◽

Single Cell ◽

Fluorescent Protein ◽

Single Cells ◽

Phenotypic Heterogeneity ◽

Single Cell Level ◽

Cell Level ◽

Multiple Gene ◽

Genetic Tools

Key microbial processes in many bacterial species are heterogeneously expressed in single cells of bacterial populations. However, the paucity of adequate molecular tools for live, real-time monitoring of multiple gene expression at the single cell level has limited the understanding of phenotypic heterogeneity. In order to investigate phenotypic heterogeneity in the ubiquitous opportunistic pathogen Pseudomonas aeruginosa, a genetic tool that allows gauging multiple gene expression at the single cell level has been generated. This tool, named pRGC, consists in a promoter-probe vector for transcriptional fusions that carries three reporter genes coding for the fluorescent proteins mCherry, green fluorescent protein (GFP) and cyan fluorescent protein (CFP). The pRGC vector has been characterized and validated via single cell gene expression analysis of both constitutive and iron-regulated promoters, showing clear discrimination of the three fluorescence signals in single cells of a P. aeruginosa population, without the need of image-processing for spectral crosstalk correction. In addition, two pRGC variants have been generated for either i) integration of the reporter gene cassette into a single neutral site of P. aeruginosa chromosome, that is suitable for long-term experiments in the absence of antibiotic selection, or ii) replication in bacterial genera other than Pseudomonas. The easy-to-use genetic tools generated in this study will allow rapid and cost-effective investigation of multiple gene expression in populations of environmental and pathogenic bacteria, hopefully advancing the understanding of microbial phenotypic heterogeneity. IMPORTANCE Within a bacterial population single cells can differently express some genes, even though they are genetically identical and experience the same chemical and physical stimuli. This phenomenon, known as phenotypic heterogeneity, is mainly driven by gene expression noise and results in the emergence of bacterial sub-populations with distinct phenotypes. The analysis of gene expression at the single cell level has shown that phenotypic heterogeneity is associated with key bacterial processes, including competence, sporulation and persistence. In this study, new genetic tools have been generated that allow easy cloning of up to three promoters upstream of distinct fluorescent genes, making it possible to gauge multiple gene expression at the single cell level by fluorescent microscopy, without the need of advanced image-processing procedures. A proof of concept has been provided by investigating iron-uptake and iron-storage gene expression in response to iron availability in P. aeruginosa.

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Monitoring the Single-Cell Stress Response of the Diatom Thalassiosira pseudonana by Quantitative Real-Time Reverse Transcription-PCR

Applied and Environmental Microbiology ◽

10.1128/aem.03399-12 ◽

2013 ◽

Vol 79 (6) ◽

pp. 1850-1858 ◽

Cited By ~ 16

Author(s):

Xu Shi ◽

Weimin Gao ◽

Shih-hui Chao ◽

Weiwen Zhang ◽

Deirdre R. Meldrum

Keyword(s):

Gene Expression ◽

Stress Response ◽

Single Cell ◽

Real Time ◽

Reverse Transcription ◽

Thalassiosira Pseudonana ◽

Single Cell Level ◽

Cell Level ◽

Content Type ◽

Reverse Transcription Pcr

ABSTRACTDirectly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatomThalassiosira pseudonanaas a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels inT. pseudonanaand demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.

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Gene expression profiling of the different stages of Arabidopsis thaliana trichome development on the single cell level

Plant Physiology and Biochemistry ◽

10.1016/j.plaphy.2007.11.001 ◽

2008 ◽

Vol 46 (2) ◽

pp. 160-173 ◽

Cited By ~ 26

Author(s):

Sergiy Kryvych ◽

Victoria Nikiforova ◽

Michel Herzog ◽

Daniel Perazza ◽

Joachim Fisahn

Keyword(s):

Gene Expression ◽

Arabidopsis Thaliana ◽

Single Cell ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Single Cell Level ◽

Cell Level ◽

Trichome Development

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Unmasking cellular response of a bloom-forming alga to virus infection by resolving expression profiling at a single-cell level

10.1101/186981 ◽

2017 ◽

Author(s):

Shilo Rosenwasser ◽

Miguel J. Frada ◽

David Pilzer ◽

Ron Rotkopf ◽

Assaf Vardi

Keyword(s):

Gene Expression ◽

Viral Infection ◽

Single Cell ◽

Host Defense ◽

Host Response ◽

Expression Profiles ◽

Life Cycles ◽

Viral Gene ◽

Single Cell Level ◽

Cell Level

AbstractMarine viruses are major evolutionary and biogeochemical drivers of microbial life in the ocean. Host response to viral infection typically includes virus-induced rewiring of metabolic network to supply essential building blocks for viral assembly, as opposed to activation of anti-viral host defense. Nevertheless, there is a major bottleneck to accurately discern between viral hijacking strategies and host defense responses when averaging bulk population response. Here we use Emiliania huxleyi, a bloom-forming alga and its specific virus (EhV), as one of the most ecologically important host-virus model system in the ocean. Using automatic microfluidic setup to capture individual algal cells, we quantified host and virus gene expression on a single-cell resolution during the course of infection. We revealed high heterogeneity in viral gene expression among individual cells. Simultaneous measurements of expression profiles of host and virus genes at a single-cell level allowed mapping of infected cells into newly defined infection states and uncover a yet unrecognized early phase in host response that occurs prior to viral expression. Intriguingly, resistant cells emerged during viral infection, showed unique expression profiles of metabolic genes which can provide the basis for discerning between viral resistant and sensitive cells within heterogeneous populations in the marine environment. We propose that resolving host-virus arms race at a single-cell level will provide important mechanistic insights into viral life cycles and will uncover host defense strategies.

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Investigating DNA Replication in Escherichia Coli on the Single Cell Level Utilizing Microfluidics and Single-Molecule Fluorescence Microscopy

Biophysical Journal ◽

10.1016/j.bpj.2012.11.2048 ◽

2013 ◽

Vol 104 (2) ◽

pp. 368a-369a

Author(s):

Charl Moolman ◽

Sriram T. Krishnan ◽

Jacob W.K. Kerssemakers ◽

Susanne Hage ◽

Rodrigo Reyes-Lamothe ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Fluorescence Microscopy ◽

Single Cell ◽

Single Molecule ◽

Single Cell Level ◽

Single Molecule Fluorescence ◽

Cell Level ◽

Single Molecule Fluorescence Microscopy

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Detection of PLGA-based nanoparticles at a single-cell level by synchrotron radiation FTIR spectromicroscopy and correlation with X-ray fluorescence microscopy

International Journal of Nanomedicine ◽

10.2147/ijn.s58685 ◽

2014 ◽

pp. 2791 ◽

Cited By ~ 5

Author(s):

Giovanni Maria Severini ◽

Lorella Pascolo ◽

Barbara Bortot ◽

Nuria Benseny-Cases ◽

Alessandra Gianoncelli ◽

...

Keyword(s):

Synchrotron Radiation ◽

Fluorescence Microscopy ◽

Single Cell ◽

Single Cell Level ◽

Cell Level ◽

X Ray

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Circulating melanoma cells isolated from clinical blood samples and characterized by full-length mRNA sequencing at single-cell level.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.10539 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 10539-10539 ◽

Cited By ~ 1

Author(s):

Yu-Chieh Wang ◽

Daniel Ramskold ◽

Shujun Luo ◽

Robin Li ◽

Qiaolin Deng ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Expression Patterns ◽

Melanoma Cells ◽

Clinical Samples ◽

Single Cell Level ◽

Cell Level ◽

Blood Samples

10539 Background: Melanoma is the most aggressive type of skin cancer. Late-stage melanoma is highly metastatic and currently lacks effective treatment. This discouraging clinical observation highlights the need for a better understanding of the molecular mechanisms underlying melanoma initiation and progression and for developing new therapeutic approaches based on novel targets. Although genome-wide transcriptome analyses have been frequently used to study molecular alterations in clinical samples, it has been technically challenging to obtain the transcriptomic profiles at single-cell level. Methods: Using antibody-mediated magnetic activated cell separation (MACS), we isolated and individualized putative circulating melanoma cells (CMCs) from the blood samples of the melanoma patients at advance stages. The transcriptomic analysis based on a novel and robust mRNA-Seq protocol (Smart-Seq) was established and applied to the putative CMCs for single-cell profiling. Results: We have discovered distinct gene expression patterns, including new putative markers for CMCs. Meanwhile, the gene expression profiles derived of the CMC candidates isolated from the patient’s blood samples are closely-related to the expression profiles of other cells originated from human melanocytes, including normal melanocytes in primary culture and melanoma cell lines. Compared with existing methods, Smart-Seq has improved read coverage across transcripts, which provides advantage for better analyzing transcript isoforms and SNPs. Conclusions: Our results suggest that the techniques developed in this research for cell isolation and transcriptomic analyses can potentially be used for addressing many biological and clinical questions requiring genomewide transcriptome profiling in rare cells.

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