A novel method for measuring lag times in division of individual bacterial cells using image analysis

A method is presented for determining the time to first division of individual bacterial cells growing on agar media. Bacteria were inoculated onto agar-coated slides and viewed by phase-contrast microscopy. Digital images of the growing bacteria were captured at intervals and the time to first division estimated by calculating the "box area ratio". This is the area of the smallest rectangle that can be drawn around an object, divided by the area of the object itself. The box area ratios of cells were found to increase suddenly during growth at a time that correlated with cell division as estimated by visual inspection of the digital images. This was caused by a change in the orientation of the two daughter cells that occurred when sufficient flexibility arose at their point of attachment. This method was used successfully to generate lag time distributions for populations of Escherichia coli, Listeria monocytogenes and Pseudomonas aeruginosa, but did not work with the coccoid organism Staphylococcus aureus. This method provides an objective measure of the time to first cell division, whilst automation of the data processing allows a large number of cells to be examined per experiment.     

RT-qPCR based quantitative analysis of gene expression in single bacterial cells

Recent evidence suggests that cell-to-cell difference at the gene expression level is an order of magnitude greater than previously thought even for isogenic bacterial populations. Such gene expression heterogeneity determines the fate of individual bacterial cells in populations and could also affect the ultimate fate of populations themselves. To quantify the heterogeneity and its biological significance, quantitative methods to measure gene expression in single bacterial cells are needed. In this work, we developed two SYBR Green-based RT-qPCR methods to determine gene expression directly in single bacterial cells. The first method involves a single-tube operation that can analyze one gene from each bacterial cell. The second method is featured by a two-stage protocol that consists of RNA isolation from a single bacterial cell and cDNA synthesis in the first stage, and qPCR in the second stage, which allows determination of expression level of multiple genes simultaneously for single bacterial cells of both gram-positive and negative. We applied the methods to stress-treated (i.e. low pH and high temperature) Escherichia coli populations. The reproducible results demonstrated that the method is sensitive enough not only for measuring cellular responses at the single-cell level, but also for revealing gene expression heterogeneity among the bacterial cells. Furthermore, our results showed that the two-stage method can reproducibly measure multiple highly expressed genes from a single E. coli cell, which exhibits important foundation for future development of a high throughput and lab-on-chips whole-genome RT-qPCR methodology for single bacterial cells.     

Analysis of gene expression in single cells

A cell's structural and functional characteristics are dependent on the specific complement of genes it expresses. The ability to study and compare gene usage at the cellular level will therefore provide valuable insights into cell physiology. Such analyses are complicated by problems associated with sample collection, sample size and the limited sensitivity of expression assays. Advances have been made in approaches to the collection of cellular material and the performance of single-cell gene expression analysis. Recent development in global amplification of mRNA may soon permit expression analyses of single cells to be performed on DNA microarrays.     

Analysis of pigmentation in individual cultured plant cells using an image processing system

Anthocyanin accumulation in strawberry (Fragaria ananassa) cells cultured on a solid medium was monitored using an image-processing system that did not require direct sampling or destruction of the cells. Because of the intercellular heterogeneity of secondary metabolite production in plant cell cultures, the maximum metabolite concentration in individual cells is often more than 10 times higher than that of the average concentration. An image-processing based method enabled the growth and the pigmentation behavior of individual cells to be traced. Changes in the time courses of the anthocyanin content of individual cells differed from each other, although the average anthocyanin contents increased gradually with time in a batch culture. However, these various changing patterns in the anthocyanin content of each cell were independent of the cell cycle. In addition, image analysis revealed that the two cells just after cell division were almost identical to each other both in size and anthocyanin content. The proposed method which uses an image-processing system provides a useful tool for analyzing the secondary metabolism in individual cultured plant cells.     

Factors governing the expression of a bacterial gene in mammalian cells.

Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.     

Effects of inducer levels on a recombinant bacterial biofilm formation and gene expression

Summary A segregationally stable host-plasmid system, E. coli DH5 (pTKW106), was used to study the effect of induction on the accumulation rate of cells and gene expression in biofilm cultures. Isopropyl -D-thiogalactoside (IPTG) was used to induce the expression of -galactosidase from the plasmid. The biofilm cell net accumulation rates decreased with increasing induction levels. At 0.17 and 0.34 mM of IPTG, the biofilm cell net accumulation rates ranged between 17 and 30% when compared to the uninduced case. At 0.51 mM of IPTG, the biofilm cell density never increased. At 0.17 and 0.34 mM of IPTG, -galactosidase contents reached maxima 36 hours after induction with both amounts representing about 7.5% of total protein. At 0.51 mM of IPTG, -galactosidase production reached its maximum, about 16% of total protein, 48 hours after induction. The -galactosidase mRNA synthesis rates increased with increasing inducer levels. Maximum -galactosidase mRNA synthesis rates were reached 36 hours after induction for each IPTG concentration.     

Abscisic add levels of individual leaf cells

Abscisic acid (ABA) concentrations of guard cells, subsidiary cells and epidermis cells of Comrnelina communis L. and Tradescantia virginiana L. were determined in cell sap samples extracted by means of micro glass capillaries. Concentrations up to 6.7 m M were indicated by commercial immunoassay test kits. A gradient of ABA concentration was found between guard cells (2,49 ± 1.81 m M , n = 25), subsidiary cells (1.25 ± 1.46 m M , n = 21) and epidermis cells (0.86 ± 0.76 m M , n = 20; mean values ± SD).     

Simultaneous monitoring of pituitary hormone secretion and gene expression within individual cells.

We have recently developed a method to simultaneously quantitate the level of gene expression and the level of secretion of a peptide from individual cells. Our approach has been to combine the reverse hemolytic plaque assay sequentially with in situ hybridization. We present data to show how we have used the pituitary lactotroph as a model to demonstrate the power of this technique. However, we are particularly excited about the potential application of this strategy to approach a broad spectrum of questions regarding the cellular and molecular mechanisms that regulate the coupling of peptide secretion and gene expression at the single cell level. The method can be used in any system in which an appropriate antibody for the reverse hemolytic plaque assay and probes complementary to the mRNA of interest are available.     

Automated image analysis of bacterial colony growth as a tool to study individual lag time distributions of immobilized cells

A method to determine the individual lag time (lag) distributions of immobilized bacteria was presented. The method was based on the image analysis of the bacterial colony growth. The lag distributions were retrieved from the distributions of the detection times (Td) required to form macroscopically visible colonies. Using this method, the lag distributions on agar for Listeria monocytogenes cells previously subjected to two situations reproducing conditions encountered during the contamination of cheese, were determined. The results were presented and compared with lag distributions obtained with an established method based on the time to detection of turbidity in broth. An original method to retrieve lag in broth and agar without any knowledge of the growth rate was also proposed. In order not to bias the distributions of lag on agar the impact of spatial separation between colonies on colony growth rates was quantified. Means and standard deviations of lag distributions for the two different stresses were found to be similar in broth and on agar. Extreme Value type II distributions fitted the best the different datasets of lag distributions.     

Heterogeneity of stress gene expression and stress resistance among individual cells of Saccharomyces cerevisiae

Knowledge of gene expression and cellular responses in microorganisms is derived from analyses of populations consisting of millions of cells. Analytical techniques that provide data as population averages fail to inform of culture heterogeneity. Flow cytometry and fluorescence techniques were used to provide information on the heterogeneity of stress-responsive gene expression and stress tolerance in individual cells within populations. A sequence of DNA encoding the heat shock and stress response elements of the Saccharomyces cerevisiae HSP104 gene was used to express enhanced green fluorescent protein (EGFP). When integrated into the genome of yeast strain W303-1A, intrinsic expression of EGFP increased about twofold as cells progressed from growth on glucose to ethanol utilization in aerobic batch cultures. Staining of cells with orange/red fluorescent propidium iodide (PI), which only enters cells that have compromised membrane integrity, revealed that the population became more tolerant to 52 degrees C heat stress as it progressed from growth on glucose and through the ethanol utilization phase of aerobic batch culture. Exposure of cultures growing on glucose to a mild heat shock (shift from 25 degrees C to 37 degrees C) resulted in significantly increased expression of EGFP in the population. However, there was heterogeneity in the intensity of fluorescence of individual cells from heat-shocked cultures, indicating variability in the strength of stress response in the clonal population. Detailed analysis of the heterogeneity showed a clear positive trend between intensity of stress response and individual cell resistance, measured in terms of PI exclusion, to heat stress at 52 degrees C. Further experiments indicated that, although the mean gene expression by a population is influenced by the genetic background, the heterogeneity among individual cells in clonal populations is largely physiologically based.     

Modulating gene expression in stem cells without recombinant DNA and permanent genetic modification

Future therapeutic applications of stem cells in regenerative medicine require efficient techniques for modulating gene expression. Conventionally, this is achieved through the use of recombinant DNA, which invariably leads to permanent genetic alteration to the cell. Overwhelming safety and ethical concerns are likely to preclude the application of genetically modified stem cells in human clinical therapy for the foreseeable near future. An alternative may be to adopt a milieu-based approach to influence gene expression, by exposing stem cells to a cocktail of exogenous cytokines, growth factors, and extracellular matrix. Nevertheless, the non-specific pleiotropic effects exerted by various cytokines, growth factors, and extracellular matrix would make this a relatively inefficient approach. Moreover, a milieu-based approach is likely to require extended durations of in vitro culture, which might delay autologous transplantation of adult stem cells to the patient and might alter their immunogenicity through prolonged exposure to xenogenic proteins within the culture milieu. The obvious solution would be to deliver proteins, RNA, or their synthetic analogs, such as peptide nucleic acid, directly into the cell to modulate gene expression. Currently, two promising delivery platforms are available: (1) protein transduction domains, and (2) immunoliposomes. Because such molecules have a limited active half-life in the cytosol and are obviously not incorporated into the genetic code of the cell, these would only exert a transient modulatory effect on gene expression. Nevertheless, a transient effect may be preferable for clinical therapy, since this would ultimately avoid permanent genetic alteration to the cell.     

RNA sequencing reveals two major classes of gene expression levels in metazoan cells

The expression level of a gene is often used as a proxy for determining whether the protein or RNA product is functional in a cell or tissue. Therefore, it is of fundamental importance to understand the global distribution of gene expression levels, and to be able to interpret it mechanistically and functionally. Here we use RNA sequencing (RNA‐seq) of mouse Th2 cells, coupled with a range of other techniques, to show that all genes can be separated, based on their expression abundance, into two distinct groups: one group comprised of lowly expressed and putatively non‐functional mRNAs, and the other of highly expressed mRNAs with active chromatin marks at their promoters. These observations are confirmed in many other microarray and RNA‐seq data sets of metazoan cell types.