title: Heterogeneity of stress gene expression and stress resistance among individual cells of Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Centre for Fluorimetric Applications in Biotechnology, Department of Biological Sciences, Macquarie University, Sydney, New South Wales, Australia. pattfiel@ma.bio.mq.edu.au
authors.forename: P V
authors.initials: PV
authors.lastname: Attfield
authors.forename: H Y
authors.initials: HY
authors.lastname: Choi
authors.forename: D A
authors.initials: DA
authors.lastname: Veal
authors.forename: P J
authors.initials: PJ
authors.lastname: Bell
mesh_terms: D001483:Base Sequence; D002455:Cell Division; D005285:Fermentation; D015966:Gene Expression Regulation, Fungal; D018740:Genetic Heterogeneity; D005947:Glucose / Q000378:metabolism; D049452:Green Fluorescent Proteins; D018869:Heat-Shock Response / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011419:Propidium / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D011419:Propidium; D005947:Glucose