Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria

ABSTRACT We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter PA1/O4/O3 in response to increasing concentrations of the inducer IPTG (isopropyl-β-d-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within PA1/O4/O3. Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfpfusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.

[1]  S. Lindow,et al.  Appetite of an epiphyte: Quantitative monitoring of bacterial sugar consumption in the phyllosphere , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Bjarke Bak Christensen,et al.  gfp-Based N-Acyl Homoserine-Lactone Sensor Systems for Detection of Bacterial Communication , 2001, Applied and Environmental Microbiology.

[3]  S. Sørensen,et al.  Detection of Oxytetracycline Production by Streptomyces rimosus in Soil Microcosms by Combining Whole-Cell Biosensors and Flow Cytometry , 2001, Applied and Environmental Microbiology.

[4]  S. Sørensen,et al.  Versatile biosensor vectors for detection and quantification of mercury. , 2000, FEMS microbiology letters.

[5]  J. Leveau,et al.  Improved gfp and inaZ broad-host-range promoter-probe vectors. , 2000, Molecular plant-microbe interactions : MPMI.

[6]  S. Lindow,et al.  Heterogeneity of iron bioavailability on plants assessed with a whole-cell GFP-based bacterial biosensor. , 2000, Microbiology.

[7]  K. Geider,et al.  Molecular Analysis of Sucrose Metabolism ofErwinia amylovora and Influence on Bacterial Virulence , 2000, Journal of bacteriology.

[8]  T. Baker,et al.  A specificity-enhancing factor for the ClpXP degradation machine. , 2000, Science.

[9]  G. Geesey,et al.  Differentiation of Chitinase-Active and Non-Chitinase-Active Subpopulations of a Marine Bacterium during Chitin Degradation , 2000, Applied and Environmental Microbiology.

[10]  W. Hillen,et al.  Quantitative analysis of gene expression with an improved green fluorescent protein. p6. , 2000, European journal of biochemistry.

[11]  S. Molin,et al.  Bacterial Activity in the Rhizosphere Analyzed at the Single-Cell Level by Monitoring Ribosome Contents and Synthesis Rates , 2000, Applied and Environmental Microbiology.

[12]  P. Dehaseth,et al.  Green fluorescent protein as a quantitative reporter of relative promoter activity in E. coli. , 2000, BioTechniques.

[13]  G. Tsujimoto,et al.  Spatial and temporal regulation of protein expression by bldA within a Streptomyces lividans colony , 1999, FEBS letters.

[14]  D. Sherman,et al.  Time‐lapsed confocal microscopy reveals temporal and spatial expression of the lysine ɛ‐aminotransferase gene in Streptomyces clavuligerus , 1999, Molecular microbiology.

[15]  M. Ehrenberg,et al.  Activities of constitutive promoters in Escherichia coli. , 1999, Journal of molecular biology.

[16]  B. Christensen,et al.  Distribution of Bacterial Growth Activity in Flow-Chamber Biofilms , 1999, Applied and Environmental Microbiology.

[17]  W. Bentley,et al.  Green Fluorescent Protein as a Noninvasive Stress Probe in Resting Escherichia coli Cells , 1999, Applied and Environmental Microbiology.

[18]  I. Badr,et al.  Green fluorescent protein in the design of a living biosensing system for L-arabinose. , 1999, Analytical chemistry.

[19]  A. Rakin,et al.  In vitro and in vivo expression studies of yopE from Yersinia enterocolitica using the gfp reporter gene , 1998, Molecular microbiology.

[20]  D. Brooks,et al.  The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein , 1998, Molecular microbiology.

[21]  S. Techkarnjanaruk,et al.  Use of Green Fluorescent Protein To Tag and Investigate Gene Expression in Marine Bacteria , 1998, Applied and Environmental Microbiology.

[22]  L. Poulsen,et al.  New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria , 1998, Applied and Environmental Microbiology.

[23]  R. Sauer,et al.  The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system. , 1998, Genes & development.

[24]  H. Mobley,et al.  Use of Green Fluorescent Protein To Assess Urease Gene Expression by Uropathogenic Proteus mirabilis during Experimental Ascending Urinary Tract Infection , 1998, Infection and Immunity.

[25]  C. Albano,et al.  Green Fluorescent Protein as a Real Time Quantitative Reporter of Heterologous Protein Production , 1998, Biotechnology progress.

[26]  G. Patterson,et al.  Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. , 1997, Biophysical journal.

[27]  H. Bujard,et al.  Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. , 1997, Nucleic acids research.

[28]  Govind Rao,et al.  Quantitative measurement of green fluorescent protein expression , 1996 .

[29]  S. Lindow,et al.  Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola , 1996, Applied and environmental microbiology.

[30]  S. Falkow,et al.  Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid‐inducible promoters by differential fluorescence induction , 1996, Molecular microbiology.

[31]  F. Srienc,et al.  Quantitative analysis of transient gene expression in mammalian cells using the green fluorescent protein. , 1996, Journal of biotechnology.

[32]  R. Sauer,et al.  Role of a Peptide Tagging System in Degradation of Proteins Synthesized from Damaged Messenger RNA , 1996, Science.

[33]  S. Falkow,et al.  Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions. , 1996, Gene.

[34]  S Falkow,et al.  FACS-optimized mutants of the green fluorescent protein (GFP). , 1996, Gene.

[35]  M. Chalfie.  GREEN FLUORESCENT PROTEIN , 1995, Photochemistry and photobiology.

[36]  Roger Y. Tsien,et al.  Improved green fluorescence , 1995, Nature.

[37]  R. Christen,et al.  Assessment of the state of activity of individual bacterial cells by hybridization with a ribosomal RNA targeted fluorescently labelled oligonucleotidic probe , 1994 .

[38]  M. Saier,et al.  Sequence of the fruB gene of Escherichia coli encoding the diphosphoryl transfer protein (DTP) of the phosphoenolpyruvate: sugar phosphotransferase system. , 1994, FEMS microbiology letters.

[39]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[40]  Pedro Mendes,et al.  GEPASI: a software package for modelling the dynamics, steady states and control of biochemical and other systems , 1993, Comput. Appl. Biosci..

[41]  R. Amann,et al.  Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. , 1993, Cytometry.

[42]  V. de Lorenzo,et al.  Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria , 1990, Journal of bacteriology.

[43]  V. de Lorenzo,et al.  Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria , 1990, Journal of bacteriology.

[44]  F. Neidhardt,et al.  Physiology of the bacterial cell : a molecular approach , 1990 .

[45]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[46]  D. Helinski,et al.  Isolation and characterization of the recA gene of Rhizobium meliloti , 1983, Journal of bacteriology.

[47]  W. Gilbert,et al.  Mutants that make more lac repressor. , 1968, Proceedings of the National Academy of Sciences of the United States of America.