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Monitoring of gene expression in bacteria during infections using an adaptable set of bioluminescent, fluorescent and colorigenic fusion vectors.

Uliczka F, Pisano F, Kochut A, Opitz W, Herbst K, Stolz T, Dersch P - PLoS ONE (2011)

Bottom Line: The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis.The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs.A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Lower Saxony, Germany.

ABSTRACT
A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfp(mut3.1), amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.

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Comparison of rovA-gfpmut3.1 and rovA-gfpmut3.1-LVA expression.Y. pseudotuberculosis strain YPIII pKH59 (rovA-gfpmut3.1) and YPIII pKH83 (rovA-gfpmut3.1-LVA) were grown overnight and fluorescence was determined as described in Material and Methods and is given as relative fluorescence units (RFU) per optical density at 600 nm (OD).
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pone-0020425-g004: Comparison of rovA-gfpmut3.1 and rovA-gfpmut3.1-LVA expression.Y. pseudotuberculosis strain YPIII pKH59 (rovA-gfpmut3.1) and YPIII pKH83 (rovA-gfpmut3.1-LVA) were grown overnight and fluorescence was determined as described in Material and Methods and is given as relative fluorescence units (RFU) per optical density at 600 nm (OD).

Mentions: In order to allow the analysis of transient expression events, we also constructed a derivative of pFU31 encoding unstable variants of GFPmut3.1 tagged with a C-terminal extension, which is a target for tail-specific proteases [32]. Addition of an ASV-, AAV- and LVA -tag reduces the half-life of GFPmut3.1 from 24 hours to 110 min, 60 min and 40 min, respectively [33]. To demonstrate useful application of our vector system for the analysis of transient gene expression, we analyzed the activity of the rovA promoter of Y. pseudotuberculosis cloned upstream of the gfpmut3.1 gene or the gfpmut3.1-LVA variant. It has been previously demonstrated that the rovA gene of Y. pseudotuberculosis encodes a MarR-type virulence regulator that acts as a protein thermometer [34]. A thermal upshift from 25°C to 37°C leads to a reversible conformational change of the regulator which reduces its DNA-binding function and renders it more susceptible for proteolysis. As a result cooperative binding to its own promoter region and autoactivation are significantly reduced at 37°C [34]. To test our vector system for transient gene expression Y. pseudotuberculosis YPIII carrying the rovA-gfpmut3.1 or the rovA-gfpmut3.1-LVA fusion was grown at 25°C and shifted to 37°C for several hours. As shown in Figure 4, rovA-gfpmut3.1-LVA expression was more rapidly repressed than the rovA-gfpmut3.1 fusion after a thermal upshift from 25°C to 37°C. In addition to these experiments, we also monitored the luciferase activity of the constitutive gapA-luxCDABE fusion of Y. pseudotuberculosis YPIII pFU166 after transcription was stopped by the addition of rifampicin. As shown in Figure S4, luciferase activity declined rapidly after growth arrest, demonstrating that also the luxCDABE reporter is useful for the analysis of transient gene expression.


Monitoring of gene expression in bacteria during infections using an adaptable set of bioluminescent, fluorescent and colorigenic fusion vectors.

Uliczka F, Pisano F, Kochut A, Opitz W, Herbst K, Stolz T, Dersch P - PLoS ONE (2011)

Comparison of rovA-gfpmut3.1 and rovA-gfpmut3.1-LVA expression.Y. pseudotuberculosis strain YPIII pKH59 (rovA-gfpmut3.1) and YPIII pKH83 (rovA-gfpmut3.1-LVA) were grown overnight and fluorescence was determined as described in Material and Methods and is given as relative fluorescence units (RFU) per optical density at 600 nm (OD).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108616&req=5

pone-0020425-g004: Comparison of rovA-gfpmut3.1 and rovA-gfpmut3.1-LVA expression.Y. pseudotuberculosis strain YPIII pKH59 (rovA-gfpmut3.1) and YPIII pKH83 (rovA-gfpmut3.1-LVA) were grown overnight and fluorescence was determined as described in Material and Methods and is given as relative fluorescence units (RFU) per optical density at 600 nm (OD).
Mentions: In order to allow the analysis of transient expression events, we also constructed a derivative of pFU31 encoding unstable variants of GFPmut3.1 tagged with a C-terminal extension, which is a target for tail-specific proteases [32]. Addition of an ASV-, AAV- and LVA -tag reduces the half-life of GFPmut3.1 from 24 hours to 110 min, 60 min and 40 min, respectively [33]. To demonstrate useful application of our vector system for the analysis of transient gene expression, we analyzed the activity of the rovA promoter of Y. pseudotuberculosis cloned upstream of the gfpmut3.1 gene or the gfpmut3.1-LVA variant. It has been previously demonstrated that the rovA gene of Y. pseudotuberculosis encodes a MarR-type virulence regulator that acts as a protein thermometer [34]. A thermal upshift from 25°C to 37°C leads to a reversible conformational change of the regulator which reduces its DNA-binding function and renders it more susceptible for proteolysis. As a result cooperative binding to its own promoter region and autoactivation are significantly reduced at 37°C [34]. To test our vector system for transient gene expression Y. pseudotuberculosis YPIII carrying the rovA-gfpmut3.1 or the rovA-gfpmut3.1-LVA fusion was grown at 25°C and shifted to 37°C for several hours. As shown in Figure 4, rovA-gfpmut3.1-LVA expression was more rapidly repressed than the rovA-gfpmut3.1 fusion after a thermal upshift from 25°C to 37°C. In addition to these experiments, we also monitored the luciferase activity of the constitutive gapA-luxCDABE fusion of Y. pseudotuberculosis YPIII pFU166 after transcription was stopped by the addition of rifampicin. As shown in Figure S4, luciferase activity declined rapidly after growth arrest, demonstrating that also the luxCDABE reporter is useful for the analysis of transient gene expression.

Bottom Line: The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis.The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs.A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Lower Saxony, Germany.

ABSTRACT
A family of versatile promoter-probe plasmids for gene expression analysis was developed based on a modular expression plasmid system (pZ). The vectors contain different replicons with exchangeable antibiotic cassettes to allow compatibility and expression analysis on a low-, midi- and high-copy number basis. Suicide vector variants also permit chromosomal integration of the reporter fusion and stable vector derivatives can be used for in vivo or in situ expression studies under non-selective conditions. Transcriptional and translational fusions to the reporter genes gfp(mut3.1), amCyan, dsRed2, luxCDABE, phoA or lacZ can be constructed, and presence of identical multiple cloning sites in the vector system facilitates the interchange of promoters or reporter genes between the plasmids of the series. The promoter of the constitutively expressed gapA gene of Escherichia coli was included to obtain fluorescent and bioluminescent expression constructs. A combination of the plasmids allows simultaneous detection and gene expression analysis in individual bacteria, e.g. in bacterial communities or during mouse infections. To test our vector system, we analyzed and quantified expression of Yersinia pseudotuberculosis virulence genes under laboratory conditions, in association with cells and during the infection process.

Show MeSH
Related in: MedlinePlus