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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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Comparative gene expression analysis using pFU plasmids.Y. pseudotuberculosis strains YPIII pTS39 and YPIII pTS40 were diluted 1∶50 in fresh LB from overnight cultures and grown to late exponential phase at 25°C or 37°C. Bacteria were detected by bright field microscopy and expression of the yadA-gfpmut3.1 (A) and yadA-dsRed2 (B) fusion was visualized by fluorescence microscopy. White bar indicates 5 µm.
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pone-0020425-g003: Comparative gene expression analysis using pFU plasmids.Y. pseudotuberculosis strains YPIII pTS39 and YPIII pTS40 were diluted 1∶50 in fresh LB from overnight cultures and grown to late exponential phase at 25°C or 37°C. Bacteria were detected by bright field microscopy and expression of the yadA-gfpmut3.1 (A) and yadA-dsRed2 (B) fusion was visualized by fluorescence microscopy. White bar indicates 5 µm.

Mentions: It has previously been shown that expression of the yadA gene encoding the well-characterized Yersinia adhesin A is thermoregulated and is expressed only at 37°C by the temperature-regulated virulence gene activator LcrF [29], [30]. In order to test the different vectors, the yadA promoter region of Y. pseudotuberculosis strain YPIII was cloned into different pFU fusion vectors to generate transcriptional yadA-luxCDABE fusions on compatible low, medium and high copy vectors (pTS31, pTS37, pTS36). Furthermore, a pSC101*-based vector set was used to generate yadA-lacZ, yadA-phoA, yadA-dsRed2 and yadA-gfpmut3.1 fusions. The plasmids were transformed into Y. pseudotuberculosis strain YPIII and expression of the fusion was determined at 25°C (repressing conditions) and 37°C (inducing conditions) by bioluminescence, colorimetric and fluorescence detection assays (for details see Material and Methods). As shown in Figure 2A, a 9–10fold difference in the yadA expression levels could be detected with the luxCDABE fusions (Figure 2A), and a 4–5fold increase could be observed with the colorimetric reporters (Figure 2B–C). In addition, both tested fluorescent reporters (gfpmut3.1 and dsRed2) were highly expressed in the bacteria at 37°C, but no fluorescence was detectable at 25°C with the same photographic adjustments (Figure 3). The fastest and most sensitive response with almost no background activity was observed with the luxCDABE reporter plasmids making these vectors very suitable for the analysis of weakly expressed genes. This is in agreement with a previous study demonstrating that the luxCDABE reporter is more reactive and achieves a more rapid response than the fluorescent proteins GFP and DsRed [31]. Furthermore, transcription levels of the yadA-luxCDABE reporter fusion were found to correlate with the copy number of the promoter probe vector (Figure 2A). Ratio of temperature-dependent activation of yadA expression was very similar, but the expression level was strongly dependent on the copy-number of the fusion plasmids. A 2.5-fold higher expression was measured with the ColE1-based plasmid compared to the p29807 derivative, and a 3-fold higher activity was detectable with the p29807-based plasmid compared to the pSC101*-based vector, demonstrating that the vector system can be used for (simultaneous) expression analysis of genes with significantly different transcription levels. As bacterial luciferase activity depends on NADH and ATP, we also tested the influence of the metabolic activity of the bacterial cells on expression of the luxCDABE fusion system. To do so, we followed expression of a constitutive gapA-luxCDABE fusion expressed by Y. pseudotuberculosis YPIII pFU166 along the bacterial growth curve and compared luciferase activity with the optical density and the number of bacteria in the culture. As shown in Figure S3, luciferase activity increased during exponential phase reflecting the increase of the number of bacteria in the culture. However, bioluminescence declined continuously after the bacteria entered stationary phase, whereas an equivalent gapA-gfp fusion remained highly fluorescent (data not shown). This demonstrated that luxCDABE fusion systems are highly sensitive and very useful for the quantification of gene expression when the bacteria are metabolically active, whereas colorigenic and fluorescent reporter vector systems are more appropriate for the analysis of gene expression in resting/starved bacteria (e.g. during stationary phase).


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)

Comparative gene expression analysis using pFU plasmids.Y. pseudotuberculosis strains YPIII pTS39 and YPIII pTS40 were diluted 1∶50 in fresh LB from overnight cultures and grown to late exponential phase at 25°C or 37°C. Bacteria were detected by bright field microscopy and expression of the yadA-gfpmut3.1 (A) and yadA-dsRed2 (B) fusion was visualized by fluorescence microscopy. White bar indicates 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020425-g003: Comparative gene expression analysis using pFU plasmids.Y. pseudotuberculosis strains YPIII pTS39 and YPIII pTS40 were diluted 1∶50 in fresh LB from overnight cultures and grown to late exponential phase at 25°C or 37°C. Bacteria were detected by bright field microscopy and expression of the yadA-gfpmut3.1 (A) and yadA-dsRed2 (B) fusion was visualized by fluorescence microscopy. White bar indicates 5 µm.
Mentions: It has previously been shown that expression of the yadA gene encoding the well-characterized Yersinia adhesin A is thermoregulated and is expressed only at 37°C by the temperature-regulated virulence gene activator LcrF [29], [30]. In order to test the different vectors, the yadA promoter region of Y. pseudotuberculosis strain YPIII was cloned into different pFU fusion vectors to generate transcriptional yadA-luxCDABE fusions on compatible low, medium and high copy vectors (pTS31, pTS37, pTS36). Furthermore, a pSC101*-based vector set was used to generate yadA-lacZ, yadA-phoA, yadA-dsRed2 and yadA-gfpmut3.1 fusions. The plasmids were transformed into Y. pseudotuberculosis strain YPIII and expression of the fusion was determined at 25°C (repressing conditions) and 37°C (inducing conditions) by bioluminescence, colorimetric and fluorescence detection assays (for details see Material and Methods). As shown in Figure 2A, a 9–10fold difference in the yadA expression levels could be detected with the luxCDABE fusions (Figure 2A), and a 4–5fold increase could be observed with the colorimetric reporters (Figure 2B–C). In addition, both tested fluorescent reporters (gfpmut3.1 and dsRed2) were highly expressed in the bacteria at 37°C, but no fluorescence was detectable at 25°C with the same photographic adjustments (Figure 3). The fastest and most sensitive response with almost no background activity was observed with the luxCDABE reporter plasmids making these vectors very suitable for the analysis of weakly expressed genes. This is in agreement with a previous study demonstrating that the luxCDABE reporter is more reactive and achieves a more rapid response than the fluorescent proteins GFP and DsRed [31]. Furthermore, transcription levels of the yadA-luxCDABE reporter fusion were found to correlate with the copy number of the promoter probe vector (Figure 2A). Ratio of temperature-dependent activation of yadA expression was very similar, but the expression level was strongly dependent on the copy-number of the fusion plasmids. A 2.5-fold higher expression was measured with the ColE1-based plasmid compared to the p29807 derivative, and a 3-fold higher activity was detectable with the p29807-based plasmid compared to the pSC101*-based vector, demonstrating that the vector system can be used for (simultaneous) expression analysis of genes with significantly different transcription levels. As bacterial luciferase activity depends on NADH and ATP, we also tested the influence of the metabolic activity of the bacterial cells on expression of the luxCDABE fusion system. To do so, we followed expression of a constitutive gapA-luxCDABE fusion expressed by Y. pseudotuberculosis YPIII pFU166 along the bacterial growth curve and compared luciferase activity with the optical density and the number of bacteria in the culture. As shown in Figure S3, luciferase activity increased during exponential phase reflecting the increase of the number of bacteria in the culture. However, bioluminescence declined continuously after the bacteria entered stationary phase, whereas an equivalent gapA-gfp fusion remained highly fluorescent (data not shown). This demonstrated that luxCDABE fusion systems are highly sensitive and very useful for the quantification of gene expression when the bacteria are metabolically active, whereas colorigenic and fluorescent reporter vector systems are more appropriate for the analysis of gene expression in resting/starved bacteria (e.g. during stationary phase).

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