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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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Cell contact dependent gene expression analysis using pFU plasmids.(A) About 5×104 bacteria of Y. pseudotuberculosis strain YPIII pTS28 (yopE-gfpmut3.1) were used to infect 103 HEp-2 cells. Bacteria were centrifuged onto the cell monolayer. Expression of the yopE-gfpmut3.1 fusion was monitored by phase contrast and fluorescence microscopy directly after centrifugation (0 h) and after four hours (4 h). (B,C) About 106 bacteria of Y. pseudotuberculosis strain YPIII pFU98 (empty vector) and YPIII pWO34 (yopE-luxCDABE) resuspended in PBS were incubated in the absence or in the presence of 104 HEp-2 cells for 4 hours as described above. Bioluminescence emitted by cultures was monitored after 4 hours of infection (B), or was determined every 20 min after infection to monitor the kinetic of host cell contact-dependent yopE-luxCDABE induction (C). Bioluminescence is given as relative luminescence units (RLU) per colony forming units (CFU) and represents the mean of three independent experiments done in triplicate. White bar indicates 5 µm.
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pone-0020425-g005: Cell contact dependent gene expression analysis using pFU plasmids.(A) About 5×104 bacteria of Y. pseudotuberculosis strain YPIII pTS28 (yopE-gfpmut3.1) were used to infect 103 HEp-2 cells. Bacteria were centrifuged onto the cell monolayer. Expression of the yopE-gfpmut3.1 fusion was monitored by phase contrast and fluorescence microscopy directly after centrifugation (0 h) and after four hours (4 h). (B,C) About 106 bacteria of Y. pseudotuberculosis strain YPIII pFU98 (empty vector) and YPIII pWO34 (yopE-luxCDABE) resuspended in PBS were incubated in the absence or in the presence of 104 HEp-2 cells for 4 hours as described above. Bioluminescence emitted by cultures was monitored after 4 hours of infection (B), or was determined every 20 min after infection to monitor the kinetic of host cell contact-dependent yopE-luxCDABE induction (C). Bioluminescence is given as relative luminescence units (RLU) per colony forming units (CFU) and represents the mean of three independent experiments done in triplicate. White bar indicates 5 µm.

Mentions: Autofluorescent and bioluminescent proteins are excellent reporters for monitoring gene expression in single bacteria in association with host cells, and they were successfully used to study bacterial gene expression within or onto host cells [35], [36]. For example, expression of the pathogenicity factor YopE of Yersinia was shown to be induced upon host cell contact [35]. In order to test our reporter plasmids we challenged cells with YPIII pTS28 and determined yopE-gfpmut3.1 expression for four hours after infection. We found that yersiniae bound to HEp-2 cells were significantly more fluorescent than bacteria without host cell contact (Figure 5A). A quantitative analysis of HEp-2 cells infected with YPIII harboring the yopE-luxCDABE fusion gave similar results and demonstrated a very strong (28-fold) increase of luciferase activity in the presence of host cells four hours post infection (Figure 5B). A kinetic analysis of host cell contact-dependent activation of the yopE-luxCDABE fusion revealed a very strong increase of yopE expression during the first 30 min after infection which is followed by a smaller but steady increase of yopE expression (Figure 5C). This clearly demonstrates that the different vector systems harboring fluorescent and bioluminescent reporter genes are suitable to follow and quantify gene expression of bacteria in association with cells.


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)

Cell contact dependent gene expression analysis using pFU plasmids.(A) About 5×104 bacteria of Y. pseudotuberculosis strain YPIII pTS28 (yopE-gfpmut3.1) were used to infect 103 HEp-2 cells. Bacteria were centrifuged onto the cell monolayer. Expression of the yopE-gfpmut3.1 fusion was monitored by phase contrast and fluorescence microscopy directly after centrifugation (0 h) and after four hours (4 h). (B,C) About 106 bacteria of Y. pseudotuberculosis strain YPIII pFU98 (empty vector) and YPIII pWO34 (yopE-luxCDABE) resuspended in PBS were incubated in the absence or in the presence of 104 HEp-2 cells for 4 hours as described above. Bioluminescence emitted by cultures was monitored after 4 hours of infection (B), or was determined every 20 min after infection to monitor the kinetic of host cell contact-dependent yopE-luxCDABE induction (C). Bioluminescence is given as relative luminescence units (RLU) per colony forming units (CFU) and represents the mean of three independent experiments done in triplicate. White bar indicates 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108616&req=5

pone-0020425-g005: Cell contact dependent gene expression analysis using pFU plasmids.(A) About 5×104 bacteria of Y. pseudotuberculosis strain YPIII pTS28 (yopE-gfpmut3.1) were used to infect 103 HEp-2 cells. Bacteria were centrifuged onto the cell monolayer. Expression of the yopE-gfpmut3.1 fusion was monitored by phase contrast and fluorescence microscopy directly after centrifugation (0 h) and after four hours (4 h). (B,C) About 106 bacteria of Y. pseudotuberculosis strain YPIII pFU98 (empty vector) and YPIII pWO34 (yopE-luxCDABE) resuspended in PBS were incubated in the absence or in the presence of 104 HEp-2 cells for 4 hours as described above. Bioluminescence emitted by cultures was monitored after 4 hours of infection (B), or was determined every 20 min after infection to monitor the kinetic of host cell contact-dependent yopE-luxCDABE induction (C). Bioluminescence is given as relative luminescence units (RLU) per colony forming units (CFU) and represents the mean of three independent experiments done in triplicate. White bar indicates 5 µm.
Mentions: Autofluorescent and bioluminescent proteins are excellent reporters for monitoring gene expression in single bacteria in association with host cells, and they were successfully used to study bacterial gene expression within or onto host cells [35], [36]. For example, expression of the pathogenicity factor YopE of Yersinia was shown to be induced upon host cell contact [35]. In order to test our reporter plasmids we challenged cells with YPIII pTS28 and determined yopE-gfpmut3.1 expression for four hours after infection. We found that yersiniae bound to HEp-2 cells were significantly more fluorescent than bacteria without host cell contact (Figure 5A). A quantitative analysis of HEp-2 cells infected with YPIII harboring the yopE-luxCDABE fusion gave similar results and demonstrated a very strong (28-fold) increase of luciferase activity in the presence of host cells four hours post infection (Figure 5B). A kinetic analysis of host cell contact-dependent activation of the yopE-luxCDABE fusion revealed a very strong increase of yopE expression during the first 30 min after infection which is followed by a smaller but steady increase of yopE expression (Figure 5C). This clearly demonstrates that the different vector systems harboring fluorescent and bioluminescent reporter genes are suitable to follow and quantify gene expression of bacteria in association with cells.

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