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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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Related in: MedlinePlus

Detection of Yersinia in infected tissue and simultaneous analysis of yadA-gfpmut3.1 expression.1×109 bacteria of Y. pseudotuberculosis strain YPIII pFU96/pTS39 (gapA-dsRed2; yadA-gfpmut3.1) were used to orally infect Balb/c mice. 3 days post infection, mice were sacrificed, PPs and MLNs were isolated. Histological slides were prepared and analyzed by fluorescence microscopy to detect the bacteria in the tissues by expression of the reporter protein DsRed2. In parallel yadA expression in the bacteria was monitored by GFPmut3.1 mediated fluorescence. White bar indicates 10 µm.
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pone-0020425-g008: Detection of Yersinia in infected tissue and simultaneous analysis of yadA-gfpmut3.1 expression.1×109 bacteria of Y. pseudotuberculosis strain YPIII pFU96/pTS39 (gapA-dsRed2; yadA-gfpmut3.1) were used to orally infect Balb/c mice. 3 days post infection, mice were sacrificed, PPs and MLNs were isolated. Histological slides were prepared and analyzed by fluorescence microscopy to detect the bacteria in the tissues by expression of the reporter protein DsRed2. In parallel yadA expression in the bacteria was monitored by GFPmut3.1 mediated fluorescence. White bar indicates 10 µm.

Mentions: In order to identify bacteria in host tissues and study virulence gene expression in parallel, YPIII harboring pFU96 (gapA-dsRed2) and a compatible yadA-gfpmut3.1 fusion plasmid (pTS39) was used to infect Balb/c mice. Cryosections were prepared of PPs and MLNs 3 days post infection. The bacteria in the tissues were visualized by monitoring DsRed2, and then tested for yadA-gfpmut3.1 expression with a fluorescent microscope. As shown in Figure 8, red-fluorescent bacteria could easily be detected within the tissues. By switching the fluorescence filter we could demonstrate that bacteria identified in these tissues also express the yadA-gfpmut3.1 fusion construct (Figure 8). Interestingly, yadA expression was highly induced in many, but not in all bacteria detectable in the PPs, indicating heterogeneous yadA expression, whereas all yersiniae in the microcolonies found in the MLNs expressed yadA.


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)

Detection of Yersinia in infected tissue and simultaneous analysis of yadA-gfpmut3.1 expression.1×109 bacteria of Y. pseudotuberculosis strain YPIII pFU96/pTS39 (gapA-dsRed2; yadA-gfpmut3.1) were used to orally infect Balb/c mice. 3 days post infection, mice were sacrificed, PPs and MLNs were isolated. Histological slides were prepared and analyzed by fluorescence microscopy to detect the bacteria in the tissues by expression of the reporter protein DsRed2. In parallel yadA expression in the bacteria was monitored by GFPmut3.1 mediated fluorescence. White bar indicates 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020425-g008: Detection of Yersinia in infected tissue and simultaneous analysis of yadA-gfpmut3.1 expression.1×109 bacteria of Y. pseudotuberculosis strain YPIII pFU96/pTS39 (gapA-dsRed2; yadA-gfpmut3.1) were used to orally infect Balb/c mice. 3 days post infection, mice were sacrificed, PPs and MLNs were isolated. Histological slides were prepared and analyzed by fluorescence microscopy to detect the bacteria in the tissues by expression of the reporter protein DsRed2. In parallel yadA expression in the bacteria was monitored by GFPmut3.1 mediated fluorescence. White bar indicates 10 µm.
Mentions: In order to identify bacteria in host tissues and study virulence gene expression in parallel, YPIII harboring pFU96 (gapA-dsRed2) and a compatible yadA-gfpmut3.1 fusion plasmid (pTS39) was used to infect Balb/c mice. Cryosections were prepared of PPs and MLNs 3 days post infection. The bacteria in the tissues were visualized by monitoring DsRed2, and then tested for yadA-gfpmut3.1 expression with a fluorescent microscope. As shown in Figure 8, red-fluorescent bacteria could easily be detected within the tissues. By switching the fluorescence filter we could demonstrate that bacteria identified in these tissues also express the yadA-gfpmut3.1 fusion construct (Figure 8). Interestingly, yadA expression was highly induced in many, but not in all bacteria detectable in the PPs, indicating heterogeneous yadA expression, whereas all yersiniae in the microcolonies found in the MLNs expressed yadA.

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