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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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Monitoring of a Y. pseudotuberculosis YPIII infection expressing a constitutive gapA-luxCDABE fusion.1×109 bacteria of Y. pseudotuberculosis strain YPIII pFU166 (gapA-luxCDABE) were used to orally infect Balb/c mice. (A) 1–5 days post infection, mice were anesthesized and bioluminescence was determined with an IVIS camera on the ventral side. (B) On day 3, one mouse was sacrificed and bioluminescence of the gastrointestinal tract, including Peyer's patches (PP), caecum, and associated mesenterial lymph nodes (MLN) were analyzed with the IVIS system. (C) At the indicated time points 3 mice were killed, the caecum and the MLNs were prepared, homogenized and the number of bacteria in the tissues (CFU) per g organ were determined.
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pone-0020425-g007: Monitoring of a Y. pseudotuberculosis YPIII infection expressing a constitutive gapA-luxCDABE fusion.1×109 bacteria of Y. pseudotuberculosis strain YPIII pFU166 (gapA-luxCDABE) were used to orally infect Balb/c mice. (A) 1–5 days post infection, mice were anesthesized and bioluminescence was determined with an IVIS camera on the ventral side. (B) On day 3, one mouse was sacrificed and bioluminescence of the gastrointestinal tract, including Peyer's patches (PP), caecum, and associated mesenterial lymph nodes (MLN) were analyzed with the IVIS system. (C) At the indicated time points 3 mice were killed, the caecum and the MLNs were prepared, homogenized and the number of bacteria in the tissues (CFU) per g organ were determined.

Mentions: Vector pFU166 in which the bioluminescent reporter genes luxCDABE were constitutively expressed under the control of PgapA was used to follow the course of a Yersinia infection in Balb/c mice. A set of mice was intragastrically inoculated with 1×109 bacteria of Y. pseudotuberculosis strain YPIII pFU166 (gapA-luxCDABE), and the bioluminescent signal was monitored in living animals for 5 days after infection. One day post-feeding a strong bioluminescent signal was observed in the intestinal tract of the infected mice. The signal increased to a plateau at day 3–4 and decreased again at day 5 (Figure 7A). Preparation of the intestinal tract and gut-associated tissues further showed that the highest bioluminescent signal occured in the PPs, the MLNs and the caecum (Figure 7B). In parallel, we determined the colony forming units of bacteria isolated from the intestinal tract (caecum) and the MLNs (Figure 7C). Consistently to the bioluminescent intensity pictures, the highest number of bacteria was recovered from the caecum 2–4 days and from the MLNs 4 days post infection. These results indicated that colonization of the intestine and associated lymphatic tissues can be detected directly by simple recording of the bioluminescent signal in living animals harboring the luxCDABE expression plasmid. This constitutes an advantage compared to other inducible systems such as the PlacZ- or PxylE-dependent fusion constructs or the PBAD-luxCDABE expression system in which 150 mg L-arabinose needs to be applied intraperitoneally to induce luciferase expression 2–5 hours before monitoring of the luciferase activity during infection [37], [38].


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)

Monitoring of a Y. pseudotuberculosis YPIII infection expressing a constitutive gapA-luxCDABE fusion.1×109 bacteria of Y. pseudotuberculosis strain YPIII pFU166 (gapA-luxCDABE) were used to orally infect Balb/c mice. (A) 1–5 days post infection, mice were anesthesized and bioluminescence was determined with an IVIS camera on the ventral side. (B) On day 3, one mouse was sacrificed and bioluminescence of the gastrointestinal tract, including Peyer's patches (PP), caecum, and associated mesenterial lymph nodes (MLN) were analyzed with the IVIS system. (C) At the indicated time points 3 mice were killed, the caecum and the MLNs were prepared, homogenized and the number of bacteria in the tissues (CFU) per g organ were determined.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020425-g007: Monitoring of a Y. pseudotuberculosis YPIII infection expressing a constitutive gapA-luxCDABE fusion.1×109 bacteria of Y. pseudotuberculosis strain YPIII pFU166 (gapA-luxCDABE) were used to orally infect Balb/c mice. (A) 1–5 days post infection, mice were anesthesized and bioluminescence was determined with an IVIS camera on the ventral side. (B) On day 3, one mouse was sacrificed and bioluminescence of the gastrointestinal tract, including Peyer's patches (PP), caecum, and associated mesenterial lymph nodes (MLN) were analyzed with the IVIS system. (C) At the indicated time points 3 mice were killed, the caecum and the MLNs were prepared, homogenized and the number of bacteria in the tissues (CFU) per g organ were determined.
Mentions: Vector pFU166 in which the bioluminescent reporter genes luxCDABE were constitutively expressed under the control of PgapA was used to follow the course of a Yersinia infection in Balb/c mice. A set of mice was intragastrically inoculated with 1×109 bacteria of Y. pseudotuberculosis strain YPIII pFU166 (gapA-luxCDABE), and the bioluminescent signal was monitored in living animals for 5 days after infection. One day post-feeding a strong bioluminescent signal was observed in the intestinal tract of the infected mice. The signal increased to a plateau at day 3–4 and decreased again at day 5 (Figure 7A). Preparation of the intestinal tract and gut-associated tissues further showed that the highest bioluminescent signal occured in the PPs, the MLNs and the caecum (Figure 7B). In parallel, we determined the colony forming units of bacteria isolated from the intestinal tract (caecum) and the MLNs (Figure 7C). Consistently to the bioluminescent intensity pictures, the highest number of bacteria was recovered from the caecum 2–4 days and from the MLNs 4 days post infection. These results indicated that colonization of the intestine and associated lymphatic tissues can be detected directly by simple recording of the bioluminescent signal in living animals harboring the luxCDABE expression plasmid. This constitutes an advantage compared to other inducible systems such as the PlacZ- or PxylE-dependent fusion constructs or the PBAD-luxCDABE expression system in which 150 mg L-arabinose needs to be applied intraperitoneally to induce luciferase expression 2–5 hours before monitoring of the luciferase activity during infection [37], [38].

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