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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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In vivo expression analysis of a yadA-luxCDABE fusion.(A) 1×109 bacteria of Y. pseudotuberculosis strain YPIII pTS31 (yadA-luxCDABE) were used to orally infect Balb/c mice. Three days post infection, mice were anesthesized and bioluminescence was determined with an IVIS camera on the ventral or dorsal side. (B) Subsequently, mice were sacrifized and the gastrointestinal tract (G), the mesenterial lymph nodes (MLN) and the organs such as kidneys (K) and liver (L) were prepared and bioluminescence was determined with the IVIS system.
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pone-0020425-g006: In vivo expression analysis of a yadA-luxCDABE fusion.(A) 1×109 bacteria of Y. pseudotuberculosis strain YPIII pTS31 (yadA-luxCDABE) were used to orally infect Balb/c mice. Three days post infection, mice were anesthesized and bioluminescence was determined with an IVIS camera on the ventral or dorsal side. (B) Subsequently, mice were sacrifized and the gastrointestinal tract (G), the mesenterial lymph nodes (MLN) and the organs such as kidneys (K) and liver (L) were prepared and bioluminescence was determined with the IVIS system.

Mentions: Y. pseudotuberculosis is known to replicate in lymphatic tissues (e.g. Peyer's patches (PPs), MLNs) and organs such as spleen, and the YadA adhesin was found to be crucial for the colonization of these tissues and organs [29]. In order to test our luminescent reporter plasmids expression of the yadA virulence gene was analyzed during infection of mice. To do so, we orally infected Balb/c mice with 1×109 bacteria of the Y. pseudotuberculosis strain YPIII pTS31 harboring the yadA-luxCDABE fusion. Three days after infection mice were anesthetized and their bioluminescence was measured using an IVIS camera. As shown in Figure 6A yadA-luxCDABE expression was detected in the intestinal tract and spleen demonstrating yadA expression in the bacteria during murine infections. Interestingly, the nasal cavity and the cervical lymph nodes of the mice were also luminescent after 3 days of infection, indicating colonization and yadA expression of the nasal-associated lymphoid tissue (NALT) by Y. pseudotuberculosis. In order to analyze colonization of the mice in more detail, mice were sacrificed at day three post infection and the small intestine, the MLNs, kidney and liver were removed and analyzed using the IVIS system. We found that multiple PPs were luminescent and luminescent bacteria were also observed within the MLNs (Figure 6B). In contrast, no bioluminescence was detectable in mice infected with empty luxCDABE fusion plasmids (data not shown).


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)

In vivo expression analysis of a yadA-luxCDABE fusion.(A) 1×109 bacteria of Y. pseudotuberculosis strain YPIII pTS31 (yadA-luxCDABE) were used to orally infect Balb/c mice. Three days post infection, mice were anesthesized and bioluminescence was determined with an IVIS camera on the ventral or dorsal side. (B) Subsequently, mice were sacrifized and the gastrointestinal tract (G), the mesenterial lymph nodes (MLN) and the organs such as kidneys (K) and liver (L) were prepared and bioluminescence was determined with the IVIS system.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020425-g006: In vivo expression analysis of a yadA-luxCDABE fusion.(A) 1×109 bacteria of Y. pseudotuberculosis strain YPIII pTS31 (yadA-luxCDABE) were used to orally infect Balb/c mice. Three days post infection, mice were anesthesized and bioluminescence was determined with an IVIS camera on the ventral or dorsal side. (B) Subsequently, mice were sacrifized and the gastrointestinal tract (G), the mesenterial lymph nodes (MLN) and the organs such as kidneys (K) and liver (L) were prepared and bioluminescence was determined with the IVIS system.
Mentions: Y. pseudotuberculosis is known to replicate in lymphatic tissues (e.g. Peyer's patches (PPs), MLNs) and organs such as spleen, and the YadA adhesin was found to be crucial for the colonization of these tissues and organs [29]. In order to test our luminescent reporter plasmids expression of the yadA virulence gene was analyzed during infection of mice. To do so, we orally infected Balb/c mice with 1×109 bacteria of the Y. pseudotuberculosis strain YPIII pTS31 harboring the yadA-luxCDABE fusion. Three days after infection mice were anesthetized and their bioluminescence was measured using an IVIS camera. As shown in Figure 6A yadA-luxCDABE expression was detected in the intestinal tract and spleen demonstrating yadA expression in the bacteria during murine infections. Interestingly, the nasal cavity and the cervical lymph nodes of the mice were also luminescent after 3 days of infection, indicating colonization and yadA expression of the nasal-associated lymphoid tissue (NALT) by Y. pseudotuberculosis. In order to analyze colonization of the mice in more detail, mice were sacrificed at day three post infection and the small intestine, the MLNs, kidney and liver were removed and analyzed using the IVIS system. We found that multiple PPs were luminescent and luminescent bacteria were also observed within the MLNs (Figure 6B). In contrast, no bioluminescence was detectable in mice infected with empty luxCDABE fusion plasmids (data not shown).

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