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A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus.

Thanbichler M, Iniesta AA, Shapiro L - Nucleic Acids Res. (2007)

Bottom Line: This study reports the identification and functional characterization of a vanillate-regulated promoter (P(van)) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (P(xyl)).Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter.Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany. thanbichler@mpi-marburg.mpg.de

ABSTRACT
Caulobacter crescentus is widely used as a powerful model system for the study of prokaryotic cell biology and development. Analysis of this organism is complicated by a limited selection of tools for genetic manipulation and inducible gene expression. This study reports the identification and functional characterization of a vanillate-regulated promoter (P(van)) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (P(xyl)). Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter. Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins. Since many of these constructs are also suitable for use in other bacteria, this work provides a comprehensive collection of tools that will enrich many areas of microbiological research.

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Response of the vanAB promoter to different inducers and inducer concentrations. (A) Reporter constructs used to determine Pvan promoter activity. The 5′ region of vanA, which was fused in frame to the lacZ gene, is shown in black. (B) Activity of Pvan in the presence of different inducers. Wild-type strain CB15N was transformed with reporter plasmid pMT122 and grown in M2G minimal medium. In mid-exponential phase, vanillate, vanillin or vanillyl alcohol (vanOH) were added to a final concentration of 0.5 mM, respectively, and the activity of Pvan was determined. The inset shows the structural formulas of the inducers used. (C and D) Response of Pvan to different concentrations of vanillate. Cells of strain MT231 (▵vanA) transformed with reporter plasmid pMT122 were grown in M2G minimal medium (C) or PYE rich medium (D), exposed for 3 h to different concentrations of vanillate, and used to determine the activity of Pvan. The inset in (C) shows Pvan promoter activity at vanillate concentrations of 0–5 µM. Data depicted in panels (B), (C) and (D) represent the average of two independent experiments, each performed in triplicate. Standard deviations were smaller than 11% throughout.
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Figure 3: Response of the vanAB promoter to different inducers and inducer concentrations. (A) Reporter constructs used to determine Pvan promoter activity. The 5′ region of vanA, which was fused in frame to the lacZ gene, is shown in black. (B) Activity of Pvan in the presence of different inducers. Wild-type strain CB15N was transformed with reporter plasmid pMT122 and grown in M2G minimal medium. In mid-exponential phase, vanillate, vanillin or vanillyl alcohol (vanOH) were added to a final concentration of 0.5 mM, respectively, and the activity of Pvan was determined. The inset shows the structural formulas of the inducers used. (C and D) Response of Pvan to different concentrations of vanillate. Cells of strain MT231 (▵vanA) transformed with reporter plasmid pMT122 were grown in M2G minimal medium (C) or PYE rich medium (D), exposed for 3 h to different concentrations of vanillate, and used to determine the activity of Pvan. The inset in (C) shows Pvan promoter activity at vanillate concentrations of 0–5 µM. Data depicted in panels (B), (C) and (D) represent the average of two independent experiments, each performed in triplicate. Standard deviations were smaller than 11% throughout.

Mentions: In order to investigate the regulation of vanAB expression in more detail, a fragment comprising the vanR gene, the vanR-vanAB intergenic region, and the 5′ part of the vanA gene was cloned into the low-copy promoter probe vector pPR9TT (13), generating a translational fusion between vanA and the plasmid-borne lacZ reporter gene (Figure 3A). When wild-type strain CB15N was transformed with the resulting plasmid (pMT122) and grown in glucose minimal medium lacking any inducer, no transcriptional activity was detectable. Addition of vanillate, by contrast, strongy activated the vanAB promoter, thus confirming the regulatory pattern suggested by primer extension analysis (Figure 3B). A similar response was elicited by vanillin, another common product of lignin biodegradation (35). As a structural relative of vanillate, vanillin might directly interact with the effector-binding site of VanR. Alternatively, since Caulobacter contains a gene (CC2402) whose product shows 58% identity with vanillin dehydrogenase from Pseudomonas sp. HR199 (47), the stimulating effect of vanillin could be indirect, resulting from its oxidation to vanillate, which then serves as the actual inducer. In contrast to the carboxy and aldehyde forms, vanillyl alcohol only slightly activated the vanAB promoter, possibly due to weak interaction with VanR or slow metabolic conversion into vanillate as observed in Pseudomonas sp. PN-1 (48).Figure 3.


A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus.

Thanbichler M, Iniesta AA, Shapiro L - Nucleic Acids Res. (2007)

Response of the vanAB promoter to different inducers and inducer concentrations. (A) Reporter constructs used to determine Pvan promoter activity. The 5′ region of vanA, which was fused in frame to the lacZ gene, is shown in black. (B) Activity of Pvan in the presence of different inducers. Wild-type strain CB15N was transformed with reporter plasmid pMT122 and grown in M2G minimal medium. In mid-exponential phase, vanillate, vanillin or vanillyl alcohol (vanOH) were added to a final concentration of 0.5 mM, respectively, and the activity of Pvan was determined. The inset shows the structural formulas of the inducers used. (C and D) Response of Pvan to different concentrations of vanillate. Cells of strain MT231 (▵vanA) transformed with reporter plasmid pMT122 were grown in M2G minimal medium (C) or PYE rich medium (D), exposed for 3 h to different concentrations of vanillate, and used to determine the activity of Pvan. The inset in (C) shows Pvan promoter activity at vanillate concentrations of 0–5 µM. Data depicted in panels (B), (C) and (D) represent the average of two independent experiments, each performed in triplicate. Standard deviations were smaller than 11% throughout.
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Related In: Results  -  Collection

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Figure 3: Response of the vanAB promoter to different inducers and inducer concentrations. (A) Reporter constructs used to determine Pvan promoter activity. The 5′ region of vanA, which was fused in frame to the lacZ gene, is shown in black. (B) Activity of Pvan in the presence of different inducers. Wild-type strain CB15N was transformed with reporter plasmid pMT122 and grown in M2G minimal medium. In mid-exponential phase, vanillate, vanillin or vanillyl alcohol (vanOH) were added to a final concentration of 0.5 mM, respectively, and the activity of Pvan was determined. The inset shows the structural formulas of the inducers used. (C and D) Response of Pvan to different concentrations of vanillate. Cells of strain MT231 (▵vanA) transformed with reporter plasmid pMT122 were grown in M2G minimal medium (C) or PYE rich medium (D), exposed for 3 h to different concentrations of vanillate, and used to determine the activity of Pvan. The inset in (C) shows Pvan promoter activity at vanillate concentrations of 0–5 µM. Data depicted in panels (B), (C) and (D) represent the average of two independent experiments, each performed in triplicate. Standard deviations were smaller than 11% throughout.
Mentions: In order to investigate the regulation of vanAB expression in more detail, a fragment comprising the vanR gene, the vanR-vanAB intergenic region, and the 5′ part of the vanA gene was cloned into the low-copy promoter probe vector pPR9TT (13), generating a translational fusion between vanA and the plasmid-borne lacZ reporter gene (Figure 3A). When wild-type strain CB15N was transformed with the resulting plasmid (pMT122) and grown in glucose minimal medium lacking any inducer, no transcriptional activity was detectable. Addition of vanillate, by contrast, strongy activated the vanAB promoter, thus confirming the regulatory pattern suggested by primer extension analysis (Figure 3B). A similar response was elicited by vanillin, another common product of lignin biodegradation (35). As a structural relative of vanillate, vanillin might directly interact with the effector-binding site of VanR. Alternatively, since Caulobacter contains a gene (CC2402) whose product shows 58% identity with vanillin dehydrogenase from Pseudomonas sp. HR199 (47), the stimulating effect of vanillin could be indirect, resulting from its oxidation to vanillate, which then serves as the actual inducer. In contrast to the carboxy and aldehyde forms, vanillyl alcohol only slightly activated the vanAB promoter, possibly due to weak interaction with VanR or slow metabolic conversion into vanillate as observed in Pseudomonas sp. PN-1 (48).Figure 3.

Bottom Line: This study reports the identification and functional characterization of a vanillate-regulated promoter (P(van)) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (P(xyl)).Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter.Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany. thanbichler@mpi-marburg.mpg.de

ABSTRACT
Caulobacter crescentus is widely used as a powerful model system for the study of prokaryotic cell biology and development. Analysis of this organism is complicated by a limited selection of tools for genetic manipulation and inducible gene expression. This study reports the identification and functional characterization of a vanillate-regulated promoter (P(van)) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (P(xyl)). Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter. Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins. Since many of these constructs are also suitable for use in other bacteria, this work provides a comprehensive collection of tools that will enrich many areas of microbiological research.

Show MeSH
Related in: MedlinePlus