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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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Vanillate degradation by Caulobacter. (A) Conversion of vanillate into protocatechuate, as catalyzed by the vanillate demethylase (VanAB) complex. (B) Arrangement of the vanR, vanA and vanB genes in C. crescentus, Acinetobacter sp. ADP1 and Pseudomonas sp. HR199. (C) Cell densities achieved with vanillate or glucose as the sole carbon source. Minimal media composed of 0.5 mM vanillate (light bars) or glucose (dark bars), respectively, in M2 salts (6) were inoculated with washed cells of wild-type strain CB15N. The cultures were grown until the carbon source was depleted, and their optical densities were determined (growth cycle 1). Subsequently, the cells were subjected to another five growth cycles, each of which started with replenishment of the carbon source (0.5 mM final concentration), followed by growth to stationary phase and determination of the respective cell densities. Note: Stepwise addition of the carbon source was necessary due to a negative effect of vanillate on growth at concentrations higher than 0.5 mM. (D) Involvement of CC2393 (VanA) in vanillate degradation. M2G minimal medium containing no (−) or 0.5 mM (+) vanillate, respectively, was inoculated with wild-type strain CB15N (WT) or its ▵vanA-derivative MT231 and incubated for 24 h. Subsequently, the cells were pelleted, and the supernatant was analyzed spectrophotometrically at 286 nm (the absorption maximum of vanillate) to determine the amount of vanillate left in the medium. Sterile medium (w/o) was used as a control. Data represent the mean of three independent experiments (±SD). (E) Induction kinetics of vanillate degradation. Cells of wild-type strain CB15N were grown to exponential phase in M2G minimal medium, washed, and resuspended in M2G medium containing 0.5 mM vanillate to an OD600 of 0.11. Samples were taken at one-hour intervals and analyzed for cell density (OD600, open triangle) and vanillate content (E286, filled circle).
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Figure 1: Vanillate degradation by Caulobacter. (A) Conversion of vanillate into protocatechuate, as catalyzed by the vanillate demethylase (VanAB) complex. (B) Arrangement of the vanR, vanA and vanB genes in C. crescentus, Acinetobacter sp. ADP1 and Pseudomonas sp. HR199. (C) Cell densities achieved with vanillate or glucose as the sole carbon source. Minimal media composed of 0.5 mM vanillate (light bars) or glucose (dark bars), respectively, in M2 salts (6) were inoculated with washed cells of wild-type strain CB15N. The cultures were grown until the carbon source was depleted, and their optical densities were determined (growth cycle 1). Subsequently, the cells were subjected to another five growth cycles, each of which started with replenishment of the carbon source (0.5 mM final concentration), followed by growth to stationary phase and determination of the respective cell densities. Note: Stepwise addition of the carbon source was necessary due to a negative effect of vanillate on growth at concentrations higher than 0.5 mM. (D) Involvement of CC2393 (VanA) in vanillate degradation. M2G minimal medium containing no (−) or 0.5 mM (+) vanillate, respectively, was inoculated with wild-type strain CB15N (WT) or its ▵vanA-derivative MT231 and incubated for 24 h. Subsequently, the cells were pelleted, and the supernatant was analyzed spectrophotometrically at 286 nm (the absorption maximum of vanillate) to determine the amount of vanillate left in the medium. Sterile medium (w/o) was used as a control. Data represent the mean of three independent experiments (±SD). (E) Induction kinetics of vanillate degradation. Cells of wild-type strain CB15N were grown to exponential phase in M2G minimal medium, washed, and resuspended in M2G medium containing 0.5 mM vanillate to an OD600 of 0.11. Samples were taken at one-hour intervals and analyzed for cell density (OD600, open triangle) and vanillate content (E286, filled circle).

Mentions: Apart from polysaccharides, lignin represents the second most abundant constituent of the vascular plant cell wall. It is a heterogeneous, highly crosslinked polymer derived from the aromatic monolignols coniferyl, coumaryl and sinapyl alcohol (34). Due to its complex chemical structure, its initial, oxidative cleavage can only be performed by a small group of organisms, most importantly fungi (35). The soluble phenolic intermediates, however, released during this process are subsequently metabolized by a variety of different bacterial species. One of the compounds commonly produced from lignin is vanillate (Figure 1A). To allow its utilization as a carbon source, it first has to be converted into protocatechuate by removal of its O-methyl group. Cleavage of the aromatic ring system and further degradation via the β-ketoadipate pathway then yield succinyl-CoA and acetyl-CoA, which can directly enter the citric acid cycle (36). In many bacteria, O-demethylation of vanillate is carried out by a two-component monooxygenase, composed of a terminal oxygenase (VanA) and a ferredoxin-like reductase (VanB) subunit. Usually, these two proteins are encoded in an operon, with their synthesis being regulated by a transcriptional repressor, VanR, whose gene is transcribed together with or divergently from the vanAB trancriptional unit (37–40) (Figure 1B).Figure 1.


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)

Vanillate degradation by Caulobacter. (A) Conversion of vanillate into protocatechuate, as catalyzed by the vanillate demethylase (VanAB) complex. (B) Arrangement of the vanR, vanA and vanB genes in C. crescentus, Acinetobacter sp. ADP1 and Pseudomonas sp. HR199. (C) Cell densities achieved with vanillate or glucose as the sole carbon source. Minimal media composed of 0.5 mM vanillate (light bars) or glucose (dark bars), respectively, in M2 salts (6) were inoculated with washed cells of wild-type strain CB15N. The cultures were grown until the carbon source was depleted, and their optical densities were determined (growth cycle 1). Subsequently, the cells were subjected to another five growth cycles, each of which started with replenishment of the carbon source (0.5 mM final concentration), followed by growth to stationary phase and determination of the respective cell densities. Note: Stepwise addition of the carbon source was necessary due to a negative effect of vanillate on growth at concentrations higher than 0.5 mM. (D) Involvement of CC2393 (VanA) in vanillate degradation. M2G minimal medium containing no (−) or 0.5 mM (+) vanillate, respectively, was inoculated with wild-type strain CB15N (WT) or its ▵vanA-derivative MT231 and incubated for 24 h. Subsequently, the cells were pelleted, and the supernatant was analyzed spectrophotometrically at 286 nm (the absorption maximum of vanillate) to determine the amount of vanillate left in the medium. Sterile medium (w/o) was used as a control. Data represent the mean of three independent experiments (±SD). (E) Induction kinetics of vanillate degradation. Cells of wild-type strain CB15N were grown to exponential phase in M2G minimal medium, washed, and resuspended in M2G medium containing 0.5 mM vanillate to an OD600 of 0.11. Samples were taken at one-hour intervals and analyzed for cell density (OD600, open triangle) and vanillate content (E286, filled circle).
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Figure 1: Vanillate degradation by Caulobacter. (A) Conversion of vanillate into protocatechuate, as catalyzed by the vanillate demethylase (VanAB) complex. (B) Arrangement of the vanR, vanA and vanB genes in C. crescentus, Acinetobacter sp. ADP1 and Pseudomonas sp. HR199. (C) Cell densities achieved with vanillate or glucose as the sole carbon source. Minimal media composed of 0.5 mM vanillate (light bars) or glucose (dark bars), respectively, in M2 salts (6) were inoculated with washed cells of wild-type strain CB15N. The cultures were grown until the carbon source was depleted, and their optical densities were determined (growth cycle 1). Subsequently, the cells were subjected to another five growth cycles, each of which started with replenishment of the carbon source (0.5 mM final concentration), followed by growth to stationary phase and determination of the respective cell densities. Note: Stepwise addition of the carbon source was necessary due to a negative effect of vanillate on growth at concentrations higher than 0.5 mM. (D) Involvement of CC2393 (VanA) in vanillate degradation. M2G minimal medium containing no (−) or 0.5 mM (+) vanillate, respectively, was inoculated with wild-type strain CB15N (WT) or its ▵vanA-derivative MT231 and incubated for 24 h. Subsequently, the cells were pelleted, and the supernatant was analyzed spectrophotometrically at 286 nm (the absorption maximum of vanillate) to determine the amount of vanillate left in the medium. Sterile medium (w/o) was used as a control. Data represent the mean of three independent experiments (±SD). (E) Induction kinetics of vanillate degradation. Cells of wild-type strain CB15N were grown to exponential phase in M2G minimal medium, washed, and resuspended in M2G medium containing 0.5 mM vanillate to an OD600 of 0.11. Samples were taken at one-hour intervals and analyzed for cell density (OD600, open triangle) and vanillate content (E286, filled circle).
Mentions: Apart from polysaccharides, lignin represents the second most abundant constituent of the vascular plant cell wall. It is a heterogeneous, highly crosslinked polymer derived from the aromatic monolignols coniferyl, coumaryl and sinapyl alcohol (34). Due to its complex chemical structure, its initial, oxidative cleavage can only be performed by a small group of organisms, most importantly fungi (35). The soluble phenolic intermediates, however, released during this process are subsequently metabolized by a variety of different bacterial species. One of the compounds commonly produced from lignin is vanillate (Figure 1A). To allow its utilization as a carbon source, it first has to be converted into protocatechuate by removal of its O-methyl group. Cleavage of the aromatic ring system and further degradation via the β-ketoadipate pathway then yield succinyl-CoA and acetyl-CoA, which can directly enter the citric acid cycle (36). In many bacteria, O-demethylation of vanillate is carried out by a two-component monooxygenase, composed of a terminal oxygenase (VanA) and a ferredoxin-like reductase (VanB) subunit. Usually, these two proteins are encoded in an operon, with their synthesis being regulated by a transcriptional repressor, VanR, whose gene is transcribed together with or divergently from the vanAB trancriptional unit (37–40) (Figure 1B).Figure 1.

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