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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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Characteristics of the vanAB promoter. (A) Overview of the vanAB promoter region. The vanR-vanAB intergenic region is shown in normal print, whereas the 5′ ends of the flanking vanR and vanA genes are given in boldface, with their orientations indicated by arrows. The transcriptional start site of vanA (see Figure 2B) and the putative −10 and −35 motifs of the vanA promoter are labeled. Diverging arrows and italic letters mark two copies of a perfectly palindromic sequence, which is likely to represent the VanR target site. (B) Determination of the vanAB transcriptional start site. Primer extension analysis was conducted on RNA extracted from cells of wild-type strain CB15N which had been grown in M2G medium in the presence (lane 5) or absence (lane 6) of 0.5 mM vanillate. In parallel, sequencing reactions were performed (lanes 1–4). The two reaction products and the corresponding +1 sites are indicated by arrows.
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Figure 2: Characteristics of the vanAB promoter. (A) Overview of the vanAB promoter region. The vanR-vanAB intergenic region is shown in normal print, whereas the 5′ ends of the flanking vanR and vanA genes are given in boldface, with their orientations indicated by arrows. The transcriptional start site of vanA (see Figure 2B) and the putative −10 and −35 motifs of the vanA promoter are labeled. Diverging arrows and italic letters mark two copies of a perfectly palindromic sequence, which is likely to represent the VanR target site. (B) Determination of the vanAB transcriptional start site. Primer extension analysis was conducted on RNA extracted from cells of wild-type strain CB15N which had been grown in M2G medium in the presence (lane 5) or absence (lane 6) of 0.5 mM vanillate. In parallel, sequencing reactions were performed (lanes 1–4). The two reaction products and the corresponding +1 sites are indicated by arrows.

Mentions: Having identified a putative vanillate degradation gene cluster, we determined if the promoter driving transcription of the vanAB operon (Pvan) could be exploited for inducible gene expression in Caulobacter. For this purpose, a detailed functional analysis of the 126 bp intergenic region shared by the vanR gene and vanAB genes (Figure 2A) was performed. Primer extension analysis of RNA isolated from wild-type strain CB15N grown in the presence of vanillate showed that transcription of vanA mostly initiates at an adenosine residue 35 bp upstream of the vanAB translational start site (Figure 2B, lane 5). In addition, a minor transcript species carrying an additional 5′ cytosine residue could be detected. The primer extension reaction consistently failed to yield a product when performed on RNA from cells that had been grown in medium lacking vanillate (Figure 2B, lane 6), indicating that expression of vanAB is induced by vanillate or one of its degradation products. Upstream of the experimentally determined +1 site, sequences corresponding to the characteristic -35 and -10 boxes of a housekeeping, σ73-dependent promoter can be identified (44,45) (Figure 2A). Their relative spacing and distance to the transcriptional start site suggests that they represent the core promoter elements responsible for vanAB transcription. The vanAB upstream region further contains two copies of a perfect inverted repeat (5′-ATTGGATCCAAT-3′), which cannot be found anywhere else in the Caulobacter genome (32). One of them immediately precedes the –35 box, whereas the other one overlaps the –10 box and the +1 site (Figure 2A). Palindromic sequences arranged in such a manner frequently reflect GntR-type transcription factor binding sites (46). The two repeats are, therefore, likely to interact with the Caulobacter VanR homolog, mediating repression of vanAB transcription in the absence of vanillate.Figure 2.


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)

Characteristics of the vanAB promoter. (A) Overview of the vanAB promoter region. The vanR-vanAB intergenic region is shown in normal print, whereas the 5′ ends of the flanking vanR and vanA genes are given in boldface, with their orientations indicated by arrows. The transcriptional start site of vanA (see Figure 2B) and the putative −10 and −35 motifs of the vanA promoter are labeled. Diverging arrows and italic letters mark two copies of a perfectly palindromic sequence, which is likely to represent the VanR target site. (B) Determination of the vanAB transcriptional start site. Primer extension analysis was conducted on RNA extracted from cells of wild-type strain CB15N which had been grown in M2G medium in the presence (lane 5) or absence (lane 6) of 0.5 mM vanillate. In parallel, sequencing reactions were performed (lanes 1–4). The two reaction products and the corresponding +1 sites are indicated by arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175322&req=5

Figure 2: Characteristics of the vanAB promoter. (A) Overview of the vanAB promoter region. The vanR-vanAB intergenic region is shown in normal print, whereas the 5′ ends of the flanking vanR and vanA genes are given in boldface, with their orientations indicated by arrows. The transcriptional start site of vanA (see Figure 2B) and the putative −10 and −35 motifs of the vanA promoter are labeled. Diverging arrows and italic letters mark two copies of a perfectly palindromic sequence, which is likely to represent the VanR target site. (B) Determination of the vanAB transcriptional start site. Primer extension analysis was conducted on RNA extracted from cells of wild-type strain CB15N which had been grown in M2G medium in the presence (lane 5) or absence (lane 6) of 0.5 mM vanillate. In parallel, sequencing reactions were performed (lanes 1–4). The two reaction products and the corresponding +1 sites are indicated by arrows.
Mentions: Having identified a putative vanillate degradation gene cluster, we determined if the promoter driving transcription of the vanAB operon (Pvan) could be exploited for inducible gene expression in Caulobacter. For this purpose, a detailed functional analysis of the 126 bp intergenic region shared by the vanR gene and vanAB genes (Figure 2A) was performed. Primer extension analysis of RNA isolated from wild-type strain CB15N grown in the presence of vanillate showed that transcription of vanA mostly initiates at an adenosine residue 35 bp upstream of the vanAB translational start site (Figure 2B, lane 5). In addition, a minor transcript species carrying an additional 5′ cytosine residue could be detected. The primer extension reaction consistently failed to yield a product when performed on RNA from cells that had been grown in medium lacking vanillate (Figure 2B, lane 6), indicating that expression of vanAB is induced by vanillate or one of its degradation products. Upstream of the experimentally determined +1 site, sequences corresponding to the characteristic -35 and -10 boxes of a housekeeping, σ73-dependent promoter can be identified (44,45) (Figure 2A). Their relative spacing and distance to the transcriptional start site suggests that they represent the core promoter elements responsible for vanAB transcription. The vanAB upstream region further contains two copies of a perfect inverted repeat (5′-ATTGGATCCAAT-3′), which cannot be found anywhere else in the Caulobacter genome (32). One of them immediately precedes the –35 box, whereas the other one overlaps the –10 box and the +1 site (Figure 2A). Palindromic sequences arranged in such a manner frequently reflect GntR-type transcription factor binding sites (46). The two repeats are, therefore, likely to interact with the Caulobacter VanR homolog, mediating repression of vanAB transcription in the absence of vanillate.Figure 2.

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