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Positively regulated bacterial expression systems.

Brautaset T, Lale R, Valla S - Microb Biotechnol (2008)

Bottom Line: The available systems that have been applied to express heterologous genes are regulated either by sugars (L-arabinose, L-rhamnose, xylose and sucrose), substituted benzenes, cyclohexanone-related compounds, ε-caprolactam, propionate, thiostrepton, alkanes or peptides.It is of applied interest that some of the inducers require the presence of transport systems, some are more prone than others to become metabolized by the host and some have been applied mainly in one or a limited number of species.Based on bioinformatics analyses, the AraC-XylS family of regulators contains a large number of different members (currently over 300), but only a small fraction of these, the XylS/Pm, AraC/P(BAD), RhaR-RhaS/rhaBAD, NitR/PnitA and ChnR/Pb regulator/promoter systems, have so far been explored for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Sintef Materials and Chemistry, Sintef, Trondheim, Norway. trygve.brautaset@sintef.no

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AraC‐XylS family regulators' characteristic HTH DNA binding motif is shown by using the member MarA as a model (α‐helixes 3 and 6 in red colour). Conserved amino acid residues are depicted in the bottom row. The alignment was derived from the full‐length primary sequences of the given TFs by using the PROMALS3D web server (Pei et al., 2008). Parameters were left at default values. The figure was prepared by using PyMOL (DeLano, 2003). Note that MarA binds DNA as a monomer.
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f2: AraC‐XylS family regulators' characteristic HTH DNA binding motif is shown by using the member MarA as a model (α‐helixes 3 and 6 in red colour). Conserved amino acid residues are depicted in the bottom row. The alignment was derived from the full‐length primary sequences of the given TFs by using the PROMALS3D web server (Pei et al., 2008). Parameters were left at default values. The figure was prepared by using PyMOL (DeLano, 2003). Note that MarA binds DNA as a monomer.

Mentions: Experimental work involving functional expression, purification and biochemical characterization of AraC is technically very difficult, as reviewed by Schleif (2003). This property also holds for a large number of the AraC‐XylS family members; they are extremely insoluble. The major reason is predicted to be due to the unusually long contact area between the protein DNA binding domain and its target DNA (almost 40 base pairs). Each monomer contacts two major groove regions of the DNA (see Fig. 1) using two helix–turn–helix motifs (HTH) (Ogden et al., 1980; Hendricksen and Schleif, 1985; Brunelle and Schleif, 1989; Carra and Schleif, 1993). It has been proposed that the AraC protein, in particular the DNA binding domain, does not complete folding in the absence of DNA (Schleif, 2003). A partially folded DNA binding domain is sensitive to proteases and with an excessive number of hydrophobic residues exposed, this may also lead to aggregation. Due to all these problems, the AraC protein crystal structure was determined more than 20 years after its discovery (Soisson et al., 1997a,b). The technical difficulties experienced with AraC are most likely one major reason why few of the large number of TFs of this family have not yet been characterized biochemically (Martin and Rosner, 2001). Currently, 3D structures have been experimentally solved for only three members; AraC, and the monomeric regulators RobA, and MarA (Rhee et al., 1998; Kwon et al., 2000). The structure of the MarA protein is shown in Fig. 2. The conserved C‐terminal domain of the TF contains the characteristic HTH motif critical for DNA binding. In contrast, the N‐terminal domains among AraC‐XylS family TFs are structurally highly divergent, supporting the assumption that the insolubility properties of these proteins can be mainly assigned to the DNA binding domain.


Positively regulated bacterial expression systems.

Brautaset T, Lale R, Valla S - Microb Biotechnol (2008)

AraC‐XylS family regulators' characteristic HTH DNA binding motif is shown by using the member MarA as a model (α‐helixes 3 and 6 in red colour). Conserved amino acid residues are depicted in the bottom row. The alignment was derived from the full‐length primary sequences of the given TFs by using the PROMALS3D web server (Pei et al., 2008). Parameters were left at default values. The figure was prepared by using PyMOL (DeLano, 2003). Note that MarA binds DNA as a monomer.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815419&req=5

f2: AraC‐XylS family regulators' characteristic HTH DNA binding motif is shown by using the member MarA as a model (α‐helixes 3 and 6 in red colour). Conserved amino acid residues are depicted in the bottom row. The alignment was derived from the full‐length primary sequences of the given TFs by using the PROMALS3D web server (Pei et al., 2008). Parameters were left at default values. The figure was prepared by using PyMOL (DeLano, 2003). Note that MarA binds DNA as a monomer.
Mentions: Experimental work involving functional expression, purification and biochemical characterization of AraC is technically very difficult, as reviewed by Schleif (2003). This property also holds for a large number of the AraC‐XylS family members; they are extremely insoluble. The major reason is predicted to be due to the unusually long contact area between the protein DNA binding domain and its target DNA (almost 40 base pairs). Each monomer contacts two major groove regions of the DNA (see Fig. 1) using two helix–turn–helix motifs (HTH) (Ogden et al., 1980; Hendricksen and Schleif, 1985; Brunelle and Schleif, 1989; Carra and Schleif, 1993). It has been proposed that the AraC protein, in particular the DNA binding domain, does not complete folding in the absence of DNA (Schleif, 2003). A partially folded DNA binding domain is sensitive to proteases and with an excessive number of hydrophobic residues exposed, this may also lead to aggregation. Due to all these problems, the AraC protein crystal structure was determined more than 20 years after its discovery (Soisson et al., 1997a,b). The technical difficulties experienced with AraC are most likely one major reason why few of the large number of TFs of this family have not yet been characterized biochemically (Martin and Rosner, 2001). Currently, 3D structures have been experimentally solved for only three members; AraC, and the monomeric regulators RobA, and MarA (Rhee et al., 1998; Kwon et al., 2000). The structure of the MarA protein is shown in Fig. 2. The conserved C‐terminal domain of the TF contains the characteristic HTH motif critical for DNA binding. In contrast, the N‐terminal domains among AraC‐XylS family TFs are structurally highly divergent, supporting the assumption that the insolubility properties of these proteins can be mainly assigned to the DNA binding domain.

Bottom Line: The available systems that have been applied to express heterologous genes are regulated either by sugars (L-arabinose, L-rhamnose, xylose and sucrose), substituted benzenes, cyclohexanone-related compounds, ε-caprolactam, propionate, thiostrepton, alkanes or peptides.It is of applied interest that some of the inducers require the presence of transport systems, some are more prone than others to become metabolized by the host and some have been applied mainly in one or a limited number of species.Based on bioinformatics analyses, the AraC-XylS family of regulators contains a large number of different members (currently over 300), but only a small fraction of these, the XylS/Pm, AraC/P(BAD), RhaR-RhaS/rhaBAD, NitR/PnitA and ChnR/Pb regulator/promoter systems, have so far been explored for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Sintef Materials and Chemistry, Sintef, Trondheim, Norway. trygve.brautaset@sintef.no

Show MeSH