Limits...
Regulation of the expression level of transcription factor XylS reveals new functional insight into its induction mechanism at the Pm promoter.

Zwick F, Lale R, Valla S - BMC Microbiol. (2013)

Bottom Line: Only the dimers are active and able to induce expression from Pm.Maximization of the induction ratio at Pm can be obtained by expression of XylS at the level where aggregation occurs, which might be exploited for recombinant gene expression.The results described here also indicate that there might exist variants of XylS which can exist at higher active dimer concentrations and thus lead to increased expression levels from Pm.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands Vei 6/8, Trondheim N-7491, Norway. rahmi.lale@ntnu.no.

ABSTRACT

Background: XylS is the positive regulator of the inducible Pm promoter, originating from Pseudomonas putida, where the system controls a biochemical pathway involved in degradation of aromatic hydrocarbons, which also act as inducers. The XylS/Pm positive regulator/promoter system is used for recombinant gene expression and the output from Pm is known to be sensitive to the intracellular XylS concentration.

Results: By constructing a synthetic operon consisting of xylS and luc, the gene encoding luciferase, relative XylS expression levels could be monitored indirectly at physiological concentrations. Expression of XylS from inducible promoters allowed control over a more than 800-fold range, however, the corresponding output from Pm covered only an about five-fold range. The maximum output from Pm could not be increased by introducing more copies of the promoter in the cells. Interestingly, a previously reported XylS variant (StEP-13), known to strongly stimulate expression from Pm, caused the same maximum activity from Pm as wild-type XylS at high XylS expression levels. Under uninduced conditions expression from Pm also increased as a function of XylS expression levels, and at very high concentrations the maximum activity from Pm was the same as in the presence of inducer.

Conclusion: According to our proposed model, which is in agreement with current knowledge, the regulator, XylS, can exist in three states: monomers, dimers, and aggregates. Only the dimers are active and able to induce expression from Pm. Their maximum intracellular concentration and the corresponding output from Pm are limited by the concentration-dependent conversion into inactive aggregates. Maximization of the induction ratio at Pm can be obtained by expression of XylS at the level where aggregation occurs, which might be exploited for recombinant gene expression. The results described here also indicate that there might exist variants of XylS which can exist at higher active dimer concentrations and thus lead to increased expression levels from Pm.

Show MeSH

Related in: MedlinePlus

Effects of XylS expression variations on induced and uninduced Pm activity. Upper host ampicillin tolerance levels as a function of the expression level of XylS in the absence (white squares) and presence (grey squares) of Pm induction (0/1 mM m-toluate). The shape that is half grey and half white represents an identical data point for both induced and uninduced. Relative expression from Pm and relative XylS expression were determined in the same way as described in Figure 3. The data points were collected from cells containing the Pm-bearing plasmid pFS15 in all cases and a: pFZ2B1, inducer concentrations as in Figure 3 (the grey data points are the same as the corresponding points in Figure 3); b: pET16.xylS, 0 mM IPTG; c: pFZ2B2, 0.25 and 0.5 mM cyclohexanone (from left to right); d: pFZ2B3, 0.25 and 0.5 mM cyclohexanone (from left to right); e: pET16.xylS, 0.5 mM IPTG.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4225500&req=5

Figure 4: Effects of XylS expression variations on induced and uninduced Pm activity. Upper host ampicillin tolerance levels as a function of the expression level of XylS in the absence (white squares) and presence (grey squares) of Pm induction (0/1 mM m-toluate). The shape that is half grey and half white represents an identical data point for both induced and uninduced. Relative expression from Pm and relative XylS expression were determined in the same way as described in Figure 3. The data points were collected from cells containing the Pm-bearing plasmid pFS15 in all cases and a: pFZ2B1, inducer concentrations as in Figure 3 (the grey data points are the same as the corresponding points in Figure 3); b: pET16.xylS, 0 mM IPTG; c: pFZ2B2, 0.25 and 0.5 mM cyclohexanone (from left to right); d: pFZ2B3, 0.25 and 0.5 mM cyclohexanone (from left to right); e: pET16.xylS, 0.5 mM IPTG.

Mentions: The results presented above might indicate that expression from Pm could not be stimulated more by further increasing the XylS expression. We have on the other hand observed that 2 mM cyclohexanone is not so far from concentrations that have observable negative effects on cell growth [34], and we therefore wanted to create conditions at which XylS expression could be increased further without using near-toxic concentrations of cyclohexanone. In a parallel ongoing project we had observed that the expression level from the Pb promoter is, like Pm, very sensitive to the amounts of its regulator, ChnR. This was taken advantage of by substituting the chnR native promoter with constitutive promoters from the Registry of Standard Biological Parts, which were identified by a library screening [35]. Two promising variants were used to drive chnR expression in derivatives of pFZ2B1, namely pFZ2B2 and pFZ2B3, such that XylS expression could be controlled by cyclohexanone, as above, but hopefully at higher levels. As expected this resulted in increased XylS expression (measured as luciferase activity), up to 50-fold (pFZ2B3) above the maximum for pFZ2B1. In spite of this, the expression from Pm (in pFS15) was not higher than when pFZ2B1 was used for expression of XylS (Figure 4a,c and d, grey squares).


Regulation of the expression level of transcription factor XylS reveals new functional insight into its induction mechanism at the Pm promoter.

Zwick F, Lale R, Valla S - BMC Microbiol. (2013)

Effects of XylS expression variations on induced and uninduced Pm activity. Upper host ampicillin tolerance levels as a function of the expression level of XylS in the absence (white squares) and presence (grey squares) of Pm induction (0/1 mM m-toluate). The shape that is half grey and half white represents an identical data point for both induced and uninduced. Relative expression from Pm and relative XylS expression were determined in the same way as described in Figure 3. The data points were collected from cells containing the Pm-bearing plasmid pFS15 in all cases and a: pFZ2B1, inducer concentrations as in Figure 3 (the grey data points are the same as the corresponding points in Figure 3); b: pET16.xylS, 0 mM IPTG; c: pFZ2B2, 0.25 and 0.5 mM cyclohexanone (from left to right); d: pFZ2B3, 0.25 and 0.5 mM cyclohexanone (from left to right); e: pET16.xylS, 0.5 mM IPTG.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225500&req=5

Figure 4: Effects of XylS expression variations on induced and uninduced Pm activity. Upper host ampicillin tolerance levels as a function of the expression level of XylS in the absence (white squares) and presence (grey squares) of Pm induction (0/1 mM m-toluate). The shape that is half grey and half white represents an identical data point for both induced and uninduced. Relative expression from Pm and relative XylS expression were determined in the same way as described in Figure 3. The data points were collected from cells containing the Pm-bearing plasmid pFS15 in all cases and a: pFZ2B1, inducer concentrations as in Figure 3 (the grey data points are the same as the corresponding points in Figure 3); b: pET16.xylS, 0 mM IPTG; c: pFZ2B2, 0.25 and 0.5 mM cyclohexanone (from left to right); d: pFZ2B3, 0.25 and 0.5 mM cyclohexanone (from left to right); e: pET16.xylS, 0.5 mM IPTG.
Mentions: The results presented above might indicate that expression from Pm could not be stimulated more by further increasing the XylS expression. We have on the other hand observed that 2 mM cyclohexanone is not so far from concentrations that have observable negative effects on cell growth [34], and we therefore wanted to create conditions at which XylS expression could be increased further without using near-toxic concentrations of cyclohexanone. In a parallel ongoing project we had observed that the expression level from the Pb promoter is, like Pm, very sensitive to the amounts of its regulator, ChnR. This was taken advantage of by substituting the chnR native promoter with constitutive promoters from the Registry of Standard Biological Parts, which were identified by a library screening [35]. Two promising variants were used to drive chnR expression in derivatives of pFZ2B1, namely pFZ2B2 and pFZ2B3, such that XylS expression could be controlled by cyclohexanone, as above, but hopefully at higher levels. As expected this resulted in increased XylS expression (measured as luciferase activity), up to 50-fold (pFZ2B3) above the maximum for pFZ2B1. In spite of this, the expression from Pm (in pFS15) was not higher than when pFZ2B1 was used for expression of XylS (Figure 4a,c and d, grey squares).

Bottom Line: Only the dimers are active and able to induce expression from Pm.Maximization of the induction ratio at Pm can be obtained by expression of XylS at the level where aggregation occurs, which might be exploited for recombinant gene expression.The results described here also indicate that there might exist variants of XylS which can exist at higher active dimer concentrations and thus lead to increased expression levels from Pm.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands Vei 6/8, Trondheim N-7491, Norway. rahmi.lale@ntnu.no.

ABSTRACT

Background: XylS is the positive regulator of the inducible Pm promoter, originating from Pseudomonas putida, where the system controls a biochemical pathway involved in degradation of aromatic hydrocarbons, which also act as inducers. The XylS/Pm positive regulator/promoter system is used for recombinant gene expression and the output from Pm is known to be sensitive to the intracellular XylS concentration.

Results: By constructing a synthetic operon consisting of xylS and luc, the gene encoding luciferase, relative XylS expression levels could be monitored indirectly at physiological concentrations. Expression of XylS from inducible promoters allowed control over a more than 800-fold range, however, the corresponding output from Pm covered only an about five-fold range. The maximum output from Pm could not be increased by introducing more copies of the promoter in the cells. Interestingly, a previously reported XylS variant (StEP-13), known to strongly stimulate expression from Pm, caused the same maximum activity from Pm as wild-type XylS at high XylS expression levels. Under uninduced conditions expression from Pm also increased as a function of XylS expression levels, and at very high concentrations the maximum activity from Pm was the same as in the presence of inducer.

Conclusion: According to our proposed model, which is in agreement with current knowledge, the regulator, XylS, can exist in three states: monomers, dimers, and aggregates. Only the dimers are active and able to induce expression from Pm. Their maximum intracellular concentration and the corresponding output from Pm are limited by the concentration-dependent conversion into inactive aggregates. Maximization of the induction ratio at Pm can be obtained by expression of XylS at the level where aggregation occurs, which might be exploited for recombinant gene expression. The results described here also indicate that there might exist variants of XylS which can exist at higher active dimer concentrations and thus lead to increased expression levels from Pm.

Show MeSH
Related in: MedlinePlus