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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.

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SDS-PAGE gel for XylS produced from the T7 promoter. Samples were crude bacterial lysates from cells containing vector pET16b.xylS, grown in the presence or absence of inducer. Samples were split into soluble and insoluble fractions. Sizes of the protein ladder in kDa are given on the left site.
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Figure 5: SDS-PAGE gel for XylS produced from the T7 promoter. Samples were crude bacterial lysates from cells containing vector pET16b.xylS, grown in the presence or absence of inducer. Samples were split into soluble and insoluble fractions. Sizes of the protein ladder in kDa are given on the left site.

Mentions: Based on the luciferase activity measurements over 800 times more XylS was expressed from the T7 promoter than from Ps2. If previous estimates of about 200 molecules per cell [5] are reasonably close to the true value, simple calculations indicated that an over 800-fold increase would yield a band directly visible on SDS-PAGE. A bacterial cell culture containing plasmid pET16.xylS was split into two such that one was induced by IPTG (0.5 mM), the other was not. Cells were harvested by centrifugation, lysed and split into a soluble and an insoluble fraction by centrifugation and the resulting samples were separated on an SDS-PAGE gel. Inspection of the band patterns (Figure 5) clearly demonstrated a unique and strong band in only the sample from the induced insoluble fraction. The distance of migration also matched to the expected position for XylS (36 kDa). The weaker band representing a similar size protein in the insoluble fraction of the uninduced culture seems to originate from a host-derived protein, as the same band was observed for samples from cells containing plasmid without xylS both in the presence and absence of inducer (data not shown). Thus, the vast majority of the XylS protein expressed from pET16.xylS is produced in an aggregated and presumably inactive form.


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)

SDS-PAGE gel for XylS produced from the T7 promoter. Samples were crude bacterial lysates from cells containing vector pET16b.xylS, grown in the presence or absence of inducer. Samples were split into soluble and insoluble fractions. Sizes of the protein ladder in kDa are given on the left site.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: SDS-PAGE gel for XylS produced from the T7 promoter. Samples were crude bacterial lysates from cells containing vector pET16b.xylS, grown in the presence or absence of inducer. Samples were split into soluble and insoluble fractions. Sizes of the protein ladder in kDa are given on the left site.
Mentions: Based on the luciferase activity measurements over 800 times more XylS was expressed from the T7 promoter than from Ps2. If previous estimates of about 200 molecules per cell [5] are reasonably close to the true value, simple calculations indicated that an over 800-fold increase would yield a band directly visible on SDS-PAGE. A bacterial cell culture containing plasmid pET16.xylS was split into two such that one was induced by IPTG (0.5 mM), the other was not. Cells were harvested by centrifugation, lysed and split into a soluble and an insoluble fraction by centrifugation and the resulting samples were separated on an SDS-PAGE gel. Inspection of the band patterns (Figure 5) clearly demonstrated a unique and strong band in only the sample from the induced insoluble fraction. The distance of migration also matched to the expected position for XylS (36 kDa). The weaker band representing a similar size protein in the insoluble fraction of the uninduced culture seems to originate from a host-derived protein, as the same band was observed for samples from cells containing plasmid without xylS both in the presence and absence of inducer (data not shown). Thus, the vast majority of the XylS protein expressed from pET16.xylS is produced in an aggregated and presumably inactive form.

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