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

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.

Figure 1

Map of plasmid pFS7.Ps2: constitutive promoter; xylS: gene encoding Pm activator; luc: gene encoding luciferase; Pm: positively regulated promoter; bla: ampicillin resistance gene encoding β-lactamase; t₁: rrnBT₁T₂ bidirectional transcriptional terminator; trfA: gene encoding the replication protein; oriV: origin of vegetative replication; kan: kanamycin resistance gene; oriT: origin of conjugal transfer. The DNA sequence of the overlapping stop-start codon is depicted.

Figure 2

Expression levels from pFS7 for different variants of xylS with silent mutations. Relative expression levels from Pm (measured as maximum ampicillin tolerance at 1 mM m-toluate) are given in grey (error bars = lowest ampicillin concentrations in test on which no growth was observed) and relative luciferase activity as a measure for XylS amounts in black (values from at least two biological replicas). All values (relative ampicillin tolerance and luciferase expression) refer to those of wild type XylS (tolerating 350 μg mL^-1), which are both arbitrarily set to 1. Mutations in the variants (1 to 9), the number stands for the base position that has been changed, relative to the translational start site, the character tells the base in the variant. 1: 6- > C; 2: 13- > C; 3: 15- > G; 4: 16- > C; 5: 27- > G; 6: 30- > C; 7: 36- > T; 8: 42- > T; 9: all of the eight mutations.

Figure 3

Effects of variations in wild type or variant XylS expression on Pm activity. Upper host ampicillin tolerance levels as a function of the expression level of wild type XylS (pFZ2B1) or variant StEP-13 (pFZ2B1.StEP-13), using two different copy number variants (pFS15 and pFS15.271) of the target plasmid. Pm activity was measured as upper relative ampicillin tolerance on agar medium. The tolerance for cells containing pFZ2B1 + pFS15, no cyclohexanone, was arbitrarily set to 1 and corresponds to about 650 μg mL^-1 ampicillin resistance. The relative XylS expression was measured as luciferase activity and was also set to 1 for the same data point. The data points indicate the highest ampicillin concentration on which growth occurred, while the lowest concentration on which no growth was observed is indicated by error bars. Shapes that are half grey and half black indicate identical data points for both wild type and StEP-13. 1 mM m-toluate was added to all samples, cyclohexanone concentrations leading to the measured XylS expression levels (from left to right): 0, 0.25, 0.5, 1 and 2 mM, respectively.

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.

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.

Figure 6

Visualization of the hypothesis explaining XylS behaviour at various intracellular concentrations. The numbers of DNA or XylS molecules are not meant to represent the actual numbers in the cells. Only aggregates formed from active dimers of the protein are considered. At low XylS concentrations a certain percentage of the dimerized XylS molecules will activate transcription (a); the amount of activated Pm promoters will increase proportionally to XylS amounts up to a certain treshold value (b); when the threshold value is exceeded, XylS dimers will aggregate and become inactive, while the amount of active dimers remains constant (c). For StEP-13 a higher percentage of XylS molecules will dimerize at low XylS concentrations, resulting in more transcribed DNA (d); when the saturating concentration for wild type XylS is reached, there will already be some aggregation of dimers in case of StEP-13 (e), and as for wild type this will increase further as more XylS is expressed (f). In the absence of m-toluate, only a very small fraction of the XylS molecules will form dimers and these will activate transcription from Pm, aggregation does not start at the XylS expression levels depicted here (g, h, i).