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Expression of the pstS gene of Streptomyces lividans is regulated by the carbon source and is partially independent of the PhoP regulator.

Esteban A, Díaz M, Yepes A, Santamaría RI - BMC Microbiol. (2008)

Bottom Line: Functionality experiments revealed that this extracellular PstS protein does not have the capacity to capture phosphate and transfer it to the cell.We observed that the pstS gene was expressed in cultures containing glucose or fructose, but not in complex basal medium.Additionally, deletion of the fragment corresponding to the Pho boxes recognized by the PhoP regulator (from nucleotide -141 to -113) resulted in constitutive pstS expression that was independent of this regulator.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Microbiología Bioquímica/Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain. aesteban@usal.es

ABSTRACT

Background: PstS is a phosphate-binding lipoprotein that is part of the high-affinity phosphate transport system. Streptomyces lividans accumulates high amounts of the PstS protein in the supernatant of liquid cultures grown in the presence of different carbon sources, such as fructose or mannose, but not in the presence of glucose or in basal complex medium.

Results: Functionality experiments revealed that this extracellular PstS protein does not have the capacity to capture phosphate and transfer it to the cell. Regulation of the pstS promoter was studied with Northern blot experiments, and protein levels were detected by Western blot analysis. We observed that the pstS gene was expressed in cultures containing glucose or fructose, but not in complex basal medium. Northern blot analyses revealed that the pst operon (pstSCAB) was transcribed as a whole, although higher transcript levels of pstS relative to those of the other genes of the operon (pstC, pstA and pstB) were observed. Deletion of the -329/-144 fragment of the pstS promoter, including eight degenerated repeats of a sequence of 12 nucleotides, resulted in a two-fold increase in the expression of this promoter, suggesting a regulatory role for this region. Additionally, deletion of the fragment corresponding to the Pho boxes recognized by the PhoP regulator (from nucleotide -141 to -113) resulted in constitutive pstS expression that was independent of this regulator. Thus, the PhoP-independent expression of the pstS gene makes this system different from all those studied previously.

Conclusion: 1.- In S. lividans, only the PstS protein bound to the cell has the capacity to bind phosphate and transfer it there, whereas the PstS form accumulated in the supernatant lacks this capacity. 2.- The stretch of eight degenerated repeats present in the pstS promoter may act as a binding site for a repressor. 3.- There is a basal expression of the pstS gene that is not controlled by the main regulator: PhoP.

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Xylanase production under control of the pstS promoter. A) Coomassie-Blue-R-stained SDS-PAGE showing the production of the Xys1 xylanase in supernatants of the S. lividans TK24 (wild-type) transformed with different plasmids: pNX30, the xylanase gene has no promoter; pNUF5, the xylanase gene is under the control of the full length pstS promoter; pNUF13, the xylanase is under the 186-bp-deleted pstS promoter (from -329 to -144). 5 μl of culture supernatant was loaded per track. B) Histogram showing the xylanase activity detected in the supernatant of the indicated strains. G, glucose; F, fructose. The results presented are means of three independent experiments.
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Figure 3: Xylanase production under control of the pstS promoter. A) Coomassie-Blue-R-stained SDS-PAGE showing the production of the Xys1 xylanase in supernatants of the S. lividans TK24 (wild-type) transformed with different plasmids: pNX30, the xylanase gene has no promoter; pNUF5, the xylanase gene is under the control of the full length pstS promoter; pNUF13, the xylanase is under the 186-bp-deleted pstS promoter (from -329 to -144). 5 μl of culture supernatant was loaded per track. B) Histogram showing the xylanase activity detected in the supernatant of the indicated strains. G, glucose; F, fructose. The results presented are means of three independent experiments.

Mentions: We have previously proposed that the sequence ACTCACCCCCGC, repeated several times in the S. coelicolor pstS promoter and -with some discrepancies- up to eight times in the pstS promoter of S. lividans, might be involved in the carbon regulation of the expression of this promoter [6]. To study this in more detail, we deleted the portion of the S. lividans pstS promoter that contains the eight-times repeated degenerated sequence with the consensus sequence ACYCASCCMNSV. To do so, the -329/-144 region of the pstS promoter was deleted and the rest of the promoter was used to drive the expression of the ORF of the xysA xylanase gene [13] and used as reporter in a multicopy plasmid designated pNUF13 (Methods and Table 1). This plasmid (pNUF13), plasmid pNX30 (negative control: xysA without promoter), and plasmid pNUF5 (full-length pstS promoter controlling xysA) (Table 1) were introduced into S. lividans TK24 and cultures were grown in YE supplemented with 5% glucose or with 5% fructose in the presence of neomycin (20 μg.ml-1) for 72 h. The production of xylanase in the culture supernatants was studied by Coomassie blue-stained SDS-PAGE and by measuring the xylanase activity. The xylanase band obtained in the strain harbouring pNUF13 was significantly more intense than that obtained with pNUF5 in the presence of both carbon sources (Fig. 3A). Xylanase activity was quantified in all the supernatants, and we observed that no xylanase activity was detected in the cultures of the S. lividans TK24 strain transformed with pNX30 under both conditions (data not shown). However, xylanase activity was detected in the strain transformed with pNUF5 or with pNUF13 (Fig. 3B). Clearly, there was an increase in the xylanase activity detected in the strain carrying the pstS truncated promoter (pNUF13) under both culture conditions. This increase was more than two-fold when the strain was grown in the presence of glucose and 1.7-fold in the case of the cultures performed with fructose (Fig. 3B). In addition, we observed a higher expression in presence of fructose than in the presence of glucose for both truncated and complete pstS promoters. Thus, when the S. lividans TK24 strain was transformed with pNUF5, 2.15-fold more xylanase was produced with fructose than with glucose. When the plasmid used was pNUF13, the overproduction obtained with fructose was 1.7 fold, values of 340 U/ml of xylanase being attained. These results clearly indicate that the region containing the eight-times repeats may play an important role in controlling the level of expression of the pstS promoter in the presence of the different carbon sources.


Expression of the pstS gene of Streptomyces lividans is regulated by the carbon source and is partially independent of the PhoP regulator.

Esteban A, Díaz M, Yepes A, Santamaría RI - BMC Microbiol. (2008)

Xylanase production under control of the pstS promoter. A) Coomassie-Blue-R-stained SDS-PAGE showing the production of the Xys1 xylanase in supernatants of the S. lividans TK24 (wild-type) transformed with different plasmids: pNX30, the xylanase gene has no promoter; pNUF5, the xylanase gene is under the control of the full length pstS promoter; pNUF13, the xylanase is under the 186-bp-deleted pstS promoter (from -329 to -144). 5 μl of culture supernatant was loaded per track. B) Histogram showing the xylanase activity detected in the supernatant of the indicated strains. G, glucose; F, fructose. The results presented are means of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Xylanase production under control of the pstS promoter. A) Coomassie-Blue-R-stained SDS-PAGE showing the production of the Xys1 xylanase in supernatants of the S. lividans TK24 (wild-type) transformed with different plasmids: pNX30, the xylanase gene has no promoter; pNUF5, the xylanase gene is under the control of the full length pstS promoter; pNUF13, the xylanase is under the 186-bp-deleted pstS promoter (from -329 to -144). 5 μl of culture supernatant was loaded per track. B) Histogram showing the xylanase activity detected in the supernatant of the indicated strains. G, glucose; F, fructose. The results presented are means of three independent experiments.
Mentions: We have previously proposed that the sequence ACTCACCCCCGC, repeated several times in the S. coelicolor pstS promoter and -with some discrepancies- up to eight times in the pstS promoter of S. lividans, might be involved in the carbon regulation of the expression of this promoter [6]. To study this in more detail, we deleted the portion of the S. lividans pstS promoter that contains the eight-times repeated degenerated sequence with the consensus sequence ACYCASCCMNSV. To do so, the -329/-144 region of the pstS promoter was deleted and the rest of the promoter was used to drive the expression of the ORF of the xysA xylanase gene [13] and used as reporter in a multicopy plasmid designated pNUF13 (Methods and Table 1). This plasmid (pNUF13), plasmid pNX30 (negative control: xysA without promoter), and plasmid pNUF5 (full-length pstS promoter controlling xysA) (Table 1) were introduced into S. lividans TK24 and cultures were grown in YE supplemented with 5% glucose or with 5% fructose in the presence of neomycin (20 μg.ml-1) for 72 h. The production of xylanase in the culture supernatants was studied by Coomassie blue-stained SDS-PAGE and by measuring the xylanase activity. The xylanase band obtained in the strain harbouring pNUF13 was significantly more intense than that obtained with pNUF5 in the presence of both carbon sources (Fig. 3A). Xylanase activity was quantified in all the supernatants, and we observed that no xylanase activity was detected in the cultures of the S. lividans TK24 strain transformed with pNX30 under both conditions (data not shown). However, xylanase activity was detected in the strain transformed with pNUF5 or with pNUF13 (Fig. 3B). Clearly, there was an increase in the xylanase activity detected in the strain carrying the pstS truncated promoter (pNUF13) under both culture conditions. This increase was more than two-fold when the strain was grown in the presence of glucose and 1.7-fold in the case of the cultures performed with fructose (Fig. 3B). In addition, we observed a higher expression in presence of fructose than in the presence of glucose for both truncated and complete pstS promoters. Thus, when the S. lividans TK24 strain was transformed with pNUF5, 2.15-fold more xylanase was produced with fructose than with glucose. When the plasmid used was pNUF13, the overproduction obtained with fructose was 1.7 fold, values of 340 U/ml of xylanase being attained. These results clearly indicate that the region containing the eight-times repeats may play an important role in controlling the level of expression of the pstS promoter in the presence of the different carbon sources.

Bottom Line: Functionality experiments revealed that this extracellular PstS protein does not have the capacity to capture phosphate and transfer it to the cell.We observed that the pstS gene was expressed in cultures containing glucose or fructose, but not in complex basal medium.Additionally, deletion of the fragment corresponding to the Pho boxes recognized by the PhoP regulator (from nucleotide -141 to -113) resulted in constitutive pstS expression that was independent of this regulator.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Microbiología Bioquímica/Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain. aesteban@usal.es

ABSTRACT

Background: PstS is a phosphate-binding lipoprotein that is part of the high-affinity phosphate transport system. Streptomyces lividans accumulates high amounts of the PstS protein in the supernatant of liquid cultures grown in the presence of different carbon sources, such as fructose or mannose, but not in the presence of glucose or in basal complex medium.

Results: Functionality experiments revealed that this extracellular PstS protein does not have the capacity to capture phosphate and transfer it to the cell. Regulation of the pstS promoter was studied with Northern blot experiments, and protein levels were detected by Western blot analysis. We observed that the pstS gene was expressed in cultures containing glucose or fructose, but not in complex basal medium. Northern blot analyses revealed that the pst operon (pstSCAB) was transcribed as a whole, although higher transcript levels of pstS relative to those of the other genes of the operon (pstC, pstA and pstB) were observed. Deletion of the -329/-144 fragment of the pstS promoter, including eight degenerated repeats of a sequence of 12 nucleotides, resulted in a two-fold increase in the expression of this promoter, suggesting a regulatory role for this region. Additionally, deletion of the fragment corresponding to the Pho boxes recognized by the PhoP regulator (from nucleotide -141 to -113) resulted in constitutive pstS expression that was independent of this regulator. Thus, the PhoP-independent expression of the pstS gene makes this system different from all those studied previously.

Conclusion: 1.- In S. lividans, only the PstS protein bound to the cell has the capacity to bind phosphate and transfer it there, whereas the PstS form accumulated in the supernatant lacks this capacity. 2.- The stretch of eight degenerated repeats present in the pstS promoter may act as a binding site for a repressor. 3.- There is a basal expression of the pstS gene that is not controlled by the main regulator: PhoP.

Show MeSH
Related in: MedlinePlus