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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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Extracellular PstS does not participate in phosphate incorporation. A) Western blot to detect extracellular (S) and cell-bound (C) PstS in the indicated S. lividans strains. (20 μg of total protein were loaded per lane). B) Uptake of 32P-labeled phosphate after 1 hour at 30°C. Strains assayed: wild-type S. lividans (wt); the ΔpstS deletion mutant (ΔpstS); the complemented transformant ΔpstS (ΔpstS+pINTUF5), and the same mutant containing the integrative fusion xylanase signal peptide-PstS (ΔpstS+pINTUF9). The results presented are the means of three independent experiments.
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Figure 1: Extracellular PstS does not participate in phosphate incorporation. A) Western blot to detect extracellular (S) and cell-bound (C) PstS in the indicated S. lividans strains. (20 μg of total protein were loaded per lane). B) Uptake of 32P-labeled phosphate after 1 hour at 30°C. Strains assayed: wild-type S. lividans (wt); the ΔpstS deletion mutant (ΔpstS); the complemented transformant ΔpstS (ΔpstS+pINTUF5), and the same mutant containing the integrative fusion xylanase signal peptide-PstS (ΔpstS+pINTUF9). The results presented are the means of three independent experiments.

Mentions: In principle, lipoproteins such as PstS are attached to the cell membranes, where they exert their function. However, our previous observations showed that the PstS protein was strongly accumulated in the supernatants of S. lividans cultures grown in the presence of certain carbon sources. We therefore decided to study whether this fraction of the protein also had the capacity to bind extracellular phosphate and transfer it to the cell. To address this issue, a construction expressing a [Xys1]-PstS fusion protein, which was completely secreted to the supernatant, was obtained (Methods). In this construction, the pstS promoter drives the expression of an in-frame fusion between the DNA fragment of the xysA gene encoding the signal peptide of the xylanase Xys1 from S. halstedii JM8 [13] and the region of the pstS gene that encodes the secreted form of the PstS protein. This fusion gene was cloned into a Streptomyces integrative plasmid to obtain plasmid pINTUF9 (Table 1), and this was introduced into the S. lividans pstS mutant (Table 2). As controls, S. lividans wild-type, the pstS mutant, and the pstS mutant transformed with plasmid pINTUF5 (Table 1), which produces the wild-type PstS protein, were used. The expression and location of the PstS protein were followed by Western blot analysis of the supernatants and cellular fractions of the different strains after 72 hours of culture. The original PstS protein was detected in the supernatants and in the cell extracts of the wild-type strain and in the pstS mutant transformed with pINTUF5. However, the PstS fusion protein, produced from pINTUF9 in the pstS mutant, was only detected in the culture supernatant (Fig. 1A). This result clearly demonstrates the capacity of the 45-amino acid signal peptide of the xylanase encoded by the xysA gene to secrete other proteins: in this case, PstS. The N-terminus of the secreted PstS protein obtained from the strain carrying pINTUF9 was identical to the wild-type PstS extracellular protein [6], except that it had two extra amino acids (A, G) at its N-terminus in order to keep the signal peptide processing site present in the original xylanase. Clearly, the size of the PstS protein observed in the cells and in the supernatant of the strains carrying the original pstS gene was different (Fig. 1A). This is due to the fact that the protein released to the supernatant does not have the first 41 amino acids [6].


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)

Extracellular PstS does not participate in phosphate incorporation. A) Western blot to detect extracellular (S) and cell-bound (C) PstS in the indicated S. lividans strains. (20 μg of total protein were loaded per lane). B) Uptake of 32P-labeled phosphate after 1 hour at 30°C. Strains assayed: wild-type S. lividans (wt); the ΔpstS deletion mutant (ΔpstS); the complemented transformant ΔpstS (ΔpstS+pINTUF5), and the same mutant containing the integrative fusion xylanase signal peptide-PstS (ΔpstS+pINTUF9). The results presented are the means of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2605767&req=5

Figure 1: Extracellular PstS does not participate in phosphate incorporation. A) Western blot to detect extracellular (S) and cell-bound (C) PstS in the indicated S. lividans strains. (20 μg of total protein were loaded per lane). B) Uptake of 32P-labeled phosphate after 1 hour at 30°C. Strains assayed: wild-type S. lividans (wt); the ΔpstS deletion mutant (ΔpstS); the complemented transformant ΔpstS (ΔpstS+pINTUF5), and the same mutant containing the integrative fusion xylanase signal peptide-PstS (ΔpstS+pINTUF9). The results presented are the means of three independent experiments.
Mentions: In principle, lipoproteins such as PstS are attached to the cell membranes, where they exert their function. However, our previous observations showed that the PstS protein was strongly accumulated in the supernatants of S. lividans cultures grown in the presence of certain carbon sources. We therefore decided to study whether this fraction of the protein also had the capacity to bind extracellular phosphate and transfer it to the cell. To address this issue, a construction expressing a [Xys1]-PstS fusion protein, which was completely secreted to the supernatant, was obtained (Methods). In this construction, the pstS promoter drives the expression of an in-frame fusion between the DNA fragment of the xysA gene encoding the signal peptide of the xylanase Xys1 from S. halstedii JM8 [13] and the region of the pstS gene that encodes the secreted form of the PstS protein. This fusion gene was cloned into a Streptomyces integrative plasmid to obtain plasmid pINTUF9 (Table 1), and this was introduced into the S. lividans pstS mutant (Table 2). As controls, S. lividans wild-type, the pstS mutant, and the pstS mutant transformed with plasmid pINTUF5 (Table 1), which produces the wild-type PstS protein, were used. The expression and location of the PstS protein were followed by Western blot analysis of the supernatants and cellular fractions of the different strains after 72 hours of culture. The original PstS protein was detected in the supernatants and in the cell extracts of the wild-type strain and in the pstS mutant transformed with pINTUF5. However, the PstS fusion protein, produced from pINTUF9 in the pstS mutant, was only detected in the culture supernatant (Fig. 1A). This result clearly demonstrates the capacity of the 45-amino acid signal peptide of the xylanase encoded by the xysA gene to secrete other proteins: in this case, PstS. The N-terminus of the secreted PstS protein obtained from the strain carrying pINTUF9 was identical to the wild-type PstS extracellular protein [6], except that it had two extra amino acids (A, G) at its N-terminus in order to keep the signal peptide processing site present in the original xylanase. Clearly, the size of the PstS protein observed in the cells and in the supernatant of the strains carrying the original pstS gene was different (Fig. 1A). This is due to the fact that the protein released to the supernatant does not have the first 41 amino acids [6].

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