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Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885).

Ueda A, Wood TK - PLoS Pathog. (2009)

Bottom Line: Expression of PA3885 in trans reduced biofilm formation and abolished aggregation.These results show that the PA3885 protein responds to AHL signals and likely dephosphorylates PA1120, which leads to reduced c-di-GMP production.This inhibits matrix exopolysaccharide formation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilm formation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilm formation and PA1120 should be TpbB.

View Article: PubMed Central - PubMed

Affiliation: Artie McFerrin Department of Chemical Engineering, Texas A & M University, College Station, Texas, United States of America.

ABSTRACT
With the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing based on homoserine lactones was found to influence biofilm formation. Here we discern a mechanism by which quorum sensing controls biofilm formation by screening 5850 transposon mutants of P. aeruginosa PA14 for altered biofilm formation. This screen identified the PA3885 mutant, which had 147-fold more biofilm than the wild-type strain. Loss of PA3885 decreased swimming, abolished swarming, and increased attachment, although this did not affect production of rhamnolipids. The PA3885 mutant also had a wrinkly colony phenotype, formed pronounced pellicles, had substantially more aggregation, and had 28-fold more exopolysaccharide production. Expression of PA3885 in trans reduced biofilm formation and abolished aggregation. Whole transcriptome analysis showed that loss of PA3885 activated expression of the pel locus, an operon that encodes for the synthesis of extracellular matrix polysaccharide. Genetic screening identified that loss of PelABDEG and the PA1120 protein (which contains a GGDEF-motif) suppressed the phenotypes of the PA3885 mutant, suggesting that the function of the PA3885 protein is to regulate 3,5-cyclic diguanylic acid (c-di-GMP) concentrations as a phosphatase since c-di-GMP enhances biofilm formation by activating PelD, and c-di-GMP inhibits swarming. Loss of PA3885 protein increased cellular c-di-GMP concentrations; hence, PA3885 protein is a negative regulator of c-di-GMP production. Purified PA3885 protein has phosphatase activity against phosphotyrosine peptides and is translocated to the periplasm. Las-mediated quorum sensing positively regulates expression of the PA3885 gene. These results show that the PA3885 protein responds to AHL signals and likely dephosphorylates PA1120, which leads to reduced c-di-GMP production. This inhibits matrix exopolysaccharide formation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilm formation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilm formation and PA1120 should be TpbB.

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TpbA has phosphatase activity against tyrosine residues.Purification of TpbA-cHis (lane 1: protein marker, lane 2: whole cell lysate from E. coli BL21(DE3)/pET28b-13660c after 3 h of IPTG induction, lane 3: purified TpbA-cHis) (A). p-Nitrophenyl phosphate phosphatase assay with TpbA-cHis protein (B). Phosphatase reaction was performed at 37°C for 1 h with the indicated amount of protein. Na3VO4 (10 mM) was used as an inhibitor specific for tyrosine phosphatases. Protein tyrosine phosphatase assay with TpbA-cHis (C). Phosphatase reaction was performed with synthetic phosphotyrosine peptides (type I: END(pY)INASL and type II: DADE(pY)LIPQQG) at 37°C for 3 h. Na3VO4 (50 mM) was used as an inhibitor.
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ppat-1000483-g006: TpbA has phosphatase activity against tyrosine residues.Purification of TpbA-cHis (lane 1: protein marker, lane 2: whole cell lysate from E. coli BL21(DE3)/pET28b-13660c after 3 h of IPTG induction, lane 3: purified TpbA-cHis) (A). p-Nitrophenyl phosphate phosphatase assay with TpbA-cHis protein (B). Phosphatase reaction was performed at 37°C for 1 h with the indicated amount of protein. Na3VO4 (10 mM) was used as an inhibitor specific for tyrosine phosphatases. Protein tyrosine phosphatase assay with TpbA-cHis (C). Phosphatase reaction was performed with synthetic phosphotyrosine peptides (type I: END(pY)INASL and type II: DADE(pY)LIPQQG) at 37°C for 3 h. Na3VO4 (50 mM) was used as an inhibitor.

Mentions: tpbA encodes a 218 aa protein that has the conserved domain for a protein tyrosine phosphatase [28],[29] since it has the C(X)5R(S/T) motif beginning at aa 132 (CKHGNNRT). To confirm it is a tyrosine phosphatase, we purified TpbA by adding a polyhistidine tag at either the N-terminus (TpbA-nHis) or the C-terminus (TpbA-cHis) (note only the C-terminus fusion protein was active). Expression of recombinant TpbA was confirmed in E. coli by clear expression of a band at 24 kD (Fig. 6A). The purified TpbA protein had phosphatase activity with p-nitrophenyl phosphate (pNPP) that is often used as a general phosphatase substrate [29] (Fig. 6B). Further proof that TpbA is a tyrosine phosphatase was found using a tyrosine phosphatase specific inhibitor, trisodium orthovanadate [30], that completely inhibited the phosphatase activity of TpbA-cHis (Fig. 6B). The third and fourth lines of evidence that TpbA is a tyrosine phosphatase were found using tyrosine specific substrates; TpbA-cHis dephosphorylated both phosphotyrosine peptides, END(pY)INASL (peptide type I) and DADE(pY)LIPQQG (peptide type II) (Fig. 6C), and this activity was inhibited by trisodium orthovanadate. These results show conclusively that TpbA encodes a tyrosine phosphatase.


Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885).

Ueda A, Wood TK - PLoS Pathog. (2009)

TpbA has phosphatase activity against tyrosine residues.Purification of TpbA-cHis (lane 1: protein marker, lane 2: whole cell lysate from E. coli BL21(DE3)/pET28b-13660c after 3 h of IPTG induction, lane 3: purified TpbA-cHis) (A). p-Nitrophenyl phosphate phosphatase assay with TpbA-cHis protein (B). Phosphatase reaction was performed at 37°C for 1 h with the indicated amount of protein. Na3VO4 (10 mM) was used as an inhibitor specific for tyrosine phosphatases. Protein tyrosine phosphatase assay with TpbA-cHis (C). Phosphatase reaction was performed with synthetic phosphotyrosine peptides (type I: END(pY)INASL and type II: DADE(pY)LIPQQG) at 37°C for 3 h. Na3VO4 (50 mM) was used as an inhibitor.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691606&req=5

ppat-1000483-g006: TpbA has phosphatase activity against tyrosine residues.Purification of TpbA-cHis (lane 1: protein marker, lane 2: whole cell lysate from E. coli BL21(DE3)/pET28b-13660c after 3 h of IPTG induction, lane 3: purified TpbA-cHis) (A). p-Nitrophenyl phosphate phosphatase assay with TpbA-cHis protein (B). Phosphatase reaction was performed at 37°C for 1 h with the indicated amount of protein. Na3VO4 (10 mM) was used as an inhibitor specific for tyrosine phosphatases. Protein tyrosine phosphatase assay with TpbA-cHis (C). Phosphatase reaction was performed with synthetic phosphotyrosine peptides (type I: END(pY)INASL and type II: DADE(pY)LIPQQG) at 37°C for 3 h. Na3VO4 (50 mM) was used as an inhibitor.
Mentions: tpbA encodes a 218 aa protein that has the conserved domain for a protein tyrosine phosphatase [28],[29] since it has the C(X)5R(S/T) motif beginning at aa 132 (CKHGNNRT). To confirm it is a tyrosine phosphatase, we purified TpbA by adding a polyhistidine tag at either the N-terminus (TpbA-nHis) or the C-terminus (TpbA-cHis) (note only the C-terminus fusion protein was active). Expression of recombinant TpbA was confirmed in E. coli by clear expression of a band at 24 kD (Fig. 6A). The purified TpbA protein had phosphatase activity with p-nitrophenyl phosphate (pNPP) that is often used as a general phosphatase substrate [29] (Fig. 6B). Further proof that TpbA is a tyrosine phosphatase was found using a tyrosine phosphatase specific inhibitor, trisodium orthovanadate [30], that completely inhibited the phosphatase activity of TpbA-cHis (Fig. 6B). The third and fourth lines of evidence that TpbA is a tyrosine phosphatase were found using tyrosine specific substrates; TpbA-cHis dephosphorylated both phosphotyrosine peptides, END(pY)INASL (peptide type I) and DADE(pY)LIPQQG (peptide type II) (Fig. 6C), and this activity was inhibited by trisodium orthovanadate. These results show conclusively that TpbA encodes a tyrosine phosphatase.

Bottom Line: Expression of PA3885 in trans reduced biofilm formation and abolished aggregation.These results show that the PA3885 protein responds to AHL signals and likely dephosphorylates PA1120, which leads to reduced c-di-GMP production.This inhibits matrix exopolysaccharide formation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilm formation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilm formation and PA1120 should be TpbB.

View Article: PubMed Central - PubMed

Affiliation: Artie McFerrin Department of Chemical Engineering, Texas A & M University, College Station, Texas, United States of America.

ABSTRACT
With the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing based on homoserine lactones was found to influence biofilm formation. Here we discern a mechanism by which quorum sensing controls biofilm formation by screening 5850 transposon mutants of P. aeruginosa PA14 for altered biofilm formation. This screen identified the PA3885 mutant, which had 147-fold more biofilm than the wild-type strain. Loss of PA3885 decreased swimming, abolished swarming, and increased attachment, although this did not affect production of rhamnolipids. The PA3885 mutant also had a wrinkly colony phenotype, formed pronounced pellicles, had substantially more aggregation, and had 28-fold more exopolysaccharide production. Expression of PA3885 in trans reduced biofilm formation and abolished aggregation. Whole transcriptome analysis showed that loss of PA3885 activated expression of the pel locus, an operon that encodes for the synthesis of extracellular matrix polysaccharide. Genetic screening identified that loss of PelABDEG and the PA1120 protein (which contains a GGDEF-motif) suppressed the phenotypes of the PA3885 mutant, suggesting that the function of the PA3885 protein is to regulate 3,5-cyclic diguanylic acid (c-di-GMP) concentrations as a phosphatase since c-di-GMP enhances biofilm formation by activating PelD, and c-di-GMP inhibits swarming. Loss of PA3885 protein increased cellular c-di-GMP concentrations; hence, PA3885 protein is a negative regulator of c-di-GMP production. Purified PA3885 protein has phosphatase activity against phosphotyrosine peptides and is translocated to the periplasm. Las-mediated quorum sensing positively regulates expression of the PA3885 gene. These results show that the PA3885 protein responds to AHL signals and likely dephosphorylates PA1120, which leads to reduced c-di-GMP production. This inhibits matrix exopolysaccharide formation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilm formation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilm formation and PA1120 should be TpbB.

Show MeSH
Related in: MedlinePlus