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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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Las QS activates transcription of tpbA.β-galactosidase activity of ptpbA was measured with biofilm cells of PA14 and the mutants lasI, rhlI, and lasR rhlR using pLP-ptpbA. Data show the average of the two independent experiments±s.d.
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ppat-1000483-g007: Las QS activates transcription of tpbA.β-galactosidase activity of ptpbA was measured with biofilm cells of PA14 and the mutants lasI, rhlI, and lasR rhlR using pLP-ptpbA. Data show the average of the two independent experiments±s.d.

Mentions: QS regulates many genes in P. aeruginosa via a conserved cis-element in the promoter of each gene. N-(3-oxododecanoyl)-L-homoserine lactone binds to the LasR transcriptional regulator [35], and this complex interacts with the las-box, defined as CT-(N)12-AG sequence [36]. The Las-box is conserved among the promoters of the Las-QS regulated genes including lasB, rhlAB, and rhlI [36]. Another class of transcriptional regulation is governed by the lys-box, that is defined as a palindromic sequence, T-(N)11-A [37], and MvfR is a LysR-type transcription factor that binds to the lys-box [38]. We found that the promoter of tpbA (ptpbA) has a putative las-box 220 bp upstream of the start codon (CTCGCCTCGCTGAAAG) and a putative lys-box 90 bp upstream of the start codon (TGAAGCTGCCTCA). In order to examine if expression of tpbA is regulated by QS, we constructed a ptpbA::lacZ fusion plasmid (pLP-ptpbA) and transformed this into QS-related PA14 mutants (lasI, rhlI, and lasR rhlR). Expression of tpbA gene in biofilm cells was reduced by 42% in the lasI mutant, but not in the rhlI mutant (Fig. 7). Corroborating these results, inactivation of both lasR and rhlR also decreased expression of tpbA gene by 39% (Fig. 7). Similar results were obtained when the activity of ptpbA::lacZ was examined in planktonic cells (50% reduction in transcription for the lasI mutant and 37% reduction for the lasR rhlR mutant). Since loss of QS only affected expression of tpbA by 50%, other factors may also participate in the regulation of tpbA. These results suggest that Las-QS, rather than Rhl-QS, is an activator of tpbA expression with other unknown regulators.


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)

Las QS activates transcription of tpbA.β-galactosidase activity of ptpbA was measured with biofilm cells of PA14 and the mutants lasI, rhlI, and lasR rhlR using pLP-ptpbA. Data show the average of the two independent experiments±s.d.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000483-g007: Las QS activates transcription of tpbA.β-galactosidase activity of ptpbA was measured with biofilm cells of PA14 and the mutants lasI, rhlI, and lasR rhlR using pLP-ptpbA. Data show the average of the two independent experiments±s.d.
Mentions: QS regulates many genes in P. aeruginosa via a conserved cis-element in the promoter of each gene. N-(3-oxododecanoyl)-L-homoserine lactone binds to the LasR transcriptional regulator [35], and this complex interacts with the las-box, defined as CT-(N)12-AG sequence [36]. The Las-box is conserved among the promoters of the Las-QS regulated genes including lasB, rhlAB, and rhlI [36]. Another class of transcriptional regulation is governed by the lys-box, that is defined as a palindromic sequence, T-(N)11-A [37], and MvfR is a LysR-type transcription factor that binds to the lys-box [38]. We found that the promoter of tpbA (ptpbA) has a putative las-box 220 bp upstream of the start codon (CTCGCCTCGCTGAAAG) and a putative lys-box 90 bp upstream of the start codon (TGAAGCTGCCTCA). In order to examine if expression of tpbA is regulated by QS, we constructed a ptpbA::lacZ fusion plasmid (pLP-ptpbA) and transformed this into QS-related PA14 mutants (lasI, rhlI, and lasR rhlR). Expression of tpbA gene in biofilm cells was reduced by 42% in the lasI mutant, but not in the rhlI mutant (Fig. 7). Corroborating these results, inactivation of both lasR and rhlR also decreased expression of tpbA gene by 39% (Fig. 7). Similar results were obtained when the activity of ptpbA::lacZ was examined in planktonic cells (50% reduction in transcription for the lasI mutant and 37% reduction for the lasR rhlR mutant). Since loss of QS only affected expression of tpbA by 50%, other factors may also participate in the regulation of tpbA. These results suggest that Las-QS, rather than Rhl-QS, is an activator of tpbA expression with other unknown regulators.

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