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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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Reduction in biofilm formation by tpbA phenotype reversal mutations.Biofilm formation of double mutants (A) and single mutants (B) identified by genetic screening for the tpbA mutation at 37°C in LB after 24 h. Six to ten wells were used for each culture. Representative data are shown in (A). Biofilm formation of each mutant was calculated relative to that of PA14 (OD540 mutant/OD540 wild-type). Data show the average of the two independent experiments±s.d.
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ppat-1000483-g005: Reduction in biofilm formation by tpbA phenotype reversal mutations.Biofilm formation of double mutants (A) and single mutants (B) identified by genetic screening for the tpbA mutation at 37°C in LB after 24 h. Six to ten wells were used for each culture. Representative data are shown in (A). Biofilm formation of each mutant was calculated relative to that of PA14 (OD540 mutant/OD540 wild-type). Data show the average of the two independent experiments±s.d.

Mentions: To investigate how TpbA regulates biofilm formation, EPS production, wrinkly colony morphology, and cell aggregation, genetic screening was conducted using Tn5-luxAB transposon mutagenesis to find suppressive loci for the phenotypes of the tpbA mutation. The double mutant library (tpbA plus random gene inactivations) was screened first for a reduction in aggregation; this step eliminated most cells with unaltered phenotypes by allowing them to aggregate and precipitate at the bottom of the tube. The cells remaining in the supernatant that failed to aggregate like the tpbA mutant were grown on Congo-red plates, incubated at 37°C for 3–4 days, and colonies displaying a white and smooth shape like the wild-type strain were chosen. After that, a third screen was performed by assaying biofilm formation using 96-well polystyrene plates to identify double mutants that had biofilm formation like the wild-type strain. Twenty-six mutants were identified that showed reduced aggregation, a white smooth colony, and reduced biofilm formation like the wild-type strain, and 19 of these mutations were in the pel locus (Fig. 5A, Table 1). Four of the other mutants have the Tn5-luxAB insertion in the TpbB gene (encodes a GGDEF-motif protein) and in the PA1121 gene (encodes a hypothetical protein). In addition, insertions were found in the PA1678 gene (encodes a putative DNA methylase) and in the promoter of the PA5132 gene (encodes a putative protease) (Table 1). Like the double mutants, all of the single mutants lacking the gene identified by genetic screening were tested for biofilm formation, and all of these mutants formed less biofilm as reported previously (Fig. 5) [3],[4].


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)

Reduction in biofilm formation by tpbA phenotype reversal mutations.Biofilm formation of double mutants (A) and single mutants (B) identified by genetic screening for the tpbA mutation at 37°C in LB after 24 h. Six to ten wells were used for each culture. Representative data are shown in (A). Biofilm formation of each mutant was calculated relative to that of PA14 (OD540 mutant/OD540 wild-type). 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-g005: Reduction in biofilm formation by tpbA phenotype reversal mutations.Biofilm formation of double mutants (A) and single mutants (B) identified by genetic screening for the tpbA mutation at 37°C in LB after 24 h. Six to ten wells were used for each culture. Representative data are shown in (A). Biofilm formation of each mutant was calculated relative to that of PA14 (OD540 mutant/OD540 wild-type). Data show the average of the two independent experiments±s.d.
Mentions: To investigate how TpbA regulates biofilm formation, EPS production, wrinkly colony morphology, and cell aggregation, genetic screening was conducted using Tn5-luxAB transposon mutagenesis to find suppressive loci for the phenotypes of the tpbA mutation. The double mutant library (tpbA plus random gene inactivations) was screened first for a reduction in aggregation; this step eliminated most cells with unaltered phenotypes by allowing them to aggregate and precipitate at the bottom of the tube. The cells remaining in the supernatant that failed to aggregate like the tpbA mutant were grown on Congo-red plates, incubated at 37°C for 3–4 days, and colonies displaying a white and smooth shape like the wild-type strain were chosen. After that, a third screen was performed by assaying biofilm formation using 96-well polystyrene plates to identify double mutants that had biofilm formation like the wild-type strain. Twenty-six mutants were identified that showed reduced aggregation, a white smooth colony, and reduced biofilm formation like the wild-type strain, and 19 of these mutations were in the pel locus (Fig. 5A, Table 1). Four of the other mutants have the Tn5-luxAB insertion in the TpbB gene (encodes a GGDEF-motif protein) and in the PA1121 gene (encodes a hypothetical protein). In addition, insertions were found in the PA1678 gene (encodes a putative DNA methylase) and in the promoter of the PA5132 gene (encodes a putative protease) (Table 1). Like the double mutants, all of the single mutants lacking the gene identified by genetic screening were tested for biofilm formation, and all of these mutants formed less biofilm as reported previously (Fig. 5) [3],[4].

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