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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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Inactivation of tpbA increases cell aggregation.Aggregation of PA14 and the tpbA mutant after diluting with fresh LB medium (percentages indicate volume % of the starting overnight culture and fresh medium) (A). Biofilm formation of mutants lacking adhesin (PA4625) and its regulator (PA4624) at 37°C after 1, 2, and 24 h (B). Ten wells were used for each culture. Data show the average of the two independent experiments±s.d.
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ppat-1000483-g004: Inactivation of tpbA increases cell aggregation.Aggregation of PA14 and the tpbA mutant after diluting with fresh LB medium (percentages indicate volume % of the starting overnight culture and fresh medium) (A). Biofilm formation of mutants lacking adhesin (PA4625) and its regulator (PA4624) at 37°C after 1, 2, and 24 h (B). Ten wells were used for each culture. Data show the average of the two independent experiments±s.d.

Mentions: Cell aggregative behavior is also related to biofilm formation so we investigated the role of TpbA on cell aggregation and found the tpbA mutant causes cell aggregation (Fig. 4A). Autoaggregation of the tpbA mutant was also observed in 96-well polystyrene plates during biofilm formation (data not shown). Our whole-transcriptome analysis showed that inactivating tpbA induced both PA4624 (encodes for a putative hemolysin activator) and PA4625 (encodes for an adhesin/hemagglutinin) by 2.1- to 4.9-fold. In E. coli, adhesin regulates cell aggregation as well as attachment [26]. To examine whether PA4624–PA4625 control adhesive activity in P. aeruginosa, we investigated biofilm formation with these mutants. Both mutants showed decreased initial biofilm formation; i.e., initial attachment, to polystyrene plates at 1 h and 2 h (Fig. 4B), and final biofilm formation at 24 h was also decreased for both the PA4624 and PA4625 mutants, which suggests that both gene products control attachment to the surface. Therefore, TpbA decreases cell aggregation probably by repressing the PA4624 and PA4625 genes.


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)

Inactivation of tpbA increases cell aggregation.Aggregation of PA14 and the tpbA mutant after diluting with fresh LB medium (percentages indicate volume % of the starting overnight culture and fresh medium) (A). Biofilm formation of mutants lacking adhesin (PA4625) and its regulator (PA4624) at 37°C after 1, 2, and 24 h (B). Ten wells were used for each culture. 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-g004: Inactivation of tpbA increases cell aggregation.Aggregation of PA14 and the tpbA mutant after diluting with fresh LB medium (percentages indicate volume % of the starting overnight culture and fresh medium) (A). Biofilm formation of mutants lacking adhesin (PA4625) and its regulator (PA4624) at 37°C after 1, 2, and 24 h (B). Ten wells were used for each culture. Data show the average of the two independent experiments±s.d.
Mentions: Cell aggregative behavior is also related to biofilm formation so we investigated the role of TpbA on cell aggregation and found the tpbA mutant causes cell aggregation (Fig. 4A). Autoaggregation of the tpbA mutant was also observed in 96-well polystyrene plates during biofilm formation (data not shown). Our whole-transcriptome analysis showed that inactivating tpbA induced both PA4624 (encodes for a putative hemolysin activator) and PA4625 (encodes for an adhesin/hemagglutinin) by 2.1- to 4.9-fold. In E. coli, adhesin regulates cell aggregation as well as attachment [26]. To examine whether PA4624–PA4625 control adhesive activity in P. aeruginosa, we investigated biofilm formation with these mutants. Both mutants showed decreased initial biofilm formation; i.e., initial attachment, to polystyrene plates at 1 h and 2 h (Fig. 4B), and final biofilm formation at 24 h was also decreased for both the PA4624 and PA4625 mutants, which suggests that both gene products control attachment to the surface. Therefore, TpbA decreases cell aggregation probably by repressing the PA4624 and PA4625 genes.

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