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

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.

Figure 1. Inactivation of tpbA increases biofilm formation.

Total biofilm formation (at the liquid/solid and air/liquid interfaces) (A), and biofilm formation on the bottom of polystyrene plates (B) by P. aeruginosa PA14 and the tpbA mutant at 37°C in LB after 50 h. Six to ten wells were used for each culture. Data show the average of the two independent experiments±s.d.

Figure 2. TpbA regulates swarming, swimming motility, and production of rhamnolipids.

Swarming motility (A), swimming motility (B), and production of rhamnolipids (C) of P. aeruginosa PA14 and the tpbA mutant at 37°C after 24 h. Five plates were used for each swarming and swimming culture, and data show the average of two independent experiments. For the production of rhamnolipids, data show the average of the two independent experiments±s.d.

Figure 3. Inactivation of tpbA increases colony roughness and enhances EPS production.

Colony morphology of P. aeruginosa PA14, the tpbA mutant, and the pelA mutant on Congo-red plates after 6 days at 25°C or 37°C (A). EPS production of each strain after 24 h at 37°C or after 48 h at 25°C (B). Data show the average of the two independent experiments±s.d.

Figure 4. 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.

Figure 5. 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.

Figure 6. 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.

Figure 7. 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.

Figure 8. Schematic of TpbA regulation of biofilm formation in P. aeruginosa PA14.

The QS molecule, N-(3-oxododecanoyl)-L-homoserine lactone (3-oxoC12-HSL), binds to the LasR transcription factor, and this complex activates expression of tpbA. TpbA has a N-terminal signal sequence and is translocated into the periplasm. Periplasmic TpbA dephosphorylates the membrane-anchored GGDEF protein TpbB at a tyrosine reside which deactivates GGDEF protein activity. The reduced cellular c-di-GMP concentration decreases expression of the pel operon as well as adhesin genes. This leads to reduced EPS production, biofilm formation, and pellicle formation, as well as enhanced swarming motility. Production of rhamnolipids is not regulated by TpbA.