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.

Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c

“…In fact, the recombinant expression of the M. leprae dgcA was able to mimic the same phenotypes in P. aeruginosa that were induced by overexpression of a well-characterized P. aeruginosa encoded DGC, tpbB (Kulasakara et al, 2006;Ueda & Wood, 2009). DgcA from M. leprae possesses conserved DGC A-site (RFGGDEF) and I-site (RSRD) motifs.…”

“…The ml1419c DGGDEF construct was digested with EcoRI and SpeI endonucleases and cloned into the pJN105 to generate pMRLB108. The plasmid pJN1120 (Hickman & Harwood, 2008) that expresses tpbB (PA1120), a well-characterized DGC from P. aeruginosa, was used as a positive control (Kulasakara et al, 2006;Ueda & Wood, 2009) and pJN105 was used as a negative control. All constructs were confirmed by nucleotide sequencing.…”

Global Regulatory Pathways and Cross-talk ControlPseudomonas aeruginosaEnvironmental Lifestyle and Virulence Phenotype

“…A tpbA mutant displayed increased attachment to polystyrene and a defect in swarming motility, but no alteration in rhamnolipid production (Ueda, 2009a). Similar to a wspF mutant, the tpbA mutant displayed a wrinkly colony phenotype on Congo-red plates, implying increased production of EPS.…”

“…Transcriptome analysis of a tpbA mutant revealed enhanced expression of the pelACDF genes and the biofilm matrix adhesin CdrA. The expression of tpbA was also upregulated, suggesting auto-regulation (Ueda, 2009a).…”

Proteus mirabilis interkingdom swarming signals attract blow flies

“…DNA sequencing to identify transposon insertion positions Genomic DNA was isolated from the swarming mutants via the UltraClean Microbial DNA isolation kit (MO BIO, Carlsbad, CA, USA). For sequencing, arbitrary PCR (Ueda and Wood, 2009) was performed; the first round of arbitrary PCR reaction (PCR1) was performed using 100 ng of genomic DNA and arbitrary primer 1 (5 0 -GGCCAGGCCTGCAGAT GATGNNNNNNNNNNGTAT-3 0 ) along with internal specific primer (5 0 -ACGGATTCGCAAACCTGTCAC G-3 0 ). The second arbitrary PCR reaction (PCR2) was performed with the PCR1 product and arbitrary primer 2 (5 0 -GGCCAGGCCTGCAGATGATG-3 0 ) along with external specific primer I (5 0 -AGGTGGCGGAA ACATTGGATG-3 0 ).…”