Dataset Information

Flagellin (FliC) phosphorylation affects Type 2 protease secretion and biofilm dispersal in Pseudomonas aeruginosa PAO1

ABSTRACT: Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of Pseudomonas aeruginosa PAO1. The absence of phosphorylation in the conserved T27 and S28 residues of flagellin FliC, interestingly, did not affect swimming motility, but affected the secretome of type 2 secretion system (T2SS) and biofilm formation of PAO1. Flagellin phosphomutants had increased levels and activities of type 2 secretome proteins. This occurs by a possible mechanism affecting the secretion efficiency of T2SS machinery. Flagellin phosphomutants also formed reduced biofilm at 24h and had delayed biofilm dispersal under static and dynamic flow conditions, respectively. The levels of type 2 secretome and biofilm formation under static conditions had an inverse correlation. Hence, increase in the levels of type 2 secretome was accompanied by reduced biofilm formation in the flagellin phosphomutants. Altogether, we found a system of phosphorylation that co-ordinately regulates surface related processes such as proteases secretion by T2SS, biofilm formation and dispersal.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Pseudomonas Aeruginosa Pao1

SUBMITTER: Miao-Hsia Lin

LAB HEAD: Miao-Hsia Lin

PROVIDER: PXD003645 | Pride | 2022-03-02

REPOSITORIES: Pride

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Publications

Flagellin FliC Phosphorylation Affects Type 2 Protease Secretion and Biofilm Dispersal in Pseudomonas aeruginosa PAO1.

Suriyanarayanan Tanujaa T Periasamy Saravanan S Lin Miao-Hsia MH Ishihama Yasushi Y Swarup Sanjay S

PloS one 20161004 10

Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of P. aeruginosa PAO1. This is the first report of occurrence of ...[more]

PMID: 27701473

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Project description:The global transcriptional regulator Hha of Escherichia coli controls hemolysin activity, biofilm formation, and virulence expressions. Earlier, we have reported that Hha represses initial biofilm formation and disperses biofilms as well as controls prophage excision in E. coli. Since biofilm dispersal is a promising area to control biofilms, here we rewired Hha to control biofilm dispersal and formation. The Hha variant Hha13D6 was obtained to have enhanced biofilm dispersal activity along with increased toxicity compared to wild-type Hha (Hha13D6 induces dispersal 60%, whereas wild-type Hha induces dispersal at early biofilms but not at mature biofilms). Toxic Hha13D6 caused cell death probably by the activation of proteases HslUV, Lon, and PrlC, and deletion of protease gene hslV with overproducing Hh13D6 repressed biofilm dispersal, indicating Hha13D6 induces biofilm dispersal through the activity of protease HslV. Furthermore, another Hha variant Hha24E9 was also obtained to decrease biofilm formation 4-fold compared to wild-type Hha by regulation of gadW, glpT, and phnF. However, the dispersal variant Hha13D6 did not decrease biofilm formation, while the biofilm variant Hha24E9 did not induce biofilm dispersal. Hence, Hha may have evolved two ways in response to environmental factors to control biofilm dispersal and formation, but both controlling mechanisms come from different regulatory systems. For the whole-transcriptome study of BW25113 hha/pCA24N-hha13D6 versus BW25113 hha/pCA24N-hha biofilm dispersal, cells were grown in 250 mL of LB glucose (0.2%) for 16 h at 125 rpm with 10 g of glass wool (Corning Glass Works, Corning, NY, USA) in 1 L Erlenmeyer flasks to form a robust biofilm (Ren et al., 2004) and incubated an additional 1 h with 1 mM IPTG to induce wild-type Hha and Hha13D6. Similarly, for the whole transcriptome study of BW25113 hha/pCA24N-hha24E9 versus BW25113 hha/pCA24N-hha biofilm formation, cells were grown in 250 mL of LB glucose (0.2%) containing 1 mM IPTG for 7 h at 250 rpm with 10 g of glass wool to form biofilms. Biofilm cells were obtained by rinsing and sonicating the glass wool in sterile 0.85% NaCl solution at 0Â°C, and RNALater bufferÂ® (Applied Biosystems, Foster City, CA, USA) was added to stabilize RNA during the RNA preparation steps. Total RNA was isolated from biofilm cells using a bead beater (Biospec, Bartlesville, OK, USA). cDNA synthesis, fragmentation, and hybridizations to the E. coli GeneChip Genome 2.0 array (Affymetrix, Santa Clara, CA, USA; P/N 511302). Genes were identified as differentially expressed if the expression ratio was higher than the standard deviation: 2.0-fold (induced and repressed) cutoff for Hha13D6 DNA microarrays (standard deviation 1.3-fold) and 10.0-fold (induced) or 4.0-fold (repressed) for Hha24E9 DNA microarrays (standard deviation 4.0-fold), and if the p-value for comparing two chips was less than 0.05.

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Dataset Information

Flagellin (FliC) phosphorylation affects Type 2 protease secretion and biofilm dispersal in Pseudomonas aeruginosa PAO1

Publications

Flagellin FliC Phosphorylation Affects Type 2 Protease Secretion and Biofilm Dispersal in Pseudomonas aeruginosa PAO1.

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