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Comparative genomic, proteomic and exoproteomic analyses of three Pseudomonas spp. Part 5
Bacteria that inhabit the rhizosphere of agricultural crops can have a beneficial effect on crop growth, including through the solubilisation and remineralisation of complex forms of phosphorus (P). However, our understanding of the bacterial proteomic response to P stress is limited. Here, exoproteomic analysis of three Pseudomonas species was performed in unison with proteomic analysis of Pseudomonas putida BIRD-1 (BIRD-1) grown under P replete and P deplete conditions. Comparative exoproteomics revealed marked heterogeneity in the exoproteomes of each Pseudomonas species in response to low concentrations of P. In addition to well-characterised members of the PHO regulon such as alkaline phosphatases, several previously undiscovered proteins are responsive to phosphate depletion including putative nucleases, phosphotriesterases, putative phosphonate transporters and outer membrane proteins. Moreover, in BIRD-1, mutagenesis of the master regulator, phoBR, led us to confirm the addition of several novel PHO-dependent proteins. Our data expands knowledge of the Pseudomonas PHO regulon, including species that are frequently used as bioinoculants, opening up the potential for more efficient and complete use of soil complexed P.
Sample Processing Protocol
Experimental procedures Growth and maintenance of bacterial strains All three Pseudomonas strains were routinely grown in Luria Bertani (LB) medium at 30 °C. Liquid cultures were incubated with shaking at 180 rpm. Solid media was prepared by the addition of 1.5% (w/v) agar to LB medium. To investigate the effect of phosphate-depletion on the three strains, each was grown in an adapted Minimal A medium comprising: Na-Succinate 5.4 g l-1, NaCl 200 mg l-1, NH4Cl 450 mg l-1, CaCl2 200 mg l-1, KCL mg l-1 MgCl2 450 mg l-1, FeCl2 10 mg l-1, MnCl2 10 mg l-1, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.2, with KH2PO4 added to a final concentration of either 50 μM or 1.4 mM. Quantification of alkaline phosphatase activity 0.5 ml culture was incubated with 20 μl N-para-nitrophenolphosphate (PNPP) (final conc. 4mM) and incubated at room temperature for 1 h or when colour development started to occur. The reaction was stopped using 25 μl NaOH (2 mM) and incubated for 10 min. Cell debris and precipitants were removed via centrifugation (2 min, 8,000 x g) prior to spectrophotometry (optical density 405 nm). A standard curve for N-para-nitrophenol was generated using a range of known concentrations (0, 4, 8, 25, 50,75, 100 mg ml-1). Preparation of exoproteomes, trypsin in-gel proteolysis, and nano-LC-MS/MS analysis The supernatants of bacterial cultures were harvested during mid exponential growth (OD600 0.5 - 0.8). Cells were removed via centrifugation (3,200 x g, 10 min) and filtration through a 0.22 μm membrane (Fisherbrand, syringe filter PVDF 33mm). Supernatants were pre-filtered through a 0.45 μm membrane (Fisherbrand, syringe filter PVDF 33mm). Proteins in the remaining milieu were concentrated and purified by precipitation with trichloroacetic acid (75% w/v) and separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Precipitated proteins were loaded onto a 4-20 % SDS Precast gel (Expedeon). SDS-PAGE was performed by using 20X Teo-Tricine-SDS running buffer (Expedeon) for at 180 V for 45 min. Gels were stained using Instant Blue (Expedeon). Polyacrylamide gel bands containing the entire exoproteome were cut and processed for in-gel proteolysis with trypsin (Roche) as previously described (Christie-Oleza and Armengaud, 2010). Gel-dependent nanoflow liquid chromatography-tandem MS (nano LC-MS/MS) was subsequently performed as follows: An UltiMate® 3000 HPLC series (Thermo Scientific and Dionex Integrated Solutions, Sunnyvale, CA USA) was used for peptide concentration and separation. Samples were separated in Nano Series™ Standard Columns 75 µm i.d. x 15 cm, packed with C18 Acclaim PepMap100, 3 µm, 100Å Thermo Scientific and Dionex Integrated Solutions, Sunnyvale, CA USA). The gradient used was from 3% to 35% solvent B (0.1% formic acid in acetonitrile) for 30 min. Peptides were eluted directly (~ 300 nL min-1) via a Triversa Nanomate nanospray source (Advion Biosciences, NY) into a Orbitrap Fusion mass spectrometer (Q-OT-qIT, Thermo Scientific, Germany). Survey scans of peptide precursors from 350 to 1500 m/z were performed at 120 K resolution (at 200 m/z) with automatic gain control (AGC) to a target value of 4 x 105. Precursor ions with charge state 2–4 + were isolated and subjected to HCD fragmentation in the ion trap. MS/MS analysis was performed using collision energy 35, AGC 1 x 104 and max injection time 200 ms. The dynamic exclusion duration was set to 45 s with a 10 ppm tolerance for the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 2 s cycles.
Data Processing Protocol
Peptide identification through MS/MS database searching The recorded MS/MS spectra were searched against the protein sequence database (P. putida BIRD-1, NC_017530.1; P. fluorescens SBW25, NC_012660.1; P. stutzeri DSM4166, NC_017532.1). The search was carried out with MASCOT 2.2.04 software (Matrix Science). Parameters were established as follows: tryptic peptides with a maximum of 1 missed cleavage during proteolytic digestion, a mass tolerance of 5 ppm on the parent ion and 0.5 Da on the MS/MS, fixed modification for carboxyamidomethylated Cysteine and variable modification for oxidized Methionine. MASCOT results were combined and parsed using Scaffold (Proteome Software). A protein identification was considered valid when at least two different peptides were detected in the same experiment. The false-positive rate for protein identification was estimated using a reverse decoy database as below 0.1% with these parameters by peptide prophet as part of Scaffold.
Lidbury ID, Murphy AR, Scanlan DJ, Bending GD, Jones AM, Moore JD, Goodall A, Hammond JP, Wellington EM. Comparative genomic, proteomic and exoproteomic analyses of three Pseudomonas strains reveals novel insights into the phosphorus scavenging capabilities of soil bacteria. Environ Microbiol. 2016 Oct;18(10):3535-3549 PubMed: 27233093
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||62909||no assay title provided (mzIdentML)||1285||7091||3967||7014||7014||
|2||62908||no assay title provided (mzIdentML)||1161||6497||3571||6426||6426||
|3||62907||no assay title provided (mzIdentML)||1277||7308||4030||7216||7216||
|4||62912||no assay title provided (mzIdentML)||1378||8563||5231||8440||8440||
|5||62910||no assay title provided (mzIdentML)||1434||8380||4998||8273||8273||
|6||62911||no assay title provided (mzIdentML)||1371||7815||4665||7708||7708||