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Comparative Study
, 18 (10), 3535-3549

Comparative Genomic, Proteomic and Exoproteomic Analyses of Three Pseudomonas Strains Reveals Novel Insights Into the Phosphorus Scavenging Capabilities of Soil Bacteria

Affiliations
Comparative Study

Comparative Genomic, Proteomic and Exoproteomic Analyses of Three Pseudomonas Strains Reveals Novel Insights Into the Phosphorus Scavenging Capabilities of Soil Bacteria

Ian D E A Lidbury et al. Environ Microbiol.

Abstract

Bacteria that inhabit the rhizosphere of agricultural crops can have a beneficial effect on crop growth. One such mechanism is the microbial-driven solubilization and remineralization of complex forms of phosphorus (P). It is known that bacteria secrete various phosphatases in response to low P conditions. However, our understanding of their global proteomic response to P stress is limited. Here, exoproteomic analysis of Pseudomonas putida BIRD-1 (BIRD-1), Pseudomonas fluorescens SBW25 and Pseudomonas stutzeri DSM4166 was performed in unison with whole-cell proteomic analysis of BIRD-1 grown under phosphate (Pi) replete and Pi deplete conditions. Comparative exoproteomics revealed marked heterogeneity in the exoproteomes of each Pseudomonas strain in response to Pi depletion. In addition to well-characterized members of the PHO regulon such as alkaline phosphatases, several proteins, previously not associated with the response to Pi depletion, were also identified. These included 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.

Figures

Figure 1
Figure 1
A–C. Growth of the three Pseudomonas strains under either Pi replete (1.4 mM) or Pi deplete (50 μM) growth conditions. Arrows denote sampling points for exoproteomics. D. During the growth experiments, alkaline phosphatase activity was quantified as a proxy for determining the activation of the PHO regulon. The values shown represent a given time point when the maximal alkaline phosphatase activity detected for each strain was obtained. Results presented are the mean of triplicate cultures. Error bars denote standard deviation.
Figure 2
Figure 2
A qualitative assessment, using 1D‐SDS PAGE, of the exoproteomes of all three Pseudomonas strains examined prior to HPLC 2D‐MS/MS. Each gel lane represents 20 ml culture supernatant. For both Pi deplete and Pi replete growth conditions, three biological replicates were performed.
Figure 3
Figure 3
Protein expression analyses in response to Pi depletion of the three Pseudomonas exoproteomes in addition to the exoproteome of the phoBR mutant of P. putida BIRD‐1. White spaces represent the absence of genes encoding the corresponding proteins from their genomes. Each individual biological replicate is displayed. The colour key represents Log2 transformations of protein fold change.
Figure 4
Figure 4
The relative abundance of Pi scavenging proteins detected in the exoproteomes of the three Pseudomonas strains and the phoBR mutant grown in both Pi replete and Pi deplete growth conditions. The normalized spectral abundance factor (NSAF) was calculated using Scaffold 4. Values displayed are the mean of triplicate cultures.
Figure 5
Figure 5
Genomic analyses of the Pi binding proteins found in the three Pseudomonas strains (A) The genetic neighbourhood profiles of the different Pi binding proteins located in the three Pseudomonas strains (B) The diversity of proteins that contain the Pfam12849 domain using a number of genome‐sequenced soil bacteria with the inclusion of characterized Pi binding proteins. Abbreviations; pstSCAB1/2, Pi‐specific ABC transporter; psp, DING‐family Pi binding protein; psp2/3, uncharacterized Pi binding protein; nptA, NA+/Pi co‐transporter; glpD, glycerol 3‐phosphate dehydrogenase; glpR, transcriptional regulator; glycerol kinase; glpK, glycerol uptake facilitator; fhaB‐like, putative filamentous haemagglutinin; phoBR, two component regulator; gsp, type II secretion system.
Figure 6
Figure 6
Genomic and proteomic analyses of the phosphonate binding proteins found in the three Pseudomonas strains (A) The genetic neighbourhood profiles of the different phosphonate binding proteins located in the three Pseudomonas strains (BIRD‐1, SBW25, DSM4166) as well as Sinorhizobium meliloti 1021 (1021). (B) Neighbour‐joining phylogenetic analysis of the different phosphonate binding proteins detected in the Pseudomonas strains outlined in Table 1 with the addition of various Burkholderia and Flavobacteria strains. Bootstrap values (500 runs) have been omitted for clarity. IMG accession numbers have been included as a reference. Abbreviations: phnWAY, alternative 2‐aminoethylphosphonate degradation pathway; phnDCE1/2/3/4, phosphonate ABC transporter; phnF‐M, C‐P lyase.

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