Label-Free Quantitative Proteomic Analysis of the Global Response to Indole-3-Acetic Acid in Newly Isolated Pseudomonas sp. Strain LY1

Abstract

Indole-3-acetic acid (IAA), known as a common plant hormone, is one of the most distributed indole derivatives in the environment, but the degradation mechanism and cellular response network to IAA degradation are still not very clear. The objective of this study was to elucidate the molecular mechanisms of IAA degradation at the protein level by a newly isolated strain Pseudomonas sp. LY1. Label-free quantitative proteomic analysis of strain LY1 cultivated with IAA or citrate/NH₄Cl was applied. A total of 2,604 proteins were identified, and 227 proteins have differential abundances in the presence of IAA, including 97 highly abundant proteins and 130 less abundant proteins. Based on the proteomic analysis an IAA degrading (iad) gene cluster in strain LY1 containing IAA transformation genes (organized as iadHABICDEFG), genes of the β-ketoadipate pathway for catechol and protocatechuate degradation (catBCA and pcaABCDEF) were identified. The iadA, iadB, and iadE-disrupted mutants lost the ability to grow on IAA, which confirmed the role of the iad cluster in IAA degradation. Degradation intermediates were analyzed by HPLC, LC-MS, and GC-MS analysis. Proteomic analysis and identified products suggested that multiple degradation pathways existed in strain LY1. IAA was initially transformed to dioxindole-3-acetic acid, which was further transformed to isatin. Isatin was then transformed to isatinic acid or catechol. An in-depth data analysis suggested oxidative stress in strain LY1 during IAA degradation, and the abundance of a series of proteins was upregulated to respond to the stress, including reaction oxygen species (ROS) scavenging, protein repair, fatty acid synthesis, RNA protection, signal transduction, chemotaxis, and several membrane transporters. The findings firstly explained the adaptation mechanism of bacteria to IAA degradation.

Keywords: Pseudomonas sp. LY1; indole-3-acetic acid; microbial degradation; proteomics; stress response.

FIGURE 1

Indole-3-acetic acid (IAA) transformation by resting cells of strain LY1. (A) IAA (2,000 mg/L) degradation by resting cells of strain LY1 cultivated with IAA () and citrate/NH₄Cl (), respectively. The cell density of resting cells is OD600 = 6.0. The reaction was performed at 30°C 120 rpm. (B) UV scanning of the samples at interval times from IAA transformation reactions. The reactions were performed with resting cells of strain LY1 cultivated with IAA. (C) HPLC analysis of the samples at interval times from IAA transformation reactions. The reactions were performed with resting cells of strain LY1 cultivated with IAA. The signal at 254 nm was recorded. The arrow indicated the UV spectrum of the compound with a retention time of 3.60 min. The mobile phase was 35% (v/v) methanol and 65% (v/v) 0.05% formic acid. The HPLC column was Agilent XBD C18 (4.6 mm × 250 mm, 5 μm). Each value is the mean from three parallel replicates ± SD.

FIGURE 2

SDS-PAGE, Venn diagram, and volcano plot of the proteomic dataset. (A) SDS-PAGE analysis of crude proteins extracted from cell of IAA group (cultivated with IAA) and IAACon group (cultivated with citrate/NH₄Cl). (B) Venn diagram of proteins identified in the proteomes of IAA and IAACon group. (C) Volcano plot showing changes in protein abundance from IAA and IAACon group.

FIGURE 3

Overview of proteome data. (A) Pearson correlation coefficients for pair-wise comparisons of the IAA and IAACon proteome data. (B) PLS-DA of proteome data from IAA and IAACon samples.

FIGURE 4

Functional categorization analysis of upregulated DAPs in the presence of IAA. (A) Major enriched GO terms of IAA-up DAPs in molecular function (MF), cellular component (CC), and biological process (BP) terms. The statistics with more than 3 proteins at GO level 2 are shown in this figure. (B) Major enriched KEGG pathways of IAA-up DAPs with at least three DAPs.

FIGURE 5

PPI network of the IAA-up DAPs were searched for their PPI using the web resource STRING and characterized using Cytoscape. Networks with at least 3 elements are shown in the figure. The size of the circle denoted the fold-change of the protein. Fold-change of the DAPs that were unique to the IAA group was set as 2. The enriched KEGG functions of the DAPs are indicated in different colors.

FIGURE 6

Graphical representation of iad gene cluster in strain Pseudomonas sp. LY1. Arrows indicate the size and direction of transcription of each gene. The numbers above the arrows indicate the amino acid sequence identity between the marked protein and the corresponding orthologous protein from Pseudomonas sp. LY1. Highlighted in cyan are the iad (iac) genes, in blue are the regulator genes, in orange are the catBCA genes, in purple are the paaIJD genes, in gray are the genes with unknown function, and in yellow and green are the pcaABCDEF genes.

FIGURE 7

Expression level of IAA degrading relative genes in iad cluster. (A) Protein abundance levels of IAA degrading enzymes in the IAA group (gray bar) and the IAACon group (white bar). (B) qPCR analysis of the IAA degrading genes that were differentially expressed in IAA group (gray bar) and IAACon group (white bar). The least protein abundance and expression level of iadE in the IAACon group were set as 1. t-test was used to calculate statistical significance between IAA and IAACon group. *p < 0.05.

FIGURE 8

Proposed IAA degradation in strain Pseudomonas sp. LY1.

FIGURE 9

Proposed protein profile of IAA degradation response in strain LY1.