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Characterization of indole-3-acetic acid biosynthesis and the effects of this phytohormone on the proteome of the plant-associated microbe Pantoea sp. YR343 Kasey Estenson, Gregory B. Hurst, Robert F. Standaert, Amber N. Bible, David Garcia, Karuna Chourey, Mitchel J Doktycz, and Jennifer L Morrell-Falvey J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00708 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 26, 2018
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Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Proteome Research
Characterization of indole-3-acetic acid biosynthesis and the effects of this phytohormone on the proteome of the plant-associated microbe Pantoea sp. YR343
Kasey Estenson†,‡, Gregory B. Hurst§, Robert F. Standaert†,#,ǁ,┴ , Amber N. Bibleǁ, David Garcia†,¶, Karuna Chourey§, Mitchel J. Doktycz†,‡,¶, and Jennifer L. Morrell-Falvey*†,‡,ǁ †
Biosciences, §Chemical Sciences and #Neutron Scattering Divisions, Oak Ridge National Laboratory, Oak Ridge TN, USA; ‡UT-ORNL Graduate School of Genome Science and
Technology, University of Tennessee, Knoxville TN, USA; ǁDepartment of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville TN, USA; ┴Shull Wollan Center — a Joint Institute for Neutron Sciences, Oak Ridge, TN, USA; ¶Bredesen Center, University of Tennessee, Knoxville, TN
Keywords: Pantoea sp. YR343, indole-3-acetic acid, indole-3-pyruvate decarboxylase, tryptophol, poplar, plant colonization
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ABSTRACT
Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plantassociated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted Pantoea sp. YR343, a microbe isolated from Populus deltoides, synthesizes IAA using the indole-3-pyruvate (IPA) pathway. To better understand IAA biosynthesis and the effects of IAA exposure on cell physiology, we characterized proteomes of Pantoea sp. YR343 grown in the presence of tryptophan or IAA. Exposure to IAA resulted in upregulation of proteins predicted to function in carbohydrate and amino acid transport and exopolysaccharide (EPS) biosynthesis. Metabolite profiles of wildtype cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed a ∆ipdC mutant which showed elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wildtype levels). Although we failed to detect intermediates from other known IAA biosynthetic pathways, this result suggests the possibility of an alternate pathway or the production of IAA by a non-enzymatic route in Pantoea sp. YR343. The ∆ipdC mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association.
INTRODUCTION Populus deltoides (poplar) hosts a diverse microbiome that influences its growth and productivity.1-3 Some bacteria associated with P. deltoides are beneficial to the health of the host, acting as bioprotectants, biofertilizers, or biostimulants.4,5 For example, many plant-associated microbes produce phytohormones, such as indole-3-acetic acid (IAA). IAA influences plant
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hosts in different ways, including by induction of tissue differentiation, cell division, elongation, lateral-root formation, and cambial growth.6,7 Multiple IAA biosynthetic pathways have been described in microbes, most of which require tryptophan as the precursor.7-9 These tryptophan-dependent pathways include the indole-3acetonitrile (IAN) pathway, the indole-3-acetamide (IAM) pathway, the tryptophan side-chain oxidase (TSO) pathway, the indole-3-pyruvate (IPA) pathway, and the tryptamine (TA) pathway.7,9 IAA production has been studied in many microbes, including Azospirillum brasilense, Enterobacter cloacae UW5, Pantoea dispersa, Pantoea agglomerans, and Pseudomonas putida.10-14 For example, A. brasilense is a plant growth-promoting bacterium that synthesizes IAA primarily through the indole-3-pyruvate pathway.15 In A. brasilense, a deletion of the ipdC gene, which encodes indole-3-pyruvate decarboxylase, resulted in a drastic reduction in IAA production in the mutant (10% of wildtype levels).16 That the ipdC mutant strain was still capable of IAA production, however, suggested the presence of a second biosynthetic pathway.17 Moreover, the presence of IAA itself induced ipdC gene expression in A. brasilense suggesting that IAA biosynthesis is a highly regulated process.14,16,18-20 Many species within the genus Pantoea have been found associated with plants.21 While some of these species, such as P. stewartii and P. rodasii, are known pathogens of corn and eucalyptus, respectively22,23, other strains have been shown to have beneficial effects on the plant host. For example, P. agglomerans is thought to promote plant growth by enhancing root growth, which can increase water and mineral uptake.13 Indeed, this growth promotion is thought to be the result of IAA production, and genomic analyses indicated the presence of a gene (ipdC) encoding indole-3-pyvuvate decarboxylase (IpdC).13 P. dispersa was also shown to produce IAA, and a functional IPA pathway was confirmed by metabolite analyses.10
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Pantoea sp. YR343 was isolated from P. deltoides and shown to be a robust plant colonizer, with the ability to solubilize phosphate and produce IAA.24 Pantoea sp. YR343 can also colonize other plants, including Triticum aestivum and Arabidopsis thaliana, where colonization by Pantoea sp. YR343 resulted in increased lateral root production.24 As with P. agglomerans and P. dispersa, genomic analyses of Pantoea. sp. YR343 indicated the presence of an ipdC homolog (PMI39_00059), suggesting the possibility that Pantoea sp. YR343 synthesizes IAA through the IPA pathway. To test this prediction and to determine how Pantoea sp. YR343 responds to the presence of tryptophan and IAA, we characterized proteomes of Pantoea sp. YR343 grown in minimal medium supplemented with tryptophan or IAA. In addition, we constructed a mutant of Pantoea sp. YR343 defective in IpdC activity and examined its metabolite profile and ability to colonize plant roots.
MATERIALS AND METHODS Growth of Pantoea sp. YR343 Pantoea sp. YR343 was grown in either R2A (R2A Broth Premix, TEKnova, Inc.) or M9 minimal medium (per 1 L: 6 g Na2HPO4, 3 g KH2PO4, 0.5 g NaCl and 1 g NH4Cl, plus 10 mL each filter sterilized 100 mM MgSO4, 20% glucose, and 10 mM CaCl2) with shaking at 28 °C. For proteomic analyses, Pantoea sp. YR343 was grown in M9 medium supplemented with 1 mM Trp or with 5 µM, 50 µM or 500 µM IAA (Sigma Aldrich). Metabolite analysis Pantoea sp. YR343 wild type and ∆ipdC cultures were grown in M9 medium with or without Trp (1 mM) to an OD600 of 1. After centrifugation, the supernatant was collected (800 µL) and prepared for metabolite analysis. For acidic and neutral metabolites, the supernatants were
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acidified to pH 2 with hydrochloric acid after addition of 1 µg of IAA-d7 (D-IAA; Cambridge Isotope Laboratories) as an internal standard and then extracted with ethyl acetate (800 µL). For basic metabolites (specifically, tryptamine (TA)), samples were basified to pH 11 with sodium hydroxide and extracted with toluene (800 µL). Samples were vortexed vigorously and briefly centrifuged, after which a 600-µL portion of the organic (top) layer was collected and transferred to a 2-mL glass vial. Next, the solvent was evaporated under a stream of argon gas, and the residue was resuspended in 100 µL of derivatization agent (BSTFA + TMSCl 99:1). These samples were then incubated at 80 °C for 1 h. After derivatization, each sample was diluted with 900 µL of hexane in the same vial. GC/MS analysis was performed with an Agilent 7890A gas chromatograph equipped with a 7693A automatic liquid sampler, an HP-5ms capillary column (30 m long × 0.25 mm inside diameter with a 0.25-µm capillary film of 5% phenyl methylsilicone) and a 5975C masssensitive detector. Splitless injections of 1 µL were made at an inlet temperature of 270 °C with a 15-s dwell time (needle left in the inlet after injection), an initial column temperature of 60 °C and helium carrier gas at a constant flow of 1 mL/min. After 2 min at 60 °C, the temperature was ramped at 20 °C/min to 200 °C, then at 10 °C/min to 270 °C and finally 30 °C/min to 300 °C, with a 2-min hold at 300 °C. The detector was operated with a transfer line temperature of 300 °C, source temperature of 230 °C and quadrupole temperature of 150 °C. After a 5-min solvent delay, electron-impact mass spectra from 50–500 amu were collected continuously (~3/s) for the duration of the run. Metabolites were identified on the basis of comparison with authentic materials (IAA and TA) or mass spectral matches with the NIST Mass Spectral Library (IPA, ILA and TOL). Approximate IAA concentrations were determined by comparison of peak areas for IAA with those for the D-IAA standard.
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Promoter-Reporter (GFP) Construct The ipdC (PMI39_00059) promoter region was defined as the 350 bp upstream of the transcriptional
start
site
ipdCprom_(HindIII)For:
and
amplified
from
genomic
DNA
using
the
5’-CCCAAGCTTGGCTGTTATCGACGCGCG-3’
primers and
ipdCprom_(EcoRI) Rev: 5’-CCGGAATTCGCCAACGTTGGGGGTTTT-3’. The fragment was subcloned into pPROBE-NT25, and the resulting plasmid was transformed into Pantoea sp. YR343 via electroporation with selection on R2A plates containing 50 µg/mL kanamycin.24 GFP expression from cells harboring pPROBE-ipdC was monitored using a Zeiss LSM710 confocal laser scanning microscope. ImageJ was used for image processing.26 Sample preparation for proteomics All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), unless specified otherwise. High performance liquid chromatography- (HPLC-) grade water and other solvents were obtained from Burdick & Jackson (Muskegon, MI), 99% formic acid was purchased from EM Science (Darmstadt, Germany) and sequencing-grade trypsin was acquired from Promega (Madison, WI). Pantoea sp. YR343 was grown in M9 medium or in M9 medium supplemented with 1 mM Trp, 5 µM IAA, 50 µM IAA, or 500 µM IAA. Three biological replicates were collected and analyzed for each condition. Frozen cell pellets were ground to a powder under liquid nitrogen and suspended in a detergent-based cell lysis buffer (5% SDS, 50 mM Tris-HCl, 0.15 M NaCl, 0.1 mM EDTA, 1 mM MgCl2, pH 8.5).27 Cells were lysed via 20 min of heat lysis27 and transferred to fresh microcentrifuge tubes. Trichloroacetic acid was added to samples at a final concentration of 25%, and proteins were precipitated by storage at –20 °C overnight. The mixtures were thawed briefly and centrifuged at 21,000 × g for 20 min. The resulting cell pellets
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were washed with chilled acetone and centrifuged at 21,000 × g for 10 min. The acetone wash was repeated thrice, discarding supernatants. The cell pellets were air dried, dissolved in guanidine buffer [6M guanidine HCl in Tris-CaCl2 buffer, pH 8.5 (50mM Tris, 10mM CaCl2)] and incubated at 60 °C for 4 h. The total amount of extracted protein was measured using the RC/DC protein estimation kit (Bio-Rad Laboratories, Hercules, CA, USA) per the manufacturer’s instructions. Bovine serum albumin supplied with the kit was used as a standard for the assay. The samples were diluted six-fold using Tris-CaCl2 buffer. Proteolysis was carried out by adding modified sequencing grade trypsin at 40 µg/mg protein with overnight incubation at 37 °C and gentle mixing.28 Digested peptides were stored at –80 °C until MS analysis. Approximately 75 µg peptides per sample was loaded on an in-house packed biphasic column [strong cation exchange, SCX (Luna, Phenomenex, Torrance, CA) and reverse-phase C18 (Aqua, Phenomenex, Torrance, CA)] as described 28,29 and subjected to offline desalting.30 LC-MS-MS of proteome digests Peptides were analyzed using a two-dimensional liquid chromatography-tandem mass spectrometry approach (2D-LC-MS-MS)31, implemented as described previously.28,29 Peptides were eluted from the SCX trapping column by eleven successive step gradients of increasing ammonium acetate concentration, from 50 mM to 500 mM. Each SCX step gradient eluted a set of peptides onto a 15-cm long reverse-phase (C18) column, where they were separated by a twohour gradient from 100% solvent A (5% CH3CN, 0.1% formic acid in water) to 50:50 solvent A:solvent B (70% CH3CN, 0.1% formic acid in water). Peptides eluting from the reverse-phase column were introduced via a nanoelectrospray source (Proxeon, Odense, Denmark) into a linear ion trap mass spectrometer (LTQ-XL, Thermo Scientific, San Jose CA), where data were acquired in data-dependent mode. Following each full-scan mass spectrum, up to 5 tandem mass
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spectra (MS-MS) were acquired from the most intense ions in the full-scan spectrum. For collision-induced dissociation, precursor isolation width was 3.0 m/z units, and normalized collision energy was 35%. Dynamic exclusion was employed with a repeat count of 1, exclusion list size of 300, repeat duration of 60 sec, and exclusion duration of 180 sec. Proteomics data analysis Peptide identifications were obtained from MS-MS spectra using Myrimatch (version 2.1.138).32 Myrimatch settings included precursor m/z tolerance of 1.5, fragment m/z tolerance of 0.5, charge states up to +4, TIC cutoff of 98%, cleavage rule “Trypsin/P”, fully tryptic peptides only, with a maximum of two missed cleavage sites per peptide. The protein database for the Myrimatch searches was based on the predicted proteome containing 4900 Pantoea sp. YR343 protein sequences33, downloaded as 27136.faa from the Department of Energy Joint Genome Institute Integrated Microbial Genomes web site34 on Aug 25, 2014. Sequences for 44 common contaminant proteins and trypsin were appended for a total of 4945 proteins in the assembled protein fasta file.
Myrimatch employed a decoy database containing sequence-
reversed version of these proteins to provide an estimate of peptide false discovery rate. Protein identifications were assembled from peptide identifications using IDPicker (version 3.1.599).35 A protein identification required identification of at least two distinct peptides. The maximum false discovery rate for peptide-spectrum matches was set to 2%. Observed peptide and protein false discovery rates were ≤0.6% and ≤2.4%, respectively. Analysis of identified proteins for shared peptides was performed using Microsoft Access and custom scripts in R. Proteins sharing all identified peptides were combined into protein groups. Protein abundance was estimated using spectrum count (number of tandem mass spectra assigned to that protein36), adjusted for shared peptides.37 Normalized spectral abundance factor
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(NSAF) values38 were calculated for each protein from adjusted spectrum count, with adjusted spectrum count values of 0 replaced by 0.3. Abundance (NSAF) ratios for proteins for which all adjusted spectrum count values were zero across the 3 biological replicates of one treatment are therefore inaccurate, and were tracked through further analysis steps as “present/absent” proteins (i.e., detected in one treatment and not in the other). We chose criteria for differential abundance of a protein between a treatment and control to be an NSAF ratio ≥ 2 or ≤1/2, and a BenjaminiHochberg-corrected t test p-value ≤0.05. Proteins for which an average of < 2.5 tandem mass spectra were identified over the 3 biological replicates of a given treatment were identified as “low abundance” for that treatment39; the fraction of NSAF represented by low abundance proteins ranged from 3-7%. Reverse transcription polymerase chain reaction (RT-PCR) Total RNA was extracted from Pantoea sp. YR343 cultures grown in M9 minimal medium with or without Trp using the RNeasy Mini kit (Qiagen) as per manufacturer’s instructions. Following RNA elution, 2.5 units RNase-free DNaseI (1 U/µl) (NEB) were added to the RNA and incubated at 37˚C for 30 minutes followed by heat inactivation at 75˚C for 5 minutes. cDNA was transcribed from 500 ng RNA using ThermoScript RT-PCR system (Invitrogen) as per manufacturer’s instructions. 5 units of RNaseH were added to each sample and incubated for 20 minutes at 37˚C to eliminate residual RNA in the cDNA sample. After RNaseH treatment, RTPCR was performed using FailSafe PCR PreMix Selection Kit (Epicentre) and specific primers for ipdC (PMI39_00059) (RT_ipdC For: 5’-ATCCCGAAATTGCCTGGGTTG-3’ and RT_ipdCRev:
5’-GCGGTTAAGGTGTCGGTAAAC-3’)
and
16S
(RT_16s
For:
5’-
ACGATCCCTAGCTGGTCT-3’ and RT_16s Rev: 5’CTAATCCTGTTTGCTCCC-3’) as a
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control. PCR reactions were subjected to agarose electrophoresis with ethidium bromide staining, and images of the gels were analyzed with ImageJ. Construction of an ipdC Mutant To generate a disruption mutant for ipdC (PMI39_00059), we first constructed pKnock-ipdC by ligating the pKnock-Tc vector with a 600 bp internal ipdC fragment amplified from genomic DNA using ipdC_pknock for (XbaI): 5’-GCTCTAGAACTCCATCAGCAGGTTGCCGCA-3’ and ipdC-pknock rev (KpnI): 5'- CGG GGT ACCCCAAAGGCCGCAGTGCCTTGA-3’.40 The resulting plasmid was verified by restriction digests and transformed into Pantoea sp. YR343 by electroporation and selected on R2A plates containing 5 µg/mL tetracycline. Disruption of the ipdC gene was confirmed by PCR and sequencing. Plant assays Colonization of Populus trichocarpa BESC819 was performed as described previously.24 Briefly, Pantoea YR343-GFP or the ∆ipdC mutant was grown overnight in R2A medium, and then the OD600 of each culture was adjusted to 0.01 using fresh R2A.
Five plants were
inoculated for each treatment. For each plant, sterile clay soil with 1× Hoagland’s medium was inoculated with 10 mL of either sterile R2A medium (control), wild type Pantoea YR343-GFP24, or the ∆ipdC mutant by first mixing the bacterial culture with the clay soil, then planting a three week old rooted P. trichocarpa shoot tip. Plants were incubated in the growth chamber for an additional three weeks. Plant roots were harvested, weighed, and rinsed with PBS. The distribution of WT or mutant cells on root samples was imaged using a Zeiss LSM710 confocal microscope The rest of the sample was then washed with 3 mL of PBS containing a small amount of glass beads to disrupt bacterial attachment. Colony-forming units (CFUs) were counted for wildtype Pantoea YR343-GFP on R2A agar plates containing 10 µg/mL gentamycin.
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CFUs for the ∆ipdC mutant were counted on R2A agar plates containing 5 µg/mL tetracycline. Control plants showed no background contamination when plated on R2A plates with either gentamycin or tetracycline. No statistically significant differences were observed when data were analyzed using the student’s t-test. Wild type Arabidopsis thaliana seeds (Columbia) were sterilized as described previously24 and germinated on 0.25 X MS agar plates with 0.25% sucrose for 10 days. Wild type Pantoea sp. YR343 and the ∆ipdC mutant were grown overnight to similar cell densities in R2A media supplemented with 200 µg ml-1 tryptophan. After removing the cells by centrifugation, the culture supernatant was added to molten 0.5 X MS agar with 0.1% sucrose (1.5 ml culture supernatant mixed with 25 ml media per plate). Plates were treated with supernatant from wild type Pantoea sp.YR343 (n = 3), the ∆ipdC mutant (n = 3), or with R2A medium with tryptophan as a control. Germinated plants were added to the plates then incubated in a growth chamber set at 23°C with constant lighting for one week prior to imaging. Images were analyzed using ImageJ and statistics were calculated using the student’s t-test (p < 0.05).
Measurements
represent an average of five plants that were measured for the control treatment and eight plants for each experimental treatment.
RESULTS Identification of putative IAA biosynthetic enzymes by genomic analyses Our previous work showed that Pantoea sp. YR343 produces IAA following addition of tryptophan to the growth medium.24 A number of tryptophan-dependent biosynthetic pathways for IAA have been described in microbes7 (Figure 1). To determine which pathway(s) may be present in Pantoea sp. YR343, we performed genomic analyses to identify putative homologs of
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known proteins in these pathways. From these analyses, we identified a gene (PMI39_00059; ipdC) that encodes a protein with 26% identity to A. brasilense indole-3-pyruvate decarboxylase (IpdC), an enzyme in the IPA pathway that catalyzes the committed step.41 The first step in the IPA pathway (Trp→IPA) requires an aromatic aminotransferase and we identified seven genes in Pantoea sp. YR343 that are annotated to encode gene products with this function (Table 1). Likewise, the final step in this pathway (indole-3-acetaldehyde (IAAld) →IAA) requires an aldehyde dehydrogenase and we found 17 gene products annotated with this function (Table 1). Of these 17 prospective aldehyde dehydrogenases, four enzymes (encoded by PMI39_00725, PMI39_01356, PMI39_02889, and PMI39_04201) have substantial (~40%) identity to the aldehyde dehydrogenase from Ustilago maydis, which has been shown to support IAA synthesis when expressed heterologously in Escherichia coli42. These results suggested that Pantoea sp. YR343 may have a functional IPA pathway.
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Figure 1. Tryptophan-dependent IAA biosynthetic pathways in Pantoea sp. YR343. The pathways are color-coded based on genomic analyses with green lines and checks indicating the presence of genes encoding candidate enzymes for each step in the pathway, orange lines and questions marks indicating the presence of possible candidate gene products, and red lines with exes indicating the absence of genes encoding candidate enzymes for the pathway.
Similarly, we analyzed the genome for the presence of gene products found in the other IAA biosynthetic pathways. Because we were unable to find a homolog of tryptophan monooxygenase, we ruled out the presence of the IAM pathway in Pantoea sp. YR343 (Figure 1). Likewise, Pantoea sp. YR343 lacked enzymes in the IAN pathway (Figure 1). Although the sequence homologies were low, we did find genes encoding candidate enzymes in the tryptamine pathway, suggesting that this pathway may also be present in Pantoea sp. YR343. The TSO pathway has been described in only one organism, Pseudomonas florescens CHA043, and because no genes have been identified, we could not determine whether this pathway was present in Pantoea sp. YR343.
Table 1. Candidate enzymes in IAA biosynthetic pathways. Candidate Enzymes in IAA Biosynthesis Indole-3-Pyruvate Pathway log2ratio log2ratio LocusTag
Trp: Glu
log2ratio
log2ratio
IAA5µM:Glu IAA50µM:Glu IAA500µM:Glu
Aromatic aminotransferases PMI39_01811
0.8
0.4
0.8
1.1
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PMI39_01993
0.2
0.07
0.4
-0.3
PMI39_02094
-1.4
-2
-0.9
down
(PMI39_02628) NA
(up)
(up)
(up)
(PMI39_02920) NA
NA
NA
NA
(PMI39_04274) NA
NA
NA
(up)
PMI39_04560
0.7
0.2
1.1
NA
(up)
up
(PMI39_00313) NA
NA
NA
NA
(PMI39_00317) NA
NA
NA
NA
PMI39_00354
-0.4
0.3
0.4
0.2
(PMI39_00431) NA
NA
NA
NA
PMI39_00617
-2
0.3
1.1
(PMI39_00725) NA
(up)
(up)
(up)
(PMI39_00794) NA
(up)
NA
NA
(PMI39_00977) NA
NA
NA
NA
(PMI39_01356) NA
(up)
NA
NA
PMI39_02144
-0.03
1.1
1.7
0.9
PMI39_02889
0.6
0.3
0.4
1.4
PMI39_03367
-0.6
-0.4
-0.2
0.6
(PMI39_03939) NA
NA
NA
NA
PMI39_04111
0.09
0.6
0.4
(PMI39_04199) (down)
(down)
1
(down)
(PMI39_04201) (down)
(down)
(down)
(down)
PMI39_04236
0.03
-0.2
0.2
0.6
Indole-3-pyruvate decarboxylase (IpdC) PMI39_00059
up
Aldehyde Dehydrogenases
-0.7
0.4
-0.02
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Tryptamine Pathway PMI39_03119
-0.3
1.9
2
2.5
(PMI39_00935) NA
NA
NA
NA
(PMI39_02858) NA
NA
NA
NA
Proteomics data in the body of the table are log2-transformed NSAF ratios of experimental condition shown in the column header to control (minimal medium). Highlighted ratios indicate statistically significant differential abundance between treatment and control. Values of “up” or “down” are shown where a protein was not detected in either the treatment or control, so that an accurate ratio could not be calculated. Parentheses around “up” or “down” indicate that a protein fell below the low-abundance threshold for both treatment and control. Parentheses around a Locus Tag indicate that the protein fell below the low abundance threshold for all proteome measurements. “NA” indicates that the protein was not detected in either condition.
Proteome of Pantoea sp. YR343 grown in the presence of tryptophan To better understand how Pantoea sp. YR343 synthesizes IAA and related metabolites, we generated proteome profiles of cells growing in the presence or absence of tryptophan. We detected 2044 proteins from cultures grown in the presence or absence of tryptophan, with 16 proteins that were present above the low-abundance threshold and differentially abundant between the tryptophan cultures and control cultures (Table 2, Supplemental Figure S1A). Not surprisingly, we found that two enzymes (TrpE, PMI39_02719 and TrpD, PMI39_02721) in the tryptophan biosynthesis pathway (encoded by PMI39_02719-PMI39_02724) were less abundant in cultures with added tryptophan. Conversely, none of the enzymes predicted to be involved in IAA biosynthesis showed statistically significant differences in abundance in the presence of tryptophan (Table 1). Although the difference was not statistically significant (corrected p=0.06), IpdC was more abundant in cultures grown in the presence of excess tryptophan. Only four of the seven predicted aromatic aminotransferases in the IPA pathway were detected in cultures with added tryptophan, but none were differentially abundant compared to the control (Table 1). Likewise, only 9 out of 17 predicted aldehyde dehydrogenases were detected in tryptophan
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and/or control cultures (Table 1). In the tryptamine pathway, only one of the prospective enzymes (a putative homolog of Trp decarboxylase encoded by PMI39_03119) was detected under these growth conditions, and it was below the low abundance cutoff for both tryptophan and control cultures (Table 1).
Table 2. Proteins that are differentially expressed in the presence of tryptophan.
LocusTag
Description
log2ratio Trp:Glu
PMI39_02297
PAS domain S-box
up
PMI39_04926
ATPase, P-type (transporting), HAD superfamily, subfamily IC up
PMI39_00980
lysine-arginine-ornithine-binding periplasmic protein
up
PMI39_04398
Protein of unknown function (DUF1454).
up
PMI39_01931
hypothetical protein
up
PMI39_02605
Flagellar capping protein
3.4
(PMI39_01840) Glycosyltransferases involved in cell wall biogenesis
up
(PMI39_00879) conserved hypothetical protein, YceG family
up
PMI39_01718
4-aminobutyrate aminotransferase aminotransferases
and
related -1.1
(PMI39_03883) Transcriptional regulator
down
(PMI39_03872) Uncharacterized conserved protein
down
ABC-type uncharacterized (PMI39_00111) periplasmic component
transport
system, down
Short-chain dehydrogenases of various substrate down (PMI39_01285) specificities (PMI39_02165) hypothetical protein
down
PMI39_00349
down
arginine repressor
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PMI39_02721
anthranilate phosphoribosyltransferase
down
PMI39_04501
glucose-1-phosphate adenylyltransferase
down
PMI39_04638
Zn-dependent dipeptidase, microsomal dipeptidase homolog down
PMI39_02719
anthranilate synthase component I, proteobacterial subset down
PMI39_00256
DNA repair protein RadA
PMI39_02040
ABC-type amino acid transport/signal transduction systems, periplasmic component/domain down
PMI39_02676
Predicted ATPase
PMI39_02215
ABC-type branched-chain amino acid transport systems, periplasmic component down
down
down
The final column represents the log2-transformed NSAF ratios for growth with added tryptophan versus the control. Values of “up” or “down” are shown where a protein was not detected in either the treatment or control, so that an accurate ratio could not be calculated. Parentheses around a Locus Tag indicate that the protein fell below the low abundance threshold for the proteome measurements.
Proteomics of Pantoea sp. YR343 grown in the presence of IAA Because IAA itself can act as a positive regulator of ipdC gene expression in A. brasilense14,19, we sought to determine the effects of IAA exposure on Pantoea sp. YR343. For this experiment, we grew Pantoea sp. YR343 in minimal medium supplemented with 5 µM, 50 µM, or 500 µM IAA. Proteomics analyses detected a total of 2417 proteins across the three runs and revealed many proteins that were differentially abundant under these growth conditions (Table 3, and Supplemental Figures 1B, C, D). In the cultures grown with 5 µM IAA, we detected 24 differentially abundant proteins above the low-abundance threshold compared to cells grown in minimal medium (Table 3). In cultures grown with 50 µM and 500 µM IAA, we detected 16 and 53 differentially abundant proteins, respectively (Table 3).
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Among the proteins that are differentially regulated, we found several proteins involved in phosphate regulation, including PhoU (PMI39_02797) and PhoH (PMI39_02138) which were both upregulated, and PstB (PMI39_03605) which was down-regulated. In many bacterial systems, PhoU acts as a negative regulator of PhoB, which, together with PhoR, forms a twocomponent regulatory system that modulates the cellular response to environmental phosphate levels44. PhoU also plays a role in regulating the phosphate-specific transporter Pst (PstSCAB), of which PstB is a component45,46. Several proteins involved in inorganic ion transport and metabolism were also upregulated (PMI39_01007, PMI39_04031, and PMI39_04844) as were proteins annotated to be involved in transcriptional regulation (PMI39_00126 and PMI39_04198). A number of proteins involved in carbohydrate (PMI39_00875, PMI39_02090; PMI39_03310) and amino acid (PMI39_00919, PMI39_00980, PMI39_02763, PMI39_03122, PMI39_04094) transport and metabolism were also more abundant in the presence of IAA. In addition, we also observed upregulation of PMI39_00356 which has homology to E. coli AaeA, a protein involved in aromatic carboxylic acid efflux.47 It has been noted that AaeA and other efflux pump proteins are highly active in bacterial biofilms.48
Growth in the presence of IAA also resulted in
upregulation of the gene product encoded by PMI39_01840, which is part of a conserved gene cluster involved in exopolysaccharide (EPS) biosynthesis (PMI39_01835-PMI39_01848). Other gene products in that operon were also more abundant, although they did not pass criteria for differential abundance (Supplemental Table S1). In Erwinia amylovora and P. stewartii, the gene products encoded by this operon are required for the synthesis of an acidic extracellular polysaccharide, named amylovoran or stewartin, respectively.49,50
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None of the enzymes predicted to play a role in IAA biosynthesis was differentially abundant in the presence of IAA, with the exception of IpdC (PMI39_00059) which was more abundant when cells were grown in 500 µM IAA (Table 1 and 3). Of the seven predicted aromatic aminotransferases in the IPA pathway, we detected six (three above the low-abundance cutoff) when cells were grown in the presence of IAA. Likewise, we detected 11 (six above the lowabundance cutoff) of the 17 predicted aldehyde dehydrogenases under these growth conditions (Table 1). For the tryptamine pathway, only the putative Trp decarboxylase (PMI39_03113) was detected under these conditions.
Table 3. Proteins that are differentially expressed in the presence of IAA (5µM, 50µM, and 500µM).
LocusTag PMI39_00367
COG C
PMI39_04656
C
PMI39_01989 PMI39_04394 PMI39_03735
C C C
PMI39_02092
CHR
PMI39_00980
ET
PMI39_04094 PMI39_02719
E EH
PMI39_02763
E
PMI39_03606
F
Description Sulfite oxidase and related enzymes Short-chain alcohol dehydrogenase of unknown specificity NADH-quinone oxidoreductase, B subunit glycerol kinase aconitate hydratase 2 Lactate dehydrogenase and related dehydrogenases lysine-arginine-ornithine-binding periplasmic protein Predicted amino acid aldolase or racemase anthranilate synthase component I threonine ammonia-lyase, biosynthetic, long form phosphoribosylglycinamide formyltransferase,
log2ratio IAA 5µM: Glu
log2ratio IAA 50µM: Glu
log2ratio IAA 500µM: Glu
up
up
up
NA
NA
up
(up) -0.1 0.2
(up) 0.05 0.6
up 2.6 1.9
0.6
0.3
1.5
NA
NA
up
NA 1.4
(up) 2
up 2.7
0.8
1.2
1.1
(up)
(up)
up
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PMI39_00847 PMI39_01851
F F
PMI39_03310 PMI39_00059 PMI39_02090
G GHR G
PMI39_00875
G
PMI39_02999 PMI39_01799
IQ I
PMI39_00202 (PMI39_02976)
I I
PMI39_00126 PMI39_00132 (PMI39_00821)
K K L
PMI39_01840 PMI39_01856 PMI39_04745 PMI39_01007
M M M P
PMI39_02797
P
PMI39_04031
P
PMI39_04844
P
PMI39_04053
Q
PMI39_01997
S
PMI39_01459
S
(PMI39_02138)
T
PMI39_03722 PMI39_01901 PMI39_00356
V V V
formyltetrahydrofolate-dependent ADP-ribose pyrophosphatase deoxycytidine triphosphate deaminase ABC-type sugar transport system, periplasmic component Indole pyruvate decarboxylase Sugar kinases, ribokinase family PTS system, glucose-specific IIBC component beta-ketoacyl-acyl-carrier-protein synthase II 3-hydroxyisobutyrate dehydrogenase Phosphatidylserine/phosphatidylglycer ophosphate/cardiolipin synthases Lysophospholipase Transcriptional regulators ribonuclease III, bacterial integration host factor, alpha subunit Glycosyltransferases involved in cell wall biogenesis Carbohydrate-selective porin Predicted periplasmic protein Ferritin-like protein phosphate transport system regulatory protein PhoU ABC-type molybdenum transport system, ATPase component/photorepair protein PhrA Uncharacterized protein, homolog of Cu resistance protein CopC 2-keto-4-pentenoate hydratase/2oxohepta-3-ene-1,7-dioic acid hydratase (catechol pathway) Uncharacterized protein conserved in bacteria putative toxin-antitoxin system antitoxin component, TIGR02293 family Phosphate starvation-inducible protein PhoH, predicted ATPase ABC-type multidrug transport system, ATPase component S-formylglutathione hydrolase RND family efflux transporter, MFP
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up (up)
up (up)
up up
NA NA -0.09
(up) (up) -0.03
up up 1.5
0.9
0.6
1.3
up (up)
up NA
up up
(up) (up)
(up) (up)
up (up)
(up) (up) (up)
NA (up) (up)
up up (up)
up (up) up up
(up) NA up up
up up (up) up
(up)
up
up
(up)
up
up
(up)
(up)
up
NA
NA
up
(up)
(up)
up
(up)
(up)
up
NA
(up)
(up)
up up 1.4
(up) up 2.1
up up 3.3
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PMI39_01826 PMI39_04447
none none
PMI39_00352
none
PMI39_02043
E
PMI39_02040 PMI39_01330 PMI39_02311
ET ET F
PMI39_01065 PMI39_00743
H HR
PMI39_02094
KE
PMI39_02182
N
PMI39_00913
O
PMI39_03605 (PMI39_00828) PMI39_04666
P S S
PMI39_02823
TK
PMI39_01027 (PMI39_01963) PMI39_03268
W none none
PMI39_03037
none
PMI39_00439
none
PMI39_01725
CO
PMI39_04459
CR
PMI39_00919 PMI39_00140
E H
subunit hypothetical protein hypothetical protein Protein of unknown function (DUF1471). ABC-type polar amino acid transport system, ATPase component ABC-type amino acid transport/signal transduction systems, periplasmic component/domain Nitrogen regulatory protein PII hydroxyisourate hydrolase 3,4-dihydroxy-2-butanone 4phosphate synthase Amidases related to nicotinamidase Transcriptional regulators containing a DNA-binding HTH domain and an aminotransferase domain (MocR family) Flagellar basal body-associated protein Glutaredoxin, GrxB family phosphate ABC transporter, ATPbinding protein Uncharacterized conserved protein Uncharacterized protein conserved Response regulators consisting of a CheY-like receiver domain and a winged-helix DNA-binding domain P pilus assembly protein, chaperone PapD hypothetical protein S-adenosylhomocysteine hydrolase Protein of unknown function (DUF2884). GTPases - translation elongation factors formate dehydrogenase accessory protein FdhE NADPH:quinone reductase and related Zn-dependent oxidoreductases Glycine/D-amino acid oxidases (deaminating) L-aspartate oxidase
up (up)
up (up)
up up
NA
up
up
down
down
down
-1.2 -0.6 -1.1
down -1 -1.4
down down down
-0.09 0.05
-2 -0.8
down down
-2
-0.9
down
-0.2
-1.1
down
-0.3
-0.2
-1.3
-1.5 -0.5 down
down -1.1 -0.5
down (down) down
-0.2
-0.1
down
0.7 -0.1 0.3
0.4 -0.2 0.2
down (down) down
-0.4
-1
down
0.7
0.01
down
up
(up)
(up)
up
(up)
up
up (up)
up (up)
up up
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PMI39_04190
KT
(PMI39_01022)
KE
PMI39_01922 PMI39_03970
Q R
PMI39_01082 PMI39_01931
R none
PMI39_04563 PMI39_00219 (PMI39_03880)
none none none
(PMI39_03657) (PMI39_04877) (PMI39_02474) (PMI39_00836)
none none none O
(PMI39_03702)
O
(PMI39_00111)
R
(PMI39_01285) PMI39_03122
R E
PMI39_03751 PMI39_01209 (PMI39_04744)
F H H
PMI39_01125 PMI39_03045 (PMI39_04198)
J K K
(PMI39_02390) (PMI39_02372) (PMI39_04894)
L L L
PMI39_02489 PMI39_04795
L M
SOS-response transcriptional repressors (RecA-mediated autopeptidases) Transcriptional regulators containing a DNA-binding HTH domain and an aminotransferase domain (MocR family) ABC-type uncharacterized transport system, Predicted glutamine amidotransferase Short-chain dehydrogenases of various substrate specificities hypothetical protein Uncharacterized protein conserved in bacteria hypothetical protein Predicted O-methyltransferase Protein of unknown function (DUF1481). Uncharacterized conserved protein psiF repeat. FeS assembly scaffold SufA Iron-sulfur cluster assembly accessory protein ABC-type uncharacterized transport system, periplasmic component Short-chain dehydrogenases of various substrate specificities Phosphoserine phosphatase guanosine monophosphate reductase, eukaryotic lipoate-protein ligase B pyridoxamine-phosphate oxidase probable S-adenosylmethioninedependent methyltransferase, YraL family Putative transcriptional regulator Transcriptional regulator Uncharacterized protein conserved in bacteria DNA helicase II DNA gyrase inhibitor DNA recombination-dependent growth factor C alanine racemase
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(up)
up
(up)
NA
(up)
(up)
up (up)
(up) up
(up) (up)
NA (up)
up up
up up
(up) (up) (up)
up up (up)
(up) up (up)
(up) NA NA -0.3
(up) (up) (up) (down)
(up) (up) NA 0.2
(down)
(down)
-0.1
(down)
(down)
-0.3
(down) up
(down) (up)
-0.6 (up)
1.5 up (up)
1.1 up (up)
1.1 (up) (up)
up up (up)
(up) (up) (up)
(up) up (up)
(up) (up) (up)
(up) (up) NA
(up) (up) (up)
1.1 up
0.8 (up)
0.7 up
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PMI39_04926 PMI39_04643 (PMI39_00549)
P P P
PMI39_04680 PMI39_03844
S none
PMI39_01778 (PMI39_00174)
none none
PMI39_04118
E
PMI39_04647
ET
PMI39_01157
R
PMI39_03855
V
ATPase, P-type (transporting), HAD superfamily, subfamily IC TonB-dependent siderophore receptor formate transporter FocA Uncharacterized protein conserved in bacteria, putative virulence factor PAS domain S-box Lipopolysaccharide biosynthesis proteins, LPS:glycosyltransferases hypothetical protein ABC-type dipeptide transport system, periplasmic component ABC-type amino acid transport/signal transduction systems, periplasmic component/domain conserved hypothetical protein YtfJfamily, TIGR01626 Putative translation initiation inhibitor, yjgF family
up up (up)
up (up) (up)
up (up) NA
up up
(up) (up)
NA NA
up (up)
NA (up)
NA (up)
down
-0.2
1.1
down
0.3
0.6
down
-0.8
-0.9
down
-1.1
-2.1
The log2ratio is based on the normalized spectral abundance factor (NSAF) and is shows as the log2 ratio of trp to control. Highlighted ratios indicate statistically significant differential abundance between treatment and control. Values of “up” or “down” are shown where a protein was not detected in either the treatment or control, so that an accurate ratio could not be calculated. Parentheses around “up” or “down” indicate that a protein fell below the lowabundance threshold for both treatment and control. Parentheses around a Locus Tag indicate that the protein fell below the low abundance threshold for all proteome measurements. “NA” indicates that the protein was not detected in either condition. COG catagories: [A] RNA processing and modification; [B] Chromatin structure and dynamics; [C] Energy production and conversion; [D] Cell cycle control, cell division, chromosome partitioning; [E] Amino acid transport and metabolism; [F] Nucleotide transport and metabolism; [G] Carbohydrate transport and metabolism; [H] Coenzyme transport and metabolism; [I] Lipid transport and metabolism; [J] Translation, ribosomal structure and biogenesis; [K] Transcription; [L] Replication, recombination and repair; [M] Cell wall/membrane/envelope biogenesis; [N] Cell motility; [O] Post-translational modification, protein turnover, and chaperones; [P] Inorganic ion transport and metabolism; [Q] Secondary metabolites biosynthesis, transport, and catabolism; [R] General function prediction only; [S] Function unknown; [T] Signal transduction mechanisms; [U] Intracellular trafficking, secretion, and vesicular transport; [V] Defense mechanisms; [W] Extracellular structures; [Y] Nuclear structure; [Z] Cytoskeleton.
Induction of ipdC gene expression
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The proteomics analysis suggested that IpdC is more abundant in the presence of Trp and high levels of IAA. To determine whether ipdC gene expression is upregulated under these conditions, we constructed a reporter plasmid (pPROBE-ipdC) in which GFP expression is controlled by the ipdC promoter. After transforming this plasmid into wildtype Pantoea sp. YR343, we grew cultures in the presence of tryptophan or IAA and measured GFP fluorescence compared to control cultures. These results showed that the ipdC promoter was activated in the presence of both tryptophan and IAA (Figure 2). An increase in ipdC gene expression in the presence of excess tryptophan was confirmed using RT-PCR, which showed a 1.3 fold induction (Figure 2).
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Figure 2. Induction of ipdC gene expression in Pantoea sp. YR343. Images of cells harboring the pPROBE-ipdC reporter plasmid (A) before or (B) after growth in the presence of tryptophan or after growth in the (C) presence of 50 µM IAA. (D) Graph of average cell fluorescence from wildtype Pantoea sp. YR343 cells harboring pPROBE-ipdC grown under different conditions: no Trp (control),1 mM Trp, 5 µM IAA, 50 µM IAA, or 500 µM IAA. The fluorescence of 60 cells was averaged for each treatment. E) Comparison of ipdC gene expression using RT-PCR after growth in the absence or presence of tryptophan. ipdC gene expression was normalized against 16S RNA as a control.
Characterization of an ipdC mutant strain Combined, these data suggest that the IPA pathway is the major IAA biosynthesis pathway in Pantoea sp. YR343 and that ipdC expression is induced in the presence of tryptophan and IAA. To confirm the former hypothesis, we constructed a mutant strain of Pantoea sp. YR343 in which the ipdC gene was disrupted. We then analyzed culture media from both wild-type and ∆ipdC mutant cells for IAA and other metabolites using GC/MS. The two strains were grown in minimal medium overnight, with or without supplemental Trp, and the cells were removed by centrifugation. Supernatants were acidified with hydrochloric acid and extracted with ethyl acetate to recover acidic (e.g., IAA) and neutral metabolites. To recover basic metabolites (e.g., TA), the supernatants were treated with sodium hydroxide and the metabolites extracted into toluene. After evaporation of the solvents, the metabolites were converted to trimethylsilyl (TMS) derivatives as described in Materials and Methods prior to GC/MS analysis. The dominant Trp metabolites in wildtype supernatants were IPA, tryptophol (TOL), and indole-3-lactate (ILA), with smaller amounts of IAA and indole-3-carboxyaldehyde (Figure 3).
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Using deuterated-IAA as a standard, the analysis indicated that the wild-type cells produced approximately 2.5 µg/mL of IAA. Tryptophol and IAA are both derived from indole-3acetaldehyde (IAAld), by reduction or oxidation, respectively. IAAld itself was not detected in this analysis. Although IAAld is not well-determined by the analytical protocol, it appears not to accumulate to high levels and is rather converted to IAA or TOL. Importantly, we did not detect any indole-3-acetamide (IAM), indole-3-acetonitrile (IAN) or tryptamine (TA), predicted intermediates of the IAM, IAN or TA pathways, respectively, indicating that these pathways are not active at a detectable level in Pantoea sp. YR343. The metabolite profile is therefore consistent with an active IPA pathway. In the absence of ipdC, the supernatant from mutant cells showed a different metabolite profile, with the dominant metabolites being indole-3-pyruvate and indole-3-lactate (Figure 3). Tryptophol was completely absent in the supernatant from these mutant cells. Since tryptophol is produced from IAAld, we conclude that the ∆ipdC mutant lacks indole-3-pyruvate decarboxylase activity and is unable to produce IAAld. Interestingly, however, we did detect some IAA in the mutant cells, albeit at much lower levels compared to wild type cells (500 ng/ml; 20% of wildtype levels). As in the wildtype cells, we failed to detect IAN, IAM, or TA (Figure 3).
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Figure 3. Indole metabolite analysis of Pantoea sp. YR343 and Pantoea sp. YR343 ∆ipdC by GC/MS. A) Total ion current chromatogram B) scale expansion of TOL region C) scale expansion of IAA region. IAA produced variable mixtures of single and double trimethylsilyl
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(TMS derivatives), resulting in two peaks. IAA-d7 (D-IAA) at 1 µg/mL was added as an internal standard for IAA. Indolic compounds were identified by the use of standard materials (IAA and TA) or comparison of mass spectra with those in the NIST Mass Spectral Library (TOL, IPA and ILA).
Detection of IAA was unexpected since our genomic, proteomic, and metabolomic data were consistent with the IPA pathway being the only active biosynthetic pathway in Pantoea sp. YR343. The presence of IAA in the supernatant from the mutant could certainly suggest that IAA is being synthesized by Pantoea sp. YR343 by an unidentified pathway. Alternatively, it has been reported that IPA is unstable and can spontaneously degrade to IAA.51 These possibilities are not mutually exclusive, and the observed IAA may have resulted from a combination of spontaneous and as-yet unknown enzymatic processes. Arabidopsis lateral root production is induced by metabolites from Pantoea Pantoea sp. YR343 has been shown previously to stimulate lateral root production in Arabidopsis.24 Thus, we tested whether the metabolites produced from the ∆ipdC mutant induced an Arabidopsis root phenotype similar to the root phenotype induced by metabolites produced from wildtype cells. For this experiment, Arabidopsis seedlings were grown on plates made using culture supernatants from wildtype cells or the ∆ipdC mutant grown in the presence of Trp. As expected, seedlings grown in the presence of supernatant collected from wildtype cells were shorter in length and showed significant production of lateral roots compared to control plants (Figure 4A, B). Seedlings grown in the presence of supernatant collected from ∆ipdC mutant cells, however, showed an intermediate phenotype with short roots that produced significantly
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fewer lateral roots than seedlings grown in the presence of wildtype supernatant, but more lateral root production than control plants (Figure 4A, B). Loss of IpdC activity does not impair root colonization Finally, we investigated whether inactivation of the IPA pathway in Pantoea sp. YR343 influenced its ability to associate with plants. The results of these experiments showed that the ∆ipdC mutant was able to colonize poplar roots as efficiently as the wildtype cells based on plating assays (Figure 4C) and imaging experiments (Figure 4D, E). These data suggest that the ability of Pantoea sp. YR343 to associate with plants does not require a functional IPA pathway.
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Figure 4. Effects of ∆ipdC mutant on plants. (A) Lateral root density was measured as the number of lateral roots per mm of main root length. Images were analyzed using ImageJ and statistics were calculated using the Student’s t-test (p < 0.05). *, significant difference when compared to the control. **, significant difference between treatment with wild type supernatant and ∆ipdC supernatant. (B) Representative images of A. thaliana seedlings after one week treatment with culture supernatants from wild type Pantoea sp. YR343 and the ∆ipdC mutant. (C) Colonization experiments with P. trichocarpa. Wild type Pantoea sp. YR343 and the ∆ipdC mutant were measured as the log10 value of colony forming units (CFU) per gram of root material.
No significant differences were observed. (D, E) Representative images of P.
trichocarpa root colonization by (D) wildtype YR343:GFP or (E) mutant ∆ipdC:GFP. Bacteria were detected by GFP fluorescence (green) and P. trichocarpa roots were detected by autofluorescence (red).
DISCUSSION In this paper, we used a combination of genomic, metabolomic, and proteomic analyses to identify the likely IAA biosynthetic pathway in Pantoea sp. YR343 and to investigate the role of IAA on the colonization behavior and physiology of Pantoea sp. YR343. The production of IAA by some microbes has been associated with their plant growth promoting behavior.13,52-55 In the case of P. agglomerans, this growth promotion is thought to be due to enhanced root architecture, which, in turn, can result in enhanced water and nutrient uptake by the plant. The microbe benefits from this interaction by access to nutrients found in plant exudate, as well as increased surface area for colonization. A number of biosynthetic pathways have been described in microbes for IAA production. The ability to assign specific gene products to these pathways
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using genomic analyses, however, is limited to only a few well-characterized enzymes, such as IpdC and Trp monooxygenase. Other steps in these IAA biosynthesis pathways are defined by general functions such as aromatic aminotransferase and aldehyde dehydrogenase. Most bacterial genomes contain many candidate gene products with these predicted functions and there may be functional redundancy in vivo. Determining which of these candidate gene products is more likely to be involved in IAA biosynthesis is further hindered by the fact that few of the IAA biosynthetic enzymes appear to be co-expressed in operons. Thus, genomic analyses can provide an inventory of candidate gene products, but additional experimentation and/or molecular modeling are required to determine whether these gene products are involved in IAA biosynthesis. In Pantoea sp. YR343, genomic analyses identified 7 candidate aromatic aminotransferases and 17 candidate aldehyde dehydrogenases that may catalyze steps in the IPA pathway, of which only 4 aminotransferases and 9 aldehyde dehydrogenases were detected by proteomics during growth under conditions (added Trp) in which IAA is produced.
One of these aldehyde
dehydrogenases, encoded by PMI39_02889, has moderate homology to Iad1 from U. maydis
42
and appeared to be upregulated, although not significantly, in the presence of Trp and IAA, making it a plausible candidate for the primary aldehyde dehydrogenase for IAA biosynthesis in Pantoea sp. YR343. Testing this prediction will be the focus of future studies. With the assumption that enzymes involved in IAA biosynthesis should be present under conditions in which cells are producing IAA, the proteins that were identified in our proteomics analyses are more likely to be involved in IAA biosynthesis. It should be noted, however, that none of the enzymes was particularly abundant and other relevant enzymes may have been present in the cells but not detected in our analyses. Nevertheless, the proteomics results serve to
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prioritize selection of enzymes for future analyses. IpdC, on the other hand, was more abundant in the presence of Trp and IAA, suggesting that its expression is regulated. Indeed, we found that the ipdC promoter is activated in the presence of both Trp and IAA. How ipdC gene expression is regulated in Pantoea sp. YR343 will be the subject of future studies. Collectively, these data point to a model in which IAA biosynthesis in Pantoea sp. YR343 proceeds primarily via the IPA pathway and is regulated in part by ipdC gene expression levels. To better understand the changes to the proteome in the presence of IAA, we grew Pantoea sp. YR343 in 5µM, 50 µM or 500 µM IAA. The local concentration of IAA typically experienced by Pantoea sp. YR343 in the rhizosphere is unknown and determining this value presents a measurement challenge that will require advances in chemical imaging methods. Interestingly, growth of Pantoea sp. YR343 under each of these conditions produced different sets of differentially abundant proteins. Whether these conditions represent different stages in the initiation and maintenance of plant association is an intriguing possibility, although it is also possible that these results are a reflection of biological variation and measurement sensitivity. Taken together, it is clear that growth in the presence of IAA results in changes to the proteome in Pantoea sp. YR343. These changes are reflected in upregulation of gene products associated with transport of carbohydrates, amino acids, and inorganic ions. We also saw upregulation of gene products involved in transcriptional regulation and EPS biosynthesis. It is tempting to speculate that exposure to IAA induces changes in the physiology of Pantoea sp. YR343 that are advantageous for survival in the rhizosphere. For example, sugars and amino acids are primary components of plant exudates; thus, upregulation of these transporters may allow Pantoea sp. YR343 to better compete for these resources. Likewise, upregulation of EPS biosynthesis may promote attachment to plant roots and biofilm formation.
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Finally, the role of IAA in promoting plant-association was determined by construction of a ∆ipdC mutant. Disruption of the ipdC gene was confirmed by sequencing, and functional elimination of IpdC enzyme activity was shown by metabolite analysis. Specifically, it was shown that the mutant accumulated no TOL, which was the main end-product of IAAld in wildtype Pantoea sp. YR343 grown under the same conditions. The absence of TOL in the mutant also argues against the presence of an active tryptamine pathway since this pathway also produces IAAld. Nevertheless, we did detect some IAA (approximately 20% of wildtype) in the supernatant of the ∆ipdC mutant. The simplest explanation for the presence of IAA in the mutant is that Pantoea sp. YR343 harbors more than one pathway for IAA biosynthesis, as has been found in other bacteria.7
While we cannot rule out this scenario, our genomic and
metabolite data failed to identify an alternate pathway. An alternative explanation is that IAA is produced in the mutant by the known non-enzymatic degradation of IPA under these experimental conditions.51 The presence of TOL, rather than IAA, as the main product of the IPA pathway was surprising. One possible explanation is that the product balance (IAA via oxidation or TOL via reduction of IAAld) simply reflects the redox state of the cells, with growth in minimal medium rich in glucose favoring conversion to TOL. In the rhizosphere, a shift in IAAld flux toward IAA might be favored. Alternatively, IAA production could be regulated at the level of IAAld dehydrogenase, with TOL effectively being a stable storage pool for the reactive IAAld. Finally, TOL could be the intended product of the pathway. Indeed, TOL is known to have auxinic activity56 and it can be specifically oxidized back to IAAld by the plant enzyme indole-3-ethanol oxidase.57 The timing and location of the redox processes that interconvert TOL⇄IAAld⇄IAA may play an important role in establishing plant–microbe communication. As IAA itself can be
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formed from multiple biosynthetic pathways as well as from spontaneous decomposition of IPA, and most microorganisms can make IPA from Trp, TOL is, in fact, a more specific marker for IpdC activity than is IAA. Finally, we investigated whether the metabolites produced by wildtype cells and the ∆ipdC mutant had an effect on Arabidopsis root growth. We observed that treatment of Arabidopsis seedlings with supernatant collected from Pantoea sp. YR343 cells grown in the presence of Trp induced lateral root production, similar to what was observed during co-culture of Arabidopsis and Pantoea sp. YR343.24 Interestingly, Arabidopsis seedlings grown in the presence of supernatant collected from ∆ipdC mutant cells produced a different phenotype, displaying a shorter root length and an intermediate number of lateral roots. Whether this phenotype is the result of reduced IAA production or other metabolites in the supernatant from the mutant is unknown. It will be interesting to test individual indolic compounds produced by Pantoea sp. YR343 to determine the effects on root growth and architecture. Perhaps surprisingly, we found that ∆ipdC mutant cells were able to associate with poplar roots as efficiently as wildtype cells, suggesting that IAA production via the IPA pathway is not likely required for attachment to the root. Alternatively, it is possible that another metabolite mediates this interaction or that the reduced level of IAA produced by the mutant through a secondary pathway is still sufficient to promote plant association. A third possibility is that microbial IAA biosynthesis is triggered after plant association, and plays a role in the further development of plant-microbe interactions. To better understand plant-microbe association and distinguish among these possibilities, we will need to measure the local concentration and spatial distribution of metabolites produced by microbes during plant association. Indeed, identifying and measuring the temporal and spatial
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distribution of metabolites in complex systems is an exciting and challenging new area of research that will likely drive technical and scientific advances.
Supporting Information Supplementary Figure S1. Volcano plots illustrating significantly differentially abundant proteins in Pantoea sp. YR343 in the presence of tryptophan or IAA versus control. (file type, PDF) Supplemental Table S1 provides a complete listing of the Pantoea sp. YR343 proteins identified in proteome characterizations in this study. Further description of the contents is provided in the Legend tab of the spreadsheet. (file type, Excel) Supplemental Table S2 lists sequences, charge states, Q values, precursor m/z values, and numbers of identified tandem mass spectra assigned to tryptic peptides identified in proteome characterizations in this study.
The Legend tab in the spreadsheet provides further
documentation. (file type, Excel) Supplemental Table S3 shows the chromatographic and mass spectral properties of tryptophan metabolites identified in Pantoea sp. YR343 culture supernatants (file type, Word)
Corresponding Author *Jennifer L. Morrell-Falvey 865-241-2841 Email:
[email protected]
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Author Contributions In addition to assisting with the manuscript preparation, all authors contributed intellectually and performed data analyses. JM-F, AB, KE, RS, and MD developed the experimental plan. AB, RS, DG, and JM-F performed genomic and metabolomics analyses; AB, KE, KC, GH, and JM-F performed tandem-mass spectrometry characterization and analyses; KE and AB conducted reverse transcriptase polymerase chain reaction and mutant construction; and JM-F and KE wrote the paper. Notes The proteomics mass spectrometric output files in the original instrument vendor file format, Myrimatch search results, IDPicker analysis files, and the protein sequence file used for searches have
been
deposited
to
ProteomeXchange
(http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD007541 , dataset identifier PXD007541) via MassIVE (http://massive.ucsd.edu/ProteoSAFe/QueryPXD?id=PXD007541 , dataset identifier MSV000081478).
ACKNOWLEDGMENT This research was sponsored by the Genomic Science Program, U.S. Department of Energy, Office of Science, Biological and Environmental Research, as part of the Plant Microbe Interfaces Scientific Focus Area (http://pmi.ornl.gov). Oak Ridge National Laboratory is managed by UT-Battelle LLC, for the U.S. Department of Energy under contract DE-AC0500OR22725. ABBREVIATIONS
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BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; CFU, colony-forming unit; GC/MS, gas chromatography/mass spectrometry; GFP, green fluorescent protein; IAA, indole-3-acetic acid; IAAld, indole-3-acetaldehyde; IAM, indole-3-acetamide; IAN, indole-3-acetonitrile; ILA, indole-3-lactate; IPA, indole-3-pyruvate; IpdC, indole-3-pyruvate decarboxylase; NSAF, normalized spectral abundance factor; SCX, strong cation exchange; TIC, total ion current; TMSCl, trimethylsilyl chloride; TOL, tryptophol; Tris, tris(hydroxymethyl)aminomethane; Trp, L-tryptophan;
TSO, tryptophan side-chain oxidase
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(51) Bentley, J. A.; Farrar, K. R.; Housley, S.; Smith, G. F.; Taylor, W. C. Some chemical and physiological properties of 3-indolylpyruvic acid. Biochemical Journal 1956, 64 (1), 44-49. (52) Brandl, M. T.; Lindow, S. E. Contribution of indole-3-acetic acid production to the epiphytic fitness of Erwinia herbicola. Appl Environ Microb 1998, 64 (9), 3256-3263. (53) Dobbelaere, S.; Croonenborghs, A.; Thys, A.; Vande Broek, A.; Vanderleyden, J. Phytostimulatory effect of Azospirillum brasilense wild type and mutant strains altered in IAA production on wheat. Plant Soil. 1999, 212, 155-164. (54) Manulis, S.; Haviv-Chesner, A.; Brandl, M. T.; Lindow, S. E.; Barash, I. Differential involvement of indole-3-acetic acid biosynthetic pathways in pathogenicity and epiphytic fitness of Erwinia herbicola pv, gypsophilae. Mol Plant Microbe In 1998, 11 (7), 634-642. (55) Perrig, D.; Boiero, M. L.; Masciarelli, O. A.; Penna, C.; Ruiz, O. A.; Cassan, F. D.; Luna, M. V. Plant-growth-promoting compounds produced by two agronomically important strains of Azospirillum brasilense, and implications for inoculant formulation. Appl Microbiol Biotechnol 2007, 75 (5), 1143-1150. (56) Rayle, D. L.; Purves, W. K. Isolation and Identification of Indole-3-Ethanol (Tryptophol) from Cucumber Seedlings. Plant Physiology 1967, 42 (4), 520-524. (57) Percival, F. W.; Purves, W. K.; Vickery, L. E. Indole-3-ethanol Oxidase. Kinetics, Inhibition, and Regulation by Auxins 1973, 51 (4), 739-743.
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