10 research outputs found

    Overview of tryptophan-dependent indole-3 acetic acid (IAA) biosynthesis pathway(s) in bacteria.

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    <p>Enzymes with demonstrated biochemical activities are indicated. Enzyme abbreviations: tryptophan 2-monooxygenase (TMO), indole acetamide hydrolase (IAH), tryptophan side chain oxidase (TSO), indole pyruvate decarboxylase (IPDC) and aldehyde dehydrogenase (ALD). Two ALD enzymes, AldA and AldB, that catalyze conversion of IAAld to IAA are described in this study. Compound abbreviations: tryptophan (Trp), indole-3-acetaldoxime (IAOx), indole-3-acetonitrile (IAN), indole-3-acetamide (IAM), indole-3-pyruvate (IPyA), indole-3-acetaldehyde (IAAld) and tryptamine (TAM).</p

    Heterologous expression of putative DC3000 aldehyde dehydrogenases in <i>E</i>. <i>coli</i>.

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    <p>DC3000 genes encoding putative aldehyde dehydrogenase proteins were expressed in <i>E</i>. <i>coli</i> BL21(DE3) cells. A) Quantification of IAA produced by strains expressing PSPTO_0728, PSPTO_2673, and PSPTO_3644 and pET-21a as a negative control. B) Quantification of IAA produced by strains expressing PSPTO_0092, PSPTO_3064, and PSPTO_3323. PSPTO_2673 was included as a control for comparison to panel A. IAA levels were measured in supernatants 24 hrs post-induction with addition of 0.25 mM IAAld. Values are an average ┬▒ SEM (n = 3). Letters indicate significant difference between samples within a given time point (<i>p</i><0.01). Similar results were obtained from two additional independent experiments.</p

    <i>PR1</i> expression in plants inoculated by <i>ald</i> mutants and growth of <i>ald</i> mutants on SA-deficient <i>sid2-2</i> plants.

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    <p>A) <i>PR1</i> expression in Col-0 plants at 24 hrs following syringe infiltration (OD<sub>600</sub> = 1x10<sup>-5</sup>). Significant elevation of <i>PR1</i> expression in <i>aldA</i>-infected plants was observed in three independent experiments, and in two experiments for <i>aldB</i>-infected plants. B) Growth of <i>ald</i> single and double mutants on wild type <i>A</i>. <i>thaliana</i> (Col-0) and <i>sid2-2</i> mutant plants following syringe infiltration. Graph shows composite data from 4 independent experiments. Letters indicate significant difference between samples within a given time point (<i>p</i><0.02).</p

    Overall structure of AldA.

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    <p>A) The AldA tetramer is shown as a ribbon tracing with each subunit differentially colored. Two subunits (gold and white) were in the asymmetric unit of the crystal with the other two subunits (green and rose) related by crystallographic symmetry. N-termini are labeled. B) Domain organization of the AldA monomer. The view is rotated 90┬░ relative to panel A and shows the two subunits in the asymmetric unit. The catalytic (red), cofactor binding (blue), and oligomerization (rose) domains are highlighted in one monomer. The position of NAD<sup>+</sup> (space-filling model) is indicated. C) Substrate binding sites on opposite sides of the AldA monomer. The two views of an AldA monomer are rotated 180┬░ and show the locations of the NAD(H) and IAAld/IAA binding sites on each face of the monomer. D) Ligand binding tunnel. The positions of NAD<sup>+</sup> (rose) and IAA (gold) in the tunnel (grey surface) relative to the catalytic cysteine (Cys302) are shown. The position of docked IAAld (rose), which overlaps with IAA, is indicated.</p

    Quantification of IAA production in DC3000 <i>ald</i> mutants.

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    <p>A) Measurement of IAA accumulation in supernatants of DC3000 <i>ald</i> single mutants grown for 48 hrs in HSC media supplemented with 0.25 mM IAAld. B) Growth of <i>ald</i> mutants in HSC media supplemented with 0.25 mM IAAld. These cultures were used for quantification of IAA shown in panel A. C) Measurement of IAA accumulation in supernatants of <i>aldA</i>, <i>aldB</i> and the <i>aldA aldB</i> double mutant grown for 48 hrs in HSC media supplemented with 0.25 mM IAAld. The <i>aldB</i> mutant in this experiment is the <i>aldB</i>::pJP5603Tet mutant. For panels A-C, values are an average of three biological replicates ┬▒ SEM (error bars too small to see in panel B). Letters indicate significant difference between samples within a given time point (<i>p</i><0.01. c* in panel A indicates that IAA levels in <i>aldB</i> are significantly different from wt at p<0.05).</p

    Growth of <i>ald</i> mutants on <i>A</i>. <i>thaliana</i>.

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    <p>A) Growth of DC3000 and <i>ald</i> mutants following syringe infiltration of <i>A</i>. <i>thaliana</i>. Graph shows composite data from 4 independent experiments. Letters indicate significant difference between samples within a given time point (<i>p</i><0.001). B) Growth of DC3000, <i>aldA</i>, <i>aldB</i> and the <i>aldA aldB</i> double mutant following syringe infiltration of <i>A</i>. <i>thaliana</i>. Letters indicate significant difference between samples within a given time point (<i>p</i><0.05).</p
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