Thermodynamic Characterization of a Thermostable Antibiotic Resistance Enzyme, the Aminoglycoside Nucleotidyltransferase (4′)

The aminoglycoside nucleotidyltransferase (4′) (ANT) is an aminoglycoside-modifying enzyme that detoxifies antibiotics by nucleotidylating at the C4′-OH site. Previous crystallographic studies show that the enzyme is a homodimer and each subunit binds one kanamycin and one Mg-AMPCPP, where the transfer of the nucleotidyl group occurs between the substrates bound to different subunits. In this work, sedimentation velocity analysis of ANT by analytical ultracentrifugation showed the enzyme exists as a mixture of a monomer and a dimer in solution and that dimer formation is driven by hydrophobic interactions between the subunits. The binding of aminoglycosides shifts the equilibrium toward dimer formation, while the binding of the cosubstrate, Mg-ATP, has no effect on the monomer–dimer equilibrium. Surprisingly, binding of several divalent cations, including Mg²⁺, Mn²⁺, and Ca²⁺, to the enzyme also shifted the equilibrium in favor of dimer formation. Binding studies, performed by electron paramagnetic resonance spectroscopy, showed that divalent cations bind to the aminoglycoside binding site in the absence of substrates with a stoichiometry of 2:1. Energetic aspects of binding of all aminoglycosides to ANT were determined by isothermal titration calorimetry to be enthalpically favored and entropically disfavored with an overall favorable Gibbs energy. Aminoglycosides in the neomycin class each bind to the enzyme with significantly different enthalpic and entropic contributions, while those of the kanamycin class bind with similar thermodynamic parameters

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, 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 <i>Pantoea</i> 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 wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type 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 nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, 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 <i>Pantoea</i> 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 wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type 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 nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, 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 <i>Pantoea</i> 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 wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type 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 nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association

Microwell array fabrication and design.

<p>(A) Microwell fabrication process: (i,ii) Positive photoresist is patterned over parylene-coated silicon wafers using conventional photolithography. (iii) Dry etching is then used to etch parylene and then silicon to the desired well depth. (iv) The well surface is then modified with a protein layer then (v) a solution of bacterial cells. (vi) Parylene is removed from the substrate and (vii) the substrate is contacted with agar-coated coverslips loaded with the desired chemical media. (B) Dry lift-off procedure involving peel-off of the parylene mask (step vi). (C) Layout of a combinatorial microwell array substrate.</p

The distribution of bacteria seeded in microwell arrays is guided by well diameter.

<p>(A) Mosaic 10X false-color fluorescent image of a combinatorial microwell array after seeding <i>E</i>. <i>coli</i>-GFP at OD₆₀₀ = 0.3 and dry lift-off to remove background cells. The false color scale denotes fluorescent signal intensities indicative of cell densities. (B) Averaged well fluorescence intensities ± standard deviation measured from individual wells within each array (black line) and <i>CV</i>_array (red dashed line), the standard deviation divided by the average fluorescent signal for each well diameter.</p

Multi-member bacterial communities can be assembled at low or high dispersion.

<p>(A) Low dispersion pairing: Seeding a 1:9 mixture of <i>E</i>. <i>coli</i>-mCherry (red) and <i>E</i>. <i>coli</i>-GFP (green) at an overall OD₆₀₀ of 0.4 into 40 μm diameter microwell arrays. (B) High dispersion pairing: Seeding a 1:1 mixture of <i>E</i>. <i>coli</i>-mCherry and <i>E</i>. <i>coli</i>-GFP into 2 μm diameter arrays at an overall OD₆₀₀ of 1.0. (C) Scatter plot of GFP and mCherry signals after low or high dispersion pairing.</p

<i>P</i>. <i>aeruginosa</i> growth trajectories in 5 and 20 μm diameter microwell arrays.

<p>(A) Top: False-color fluorescent images of growth in 20 μm diameter arrays. Bottom: Corresponding growth trajectories. The dashed red trajectory indicates growth in an outlier well. (B) Top: False-color fluorescent images of growth in 5 μm diameter arrays. Solid black trajectories denote wells where growth and colonization occurred, dashed red trajectories denote wells where decay and extinction occurred. Data is representative of 4 independent growth experiments.</p

Growth of <i>P</i>. <i>aeruginosa</i> in confined volumes depends on inoculum levels.

<p>(A) Scatter plots of initial and final (t = 24 hrs) cell volume fraction in 10 μm diameter wells and (B) 5 μm diameter wells. Growth-decay line deciphers wells that increased or decreased in cell numbers over the incubation period. (C) Probability of well colonization with initial volume fraction of seeded cells for in 10 μm diameter and (D) 5 μm diameter wells. Data was taken from n = 256 wells for 10 μm diameter arrays and n = 840 wells for 5 μm diameter arrays from 4 independent growth experiments.</p

Bacterial well populations follow a Poisson distribution.

<p>(A) 20X false color fluorescent images of 5 μm diameter wells seeded with <i>E</i>.<i>coli</i>-GFP at OD₆₀₀ = 0.01, 0.1, and 1.0. (B) Probability distributions for cell populations at the varied seeding concentrations. Diamonds represent data and solid lines represent a Poisson distribution fit to the data according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155080#pone.0155080.e002" target="_blank">eq 2</a>. Seeding at an OD₆₀₀ of 0.01, 0.1, and 1.0 resulted in a <b><i>λ</i></b> value of 1.9, 6.2, and 68.6, respectively, and an <b><i>A</i></b> value of 1.65, 1.35, and 1.00, respectively.</p