The Phytohormone Ethylene Enhances Cellulose Production, Regulates CRP/FNRKx Transcription and Causes Differential Gene Expression within the Bacterial Cellulose Synthesis Operon of Komagataeibacter (Gluconacetobacter) xylinus ATCC 53582

Abstract

Komagataeibacter (formerly Gluconacetobacter) xylinus ATCC 53582 is a plant-associated model organism for bacterial cellulose (BC) biosynthesis. This bacterium inhabits the carposphere where it interacts with fruit through the bi-directional transfer of phytohormones. The majority of research regarding K. xylinus has been focused on identifying and characterizing structural and regulatory factors that control BC biosynthesis, but its ecophysiology has been generally overlooked. Ethylene is a phytohormone that regulates plant development in a variety of ways, but is most commonly known for its positive role on fruit ripening. In this study, we utilized ethephon (2-chloroethylphosphonic acid) to produce in situ ethylene to investigate the effects of this phytohormone on BC production and the expression of genes known to be involved in K. xylinus BC biosynthesis (bcsA, bcsB, bcsC, bcsD, cmcAx, ccpAx and bglAx). Using pellicle assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR), we demonstrate that ethephon-derived ethylene enhances BC directly in K. xylinus by up-regulating the expression of bcsA and bcsB, and indirectly though the up-regulation of cmcAx, ccpAx, and bglAx. We confirm that IAA directly decreases BC biosynthesis by showing that IAA down-regulates bcsA expression. Similarly, we confirm that ABA indirectly influences BC biosynthesis by showing it does not affect the expression of bcs operon genes. In addition, we are the first to report the ethylene and indole-3-acetic acid (IAA) induced differential expression of genes within the bacterial cellulose synthesis (bcs) operon. Using bioinformatics we have identified a novel phytohormone-regulated CRP/FNRKx transcription factor and provide evidence that it influences BC biosynthesis in K. xylinus. Lastly, utilizing current and previous data, we propose a model for the phytohormone-mediated fruit-bacteria interactions that K. xylinus experiences in nature.

Keywords: CRP/FNR; Komagataeibacter (Gluconacetobacter) xylinus; abscisic acid (ABA); bacterial cellulose; ethylene; fruit-bacteria interaction; indole-3-acetic acid (IAA); plant-microbe interaction.

FIGURE 1

Structural and genetic organization of the bacterial cellulose synthesis complex and the bcs operon. BcsA (green), activated by c-di-GMP, adds a glucose unit to the cellulose chain using UDP-glucose as a substrate in the cytoplasm; BcsB (blue) guides the glucan chain through the periplasm; BcsD (orange) crystallizes four glucan chains in the periplasm; BcsC (gray) exports the BC microfibril into the extracellular space (A). The genetic organization of the bcs operon that encodes the bacterial cellulose synthesis complex and the genes up- and downstream (B). The genes in the bcs operon (B) are color coordinated with their protein products (A).

FIGURE 2

Ethylene is released via ethephon decomposition in SH medium (pH 7). Dark-grown Arabidopsis thaliana seedlings display the triple response phenotype (shorter and thicker hypocotyl with exaggerated apical hook) when grown in the presence of ACC and ethephon-derived ethylene compared to the untreated control (A). The hypocotyl length of seedlings grown in the presence of ACC and ethephon-derived ethylene were significantly shorter than the untreated control (B). Scale bar represents 1 mm. ^∗∗∗∗p < 0.0001. Error bars show SD (n = 3).

FIGURE 3

The pH of K. xylinus cultures stays above 3.5, allowing for efficient decomposition of ethephon into ethylene. K. xylinus was grown in SH medium (pH 7) that was supplemented with ethephon and 0.2% (v/v) cellulase. The change in culture pH was monitored for 14 days. Note that the y-axis begins at pH 5, and that the final pH of all ethephon-treated cultures is higher than the untreated control. Error bars show SD (n = 3).

FIGURE 4

Ethephon-derived ethylene influences the properties and yield of K. xylinus BC pellicles. Cultures were grown statically in SH broth (pH 7) in 24-well plates, and incubated at 30°C for 7 days before pellicles were harvested and analyzed. Ethephon-derived ethylene decreases the wet weight (A), increases the dry weight (B), reduces pellicle hydration (C), and increases the crystallinity (D) of K. xylinus pellicles compared to the untreated control. The different ethephon treatments were not significantly different from each other. Data was normalized to and expressed as percent of the untreated control. Note the y-axis begins at 50%. Error bars show SD (n = 3). ^∗p < 0.05; ^∗∗p < 0.01.

FIGURE 5

Ethephon enhances K. xylinus BC production when grown on solid medium. K. xylinus was streaked onto SH agar plates (pH 7) that were untreated (A), or pre-treated with ultra-pure water (pH 2.5); (B) phosphate and chloride (C-E) or ethephon (F-H). Plates were incubated at 30°C for 7 days. Representative colonies are shown. The arrow points to BC, seen as the hazy substance surrounding the colony. Scale bar represents 0.5 mm.

FIGURE 6

The expression of genes involved in K. xylinus BC biosynthesis are regulated by phytohormones. Ethephon-derived ethylene and IAA induce differential expression of the genes within the bcs operon (A). Ethephon-derived ethylene and ABA influence the expression of genes flanking the K. xylinus bcs operon (B). Ethephon-derived ethylene, IAA and ABA regulate the expression of crp/fnr_Kx (C). Gene expression was quantified using RT-qPCR after treatment with 10 μM ethephon, 10 μM IAA, or 10 μM ABA. Expression values were made relative to the respective untreated controls and normalized using the expression values of reference genes, 23SrRNA and gyrB. The dotted line indicates the relative normalized expression value for the untreated control. Error bars show the SD (n = 3). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 7

Model for the phytohormone-mediated fruit-bacteria interactions of K. xylinus. Unripe fruit (A) contain high concentrations of IAA, zeatin (Z) and GA₃. IAA decreases K. xylinus BC production, while IAA, zeatin, and GA₃ increase bacterial cell growth enhancing endogenous production of ABA, zeatin, and GA₃ by K. xylinus. These hormones increase fruit size and induce ripening, characterized by degradation of polysaccharides and weakening of the fruit cell wall. On ripe fruit (B), plant-produced ethylene up-regulates biosynthesis and crystallization of BC. Exogenous ABA increases K. xylinus growth, allowing it to accelerate the fruit ripening process, which facilitates colonization. Green triangles and inverted red triangles indicate a process is enhanced or repressed, respectively. Only direct effects are shown.