Macroalgal-bacterial interactions: identification and role of thallusin in morphogenesis of the seaweed Ulva (Chlorophyta)

Abstract

Macroalgal microbiomes have core functions related to biofilm formation, growth, and morphogenesis of seaweeds. In particular, the growth and development of the sea lettuce Ulva spp. (Chlorophyta) depend on bacteria releasing morphogenetic compounds. Under axenic conditions, the macroalga Ulva mutabilis develops a callus-like phenotype with cell wall protrusions. However, co-culturing with Roseovarius sp. (MS2) and Maribacter sp. (MS6), which produce various stimulatory chemical mediators, completely recovers morphogenesis. This ecological reconstruction forms a tripartite community which can be further studied for its role in cross-kingdom interactions. Hence, our study sought to identify algal growth- and morphogenesis-promoting factors (AGMPFs) capable of phenocopying the activity of Maribacter spp. We performed bioassay-guided solid-phase extraction in water samples collected from U. mutabilis aquaculture systems. We uncovered novel ecophysiological functions of thallusin, a sesquiterpenoid morphogen, identified for the first time in algal aquaculture. Thallusin, released by Maribacter sp., induced rhizoid and cell wall formation at a concentration of 11 pmol l-1. We demonstrated that gametes acquired the iron complex of thallusin, thereby linking morphogenetic processes with intracellular iron homeostasis. Understanding macroalgae-bacteria interactions permits further elucidation of the evolution of multicellularity and cellular differentiation, and development of new applications in microbiome-mediated aquaculture systems.

Keywords: Algal growth; cell wall; cross-kingdom interaction; morphogenesis; morphogenesis-promoting factor; phytohormone; rhizoid; seaweed; siderophore.

Fig. 1.

Ulva mutabilis as a model organism for bacteria-induced morphogenesis (A) U. mutabilis (‘slender’) and two essential bacteria which release morphogenetic factors establish a tripartite community. (B) The axenic callus-like morphotype of U. mutabilis was compared with axenic cultures (i) inoculated with bacteria of the Roseovarius sp. only (ii), bacteria of the Maribacter sp. only (iii), or both bacterial strains (iv). Arrows indicate the typical colorless protrusions from the exterior cell walls due to lack of morphogens released by Maribacter sp. Scale bar=500 µm (i); 100 µm (ii–iv); image (iii) with permission adapted from Wichard (2015). (C) Growth curve of U. mutabilis in a land-based tank system (error bars: ±SD, n=3). The morphogenetic activity of AGMPFs in sterile-filtered water samples is given as a portion of normally developed cell walls using the ‘Ulva bioassay array’ (inset: algae in stationary phase, scale bar=1 cm). (D) Demonstration of axenicity by PCR amplification of a part of the 16S rDNA gene extracted from the supernatant of the purified gamete stock solution (lane 1) and the non-axenic culture of U. mutabilis (lane 2). Lane 3 shows the GeneRuler DNA Express Ladder (Thermo Fisher Scientific) (scale bar=100 µm). (E, F) The AGMPFs in sterile-filtered water samples derived from aquacultures on day 48 were determined using two different bioassays. Roseovarius-released factor(s) were distinguished (E) from Maribacter-released factor(s) (F) by performing a dilution series. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test (P<0.01, error bars: SE, n=60). Mean values accompanied by differing letters differed significantly. Axenic cultures (white) were inoculated with sterile-filtered water (gray) or Roseovarius sp. (purple), Maribacter sp. (orange), or both (green).

Fig. 2.

Identification of thallusin in the chemosphere of Ulva mutabilis (A) Algal morphogenesis-inducing fractions were analyzed by UHPLC-ESI-HRMS and compared with the reference standard of thallusin (red: extracted ion chromatograms of 458.2168 m/z). Samples were obtained from the supernatant of algal aquacultures and the bacterial growth medium of Maribacter sp. by solid-phase extraction. (B) MS/MS experiments of 458.2168 m/z indicate the substance identity of red-labeled peaks to coincide with that of (C) (–)-thallusin. Fragment 254.0301 m/z belongs to 2,6-dicarboxy-3-(2-carboxy-2-hydroxyvinyl)pyridine-1-ium. (D) The Fe complex of thallusin (966.3442 m/z) was identified in the extracted ion chromatogram (purple) using metal isotope-coded profiling. The artificial isotopic signature (⁵⁴Fe/⁵⁸Fe) of the complex is shown (inset, left). U. mutabilis gametes easily acquired Fe–thallusin compared with iron hydroxides and Fe-EDTA during the short-term uptake experiments (inset, right, error bars: SD).

Fig. 3.

Thallusin complements the activity of Roseovarius sp. Axenic gametes of Ulva mutabilis were inoculated with the bacteria, Roseovarius sp., alone (A, B), or with thallusin (1.1×10⁻¹⁰ mol l⁻¹; C, D). Arrows indicate the colorless protrusions from the exterior cell walls in the absence of thallusin. While no apical or basal end was recognized in the presence of Roseovarius sp. only, both the apical end (C) and the rhizoid (D) were clearly developed in the presence of thallusin. Axenic gametes developed into a callus [E, negative control; A–E, scale bar=500 µm; 200 µm (inset)]. In the presence of Roseovarius sp. and thallusin, U. mutabilis developed into the adult algae (scale bar=1 cm) (F).

Fig. 4.

Chemical mediators in cross-kingdom interactions of Ulva mutabilis and associated bacteria. Ulva mutabilis provide a glycerol boundary layer as a carbon source for Roseovarius sp., thus supporting biofilm formation (Alsufyani et al., 2017). U. mutabilis releases DMSP to attract Roseovarius sp. (Kessler et al., 2018), which then promote cell division and growth through the secretion of unknown and partially purified morphogens [Roseovarius-released factor(s), red circle] (Spoerner et al., 2012). N-Acyl homoserine lactone (AHL)-producing bacteria of the Roseobacter clade can also attract zoids of U. mutabilis through quorum-sensing signals (Joint et al., 2002). AHLs differ in the length of their R-group side chain. Following completion of the first interactions, thallusin from Maribacter sp. promotes rhizoid and cell wall formation, thus finalizing the formation of the tripartite community. Top image: germling with bacteria (scale bar=10 µm). Bottom image: both morphotypes ‘slender (sl)’ and ‘wild type (wt)’ are shown (scale bar=1 cm).