Time Course Exo-Metabolomic Profiling in the Green Marine Macroalga Ulva (Chlorophyta) for Identification of Growth Phase-Dependent Biomarkers

Abstract

The marine green macroalga Ulva (Chlorophyta) lives in a mutualistic symbiosis with bacteria that influence growth, development, and morphogenesis. We surveyed changes in Ulva’s chemosphere, which was defined as a space where organisms interact with each other via compounds, such as infochemicals, nutrients, morphogens, and defense compounds. Thereby, Ulva mutabilis cooperates with bacteria, in particular, Roseovarius sp. strain MS2 and Maribacter sp. strain MS6 (formerly identified as Roseobacter sp. strain MS2 and Cytophaga sp. strain MS6). Without this accompanying microbial flora, U. mutabilis forms only callus-like colonies. However, upon addition of the two bacteria species, in effect forming a tripartite community, morphogenesis can be completely restored. Under this strictly standardized condition, bioactive and eco-physiologically-relevant marine natural products can be discovered. Solid phase extracted waterborne metabolites were analyzed using a metabolomics platform, facilitating gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analysis, combined with the necessary acquisition of biological metadata. Multivariate statistics of the GC-MS and LC-MS data revealed strong differences between Ulva’s growth phases, as well as between the axenic Ulva cultures and the tripartite community. Waterborne biomarkers, including glycerol, were identified as potential indicators for algal carbon source and bacterial-algal interactions. Furthermore, it was demonstrated that U. mutabilis releases glycerol that can be utilized for growth by Roseovarius sp. MS2.

Macroalgal–bacterial interactions: Role of dimethylsulfoniopropionate in microbial gardening by Ulva (Chlorophyta)

“…An exo ‐metabolomic analysis of axenic U. mutabilis cultures has already revealed that glycerol functions as a carbon source for Roseovarius sp. (Alsufyani et al., ). In fact, the addition of glycerol to UCM triggered the growth of Roseovarius sp.…”

“…Additionally, Roseovarius sp. MS2 was cultivated in UCM enriched with 1% glycerol (v/v) (Alsufyani et al., ).…”

“…For the determination of DMSP in U. mutabilis slender, 4‐ to 5‐week‐old fertile algae (incubated at 4°C or 18°C) cultivated under standardized conditions (Alsufyani et al., ) were chosen. After carefully washing the algae with UCM and drying them with filter paper, the algae were ground under liquid nitrogen using a mortar and pestle, and 200 μl of methanol was added to extract the various osmolytes.…”

“…Solid phase extractions were employed according the protocol of Alsufyani et al. (). Upon conditioning the matrix with 6 ml of methanol and subsequently with 6 ml of distilled water, 100 ml of supernatant was passed through the EASY ® cartridges (flow rate = 1 L/h).…”

Exploring bacteria-induced growth and morphogenesis in the green macroalga order Ulvales (Chlorophyta)

“…The chemosphere as a part of the biocoenose where the organisms interact with each other via infochemicals (Wichard et al, 2011; Alsufyani, 2014) can be investigated within the tripartite community of U. mutabilis in its most simplistic variation (Figure 5). Within this chemosphere, algal-associated bacteria produce signal molecules, toxins, allelopathic compounds, and morphogens, and provide nutrients and quorum-sensing (QS) signals interfering with the settlement of spores (Dobretsov and Qian, 2002; Croft et al, 2005; Weinberger et al, 2007; Goecke et al, 2010).…”