Bacterial-Fungal Interactions in the Kelp Endomicrobiota Drive Autoinducer-2 Quorum Sensing

Abstract

Brown macroalgae are an essential component of temperate coastal ecosystems and a growing economic sector. They harbor diverse microbial communities that regulate algal development and health. This algal holobiont is dynamic and achieves equilibrium via a complex network of microbial and host interactions. We now report that bacterial and fungal endophytes associated with four brown algae (Ascophyllum nodosum, Pelvetia canaliculata, Laminaria digitata, and Saccharina latissima) produce metabolites that interfere with bacterial autoinducer-2 quorum sensing, a signaling system implicated in virulence and host colonization. Additionally, we performed co-culture experiments combined to a metabolomic approach and demonstrated that microbial interactions influence production of metabolites, including metabolites involved in quorum sensing. Collectively, the data highlight autoinducer-2 quorum sensing as a key metabolite in the complex network of interactions within the algal holobiont.

Keywords: AI-2; algal holobiont; bacterial–fungal interaction; kelp microbiota; quorum sensing (QS).

FIGURE 1

Phylogenetic tree of unique 16S rRNA sequences from bacteria isolated from four algal species. The tree was constructed by maximum likelihood using the K2, G+I model. The reliability of each node was assessed by bootstrapping over 500 replicates.

FIGURE 2

Abundance of cultivable bacterial genus (left) and phyla (right) by algal host and sampling site. ANR, Ascophylum nodosum from Roscoff; ANO, A. nodosum from Oban; PCO, Pelvetia canaliculata from Oban; LDR, Laminaria digitata from Roscoff; LDO, L. digitata from Oban; SLO, Saccharina latissima from Oban.

FIGURE 3

Induction of luminescence in the biosensor Vibrio campbellii MM32 by (gray bars), and DPD concentration in three bacterial supernatants (hatched bars). Luminescence induction was quantified as described in Section “Materials and Methods.” Error bars indicate standard deviation.

FIGURE 4

Induction of luminescence in the biosensor V. campbellii MM32 by fungal extracts. Error bars indicate standard deviation.

FIGURE 5

Impact of fungal extracts on quorum sensing in and viability of the biosensor V. campbellii MM32, as measured, respectively, by inhibition of luminescence in the presence of 2 μM DPD and by resazurin test. Assays are described in Section “Materials and Methods.” Error bars indicate standard deviation.

FIGURE 6

Box plot representing the variation of the luminescence and viability (fluorescence) of the biosensor V. campbellii MM32 by the bacterial or the fungal monoculture and the co-culture. Cla, Cladosporium monocultures; Cla_Co, Cladosporium-Cobetia co-cultures; Co, Cobetia monocultures; Cla_Ps, Cladosporium-Pseudoalteromonas co-cultures; Ps, Pseudoalteromonas monocultures. Error bars represent standard deviation for three replicates. Different letters indicate statistically significant differences between groups [mean ± SEM, N = 3, Van Der Waerden test followed by a post hoc test using the Fischer’s least significant difference (LSD), p < 0.05].

FIGURE 7

Visualization of samples using the first four latent variables from partial least squares discriminant analysis of 720 selected features. Ps, Pseudoalteromonas monocultures (violet); Co, Cobetia monocultures (green); Cla, Cladosporium monocultures (blue); Cla-Ps, Cladosporium-Pseudoalteromonas co-cultures (gray); Cla-Co, Cladosporium-Cobetia co-cultures (orange). Ellipses represent 95% confidence intervals.

FIGURE 8

PLS-based regression between the luminescence measurement (QS activity) and the metabolomic profiles obtained in the different culture or co-culture conditions.

FIGURE 9

Heatmap displaying both the culture clusters in rows and the clusters of variables (m/z) in columns. Ps, Pseudoalteromonas; Co, Cobetia monocultures; Cla, Cladosporium monocultures; Cla-Ps, Cladosporium-Pseudoalteromonas co-cultures; Cla-Co, Cladosporium-Cobetia co-cultures. For convenience, not all variables are labeled. In the black banner are given variables marked in yellow and pink vertical lines for Cla-Co and Cla-Ps co-cultures, respectively, which were significantly filtered according to (i) a VIP value above 1.20 (and with respective SE values lower than VIP), and (ii) an absolute value of the correlation between the variables selected at the previous step and the PLS component which is above 0.75.

FIGURE 10

10 variables displaying a correlation with the predicted luminescence response above 0.75 and a higher mean value in co-culture conditions compared to other mono-culture conditions or the alternate co-culture condition. p-Values coming from ANOVA are stringently corrected for the false discovery rate when considering the initial set of 4221 variables. Multiple comparisons of means used the Student-Newman-Keuls test.