Interplay between the microalgae Micrasterias radians and its symbiont Dyadobacter sp. HH091

Abstract

Based on previous research, related to detailed insight into mutualistic collaboration of microalga and its microbiome, we established an artificial plant-bacteria system of the microalga Micrasterias radians MZCH 672 and the bacterial isolate Dyadobacter sp. HH091. The bacteria, affiliated with the phylum Bacteroidota, strongly stimulated growth of the microalga when it was added to axenic algal cultures. For further advances, we studied the isolate HH091 and its interaction with the microalga M. radians using transcriptome and extensive genome analyses. The genome of HH091 contains predicted polysaccharide utilizing gene clusters co-working with the type IX secretion system (T9SS) and conceivably involved in the algae-bacteria liaison. Here, we focus on characterizing the mechanism of T9SS, implementing the attachment and invasion of microalga by Dyadobacter sp. HH091. Omics analysis exposed T9SS genes: gldK, gldL, gldM, gldN, sprA, sprE, sprF, sprT, porU and porV. Besides, gld genes not considered as the T9SS components but required for gliding motility and protein secretion (gldA, gldB, gldD, gldF, gldG, gldH, gldI, gldJ), were also identified at this analysis. A first model of T9SS apparatus of Dyadobacter was proposed in a course of this research. Using the combination of fluorescence labeling of Dyadobacter sp. HH091, we examined the bacterial colonisation and penetration into the cell wall of the algal host M. radians MZCH 672.

Keywords: Dyadobacter sp. HH091; Micrasterias radians; microalgaebacteria interaction; symbiotic relations; synthetic early plant-bacteria system.

Figure 1

Confocal microscope including Z-Stack images of Dyadobacter sp. HH091 expressing eGFP (yellow arrows) found in a close proximity to Micrasterias radians MZCH 672. Autofluorescence Quenching Kit was used to lower the autofluorescence of chlorophyll of the microalga. Structures: c chloroplast, n nuclear region, p pyrenoid. Scale bar = 2 μm in each micrograph. (A) First day of incubation. (B) Third day of incubation.

Figure 2

Heatmap of expression levels of differentially expressed genes (DEGs) response of Dyadobacter sp. HH091 in co-culture with Micrasterias radians. RNA-Seq analysis was performed using the Tuxedo strategy, the heatmap was generated using the Expression Import Service of the Pathosystems Resource Integration Center, PATRIC, the absolute value of log2 Ratio > 1.5. Color key: up-regulated genes, down-regulated genes.

Figure 3

DEGs in Dyadobacter sp. HH091 co-cultured with M. radians MZCH 672 compared with control dataset. (A) Volcano plot is highlighting the DEGs in Dyadobacter sp. x-axis: log2, large-scale fold changes; y-axis: –log10 of the value of p showing the statistical significance. Each point corresponds to one gene. The points above the vertical and horizontal dotted lines represent log2FC ≥ 0.58 and value of p < 0.05. A volcano plot was generated using A Shiny app ggVolcanoR. (B) Function profile of differentially expressed genes (DEGs) in Dyadobacter sp. HH091 is presenting the groups of highly active genes. Total number of genes are shown in brackets. Color key: up-regulated genes, down-regulated genes.

Figure 4

Proposed model of flexirubin or dialkylresorcinol (DARs) biosynthesis in Dyadobacter sp. HH091. In the proposed model, Dyadobacter sp. HH091 communicates via DARs and represents a novel quorum sensing (QS) circuit (Brameyer et al., 2015). It consists of the dar operon and a neighboring gene encoding a luxR solo (narL/fixJ). NarL/FixJ shares 46% identity and 47% similarity with the LuxR solo PluR of P. luminescens (IMG 2597490348), LuxR-type receptor serving for QS. The proposed QS circuit genes, adjacent to T9SS genes, genes affiliated with gliding motility and protein secretion, possibly upregulates several mechanisms, including T9SS, gliding motility and protein secretion. (A) Expression levels of DEGs involved into flexirubin biosynthesis: dar and flx clusters, and transport systems (ATP binding cassettes (ABC) and hypothetical proteins). Color key: up-regulated genes, down-regulated genes. (B) Expression levels of differentially expressed genes (DEGs) involved into flexirubin biosynthesis: dar and flx clusters, and transport systems (ATP binding cassettes (ABC)-transporters and hypothetical proteins).

Figure 5

Proposed model of T9SS in Dyadobacter sp. HH091, serving as the secretion system of cargo-proteins. PorXY-SigP signalling system upregulates several components: T9SS category (GldK, GldL, GldM, GldN, SprA, SprE, SprF, SprT, PorU, PorV), and further Gld proteins (GldA, GldB, GldD, GldF, GldG, GldH, GldI, GldJ). C-terminal domain (CTD), N-terminal signal peptide (N-terminal), outer membrane (OM), inner membrane (IM).