Microalgae and Bacteria Interaction-Evidence for Division of Diligence in the Alga Microbiota

Abstract

Microalgae are one of the most dominant forms of life on earth that is tightly associated with a distinct and specialized microbiota. We have previously shown that the microbiota of Scenedesmus quadricauda harbors less than 10 distinct microbial species. Here, we provide evidence that dominant species are affiliated with the genera of Variovorax, Porphyrobacter, and Dyadobacter. Experimental and transcriptome-based evidence implies that within this multispecies interaction, Dyadobacter is a key to alga growth and fitness and is highly adapted to live in the phycosphere. While presumably under light conditions the alga provides the energy source to the bacteria, Dyadobacter produces and releases mainly a large variety of polysaccharides modifying enzymes. This is coherent with high-level expression of the T9SS in alga cocultures. The transcriptome data further imply that quorum-quenching proteins (QQ) and biosynthesis of vitamins B₁, B₂, B₅, B₆, and B₉ are expressed by Dyadobacter at high levels in comparison to Variovorax and Porphyrobacter. Notably, Dyadobacter produces a significant number of leucine-rich repeat (LRR) proteins and enzymes involved in bacterial reactive oxygen species (ROS) tolerance. Complementary to this, Variovorax expresses the genes of the biosynthesis of vitamins B₂, B₅, B₆, B₇, B₉, and B₁₂, and Porphyrobacter is specialized in the production of vitamins B₂ and B₆. Thus, the shared currency between partners are vitamins, microalgae growth-promoting substances, and dissolved carbon. This work significantly enlarges our knowledge on alga-bacteria interaction and demonstrates physiological investigations of microalgae and associated bacteria, using microscopy observations, photosynthetic activity measurements, and flow cytometry. IMPORTANCE The current study gives a detailed insight into mutualistic collaboration of microalgae and bacteria, including the involvement of competitive interplay between bacteria. We provide experimental evidence that Gram-negative bacteria belonging to the Dyadobacter, Porphyrobacter, and Variovorax are the key players in a Scenedesmus quadricauda alga-bacteria interaction. We impart strong evidence that Dyadobacter produces and releases polysaccharides degradation enzymes and leucine-rich repeat proteins; Variovorax supplies the consortium with auxins and vitamin B₁₂, while Porphyrobacter produces a broad spectrum of B vitamins. We show not only that the microalgae collaborate with the bacteria and vice versa but also that the bacteria interact with each other via quorum-sensing and secretion system mechanisms. The shared currency between partners appears to be vitamins, microalgae growth-promoting substances, and dissolved carbon.

Keywords: Dyadobacter sp.; Scenedesmus quadricauda; microalgae and microbiota interaction; phycosphere biofilm; synthetic plant-bacteria system.

FIG 1

Confocal microscope images of the strain Dyadobacter sp. HH091 expressing enhanced green fluorescent protein (eGFP) (yellow arrows) in coculture with S. quadricauda MZCH 10104. A confocal laser scanning microscope (CLSM) Axio Observer.Z1/7 LSM 800 (Carl Zeiss Microscopy GmbH, Jena, Germany) with ZEN software (version 2.3; Carl Zeiss Microscopy GmbH) was used. (A) Three-day culture. (B) seven-day culture; (C) seven-day culture, Z-Steck image. An autofluorescence quenching kit was used to lower the autofluorescence of chlorophyll of the microalga. c = chloroplast; p = pyrenoid. Bar = 5 μm.

FIG 2

(A, B) Photosynthetic activity and growth measurement (A) and fluorescence-activated cell sorting (FACS) analyses (B) of S. quadricauda MZCH 10104 in coculture with the strain Dyadobacter sp. HH091. (A) Increased photosynthetic activity and growth rate (optical density at 750 nm [OD 750 nm]) can be observed in the coculture with HH091. (B) Comparison of populations, based on chlorophyll intensity. Orange shading indicates a stable distribution of bacteria (population I), dead and lysed algae (population II), and healthy microalgae (population III) in the coculture at the end of the experiment (13 days). Blue shading shows how the proportion of dead and lysed algae is increased in the culture without HH091. The raw data are available in Fig. S2. (C) Images of cell sorting acquired with CLSM Axio Observer.Z1/7 LSM 800 (Carl Zeiss Microscopy GmbH, Jena, Germany) and ZEN software (version 2.3; Carl Zeiss Microscopy GmbH). Bar = 5 μm. Pulse-amplitude-modulation (PAM) fluorometry and FACS analyses demonstrated the improved fitness of S. quadricauda cocultured with HH091.

FIG 3

Expressed genes of bacteria in coculture with the microalga S. quadricauda during the exponential (A) and stationary (B) growth phases. The generated sequence data (approximal 42 mio reads) were mapped to the available genomes and metagenomes (8). The most dominant species of the microbiome are affiliated with the phyla of the Proteobacteriota (Variovorax and Porphyrobacter) and Bacteroidota (Dyadobacter).

FIG 4

Heat map reflecting the relative expression of genes affiliated with overall plant-bacteria interaction pathways of the bacterial metagenome of S. quadricauda during the exponential (A) and stationary (B) growth phases. Red shading indicates a high expression level, and white indicates a low expression level. The numbers indicate the total amount of hits during RNA-Seq. Genes have more than 500 counts/gene.

FIG 5

Metatranscriptome mapping to the reference genomes of Porphyrobacter sp. AAP82 (IMG 2551306481) (A), Variovorax paradoxus S110 (IMG 644736413) (B), and Dyadobacter sp. HH091 (IMG 222279) (C). Moving inward, the subsequent two rings show coding DNA sequences (CDSs) in forward (brown) and reverse (green) strands. Blue and yellow plots indicate GC content and a GC skew ([GC]/[G+C]]. Key features of possible competitive and plant-bacteria interaction pathways are marked with the following colors: purple, transporter, efflux pumps and secretion systems; orange, signal transduction and regulation mechanisms; green, polysaccharide degradation; red, competitive interactions; blue, bacteria-plant interaction pathways.