Bacterial alkylquinolone signaling contributes to structuring microbial communities in the ocean

Abstract

Background: Marine bacteria form complex relationships with eukaryotic hosts, from obligate symbioses to pathogenic interactions. These interactions can be tightly regulated by bioactive molecules, creating a complex system of chemical interactions through which these species chemically communicate thereby directly altering the host's physiology and community composition. Quorum sensing (QS) signals were first described in a marine bacterium four decades ago, and since then, we have come to discover that QS mediates processes within the marine carbon cycle, affects the health of coral reef ecosystems, and shapes microbial diversity and bacteria-eukaryotic host relationships. Yet, only recently have alkylquinolone signals been recognized for their role in cell-to-cell communication and the orchestration of virulence in biomedically relevant pathogens. The alkylquinolone, 2-heptyl-4-quinolone (HHQ), was recently found to arrest cell growth without inducing cell mortality in selected phytoplankton species at nanomolar concentrations, suggesting QS molecules like HHQ can influence algal physiology, playing pivotal roles in structuring larger ecological frameworks.

Results: To understand how natural communities of phytoplankton and bacteria respond to HHQ, field-based incubation experiments with ecologically relevant concentrations of HHQ were conducted over the course of a stimulated phytoplankton bloom. Bulk flow cytometry measurements indicated that, in general, exposure to HHQ caused nanoplankton and prokaryotic cell abundances to decrease. Amplicon sequencing revealed HHQ exposure altered the composition of particle-associated and free-living microbiota, favoring the relative expansion of both gamma- and alpha-proteobacteria, and a concurrent decrease in Bacteroidetes. Specifically, Pseudoalteromonas spp., known to produce HHQ, increased in relative abundance following HHQ exposure. A search of representative bacterial genomes from genera that increased in relative abundance when exposed to HHQ revealed that they all have the genetic potential to bind HHQ.

Conclusions: This work demonstrates HHQ has the capacity to influence microbial community organization, suggesting alkylquinolones have functions beyond bacterial communication and are pivotal in driving microbial community structure and phytoplankton growth. Knowledge of how bacterial signals alter marine communities will serve to deepen our understanding of the impact these chemical interactions have on a global scale.

Keywords: 2-heptyl-4-quinolone; Microbiome; Phytoplankton; Pseudoalteromonas; Quorum sensing.

Fig. 1

Mesocosm chlorophyll a and microbial cell concentrations. Chlorophyll a samples were taken from nutrient replete (green circles and black arrows) and unamended control mesocosms (black circles). Samples for total bacteria/archaea (blue triangles) and total phytoplankton < 15 μm (orange triangles) cell abundances were taken from replete mesocosms. DNA sampling and 2-heptyl-4-quinolone manipulations occurred at eight experimental time points (labeled 1 through 8) during sampling (red circles). All samples were taken from a depth of 1 m and symbols represent the mean (± s.d.) of biological triplicates.

Fig. 2

Nonmetric multidimensional scaling ordination displaying eukaryotic communities over the course of the bloom. Ordination of eukaryotic phytoplankton communities based on 18S rRNA (a) and 16S chloroplast (b) amplicon sequence variants visualized by non-metric multidimensional scaling (NMDS) of Bray-Curtis distance. Triangle color indicates experimental time points. Relative abundances of eukaryotic phytoplankton divisions (≥ 1%) determined by 18S rRNA (c) and 16S chloroplast (d) amplicon sequence variants. X-axes indicate experimental time points.

Fig. 3

Nonmetric multidimensional scaling ordination displaying microbial communities over the course of the bloom. Ordination of heterotrophic prokaryote communities based on 16S rRNA amplicon sequence variants (a) visualized by non-metric multidimensional scaling (NMDS) of Bray-Curtis distance. Triangles and circles correspond to particle-associated (> 1 μm size) and free-living communities, respectively. Symbol color denotes experimental time point. Relative abundances of heterotrophic prokaryote orders (≥ 1%) determined by 16S rRNA amplicon sequence variants for particle-associated (> 1 μm size) (b) and free-living (c) communities. X-axes indicate experimental time points.

Fig. 4

Response of phytoplankton communities to HHQ exposure. Difference in growth rate (d⁻¹) in phytoplankton abundance after 24 h exposure to 410 μM (100 ng mL⁻¹) 2-heptyl-4-quinolone (HHQ) compared to the DMSO control determined by chlorophyll a concentration, or flow cytometry counts of nano- and pico-eukaryotes (a) over eight experimental time points. Bars represent the mean (± s.d.) of biological triplicates and asterisks indicate significant changes in phytoplankton growth rate between the two treatments (p < 0.05). Ordination of eukaryotic phytoplankton communities based on 18S rRNA (b) and 16S chloroplast (c) amplicon sequence variants visualized by non-metric multidimensional scaling (NMDS) of Bray-Curtis distance. Circles and triangles correspond to DMSO control and HHQ exposed communities, respectively. Symbol color denotes experimental time point.

Fig. 5

Response of microbial communities to HHQ exposure. Difference in growth rate (d⁻¹) in heterotrophic prokaryote abundance after 24 h exposure to 410 μM (100 ng mL⁻¹) 2-heptyl-4-quinolone (HHQ) compared to the DMSO control determined by flow cytometry over eight experimental time points (a). Bars represent the mean (± s. d.) of biological triplicate and asterisks indicate significant changes in the growth rate of the bulk communities between the two treatments (p < 0.05). Ordination of heterotrophic prokaryote communities based on particle-associated 16S rRNA (b) and free-living 16S rRNA (c) amplicon sequence variants visualized by non-metric multidimensional scaling (NMDS) of Bray-Curtis distance. Circles and triangles correspond to DMSO control and HHQ exposed communities, respectively. Symbol color denotes experimental time point.

Fig. 6

Heatmap showing average relative abundances of microbial communities after HHQ exposure. Relative abundance of heterotrophic prokaryotes in particle-associated (> 1 μm) (a) and free-living (b) communities for orders representing ≥ 1% of the community in at least one sample. Experimental time points and bloom phase is noted below each heatmap. Each column represents the mean of triplicate samples taken from replete mesocosms (T₀) or exposed for 24 h to DMSO or 2-hepyl-4-quinolone (HHQ). Bolded taxa contain amplicon sequence variants that changed (+/−) significantly (BH-adjusted p value < 0.1) in relative abundance after HHQ exposure compared to the DMSO control.

Fig. 7

Amplicon sequence variants showing significant changes in their relative abundance following HHQ exposure. Phylogenetic relationship of amplicon sequence variants (ASVs) that significantly (BH-adjusted p value < 0.1) increased (magenta) or decreased (gray) in relative abundance after 24 h exposure to 2-heptyl-4-quinolone (HHQ). One ASV showing both an increase and decrease at different time points is labeled in green. Sequences were aligned using MAFFT and maximum-likelihood phylogenetic inference was done using RAxML.

Fig. 8

A representative Pseudoalteromonas ASV showing increased relative abundance following HHQ exposure. Normalized counts of Pseudoalteromonas ASV_148 from replete mesocosms at time zero (T₀; black circles), and after 24 h exposure to either a solvent control (DMSO; gray circles) or 2-heptyl-4-quinolone (HHQ; magenta circles). Asterisks indicate significant differences in the relative abundance after HHQ exposure compared to the DMSO control (BH-adjusted p value < 0.1). X-axes indicate experimental time points.