Spatial organization of the kelp microbiome at micron scales

Abstract

Background: Elucidating the spatial structure of host-associated microbial communities is essential for understanding taxon-taxon interactions within the microbiota and between microbiota and host. Macroalgae are colonized by complex microbial communities, suggesting intimate symbioses that likely play key roles in both macroalgal and bacterial biology, yet little is known about the spatial organization of microbes associated with macroalgae. Canopy-forming kelp are ecologically significant, fixing teragrams of carbon per year in coastal kelp forest ecosystems. We characterized the micron-scale spatial organization of bacterial communities on blades of the kelp Nereocystis luetkeana using fluorescence in situ hybridization and spectral imaging with a probe set combining phylum-, class-, and genus-level probes to localize and identify > 90% of the microbial community.

Results: We show that kelp blades host a dense microbial biofilm composed of disparate microbial taxa in close contact with one another. The biofilm is spatially differentiated, with clustered cells of the dominant symbiont Granulosicoccus sp. (Gammaproteobacteria) close to the kelp surface and filamentous Bacteroidetes and Alphaproteobacteria relatively more abundant near the biofilm-seawater interface. A community rich in Bacteroidetes colonized the interior of kelp tissues. Microbial cell density increased markedly along the length of the kelp blade, from sparse microbial colonization of newly produced tissues at the meristematic base of the blade to an abundant microbial biofilm on older tissues at the blade tip. Kelp from a declining population hosted fewer microbial cells compared to kelp from a stable population.

Conclusions: Imaging revealed close association, at micrometer scales, of different microbial taxa with one another and with the host. This spatial organization creates the conditions necessary for metabolic exchange among microbes and between host and microbiota, such as provisioning of organic carbon to the microbiota and impacts of microbial nitrogen metabolisms on host kelp. The biofilm coating the surface of the kelp blade is well-positioned to mediate interactions between the host and surrounding organisms and to modulate the chemistry of the surrounding water column. The high density of microbial cells on kelp blades (10⁵-10⁷ cells/cm²), combined with the immense surface area of kelp forests, indicates that biogeochemical functions of the kelp microbiome may play an important role in coastal ecosystems. Video abstract.

Keywords: Biogeography; CLASI-FISH; Endophytic; Epiphytic; Host-microbe; Nereocystis luetkeana; Polymicrobial interaction; Spatial structure.

Fig. 1

Relative abundance of bacterial taxa on the kelp N. luetkeana. Sequences are grouped by phylum, class and genus-level taxonomy to show taxa detected by our CLASI-FISH probe set. 16S rRNA gene sequencing showed that bacterial composition was broadly consistent throughout the summer. The most abundant taxa were Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, and Verrucomicrobia. The gammaproteobacterial genus Granulosicoccus was highly abundant in samples from Tatoosh Island, while Alphaproteobacteria were dominant at Squaxin Island. Collection date is shown at the top, and collection site is shown at the bottom. Sample numbers and portion of the kelp blade sampled (T = tip; B = base; M = middle) are indicated in the x-axis labels

Fig. 2

Cross-sectional, whole-mount, and oblique optical section images give different views of the biofilm on kelp. N. luetkeana blades were subjected to CLASI-FISH with a probe set for the 5 major bacterial groups. A Cross-sectional image of a kelp blade embedded in methacrylate. Merged transmitted light and confocal images show the microbial biofilm on both sides of the kelp blade, with some microbial cells in the center. (i), (ii), and (iii) are enlarged images of the dashed rectangles in panel (A). Bacteroidetes rods within the autofluorescent region of kelp tissue in panel (i) fluoresce more brightly than rods in the surface biofilm and therefore appear overexposed in the image. B Whole-mount preparation imaged as a z-stack; planes 1 micrometer apart in the z dimension are shown. C Oblique optical section showing the microbial biofilm at left and kelp surface at right. Bacteroidetes rods are visible between kelp surface cells (right)

Fig. 3

Bacterial cell abundance of the surface biofilms of old and young tissue. Whole-mount images of kelp blade tip tissues (old) and base tissues (young) collected from the same kelp frond. Three individuals from different collection dates are shown. Older tissue from the tip of the kelp blade (A, B, C) is densely colonized compared to young tissue from the base of the blade (D, E, F). The same pattern is observed throughout the summer

Fig. 4

Spatial structure of the epiphytic microbial community at the tip of N. luetkeana blades. Bacteria at the tip of kelp blades form a dense biofilm. A and B show whole-mount images of samples collected on different dates; C is a cross section showing the thickness of the surface biofilm relative to the kelp cell surface. Microorganisms are intermixed, always within 10 microns of other taxa, and often directly adjacent to cells of disparate taxa or diatoms (red arrowheads). Granulosicoccus aggregate in clusters while other taxa are more dispersed. Abundant Bacteroidetes filaments appear to be lying across the other taxa in (A) and (B). In the cross section (C), filaments of Bacteroidetes and Gammaproteobacteria project into the water column. D Cross section showing diatoms embedded within the bacterial biofilm

Fig. 5

Variation of biofilm thickness. Cross-sectional images showing the variation of the biofilm thickness within and among samples. A A patch of biofilm up to 32μm thick with adjacent thinner biofilm of 3 to 6 μm. B The most common thickness observed in the kelp biofilm was 3 to 7 μm, with chains and filaments extending further into the water column. C A sparse biofilm including a region with no visible biofilm (red arrowhead)

Fig. 6

Endophytic bacteria of N. luetkeana. A Cross section showing Bacteroidetes rods colonizing intercellular spaces of brightly autofluorescent kelp surface cells. B A region in which the biofilm is directly adjacent to the kelp tissue and some Granulosicoccus are observed between kelp cells. C Bacteria were also detected colonizing deeper areas of the tissue, in this instance around 120 μm from the surface. D Enlarged image of the dashed rectangle in (C)

Fig. 7

Unipolar labeling of adherent Alphaproteobacteria by wheat germ agglutinin. Wheat germ agglutinin was used to stain N-acetylglucosamine and N-acetylmuramic acid residues. Staining was observed on Alphaproteobacteria rods at only one end, showing apparent polarity with respect to the cells. A Hybridization of a kelp sample with probes for Alphaproteobacteria (cyan) and Granulosicoccus (magenta). B Signal from fluorophore-labeled WGA (red) in the same field of view as (A). C Merged image of A and B showing that WGA staining was observed in cells hybridizing with the Granulosicoccus and Alphaproteobacteria probes. WGA staining was detected surrounding the Granulosicoccus cells while the stained Alphaproteobacteria showed fluorescence at only one end of the cell. WGA signal associated with Alphaproteobacteria was brighter and more localized than that associated with Granulosicoccus. D and E Representative images of FISH on whole-mount samples from kelp blades collected in different months from Tatoosh Island. F Cross-sectional image showing Alphaproteobacteria rods with the polar polysaccharide end adjacent to the kelp surface

Fig. 8

Low microbial density on kelp blades from a declining kelp population at Squaxin Island. A and B Whole mount FISH showing sparse bacteria on the mid-blade kelp surface. C Cross section in which no dense biofilm was observed on the surface, but a few bacteria were visible. Strong autofluorescence of kelp cells was observed. D, E, and F are enlarged images of the dashed squares in (A), (B), and (C), respectively

Fig. 9

Epibiotic bacteria associated with Granulosicoccus cells. Small bacteria identified only with the near-universal Eub338-1 probe were observed adjacent to Granulosicoccus despite being surrounded by numerous bacteria of different taxa. A-C Representative images of samples collected on different dates during the summer on Tatoosh Island