Direct interaction between marine cyanobacteria mediated by nanotubes

Abstract

Microbial associations and interactions drive and regulate nutrient fluxes in the ocean. However, physical contact between cells of marine cyanobacteria has not been studied thus far. Here, we show a mechanism of direct interaction between the marine cyanobacteria Prochlorococcus and Synechococcus, the intercellular membrane nanotubes. We present evidence of inter- and intra-genus exchange of cytoplasmic material between neighboring and distant cells of cyanobacteria mediated by nanotubes. We visualized and measured these structures in xenic and axenic cultures and in natural samples. We show that nanotubes are produced between living cells, suggesting that this is a relevant system of exchange material in vivo. The discovery of nanotubes acting as exchange bridges in the most abundant photosynthetic organisms in the ocean may have important implications for their interactions with other organisms and their population dynamics.

Fig. 1.. Nanotubes form between cyanobacterial cells.

(A and B) SEM images of Prochlorococcus sp. SB (xenic culture grown in coculture with Alteromonas). (C) Fluorescence images of Synechococcus sp. PCC 7002 stained with the lipophilic fluorescent dye FM 1-43. Three cells and one nanotube imaged by bright field (left), membranes stained by FM 1-43 (green), cyanobacterial autofluorescence (red), and merged images (right). (D to H) Transmission electron micrographs of axenic cultures of Synechococcus sp. PCC 7002 (D), sp. WH8102 (E), and sp. WH7803 (F and H) and Prochlorococcus sp. MIT9313 (G). (I to K) Ultrathin sections of Synechococcus sp. WH7803 by TEM. Arrows indicate intercellular nanotubes connecting neighboring cells. (A) to (D), (F), and (G) show narrow nanotubes, and (E), (G), and (H) to (K) show wide nanotubes. Type IV pili are shown in micrographs (A), (B), and (D) to (G).

Fig. 2.. Nanotubes connecting Synechococcus sp. PCC 7002 recombinant cells expressing sfGFP.

(A) A field of recombinant cells with pGFP. Top: An additional example of magnified cells showing the pGFP recombinant cells. (B to D) Recombinant cells with the cGFP. White arrows indicate the localization of GFP molecules within the nanotubes. The Synechococcus cells drawn in the upper left and lower right corners show the two different sfGFP locations used in the experiment.

Fig. 3.. Transfer of calcein between Synechococcus sp. PCC 7002 cells.

(A to D) Synechococcus sp. PCC 7002 calcein-labeled cells were mixed with unlabeled ones. A total of 100 cells were selected: 50 calcein-labeled cells (white circle) and 50 unlabeled cells (cyan circle). The average fluorescence intensity at t = 0 min versus t = 15 min (after the mix) was compared (Supplementary Materials and table S1). (E and F) Magnification of the orange squares in (C) and (D). White arrows highlight cells whose average fluorescence intensity decreases after 15 min. Orange arrows highlight cells whose average fluorescence intensity increases after 15 min.

Fig. 4.. Correlation between nanotube formation and vitality in xenic cultures of Synechococcus sp. PCC 7002 observed by IFC.

(A) Cytogram showing Synechococcus populations observed in cultures. The particle size and chlorophyll autofluorescence identified alive single or doublets Synechococcus cells (subpopulation P1), aggregated beads (subpopulation P2), and cellular debris with heterotrophic bacteria (subpopulation P3). (B) Cytogram obtained from SYTOX Green–stained samples. The SYTOX Green fluorescence allowed to distinguish between alive or dead cells. Subpopulation P4 includes small particles like heterotrophic bacteria (alive or dead) and dying cells of Synechococcus, and subpopulation P5 includes large size particles, which are alive and dead Synechococcus cells (solitary or into aggregates). (C) Pie chart showing the percentage of alive cells and damaged cells with nanotubes (NTs) from a culture population of 3230 cells (see Materials and Methods). (D) Bright-field images at ×60 magnification from Synechococcus alive cells [chlorophyll a (Chl a) autofluorescence; a to f]. (E) Bright-field images at ×60 magnification from aggregated calibration beads (larger size and non–Chl a autofluorescence; a and b). (F) Bright-field images at ×60 magnification from heterotrophic bacteria alive cells (smaller size and non–Chl a autofluorescence; a). (G) Bright-field images at ×60 magnification from small particles including Synechococcus dying cells (Chl a autofluorescence and SYTOX Green fluorescence; a and b), dying heterotrophic bacteria (SYTOX Green fluorescence and non–Chl a autofluorescence; c), and alive heterotrophic bacteria (non–Chl a autofluorescence; d). (H) Bright-field images of aggregated alive cells of Synechococcus (Chl a autofluorescence; a to d). Blue arrows show nanotubes in aggregated cells (see also fig. S18).

Fig. 5.. Nanotube formation in natural population of cyanobacteria observed by IFC.

(A) Cytogram obtained from the sample collected on 7 March 2023 by IFC showing the cyanobacterial population discerned by size and pigment composition (Chl a) (P1), aggregates of cyanobacterial cells (Chl a autofluorescence in the small particle area) and photosynthetic eukaryotes (Chl a autofluorescence in the large particle area) (P2), detritus and some heterotrophic bacteria (P3), and heterotrophic bacteria and aggregated beads (P4). P3 and P4 populations might also include some cyanobacterial and picoeukaryotic dead cells. (B) Bright-field images at ×60 magnification of aggregates of Synechococcus cells (Chl a autofluorescence) with other cells (dying cyanobacterial cells or heterotrophic bacteria; a and b). Blue arrows show nanotubes in aggregated cells. (C) Bright-field images at ×60 magnification of individual Synechococcus cells (Chl a autofluorescence; a to f). (D) Bright-field images at ×60 magnification of individual heterotrophic bacteria (a to e).