Scarcity of fixed carbon transfer in a model microbial phototroph–heterotroph interaction

Abstract Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtii’s capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum, a vitamin B12-producing α-proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more 13C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the alga’s fermentative degradation of starch reserves during the night would boost M. japonicum heterotrophy. 15NH4 assimilation rates and changes in cell size revealed that M. japonicum cells reduced new biomass synthesis in coculture with the alga but continued to divide—a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B12 and supported the growth of a B12-dependent C. reinhardtii mutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B12 exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal–bacterial interactions.


C. reinhardtii strain haplotype determination
The haplotype of the wild-type C. reinhardtii strain used in this study was determined by allelespecific amplification as previously described [1].Genomic DNA was extracted using CTAB (hexadecyltrimethylammonium bromide).Cell pellets were resuspended in 0.7 ml 60°C CTAB buffer (2% CTAB, 100 mM Tris-HCl, pH 8, 20 mM EDTA, 1.4 M NaCl, 0.2% betamercaptoethanol, 0.1 mg/ml proteinase K) and incubated for 1 h at 60°C.Then, DNA was extracted by addition of 0.7 ml chloroform/isoamylalcohol (24:1), inversion for 2 min, centrifugation at 16,000 xg for 10 min at 4°C, and collection of the aqueous phase.DNA was precipitated by overnight incubation in 50% isopropanol at -20°C.DNA was then collected by centrifugation at 16,000 xg for 15 min at 4°C, and was then washed with cold 70% ethanol.DNA was air-dried for 15 min at room temperature and resuspended in purified water.

Chlorophyll measurement
Chlorophyll (Chl) content was measured as previously described [2]. 1 ml of culture was collected by centrifugation at 16,000 xg for 1 min and the supernatant was discarded.Cell pellets were stored at -80°C until analysis.Pellets were thawed at ambient temperature and then resuspended in 1 ml 80:20 acetone:methanol.Samples were incubated on ice for 5 min, and then starch was collected by centrifugation at 16,000 xg for 2 min.Absorbance of the supernatant was measured at 647 nm, 664 nm, and 750 nm using a UV-6300PC Double Beam Spectrophotometer (VWR, PA, USA).As Abs750 was below the detection limit, total Chl was calculated as Chls a + b (µg/ml) = (17.76x Abs646) + (7.34 x Abs663) and was then normalized to the C. reinhardtii cells in the sample.

Fv/Fm measurement
The maximum quantum efficiency of photosystem II (Fv/Fm) was measured using a Walz IMAGING-PAM MAXI Chlorophyll Fluorescence System equipped with an IMAG-K7 CCD Camera (Heinz Walz GmbH, Effeltrich, Germany).300 µl culture was placed in a 96-well plate and dark-adapted for 15 min prior to fluorescence measurement.Fv/Fm was calculated as (Fm -Fo)/Fm, where Fm is the maximum fluorescence measured upon a saturating pulse and Fo is the minimal fluorescence of dark-adapted cells in the dark.

1.
Gallaher SD, Fitz-Gibbon ST, Glaesener AG, Pellegrini M, Merchant SS.Chlamydomonas genome resource for laboratory strains reveals a mosaic of sequence variation, identifies true strain histories, and enables strain-specific studies.

Figure 1 :
The C. reinhardtii "wild type" used in this study is closely related to strain S24-.The haplotype of the C. reinhardtii "wild type" was determined by allele-specific amplification of genomic DNA and was compared to the reported haplotypes of related strains (S24-and CC-124) and of CC-5390[1].Haplotype is indicated at 41 haplotype blocks across the C. reinhardtii genome: blue represents haplotype 1, yellow represents haplotype 2, and mating type is indicated as + or -at the mating type locus.Of the C. reinhardtii strains for which a haplotype has been reported, the wild-type strain used in this study was most similar to S24-.SIFigure 2: M. japonicum does not impact chlorophyll content nor Fv/Fm of wild-type C. reinhardtii.Triplicate cultures (n = 3) were inoculated with or without 150 µg/ml sucrose and maintained under continuous light, bubbled with air, and shaken at 110 rpm.(A) Chlorophyll content normalized to the number of C. reinhardtii cells in the cultures 72 h after inoculation.(B) Fv/Fm of cultures 72 h after inoculation.Differences were not significant (n.s.) by two-tailed Student's t-test (p > 0.05).SI Figure 3: C. reinhardtii strain CC-5390 is more prone to cell lysis and release of organic carbon into the medium.Triplicate cultures (n = 3) of C. reinhardtii wild type and cell wall reduced strain CC-5390 were maintained under continuous light, bubbled with air, and shaken at 125 rpm.(A) The degree of C. reinhardtii cell lysis 72 h after inoculation estimated by CellTox Green fluorescence, reported relative to a 100% killed control.(B) NPOC in spent medium sampled 72 h after inoculation.Error bars represent the standard deviation from the mean.Asterisks indicate significant differences by Student's t-test (p < 0.05).SI Figure 4: Light regime does not impact C. reinhardtii cell lysis.Triplicate cultures (n = 3) of cell wall reduced strain CC-5390 were maintained in semi-continuous culture in parallel photobioreactors under continuous light or diurnal light (12-h-light/12-h-dark) and bubbled with ambient air.The degree of C. reinhardtii cell lysis estimated by CellTox Green fluorescence, reported relative to a 100% killed control, was not significantly different under the two light regimes by two-tailed Student's t-tests (p = 0.86).Error bars represent the standard deviation from the mean.SI Figure 5: Representative raw nanoSIMS images.Representative raw nanoSIMS secondary electron images, 12 C 13 C, 12 C 14 N, and 12 C 15 N signals from (A) C. reinhardtii monoculture and (B) coculture with M. japonicum.Algal cells are clearly visible in the scanning electron, 12 C 13 C, and both CN images.Bacterial cells are most visible in the 12 C 14 N image.Micron-scale hotspots in the 12 C 13 C image are present in both the monoculture and coculture and do not line up with CN hotspots in the coculture; therefore, they are interpreted as algal debris (e.g., organelles, starch), rather than bacterial cells.Algal ROIs were circled using the scanning electron and 12 C 13 C images and bacterial ROIs were circled using the 12 C 14 N image.SI Figure 6: Cross-plots of stable isotope enrichment data.Cross-plots of stable isotope enrichment at the indicated times after inoculation in C. reinhardtii cells (A, C) and M. japonicum cells (B, D), under continuous (A, B) and diurnal (C, D) light, from the coculture experiment described and shown in Fig. 3 plotted alongside monocultures and killed controls.M. japonicum grown on sucrose (pink points) is used to constrain possible 13 C enrichment by direct 13 CO2 uptake during heterotrophic growth (dashed line), which is well below the maximum 13 C enrichment observed in cocultures.SI Figure 7: M. japonicum 13 C enrichment is significantly higher during growth with C. reinhardtii than during heterotrophic growth on sucrose or unlabeled C. reinhardtii cell lysate.Duplicate cultures (n = 2) of M. japonicum were grown with 150 µg/ml sucrose, in C. reinhardtii cell lysate, or in coculture with CC-5390 under diurnal light as described in Fig. 3 with 50% of their NH4 + provided as 15 N and with 13 CO2 added to the air used to bubble the cultures starting 13 h after inoculation.Isotope enrichment was measured using nanoSIMS 48 h after inoculation. 13C atom percent enrichment (APE) data of n ≥ 16 individual cells is shown.Asterisks indicate significant differences between the conditions by two-tailed Student's t-test (p < 0.05).SI Figure 8: Increase in M. japonicum density during stable isotope probing experiments informs Nnet expected under balanced growth.(A) M. japonicum cell density during the experiment described in Fig. 3, Fig. 4, SI Fig. 6, and SI Fig. 9 over time after inoculation.The grey background indicates sample timepoints that occurred during the dark phase.(B) Model of Nnet as biomass doubles (blue) was used to estimate the expected Nnet of M. japonicum cells at each timepoint (circles and triangles) if the number of doublings in CFU/ml observed at that time corresponded to doublings in biomass.SI Figure 9: C. reinhardtii cell density at the time of nanoSIMS sampling.Growth of cell wall reduced strain CC-5390 in continuous light or diurnal light (12-h-light/12-hdark) with and without M. japonicum, during the experiment described in Fig. 3, SI Fig. 6, and SI Fig. 8.The grey backgrounds indicate sample timepoints that occurred during the dark phase.SI Figure 10: Growth of C. reinhardtii mete1 strain is supported by vitamin B12 and by M. japonicum.Duplicate cultures of C. reinhardtii strains with B12 or with M. japonicum maintained under diurnal light (12-h-light/12-h-dark) and shaking at 130 rpm.(A) Maximum cell density of C. reinhardtii mete1 mutant (triangles, left) and parental wild-type strain (circles, right) 120 h after inoculation with various amounts of exogenous B12 (grey) or with 10, 100, or 1000 bacterial cells per algal cell.Error bars represent the standard deviation from the mean.(B) Growth of M. japonicum with C. reinhardtii mete1 (triangles) or wild type (circles) when inoculated at roughly 10, 100, and 1000 bacterial cells per algal cell (starting density of 5x10 4 C. reinhardtii cells/ml).