Molecular detection of abundance and activity of marine, symbiotic, N2-fixing cyanobacteria

Abstract: Marine primary productivity in large parts of the oceans is supported by nitrogen fixers (diazotrophs). Richelia, a genus of multiple closely related species of heterocystous filamentous cyanobacterial diazotrophs, are often found in symbioses with a few genera of diatoms. Several Richelia species: R. euintracellularis, R. intracellularis and R. rhizosoleniae, form stable host specific partnerships and these populations are important for the nitrogen (N) cycle, especially in the oligotrophic open oceans. In general, the gene nifH, which encodes the enzyme nitrogenase for N2 fixation, is used as a molecular marker for presence (DNA level) and activity (RNA level) of diazotrophs. However, evidence of cross-reactivity of the nifH assays between two of the Richelia species shows the risk of incorrect estimations of abundance and activity. Moreover, transcript abundance is rarely normalized to a housekeeping gene. The aims of this work were to develop and assess new assays for the detection of three symbiotic Richelia species: R. euintracellularis, R. intracellularis and R. rhizosoleniae. These new assays targeted molecular markers that were indicative nutrient acquisition. For example, one assay targets a gene encoding the high affinity phosphate transporter (pstS), a second targets a gene involved in iron (Fe) transport (exbB) (indicator of P transport, Fe transport, respectively), and a third assays a housekeeping gene encoding Ribonuclease P protein (rnpA). All assays were used to estimate abundance and expression in lab and field based samples.The new assays have high species specificity as revealed by BlastN analyses and lab-based cross-hybridization assays. We applied the assays to samples from a lab-based experiment of the facultative symbiont R. rhizosoleniae RrhiSC01 in order to investigate the temporal dynamics of expression. This revealed periodic expression of nifH and pstS related to the photoperiod with higher expression in the early and late photoperiod, respectively. The peak of pstS expression in the late photoperiod is likely to support the high P requirement of both photosynthesis and N2 fixation. Given the temporal regulation of pstS (and nifH) expression shown here, one must consider these results when sampling and/or interpreting field studies. Moreover, a comparison of the Richelia draft genomes and environmental metagenomic assembled genomes (MAGs) show that gene copy number per genome of exbB and pstS varies between strains. Given that Richelia tend to live in low nutrient environments, including N, P, and often Fe, the increased copy number per genome for pstS and exbB could be suggestive/evidence of adaptation to constant nutrient limitation.In addition, the assays were applied to natural samples containing populations of R. intracellularis and R. euintracellularis collected in the North and South Atlantic Ocean from multiple depths (5-90 meters). The DNA based gene copy abundances showed higher estimates when nifH is used compared to rnpA, which is likely due to a higher degree of cross-reactivity in the nifH assays. Gene expression was present but low for all targets. However, expression of exbB and pstS was higher in surface water – where nutrients are expected to be depleted. Lastly, bulk fixation rates of N2 and carbon (C) showed that the activity was low and fixation rates did not increase with the addition of dissolved organic phosphate (DOP) (a 1:4 mixture of 2-aminoethylphosphonic acid and beta-glycerophosphate disodium salt hydrate). This could be due to patchiness and low abundance of diazotrophs and discrepancies between samples, seasonal variation or that the populations were not P limited. In summary, the new assays constitute a better option to interspecific detection of Richelia. However, more work is required to assess how the expression relates to limitation of nutrients and other external factors to better understand the activity of these biogeochemically important populations.