Aerobic bacteria produce nitric oxide via denitrification and promote algal population collapse

The study addresses why aerobic marine bacteria in oxygenated surface waters carry denitrification genes and what roles these genes play in interactions with phytoplankton. Specifically, it tests the hypothesis that algal-secreted nitrite during growth triggers bacterial denitrification gene expression under oxic conditions, leading to bacterial nitric oxide (NO) production that induces a programmed cell death (PCD)-like cascade in the coccolithophore Gephyrocapsa huxleyi, culminating in population collapse. The work situates NO as a short-lived, diffusible signaling molecule with central roles in stress responses and PCD across taxa, and posits that inorganic nitrogen exchange in oxygenated environments represents an overlooked communication route in marine microbial ecology with implications for bloom dynamics and the nitrogen cycle.

- Denitrification (NO3−→NO2−→NO→N2O→N2) typically occurs in oxygen-deficient zones and sediments, but denitrification intermediates (NO2−, NO, N2O) are observed in marine settings. - NO functions broadly as a signaling molecule in unicellular and multicellular organisms, often linked to oxidative stress and PCD, and can diffuse between neighboring cells. - In G. huxleyi, NO production has been associated with bloom demise, particularly during viral infection, and diffusion of NO within dense algal populations has been suggested. - Marine Roseobacters (Rhodobacteraceae) commonly associate with phytoplankton and often harbor denitrification genes despite being aerobes. - Prior work in the G. huxleyi–Phaeobacter system showed a shift from mutualism to pathogenicity with aging algae mediated by bacterial metabolites (e.g., tryptophan-derived hormone, DMSP-driven virulence), but how bacteria sense algal aging was unclear. Algal p-coumaric acid has been proposed as a senescence signal, and nitrite secretion has been reported in other phytoplankton (e.g., diatoms) during exponential growth. - Reports exist of denitrification gene expression and activity under oxic conditions in cultures and environments, though mechanisms and ecological roles were not resolved.

- Culture system: Axenic Gephyrocapsa huxleyi CCMP3266 grown in L1-Si medium (18°C, 16/8 h light cycle). Phaeobacter inhibens DSM 17395 cultured in CNPS medium (30°C, shaking). Co-cultures initiated by adding 10–100 CFU/ml bacteria to 10^4 algae in 30 ml L1-Si after 4 days of algal growth; sampling days counted post bacterial addition. - Inorganic nutrient profiling: Filtrates (0.1–0.2 µm) from axenic algal cultures analyzed using an autoanalyzer for NO3−, NO2−, PO4^3−, SO4^2−, NH4^+; daily nitrite monitoring via Griess assay. Temporal linkage of nitrite peaks to algal growth phase assessed by varying initial algal inoculum densities. - Anaerobic growth tests: Assayed P. inhibens DSM 17395 and known denitrifier P. inhibens T5 (DSM 16493) in anoxic seawater-based medium with 10 mM nitrite as terminal electron acceptor; OD600 tracked up to 29 days. - Bacterial genetics: Constructed mutants by allelic exchange/electroporation: • Δ262: strain cured of native 262 kb plasmid (lacking denitrification genes). • Δnor (replacement of norC–norD operon with gentamicin cassette), Δnirk (replacement with gentamicin), and ΔnnrS→nirV (deletion of the entire 10.4 kb denitrification locus replaced with kanamycin cassette). PCR/sequencing validation. - Gene expression (qPCR): For bacteria in pure culture exposed to 10 µM nitrite (0–120 min), measured nirk and norB; for co-cultures sampled across days, measured bacterial nirk relative to day 10. For algae treated with NO donor (DEANO), measured PCD/oxidative-stress markers and noa genes. Normalization to housekeeping genes (bacteria: recA, gyrA; algae: β-tubulin, rpL13). Primer efficiencies verified; RNA/DNase treatment and rRNA depletion for co-cultures. - Intracellular NO detection: DAF-FM diacetate staining with fluorescence microscopy and flow cytometry. For bacteria: assessed NO production after nitrite addition (100 µM) in WT vs Δ262. For algae: monitored DAF-FM fluorescence after exposure to chemical NO donor (DEANO), live/dead bacteria, with/without added nitrite. - Extracellular NO quantification: Liposome-Encapsulated Spin Trap (LEST) method coupled to EPR spectroscopy. Incubated cultures with MGD/Fe(II)-loaded liposomes for 5 h; measured stabilized NO adducts by EPR; included positive (DEANO) and negative (nitrite-only) controls. - Algal mortality assays: Applied NO donor (DEANO) in single or semi-continuous dosing; rescue experiments with NO scavenger c-PTIO (20 µM). Monitored algal growth by flow cytometry; defined algal death by culture color change and collapse. - PCD-like response profiling: Compiled a PCD/oxidative stress gene set from prior transcriptomes; measured expression changes in algae after NO treatment. - Algal NO propagation visualization: Pre-treat subset of algae with DEANO, wash, mix with DAF-FM stained naïve algae, and time-lapse microscopy to visualize spread of NO production among cells. - Environmental analyses: Queried Ocean Gene Atlas for occurrences of denitrification genes (nirk, nirs, norB, norC) in deep chlorophyll maximum (DCM) samples with high oxygen; analyzed TARA Oceans metatranscriptomes for transcript abundances (napA, nirs, norB, nosZ, nirk) vs oxygen and nitrite; built phylogenies of Roseobacter MAGs and annotated denitrification gene complements. - Statistics/replication: Typically ≥3 biological replicates; error bars represent ±SD; two-sample t-tests used for specified comparisons; significance thresholds reported (e.g., p < 0.01, p < 0.05).

- G. huxleyi secretes nitrite during exponential growth, yielding a peak at the end of exponential phase; timing of the nitrite peak shifts earlier with higher initial algal inoculum. Nitrate and phosphate decrease over time; ammonium and sulfate remain unchanged. - The timing of sudden algal death in co-cultures correlates with the timing of the algal nitrite peak: denser algal inoculum leads to earlier nitrite peaks and earlier algal death, while peak nitrite concentrations are similar across inocula. - P. inhibens harbors denitrification-related genes on a 262 kb plasmid (including nirk, norCBQD, nnrS, nnrR), yet exhibits poor growth under anoxic, nitrite-respiring conditions compared to a known denitrifier, suggesting non-bioenergetic roles. - Under oxic conditions, exposure of P. inhibens to 10 µM nitrite rapidly induces denitrification genes: nirk up to ~4-fold and norB up to ~10-fold within 1 hour. - Intracellular NO is produced by bacteria upon nitrite exposure (DAF-FM fluorescence), whereas the plasmid-cured Δ262 strain does not produce NO despite similar growth. - Extracellular NO is detected by LEST/EPR from WT bacteria supplemented with nitrite but not from Δ262, indicating NO secretion under oxic conditions and partial escape from intracellular reduction. - Denitrification capacity contributes to algal killing: Δ262 bacteria fail to trigger algal death in co-culture; deletion of the entire denitrification locus (ΔnnrS→nirV) markedly delays algal death, while Δnirk alone does not, suggesting additional nitrite reductases/regulation contribute. WT and mutant bacterial growth are comparable in co-culture. - In co-cultures, bacterial nirk expression increases >10-fold during algal exponential phase, coincident with the algal nitrite peak, indicating bacterial response to algal-secreted nitrite. - Bacterial NO diffuses into algae: algae display increased intracellular DAF-FM fluorescence when incubated with live bacteria plus nitrite, or with a chemical NO donor; dead bacteria or absence of nitrite do not elicit the response. - Exogenous NO alone is sufficient to kill axenic algal cultures across growth phases; the NO scavenger c-PTIO rescues from NO-induced mortality, demonstrating NO causality. - NO triggers a PCD-like response in algae: NO treatment upregulates oxidative stress and PCD marker genes by ~3–8-fold. The algal nitric oxide-associated gene noaa2 increases >15-fold after NO donor treatment; in co-cultures noaa2 increases during stationary phase/death. - Algae produce and secrete NO during co-culture stationary phase (extracellular NO detected on day 10), whereas age-matched axenic algal cultures do not. Pre-exposed algae propagate NO production through the algal population, visualized by DAF-FM time-lapse microscopy. - Applying an NO scavenger to co-cultures just before stationary phase prevents algal death, supporting a role for extracellular NO in propagating collapse. - Environmental support: In oxygenated DCM layers (100–400 µM O2), denitrification genes (nirk/nirs/norB/norC) co-occur with high chlorophyll. TARA metatranscriptomes show napA, nirs, norB, nosZ transcripts decrease with rising oxygen, whereas nirk transcripts remain variable and frequently high in phytoplankton-rich, oxygenated waters with measurable nitrite (0.1–1.5 µM), consistent with nitrite-to-NO activity in oxic environments.

The findings demonstrate that an inorganic nitrogen exchange—algal secretion of nitrite and bacterial reduction of nitrite to NO under oxic conditions—mediates inter-kingdom communication that controls algal population fate. Bacterial NO acts as a signal that initiates a PCD-like cascade in G. huxleyi, and the algae themselves subsequently produce and release NO, amplifying and propagating the death signal within the population. This mechanism links algal growth phase (exponential nitrite leakage) to the timing of bacterial pathogenicity and bloom collapse. It reconciles the paradox of denitrification genes in aerobic, phytoplankton-associated bacteria by revealing signaling roles unrelated to anaerobic respiration. Environmental gene and transcript distributions support the ecological plausibility of nitrite-to-NO production in oxygenated, phytoplankton-rich waters. The work broadens the concept of the marine phycosphere to include reactive nitrogen gases as local signals, suggests that NO contributes to bloom dynamics and nutrient release via synchronized algal mortality, and implies that denitrification intermediates influence interspecies interactions and the nitrogen cycle beyond classical ODZ settings.

This study identifies a novel, inorganic signaling axis in algal–bacterial interactions: algal nitrite secretion during exponential growth induces aerobic bacterial denitrification gene expression and NO production, which triggers a PCD-like response and NO generation in algae, culminating in population collapse. The results establish NO as an inter-kingdom signaling molecule in oxic marine systems and provide a mechanistic link between algal growth phase and bacterial pathogenicity. Environmental data indicate that denitrification gene expression, particularly nirk, is common in oxygenated, phytoplankton-rich layers, underscoring wider ecological relevance. Future work should: determine microscale NO and nitrite concentrations in the phycosphere; resolve the molecular mechanisms of NO-induced algal death; dissect regulatory networks and redundant nitrite reductases in bacteria; test interactions under microaerophilic niches; and quantify the contribution of oxic NO signaling to bloom dynamics and marine nitrogen cycling.

- The precise molecular mechanism by which NO triggers PCD-like death in G. huxleyi remains unresolved; whether NO itself is lethal or acts upstream in a signaling cascade is unknown. - Local (phycosphere-scale) NO and nitrite concentrations experienced by cells were not directly measured; diffusion-boundary layer effects may create microenvironments not captured by bulk measurements. - The Δnirk mutant did not phenocopy the denitrification locus deletion, indicating additional nitrite reductases/regulatory elements; the full complement and regulation of NO-producing enzymes remain to be defined. - The 262 kb plasmid encodes other genes; although a targeted denitrification locus deletion was used, potential pleiotropic effects cannot be fully excluded. - Poor anaerobic growth of DSM 17395 under tested conditions does not exclude denitrification-based respiration under untested microaerophilic conditions. - Environmental analyses are correlative; gene/transcript presence and oxygen/nitrite co-occurrence do not prove mechanism or active NO signaling in situ. - Quantitative thresholds (dose, duration) of NO required to trigger algal PCD in natural settings are not established.