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

Marine bacteria inhabiting oxygenated surface waters surprisingly possess denitrification genes, a process typically associated with anaerobic respiration. Denitrification involves a multi-step reduction of nitrogen species, starting with nitrate (NO₃⁻) and proceeding to nitrite (NO₂⁻), nitric oxide (NO), nitrous oxide (N₂O), and finally dinitrogen (N₂). This process is prevalent in oxygen-deficient zones (ODZs) and ocean sediments. While nitrite, NO, and N₂O are known to accumulate in marine environments, knowledge regarding NO is limited due to its short lifespan. NO's role in oceanic processes may, therefore, be underestimated. NO's presence in the ocean stems from various sources beyond denitrification. Organisms use NO for signaling in both normal and perturbed physiological states. It's often linked to programmed cell death (PCD) and oxidative stress. NO's small size and gas-like nature allow it to readily pass through cell membranes, leading to localized responses in tissues and intercellular communication in dense populations. NO diffusion has been noted in *Gephyrocapsa huxleyi* (formerly *Emiliania huxleyi*), a widespread coccolithophore forming extensive blooms. These blooms, lasting several weeks, end with sudden collapses, with NO potentially implicated in this demise. Algal populations in these blooms and in lab settings harbor diverse bacterial communities including Roseobacters, which are metabolically versatile marine bacteria and are frequently associated with microalgae. Many Roseobacters, even though often aerobic, possess denitrification genes raising questions about their function. This study utilizes a model system consisting of *G. huxleyi* and the Roseobacter *P. inhibens* to investigate inorganic nitrogen exchange and examine the pathogenic transition of bacteria that results in algal death. Initially mutualistic, this algal-bacterial interaction eventually shifts, with bacteria becoming pathogenic and killing their algal partners. The research investigates whether *P. inhibens* activates denitrification genes and produces NO under oxic conditions, and examines the subsequent impact on algal PCD.

The literature review section of the paper highlights existing knowledge on denitrification, NO signaling in various organisms, the characteristics and bloom dynamics of *G. huxleyi*, and the role of Roseobacters in algal-bacterial interactions. Prior research established that denitrification is an anaerobic respiratory process, but the presence of denitrification genes in aerobic bacteria remained a puzzle. Studies had shown NO's involvement in PCD and oxidative stress in diverse organisms, and its diffusion capabilities for intercellular signaling. Regarding *G. huxleyi*, previous work documented its bloom formation and collapse, with hints towards NO's involvement in bloom demise, particularly in the context of viral infection. Roseobacters were known to associate with microalgae, with some harboring denitrification genes, but the full scope of their interactions remained unclear. The authors build upon existing knowledge about these individual elements to introduce a novel interaction mechanism involving inorganic nitrogen exchange.

The study employed a multi-faceted approach combining experiments with *G. huxleyi* and *P. inhibens* cultures, genetic manipulations of *P. inhibens*, chemical treatments, microscopy, flow cytometry, qPCR, EPR spectroscopy, and bioinformatic analyses of environmental data. **Algal Nitrite Secretion:** The researchers measured inorganic nutrients secreted by *G. huxleyi* cultures, observing a nitrite peak during exponential growth, unaffected by the presence of bacteria. This peak's timing correlated with algal death in co-cultures. **Bacterial Denitrification Genes:** The *P. inhibens* genome was analyzed, revealing genes involved in nitrite metabolism, including nitrite reductases (*nirk*, *msrPQ*) and a nitrite transporter. The genomic location of *nirk* revealed a denitrification locus including other genes like *nirV*, *nor* (NO reductase), *nnrs* (NO stress alleviation), and *nnrR* (NO transcriptional regulator). The bacterium showed poor growth under anoxic conditions, suggesting additional roles for these genes beyond bioenergetics. **Bacterial NO Production and Secretion:** The expression of *nirk* and *norB* genes in *P. inhibens* was monitored upon nitrite addition under oxic conditions. DAF-FM Diacetate staining and LEST method (Liposome-Encapsulated-Spin-Trap) coupled with EPR spectroscopy were used to confirm NO production and its extracellular presence, demonstrated through the comparison of wild-type and denitrification gene-deficient mutants. **Role of NO in Algal Death:** Co-cultures of *G. huxleyi* with *P. inhibens* mutants lacking denitrification genes showed delays in algal death compared to those with wild-type *P. inhibens*. The expression of *nirk* in co-cultures also mirrored the algal nitrite peak. Exposure of axenic *G. huxleyi* cultures to chemical NO donors induced algal death, which was rescued by a NO scavenger. **Algal NO Production and Propagation:** Analysis of algal NO production revealed that algal NO production in response to an external NO donor and this was propagated within the algal population, visualized with DAF-FM staining. Addition of NO scavengers prevented algal death in co-cultures. **Environmental Data Analysis:** Environmental metagenomes, metatranscriptomes, and MAGs from oxygen-rich waters were examined. The co-occurrence of bacterial denitrification genes and transcripts with phytoplankton in oxygenated regions was determined using data from Ocean Gene Atlas (OGA) and Tara Oceans metatranscriptome data. Analysis of phylogenetic relationships among marine Roseobacters helped to contextualize the findings within the natural environment.

The study's key findings centered around a novel inorganic nitrogen communication pathway between *G. huxleyi* and *P. inhibens*. 1. **Algal Nitrite Secretion:** *G. huxleyi* secretes nitrite during exponential growth; this secretion serves as a signal. The timing of nitrite secretion is independent of the presence of bacteria. 2. **Bacterial Nitrite Reduction to NO:** *P. inhibens* reduces algal-secreted nitrite to NO under oxygenated conditions using its denitrification genes. This is not primarily for energy generation, suggesting a different function. The bacterium’s *nirk* and *norB* genes increase their expression upon exposure to nitrite. The use of DAF-FM showed that NO is produced intracellularly in a heterogeneous manner. The use of LEST and EPR showed that NO is secreted extracellularly by bacteria exposed to nitrite, but not in a mutant lacking denitrification genes. 3. **NO's Role in Algal Death:** Bacterial NO triggers a PCD-like process in algae. Mutants lacking denitrification genes show delayed algal death in co-cultures, although algal death was not completely prevented. This may be due to redundant pathogenicity mechanisms. The treatment of algal cultures with NO triggers algal death, indicating a causal link. This effect is rescued with the addition of a NO scavenger. 4. **Algal NO Propagation:** Following exposure to exogenous NO, algae produce NO and secrete it extracellularly, amplifying the death signal across the algal population. This was visualized using microscopy. The addition of a NO scavenger completely prevented algal death in co-cultures. 5. **Environmental Significance:** Environmental data (OGA and Tara Oceans data) shows that bacterial denitrification genes and transcripts are present in oxygenated regions of the ocean, co-occurring with phytoplankton. Many of these genes are found in members of the Rhodobacteraceae family, which supports the relevance of the laboratory findings to the marine environment. Some MAGs grouped with aerobic bacteria and only possessed a subset of denitrification genes, hinting towards similar functions in other species.

This research unravels a novel mechanism of microbial interaction mediated by inorganic nitrogen compounds, adding to existing models based primarily on organic molecule exchange. The findings connect algal growth phase, indicated by nitrite secretion, to bacterial pathogenicity, triggered by bacterial NO production and secretion. The role of NO in algal death is supported by the delayed algal death in denitrification-deficient mutants, the ability of exogenous NO to induce death in axenic algal cultures and the rescue effect of a NO scavenger. Moreover, the observed algal NO production and propagation provide a positive feedback loop, accelerating population collapse. The environmental data extends the ecological significance of this inorganic nitrogen exchange, suggesting its relevance in marine ecosystems where aerobic bacteria may utilize denitrification for interspecies interactions rather than anaerobic respiration. The redundancy of pathogenic pathways in *P. inhibens* highlights the adaptability and complexity of microbial interactions. The study proposes that nitrite could be a general senescence signal exploited by bacteria to enhance their resource acquisition.

This study reveals a novel inorganic nitrogen exchange pathway between aerobic bacteria and algae, highlighting NO as a key inter-kingdom signaling molecule involved in algal PCD and population collapse. The findings challenge the traditional view of microbial interactions, emphasizing the importance of inorganic compounds in mediating these processes. Future research could explore the detailed molecular mechanisms underlying NO's effects on algal PCD, investigate the broader ecological implications of this interaction across diverse marine environments, and develop further technologies for directly probing the local chemical processes within the algal phycosphere.

The study focused on a specific algal-bacterial model system (*G. huxleyi* and *P. inhibens*). The generalizability of these findings to other algal-bacterial interactions needs further investigation. While the environmental data supports the ecological relevance, direct measurements of NO concentrations in the algal phycosphere during natural blooms would strengthen the conclusions. Further work is required to fully elucidate the molecular mechanisms underlying NO's effects on algal PCD and the specific selection pressures driving the maintenance of denitrification genes in aerobic bacteria.