The Invisible Molecule Shaping Microbial Life
In the unseen world of bacteria, an invisible gas wields immense power. It can spell death for some microbes, while for others, it's a signal for survival, a weapon, or even a source of food.
Nitric oxide is a reactive and highly diffusible gaseous molecule, a free radical that can easily cross bacterial membranes 1 . For a long time, its story in microbiology was one-dimensional: it was considered primarily a toxic agent used by our immune systems to halt bacterial growth 1 5 . However, this view has been dramatically expanded. Researchers now understand that bacterial relationships with NO are nuanced and multifaceted.
NO's effects are concentration-dependent. At high concentrations, it is indeed a powerful bacteriostatic agent, damaging cellular proteins, disrupting iron-sulfur clusters, and causing DNA damage 1 5 . Yet, at lower concentrations, it functions as a critical signaling molecule, influencing community behaviors like biofilm formation and dispersion 1 . Furthermore, for a specialized group of microbes, NO is not a threat but a resource—an energy source that can be "breathed" to support growth 6 .
Bacteria are ingenious chemists, and they can produce NO through several distinct biochemical routes. These are broadly classified into two pathways:
This is part of the global nitrogen cycle. During denitrification (a process that converts nitrate to nitrogen gas), specialized enzymes called nitrite reductases (NirS and NirK) carry out the one-electron reduction of nitrite (NO₂⁻) to NO 1 . Similarly, in nitrification (the oxidation of ammonia to nitrate), the enzyme hydroxylamine oxidoreductase (HAO) can generate NO as an intermediate 1 .
Many bacteria possess their own version of the enzyme bacterial nitric oxide synthase (bNOS) 1 . Similar to the human version, bNOS catalyzes the production of NO from the amino acid L-arginine, yielding L-citrulline as a byproduct 1 . This pathway is used for signaling and protection against oxidative stress.
| Enzyme | Pathway | Function | Key Product |
|---|---|---|---|
| NirS & NirK | Denitrification | Reduces nitrite (NO₂⁻) | Nitric Oxide (NO) |
| Hydroxylamine Oxidoreductase (HAO) | Nitrification | Oxidizes hydroxylamine (NH₂OH) | Nitric Oxide (NO) |
| Bacterial NOS (bNOS) | Non-respiratory | Oxidizes L-arginine | Nitric Oxide (NO) |
For years, the study of NO-reducing bacteria was hampered by a major challenge: successfully growing them in the lab with NO as the primary electron acceptor. A groundbreaking study published in Nature Microbiology in 2023 changed this 6 . Researchers successfully enriched a microbial community from a wastewater treatment plant that could not only survive on NO but thrive on it.
Researchers cultivated bacteria using NO as their sole electron acceptor for over 1,500 days, demonstrating sustained growth and efficient conversion of NO to harmless N₂ gas.
Days of continuous cultivation
The research team used a continuous bioreactor, meticulously controlling the conditions to favor organisms that could use NO.
The bioreactor was inoculated with sludge from a municipal wastewater treatment plant and continuously fed with formate (as an electron donor and carbon source) and NO (as the sole electron acceptor) for over 1,500 days 6 .
The system was constantly sparged with an argon/CO₂ mixture to maintain strict anoxic conditions, preventing the non-biological, oxygen-driven conversion of NO 6 .
The team closely monitored the consumption of NO and formate, and the production of nitrogen gases (N₂O and N₂), tracking the culture's growth through protein content 6 .
The experiment was a remarkable success, yielding profound insights into the physiology of NO-reducing bacteria.
The enriched culture grew steadily, with a doubling time of about 6.65 days, while reducing NO at a rate of 1.69 mmol per day 6 .
The culture reduced NO all the way to harmless N₂ gas, with little to no detectable accumulation of the potent greenhouse gas nitrous oxide (N₂O) 6 .
Metagenomic analysis revealed the community was dominated by two previously unknown species from the betaproteobacterial Sterolibacteriaceae family 6 .
| Parameter | Measurement | Biological Significance |
|---|---|---|
| NO Reduction Rate | 1.69 ± 0.1 mmol/day | Demonstrates robust metabolic activity directly coupled to NO consumption. |
| Formate Oxidation Rate | 2.38 ± 0.2 mmol/day | Confirms the coupling of carbon source oxidation to NO reduction for energy. |
| N₂O Production | ≤ 2.8% of converted NO | Highlights the culture's high efficiency in preventing greenhouse gas release. |
| Doubling Time | ~6.65 days | Indicates steady, though slow, growth supported by NO respiration. |
This experiment was not just about growing difficult microbes. It demonstrated that natural ecosystems harbor highly efficient organisms that play a crucial, previously underappreciated role in mitigating climate-active gases. It also provided a window into the ancient past, suggesting how early life might have used NO for respiration long before oxygen was widely available 6 .
Studying a gaseous, reactive, and short-lived molecule like NO requires a specialized toolkit. Researchers rely on a combination of chemical traps, inhibitors, and donor molecules to detect, measure, and manipulate NO in bacterial systems.
Category: Detection
A colorimetric assay that detects nitrite (NO₂⁻), a stable decomposition product of NO. Simple but limited to µM sensitivity 2 .
Category: Detection
A fluorescent probe that reacts with NO-derived species to form a fluorescent product (NAT). Offers nanomolar sensitivity 2 .
Category: NO Donor
A class of compounds that predictably release NO in solution, allowing researchers to control the timing and amount of NO delivery 3 .
The story of nitric oxide in bacteria is a powerful reminder of the complexity of the microbial world. It is a tale of a molecule with a dual nature, a Janus-faced entity that is both a poison and a nutrient, a random damaging radical and a precise signal.
To dictating the outcome of infections 5
Even potentially impacting human exercise performance through our oral microbiome 8
As research continues to untangle its intricate web of interactions, one thing is clear: this simple, invisible gas is a fundamental force in the hidden world of microbes.