Nitrogen is an essential building block of proteins, amino acids, and DNA. Without nitrogen, cells can’t function or reproduce. Nitrogen is abundant in the atmosphere as dinitrogen gas, but plants and animals can’t access atmospheric nitrogen. Instead, we rely on biologically available forms like ammonia and nitrate.
Specialized microbes can transform atmospheric nitrogen into ammonia via a process called biological nitrogen fixation. Nitrogen-fixing soil bacteria have a unique protein complex, the nitrogenase complex, that facilitates this nitrogen transformation. The nitrogenase complex is activated by a group of genes known as nif genes.
Legume plants like peanuts, peas, and beans have nitrogen-fixing bacteria in the soil zone around their roots, called the rhizosphere. The symbiotic relationship between nitrogen-fixing soil bacteria and legume plants supplies most of the biologically available nitrogen to life on Earth.
Bacteria attached to plant roots “feed” nitrogen to plants through the soil, and in turn receive organic acids for energy from plants performing photosynthesis. Source: Wikimedia Commons
However, one piece of the puzzle remains unexplored: the role of soil viruses in these nitrogen transformations, referred to as the nitrogen cycle. Viruses are microscopic particles made up of genetic material and proteins, but they can’t reproduce without a host cell. Scientists consider viruses to be biological entities that are halfway between living and non-living.
To reproduce, viruses contact a host cell and inject their genetic material into it. The virus’s genetic material takes over the host cell’s genome and forces the cell to produce proteins that assemble into new viruses. As new viruses are created, they can incorporate genes from the host cell into their own genetic material. Genes that are transferred from a host cell to a virus are known as auxiliary metabolic genes (AMGs). Viruses can carry AMGs and pass them to other hosts or use them to increase their own ecological fitness.
Scientists from China, Spain, and the Czech Republic set out to investigate whether viruses from legume roots could increase local soil nitrogen fixation by expressing nif AMGs or by transferring nif genes to other bacteria. First, they determined the global distribution of viral nitrogen-fixing genes by analyzing about 8.6 million viral genomes from the Integrated Microbial Genomes/Virus database. They looked for the DNA sequences of known nitrogen-fixing genes and the locations where the viruses were sampled. They found that only 0.003% of the viruses in the database had at least 1 nitrogen-fixing gene, but they clustered in the same locations as nitrogen-fixing bacteria.
The team also found 3 types of viruses that most commonly carried nitrogen-fixing genes: Kyanoviridae, Nudiviridae, and Bronfenbrennervirinae. Kyanoviridae viruses carried a nif gene called nifU. The researchers used a computer program called DRAM-v to confirm that nifU was a fully functional gene and a real tool that viruses could use.
Next, the team collected soil samples from a cowpea field in Nanjing, China, to test whether the cowpea roots influenced the abundance of viruses using nitrogen-fixing genes. They collected soil from the cowpea rhizospheres and soil away from the plants. They analyzed both viral and bacterial RNA in the soil samples to identify active genes, using a sequencing process called metatranscriptomics. They found that the viral nifU gene was actively expressed in the cowpea rhizosphere samples more than in the non-crop soil. Although about 96% of nifU genes were being expressed by bacteria, viruses contributed about 4%.
The researchers then assessed whether rhizospheric viruses changed the amount of nitrogen being fixed in these soils. They set up small, enclosed containers of sterile soil and added either bacteria or a mixture of bacteria and viruses extracted from the cowpea rhizospheres. They found that soil with viruses had higher total nitrogen (36 milligrams per kilogram, or mg/kg) than soil without viruses (17 mg/kg).
Finally, they adjusted the air in each container to have a different form of dinitrogen gas. Atoms of the same element that have different atomic masses are known as isotopes. Nitrogen has 2 stable isotopes: lighter nitrogen-14 and heavier nitrogen-15. Bacteria or viruses that fix nitrogen at natural isotope abundances will take up nitrogen-14. But scientists can track who in the population fixes nitrogen by adding nitrogen-15 to the air, a process called nitrogen-15 stable isotope probing. Nitrogen-fixers will incorporate the heavy nitrogen-15 into their biomass over time and increase their weight, allowing researchers to separate and identify them.
The team grew the bacteria and viruses with nitrogen-15 for 35 days, then used stable isotope probing to separate the nitrogen fixers. They detected viral nifU AMGs among the heavier biomass, confirming that rhizospheric viruses were fixing nitrogen.
The team concluded that rhizospheric viruses and the viral nifU AMG can increase nitrogen fixation in cowpea soils. The team suggested that although viruses with nitrogen-fixing genes are rare, they can influence the soil nitrogen cycle. They recommended that future researchers further test the contribution of viral nifU genes to plant nitrogen fixation with controlled infection experiments.
