1 Experimental Data Analysis

The key findings of the experiment are mainly reflected in two aspects: First, in terms of diversity and ecological environment: (1) NrfA-dependent ammonifying microorganisms are widely present in both terrestrial and aquatic environments, with a rich variety of species and broad distribution. (2) These microorganisms demonstrate the capability to reduce nitrate, especially under oxygen-limited conditions, across various environments. The second aspect concerns physiological functions: (1) The NrfA enzyme is a key biocatalyst linking nitrate reduction to ammonia, through which these microorganisms contribute to nitrogen retention in the environment. (2) The NrfA enzyme, by acting in the space between the outer membranes of bacterial cells, can directly reduce nitrite to ammonia.

Figure 1 presents a conceptual schematic of the inorganic nitrogen cycle, revealing the pathways of nitrogen transformation in the natural environment. The nitrogen cycle primarily includes: (1) atmospheric nitrogen fixation, (2) mineralization of organic nitrogen, (3) nitrification, (4) denitrification, (5) ammonification of nitrate, (6) anaerobic ammonium oxidation, and (7) assimilation of ammonia and nitrate. The products of each stage and some intermediate products are indicated by their chemical names in the diagram. Nitrogen is lost from ecosystems through leaching of nitrate and gaseous losses. This cyclic process is fundamental for understanding the flow of nitrogen in soil, water, and air and its impact on ecosystems.

Figure 1 Schematic illustration of the inorganic nitrogen cycle

Figure 2 provides a detailed depiction of the dimeric structure of the NrfA enzyme in Escherichia coli and its role in the electron transport chain from formate to nitrite. Panel A shows the three-dimensional structure of the NrfA homodimer, each monomer containing five heme groups, along with calcium ions and iron atoms. Panel B illustrates the electron transfer during the respiratory nitrite ammonification process, involving NrfH or NrfBCD as electron carriers. Through this process, formate is oxidized on the cell membrane, generating proton motive force that drives electrons through the complex, ultimately achieving the reduction of nitrite. This complex reaction process is crucial for intracellular energy conversion and material cycling.

Figure 2 Structure of NrfA and schematics of the enzyme complexes involved in the electron transport chain from formate to nitrite

Figure 3 shows the maximum likelihood phylogenetic tree inferred from the alignment of 1 150 complete NrfA sequences within 1 121 genomic assemblies, based on 350 amino acid positions. Different bacterial phyla are identified by colored bands in the outer ring, with the color coding based on genomic classification databases. Phyla represented by fewer than 10 sequences are labeled as "Others" and shown in gray. Gray branches represent those sequences that contain a Cys-X-X-Cys-His variant at the first heme-binding site. Blue stars mark known bacterial isolates that can reduce nitrate or nitrite to ammonia, while yellow stars indicate bacterial isolates with a determined NrfA crystal structure. The scale bar represents the rate of amino acid substitution (WAG+R10). Outgroups are not shown. This phylogenetic tree reveals the rich diversity of NrfA sequences and their distribution across different bacterial phyla.

Figure 3 Maximum likelihood phylogeny of 1150 full-length NrfA sequences from 1121 genome assemblies inferred from the alignment of 350 amino acid positions

Figure 4 illustrates the distribution of nrfA sequence fragments from metagenomes across different biomes through a phylogenetic tree. Part A shows the distribution in aquatic environments, while Part B displays terrestrial biomes. Bacterial phyla are color-coded in the outer ring based on genomic classification databases. In the diagram, the size of the dots represents the proportion of sequences in the corresponding biome, providing a visual reference for quantitative analysis. This combination of categorization and quantification lays the foundation for studying the ecology and evolution of NrfA-dependent ammonifying microorganisms in various biomes. The scale represents the rate of amino acid substitution, a common metric for comparing gene sequence differences. Outgroup sequences distinctly different from other biomes are not shown in the diagram.

Figure 4 Phylogenetic placement of the metagenomic nrfA sequence fragments across biomes

2 Analysis of Research Findings

This study provides a comprehensive insight into how NrfA-dependent ammonifying microorganisms facilitate the nitrogen cycle. These microorganisms not only help retain nitrogen within ecosystems but also contribute to reducing greenhouse gas emissions by minimizing nitrate loss.

3 Evaluation of the Research

This study is methodologically rigorous and addresses significant gaps in our understanding of nitrogen-cycling microorganisms. However, the research would benefit from broader geographic sampling to enhance the generalizability of the findings.

4 Conclusions

NrfA-dependent ammonifying microorganisms are crucial in maintaining nitrogen within ecosystems, impacting environmental health and agricultural practices. Further research is needed to fully leverage their potential for effective nitrogen management across various biotic communities.

5 Access the Full Text

Saghaï A., and Hallin S., 2024, Diversity and ecology of NrfA-dependent ammonifying microorganisms, Trends in Microbiology, 32(6): 602-613. DOI: 10.1016/j.tim.2024.02.007.

Acknowledgments

The authors sincerely thank the Trends in Microbiology journal for providing open access to their papers, especially the research on the diversity and ecology of NrfA-dependent ammonifying microorganisms by Aurélien Saghaï and Sara Hallin (2024). This has given us the opportunity to gain an in-depth understanding and share the latest findings in this field with our peers, contributing to the advancement of the scientific community.