Scientists unveil the hidden respiratory mechanisms of gut bacteria

In a recent study published in Nature Microbiology, researchers use a genome-mining technique to investigate respiratory electron acceptor utilization in the human gut microbiota.

Study: Dietary- and host-derived metabolites are used by diverse gut bacteria for anaerobic respiration. Image Credit: Kateryna Kon / Shutterstock.com

How microorganisms generate energy

Heterotrophic respiration involves oxidation and electron transport, which creates an ion gradient for adenosine triphosphate (ATP) production. Respiratory reductases are required for microorganisms to use anaerobic ecosystem chemicals as energy-generating electron acceptors.

Microorganisms that lack oxygen employ alternate electron acceptors. Fermentation metabolism dominates the anaerobic gut microbial community; however, certain conventional respiratory metabolism reactions also occur. Sulfate-reducing bacteria use different sulfate electron acceptors, whereas immune cells create electron acceptors in the inflamed gut.

About the study

In the present study, researchers develop a respiratory method using a vast reductase arsenal to access various metabolite electron acceptors. A total of 1,533 metagenome-assembled genomes from various human gut prokaryotes were examined to determine whether gut microbes with many reductases for each genome could breathe.

To this end, the E. lenta, S. wadsworthensis, and H. filiformis strains were selected for analysis. Compounds with electron-accepting qualities in the gastrointestinal tract were investigated, in addition to common respiratory electron donors, for their capacity to trigger urocanate-dependent growth promotion.

Thirteen flavin-type reductases detected in proteomic and transcriptomic investigations were recombinantly generated to investigate whether gene expression patterns might predict enzyme substrate selectivity. Due to low itaconate-induced reductase (IrdA) yields in E. lenta, two closely related orthologues of IrdA were expressed from Berryella wangjianweii and Adlercreutzia muris. Purified reductases against known electron acceptors were also tested.

The link between reductase evolution and substrate specificity was investigated using flavin reductases from the H. filiformis, S. wadsworthensis, and E. lenta genomes. Mechanistic investigations were performed to explore distinct evolutionary paths that may have resulted in reductases with equal cinnamate substrate specificities.

Point mutants targeting conserved amino acids in representative cinnamate reductases were generated from the four reductase clades to determine whether the various patterns of active-site conservation in the four reductase clades reflected mechanistic differences.

Study findings

Three taxonomically different human gut bacterial families of Erysipelotrichaceae, Burkholderiaceae, and Eggerthellaceae with an arsenal of respiratory-like reductase enzymes in tens to hundreds were identified. A total of 22 compounds were utilized to accept respiratory electrons in species-specific ways by screening species of every bacterial family, including Holdemania filiformis, Sutterella wadsworthensis, and Eggerthella lenta. These processes catalyze the transformation of various host- and diet-obtained metabolites, including the beneficial compounds itaconate and resveratrol.

Products of known respiratory metabolisms, such as itaconate-obtained 2-methyl succinate, highlight poorly understood molecules. Reductase substrate profiling specifies enzyme-substrate pairings and presents a complicated image of reductase development, thus demonstrating that reductases specific for similar cinnamate substrates evolved independently four or more times. Distantly related bacteria encode extensive reductase arsenals, with most flavin and molybdopterin respiratory reductases possessing an N-terminal signal peptide indicative of extra-cytosolic location.

In the Actinobacteria family Eggerthellaceae, Proteobacteria family Burkholderiaceae, and Firmicutes family Erysipelotrichaceae, high-reductase clades comprise three separate clades that span many genera. These high-reductase clades consist of bacteria with more than 200 reductases for each genome and intricate reductase gain-and-loss patterns, thereby indicating a complicated evolutionary history.

Respiratory growth was observed in bacteria producing a flavin reductase with over 50% sequence homology to a previously identified respiratory urocanate reductase (UrdA) encoded within each strain’s genome. Species-specific consumption patterns were also observed for several substrate types.

Reductase substrates and products were found in feces, with different chemicals found in mouse and human samples, although at lower amounts in antibiotic-treated groups. Respiratory electron acceptors preferentially stimulate cinnamate reductase in H. filiformis and E. lenta, thus demonstrating their evolutionary complexity.

The geographic distribution of reductase activity reflects a complex history, including cinnamate reductases exemplifying the intricate association between substrate selectivity and amino acid sequence. 

Amino acid molecules conserved at active sites across clades were required for activity and revealed unique active-site designs that functionally separated cinnamate reductases. Flavin reductases of similar substrate specificity with certain functional differences were created through parallel evolutionary processes. Broad-spectrum antibiotic exposure significantly affected the gut microbiome composition in samples with over 90% relative abundances of Enterococcus and Proteobacteria.

Conclusions

The study findings highlight a novel type of anaerobic respiration that associates microbial energy metabolism with the gut metabolome. The gut microbiome consists of different organisms with several respiratory-like reductase enzymes in their genomes, respiratory metabolisms, and strain-dependent utilization patterns. This form of respiration may be crucial to the gut by participating in the regulation of enzymes and transcription factors, as well as metabolic and immunological processes.