Dietary intake of l-carnitine can promote cardiovascular diseases in humans through microbial production of trimethylamine (TMA) and its subsequent oxidation to trimethylamine N-oxide by hepatic flavin-containing monooxygenases. Although our microbiota are responsible for TMA formation from carnitine, the underpinning molecular and biochemical mechanisms remain unclear. In this study, using bioinformatics approaches, we first identified a two-component Rieske-type oxygenase/reductase (CntAB) and associated gene cluster proposed to be involved in carnitine metabolism in representative genomes of the human microbiota. CntA belongs to a group of previously uncharacterized Rieske-type proteins and has an unusual “bridging” glutamate but not the aspartate residue, which is believed to facilitate intersubunit electron transfer between the Rieske center and the catalytic mononuclear iron center. Using Acinetobacter baumannii as the model, we then demonstrate that cntAB is essential in carnitine degradation to TMA. Heterologous overexpression of cntAB enables Escherichia coli to produce TMA, confirming that these genes are sufficient in TMA formation. Site-directed mutagenesis experiments have confirmed that this unusual “bridging glutamate” residue in CntA is essential in catalysis and neither mutant (E205D, E205A) is able to produce TMA. Taken together, the data in our study reveal the molecular and biochemical mechanisms underpinning carnitine metabolism to TMA in human microbiota and assign the role of this novel group of Rieske-type proteins in microbial carnitine metabolism. Significance Metabolism of l-carnitine, a compound abundant in human diet, to trimethylamine by human microbiota has been shown to promote atherosclerosis and subsequent development of heart disease. However, the underpinning molecular and biochemical mechanisms remain unknown. In this study, we reveal that a previously unidentified Rieske-type protein is responsible for carnitine transformation to trimethylamine from human microbiota. Knowledge gained in our study provides the opportunity not only to explore Rieske protein inhibitors in preventing trimethylamine formation in animal studies and clinical trials, but also for its use as a functional genetic marker to better understand human microbiota and their dynamics in our health and disease in future epidemiological studies and dietary interventions.

The investigation of the microbial populations of the human body, known as the microbiome, has led to a revolutionary field of science, and understanding of its impacts on human development and health. The majority of microbiome research to date has focussed on bacteria and other kingdoms of life, such as fungi. Trailing behind these is the interrogation of the gut viruses, specifically the phageome. Bacteriophages, viruses that infect bacterial hosts, are known to dictate the dynamics and diversity of bacterial populations in a number of ecosystems. However, the phageome of the human gut, while of apparent importance, remains an area of many unknowns. In this paper we discuss the role of bacteriophages within the human gut microbiome. We examine the methods used to study bacteriophage populations, how this evolved over time and what we now understand about the phageome. We review the phageome development in infancy, and factors that may influence phage populations in adult life. The role and action of the phageome is then discussed at both a biological-level, and in the broader context of human health and disease.

Introduction: Klebsiella is a clinically important pathogen causing a variety of antimicrobial resistant infections in both community and nosocomial settings, particularly pneumonia, urinary tract infection, and sepsis. Bacteriophage (phage) therapy is being considered a primary option for the treatment of drug-resistant infections of these types. Methods: We report the successful isolation and characterization of 30 novel, genetically diverse Klebsiella phages. Results: The isolated phages span six different phage families and nine genera, representing both lysogenic and lytic lifestyles. Individual Klebsiella phage isolates infected up to 11 of the 18 Klebsiella capsule types tested, and all 18 capsule-types were infected by at least one of the phages. Conclusions: Of the Klebsiella -infecting phages presented in this study, the lytic phages are most suitable for phage therapy, based on their broad host range, high virulence, short lysis period and given that they encode no known toxin or antimicrobial resistance genes. Phage isolates belonging to the Sugarlandvirus and Slopekvirus genera were deemed most suitable for phage therapy based on our characterization. Importantly, when applied alone, none of the characterized phages were able to suppress the growth of Klebsiella for more than 12 h, likely due to the inherent ease of Klebsiella to generate spontaneous phage-resistant mutants. This indicates that for successful phage therapy, a cocktail of multiple phages would be necessary to treat Klebsiella infections.

Existing metagenome datasets from many different environments contain untapped potential for understanding metabolic pathways and their biological impact. Our interest lies in the formation of trimethylamine (TMA), a key metabolite in both human health and climate change. Here, we focus on bacterial degradation pathways for choline, carnitine, glycine betaine and trimethylamine N-oxide (TMAO) to TMA in human gut and marine metagenomes. We found the TMAO reductase pathway was the most prevalent pathway in both environments. Proteobacteria were found to contribute the majority of the TMAO reductase pathway sequences, except in the stressed gut, where Actinobacteria dominated. Interestingly, in the human gut metagenomes, a high proportion of the Proteobacteria hits were accounted for by the genera Klebsiella and Escherichia. Furthermore Klebsiella and Escherichia harboured three of the four potential TMA-production pathways (choline, carnitine and TMAO), suggesting they have a key role in TMA cycling in the human gut. In addition to the intensive TMAO–TMA cycling in the marine environment, our data suggest that carnitine-to-TMA transformation plays an overlooked role in aerobic marine surface waters, whereas choline-to-TMA transformation is important in anaerobic marine sediments. Our study provides new insights into the potential key microbes and metabolic pathways for TMA formation in two contrasting environments.

The rumen contains a multi-kingdom, commensal microbiome, including protozoa, bacteria, archaea, fungi and viruses, which enables ruminant herbivores to ferment and utilize plant feedstuffs that would be otherwise indigestible. Within the rumen, virus populations are diverse and highly abundant, often out-numbering the microbial populations that they both predate on and co-exist with. To date the research effort devoted to understanding rumen-associated viral populations has been considerably less than that given to the other microbial populations, yet their contribution to maintaining microbial population balance, intra-ruminal microbial lysis, fiber breakdown, nutrient cycling and genetic transfer may be highly significant. This review follows the technological advances which have contributed to our current understanding of rumen viruses and drawing on knowledge from other environmental and animal-associated microbiomes, describes the known and potential roles and impacts viruses have on rumen function and speculates on the future directions of rumen viral research.

Background: The rhizosphere is a hotspot for microbial activity and contributes to ecosystem services including plant health and biogeochemical cycling. The activity of microbial viruses, and their influence on plant-microbe interactions in the rhizosphere, remains undetermined. Given the impact of viruses on the ecology and evolution of their host communities, determining how soil viruses influence microbiome dynamics is crucial to build a holistic understanding of rhizosphere functions. Results: Here, we aimed to investigate the influence of crop management on the composition and activity of bulk soil, rhizosphere soil, and root viral communities. We combined viromics, metagenomics, and metatranscriptomics on soil samples collected from a 3-year crop rotation field trial of oilseed rape (Brassica napus L.). By recovering 1059 dsDNA viral populations and 16,541 ssRNA bacteriophage populations, we expanded the number of underexplored Leviviricetes genomes by > 5 times. Through detection of viral activity in metatranscriptomes, we uncovered evidence of “Kill-the-Winner” dynamics, implicating soil bacteriophages in driving bacterial community succession. Moreover, we found the activity of viruses increased with proximity to crop roots, and identified that soil viruses may influence plant-microbe interactions through the reprogramming of bacterial host metabolism. We have provided the first evidence of crop rotation-driven impacts on soil microbial communities extending to viruses. To this aim, we present the novel principal of “viral priming,” which describes how the consecutive growth of the same crop species primes viral activity in the rhizosphere through local adaptation. Conclusions: Overall, we reveal unprecedented spatial and temporal diversity in viral community composition and activity across root, rhizosphere soil, and bulk soil compartments. Our work demonstrates that the roles of soil viruses need greater consideration to exploit the rhizosphere microbiome for food security, food safety, and environmental sustainability.