The gut microbial gbu gene cluster participates in red meat diet-enhanced cardiovascular disease risk

L-carnitine, a nutrient abundant in red meat, is metabolized by gut microbiota to form trimethylamine (TMA), and the pro-atherogenic and pro-thrombotic metabolite trimethylamine N-oxide (TMAO). Gut microbiota conversion of carnitine into TMA occurs in omnivores, but far less so in vegetarians or vegans. Previous studies revealed the carnitine -> TMA microbial transformation occurs via a two-step process, wherein multiple microbes can convert dietary L-carnitine into an intermediate, gamma-butyrobetaine (g-BB), but relatively few have capacity to transform g-BB into TMA. The heightened risk for cardiovascular disease (CVD) observed with omnivores compared to vegetarians or vegans is thought to be linked, in part, to gut microbiota-dependent TMAO generation from carnitine. Despite the central role of g-BB in this transformation, neither the relationship of g-BB to CVD risks in subjects, nor the gut microbial genes responsible for g-BB transformation into TMA in omnivores are known. In vitro fecal culturing alongside studies in germ free mice with defined synthetic communities, shows introduction of Emergencia timonensis, which can promote g-BB transformation into TMA, is sufficient to impart polymicrobial communities with the capacity to complete the carnitine -> g-BB -> TMA transformation, elevate TMAO levels, and enhance thrombosis potential in recipient animals following arterial injury. RNAseq analyses of E. timonensis revealed a 6 gene cluster, herein named gamma-butyrobetaine utilization gene cluster (gbu), which is uniquely upregulated in response to g-BB. Subsequent combinatorial cloning and functional expression studies identified a minimum contingent of 4 genes in this operon (gbuA, gbuB, gbuC, and gbuE) are necessary and sufficient to recapitulate the conversion of g-BB ->TMA when co-expressed in E. coli.