Evolutionary, ecological and biotechnological perspectives on plasmids resident in the human gut mobile metagenome

Numerous mobile genetic elements (MGE) are associated with the human gut microbiota and collectively referred to as the gut mobile metagenome. The role of this flexible gene pool in development and functioning of the gut microbial community remains largely unexplored, yet recent evidence suggests that at least some MGE comprising this fraction of the gut microbiome reflect the co-evolution of host and microbe in the gastro-intestinal tract. In conjunction, the high level of novel gene content typical of MGE coupled with their predicted high diversity, suggests that the mobile metagenome constitutes an immense and largely unexplored gene-space likely to encode many novel activities with potential biotechnological or pharmaceutical value, as well as being important to the development and functioning of the gut microbiota. Of the various types of MGE that comprise the gut mobile metagenome, plasmids are of particular importance since these elements are often capable of autonomous transfer between disparate bacterial species, and are known to encode accessory functions that increase bacterial fitness in a given environment facilitating bacterial adaptation. In this article current knowledge regarding plasmids resident in the human gut mobile metagenome is reviewed, and available strategies to access and characterize this portion of the gut microbiome are described. The relative merits of these methods and their present as well as prospective impact on our understanding of the human gut microbiota is discussed

Exclusion of T4 phage by the hok/sok killer locus from plasmid R1.

The hok (host killing) and sok (suppressor of killing) genes (hok/sok) efficiently maintain the low-copy-number plasmid R1. To investigate whether the hok/sok locus evolved as a phage-exclusion mechanism, Escherichia coli cells that contain hok/sok on a pBR322-based plasmid were challenged with T1, T4, T5, T7, and lambda phage. Upon infection with T4, the optical density of cells containing hok/sok on a high-copy-number plasmid continued to increase whereas the optical density for those lacking hok/sok rapidly declined. The presence of hok/sok reduced the efficiency of plating of T4 by 42% and decreased the plaque size by approximately 85%. Single-step growth experiments demonstrated that hok/sok decreased the T4 burst size by 40%, increased the time to form mature phage (eclipse time) from 22 to 30 min, and increased the time to cell lysis (latent period) from 30 to 60 min. These results further suggest that single cells exhibit altruistic behavior

In Vivo Gene Expression Dynamics of Tumor-Targeted Bacteria

The engineering of bacteria to controllably deliver therapeutics is an attractive application for synthetic biology. While most synthetic gene networks have been explored within microbes, there is a need for further characterization of in vivo circuit behavior in the context of applications where the host microbes are actively being investigated for efficacy and safety, such as tumor drug delivery. One major hurdle is that culture-based selective pressures are absent in vivo, leading to strain-dependent instability of plasmid-based networks over time. Here, we experimentally characterize the dynamics of in vivo plasmid instability using attenuated strains of S. typhimurium and real-time monitoring of luminescent reporters. Computational modeling described the effects of growth rate and dosage on live-imaging signals generated by internal bacterial populations. This understanding will allow us to harness the transient nature of plasmid-based networks to create tunable temporal release profiles that reduce dosage requirements and increase the safety of bacterial therapies.Misrock Foundation (Postdoctoral Fellowship)National Institutes of Health (U.S.) (Medical Scientist Training Program)Massachusetts Institute of Technology. Ludwig Center for Molecular Oncology (Graduate Fellowship)Marie D. and Pierre Casimir-Lambert FundHoward Hughes Medical Institute (Investigator