Recent advances in synthetic biology have made it possible to create a bacterium programmed by a synthetic genome. Past work demonstrates synthesis of a whole bacterial genome in yeast, and subsequent genome transfer into bacterial cells. However, the genome transfer has only worked for a small closely related group of Mycoplasma species whose chromosome is relatively small (roughly 1-Mb). To enable the genome transfer approach in E. coli, whose chromosome size is originally 4.6-Mb, the authors developed a E. coli strain programed by three 1-Mb split-chromosomes. Each split-chromosome was purified as supercoiled DNA molecules. Two of these split-chromosomes were then individually transferred into E. coli cells by electroporation. This method could accelerate the booting up of synthetic genomes in commonly used bacteria.
NAR’s Breakthrough Articles present high-impact studies answering long-standing questions in the field of nucleic acids research and/or opening up new areas and mechanistic hypotheses for investigation. These articles are chosen by the Editors on the recommendation of Editorial Board Members and Referees. Articles are accompanied by a brief synopsis explaining the findings of the paper and where they fit in the broader context of nucleic acids research. They represent the very best papers published at NAR.
Fungal tRNA ligases generate a 2’-PO4 splice junction that is converted into a 2’-OH in a two-step reaction by members of the Tpt1 family of NAD+-dependent 2’-phosphotransferases. Tpt1 enzymes are essential components of the fungal tRNA splicing machinery and thus promising targets for antifungal drug discovery. How Tpt1 recognizes its substrates NAD+ and 2’-PO4-modified RNA, and potential conformational changes accompanying substrate binding, are ill-defined. Here, we utilized solution NMR and complementary biophysical approaches to provide new mechanistic insights into NAD+-recognition by a Tpt1 homolog from the Gram-negative bacterium Runella slithyformis, culminating in structures of free Tpt1 and a Tpt1•NAD+ binary complex. The importance of enzymic contacts to NAD+ were validated by mutagenesis.
The emergence of multidrug resistant bacteria is fuelled by diverse mobile genetic elements including genomic islands and conjugative plasmids that often carry many antibiotic resistance genes. Mobilizable genomic islands ('MGIs') such as Salmonella Genomic Island 1 (SGI1) confer resistance to a wide range of antibacterial agents, while IncC plasmids are large, broad-host-range, and globally distributed plasmids that contribute to the propagation of multidrug resistance genes. MGIs are usually thought of as relatively passive mobile genetic elements that await the arrival of a helper self-transmissible element (often a conjugative plasmid) for horizontal transfer. To accomplish this, MGIs need to excise from the host’s chromosome and be translocated through a pore encoded by their helper element. This study describes details of a genetic regulatory mechanism by which SGI1 actively associates itself with a conjugative lncC plasmid in order to undergo horizontal transfer.
