The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens

Vibrio natriegens is known as the world's fastest growing organism with a doubling time of less than 10 minutes. This incredible growth speed empowers V. natriegens as a chassis for synthetic and molecular biology, potentially replacing E. coli in many applications. While first genetic parts have been built and tested for V. natriegens, a comprehensive toolkit containing well-characterized and standardized parts did not exist. To close this gap, we created the Marburg Collection - a highly flexible Golden Gate Assembly-based cloning toolbox optimized for the emerging chassis organism V. natriegens. The Marburg Collection overcomes the paradigm of plasmid construction - integrating inserts into a backbone - by enabling the de novo assembly of plasmids from basic genetic parts. This allows users to select the plasmid replication origin and resistance part independently, which is highly advantageous when limited knowledge about the behavior of those parts in the target organism is available. Additional design highlights of the Marburg Collection are novel connector parts, which facilitate modular circuit assembly and, optionally, the inversion of individual transcription units to reduce transcriptional crosstalk in multigene constructs. To quantitatively characterize the genetic parts contained in the Marburg Collection in V. natriegens, we developed a reliable microplate reader measurement workflow for reporter experiments and overcame organism-specific challenges. We think the Marburg Collection with its thoroughly characterized parts will provide a valuable resource for the growing V. natriegens community.

Conjugative plasmid-encoded toxin–antitoxin system PrpT/PrpA directly controls plasmid copy number

Toxin–antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin–antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

Discovery of small-molecule inhibitors of multidrug-resistance plasmid maintenance using a high-throughput screening approach

Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug-resistant pathogens for which new treatments are desperately needed. Carbapenemases and other types of antibiotic resistance genes are carried almost exclusively on large, low-copy-number plasmids (pCRE). Accordingly, small molecules that efficiently evict pCRE plasmids should restore much-needed treatment options. We therefore designed a high-throughput screen to identify such compounds. A synthetic plasmid was constructed containing the plasmid replication machinery from a representativeEscherichia coliCRE isolate as well as a fluorescent reporter gene to easily monitor plasmid maintenance. The synthetic plasmid was then introduced into anE. coliK12tolChost. We used this screening strain to test a library of over 12,000 known bioactive agents for molecules that selectively reduce plasmid levels relative to effects on bacterial growth. From 366 screen hits we further validated the antiplasmid activity of kasugamycin, an aminoglycoside; CGS 15943, a nucleoside analog; and Ro 90-7501, a bibenzimidazole. All three compounds exhibited significant antiplasmid activity including up to complete suppression of plasmid replication and/or plasmid eviction in multiple orthogonal readouts and potentiated activity of the carbapenem, meropenem, against a strain carrying the large, pCRE plasmid from which we constructed the synthetic screening plasmid. Additionally, we found kasugamycin and CGS 15943 blocked plasmid replication, respectively, by inhibiting expression or function of the plasmid replication initiation protein, RepE. In summary, we validated our approach to identify compounds that alter plasmid maintenance, confer resensitization to antimicrobials, and have specific mechanisms of action.

Increased copy number couples the evolution of plasmid horizontal transmission and antibiotic resistance

Antimicrobial resistance (AMR) in bacteria is commonly encoded on conjugative plasmids, mobile elements which can spread horizontally between hosts. Conjugative transfer disseminates AMR in communities but it remains unclear when and how high transfer rates evolve, and with which consequences. Here we studied experimentally the evolution of two antibiotic resistance encoding plasmids when confronted to different immigration rates of susceptible, plasmid-free hosts. While plasmid RP4 did not evolve detectably, plasmid R1 rapidly evolved up to 1000-fold increased transfer rates in the presence of susceptible hosts, at a cost to its host. Unexpectedly, most evolved plasmids also conferred to their hosts the ability to grow at high concentrations of antibiotics. The most common mutations in evolved plasmids were contained within the copA gene which controls plasmid replication and copy number. Evolved copA variants had elevated copy number, leading to both higher transfer rates and AMR. Due to these pleiotropic effects, host availability and antibiotics were each sufficient to select for highly transmissible plasmids conferring high levels of antibiotic resistance.

Structural Basis for Cooperative Binding of EBNA1 to the Epstein-Barr Virus Dyad Symmetry Minimal Origin of Replication

ABSTRACTEpstein-Barr virus is associated with several human malignancies, including nasopharyngeal carcinoma, gastric cancer, and lymphoma. Latently infected cells carry a circularized EBV episome where the origin of replication (oriP) is comprised of two elements: the family of repeats (FR) and dyad symmetry (DS). The viral protein Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) binds to FR and DS to promote EBV episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 binding to the DS constitutes a minimal origin of DNA replication. Here we report the crystal structure of two EBNA1 DNA-binding domain dimers bound to a DS half-site. This structure shows that the DNA is smoothly bent, allowing for stabilizing interactions between the dimers. The dimer-dimer interface requires an intricate hydrogen bonding network involving residues R491 and D581. When this interface is disrupted, we note loss of stable dimer-dimer complex formation on the DNA, compromisedoriP-containing plasmid replication in cells, and impaired recruitment of the MCM3 complex to theoriP. Surface conservation analysis reveals that these residues are part of a larger conserved surface that may be critical for recruitment of replication machinery to theoriP. Our results reveal a new region of EBNA1 critical for its activity and one that may be exploited by targeted small molecules to treat EBV-associated disease.IMPORTANCEEpstein-Barr virus (EBV) is a causative agent of various malignancies and may also contribute to autoimmune disease. The latent and episomal form of the virus is known to drive EBV-associated oncogenesis. Persistence of the viral episome in proliferating tumor cells requires the interaction of Epstein-Barr virus nuclear antigen 1 (EBNA1) with the viral origin of plasmid replication (oriP). The dyad symmetry (DS) element inoriPis the essential minimal replicator oforiP. Here we report the X-ray crystal structure of EBNA1 bound to DS. The structure reveals a previous unrecognized interface formed between dimers of EBNA1 necessary for cooperative DNA binding, recruitment of cellular replication machinery, and replication function. These findings provide new insights into the mechanism of EBNA1 function at the replication origin and new opportunities to inhibit EBV latent infection and pathogenesis.