Summary We identify the interfollicular (IF) zone as the site where germinal center B cell and T follicular helper (Tfh) cell differentiation initiates. For the first two days post-immunization, antigen-specific T and B cells remained confined within the IF zone, formed long-lived interactions, and upregulated the transcriptional repressor Bcl6. T cells also acquired the Tfh cell markers CXCR5, PD-1 and GL7. Responding B and T cells migrated to the follicle interior directly from the IF zone, T cell immigration preceding B cells by one day. Notably, in the absence of cognate B cells, Tfh cells still formed and migrated to the follicle. However, without such B cells, PD-1, ICOS and GL7 were no longer expressed on follicular Bcl6hi T cells that nevertheless persisted in the follicle. Thus, Ag-specific B cells are required for the maintenance of the PD-1hi ICOShi GL7hi Tfh cell phenotype within the follicle, but not for their initial differentiation in the IF zone.

Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels, and chemicals1. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems2,3, which include bioremediation4 and probiotics5. Although safeguards have been designed to control cell growth by essential gene regulation6, inducible toxin switches7, and engineered auxotrophies8, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations9,10. Here, we describe the construction of a series of genomically recoded organisms (GROs)11 whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase (aaRS) pair into the chromosome of a GRO that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering (MAGE)12, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harboring 3 TAG codons in conserved functional residues13 of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼1011 cells on solid media for seven days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches2,3,6-10. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternate genetic codes that impart genetic isolation by impeding horizontal gene transfer11 and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-l-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

Precolibactins and colibactins represent a family of natural products that are encoded by the clb gene cluster and are produced by certain commensal, extraintestinal, and probiotic E. coli. clb+ E. coli induce megalocytosis and DNA double-strand breaks in eukaryotic cells, but paradoxically, this gene cluster is found in the probiotic Nissle 1917. Evidence suggests precolibactins are converted to genotoxic colibactins by colibactin peptidase (ClbP)-mediated cleavage of an N-acyl- D-Asn side chain, and all isolation efforts have employed ΔclbP strains to facilitate accumulation of precolibactins. It was hypothesized that colibactins form unsaturated imines that alkylate DNA by cyclopropane ring opening (2 → 3). However, as no colibactins have been isolated, this hypothesis has not been tested experimentally. Additionally, precolibactins A–C (7–9) contain a pyridone that cannot generate the unsaturated imines that form the basis of this hypothesis. To resolve this, we prepared 13 synthetic colibactin derivatives and evaluated their DNA binding and alkylation activity. We show that unsaturated imines, but not the corresponding pyridone derivatives, potently alkylate DNA. The imine, unsaturated lactam, and cyclopropane are essential for efficient DNA alkylation. A cationic residue enhances activity. These studies suggest that precolibactins containing a pyridone are not responsible for the genotoxicity of the clb cluster. Instead, we propose that these are off-pathway fermentation products produced by a facile double cyclodehydration route that manifests in the absence of viable ClbP. The results presented herein provide a foundation to begin to connect metabolite structure with the disparate phenotypes associated with clb+ E. coli.

Genetically modified organisms (GMOs) are commonly used to produce valuable compounds in closed industrial systems. However, their emerging applications in open clinical or environmental settings require enhanced safety and security measures. Intrinsic biocontainment, the creation of bacterial hosts unable to survive in natural environments, remains a major unsolved biosafety problem. We developed a new biocontainment strategy containing overlapping ‘safeguards’—engineered riboregulators that tightly control expression of essential genes, and an engineered addiction module based on nucleases that cleaves the host genome—to restrict viability of Escherichia coli cells to media containing exogenously supplied synthetic small molecules. These multilayered safeguards maintain robust growth in permissive conditions, eliminate persistence and limit escape frequencies to <1.3 × 10−12. The staged approach to safeguard implementation revealed mechanisms of escape and enabled strategies to overcome them. Our safeguarding strategy is modular and employs conserved mechanisms that could be extended to clinically or industrially relevant organisms and undomesticated species.