Bacterial Killers Engineered to Exterminate Pathogenic Microbes.

A new study reports a synthetic bacterium that uses conjugation to transfer toxic genes that selectively kill pathogenic cells. The work represents a novel strategy for targeting pathogens, which could be the basis for a new generation of precision antimicrobials.

A suppressor tRNA-mediated feedforward loop eliminates leaky gene expression in bacteria.

Ligand-inducible genetic systems are the mainstay of synthetic biology, allowing gene expression to be controlled by the presence of a small molecule. However, 'leaky' gene expression in the absence of inducer remains a persistent problem. We developed a leak dampener tool that drastically reduces the leak of inducible genetic systems while retaining signal in Escherichia coli. Our system relies on a coherent feedforward loop featuring a suppressor tRNA that enables conditional readthrough of silent non-sense mutations in a regulated gene, and this approach can be applied to any ligand-inducible transcription factor. We demonstrate proof-of-principle of our system with the lactate biosensor LldR and the arabinose biosensor AraC, which displayed a 70-fold and 630-fold change in output after induction of a fluorescence reporter, respectively, without any background subtraction. Application of the tool to an arabinose-inducible mutagenesis plasmid led to a 540-fold change in its output after induction, with leak decreasing to the level of background mutagenesis. This study provides a modular tool for reducing leak and improving the fold-induction within genetic circuits, demonstrated here using two types of biosensors relevant to cancer detection and genetic engineering.

Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase.

Pyrrolysyl-tRNA synthetase (PylRS) is a major tool in genetic code expansion using noncanonical amino acids, yet its structure and function are not completely understood. Here we describe the crystal structure of the previously uncharacterized essential N-terminal domain of this unique enzyme in complex with tRNAPyl. This structure explains why PylRS remains orthogonal in a broad range of organisms, from bacteria to humans. The structure also illustrates why tRNAPyl recognition by PylRS is anticodon independent: the anticodon does not contact the enzyme. Then, using standard microbiological culture equipment, we established a new method for laboratory evolution-a noncontinuous counterpart of the previously developed phage-assisted continuous evolution. With this method, we evolved novel PylRS variants with enhanced activity and amino acid specificity. Finally, we employed an evolved PylRS variant to determine its N-terminal domain structure and show how its mutations improve PylRS activity in the genetic encoding of a noncanonical amino acid.

Exploring the substrate range of wild-type aminoacyl-tRNA synthetases.

We tested the substrate range of four wild-type E. coli aminoacyl-tRNA synthetases (AARSs) with a library of nonstandard amino acids (nsAAs). Although these AARSs could discriminate efficiently against the other canonical amino acids, they were able to use many nsAAs as substrates. Our results also show that E. coli tryptophanyl-tRNA synthetase (TrpRS) and tyrosyl-tRNA synthetase have overlapping substrate ranges. In addition, we found that the nature of the anticodon sequence of tRNA(Trp) altered the nsAA substrate range of TrpRS; this implies that the sequence of the anticodon affects the TrpRS amino acid binding pocket. These results highlight again that inherent AARS polyspecificity will be a major challenge in the aim of incorporating multiple different amino acids site-specifically into proteins.

Recoding the genetic code with selenocysteine.

Selenocysteine (Sec) is naturally incorporated into proteins by recoding the stop codon UGA. Sec is not hardwired to UGA, as the Sec insertion machinery was found to be able to site-specifically incorporate Sec directed by 58 of the 64 codons. For 15 sense codons, complete conversion of the codon meaning from canonical amino acid (AA) to Sec was observed along with a tenfold increase in selenoprotein yield compared to Sec insertion at the three stop codons. This high-fidelity sense-codon recoding mechanism was demonstrated for Escherichia coli formate dehydrogenase and recombinant human thioredoxin reductase and confirmed by independent biochemical and biophysical methods. Although Sec insertion at UGA is known to compete against protein termination, it is surprising that the Sec machinery has the ability to outcompete abundant aminoacyl-tRNAs in decoding sense codons. The findings have implications for the process of translation and the information storage capacity of the biological cell.

C-terminal domain of archaeal O-phosphoseryl-tRNA kinase displays large-scale motion to bind the 7-bp D-stem of archaeal tRNA(Sec).

O-Phosphoseryl-tRNA kinase (PSTK) is the key enzyme in recruiting selenocysteine (Sec) to the genetic code of archaea and eukaryotes. The enzyme phosphorylates Ser-tRNA(Sec) to produce O-phosphoseryl-tRNA(Sec) (Sep-tRNA(Sec)) that is then converted to Sec-tRNA(Sec) by Sep-tRNA:Sec-tRNA synthase. Earlier we reported the structure of the Methanocaldococcus jannaschii PSTK (MjPSTK) complexed with AMPPNP. This study presents the crystal structure (at 2.4-Å resolution) of MjPSTK complexed with an anticodon-stem/loop truncated tRNA(Sec) (Mj*tRNA(Sec)), a good enzyme substrate. Mj*tRNA(Sec) is bound between the enzyme's C-terminal domain (CTD) and N-terminal kinase domain (NTD) that are connected by a flexible 11 amino acid linker. Upon Mj*tRNA(Sec) recognition the CTD undergoes a 62-Å movement to allow proper binding of the 7-bp D-stem. This large reorganization of the PSTK quaternary structure likely provides a means by which the unique tRNA(Sec) species can be accurately recognized with high affinity by the translation machinery. However, while the NTD recognizes the tRNA acceptor helix, shortened versions of MjPSTK (representing only 60% of the original size, in which the entire CTD, linker loop and an adjacent NTD helix are missing) are still active in vivo and in vitro, albeit with reduced activity compared to the full-length enzyme.

Structure of a tRNA-dependent kinase essential for selenocysteine decoding.

Compared to bacteria, archaea and eukaryotes employ an additional enzyme for the biosynthesis of selenocysteine (Sec), the 21(st) natural amino acid (aa). An essential RNA-dependent kinase, O-phosphoseryl-tRNA(Sec) kinase (PSTK), converts seryl-tRNA(Sec) to O-phosphoseryl-tRNA(Sec), the immediate precursor of selenocysteinyl-tRNA(Sec). The sequence of Methanocaldococcus jannaschii PSTK (MjPSTK) suggests an N-terminal kinase domain (177 aa) followed by a presumed tRNA binding region (75 aa). The structures of MjPSTK complexed with ADP and AMPPNP revealed that this enzyme belongs to the P-loop kinase class, and that the kinase domain is closely related to gluconate kinase and adenylate kinase. ATP is bound by the P-loop domain (residues 11-18). Formed by antiparallel dimerization of two PSTK monomers, the enzyme structure shows a deep groove with positive electrostatic potential. Located in this groove is the enzyme's active site, which biochemical and genetic data suggest is composed of Asp-41, Arg-44, Glu-55, Tyr-82, Tyr-83, Met-86, and Met-132. Based on structural comparison with Escherichia coli adenylate kinase a docking model was generated that assigns these amino acids to the recognition of the terminal A76-Ser moieties of Ser-tRNA(Sec). The geometry and electrostatic environment of the groove in MjPSTK are perfectly complementary to the unusually long acceptor helix of tRNA(Sec).

Strategies for Improving Small-Molecule Biosensors in Bacteria.

In recent years, small-molecule biosensors have become increasingly important in synthetic biology and biochemistry, with numerous new applications continuing to be developed throughout the field. For many biosensors, however, their utility is hindered by poor functionality. Here, we review the known types of mechanisms of biosensors within bacterial cells, and the types of approaches for optimizing different biosensor functional parameters. Discussed approaches for improving biosensor functionality include methods of directly engineering biosensor genes, considerations for choosing genetic reporters, approaches for tuning gene expression, and strategies for incorporating additional genetic modules.

Improved pyrrolysine biosynthesis through phage assisted non-continuous directed evolution of the complete pathway.

Pyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improve Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we also develop Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway provides 32-fold improved yield of Pyl-containing reporter protein compared to the rationally engineered ancestor. Evolved PylB mutants are present at up to 4.5-fold elevated levels inside cells, and show up to 2.2-fold increased protease resistance. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl-proteins in E. coli.

Divergence of selenocysteine tRNA recognition by archaeal and eukaryotic O-phosphoseryl-tRNASec kinase.

Selenocysteine (Sec) biosynthesis in archaea and eukaryotes requires three steps: serylation of tRNA(Sec) by seryl-tRNA synthetase (SerRS), phosphorylation of Ser-tRNA(Sec) by O-phosphoseryl-tRNA(Sec) kinase (PSTK), and conversion of O-phosphoseryl-tRNA(Sec) (Sep-tRNA(Sec)) by Sep-tRNA:Sec-tRNA synthase (SepSecS) to Sec-tRNA(Sec). Although SerRS recognizes both tRNA(Sec) and tRNA(Ser) species, PSTK must discriminate Ser-tRNA(Sec) from Ser-tRNA(Ser). Based on a comparison of the sequences and secondary structures of archaeal tRNA(Sec) and tRNA(Ser), we introduced mutations into Methanococcus maripaludis tRNA(Sec) to investigate how Methanocaldococcus jannaschii PSTK distinguishes tRNA(Sec) from tRNA(Ser). Unlike eukaryotic PSTK, the archaeal enzyme was found to recognize the acceptor stem rather than the length and secondary structure of the D-stem. While the D-arm and T-loop provide minor identity elements, the acceptor stem base pairs G2-C71 and C3-G70 in tRNA(Sec) were crucial for discrimination from tRNA(Ser). Furthermore, the A5-U68 base pair in tRNA(Ser) has some antideterminant properties for PSTK. Transplantation of these identity elements into the tRNA(Ser)(UGA) scaffold resulted in phosphorylation of the chimeric Ser-tRNA. The chimera was able to stimulate the ATPase activity of PSTK albeit at a lower level than tRNA(Sec), whereas tRNA(Ser) did not. Additionally, the seryl moiety of Ser-tRNA(Sec) is not required for enzyme recognition, as PSTK efficiently phosphorylated Thr-tRNA(Sec).

Tuning the dynamic range of bacterial promoters regulated by ligand-inducible transcription factors.

One challenge for synthetic biologists is the predictable tuning of genetic circuit regulatory components to elicit desired outputs. Gene expression driven by ligand-inducible transcription factor systems must exhibit the correct ON and OFF characteristics: appropriate activation and leakiness in the presence and absence of inducer, respectively. However, the dynamic range of a promoter (i.e., absolute difference between ON and OFF states) is difficult to control. We report a method that tunes the dynamic range of ligand-inducible promoters to achieve desired ON and OFF characteristics. We build combinatorial sets of AraC-and LasR-regulated promoters containing -10 and -35 sites from synthetic and Escherichia coli promoters. Four sequence combinations with diverse dynamic ranges were chosen to build multi-input transcriptional logic gates regulated by two and three ligand-inducible transcription factors (LacI, TetR, AraC, XylS, RhlR, LasR, and LuxR). This work enables predictable control over the dynamic range of regulatory components.

Tunable NF-κB Oscillations in Yeast.

Studies of genetic networks in situ are confounded by unknown interactions with native networks. In Zhang et al. (2017), the authors capitalize on the fact that yeast lacks the NF-κB pathway to study the human NF-κB pathway in isolation and develop a predictive model.