Sourcing Phage-Encoded Terminators Using ONT-cappable-seq for SynBio Applications in Pseudomonas.

Efficient transcriptional terminators are essential for the performance of genetic circuitry in microbial SynBio hosts. In recent years, several libraries of characterized strong terminators have become available for model organisms such as Escherichia coli. Conversely, terminator libraries for nonmodel species remain scarce, and individual terminators are often ported over from model systems, leading to unpredictable performance in their new hosts. In this work, we mined the genomes of Pseudomonas infecting phages LUZ7 and LUZ100 for transcriptional terminators utilizing the full-length RNA sequencing technique "ONT-cappable-seq" and validated these terminators in three Gram-negative hosts using a terminator trap assay. Based on these results, we present nine terminators for E. coli, Pseudomonas putida, and Pseudomonas aeruginosa, which outperform current reference terminators. Among these, terminator LUZ7 T50 displays potent bidirectional activity. These data further support that bacteriophages, as evolutionary-adapted natural predators of the targeted bacteria, provide a valuable source of microbial SynBio parts.

Recognition of transcription terminators during retrotransposition: How to keep a group II intron quiet.

Chung et al. recently presented the structure of a primitive group IIC intron with its DNA target, which reveals the structural requirements that this class of intron uses to recognize a transcription terminator stem loop at the DNA level for insertion during retrotransposition.

Structural basis for intrinsic transcription termination.

Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.

Data-Driven and in Silico-Assisted Design of Broad Host-Range Minimal Intrinsic Terminators Adapted for Bacteria.

Efficient transcription termination relying on intrinsic terminators is critical to maintain cell fitness by avoiding unwanted read-through in bacteria. Natural intrinsic terminator (NIT) typically appears in mRNA as a hairpin followed by approximately eight conserved uridines (U-tract) at the 3' terminus. Owing to their simple structure, small size, and protein independence, assorted NITs have been redesigned as robust tools to construct gene circuits. However, most NITs exert functions to adapt to their physiological requirements rather than the demand for building synthetic gene circuits, rendering uncertain working performance when they are constructed intact in synthetic gene circuits. Here, rather than modifying NITs, we established a data-driven and in silico-assisted (DISA) design framework to forward engineer minimal intrinsic terminators (MITs). By comprehensively analyzing 75 natural intrinsic terminators from Bacillus subtilis, we revealed that two pivotal features, the length of the U-tract and the thermodynamics of the terminator hairpin, were involved in the sequence-activity relationship (SAR) of termination efficiency (TE). As per the SAR, we leveraged DISA to fabricate an array of MITs composed of in silico-assisted designed minimal hairpins and fixed U-tracts. Most of these MITs exhibited high TE in diverse Gram-positive and Gram-negative bacteria. In contrast, the TEs of the NITs were highly varied in different hosts. Moreover, TEs of MITs were flexibly tuned over a wide range by modulating the length of the U-tract. Overall, these results demonstrate an efficient framework to forward design functional and broad host-range terminators independent of tedious and iterative screening of mutagenesis libraries of natural terminators.

Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression.

In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a 'terminator catalog' by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3'-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.

CRISPR-Cas12a-Assisted Multicolor Biosensor for Semiquantitative Point-of-Use Testing of the Nopaline Synthase Terminator in Genetically Modified Crops by Unaided Eyes.

Existing methods of detecting foreign genes and their expression products from genetically modified organisms (GMOs) suffer from the requirement of professional equipment and skillful operators. The same problem stays for the CRISPR-Cas12a system, although it has been emerging as a powerful tool for nucleic acid detection due to its remarkable sensitivity and specificity. In this report, a portable platform for the visible detection of GMOs based on CRISPR-Cas12a was established, which relies on a color change of gold nanorods (GNRs) caused by the invertase-glucose oxidase cascade reaction and the Fenton reaction for signal readout. A nopaline synthase (NOS) terminator was employed as a model target commonly existing in foreign genes of GMOs. With the help of recombinase-aided amplification, this platform achieved comparable sensitivity of DNA targets (1 aM) with that of a fluorescence reporting assay. As low as 0.1 wt % genetically modified (GM) content in Bt-11 maize was visually observed by unaided eyes, and the semiquantitation of GM ingredients can be obtained within the range of 0.1 to 40 wt % through the absorption measurement of GNRs. Furthermore, five real samples were tested by our method, and the results indicated that the GM ingredient percentages of GMO samples were 2.24 and 24.08 wt %, respectively, while the other three samples were GMO-free. With the advantages of a simple procedure, no need for large or professional instruments, high sensitivity, and selectivity, this platform is expected to provide reasonable technical support for the safe supervision of GMOs.

Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites.

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.

Evaluating Terminator Strength Based on Differentiating Effects on Transcription and Translation.

Transcription terminators play a role in terminating the progress of gene transcription, and are thus essential elements in the gene circuit. Terminators have two main functions: terminating gene transcription and improving the stability of gene transcripts during translation. We therefore considered the detailed characteristics of terminators in relation to their different roles in gene transcription and translation, including transcription shut-down degree (α) and upstream mRNA protection capacity (ß), and apparent termination efficiency (η) reflecting the overall regulatory effect of the terminator. Based on a dual-reporter gene system, we constructed three terminator-probe plasmids to investigate each characteristic in Escherichia coli. According to multiple regression analysis, the transcription shut-down degree and the upstream mRNA protection capacity contributed almost equally to the apparent termination efficiency. Sequence analysis of 12 terminators demonstrated that the terminator sequence was dominated by GC bases, and that a high ratio of GC bases in the stem structure of terminators might be associated with a high degree of transcription shut-down. This comprehensive characterization of terminators furthers our understanding of the role of terminators in gene expression and provides a guide for synthetic terminator design.

Structural basis for transcription antitermination at bacterial intrinsic terminator.

Bacteriophages typically hijack the host bacterial transcriptional machinery to regulate their own gene expression and that of the host bacteria. The structural basis for bacteriophage protein-mediated transcription regulation-in particular transcription antitermination-is largely unknown. Here we report the 3.4 Å and 4.0 Å cryo-EM structures of two bacterial transcription elongation complexes (P7-NusA-TEC and P7-TEC) comprising the bacteriophage protein P7, a master host-transcription regulator encoded by bacteriophage Xp10 of the rice pathogen Xanthomonas oryzae pv. Oryzae (Xoo) and discuss the mechanisms by which P7 modulates the host bacterial RNAP. The structures together with biochemical evidence demonstrate that P7 prevents transcription termination by plugging up the RNAP RNA-exit channel and impeding RNA-hairpin formation at the intrinsic terminator. Moreover, P7 inhibits transcription initiation by restraining RNAP-clamp motions. Our study reveals the structural basis for transcription antitermination by phage proteins and provides insights into bacterial transcription regulation.

Design and Evaluation of Synthetic Terminators for Regulating Mammalian Cell Transgene Expression.

Tuning heterologous gene expression in mammalian production hosts has predominantly relied upon engineering the promoter elements driving the transcription of the transgene. Moreover, most regulatory elements have borrowed genetic sequences from viral elements. Here, we generate a set of 10 rational and 30 synthetic terminators derived from nonviral elements and evaluate them in the HT1080 and HEK293 cell lines to demonstrate that they are comparable in terms of tuning gene expression/protein output to the viral SV40 element and often require less sequence footprint. The mode of action of these terminators is determined to be an increase in mRNA half-life. Furthermore, we demonstrate that constructs comprising completely nonviral regulatory elements ( i.e., promoters and terminators) can outperform commonly used, strong viral based elements by nearly 2-fold. Ultimately, this novel set of terminators expanded our genetic toolkit for engineering mammalian host cells.

Expanding the Toolbox of Broad Host-Range Transcriptional Terminators for Proteobacteria through Metagenomics.

As the field of synthetic biology moves toward the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacterium Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria, for instance Pseudomonas putida. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-range plasmid, we identified 20 clones with potential terminator activity in P. putida. Further characterization allowed the identification of 4 unique sequences ranging from 58 to 181 bp long that efficiently terminate transcription in P. putida, E. coli, Burkholderia phymatum, and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

RNA Polymerase Clamp Movement Aids Dissociation from DNA but Is Not Required for RNA Release at Intrinsic Terminators.

In bacteria, disassembly of elongating transcription complexes (ECs) can occur at intrinsic terminators in a 2- to 3-nucleotide window after transcription of multiple kilobase pairs of DNA. Intrinsic terminators trigger pausing on weak RNA-DNA hybrids followed by formation of a strong, GC-rich stem-loop in the RNA exit channel of RNA polymerase (RNAP), inactivating nucleotide addition and inducing dissociation of RNA and RNAP from DNA. Although the movements of RNA and DNA during intrinsic termination have been studied extensively leading to multiple models, the effects of RNAP conformational changes remain less well defined. RNAP contains a clamp domain that closes around the nucleic acid scaffold during transcription initiation and can be displaced by either swiveling or opening motions. Clamp opening is proposed to promote termination by releasing RNAP-nucleic acid contacts. We developed a cysteine crosslinking assay to constrain clamp movements and study effects on intrinsic termination. We found that biasing the clamp into different conformations perturbed termination efficiency, but that perturbations were due primarily to changes in elongation rate, not the competing rate at which ECs commit to termination. After commitment, however, inhibiting clamp movements slowed release of DNA but not of RNA from the EC. We also found that restricting trigger-loop movements with the RNAP inhibitor microcin J25 prior to commitment inhibits termination, in agreement with a recently proposed multistate-multipath model of intrinsic termination. Together our results support views that termination commitment and DNA release are separate steps and that RNAP may remain associated with DNA after termination.

Cloning and expression of enterovirus 71 capsid protein 1 in a probiotic Bifidobacterium pseudocatenulatum.

This study investigated cloning and expression of enterovirus 71 viral capsid protein 1 (EV71-VP1) in Bifidobacterium pseudocatenulatum (B. pseudocatenulatum) M115. To achieve this, a codon-optimized gene coding for EV71-VP1 was analysed, designed, synthesized and cloned into a plasmid vector flanked by a transcriptional promoter and terminator sequences. The promoter was based on that of P919, a constitutive promoter of the gene encoding the large ribosomal protein of B. bifidum BGN4, while the terminator was based on that of the peptidase N gene of Lactococcus lactis. The construct was amplified in Escherichia coli XL1-blue and then transferred into B. pseudocatenulatum M115 by electrotransformation. Western blot analysis revealed that the EV71-VP1 was intracellularly expressed in B. pseudocatenulatum M115 under the control of the selected heterologous promoter. In addition, plasmid stability analysis showed the construct was maintained stably for more than 160 generations, enough for most future applications. The results derived from this study open the possibility to utilize the bacterium carrying a specific expression plasmid as cell factory for the production of proteins with high commercial and health-promoting value. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the first successful expression of a codon-optimized gene coding for enterovirus 71 viral capsid protein 1 (EV71-VP1) in Bifidobacterium pseudocatenulatum M115, a novel probiotic strain isolated from human intestines. The EV71-VP1 was constitutively expressed under the control of P919 promoter derived from B. bifidum BGN4 in the cytoplasm of bacterial cells supporting the use of heterologous promoter and terminator sequences for viral gene expression in Bifidobacterium species.

A nebulin super-repeat panel reveals stronger actin binding toward the ends of the super-repeat region.

INTRODUCTION: Nebulin is a giant actin-binding protein in the thin filament of the skeletal muscle sarcomere. Studies of nebulin interactions are limited by the size, complexity, and poor solubility of the protein. We divided the nebulin super-repeat region into a super-repeat panel, and studied nebulin/actin interactions. METHODS: Actin binding was studied using a co-sedimentation assay with filamentous actin and 26 different nebulin super-repeats. RESULTS: The panel revealed notable differences in actin binding between the super-repeats. Both ends of the super-repeat region bound actin significantly more strongly, whereas the central part of the protein bound actin weakly. Thus, the binding between nebulin and actin formed a location-dependent pattern of strong vs. weak binding. DISCUSSION: The nebulin super-repeat panel allowed us to study the actin binding of each super-repeat individually. The panel will be a powerful tool in elucidating nebulin function in health and disease. Muscle Nerve 59:116-121, 2019.

Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART).

Loop-mediated amplification (LAMP) has been widely used to amplify and hence detect nucleic acid target sequences from various pathogens, viruses and genetic modifications. Two distinct types of primer are required for LAMP; hairpin-forming LAMP and displacement. High specificity arises from this use of multiple primers, but without optimal conditions for LAMP, sensitivity can be poor. We confirm here the importance of LAMP primer design, concentrations and ratios for efficient LAMP amplification. We further show that displacement primers are non-essential to the LAMP reaction at certain concentrations providing accelerating loop primers are present. We investigate various methods to quantify DNA extracts from GM maize certified reference materials to calculate the target copy numbers of template presented to the LAMP reaction, and show that LAMP can amplify transgenic promoter/terminator sequences in DNA extracted from various maize GM events using primers designed to target the 35S promoter (35Sp) or NOS terminator (NOSt) sequences, detection with both bioluminescence in real-time (BART) and fluorescent methods. With prior denaturation and HPLC grade LAMP primers single copy detection was achieved, showing that optimised LAMP conditions can be combined with BART for single copy targets, with simple and cost efficient light detection electronics over fluorescent alternatives.

Influenza virus infection causes global RNAPII termination defects.

Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3' ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3' extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.

Essential Nucleoid Associated Protein mIHF (Rv1388) Controls Virulence and Housekeeping Genes in Mycobacterium tuberculosis.

Tight control of gene expression is crucial for Mycobacterium tuberculosis to adapt to the changing environments encountered when infecting or exiting human cells. While three nucleoid associated proteins (NAPs) EspR, HupB and Lsr2 have been investigated, the role of a fourth, the mycobacterial integration host factor (mIHF), remains elusive. Here, we report a multidisciplinary functional analysis that exploits a conditional mIHF mutant. Gene silencing was bactericidal and resulted in elongated cells devoid of septa, with only one nucleoid. ChIP-sequencing identified 153 broad peaks distributed around the chromosome, which were often situated upstream of transcriptional start sites where EspR also bound. RNA-sequencing showed expression of 209 genes to be heavily affected upon mIHF depletion, including those for many tRNAs, DNA synthesis and virulence pathways. Consistent with NAP function, mIHF acts as a global regulator by directly and indirectly controlling genes required for pathogenesis and for housekeeping functions.

Topoisomerases I and III inhibit R-loop formation to prevent unregulated replication in the chromosomal Ter region of Escherichia coli.

Type 1A topoisomerases (topos) are the only ubiquitous topos. E. coli has two type 1A topos, topo I (topA) and topo III (topB). Topo I relaxes negative supercoiling in part to inhibit R-loop formation. To grow, topA mutants acquire compensatory mutations, base substitutions in gyrA or gyrB (gyrase) or amplifications of a DNA region including parC and parE (topo IV). topB mutants grow normally and topo III binds tightly to single-stranded DNA. What functions topo I and III share in vivo and how cells lacking these important enzymes can survive is unclear. Previously, a gyrB(Ts) compensatory mutation was used to construct topA topB null mutants. These mutants form very long filaments and accumulate diffuse DNA, phenotypes that appears to be related to replication from R-loops. Here, next generation sequencing and qPCR for marker frequency analysis were used to further define the functions of type 1A topos. The results reveal the presence of a RNase HI-sensitive origin of replication in the terminus (Ter) region of the chromosome that is more active in topA topB cells than in topA and rnhA (RNase HI) null cells. The S9.6 antibodies specific to DNA:RNA hybrids were used in dot-blot experiments to show the accumulation of R-loops in rnhA, topA and topA topB null cells. Moreover topA topB gyrB(Ts) strains, but not a topA gyrB(Ts) strain, were found to carry a parC parE amplification. When a topA gyrB(Ts) mutant carried a plasmid producing topo IV, topB null transductants did not have parC parE amplifications. Altogether, the data indicate that in E. coli type 1A topos are required to inhibit R-loop formation/accumulation mostly to prevent unregulated replication in Ter, and that they are essential to prevent excess negative supercoiling and its detrimental effects on cell growth and survival.

Transformation of an Exotic Yeast Species into a Platform Organism: A Case Study for Engineering Glycolipid Production in the Yeast Starmerella bombicola.

In this chapter, a step-by-step approach on how to transform non-conventional yeasts or fungi into platform organisms is described. The non-conventional glycolipid producing yeast Starmerella bombicola (and in some cases also Pseudohyphozyma bogoriensis) is used as a case study. And more specifically how to engineer it toward production of new-to-nature glycolipids like bola sophorolipids. When starting genetic engineering efforts for non-lab strains, one should start at the very basis: identifying selection markers and possibly developing auxotrophic strains. Once this is done, the quest for the development of an optimal transformation method can be started. After optimization thereof, knock-out and knock-in strains can be generated based upon the specific strategy/aim. Sometimes this can lead to unexpected, but yet very interesting findings. To fully and efficiently expand the potential as a production platform of these yeast strains, a range of additional molecular tools are required. A well-equipped molecular toolbox should contain a set of characterized promotors, terminators, and defined genomic landing paths. The availability of an episomal system greatly facilitates engineering and screening efforts, but also offers the possibility of developing more advanced engineering techniques such as Crispr-Cas. InBio.be is a world leading pioneer to do this for the yeast S. bombicola and combined, these efforts will result in the commercialization of new types of glycolipids in the next few years.

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.

Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway. A secondary module cloning site is also available for pathway optimization, in order to limit library size if necessary. Here, we show the utility of EcoFlex using the violacein biosynthesis pathway as an example.