Introduction of Modified BglBrick System in Lactococcus lactis for Straightforward Assembly of Multiple Gene Cassettes

Genetic modification of lactic acid bacteria is an evolving and highly relevant field of research that allows the engineered bacteria to be equipped with the desired functions through the controlled expression of the recombinant protein. Novel genetic engineering techniques offer the advantage of being faster, easier and more efficient in incorporating modifications to the original bacterial strain. Here, we have developed a modified BglBrick system, originally introduced in Escherichia coli and optimized it for the lactic acid bacterium Lactococcus lactis. Six different expression cassettes, encoding model proteins, were assembled in different order as parts of a modified BglBrick system in a novel plasmid pNBBX. All cassettes included nisin promoter, protein encoding gene and transcription terminator. We demonstrated successful intracellular expression of the two fluorescent proteins and display of the four protein binders on the bacterial surface. These were expressed either alone or concomitantly, in combinations of three model proteins. Thus, a modified BglBrick system developed herein enables simple and modular construction of multigene plasmids and controlled simultaneous expression of three proteins in L. lactis.

Internal Promoters and Their Effects on the Transcription of Operon Genes for Epothilone Production in Myxococcus xanthus

The biosynthetic genes for secondary metabolites are often clustered into giant operons with no transcription terminator before the end. The long transcripts are frangible and the transcription efficiency declines along with the process. Internal promoters might occur in operons to coordinate the transcription of individual genes, but their effects on the transcription of operon genes and the yield of metabolites have been less investigated. Epothilones are a kind of antitumor polyketides synthesized by seven multifunctional enzymes encoded by a 56-kb operon. In this study, we identified multiple internal promoters in the epothilone operon. We performed CRISPR-dCas9–mediated transcription activation of internal promoters, combined activation of different promoters, and activation in different epothilone-producing M. xanthus strains. We found that activation of internal promoters in the operon was able to promote the gene transcription, but the activation efficiency was distinct from the activation of separate promoters. The transcription of genes in the operon was influenced by not only the starting promoter but also internal promoters of the operon; internal promoters affected the transcription of the following and neighboring upstream/downstream genes. Multiple interferences between internal promoters thus changed the transcriptional profile of operon genes and the production of epothilones. Better activation efficiency for the gene transcription and the epothilone production was obtained in the low epothilone-producing strains. Our results highlight that interactions between promoters in the operon are critical for the gene transcription and the metabolite production efficiency.

Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, β, β’ and ω are simultaneously coexpressed. Appending a 3′ UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

Analysis of SINE Families B2, Dip, and Ves with Special Reference to Polyadenylation Signals and Transcription Terminators

Short Interspersed Elements (SINEs) are eukaryotic non-autonomous retrotransposons transcribed by RNA polymerase III (pol III). The 3′-terminus of many mammalian SINEs has a polyadenylation signal (AATAAA), pol III transcription terminator, and A-rich tail. The RNAs of such SINEs can be polyadenylated, which is unique for pol III transcripts. Here, B2 (mice and related rodents), Dip (jerboas), and Ves (vespertilionid bats) SINE families were thoroughly studied. They were divided into subfamilies reliably distinguished by relatively long indels. The age of SINE subfamilies can be estimated, which allows us to reconstruct their evolution. The youngest and most active variants of SINE subfamilies were given special attention. The shortest pol III transcription terminators are TCTTT (B2), TATTT (Ves and Dip), and the rarer TTTT. The last nucleotide of the terminator is often not transcribed; accordingly, the truncated terminator of its descendant becomes nonfunctional. The incidence of complete transcription of the TCTTT terminator is twice higher compared to TTTT and thus functional terminators are more likely preserved in daughter SINE copies. Young copies have long poly(A) tails; however, they gradually shorten in host generations. Unexpectedly, the tail shortening below A10 increases the incidence of terminator elongation by Ts thus restoring its efficiency. This process can be critical for the maintenance of SINE activity in the genome.

Multilevel Gene Regulation Using Switchable Transcription Terminator and Toehold Switch in Escherichia coli

Nucleic acid-based regulatory components provide a promising toolbox for constructing synthetic biological circuits due to their design flexibility and seamless integration towards complex systems. In particular, small-transcriptional activating RNA (STAR) and toehold switch as regulators of transcription and translation steps have shown a large library size and a wide dynamic range, meeting the criteria to scale up genetic circuit construction. Still, there are limited attempts to integrate the heterogeneous regulatory components for multilevel regulatory circuits in living cells. In this work, inspired by the design principle of STAR, we designed several switchable transcription terminators starting from natural and synthetic terminators. These switchable terminators could be designed to respond to specific RNA triggers with minimal sequence constraints. When combined with toehold switches, the switchable terminators allow simultaneous control of transcription and translation processes to minimize leakage in Escherichia coli. Further, we demonstrated a set of logic gates implementing 2-input AND circuits and multiplexing capabilities to control two different output proteins. This study shows the potential of novel switchable terminator designs that can be computationally designed and seamlessly integrated with other regulatory components, promising to help scale up the complexity of synthetic gene circuits in living cells.

Characterization of five purine riboswitches in cellular and cell-free expression systems

Bacillus subtilis employs five purine riboswitches for the control of purine de novo synthesis and transport at the transcription level. All of them are formed by a structurally conserved aptamer, and a variable expression platform harboring a rho-independent transcription terminator. In this study, we characterized all five purine riboswitches under the context of active gene expression processes both in vitro and in vivo. We identified transcription pause sites located in the expression platform upstream of the terminator of each riboswitch. Moreover, we defined a correlation between in vitro transcription readthrough and in vivo gene expression. Our in vitro assay demonstrated that the riboswitches operate in the micromolar range of concentration for the cognate metabolite. Our in vivo assay showed the dynamics of control of gene expression by each riboswitch. This study deepens the knowledge of the regulatory mechanism of purine riboswitches.

Internal promoters of the epothilone biosynthetic gene cluster and their activation in Myxococcus xanthus by CRISPR-dCas9

Background Multiple genes involving in a complex pathway are often clustered into a giant operon with no transcription terminator before the end, and this leads to frangibility of the transcriptional process and arduous engineering work to control the transcription of operon genes. Internal promoters might occur in operon to coordinate the transcription of individual genes, but their effects on the transcription of operon genes have been less investigated. Results Epothilones are a kind of polyketides synthesized by seven multifunctional enzymes, which are encoded by a 56-kb operon of the myxobacterium Sorangium cellulosum. In this study, we determined that the epothilone operon contained multiple internal promoters. These promoters were activatable by the CRISPRa technique, and the yields of epothilones were accordingly increased. However, the activation efficiencies of promoters in operon and separate forms were greatly different. Further, we found that the transcriptional levels of the epothilone genes were always increased at a greater extent than the epothilone yields, which suggested that the transcriptional activation of single genes probably had a weak effect on the final epothilone yield, and higher yield required an overall transcriptional increase of the multiple operonic genes. Finally, we combined the activation of the starting promoter PepoA together with internal promoters in different epothilone-producing strains, and obtained the highest 15-fold increase of epothilone yield in Myxococcus xanthus ZE5. Conclusions This is the first time to report the internal promoters in epothilone gene clusters in Myxococcus xanthus and the first time to assay the activation effects of these internal promoters by CRISPR-dCas9. Our results highlight that tuning internal promoter activities is critical to control the transcription of operon genes and the production efficiency of microbial secondary metabolites.

Organization of 5S ribosomal DNA of Poa pratensis L.

5S rDNA, which belongs to the class of repeated sequences, represents a convenient model for studying the molecular evolution of plants. The 5S rDNA repeated unit consists of a conserved region encoding 5S rRNA and variable intergenic spacer (IGS) that contains the motifs required for initiation and termination of transcription. The IGS sequences can be used as a molecular marker for elucidation of the phylogenetic relationships of low-ranking taxa. Today, the molecular organization of 5S rDNA in species of the Poaceae family, which includes many economically important crops, is still poorly understood. Therefore, the aim of the study was to investigate the organization and polymorphism of 5S rDNA IGS in the genome of Poa pratensis L., a member of one of the largest genera of the Poaceae family. Using PCR amplification, cloning, sequencing and analysis of the SRA database, two variants of the 5S rDNA repeated units were found in the genome of P. pratensis. The two variants possess 119 bp-long coding regions, whereas the length of IGS ranges from 169 to 185 bp. At the beginning of IGS, the oligo-T sequence of the RNA polymerase III transcription terminator is present. In members of the Poaceae family, the putative external elements of the 5S rDNA promoter differ from those in previously studied groups of plants.

Adaptation of Bacillus thuringiensis to plant colonisation affects differentiation and toxicity

Although certain isolates from the Bacillus cereus group (Bacillus cereus sensu lato) are used as probiotics, safety concerns remain due to pathogenic traits. For example, toxin production might shift as an adaptive survival strategy in natural niches (the soil and plant rhizosphere). Therefore, it is crucial to explore bacterial evolutionary adaptation to the environment. Herein, we investigated Bacillus thuringiensis (Cry-) adaptation to the colonisation of Arabidopsis thaliana roots, and monitored changes in cellular differentiation in experimentally evolved isolates. Isolates from two populations displayed improved iterative ecesis on roots and increased toxicity against insect larvae. Molecular dissection and recreation of a causative mutation revealed the importance of a non-sense mutation in the rho transcription terminator gene. Transcriptome analysis revealed how Rho impacts various B. thuringiensis genes involved in carbohydrate metabolism and virulence. Our work suggests that evolved multicellular aggregates have a fitness advantage over single cells when colonising plants, creating a trade-off between swimming and multicellularity in evolved lineages, in addition to unrelated alterations in pathogenicity.