Engineering of a Promoter Repressed by a Light-Regulated Transcription Factor in Escherichia coli

Light-regulated gene expression systems allow controlling gene expression in space and time with high accuracy. Contrary to previous synthetic light sensors that incorporate two-component systems which require localization at the plasma membrane, soluble one-component repression systems provide several advantageous characteristics. Firstly, they are soluble and able to diffuse across the cytoplasm. Secondly, they are smaller and of lower complexity, enabling less taxing expression and optimization of fewer parts. Thirdly, repression through steric hindrance is a widespread regulation mechanism that does not require specific interaction with host factors, potentially enabling implementation in different organisms. Herein, we present the design of the synthetic promoter PEL that in combination with the light-regulated dimer EL222 constitutes a one-component repression system. Inspired by previously engineered synthetic promoters and the Escherichia coli lacZYA promoter, we designed PEL with two EL222 operators positioned to hinder RNA polymerase binding when EL222 is bound. PEL is repressed by EL222 under conditions of white light with a light-regulated repression ratio of five. Further, alternating conditions of darkness and light in cycles as short as one hour showed that repression is reversible. The design of the PEL-EL222 system herein presented could aid the design and implementation of analogous one-component optogenetic repression systems. Finally, we compare the PEL-EL222 system with similar systems and suggest general improvements that could optimize and extend the functionality of EL222-based as well as other one-component repression systems.

A Proteomics Approach to Identify Possible Biomarkers of Early and Late Stages of E. coli-induced Urinary Tract Infections

As one of the most common bacterial infections globally, urinary tract infections (UTI)s affect the bladder and kidneys of many the bladders and kidneys of many. Along with gram-negative bacteria, Escherichia coli (E. coli) causes nearly 40% of nosocomial UTIs, 25% of recurrent infections, and between 80 to 90% of community-acquired infections. Proteomics, commonly used to study changes in protein expression of organisms, can be used to explore candidate biomarkers useful for the diagnosis of pathological conditions. Here, protein profiles of samples from patients diagnosed with E. coli-induced UTI were compared to identify distinctive proteins. Extracted proteins from bacteria from patients’ urine samples were separated into excisable spots using 2D-gel electrophoresis. The gels were then analyzed using Progenesis SameSpot software to select uniquely expressed protein spots, excised, and analyzed by LC/MS. The results were then compared against a database of known proteins. We identified two proteins, outer membrane protein A (OmpA) and RNA polymerase-binding transcription factor (DksA), involved in the survival of E. coli in the harsh environment of the host. We suggest their use as a part of a battery of possible biomarkers proteins for E. coli-induced UTI, and suggest that their overexpression is possibly associated with the stage of infection, early or late.

A Structural and Dynamic Analysis of the Partially Disordered Polymerase-Binding Domain in RSV Phosphoprotein

The phosphoprotein P of Mononegavirales (MNV) is an essential co-factor of the viral RNA polymerase L. Its prime function is to recruit L to the ribonucleocapsid composed of the viral genome encapsidated by the nucleoprotein N. MNV phosphoproteins often contain a high degree of disorder. In Pneumoviridae phosphoproteins, the only domain with well-defined structure is a small oligomerization domain (POD). We previously characterized the differential disorder in respiratory syncytial virus (RSV) phosphoprotein by NMR. We showed that outside of RSV POD, the intrinsically disordered N-and C-terminal regions displayed a structural and dynamic diversity ranging from random coil to high helical propensity. Here we provide additional insight into the dynamic behavior of PCα, a domain that is C-terminal to POD and constitutes the RSV L-binding region together with POD. By using small phosphoprotein fragments centered on or adjacent to POD, we obtained a structural picture of the POD–PCα region in solution, at the single residue level by NMR and at lower resolution by complementary biophysical methods. We probed POD–PCα inter-domain contacts and showed that small molecules were able to modify the dynamics of PCα. These structural properties are fundamental to the peculiar binding mode of RSV phosphoprotein to L, where each of the four protomers binds to L in a different way.

Epigenetic Mechanisms Mediate Nicotine-Induced Reward and Behaviour in Zebrafish

: Nicotine induces long-term changes in the neural activity of the mesocorticolimbic reward pathway structures. The mechanisms involved in this process have not been fully characterized. The hypothesis discussed here proposed that epigenetic regulation participates in installing persistent adaptations and long-lasting synaptic plasticity generated by nicotine action on the mesolimbic dopamine neurons of zebrafish. The epigenetic mechanisms induced by nicotine entail histone and DNA chemical modifications, which have been described to lead to changes in gene expression. Among the enzymes that catalyze epigenetic chemical modifications, histone deacetylases (HDACs) remove acetyl groups from histones, thereby facilitating DNA relaxation and making DNA more accessible to gene transcription. DNA methylation, which is dependent on DNA methyltransferase (DNMTs) activity, inhibits gene expression by recruiting several methyl binding proteins that prevent RNA polymerase binding to DNA. In zebrafish, phenylbutyrate (PhB), an HDAC inhibitor, abolishes nicotine rewarding properties together with a series of typical reward-associated behaviors. Furthermore, PhB and nicotine alter long- and short-term object recognition memory in zebrafish, respectively. Regarding DNA methylation effects, a methyl group donor L-methionine (L-met) was found to dramatically reduce nicotine-induced conditioned place preference (CPP) in zebrafish. Simultaneous treatment with DNMT inhibitor 5-aza-2’-deoxycytidine (AZA) was found to reverse the L-met effect on nicotine-induced CPP as well as nicotine reward-specific impact on genetic expression in zebrafish. Therefore, pharmacological interventions that modulate gene expression epigenetic regulation should be considered a potential therapeutic method to treat nicotine addiction.

The σ54 system directly regulates bacterial natural product genes

Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutics and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery controlling their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explored this idea by analyzing a selected group of putative σ54 promoters identified in Myxococcus xanthus natural product gene clusters. We show that mutations in putative σ54-RNA polymerase binding regions and in putative Nla28 EBP binding sites dramatically reduce in vivo promoter activities in growing and developing cells. We also show in vivo promoter activities are reduced in a nla28 mutant, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the Nla28 binding sites are mutated. Together, our results indicate that M. xanthus uses σ54 promoters for transcription of at least some of its natural product genes. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product loci are located within genes and not in intergenic sequences.

The RNA Architecture of the SARS-CoV-2 3′-Untranslated Region

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3′ untranslated region (UTR) of this β-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3′ UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3′ UTRs in the infectious virions and the minigene-transfected cells are almost identical.

The σ54 System Directly Regulates Bacterial Natural Product Genes

Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutic agents and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery that controls their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explore this idea by analyzing four putative σ54 promoters identified in the sequences of Myxococcus xanthus natural product gene clusters. We show that mutations in the putative σ54-RNA polymerase binding regions reduce in vivo promoter activities during growth and development. We also show that the EBP Nla28 is important for the in vivo activities of three natural product promoters, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the putative Nla28 binding sites are mutated. These results indicate that the natural product promoters are bona fide σ54 promoter elements and three are direct targets of Nla28. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product clusters are located within genes and not in intergenic sequences.

Deep learning approach for predicting functional Z-DNA regions using omics data

Computational methods to predict Z-DNA regions are in high demand to understand the functional role of Z-DNA. The previous state-of-the-art method Z-Hunt is based on statistical mechanical and energy considerations about B- to Z-DNA transition using sequence information. Z-DNA CHiP-seq experiment results showed little overlap with Z-Hunt predictions implying that sequence information only is not sufficient to explain emergence of Z-DNA at different genomic locations. Adding epigenetic and other functional genomic mark-ups to DNA sequence level can help revealing the functional Z-DNA sites. Here we take advantage of the deep learning approach that can analyze and extract information from large volumes of molecular biology data. We developed a machine learning approach DeepZ that aggregates information from genome-wide maps of epigenetic markers, transcription factor and RNA polymerase binding sites, and chromosome accessibility maps. With the developed model we not only verify the experimental Z-DNA predictions, but also generate the whole-genome annotation, introducing new possible Z-DNA regions, which have not yet been found in experiments and can be of interest to the researchers from various fields.