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1. Promoter Architecture Differences among Alphaproteobacteria and Other Bacterial Taxa

   https://doi.org/10.1128/mSystems.00526-21  

   Myers, Kevin S.; Noguera, Daniel R.; Donohue, Timothy J. (August 2021, mSystems)

   Faust, Karoline (Ed.)

   ABSTRACT Much of our knowledge of bacterial transcription initiation has been derived from studying the promoters of Escherichia coli and Bacillus subtilis . Given the expansive diversity across the bacterial phylogeny, it is unclear how much of this knowledge can be applied to other organisms. Here, we report on bioinformatic analyses of promoter sequences of the primary σ factor (σ 70 ) by leveraging publicly available transcription start site (TSS) sequencing data sets for nine bacterial species spanning five phyla. This analysis identifies previously unreported differences in the −35 and −10 elements of σ 70 -dependent promoters in several groups of bacteria. We found that Actinobacteria and Betaproteobacteria σ 70 -dependent promoters lack the TTG triad in their −35 element, which is predicted to be conserved across the bacterial phyla. In addition, the majority of the Alphaproteobacteria σ 70 -dependent promoters analyzed lacked the thymine at position −7 that is highly conserved in other phyla. Bioinformatic examination of the Alphaproteobacteria σ 70 -dependent promoters identifies a significant overrepresentation of essential genes and ones encoding proteins with common cellular functions downstream of promoters containing an A, C, or G at position −7. We propose that transcription of many σ 70 -dependent promoters in Alphaproteobacteria depends on the transcription factor CarD, which is an essential protein in several members of this phylum. Our analysis expands the knowledge of promoter architecture across the bacterial phylogeny and provides new information that can be used to engineer bacteria for use in medical, environmental, agricultural, and biotechnological processes. IMPORTANCE Transcription of DNA to RNA by RNA polymerase is essential for cells to grow, develop, and respond to stress. Understanding the process and control of transcription is important for health, disease, the environment, and biotechnology. Decades of research on a few bacteria have identified promoter DNA sequences that are recognized by the σ subunit of RNA polymerase. We used bioinformatic analyses to reveal previously unreported differences in promoter DNA sequences across the bacterial phylogeny. We found that many Actinobacteria and Betaproteobacteria promoters lack a sequence in their −35 DNA recognition element that was previously assumed to be conserved and that Alphaproteobacteria lack a thymine residue at position −7, also previously assumed to be conserved. Our work reports important new information about bacterial transcription, illustrates the benefits of studying bacteria across the phylogenetic tree, and proposes new lines of future investigation. 

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2. Elucidation of Sequence–Function Relationships for an Improved Biobutanol In Vivo Biosensor in E. coli

   https://doi.org/10.3389/fbioe.2022.821152  

   Kim, Nancy M.; Sinnott, Riley W.; Rothschild, Lily N.; Sandoval Nicholas R. (October 2022, Frontiers in bioengineering and biotechnology)

   Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ⁵⁴-dependent, butanol-responsive TF–promoter pair, BmoR-P_BMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized P_BMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in P_BMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ⁵⁴-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development. 

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3. Biophysical modeling reveals the transcriptional regulatory mechanism of Spo0A, the master regulator in starving Bacillus subtilis

   https://doi.org/10.1128/msystems.00072-25  

   Zhang, Yujia; Palma, Cristina_S D; Chen, Zhuo; Zarazúa-Osorio, Brenda; Fujita, Masaya; Igoshin, Oleg A; Eichenberger, Patrick (May 2025, mSystems)

   Svensson, Sarah L (Ed.)

   ABSTRACT In starvingBacillus subtilisbacteria,the initiation of two survival programs—biofilm formation and sporulation—is controlled by the same phosphorylated master regulator, Spo0A~P. Its gene,spo0A,is transcribed from two promoters, Pvand Ps,that are, respectively, regulated by RNA polymerase (RNAP) holoenzymes bearing σ^Aand σ^H. Notably, transcription is directly autoregulated by Spo0A~P binding sites known as 0A1, 0A2, and 0A3 box, located in between the two promoters. It remains unclear whether, at the onset of starvation, these boxes activate or repressspo0Aexpression, and whether the Spo0A~P transcriptional feedback plays a role in the increase inspo0Aexpression. Based on the experimental data of the promoter activities under systematic perturbation of the promoter architecture, we developed a biophysical model of transcriptional regulation ofspo0Aby Spo0A~P binding to each of the 0A boxes. The model predicts that Spo0A~P binding to its boxes does not affect the RNAP recruitment to the promoters but instead affects the transcriptional initiation rate. Moreover, the effects of Spo0A~P binding to 0A boxes are mainly repressive and saturated early at the onset of starvation. Therefore, the increase inspo0Aexpression is mainly driven by the increase in RNAP holoenzyme levels. Additionally, we reveal that Spo0A~P affinity to 0A boxes is strongest at 0A3 and weakest at 0A2 and that there are attractive forces between the occupied 0A boxes. Our findings, in addition to clarifying how the sporulation master regulator is controlled, offer a framework to predict regulatory outcomes of complex gene-regulatory mechanisms. IMPORTANCECell differentiation is often critical for survival. In bacteria, differentiation decisions are controlled by transcriptional master regulators under transcriptional feedback control. Therefore, understanding how master regulators are transcriptionally regulated is required to understand differentiation. However, in many cases, the underlying regulation is complex, with multiple transcription factor binding sites and multiple promoters, making it challenging to dissect the exact mechanisms. Here, we address this problem for theBacillus subtilismaster regulator Spo0A. Using a biophysical model, we quantitatively characterize the effect of individual transcription factor binding sites on eachspo0Apromoter. Furthermore, the model allows us to identify the specific transcription step that is affected by transcription factor binding. Such a model is promising for the quantitative study of a wide range of master regulators involved in transcriptional feedback. 

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4. Nucleotide-level characterization and improvement of l -arabinose- and l -rhamnose-inducible systems in E. coli using a high-throughput approach

   https://doi.org/10.1093/nar/gkaf224  

   Kim, Nancy M.; Peng, Danqia; Sandoval, Nicholas R. (April 2025, Nucleic Acids Research)

   Abstract The commonly used arabinose- and rhamnose-inducible Escherichia coli promoters, PBAD and PRha, exhibit tight regulation through activation via their respective transcription factors, AraC and RhaS, alongside the cyclic AMP receptor protein. The mechanisms of these promoters have been characterized on a parts level, but nucleotide-level analysis has yet to be elucidated. Therefore, we describe here a massively parallel reporter assay that maps regulatory sites at the nucleotide level. The relative importance of nucleotides in each binding site is revealed, including loci not included in previous annotations. For PBAD, we confirm known sites and reveal novel binding sites involved in modulating gene expression. In PRha, we refine the length and sequence specificity of rhaI half-sites, updating previous annotations and providing nucleotide level insights into RhaS-mediated regulation. Mutations that lead to increased promoter strength, wider dynamic range, and altered basal expression are identified for both promoters. Engineered versions of PBAD and PRha promoters based on this data show improvements in dynamic range alongside a seven- and three-fold increase in promoter strength, respectively, with a slight increase in basal expression for the PBAD promoters and no significant increase for PRha. This work expands the genetic parts “toolkit” and increases the understanding of these important commonly used promoters. 

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5. Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors

   https://doi.org/10.1371/journal.pgen.1009550  

   Duddy, Olivia P.; Huang, Xiuliang; Silpe, Justin E.; Bassler, Bonnie L. (July 2021, PLOS Genetics)

   Crosson, Sean (Ed.)

   Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae , one quorum-sensing receptor and transcription factor, called VqmA (VqmA Vc ), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmA Phage that activates transcription of the phage gene qtip , and Qtip launches the phage lytic program. Curiously, VqmA Phage can activate vqmR expression but VqmA Vc cannot activate expression of qtip . Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmA Phage and A192, the equivalent residue in VqmA Vc , in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmA Phage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmA Phage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmA Phage and VqmA Vc . A second, targeted genetic screen revealed a region located in the VqmA Vc DNA-binding domain that is necessary to prevent VqmA Vc from binding the qtip promoter, thus restricting DNA binding to the vqmR promoter. We propose that the VqmA Vc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmA Vc from binding the qtip promoter. 

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