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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. Identifying Small Molecules That Promote Quasipalindrome-Associated Template-Switch Mutations in Escherichia coli

   https://doi.org/10.1534/g3.120.401106  

   Klaric, Julie A.; Perr, Eli L.; Lovett, Susan T. (May 2020, G3: Genes|Genomes|Genetics)

   DNA can assemble into non-B form structures that stall replication and cause genomic instability. One such secondary structure results from an inverted DNA repeat that can assemble into hairpin and cruciform structures during DNA replication. Quasipalindromes (QP), imperfect inverted repeats, are sites of mutational hotspots. Quasipalindrome-associated mutations (QPMs) occur through a template-switch mechanism in which the replicative polymerase stalls at a QP site and uses the nascent strand as a template instead of the correct template strand. This mutational event causes the QP to become a perfect or more perfect inverted repeat. Since it is not fully understood how template-switch events are stimulated or repressed, we designed a high-throughput screen to discover drugs that affect these events. QP reporters were engineered in the Escherichia coli lacZ gene to allow us to study template-switch events specifically. We tested 700 compounds from the NIH Clinical Collection through a disk diffusion assay and identified 11 positive hits. One of the hits was azidothymidine (zidovudine, AZT), a thymidine analog and DNA chain terminator. The other ten were found to be fluoroquinolone antibiotics, which induce DNA-protein crosslinks. This work shows that our screen is useful in identifying small molecules that affect quasipalindrome-associated template-switch mutations. We are currently assessing more small molecule libraries and applying this method to study other types of mutations. 

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3. 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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4. High UV damage and low repair, but not cytosine deamination, stimulate mutation hotspots at ETS binding sites in melanoma

   https://doi.org/10.1073/pnas.2310854121  

   Duan, Mingrui; Song, Shenghan; Wasserman, Hana; Lee, Po-Hsuen; Liu, Ke Jian; Gordân, Raluca; He, Yi; Mao, Peng (January 2024, Proceedings of the National Academy of Sciences)

   Noncoding mutation hotspots have been identified in melanoma and many of them occur at the binding sites of E26 transformation-specific (ETS) proteins; however, their formation mechanism and functional impacts are not fully understood. Here, we used UV (Ultraviolet) damage sequencing data and analyzed cyclobutane pyrimidine dimer (CPD) formation, DNA repair, and CPD deamination in human cells at single-nucleotide resolution. Our data show prominent CPD hotspots immediately after UV irradiation at ETS binding sites, particularly at sites with a conserved TTCCGG motif, which correlate with mutation hotspots identified in cutaneous melanoma. Additionally, CPDs are repaired slower at ETS binding sites than in flanking DNA. Cytosine deamination in CPDs to uracil is suggested as an important step for UV mutagenesis. However, we found that CPD deamination is significantly suppressed at ETS binding sites, particularly for the CPD hotspot on the 5′ side of the ETS motif, arguing against a role for CPD deamination in promoting ETS-associated UV mutations. Finally, we analyzed a subset of frequently mutated promoters, including the ribosomal protein genesRPL13AandRPS20, and found that mutations in the ETS motif can significantly reduce the promoter activity. Thus, our data identify high UV damage and low repair, but not CPD deamination, as the main mechanism for ETS-associated mutations in melanoma and uncover important roles of often-overlooked mutation hotspots in perturbing gene transcription. 

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5. Identifying the Gene Regulatory Network of the Starvation-Induced Transcriptional Activator Nla28

   https://doi.org/10.1128/jb.00265-22  

   Ma, Muqing; Garza, Anthony G.; Lemon, David J.; Caro, Eduardo A.; Ritchie, Linnea; Ryan, Charles; Spearing, Victoria M.; Murphy, Kimberly A.; Welch, Roy D. (December 2022, Journal of Bacteriology)

   O'Toole, George (Ed.)

   ABSTRACT Myxococcus xanthus copes with starvation by producing fruiting bodies filled with dormant and stress-resistant spores. Here, we aimed to better define the gene regulatory network associated with Nla28, a transcriptional activator/enhancer binding protein (EBP) and a key regulator of the early starvation response. Previous work showed that Nla28 directly regulates EBP genes that are important for fruiting body development. However, the Nla28 regulatory network is likely to be much larger because hundreds of starvation-induced genes are downregulated in a nla28 mutant strain. To identify candidates for direct Nla28-mediated transcription, we analyzed the downregulated genes using a bioinformatics approach. Nine potential Nla28 target promoters (29 genes) were discovered. The results of in vitro promoter binding assays, coupled with in vitro and in vivo mutational analyses, suggested that the nine promoters along with three previously identified EBP gene promoters were indeed in vivo targets of Nla28. These results also suggested that Nla28 used tandem, imperfect repeats of an 8-bp sequence for promoter binding. Interestingly, eight of the new Nla28 target promoters were predicted to be intragenic. Based on mutational analyses, the newly identified Nla28 target loci contained at least one gene that was important for starvation-induced development. Most of these loci contained genes predicted to be involved in metabolic or defense-related functions. Using the consensus Nla28 binding sequence, bioinformatics, and expression profiling, 58 additional promoters and 102 genes were tagged as potential Nla28 targets. Among these putative Nla28 targets, functions, such as regulatory, metabolic, and cell envelope biogenesis, were assigned to many genes. IMPORTANCE In bacteria, starvation leads to profound changes in behavior and physiology. Some of these changes have economic and health implications because the starvation response has been linked to the formation of biofilms, virulence, and antibiotic resistance. To better understand how starvation contributes to changes in bacterial physiology and resistance, we identified the putative starvation-induced gene regulatory network associated with Nla28, a transcriptional activator from the bacterium Myxoccocus xanthus . We determined the mechanism by which starvation-responsive genes were activated by Nla28 and showed that several of the genes were important for the formation of a highly resistant cell type. 

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