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Clostridioides difficile is a bacterial pathogen that causes a range of clinical disease from mild to moderate diarrhea, pseudomembranous colitis, and toxic megacolon. Typically, C. difficile infections (CDIs) occur after antibiotic treatment, which alters the gut microbiota, decreasing colonization resistance against C. difficile . Disease is mediated by two large toxins and the expression of their genes is induced upon nutrient depletion via the alternative sigma factor TcdR. Here, we use tcdR mutants in two strains of C. difficile and omics to investigate how toxin-induced inflammation alters C. difficile metabolism, tissue gene expression and the gut microbiota, and to determine how inflammation by the host may be beneficial to C. difficile . We show that C. difficile metabolism is significantly different in the face of inflammation, with changes in many carbohydrate and amino acid uptake and utilization pathways. Host gene expression signatures suggest that degradation of collagen and other components of the extracellular matrix by matrix metalloproteinases is a major source of peptides and amino acids that supports C. difficile growth in vivo. Lastly, the inflammation induced by C. difficile toxin activity alters the gut microbiota, excluding members from the genus Bacteroides that are able to utilize the same essential nutrients released from collagen degradation. The effects of antibiotics on the gut microbiota can lead to enhanced colonization of Clostridioides difficile ( C. difficile ) and toxin-mediated pathogenesis. Here, using defined toxin-mutant strains and a murine model, the authors provide insights into how toxin-induced inflammation alters C. difficile metabolism, host tissue gene expression and gut microbiota, together influencing a beneficial niche for infection.

Fusobacterium nucleatum, long known as a common oral microbe, has recently garnered attention for its ability to colonize tissues and tumors elsewhere in the human body. Clinical and epidemiological research has now firmly established F. nucleatum as an oncomicrobe associated with several major cancer types. However, with the current research focus on host associations, little is known about gene regulation in F. nucleatum itself, including global stress-response pathways that typically ensure the survival of bacteria outside their primary niche. This is due to the phylogenetic distance of Fusobacteriota to most model bacteria, their limited genetic tractability, and paucity of known gene functions. Here, we characterize a global transcriptional stress-response network governed by the extracytoplasmic function sigma factor, σE. To this aim, we developed several genetic tools for this anaerobic bacterium, including four different fluorescent marker proteins, inducible gene expression, scarless gene deletion, and transcriptional and translational reporter systems. Using these tools, we identified a σE response partly reminiscent of phylogenetically distant Proteobacteria but induced by exposure to oxygen. Although F. nucleatum lacks canonical RNA chaperones, such as Hfq, we uncovered conservation of the noncoding arm of the σE response in form of the noncoding RNA FoxI. This regulatory small RNA acts as an mRNA repressor of several membrane proteins, thereby supporting the function of σE. In addition to the characterization of a global stress response in F. nucleatum, the genetic tools developed here will enable further discoveries and dissection of regulatory networks in this early-branching bacterium.

After an RNA polymerase reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA and recommence RNA synthesis from the previous or a different promoter. Magnetic tweezers were used to monitor such secondary transcription and determine the effects of low forces assisting or opposing translocation, protein roadblocks, and transcription factors. Remarkably, up to 50% of Escherichia coli ( E. coli ) RNA polymerases diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 ( σ 70 ) likely remained associated with the DNA promoting sliding and enabling re-initiation from promoters in either orientation. However, deletions of the α -C-terminal domains severely limited the ability of RNAP to turn around between successive rounds of transcription. The addition of elongation factor NusG, which competes with σ 70 for binding to RNAP, limited additional rounds of transcription. Surprisingly, sliding RNA polymerases blocked by a DNA-bound lac repressor could slowly re-initiate transcription and were not affected by NusG, suggesting a σ -independent pathway. Low forces effectively biased promoter selection suggesting a prominent role for topological entanglements that affect RNA polymerase translocation. Tiny forces cause E. coli RNA polymerase to slide along DNA after transcription termination at up to 700 bp/s. In this paper, the authors use magnetic tweezers to show that sliding complexes containing complete alpha C-terminal domains repeat transcription from protein roadblocks sites and promoters in either orientation.

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ 70 –RNAP and the λP R promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand ‘read-out’ extends, the RNAP clamp closes, expelling an inhibitory σ 70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation. Time-resolved cryo-EM captured transient intermediates during E. coli RNAP promoter melting, revealing conformational changes affecting stepwise transcription bubble opening. Results inform how DNA sequence controls bacterial transcription initiation.

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time. Bacterial transcription is terminated when RNA polymerases encounter terminator sequences. Using a single-molecule fluorescence assay, here the authors show that the release of transcript RNA precedes RNA polymerase dissociation and that the remaining RNA polymerase can reinitiate at nearby promoters.

Promoter sequences are important genetic control elements. Through their interaction with RNA polymerase they determine transcription strength and specificity, thereby regulating the first step in gene expression. Consequently, they can be targeted as elements to control predictability and tuneability of a genetic circuit, which is essential in applications such as the development of robust microbial cell factories. This review considers the promoter elements implicated in the three stages of transcription initiation, detailing the complex interplay of sequence-specific interactions that are involved, and highlighting that DNA sequence features beyond the core promoter elements work in a combinatorial manner to determine transcriptional strength. In particular, we emphasize that, aside from promoter recognition, transcription initiation is also defined by the kinetics of open complex formation and promoter escape, which are also known to be highly sequence specific. Significantly, we focus on how insights into these interactions can be manipulated to lay the foundation for a more rational approach to promoter engineering. Unravelling the intricate and sequence-specific regulation of transcription initiation by promoter sequences is crucial, as an in-depth understanding of promoter–RNA polymerase interactions can pave the way to a more rational approach to promoter engineering.

Transcription rates are regulated by the interactions between RNA polymerase, sigma factor, and promoter DNA sequences in bacteria. However, it remains unclear how non-canonical sequence motifs collectively control transcription rates. Here, we combine massively parallel assays, biophysics, and machine learning to develop a 346-parameter model that predicts site-specific transcription initiation rates for any σ 70 promoter sequence, validated across 22132 bacterial promoters with diverse sequences. We apply the model to predict genetic context effects, design σ 70 promoters with desired transcription rates, and identify undesired promoters inside engineered genetic systems. The model provides a biophysical basis for understanding gene regulation in natural genetic systems and precise transcriptional control for engineering synthetic genetic systems. Transcription rates are regulated by the interactions between RNA polymerase, sigma factor, and promoter DNA sequences in bacteria. Here the authors combine massively parallel experiments & machine learning to develop a predictive biophysical model of transcription, validated across 22132 bacterial promoters, and apply it to the design and debugging of genetic circuits.

Background Bacterial RNA polymerase holoenzyme requires sigma70 factors to start transcription by identifying promoter elements. Cyanobacteria possess multiple sigma70 factors to adapt to a wide variety of ecological niches. These factors are grouped into two categories: primary sigma factor initiates transcription of housekeeping genes during normal growth conditions, while alternative sigma factors initiate transcription of specific genes under particular conditions. However, the present classification does not consider the modular organization of their structural domains, introducing therefore multiple functional and structural biases. A comprehensive analysis of this protein family in cyanobacteria is needed to address these limitations. Results We investigated the structure and evolution of sigma70 factors in cyanobacteria, analyzing their modular architecture and variation among unicellular, filamentous, and heterocyst-forming morphotypes. 4,193 sigma70 homologs were found with 59 distinct modular patterns, including six essential and 29 accessory domains, such as DUF6596. 90% of cyanobacteria typically have 5 to 17 sigma70 homologs and this number likely depends on the strain morphotype, the taxonomic order and the genome size. We classified sigma70 factors into 12 clans and 36 families. According to taxonomic orders and phenotypic traits, the number of homologs within the 14 main families was variable, with the A.1 family including the primary sigma factor since this family was found in all cyanobacterial species. The A.1, A.5, C.1, E.1, J.1, and K.1 families were found to be key sigma families that distinguish heterocyst-forming strains. To explain the diversification and evolution of sigma70, we propose an evolutionary scenario rooted in the diversification of a common ancestor of the A1 family. This scenario is characterized by evolutionary events including domain losses, gains, insertions, and modifications. The high occurrence of the DUF6596 domain in bacterial sigma70 proteins, and its association with the highest prevalence observed in Actinobacteria, suggests that this domain might be important for sigma70 function. It also implies that the domain could have emerged in Actinobacteria and been transferred through horizontal gene transfer. Conclusion Our analysis provides detailed insights into the modular domain architecture of sigma70, introducing a novel robust classification. It also proposes an evolutionary scenario explaining their diversity across different taxonomical orders.

Photosynthesis is sensitive to environmental stress and must be efficiently modulated in response to abiotic stress. However, the underlying mechanisms are not well understood. Here we report that ARABIDOPSIS THALIANA HOMEOBOX 17 (ATHB17), an Arabidopsis HD-Zip transcription factor, regulated the expression of a number of photosynthesis associated nuclear genes (PhANGs) involved in the light reaction and ATSIG5 in response to abiotic stress. ATHB17 was responsive to ABA and multiple stress treatments. ATHB17 -overexpressing plants displayed enhanced stress tolerance, whereas its knockout mutant was more sensitive compared to the wild type. Through RNA-seq and quantitative real-time reverse transcription PCR (qRT-PCR) analysis, we found that ATHB17 did not affect the expression of many known stress-responsive marker genes. Interestingly, we found that ATHB17 down-regulated many PhANGs and could directly modulate the expression of several PhANGs by binding to their promoters. Moreover, we identified ATSIG5 , encoding a plastid sigma factor, as one of the target genes of ATHB17. Loss of ATSIG5 reduced salt tolerance while overexpression of ATSIG5 enhanced salt tolerance, similar to that of ATHB17 . ATHB17 can positively modulate the expression of many plastid encoded genes (PEGs) through regulation of ATSIG5 . Taken together, our results suggest that ATHB17 may play an important role in protecting plants by adjusting expression of PhANGs and PEGs in response to abiotic stresses.

Necrotrophic pathogens are important plant pathogens that cause many devastating plant diseases. Despite their impact, our understanding of the plant defense response to necrotrophic pathogens is limited. The WRKY33 transcription factor is important for plant resistance to necrotrophic pathogens; therefore, elucidation of its functions will enhance our understanding of plant immunity to necrotrophic pathogens. Here, we report the identification of two WRKY33-interacting proteins, nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2, which also interact with plastid-encoded plastid RNA polymerase SIGMA FACTOR1. Both SIB1 and SIB2 contain an N-terminal chloroplast targeting signal and a putative nuclear localization signal, suggesting that they are dual targeted. Bimolecular fluorescence complementation indicates that WRKY33 interacts with SIBs in the nucleus of plant cells. Both SIB1 and SIB2 contain a short VQ motif that is important for interaction with WRKY33. The two VQ motif-containing proteins recognize the C-terminal WRKY domain and stimulate the DNA binding activity of WRKY33. Like WRKY33, both SIB1 and SIB2 are rapidly and strongly induced by the necrotrophic pathogen Botrytis cinerea. Resistance to B. cinerea is compromised in the sib1 and sib2 mutants but enhanced in SIB1-overexpressing transgenic plants. These results suggest that dual-targeted SIB1 and SIB2 function as activators of WRKY33 in plant defense against necrotrophic pathogens.