Bacterial Gene Regulation Research Articles

Transcriptional Pausing as a Mediator of Bacterial Gene Regulation.

Cellular life depends on transcription of DNA by RNA polymerase to express genetic information. RNA polymerase has evolved not just to read information from DNA and write it to RNA but also to sense and process information from the cellular and extracellular environments. Much of this information processing occurs during transcript elongation, when transcriptional pausing enables regulatory decisions. Transcriptional pauses halt RNA polymerase in response to DNA and RNA sequences and structures at locations and times that help coordinate interactions with small molecules and transcription factors important for regulation. Four classes of transcriptional pause signals are now evident after decades of study: elemental pauses, backtrack pauses, hairpin-stabilized pauses, and regulator-stabilized pauses. In this review, I describe current understanding of the molecular mechanisms of these four classes of pause signals, remaining questions about how RNA polymerase responds to pause signals, and the many exciting directions now open to understand pausing and the regulation of transcript elongation on a genome-wide scale.

STAS Domain Only Proteins in Bacterial Gene Regulation.

Sulfate Transport Anti-Sigma antagonist domains (Pfam01740) are found in all branches of life, from eubacteria to mammals, as a conserved fold encoded by highly divergent amino acid sequences. These domains are present as part of larger SLC26/SulP anion transporters, where the STAS domain is associated with transmembrane anchoring of the larger multidomain protein. Here, we focus on STAS Domain only Proteins (SDoPs) in eubacteria, initially described as part of the Bacillus subtilis Regulation of Sigma B (RSB) regulatory system. Since their description in B. subtilis, SDoPs have been described to be involved in the regulation of sigma factors, through partner-switching mechanisms in various bacteria such as: Mycobacterium. tuberculosis, Listeria. monocytogenes, Vibrio. fischeri, Bordetella bronchiseptica, among others. In addition to playing a canonical role in partner-switching with an anti-sigma factor to affect the availability of a sigma factor, several eubacterial SDoPs show additional regulatory roles compared to the original RSB system of B. subtilis. This is of great interest as these proteins are highly conserved, and often involved in altering gene expression in response to changes in environmental conditions. For many of the bacteria we will examine in this review, the ability to sense environmental changes and alter gene expression accordingly is critical for survival and colonization of susceptible hosts.

Prediction and analysis of metagenomic operons via MetaRon: a pipeline for prediction of Metagenome and whole-genome opeRons

BackgroundEfficient regulation of bacterial genes in response to the environmental stimulus results in unique gene clusters known as operons. Lack of complete operonic reference and functional information makes the prediction of metagenomic operons a challenging task; thus, opening new perspectives on the interpretation of the host-microbe interactions.ResultsIn this work, we identified whole-genome and metagenomic operons via MetaRon (Metagenome and whole-genome opeRon prediction pipeline). MetaRon identifies operons without any experimental or functional information. MetaRon was implemented on datasets with different levels of complexity and information. Starting from its application on whole-genome to simulated mixture of three whole-genomes (E. coli MG1655, Mycobacterium tuberculosis H37Rv and Bacillus subtilis str. 16), E. coli c20 draft genome extracted from chicken gut and finally on 145 whole-metagenome data samples from human gut. MetaRon consistently achieved high operon prediction sensitivity, specificity and accuracy across E. coli whole-genome (97.8, 94.1 and 92.4%), simulated genome (93.7, 75.5 and 88.1%) and E. coli c20 (87, 91 and 88%,), respectively. Finally, we identified 1,232,407 unique operons from 145 paired-end human gut metagenome samples. We also report strong association of type 2 diabetes with Maltose phosphorylase (K00691), 3-deoxy-D-glycero-D-galacto-nononate 9-phosphate synthase (K21279) and an uncharacterized protein (K07101).ConclusionWith MetaRon, we were able to remove two notable limitations of existing whole-genome operon prediction methods: (1) generalizability (ability to predict operons in unrelated bacterial genomes), and (2) whole-genome and metagenomic data management. We also demonstrate the use of operons as a subset to represent the trends of secondary metabolites in whole-metagenome data and the role of secondary metabolites in the occurrence of disease condition. Using operonic data from metagenome to study secondary metabolic trends will significantly reduce the data volume to more precise data. Furthermore, the identification of metabolic pathways associated with the occurrence of type 2 diabetes (T2D) also presents another dimension of analyzing the human gut metagenome. Presumably, this study is the first organized effort to predict metagenomic operons and perform a detailed analysis in association with a disease, in this case type 2 diabetes. The application of MetaRon to metagenomic data at diverse scale will be beneficial to understand the gene regulation and therapeutic metagenomics.

Selected Aspects Of The Crispr-Cas Biology And Applications

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins are components of the adaptive immunity system, protecting against foreign DNA, which are present in many bacteria species. Recent years have brought extensive research on this system however, not all of its biological properties have been discovered so far. It was recently discovered that CRISPR-Cas can regulate the formation of biofilm and is closely associated with the DNA repair system in bacterial cells. It is also likely that some of the spacer sequences are complementary to short sequences in the bacterial genome, which may have an influence on regulation of bacterial genes, e.g. virulence factors. Besides, phages can synthesize anti-CRISPR genes, which could be of use in the future for the purpose of development of an alternative therapy against multi-drug resistant bacterial strains. Here we present an elementary characteristic of CRISPR-Cas system, including the structure and the brief mechanism of action, systematic classification and its importance for medicine and biotechnology issues. We would like to stress the huge potential of CRISPR-Cas by discussing the selected but varied aspects. 1. Introduction. 2. Structure, operation and differences. 3. Bacterial typing. 4. Correlation with bacterial pathogenicity. 5. Potential tool for medicine. 5.1. CRISPR-tool for genome editing. 5.2. Instances of CRISPR-tool strategies in medicine. 6. Phage response. 7. Conclusions

Rewiring the specificity of extracytoplasmic function sigma factors

Bacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators' DNA binding and precise determination of biologically significant binding sites. Here we address these challenges by solving the DNA-specificity code of extracytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their putative regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the autoregulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid-nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic footprinting pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that some ECF σs are conserved global regulators controlling many genes throughout the genome, which are important under many conditions, while others are local regulators, controlling a few closely linked genes in response to specific stimuli in select species. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.

Redefining fundamental concepts of transcription initiation in bacteria.

Despite enormous progress in understanding the fundamentals of bacterial gene regulation, our knowledge remains limited when compared with the number of bacterial genomes and regulatory systems to be discovered. Derived from a small number of initial studies, classic definitions for concepts of gene regulation have evolved as the number of characterized promoters has increased. Together with discoveries made using new technologies, this knowledge has led to revised generalizations and principles. In this Expert Recommendation, we suggest precise, updated definitions that support a logical, consistent conceptual framework of bacterial gene regulation, focusing on transcription initiation. The resulting concepts can be formalized by ontologies for computational modelling, laying the foundation for improved bioinformatics tools, knowledge-based resources and scientific communication. Thus, this work will help researchers construct better predictive models, with different formalisms, that will be useful in engineering, synthetic biology, microbiology and genetics.

Programmable CRISPR-Cas transcriptional activation in bacteria.

Programmable gene activation enables fine‐tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR‐Cas‐mediated gene activation has been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR‐Cas transcription activators. Using this platform, we identified a novel CRISPR activator, dCas9‐AsiA, that could activate gene expression by more than 200‐fold across genomic and plasmid targets with diverse promoters after directed evolution. The evolved dCas9‐AsiA can simultaneously mediate activation and repression of bacterial regulons in E. coli. We further identified hundreds of promoters with varying basal expression that could be induced by dCas9‐AsiA, which provides a rich resource of genetic parts for inducible gene activation. Finally, we show that dCas9‐AsiA can be ported to other bacteria of clinical and bioindustrial relevance, thus enabling bacterial CRISPRa in more application areas. This work expands the toolbox for programmable gene regulation in bacteria and provides a useful resource for future engineering of other bacterial CRISPR‐based gene regulators.

Multidimensional gene regulatory landscape of a bacterial pathogen in plants

Understanding the gene regulation of plant pathogens is crucial for pest control and thus global food security. An integrated understanding of bacterial gene regulation in the host is dependent on multi-omic datasets, but these are largely lacking. Here, we simultaneously characterized the transcriptome and proteome of a bacterial pathogen in plants. We found a number of bacterial processes affected by plant immunity at the transcriptome and proteome levels. For instance, salicylic acid-mediated plant immunity suppressed the accumulation of proteins comprising the tip component of the bacterial type III secretion system. Interestingly, there were instances of concordant and discordant regulation of bacterial messenger RNAs and proteins. Gene co-expression analysis uncovered previously unknown gene regulatory modules underlying virulence. This study provides molecular insights into the multiple layers of gene regulation that contribute to bacterial growth in planta, and elucidates the role of plant immunity in affecting pathogen responses.

Identifying species‐specific Hfq‐RNA Interactions using a bacterial three‐hybrid assay

Bacteria use non‐coding small RNAs (sRNAs) to regulate expression of target mRNAs, often with the help of protein chaperones. About half of bacterial species possess a well‐characterized globally‐acting RNA‐binding protein, a hexameric protein called Hfq, which contributes to virulence in many pathogenic species. While much is known about the mechanism of RNA interactions by Hfq proteins in certain well‐studied model bacteria, there are significant gaps in our understanding of how conserved the RNA‐binding behavior of Hfq homologs from diverse bacterial organisms. Here, we use a recently developed bacterial three‐hybrid (B3H) assay to further define the interactions between Hfq and its RNA substrates. This assay connects the interaction between a DNA‐bound RNA and a RNA polymerase (RNAP)‐bound protein Hfq to the expression of a reporter gene, providing a genetic route to the analysis of RNA interactions from a heterologously expressed RNA‐binding protein. This provides a facile route to study the behavior of diverse RNA‐binding proteins in an innocuous laboratory strain of E. coli. Here we present data collected using the B3H assay to compare the RNA interactions of Hfq from several bacterial species, including Listeria monocytogenes, Caulobacter crescentus, and Escherichia coli. We have observed distinct RNA‐binding profiles between these Hfq proteins. Sequence alignments of these three proteins led to a hypothesis that charged residues along the rim surface of Hfq may underlie the differences in binding preferences for different sRNAs. Data will be presented that test this model using both chimeric and site‐directed mutants. Our long‐term goal is to gain a better understanding of what structural elements of an RNA‐binding protein such as Hfq determine its RNA binding and function in driving bacterial gene regulation.Support or Funding InformationThis work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.

Getting the message: expansion of a bacterial three‐hybrid assay to mRNA‐protein interactions

Non‐coding small RNAs (sRNAs) contribute to bacterial biofilm formation and virulence, but there is much we do not understand about their molecular mechanisms. Hfq is the best‐studied bacterial RNA chaperone protein and has become the paradigmatic example for how RNA‐binding proteins facilitate mRNA regulation by sRNAs. Here we present progress on establishing and broadening the capabilities of a recently developed bacterial three‐hybrid (B3H) assay for studying interactions between Hfq and its RNA targets. While Hfq interactions with both mRNAs and sRNAs are critical for its function, only sRNA‐Hfq interactions have been accessible in the B3H assay to date. In order to expand the assay to mRNA‐Hfq interactions, we first demonstrated the assay’s capacity to detect interactions between synthetic mRNA mimics and Escherichia coli Hfq. Furthermore, we have developed constructs that support detection of interactions between mRNA 5′UTRs and two E. coli RNA‐chaperone proteins: Hfq and ProQ. This development points to the potential of the B3H assay to detect a wider variety of RNA‐protein interactions than previously possible, and will allow us to dissect the molecular mechanisms of these interactions. Current efforts aim to understand the relationship between the translational status of an mRNA fragment and its ability to bind protein partners in the B3H assay. In addition, we are working to understand the relationship between the in vivo B3H data on RNA‐protein interactions and the binding energetics of these interactions, which are typically measured in vitro. The results of these efforts will provide a deeper understanding of the implications of B3H data for RNA‐protein binding and regulatory mechanisms. By expanding both the capabilities of this bacterial three‐hybrid assay and our understanding of the data it provides, we aim to broaden its utility in studying bacterial gene regulation and RNA‐protein interactions.Support or Funding InformationThis work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.

Identification of Quorum Sensing Activators and Inhibitors in The Marine Sponge Sarcotragus spinosulus.

Marine sponges, a well-documented prolific source of natural products, harbor highly diverse microbial communities. Their extracts were previously shown to contain quorum sensing (QS) signal molecules of the N-acyl homoserine lactone (AHL) type, known to orchestrate bacterial gene regulation. Some bacteria and eukaryotic organisms are known to produce molecules that can interfere with QS signaling, thus affecting microbial genetic regulation and function. In the present study, we established the production of both QS signal molecules as well as QS inhibitory (QSI) molecules in the sponge species Sarcotragus spinosulus. A total of eighteen saturated acyl chain AHLs were identified along with six unsaturated acyl chain AHLs. Bioassay-guided purification led to the isolation of two brominated metabolites with QSI activity. The structures of these compounds were elucidated by comparative spectral analysis of 1HNMR and HR-MS data and were identified as 3-bromo-4-methoxyphenethylamine (1) and 5,6-dibromo-N,N-dimethyltryptamine (2). The QSI activity of compounds 1 and 2 was evaluated using reporter gene assays for long- and short-chain AHL signals (Escherichia coli pSB1075 and E. coli pSB401, respectively). QSI activity was further confirmed by measuring dose-dependent inhibition of proteolytic activity and pyocyanin production in Pseudomonas aeruginosa PAO1. The obtained results show the coexistence of QS and QSI in S. spinosulus, a complex signal network that may mediate the orchestrated function of the microbiome within the sponge holobiont.

UV oxidation of cyclic AMP receptor protein, a global bacterial gene regulator, decreases DNA binding and cleaves DNA at specific sites

UV light is a widely-employed, and environmentally-sensitive bactericide but its mechanism of action is not fully defined. Proteins are major chromophores and targets for damage due to their abundance, but the role of proteins in inducing damage to bound DNA, and the effects on DNA-protein interactions is less well characterized. In E. coli (and other Gram-negative bacteria) the cyclic AMP receptor protein (CRP/CAP) regulates more than 500 genes. In this study we show that exposure of isolated dimeric CRP-cAMP to UV modifies specific Met, Trp, Tyr, and Pro side-chains, induces inter-protein Tyr63-Tyr41 cross-links, and decreases DNA binding via oxidation of Met114/Pro110 residues in close proximity at the CRP dimer interface. UV exposure also modifies DNA-bound cAMP-CRP, with this resulting in DNA cleavage at specific G/C residues within the sequence bound to CRP, but not at other G/C sites. Oxidation also increases CRP dissociation from DNA. The modifications at the CRP dimer interface, and the site-specific DNA strand cleavage are proposed to occur via oxidation of two species Met residues (Met114 and Met189, respectively) to reactive persulfoxides that damage neighbouring amino acids and DNA bases. These data suggest that modification to CRP, and bound DNA, contributes to UV sensitivity.

Chromosomal origin of replication coordinates logically distinct types of bacterial genetic regulation

For a long time it has been hypothesized that bacterial gene regulation involves an intricate interplay of the transcriptional regulatory network (TRN) and the spatial organization of genes in the chromosome. Here we explore this hypothesis both on a structural and on a functional level. On the structural level, we study the TRN as a spatially embedded network. On the functional level, we analyze gene expression patterns from a network perspective (“digital control”), as well as from the perspective of the spatial organization of the chromosome (“analog control”). Our structural analysis reveals the outstanding relevance of the symmetry axis defined by the origin (Ori) and terminus (Ter) of replication for the network embedding and, thus, suggests the co-evolution of two regulatory infrastructures, namely the transcriptional regulatory network and the spatial arrangement of genes on the chromosome, to optimize the cross-talk between two fundamental biological processes: genomic expression and replication. This observation is confirmed by the functional analysis based on the differential gene expression patterns of more than 4000 pairs of microarray and RNA-Seq datasets for E. coli from the Colombos Database using complex network and machine learning methods. This large-scale analysis supports the notion that two logically distinct types of genetic control are cooperating to regulate gene expression in a complementary manner. Moreover, we find that the position of the gene relative to the Ori is a feature of very high predictive value for gene expression, indicating that the Ori–Ter symmetry axis coordinates the action of distinct genetic control mechanisms.

Protein Activity Sensing in Bacteria in Regulating Metabolism and Motility.

Bacteria have evolved complex sensing and signaling systems to react to their changing environments, most of which are present in all domains of life. Canonical bacterial sensing and signaling modules, such as membrane-bound ligand-binding receptors and kinases, are very well described. However, there are distinct sensing mechanisms in bacteria that are less studied. For instance, the sensing of internal or external cues can also be mediated by changes in protein conformation, which can either be implicated in enzymatic reactions, transport channel formation or other important cellular functions. These activities can then feed into pathways of characterized kinases, which translocate the information to the DNA or other response units. This type of bacterial sensory activity has previously been termed protein activity sensing. In this review, we highlight the recent findings about this non-canonical sensory mechanism, as well as its involvement in metabolic functions and bacterial motility. Additionally, we explore some of the specific proteins and protein-protein interactions that mediate protein activity sensing and their downstream effects. The complex sensory activities covered in this review are important for bacterial navigation and gene regulation in their dynamic environment, be it host-associated, in microbial communities or free-living.

TetR Regulated in vivo Repression Technology to Identify Conditional Gene Silencing in Genetically Engineerable Bacteria Using Vibrio cholerae Murine Infections as Model System.

Investigation of bacterial gene regulation upon environmental changes is still a challenging task. For example, Vibrio cholerae, a pathogen of the human gastrointestinal tract, faces diverse transient conditions in different compartments upon oral ingestion. Genetic reporter systems have been demonstrated to be extremely powerful tools to unravel gene regulation events in complex conditions, but so far focused mainly on gene induction. Herein, we describe the TetR-controlled recombination-based in vivo expression technology TRIVET, which allows detection of gene silencing events. TRIVET resembles a modified variant of the in vivo expression technology (IVET) as well as recombination-based in vivo expression technology (RIVET), which were used to identify conditional gene induction in several bacteria during host colonization. Like its predecessors, TRIVET is a single cell based reporter system, which allows the analysis of bacterial gene repression in a spatiotemporal manner via phenotypical changes in the resistance profile. Briefly, a promoterless tetR (encoding the transcriptional repressor TetR) can be integrated randomly into the bacterial genome via transposon mutagenesis or site-specific downstream of a promoter of interest via homologous recombination. Reduction of transcriptional expression of TetR results in a de-repression of the TetR-controlled resolvase TnpR, which in turn leads to excision of an antibiotic resistance cassette (also known as res-cassette) and altered resistance profile observable via streaking on ampicillin and kanamycin plates. This alteration can then be quantified as the ratio between resistant and non-resistant isolates. Furthermore, the newly introduced second reporter gene, a promoterless phoA (encoding the alkaline phosphatase PhoA) offers an additional validation step of the results via an independent colorimetric assay to measure enzyme activity. The protocol presented herein also offers an approach to identify the gene locus in case of the random screen for gene repression as well as a quantification of the conditional repression of a gene of interest. Although the current protocol is established for gene repression during host colonization, it can likely be adapted to study gene silencing under various conditions faced by a bacterium.

Carbohydrate Metabolism Regulated by Antisense vicR RNA in Cariogenicity

Streptococcus mutans is a major cariogenic pathogen that resides in multispecies oral microbial biofilms. The VicRK 2-component system is crucial for bacterial adaptation, virulence, and biofilm organization and contains a global and vital response regulator, VicR. Notably, we identified an antisense vicR RNA (ASvicR) associated with an adjacent RNase III–encoding (rnc) gene that was relevant to microRNA-size small RNAs (msRNAs). Here, we report that ASvicR overexpression significantly impeded bacterial growth, biofilm exopolysaccharide synthesis, and cariogenicity in vivo. Transcriptome analysis revealed that the ASvicR RNA mainly regulated carbohydrate metabolism. In particular, overproducing ASvicR demonstrated a reduction in galactose and glucose metabolism by monosaccharide composition analysis. The results of high-performance gel permeation chromatography revealed that the water-insoluble glucans isolated from ASvicR presented much lower molecular weights. Furthermore, direct evidence showed that total RNAs were disrupted by rnc-encoded RNase III. With the coexpression of T4 RNA ligase, putative msRNA1657, which is an rnc-related messenger RNA, was verified to bind to the 5′-UTR regions of the vicR gene. Furthermore, ASvicR regulation revealed a sponge regulatory-mediated network for msRNA associated with adjacent RNase III–encoding genes. There was an increase in ASvicR transcript levels in clinical S. mutans strains from caries-free children, while the expression of ASvicR was decreased in early childhood caries patients; this outcome may be explored as a potential strategy contributing to the management of dental caries. Taken together, our findings suggest an important role of ASvicR-mediated sponge regulation in S. mutans, indicating the characterization of lactose metabolism by a vital response regulator in cariogenicity. These findings have a number of implications and have reshaped our understanding of bacterial gene regulation from its transcriptional conception to the key roles of regulatory RNAs.

Non-typeable Haemophilus influenzae isolates from patients with chronic obstructive pulmonary disease contain new phase-variable modA methyltransferase alleles controlling phasevarions

Phasevarions (phase-variable regulons) are emerging as an important area of bacterial gene regulation. Many bacterial pathogens contain phasevarions, with gene expression controlled by the phase-variable expression of DNA methyltransferases via epigenetic mechanisms. Non-typeable Haemophilus influenzae (NTHi) contains the phase-variable methyltransferase modA, of which multiple allelic variants exist (modA1-21). We have previously demonstrated 5 of 21 these modA alleles are overrepresented in NTHi strains isolated from children with middle ear infections. In this study we investigated the modA allele distribution in NTHi strains isolated from patients with chronic obstructive pulmonary disease, COPD. We demonstrate that the distribution of modA alleles in a large panel of COPD isolates is different to the distribution seen in middle ear infections, suggesting different modA alleles may provide distinct advantages in the differing niches of the middle ear and COPD airways. We also identified two new phase-variable modA alleles – modA15 and modA18 – and demonstrate that these alleles methylate distinct DNA sequences and control unique phasevarions. The modA15 and modA18 alleles have only been observed in COPD isolates, indicating that these two alleles may be markers for isolates likely to cause exacerbations of COPD.

Mechanisms by Which Small RNAs Affect Bacterial Activity

The oral cavity contains a distinct habitat that supports diverse bacterial flora. Recent observations have provided additional evidence that sRNAs are key regulators of bacterial physiology and pathogenesis. These sRNAs have been divided into 5 functional groups: cis-encoded RNAs, trans-encoded RNAs, RNA regulators of protein activity, bacterial CRISPR (clustered regularly interspaced short palindromic repeat) RNAs, and a novel category of miRNA-size small RNAs (msRNAs). In this review, we discuss a critical group of key commensal and opportunistic oral pathogens. In general, supragingival bacterial sRNAs function synergistically to fine-tune the regulation of cellular processes and stress responses in adaptation to environmental changes. Particularly in the cariogenic bacteria Streptococcus mutans, both the antisense vicR RNA and msRNA1657 can impede the metabolism of bacterial exopolysaccharides, prevent biofilm formation, and suppress its cariogenicity. In Enterococcus faecalis, selected sRNAs control the expression of proteins involved in diverse cellular processes and stress responses. In subgingival plaques, sRNAs from periodontal pathogens can function as novel bacterial signaling molecules that mediate bacterial-human interactions in periodontal homeostasis. In Porphyromonas gingivalis, the expression profiles of putative sRNA101 and sRNA42 were found to respond to hemin availability after hemin starvation. Regarding Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), a major periodontal pathogen associated with aggressive periodontitis, the predicted sRNAs interact with several virulence genes, including those encoding leukotoxin and cytolethal distending toxin. Furthermore, in clinical isolates, these associated RNAs could be explored not only as potential biomarkers for oral disease monitoring but also as alternative types of regulators for drug design. Thus, this emerging subspecialty of bacterial regulatory RNAs could reshape our understanding of bacterial gene regulation from their key roles of endogenous regulatory RNAs to their activities in pathologic processes.

Pleiades Toolkit: Automatic rule-based modeling of bacterial gene regulation enables simulation, prediction, and perturbation of gene responses

Pleiades Toolkit: Automatic rule-based modeling of bacterial gene regulation enables simulation, prediction, and perturbation of gene responses

Novel Heat-Promoted Folding Dynamics of the yybP-ykoY Manganese Riboswitch: Kinetic and Thermodynamic Studies at the Single-Molecule Level

Riboswitches are highly structured RNA elements that regulate gene expressions by undergoing conformational changes in response to their cognate ligands. Such regulatory strategies are particularly prevalent among bacteria, which need to be evolutionarily responsive to thermal fluctuations in the surrounding environment, for example, generating extremophiles evolved to survive anomalously high or low temperatures. As a consequence, the response of such riboswitches to thermal stress becomes of considerable interest. In this study, the temperature-dependent folding kinetics and thermodynamics of the manganese riboswitch ( yybP-ykoY) is studied by single-molecule FRET spectroscopy under external thermal control. Surprisingly, the folding of the manganese riboswitch is found to be strongly promoted by temperature. Detailed thermodynamic analysis of the equilibrium and forward/reverse kinetic rate constants reveal folding to be a strongly endothermic process (Δ H0) made feasible by an overall entropic lowering (- TΔ S0) in free energy. This is in contrast to a more typical picture of RNA folding achieving a more compact, highly ordered state (Δ S0) and clearly speaks to the significant role of solvent/cation reorganization in the folding thermodynamics. With the help of the transition-state theory, free energy landscapes for the manganese riboswitch are constructed from the temperature-dependent kinetic data, revealing two distinctive folding mechanisms promoted by Mg2+ and Mn2+, respectively. It is speculated that this unconventional temperature dependence for folding of the manganese riboswitch may reflect evolution of bacterial gene regulation strategies to survive environments with large-temperature variations.