MucR is a conserved global regulator in α-proteobacteria, essential for virulence in Brucella and symbiotic adaptation in rhizobia. It primarily acts as a xenogeneic silencer, repressing AT-rich horizontally acquired genes. Structurally, MucR comprises an N-terminal oligomerization domain, a linker region, and a C-terminal zinc finger DNA-binding domain. Here, we report the optimization of NMR experimental condition and NMR resonance assignments for the complex formed by the C-terminal DNA-binding domain of MucR and 3A3T-DNA. Nearly complete assignments are obtained for 1H, 13C, and 15N resonances of the protein and 1H resonances of 3A3T-DNA. Histidine tautomeric state analysis confirms that the two zinc-coordinating histidine residues adopt Nδ1–H tautomeric state. Additional NMR-based analyses provided structural insights into the MucRCTD and DNA complex formation. The NMR assignments provide a good basis for further structural study of the DNA binding and recognition mechanism of MucR.

Protein functions are closely linked to their dynamic behaviors, particularly involving low-abundance transient states. Quantifying these interconversions remains experimentally challenging, especially for proteins containing intrinsically disordered regions (IDRs). In this study, we utilize low-dose electron microscopy using graphene liquid cells (GLCs) to image and quantify interconversions between protein conformational states under optimized electron beam conditions and confinement effects. Applied to four proteins in solution: CRISPR-associated protein (Cas9), DNA binding protein (GapR), restriction enzyme (EcoR1), and an RNA binding protein with IDRs (FUS), we visualized low abundance transient structures distinct from crystalline forms, and elucidated transition pathways which are validated by cryo-electron microscopy (cryo-EM), revealing differences in their energy landscapes through single molecule imaging. We analyzed domain motions at nanometer resolution, cross-validated against independent computational and experimental methods. Shifts in state equilibria and conformational adaptations were observed when proteins are mutated or during biomolecular interactions, such as target searching, binding, and cleavage with nucleic acids. Notably, changes in the energy landscape were observed when proteins with IDRs and RNA were within close proximity but not in apparent contact, suggesting that conformational redistribution may precede complete binding. This study shows how to characterize protein conformations and their time-dependent changes using instrumentation that is widely available at research universities but not yet used for these purposes.

The locus of enterocyte effacement-encoded regulator (Ler) is a master transcriptional activator essential for the virulence of enterohemorrhagic and enteropathogenic Escherichia coli. Although Ler shares homology with the global silencer H-NS, it functions uniquely as an anti-silencer, a role strictly dependent on its oligomerization state. However, the structural mechanism governing Ler assembly remains poorly understood. In this study, we have characterized the N-terminal oligomerization domain (Ler1-74) of Ler using solution NMR spectroscopy and biophysical assays, and found that Ler1-74 shows concentration-dependent oligomerization. We demonstrate that Ler oligomerization is driven by two distinct interfaces with contrasting dynamic properties. We determined the solution structure of the Ler18-74 dimer, revealing a stable, anti-parallel "tail-to-tail" interface (dimer Site-2, residues 35-66) stabilized by a hydrophobic core. In contrast, the N-terminal interface (dimer Site-1, residues 12-33) forms a highly dynamic "head-to-head" dimer, which undergoes significant conformational exchange and exhibits concentration- and temperature-dependent dimerization. Based on these findings, we propose a structural model wherein Ler forms supramolecular assemblies through the propagation of alternating stable (Site-2) and dynamic (Site-1) interactions. This architecture, while reminiscent of H-NS, displays distinct stability features that may underlie Ler's specific anti-silencing function in bacterial pathogenesis.

>Dear Editor,The in vitro transcription(IVT) assay is a powerful tool frequently used in dissecting the molecular mechanism of transcriptional regulation and also plays an important role in the field of drug discovery and RNA-based therapeutics(Yang and Ma, 2016).

Staphylococcus aureus infections have long been a significant challenge to public health, particularly due to the emergence of multiple drug-resistant strains. SarA is a critical global regulator in S. aureus which binds to AT-rich sequences in the promoter regions of various genes, but the DNA-binding mechanism of SarA remains unclear. Here, we determined the solution structures of a monomeric DNA binding domain of SarA (SarAΔN19) and its complex with an AT-rich double-stranded DNA. The winged helix domain of SarAΔN19 binds to DNA in a classic way, with the α4 helix binding to the major groove of DNA, while the L5 loop binding to the minor groove, covering 10 AT base pairs. Residues L53, P65, and V68 of the α4 helix have hydrophobic interactions with thymine bases and sugar rings. The side chains of Arg90 and Arg84 from the wing are inserted into the minor groove, forming hydrogen bonds with A/T bases. Multiple positively charged or hydrophilic residues, including Lys54, Lys63, Lys69, Lys72, Lys82, and Gln64, interact with the phosphate groups on the DNA backbones. This complex structure provides an in-depth understanding of the molecular mechanism for SarA to bind DNA, and a better structure basis for future anti-bacterial drug design targeting SarA.

>In a recent paper, solution-state 19 F NMR spectroscopy was used to probe the conformational dynamics of β-arrestin-1, an essential adaptor and signaling component of the G-protein couple receptor （GPCR） signaling pathway. This work reveals a highly complex conformational energy landscape of β-arrestin-1, and illuminates the molecular mechanism of the membrane phosphoinositide PIP2-induced β-arrestin-1 activation at residue level.（https://doi.org/10.1038/s41467-023-43694-1）.

Abstract This study focused on the biosynthetic gene cluster BGC3 of Streptococcus mutans, a primary pathogen in dental caries, and its effects on the cariogenic virulence of Streptococcus mutans. BGC3 and ∆BGC3 Streptococcus mutans strains were constructed, and their growth curves were evaluated. Further, their acid production capacity was assessed by comparing their pH reduction levels. The survival of bacteria in phosphate citrate buffer solution (pH 3.0) was quantified. The expression levels of virulence genes (atpF, gtfC, gtfD, spaP, vicR, and ftf) were analysed using the reverse transcription-quantitative polymerase chain reaction. Bacterial viability was determined by microscopical examination of live/dead staining. Co-culture experiments were conducted to evaluate bacterial adaptability. The elimination of BGC3 did not significantly impact Streptococcus mutans growth or acid production in biofilms. The ∆BGC3 strain exhibited enhanced acid resistance and higher expression levels of virulence genes. In co-culture, ∆BGC3 exhibited superior bacterial viability. BGC3 reduced the cariogenic virulence of Streptococcus mutans in terms of acid tolerance and the expression of related genes. The knockout strain exhibited a more robust survival capability than the wild-type strain.

The widespread use of carbamate pesticides has led to numerous environmental and health concerns, including water contamination and perturbation of endocrine homeostasis among organisms. However, there remains a paucity of research elucidating the specific effects of methomyl on gut microbial composition and physiological functions. This study aimed to investigate the intricate relationship between changes in zebrafish bacterial communities and intestinal function after 56 days of sub-chronic methomyl exposure at environmentally relevant concentrations (0, 0.05, 0.10, and 0.20 mg/L). Our findings reveal significant methomyl-induced morphological changes in zebrafish intestines, characterized by villi shortening and breakage. Notably, methomyl exposure down-regulated nutrient and energy metabolism, and drug metabolism at 0.05-0.10 mg/L, while up-regulating cortisol, inflammation-related genes, and apoptotic markers at 0.20 mg/L. These manifestations indicate physiological stress imposition and disruption of gut microbiota equilibrium, impacting metabolic processes and instigating low-grade inflammatory responses and apoptotic cascades. Importantly, changes in intestinal function significantly correlated with shifts in specific bacterial taxa abundance, including Shewanella, Rubrobacter, Acinetobacter, Bacillus, Luteolibacter, Nocardia, Defluviimonas, and Bacteroides genus. In summary, our study underscores the potential adverse effects of environmental methomyl exposure on aquatic organisms, emphasizing the necessity for further research to mitigate its repercussions on environmental health and ecosystem stability.

Assessing the ergodicity of graphene liquid cell electron microscope measurements, we report that loop states of circular DNA interconvert reversibly and that loop numbers follow the Boltzmann distribution expected for this molecule in bulk solution, provided that the electron dose is low (80-keV electron energy and electron dose rate 1–20 e − Å −2 s −1 ). This imaging technique appears to act as a “slow motion” camera that reveals equilibrated distributions by imaging the time average of a few molecules without the need to image a spatial ensemble.

Background Tetramates or tetramic acid-containing compounds (TACs) are a group of bioactive natural products featuring a pyrrolidine-2,4-dione ring acknowledged being closed via Dieckmann cyclization. The cariogenic Streptococcus mutans strains bearing a muc biosynthetic gene cluster (BGC) can synthesize mutanocyclin (MUC), a 3-acetylated TAC that can inhibit both leukocyte chemotaxis and filamentous development in Candida albicans. Some strains can also accumulate reutericyclins (RTCs), the intermediates of MUC biosynthesis with antibacterial activities. However, the formation mechanism of the pyrrolidine-2,4-dione ring of MUC and the distribution of muc-like BGCs along with their ecological functions has not been explored extensively.Results We demonstrated that a key intermediate of MUC biosynthesis, M-307, is installed by a hybrid nonribosomal peptide synthetase-polyketide synthase assembly line and its pyrrolidine-2,4-dione ring is closed via an unprecedented lactam bond formation style. Subsequent C-3 acetylation will convert M-307 to RTCs, which is then hydrolyzed by a deacylase, MucF, to remove the N-1 fatty acyl appendage to generate MUC. Distribution analysis showed that the muc-like BGCs distribute predominantly in human-associated bacteria. Interestingly, most of the muc-like BGCs possessing a mucF gene were isolated from human or livestock directly, indicating their involvement in alleviating the host's immune attacks by synthesizing MUC; while those BGCs lacking mucF gene distribute mainly in bacteria from fermented products, suggesting that they tend to synthesize RTCs to compete with neighboring bacteria. It is noteworthy that many bacteria in the same habitats (e.g., the oral cavity) lack the muc-like BGC, but possess functional MucF homologues to "detoxify" RTCs to MUC, including several competitive bacteria of S. mutans. We also comparably studied the distribution of TAS1, a fungal enzyme responsible for the production of phytotoxic tenuazonic acids (TeAs), a class of 3-acetylated TACs with similar structure but distinct biosynthetic mechanism to MUC, and found that it mainly exists in plants or crops.Conclusions The in vivo and in vitro experiments revealed that the pyrrolidine-2,4-dione ring of MUC is closed via lactam bond formation, which may be adopted by many TACs without 3-acyl decorations. Besides, we found that muc-like BGCs are widespread in human-associated bacteria and their shapes and main products can be influenced by the habitat environment and vice versa. By comparing with TeAs, we provided thought-provoking insights into how ecological and evolutionary forces drive bacteria and fungi to construct a common 3-acetylated pyrrolidine-2,4-dione core through different routes, and how the biosynthetic processes are delicately controlled to generate diverse 3-acetylated TACs for environmental adaptation.

SarA is a global transcription regulator in S. aureus which regulates the expression of over 120 genes related to quorum sensing, biofilm synthesis, drug resistance and many other important physiological processes during host infection. SarA can bind to the promoter region of agr and other target genes to activate or repress the transcription. The crystal structure of SarA uncovered a MarR protein-like conformation with two symmetrical winged helix domains, while its DNA binding mechanism is still unknown. We have constructed a monomeric DNA binding domain of SarA (SarAΔN19) for the study of the interaction between SarA and DNA with NMR spectroscopy. Here, we report the 1H, 13C and 15N NMR assignment of SarAΔN19/DNA complex which is the first step towards further structure and function analysis.

Ler is a master regulator for the gene regulation of the locus of enterocyte effacement (LEE) pathogenicity island (PAI), which could activate the transcription of LEE2 to LEE5 by counteracting the repression of H-NS. Ler contains an N-terminal oligomerization domain, a linker region, and a C-terminal DNA binding domain. However, the DNA binding mechanism of Ler remains unclear. Here, we report the 1H, 13C, and 15N NMR assignments of LerCTD/3A3T-DNA complex. We have achieved 97.8

The C-terminal domain of SARS-CoV main protease (Mpro-C) can form 3D domain-swapped dimer by exchanging the α1-helices fully buried inside the protein hydrophobic core, under non-denaturing conditions. Here, we report that Mpro-C can also form amyloid fibrils under the 3D domain-swappable conditions in vitro, and the fibrils are not formed through runaway/propagated domain swapping. It is found that there are positive correlations between the rates of domain swapping dimerization and amyloid fibrillation at different temperatures, and for different mutants. However, some Mpro-C mutants incapable of 3D domain swapping can still form amyloid fibrils, indicating that 3D domain swapping is not essential for amyloid fibrillation. Furthermore, NMR H/D exchange data and molecular dynamics simulation results suggest that the protofibril core region tends to unpack at the early stage of 3D domain swapping, so that the amyloid fibrillation can proceed during the 3D domain swapping process. We propose that 3D domain swapping makes it possible for the unpacking of the amyloidogenic fragment of the protein and thus accelerates the amyloid fibrillation process kinetically, which explains the well-documented correlations between amyloid fibrillation and 3D domain swapping observed in many proteins.

Granulomas are the pathological hallmark of tuberculosis (TB). In individuals with latent TB infection, Mycobacterium tuberculosis cells reside within granulomas in a nonreplicating dormant state, and a portion of them will develop active TB. Little is known on the bacterial mechanisms/factors involved in this process. In this study, we found that WhiB4, an oxygen sensor and a transcription factor, plays a critical role in disease progression and reactivation of Mycobacterium marinum (M. marinum) infection in zebrafish. We show that the whiB4::Tn mutant of M. marinum caused persistent infection in adult zebrafish, which is characterized by the lower but stable bacterial loads, constant number of nonnecrotized granulomas in fewer organs, and reduced inflammation compared to those of zebrafish infected with the wild-type bacteria or the complemented strain. The mutant bacteria in zebrafish were also less responsive to antibiotic treatments. Moreover, the whiB4::Tn mutant was defective in resuscitation from hypoxia-induced dormancy and the DosR regulon was dysregulated in the mutant. Taken together, our results suggest that WhiB4 is a major driver of reactivation from persistent infection. IMPORTANCE About one-quarter of the world's population has latent TB infection, and 5 to 10% of those individuals will fall ill with TB. Our finding suggests that WhiB4 is an attractive target for the development of novel therapeutics, which may help to prevent the reactivation of latent infection, thereby reducing the incidences of active TB.

Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis, has latently infected about one-third of the world's population and may lead to severe clinical symptoms and death. The WhiB4 protein, a transcription factor, plays a crucial role in the survival and pathology of Mtb. WhiB4 leads to the condensation of mycobacterial nucleoids and regulates the expression of genes involved in central metabolism, respiration, and maintaining redox homeostasis. Here, we report the solution structure of reduced apo-WhiB4 monomer, which consists of an unstructured N-terminal domain with four cysteine residues and a helix-turn-helix C-terminal domain that plays a major role in DNA binding. The C-terminal domain of WhiB4 binds DNA at the minor groove, with five positively charged lysine/arginine residues contacting DNA sugar-phosphate backbones through electrostatic interactions. AT-rich DNA sequences with narrower minor grooves are more preferred by WhiB4. The binding affinity of a single C-terminal domain of WhiB4 is weak. When oxidized, WhiB4 can form dimers and oligomers in different forms through disulfide bonds, which should significantly enhance its DNA binding ability through multivalent effect and change the local structure of target genes and influence their transcription. These structural features form the basis for WhiB4 to function as a redox-sensitive transcription factor in Mtb.

Horizontal gene transfer (HGT) is a major driving force for bacterial evolution. To avoid the deleterious effects due to the unregulated expression of newly acquired foreign genes, bacteria have evolved specific proteins named xenogeneic silencers to recognize foreign DNA sequences and suppress their transcription. As there is considerable diversity in genomic base compositions among bacteria, how xenogeneic silencers distinguish self- from nonself DNA in different bacteria remains poorly understood. This review summarizes the progress in studying the DNA binding preferences and the underlying molecular mechanisms of known xenogeneic silencer families, represented by H-NS of Escherichia coli, Lsr2 of Mycobacterium, MvaT of Pseudomonas, and Rok of Bacillus. Comparative analyses of the published data indicate that the differences in DNA recognition mechanisms enable these xenogeneic silencers to have clear characteristics in DNA sequence preferences, which are further correlated with different host genomic features. These correlations provide insights into the mechanisms of how these xenogeneic silencers selectively target foreign DNA in different genomic backgrounds. Furthermore, it is revealed that the genomic AT contents of bacterial species with the same xenogeneic silencer family proteins are distributed in a limited range and are generally lower than those species without any known xenogeneic silencers in the same phylum/class/genus, indicating that xenogeneic silencers have multifaceted roles on bacterial genome evolution. In addition to regulating horizontal gene transfer, xenogeneic silencers also act as a selective force against the GC to AT mutational bias found in bacterial genomes and help the host genomic AT contents maintained at relatively low levels.

The WhiB4 protein, a member of WhiB-like proteins, plays an important role in the survival and pathology of Mycobacterium tuberculosis ( Mtb ). As a transcription factor, WhiB4 regulates the expression of genes involved in maintaining redox homeostasis, central metabolism, and respiration. Furthermore, WhiB4 leads to the condensation of mycobacterial nucleoids and is capable of binding to DNA. WhiB4 contains four cysteine residues and exists in multiple forms under different redox environments, including a dimeric holo form with iron-sulfur cluster, multimeric disulfide-linked oxidized apo forms and monomeric reduced apo form. Here, we report the 1 H, 13 C, 15 N chemical shifts of WhiB4 protein in its reduced apo state, providing a basis for the determination of its solution structure.

Methylation of DNA at CpG sites is a major mark for epigenetic regulation, but how transcription factors are influenced by CpG methylation is not well understood. Here, we report the molecular mechanisms of how the TCF (T-cell factor) and GEF (glucose transporter 4 enhancer factor) families of proteins selectively target unmethylated DNA sequences with a C-clamp type zinc finger domain. The structure of the C-clamp domain from human GEF family protein HDBP1 (C-clampHDBP1) in complex with DNA was determined using NMR spectroscopy, which adopts a unique zinc finger fold and selectively binds RCCGG (R = A/G) DNA sequences with an "Arg···Trp-Lys-Lys" DNA recognition motif inserted in the major groove. The CpG base pairs are central to the binding due to multiple hydrogen bonds formed with the backbone carbonyl groups of Trp378 and Lys379, as well as the side chain ε-amino groups of Lys379 and Lys380 from C-clampHDBP1. Consequently, methylation of the CpG dinucleotide almost abolishes the binding. Homology modeling reveals that the C-clamp domain from human TCF1E (C-clampTCF1E) binds DNA through essentially the same mechanism, with a similar "Arg···Arg-Lys-Lys" DNA recognition motif. The substitution of tryptophan by arginine makes C-clampHDBP1 prefer RCCGC DNA sequences. The two signature DNA recognition motifs are invariant in the GEF and TCF families of proteins, respectively, from fly to human. The recognition of the CpG dinucleotide through two consecutive backbone carbonyl groups is the same as that of the CXXC type unmethylated CpG DNA binding domains, suggesting a common mechanism shared by unmethylated CpG binding proteins.

The nucleoid-associated protein GapR found in Caulobacter crescentus is crucial for DNA replication, transcription, and cell division. Associated with overtwisted DNA in front of replication forks and the 3′ end of highly-expressed genes, GapR can stimulate gyrase and topo IV to relax (+) supercoils, thus facilitating the movement of the replication and transcription machines. GapR forms a dimer-of-dimers structure in solution that can exist in either an open or a closed conformation. It initially binds DNA through the open conformation and then undergoes structural rearrangement to form a closed tetramer, with DNA wrapped in the central channel. Here, we show that the DNA binding domain of GapR (residues 1–72, GapRΔC17) exists as a dimer in solution and adopts the same fold as the two dimer units in the full-length tetrameric protein. It binds DNA at the minor groove and reads the spatial distribution of DNA phosphate groups through a lysine/arginine network, with a preference towards AT-rich overtwisted DNA. These findings indicate that the dimer unit of GapR has an intrinsic DNA binding preference. Thus, at the initial binding step, the open tetramer of GapR with two relatively independent dimer units can be more efficiently recruited to overtwisted regions.