Bacterial Transcription Continues to Surprise: Activation by Alarmone-Mediated σ-Factor Tethering.

Travis et al. (2020) reveal how Francisella tularensis uses stress-induced ppGpp to activate its virulent pathogenesis program by tethering an αCTD-DNA organizer (PigR) to a σ-organizing heterodimer (MglA-SspA), highlighting the remarkable diversity of transcriptional mechanisms in under-studied bacteria.

Basis of narrow-spectrum activity of fidaxomicin on Clostridioides difficile.

Fidaxomicin (Fdx) is widely used to treat Clostridioides difficile (Cdiff) infections, but the molecular basis of its narrow-spectrum activity in the human gut microbiome remains unknown. Cdiff infections are a leading cause of nosocomial deaths1. Fidaxomicin, which inhibits RNA polymerase, targets Cdiff with minimal effects on gut commensals, reducing recurrence of Cdiff infection2,3. Here we present the cryo-electron microscopy structure of Cdiff RNA polymerase in complex with fidaxomicin and identify a crucial fidaxomicin-binding determinant of Cdiff RNA polymerase that is absent in most gut microbiota such as Proteobacteria and Bacteroidetes. By combining structural, biochemical, genetic and bioinformatic analyses, we establish that a single residue in Cdiff RNA polymerase is a sensitizing element for fidaxomicin narrow-spectrum activity. Our results provide a blueprint for targeted drug design against an important human pathogen.

RNA polymerase SI3 domain modulates global transcriptional pausing and pause-site fluctuations.

Transcriptional pausing aids gene regulation by cellular RNA polymerases (RNAPs). A surface-exposed domain inserted into the catalytic trigger loop (TL) of Escherichia coli RNAP, called SI3, modulates pausing and is essential for growth. Here we describe a viable E. coli strain lacking SI3 enabled by a suppressor TL substitution (ß'Ala941→Thr; ΔSI3*). ΔSI3* increased transcription rate in vitro relative to ΔSI3, possibly explaining its viability, but retained both positive and negative effects of ΔSI3 on pausing. ΔSI3* inhibited pauses stabilized by nascent RNA structures (pause hairpins; PHs) but enhanced other pauses. Using NET-seq, we found that ΔSI3*-enhanced pauses resemble the consensus elemental pause sequence whereas sequences at ΔSI3*-suppressed pauses, which exhibited greater association with PHs, were more divergent. ΔSI3*-suppressed pauses also were associated with apparent pausing one nucleotide upstream from the consensus sequence, often generating tandem pause sites. These '-2 pauses' were stimulated by pyrophosphate in vitro and by addition of apyrase to degrade residual NTPs during NET-seq sample processing. We propose that some pauses are readily reversible by pyrophosphorolysis or single-nucleotide cleavage. Our results document multiple ways that SI3 modulates pausing in vivo and may explain discrepancies in consensus pause sequences in some NET-seq studies.

Fidaxomicin resistance in Clostridioides difficile: a systematic review and predictive modeling with RNA polymerase binding sites.

Fidaxomicin (FDX), an RNA polymerase (RNAP) inhibitor antibiotic, is a guideline-recommended therapy for Clostridioides difficile infection. Mutations associated with reduced FDX minimum inhibitory concentrations (MICs) have been identified. However, the molecular characterization of these mutations on FDX binding and the development of FDX resistance have not been studied. The purpose of this systematic review was to identify FDX resistance in C. difficile isolates and determine whether single nucleotide polymorphisms associated with increased FDX MIC aligned with the RNAP binding pocket interacting residues. A systematic literature search was done in PubMed (1991-2023) with identified articles and their bibliographies searched for papers that included C. difficile genetic mutations and increased FDX MIC. Visualization of FDX-RNAP interactions was performed on Schrödinger Maestro using the publicly available C. difficile RNAP with fidaxomicin sequence (code 7L7B) on the Protein Data Bank. Seven articles were identified after applying inclusion and exclusion criteria. The most common mutation in clinical and laboratory isolates was at position V1143 of the ß subunit, which accounted for approximately 50% of the identified mutations. Most other mutations occurred within the ß' subunit of RNAP. Approximately one-third of the identified mutation aligned directly with FDX interacting residues with C. difficile RNAP (7/20) with most of the remainder occurring within 5 Å of the binding residues. C. difficile strains with elevated FDX MIC align closely with the known RNAP binding residues. These data demonstrate the potential to identify genomic methods to identify emerging FDX resistance.

Dragging but not tapping promotes preschoolers' numerical estimating with touchscreens.

When solving mathematical problems, young children will perform better when they can use gestures that match mental representations. However, despite their increasing prevalence in educational settings, few studies have explored this effect in touchscreen-based interactions. Thus, we investigated the impact on young children's performance of dragging (where a continuous gesture is performed that is congruent with the change in number) and tapping (involving a discrete gesture that is incongruent) on a touchscreen device when engaged in a continuous number line estimation task. By examining differences in the set size and position of the number line estimation, we were also able to explore the boundary conditions for the superiority effect of congruent gestures. We used a 2 (Gesture Type: drag or tap) × 2 (Set Size: Set 0-10 or Set 0-20) × 2 (Position: left of midpoint or right of midpoint) mixed design. A total of 70 children aged 5 and 6 years (33 girls) were recruited and randomly assigned to either the Drag or Tap group. We found that the congruent gesture (drag) generally facilitated better performance with the touchscreen but with boundary conditions. When completing difficult estimations (right side in the large set size), the Drag group was more accurate, responded to the stimulus faster, and spent more time manipulating than the Tap group. These findings suggest that when children require explicit scaffolding, congruent touchscreen gestures help to release mental resources for strategic adjustments, decrease the difficulty of numerical estimation, and support constructing mental representations.

Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex.

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.

Correction to: YY1 inactivated transcription co-regulator PGC-1α to promote mitochondrial dysfunction of early diabetic nephropathy-associated tubulointerstitial fibrosis.

The development of diabetic nephropathy (DN) could be promoted by the occurrence of tubulointerstitial fibrosis (TIF), which has a close relationship with mitochondrial dysfunction of renal tubular epithelial cells (RTECs). As a key regulator of metabolic homeostasis, Yin Yang 1 (YY1) plays an important role not only in regulating the fibrosis process but also in maintaining the mitochondrial function of pancreatic ß-cells. However, it was not clear whether YY1 participated in maintaining mitochondrial function of RTECs in early DN-associated TIF. In this study, we dynamically detected mitochondrial functions and protein expression of YY1 in db/db mice and high glucose (HG)-cultured HK-2 cells. Our results showed that comparing with the occurrence of TIF, the emergence of mitochondrial dysfunction of RTECs was an earlier even, besides the up-regulated and nuclear translocated YY1. Correlation analysis showed YY1 expressions were negatively associated with PGC-1α in vitro and in vivo. Further mechanism research demonstrated the formation of mTOR-YY1 heterodimer induced by HG up-regulated YY1, the nuclear translocation of which inactivated PGC-1α by binding to the PGC-1α promoter. Overexpression of YY1 induced mitochondrial dysfunctions in normal glucose-cultured HK-2 cells and 8-weeks-old db/m mice. While, dysfunctional mitochondria induced by HG could be improved by knockdown of YY1. Finally, downregulation of YY1 could retard the progression of TIF by preventing mitochondrial functions, resulting in the improvement of epithelial-mesenchymal transition (EMT) in early DN. These findings suggested that YY1 was a novel regulator of mitochondrial function of RTECs and contributed to the occurrence of early DN-associated TIF.

YY1 inactivated transcription co-regulator PGC-1α to promote mitochondrial dysfunction of early diabetic nephropathy-associated tubulointerstitial fibrosis.

The development of diabetic nephropathy (DN) could be promoted by the occurrence of tubulointerstitial fibrosis (TIF), which had a closely relationship with mitochondrial dysfunction of renal tubular epithelial cells (RTECs). As a key regulator of metabolic homeostasis, Yin Yang 1 (YY1) played an important role not only in regulating fibrosis process, but also in maintaining mitochondrial function of pancreatic ß cells. However, it was not clear whether YY1 participated in maintaining mitochondrial function of RTECs in early DN-associated TIF. In this study, we dynamically detected mitochondrial functions and protein expression of YY1 in db/db mice and high glucose (HG)-cultured HK-2 cells. Our results showed that comparing with the occurrence of TIF, the emergence of mitochondrial dysfunction of RTECs was an earlier even, besides the up-regulated and nuclear translocated YY1. Correlation analysis showed YY1 expressions were negatively associated with PGC-1α in vitro and in vivo. Further mechanism research demonstrated the formation of mTOR-YY1 heterodimer induced by HG upregulated YY1, the nuclear translocation of which inactivated PGC-1α by binding to the PGC-1α promoter. Overexpression of YY1 induced mitochondrial dysfunctions in normal glucose cultured HK-2 cells and 8-week-old db/m mice. While, dysfunctional mitochondria induced by HG could be improved by knockdown of YY1. Finally, downregulation of YY1 could retard the progression of TIF by preventing mitochondrial functions, resulting in the improvement of epithelial-mesenchymal transition (EMT) in early DN. These findings suggested that YY1 was a novel regulator of mitochondrial function of RTECs and contributed to the occurrence of early DN-associated TIF .

Protein moonlighting elucidates the essential human pathway catalyzing lipoic acid assembly on its cognate enzymes.

The lack of attachment of lipoic acid to its cognate enzyme proteins results in devastating human metabolic disorders. These mitochondrial disorders are evident soon after birth and generally result in early death. The mutations causing specific defects in lipoyl assembly map in three genes, LIAS, LIPT1, and LIPT2 Although physiological roles have been proposed for the encoded proteins, only the LIPT1 protein had been studied at the enzyme level. LIPT1 was reported to catalyze only the second partial reaction of the classical lipoate ligase mechanism. We report that the physiologically relevant LIPT1 enzyme activity is transfer of lipoyl moieties from the H protein of the glycine cleavage system to the E2 subunits of the 2-oxoacid dehydrogenases required for respiration (e.g., pyruvate dehydrogenase) and amino acid degradation. We also report that LIPT2 encodes an octanoyl transferase that initiates lipoyl group assembly. The human pathway is now biochemically defined.

Development and retention of a primordial moonlighting pathway of protein modification in the absence of selection presents a puzzle.

Lipoic acid is synthesized by a remarkably atypical pathway in which the cofactor is assembled on its cognate proteins. An octanoyl moiety diverted from fatty acid synthesis is covalently attached to the acceptor protein, and sulfur insertion at carbons 6 and 8 of the octanoyl moiety form the lipoyl cofactor. Covalent attachment of this cofactor is required for function of several central metabolism enzymes, including the glycine cleavage H protein (GcvH). In Bacillus subtilis, GcvH is the sole substrate for lipoate assembly. Hence lipoic acid-requiring 2-oxoacid dehydrogenase (OADH) proteins acquire the cofactor only by transfer from lipoylated GcvH. Lipoyl transfer has been argued to be the primordial pathway of OADH lipoylation. The Escherichia coli pathway where lipoate is directly assembled on both its GcvH and OADH proteins, is proposed to have arisen later. Because roughly 3 billion years separate the divergence of these bacteria, it is surprising that E. coli GcvH functionally substitutes for the B. subtilis protein in lipoyl transfer. Known and putative GcvHs from other bacteria and eukaryotes also substitute for B. subtilis GcvH in OADH modification. Because glycine cleavage is the primary GcvH role in ancestral bacteria that lack OADH enzymes, lipoyl transfer is a "moonlighting" function: that is, development of a new function while retaining the original function. This moonlighting has been conserved in the absence of selection by some, but not all, GcvH proteins. Moreover, Aquifex aeolicus encodes five putative GcvHs, two of which have the moonlighting function, whereas others function only in glycine cleavage.

Understanding and Engineering Distributed Biochemical Pathways in Microbial Communities.

Microbiomes impact nearly every environment on Earth by modulating the molecular composition of the environment. Temporally changing environmental stimuli and spatial organization are major variables shaping the structure and function of microbiomes. The web of interactions among members of these communities and between the organisms and the environment dictates microbiome functions. Microbial interactions are major drivers of microbiomes and are modulated by spatiotemporal parameters. A mechanistic and quantitative understanding of ecological, molecular, and environmental forces shaping microbiomes could inform strategies to control microbiome dynamics and functions. Major challenges for harnessing the potential of microbiomes for diverse applications include the development of predictive modeling frameworks and tools for precise manipulation of microbiome behaviors.

An Atypical α/ß-Hydrolase Fold Revealed in the Crystal Structure of Pimeloyl-Acyl Carrier Protein Methyl Esterase BioG from Haemophilus influenzae.

Pimeloyl-acyl carrier protein (ACP) methyl esterase is an α/ß-hydrolase that catalyzes the last biosynthetic step of pimeloyl-ACP, a key intermediate in biotin biosynthesis. Intriguingly, multiple nonhomologous isofunctional forms of this enzyme that lack significant sequence identity are present in diverse bacteria. One such esterase, Escherichia coli BioH, has been shown to be a typical α/ß-hydrolase fold enzyme. To gain further insights into the role of this step in biotin biosynthesis, we have determined the crystal structure of another widely distributed pimeloyl-ACP methyl esterase, Haemophilus influenzae BioG, at 1.26 Å. The BioG structure is similar to the BioH structure and is composed of an α-helical lid domain and a core domain that contains a central seven-stranded ß-pleated sheet. However, four of the six α-helices that flank both sides of the BioH core ß-sheet are replaced with long loops in BioG, thus forming an unusual α/ß-hydrolase fold. This structural variation results in a significantly decreased thermal stability of the enzyme. Nevertheless, the lid domain and the residues at the lid-core interface are well conserved between BioH and BioG, in which an analogous hydrophobic pocket for pimelate binding as well as similar ionic interactions with the ACP moiety are retained. Biochemical characterization of site-directed mutants of the residues hypothesized to interact with the ACP moiety supports a similar substrate interaction mode for the two enzymes. Consequently, these enzymes package the identical catalytic function under a considerably different protein surface.

The Streptomyces coelicolor lipoate-protein ligase is a circularly permuted version of the Escherichia coli enzyme composed of discrete interacting domains.

Lipoate-protein ligases are used to scavenge lipoic acid from the environment and attach the coenzyme to its cognate proteins, which are generally the E2 components of the 2-oxoacid dehydrogenases. The enzymes use ATP to activate lipoate to its adenylate, lipoyl-AMP, which remains tightly bound in the active site. This mixed anhydride is attacked by the ϵ-amino group of a specific lysine present on a highly conserved acceptor protein domain, resulting in the amide-linked coenzyme. The Streptomyces coelicolor genome encodes only a single putative lipoate ligase. However, this protein had only low sequence identity (<25%) to the lipoate ligases of demonstrated activity and appears to be a circularly permuted version of the known lipoate ligase proteins in that the canonical C-terminal domain seems to have been transposed to the N terminus. We tested the activity of this protein both by in vivo complementation of an Escherichia coli ligase-deficient strain and by in vitro assays. Moreover, when the domains were rearranged into a protein that mimicked the arrangement found in the canonical lipoate ligases, the enzyme retained complementation activity. Finally, when the two domains were separated into two proteins, both domain-containing proteins were required for complementation and catalysis of the overall ligase reaction in vitro. However, only the large domain-containing protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins, whereas both domain-containing proteins were required to form lipoyl-AMP.

Programming bacteria for multiplexed DNA detection.

DNA is a universal and programmable signal of living organisms. Here we develop cell-based DNA sensors by engineering the naturally competent bacterium Bacillus subtilis (B. subtilis) to detect specific DNA sequences in the environment. The DNA sensor strains can identify diverse bacterial species including major human pathogens with high specificity. Multiplexed detection of genomic DNA from different species in complex samples can be achieved by coupling the sensing mechanism to orthogonal fluorescent reporters. We also demonstrate that the DNA sensors can detect the presence of species in the complex samples without requiring DNA extraction. The modularity of the living cell-based DNA-sensing mechanism and simple detection procedure could enable programmable DNA sensing for a wide range of applications.

Targeting mTOR/YY1 signaling pathway by quercetin through CYP7A1-mediated cholesterol-to-bile acids conversion alleviated type 2 diabetes mellitus induced hepatic lipid accumulation.

BACKGROUND: Hepatic lipid accumulation was a major promoter for the further development of non-alcoholic fatty liver disease (NAFLD) in type 2 diabetes (T2DM). mTOR/YY1 signaling pathway regulated many metabolic processes in different organs, and played an important role in hepatic lipid metabolism. Thus, targeting mTOR/YY1 signaling pathway might be a novel therapeutic strategy of T2DM-associated NALFD. PURPOSE: To investigate the effects and the mechanism of quercetin against T2DM-associated NAFLD. STUDY DESIGN AND METHODS: The combine abilities of 24 flavonoid compounds with mTOR were detected by computer virtual screening (VS) and molecular modeling. mTOR/YY1 signaling pathway was examined in the liver of db/db mice, and high glucose (HG) and free fatty acid (FFA) co-cultured HepG2 cells. YY1 overexpression lentivirus vector and mTOR specific inhibitor rapamycin were used to further identify the indispensable role of mTOR/YY1 signaling pathway in quercetin's amelioration effect of hepatic lipid accumulation in vitro. Clinical studies, luciferase assay and chromatin immunoprecipitation (ChIP) assay were all carried out to investigate the potential mechanisms by which quercetin exerted its amelioration effect of hepatic lipid accumulation. RESULTS: Quercetin had the strongest ability to combine with mTOR and could competitively occupy its binding pocked. Along with the alleviated hepatic injury by quercetin, mTOR/YY1 signaling pathway was down-regulated in vivo and in vitro. However, the alleviation effect of quercetin against hepatic lipid accumulation was inhibited by YY1 overexpression in vitro. Mechanistically, the down-regulated nuclear YY1 induced by quercetin directly bound to CYP7A1 promoter and activated its transcription, resulting in the restoration of cholesterol homeostasis via the conversion of cholesterol-to-bile acids (BAs). CONCLUSION: The hepatoprotective effect of quercetin on T2DM-associated NAFLD was linked to the restoration of cholesterol homeostasis by the conversion of cholesterol-to-BAs via down-regulating mTOR/YY1 signaling pathway, leading to the increased CYP7A1 activity.

Interactive analysis of single-cell data using flexible workflows with SCTK2.

Analysis of single-cell RNA sequencing (scRNA-seq) data can reveal novel insights into the heterogeneity of complex biological systems. Many tools and workflows have been developed to perform different types of analyses. However, these tools are spread across different packages or programming environments, rely on different underlying data structures, and can only be utilized by people with knowledge of programming languages. In the Single-Cell Toolkit 2 (SCTK2), we have integrated a variety of popular tools and workflows to perform various aspects of scRNA-seq analysis. All tools and workflows can be run in the R console or using an intuitive graphical user interface built with R/Shiny. HTML reports generated with Rmarkdown can be used to document and recapitulate individual steps or entire analysis workflows. We show that the toolkit offers more features when compared with existing tools and allows for a seamless analysis of scRNA-seq data for non-computational users.

A roadmap for designing narrow-spectrum antibiotics targeting bacterial pathogens.

Clostridioides difficile (Cdiff) infection (CDI) continues to be the leading threat of nosocomial deaths worldwide and a major burden on health-care systems. Broad-spectrum antibiotics eradicate the normal gut microbiome, killing protective commensal bacteria and increasing CDI recurrence. In contrast, Fidaxomicin (Fdx) is a narrow-spectrum antibiotic that inhibits Cdiff growth without affecting crucial gut microbes. However, the basis of the narrow-spectrum activity of Fdx on its target, RNA polymerase (RNAP), in Cdiff has been enigmatic. Recently, Cao et al. (Nature, doi: 10.1038/s41586-022-04545-z) combined transgenic RNAP design and synthesis with cryo-electron microscopy (cryo-EM) to identify a key determinant of Fdx inhibition of Cdiff RNAP. This finding was further corroborated by biochemical, bioinformatics, and genetic analysis. This microreview describes implications of this work for lineage-specific antibiotic design and new directions toward understanding transcription and regulation in Cdiff and other bacterial pathogens.

Comprehensive generation, visualization, and reporting of quality control metrics for single-cell RNA sequencing data.

Single-cell RNA sequencing (scRNA-seq) can be used to gain insights into cellular heterogeneity within complex tissues. However, various technical artifacts can be present in scRNA-seq data and should be assessed before performing downstream analyses. While several tools have been developed to perform individual quality control (QC) tasks, they are scattered in different packages across several programming environments. Here, to streamline the process of generating and visualizing QC metrics for scRNA-seq data, we built the SCTK-QC pipeline within the singleCellTK R package. The SCTK-QC workflow can import data from several single-cell platforms and preprocessing tools and includes steps for empty droplet detection, generation of standard QC metrics, prediction of doublets, and estimation of ambient RNA. It can run on the command line, within the R console, on the cloud platform or with an interactive graphical user interface. Overall, the SCTK-QC pipeline streamlines and standardizes the process of performing QC for scRNA-seq data.

Jujuboside A ameliorates tubulointerstitial fibrosis in diabetic mice through down-regulating the YY1/TGF-ß1 signaling pathway.

Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus, which is characterized in renal tubulointerstitial fibrosis (TIF). The current study was designed to investigate the protective effect of Jujuboside A (Ju A) on TIF in type 2 diabetes (T2DM) mice, and explore its underlying anti-fibrosis mechanism. A mouse T2DM model was established using high fat diet (HFD) feeding combined with intraperitoneal injection of streptozotocin (STZ). Then, diabetic mice were treated with Ju A (10, 20 and 40 mg·kg-1·d-1, i.g.) for 12 weeks. Results showed that administration of Ju A not only down-regulated fasting blood glucose (FBG) levels, but also improved hyperlipidemia and renal function in diabetic mice. Moreover, the reduced ECM accumulation was observed in the renal cortex of Ju A treated diabetic mice, while the TIF progression was also attenuated by Ju A through blocking the epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (RTECs). Further mechanism studies showed that Ju A treatment effectively down-regulated the protein expression and subsequent nuclear translocation of Yin Yang 1 (YY1) in the renal cortex of diabetic mice, and reduced the levels of transforming growth factor-ß1 (TGF-ß1) in the serum and renal cortex of Ju A treated mice. According to invitro studies, the up-regulated YY1/TGF-ß1 signaling pathway was restored by Ju A in high glucose (HG) cultured HK-2 cells. Taken together, these findings demonstrated that Ju A can ameliorate the TIF of DN through down-regulating the YY1/TGF-ß1 signaling pathway.

Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex.

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein crosslinking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3'-segment of the product-RNA generated by backtracking extrudes through the RdRp NTP-entry tunnel, that a mismatched nucleotide at the product-RNA 3'-end frays and enters the NTP-entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.