Biology subjects

Tai, C.-H.

Publications and source records attributed to Tai, C.-H..

9 recordsLinked to original sources

bioRxiv2026

Structural and functional insights into the Rcs phosphorelay

The Rcs phosphorelay regulates gene expression in response to cell envelope stress and is critical for the virulence of pathogenic bacteria, including Klebsiella pneumoniae, due to its regulation of genes related to extracellular capsule, cell division, and motility. The RcsC histidine kinase, RcsD phosphotransfer protein and RcsB response regulator, which form the core of the Rcs phosphorelay, are negatively regulated by the unique inner membrane protein IgaA via interaction with RcsD. An outer membrane lipoprotein, RcsF, activates signaling by interaction with IgaA, but the precise activation mechanisms remain unclear. In this study, we determined the structures of IgaA and the IgaA/RcsF complex using Cryo-electron microscopy (Cryo-EM). We also determined the structures of RcsC and RcsD, which both form homodimers stabilized by hydrophobic interactions, creating ladder-like structures. Combining the Cryo-EM structures, AlphaFold3 structure predictions of IgaA/RcsD and RcsF/IgaA/RcsD, and genetic studies, we describe a model for how RcsF modifies the IgaA/RcsD interaction, lifting negative regulation and activating the Rcs phosphorelay. Our findings provide a high-resolution depiction of the Rcs stress response system and suggest potential targets for small molecule inhibitors.

biophysics↗

bioRxiv2026

16S ribosomal RNA modification drives transcript-specific translation efficiency

Bacterial ribosomal RNAs (rRNAs) are decorated with conserved nucleotide modifications, but the functionality of these modifications is often underexplored. MraW (RsmH) is a 16S rRNA methyltransferase that fine-tunes ribosomal function. We identified a loss-of-function allele in mraW that corrected a late-stage sporulation defect in Bacillus subtilis by bypassing a key sporulation checkpoint via altered translational regulation. Purified ribosomes isolated from {Delta}mraW cells displayed a [~]2-fold decrease in translation efficiency; in vivo, {Delta}mraW cells produced decreased levels of the sporulation checkpoint protein CmpA. This regulation was mediated by sequences from the 5 untranslated region and the coding sequence of cmpA, which form a step-loop structure that occlude early codons of the mRNA. Proteomic analysis revealed that MraW directly or indirectly regulates the production of multiple proteins, some of which form similar structural elements as the cmpA transcript. We propose that MraW modification of 16S rRNA enhances translation efficiency in general, and that specific transcripts, whose gene products are likely required in limiting quantities, have evolved structural features that act as a regulatory mechanism to govern protein levels. This type of regulation may be most apparent in bacteria which exhibit uncoupled transcription and translation. HIGHLIGHTSO_LIA conserved 16S rRNA modification enhances translation of structured mRNAs C_LIO_LIEarly mRNA stem-loops impose translational control dependent on ribosome modification C_LIO_LImRNA structure and rRNA modifications likely co-evolved to fine-tune protein dosage C_LI

microbiology↗

bioRxiv2025

Stenotrophomonas maltophilia exhibits defensive multicellularity in response to a Pseudomonas aeruginosa quorum sensing molecule

Microorganisms commonly exist in polymicrobial communities, where they can respond to interspecies secreted molecules by altering behaviors and physiology, however, the underlying mechanisms remain underexplored. Here we investigated interactions between Stenotrophomonas maltophilia and Pseudomonas aeruginosa, co-infecting opportunistic pathogens found in pneumonia and chronic lung infections, including in cystic fibrosis. We found that S. maltophilia forms robust protective multicellular aggregates upon exposure to P. aeruginosa secreted factors. Experimental evolution for lack of aggregation selected for fimbrial mutations and we found that fimbriae are required on both interacting S. maltophilia cells for aggregation. Untargeted metabolomics and targeted validations revealed that the quorum sensing molecule Pseudomonas quinolone signal (PQS) directly induced S. maltophilia aggregation, and co-localized with the aggregates. Further, in co-culture with P. aeruginosa, wild-type S. maltophilia formed aggregates, resulting in up to 75-fold increased survival from P. aeruginosa competition compared to fimbrial mutants. Finally, multiple other bacterial species similarly aggregated upon exposure to P. aeruginosa exoproducts, indicating a more general response. Collectively, our work identifies a novel multispecies interaction where a quorum sensing molecule from a co-infecting pathogen is sensed as a danger signal, thereby inducing a protective multicellular response.

microbiology↗

bioRxiv2024

A highly conserved sRNA downregulates multiple genes, including a sigma54 transcriptional activator, in the virulence mode of Bordetella pertussis

Bacterial sRNAs together with the RNA chaperone Hfq post-transcriptionally regulate gene expression by affecting ribosome binding or mRNA stability. In the human pathogen Bordetella pertussis, the causative agent of whooping cough, hundreds of sRNAs have been identified, but their roles in B. pertussis biology are mostly unknown. Here we characterize a Hfq-dependent sRNA (S17), whose level is dramatically higher in the virulence (Bvg+) mode. We show that transcription from a {sigma}A-dependent promoter yields a long form of 190 nucleotides (nts) that is processed by RNase E to generate a shorter, more stable form (S17S) of 67 nts. Using RNA-seq and RT-qPCR, we identify 92 genes whose expression significantly increases in the absence of S17. Of these genes, 70 contain sequences at/near their ribosome binding sites (RBSs) that are complementary to single-stranded (ss) regions (Sites 1 or 2) of S17S. The identified genes include those encoding multiple transporters and 3 transcriptional regulators. Using a lacZ translational reporter system, we demonstrate that S17S directly represses one of these genes, BP2158, a {sigma}54- dependent transcriptional regulator, suggesting the repression of a {sigma}54 regulon in the Bvg+ mode. We find that the S17S region containing Sites 1 and 2 is 100% conserved throughout various Betaproteobacteria species, and the S17S target sites are often conserved in the homologs of the B. pertussis target genes. We speculate that S17S regulation represents a highly conserved process that fine-tunes gene expression in the Bvg+ mode of B. pertussis and perhaps under other conditions in related bacteria. IMPORTANCERegulation of gene expression involves controlling transcription, translation, and transcript degradation. sRNAs with short sequences complementary to an mRNA sequence are involved in post-transcriptional regulation by aiding or interfering with either ribosome binding or nuclease attack. In the human pathogen Bordetella pertussis, the causative agent of whooping cough, hundreds of sRNA have been identified, but their functions remain largely unknown. We have characterized a sRNA that is abundant in the virulence mode of B. pertussis and serves to down-regulate multiple genes including transcriptional regulators and various transporters. We demonstrate that this sRNA directly represses a transcriptional factor, suggesting that it influences the regulation of specific B. pertussis regulons. The high conservation of this sRNA and its targets within Betaproteobacteria suggests a conserved pathway for gene regulation.

microbiology↗

bioRxiv2024

Telomeric Repeat-Containing RNA Increases in Aged Human Cells

Telomeric repeat-containing RNA (TERRA), transcribed from subtelomeric regions towards telomeric ends, poses challenges in deciphering its complete sequences. Utilizing TERRA-capture RNA-seq and Oxford Nanopore direct RNA sequencing to acquire full-length TERRA, we annotate TERRA transcription regions in the human T2T-CHM13 reference genome. TERRA transcripts encompass hundreds to over a thousand nucleotides of telomeric repeats, predominantly originating from 61-29-37 bp repeat promoters enriched with H3K4me3, RNA pol II, CTCF, and R-loops. We develop a bioinformatics tool, TERRA-QUANT, for quantifying TERRA using RNA-seq datasets and find that TERRA increases with age in blood, brain, and fibroblasts. TERRA upregulation in aged leukocytes is confirmed by RT-qPCR. Single-cell RNA-seq analysis demonstrates TERRA expression across various cell types, with upregulation observed in neurons during human embryonic stem cell differentiation. Additionally, TERRA levels are elevated in brain cells in the early stage of Alzheimers disease. Our study provides evidence linking TERRA to human aging and diseases.

genomics↗

bioRxiv2023

Protein coopted from a phage restriction system dictates orthogonal cell division plane selection in Staphylococcus aureus

The spherical bacterium Staphylococcus aureus, a leading cause of nosocomial infections, undergoes binary fission by dividing in two alternating orthogonal planes, but the mechanism by which S. aureus correctly selects the next cell division plane is not known. To identify cell division placement factors, we performed a chemical genetic screen that revealed a gene which we termed pcdA. We show that PcdA is a member of the McrB family of AAA+ NTPases that has undergone structural changes and a concomitant functional shift from a restriction enzyme subunit to an early cell division protein. PcdA directly interacts with the tubulin-like central divisome component FtsZ and localizes to future cell division sites before membrane invagination initiates. This parallels the action of another McrB family protein, CTTNBP2, which stabilizes microtubules in animals. We show that PcdA also interacts with the structural protein DivIVA and propose that the DivIVA/PcdA complex recruits unpolymerized FtsZ to assemble along the proper cell division plane. Deletion of pcdA conferred abnormal, non-orthogonal division plane selection, increased sensitivity to cell wall-targeting antibiotics, and reduced virulence in a murine infection model. Targeting PcdA could therefore highlight a treatment strategy for combatting antibiotic-resistant strains of S. aureus.

microbiology↗

bioRxiv2021

iCn3D: from Web-based 3D Viewer to Structure Analysis Tool in Batch Mode

iCn3D was initially developed as a web-based 3D molecular viewer. It then evolved from visualization into a full-featured interactive structural analysis software. It became a collaborative research instrument through the sharing of permanent, shortened URLs that encapsulate not only annotated visual molecular scenes, but also all underlying data and analysis scripts in a FAIR manner. More recently, with the growth of structural databases, the need to analyze large structural datasets systematically led us to use Python scripts and convert the code to be used in Node.js scripts. We showed a few examples of Python scripts at https://github.com/ncbi/icn3d/tree/master/icn3dpython to export secondary structures or PNG images from iCn3D. Users just need to replace the URL in the Python scripts to export other annotations from iCn3D. Furthermore, any interactive iCn3D feature can be converted into a Node.js script to be run in batch mode, enabling an interactive analysis performed on one or a handful of protein complexes to be scaled up to analysis features of large ensembles of structures. Currently available Node.js analysis scripts examples are available at https://github.com/ncbi/icn3d/tree/master/icn3dnode. This development will enable ensemble analyses on growing structural databases such as AlphaFold or RoseTTAFold on one hand and Electron Microscopy on the other. In this paper, we also review new features such as DelPhi electrostatic potential, 3D view of mutations, alignment of multiple chains, assembly of multiple structures by realignment, dynamic symmetry calculation, 2D cartoons at different levels, interactive contact maps, and use of iCn3D in Jupyter Notebook as described at https://pypi.org/project/icn3dpy.

bioinformatics↗

bioRxiv2021

Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq

Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD deletion in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

microbiology↗

bioRxiv2021

A fluorescence-based genetic screen reveals diverse mechanisms silencing small RNA signaling in E. coli

As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation as well as virulence. sRNAs can alter target mRNA translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify new regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in E. coli. The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX and McaS, previously found to act through Hfq competition, RNase T, a 3'-5' exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two new specific regulators were identified. AspX, a novel 3'end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria. SignificanceThe ability to promptly switch genes on and off allows bacteria to adapt rapidly to changing environments for better survival. Many sRNAs, including RyhB, a sRNA made in response to iron starvation, are important switches in bacteria. We discovered new factors that can keep the sRNA switch off by using a facile genetic screen platform. These new factors include a new RNA sponge and an adaptor protein for a ribonuclease, providing new perspectives on controlling sRNA signaling in bacteria.

microbiology↗