2025-05-27 | | Total: 9
Transcriptional regulation is a fundamental mechanism in bacteria and is often mediated by repressor proteins. In the type II toxin-antitoxin (TA) system. xre-res, the Xre antitoxin contains a putative helix-turn-helix (HTH) DNA-binding domain and could thus potentially bind to and repress transcription from the xre-res promoter. The structure of the Pseudomonas putida Xre-RES TA complex revealed an unusual 4:2 stoichiometry with two potential DNA-binding sites, suggesting a non-canonical mechanism of transcriptional autoregulation. Here, we show that the activity of the xre-res promoter requires both an intact σ70 element and the transcriptional start site, and that the Xre–RES complex represses transcription via binding to an imperfect inverted repeat region downstream of the σ70 element. We furthermore confirm the presence of the unusual 4:2 TA complex in solution and show that it preferably binds the imperfect inverted repeat in a 1:1 ratio. In addition, we show that the isolated Xre antitoxin is an aggregation-prone monomer in vitro and a weak repressor in vivo. We find that the Xre dimer can dissociates within the 4:2 complex and result in a 2:2 TA complex that still neutralizes the RES toxin but cannot bind DNA. Together, our data suggests that the asymmetry of the promoter is important for both transcription and repression, and we propose a model in which the Xre-RES complex regulates transcription through a dynamic and concentration-dependent equilibrium between a non-binding (2:2) and a DNA-binding (4:2) form. IMPORTANCE Transcriptional regulation is fundamental for bacteria to survive in a constantly changing environment and requires the RNA polymerase, which recognizes and binds to a DNA element (the promoter) located upstream of the gene and initiates transcription. Transcriptional repressors can bind DNA and inhibit transcription by the RNA polymerase, but the exact recognition of DNA binding sites remains unclear. The Xre antitoxin from the Pseudomonas putida toxin-antitoxin system Xre-RES contains a putative DNA-binding domain, and the unusual ratio of antitoxin to toxin within the protein complex suggests two potential DNA-binding sites. In this work, we show that Xre-RES auto-regulates its own transcription through an asymmetrical DNA binding-site, optimized to bind both the RNA polymerase and the Xre-RES complex, as well as a concentration-dependent ratio of toxin to antitoxin within the complex. Our work provides insight on how promoters can evolve to fine-tune the regulation of bacterial transcription.
Candida albicans, a commensal opportunistic pathogen, exhibits remarkable metabolic flexibility and adaptability to environmental changes. In glucose-limited niches, it utilizes alternative carbon sources such as carboxylic acids, which may influence its pathogenicity. In Saccharomyces cerevisiae, the uptake of monocarboxylates occurs through regulated plasma membrane (PM) transport proteins, such as Ato1 (Ady2), which belongs to the Acetate Uptake Transporter (AceTr) family. In C. albicans, these proteins are notably expanded, consisting of ten Ato-like proteins (ATO1-ATO10), whose functions remain unknown. Here, we investigated the role of Ato proteins in carboxylic acid utilization by C. albicans using in-silico and functional analysis. Our data revealed that several C. albicans Atos retain conserved AceTr motifs but possess distinct structural features, including differences in pore radius and binding sites for acetate and lactate. Expression analysis revealed that Ato1, Ato2, Ato3, and Ato6 exhibit distinct cellular localization and expression levels on the plasma membrane, depending on the presence or absence of monocarboxylates. Remarkably, deletion of ATO1 impaired ATO2 and ATO3 expression and caused ER retention of a distinct form of Ato2, suggesting a central regulatory role for Ato1 in the Ato transport system. Finally, we identified a novel Ato-related protein family in vertebrates. This family has three consecutive 6-helix transport domains and a unique C-terminal fusion with Sua5/YciO/YrdC, an enzyme involved in tRNA modification. Overall, our data suggests that the Ato protein family might play a critical role in the utilization of acetic or lactic acids in C. albicans. It also proposes potential functional redundancy among its members, which may contribute to rapid environmental adaptation and pathogenicity.
Phage infection begins with physical interaction and host cell entry, occurring through various strategies including exploitation of surface receptors, pili, or porins. Research has predominantly emphasized structural compatibility between phage and host, particularly attachment and recognition mechanisms. However, one critical layer remains underexplored: how host physiological state, particularly metabolic activity, influences entry likelihood. Here, we quantitatively compared phage adsorption efficiencies across diverse Escherichia coli phages under controlled metabolic conditions. Using a standardized protocol, we measured adsorption rates (η) for five phages representing various life cycles (lytic, lysogenic, chronic) and entry pathways (LamB, FhuA, pilus, Tsx). Four of the five phages exhibited significantly reduced commitment to infection under energy-limited conditions. Notably, this effect varied. Phages with high baseline η were less sensitive to metabolic inhibition than phages with low baseline η. This observation suggests weak-binding phages may disengage under unfavorable conditions to avoid non-productive infections. Our findings support a twostep infection model, where host energy availability modulates the transition from reversible binding to irreversible commitment to infection. Our study highlights the importance of considering host physiology when studying virus-host dynamics, especially in energy-limited environments.
HIV-1 proviral landscapes were investigated using near full-length HIV single-genome sequencing on blood samples from 5 children with vertically acquired infection and on ART for ~7-9 years. Proviral structures were compared to published datasets in children prior to ART, children on short-term ART, and adults on ART. We found a strong selection for large internal proviral deletions in children, especially deletions of the env gene. Only 2.5% of the proviruses were sequence-intact, lower than in the comparative datasets from adults. Of the proviruses that retained the env gene, >80% contained two or more defects, most commonly stop codons and/or gag start mutations. Significantly fewer defects in the major splice donor site (MSD) and packaging signal were found in the children on short or long-term ART compared to the adults, and tat was more frequently defective in children. These results suggest that different selection pressures shape the proviral landscape in children compared to adults and reveal potentially different genetic regions to target for measuring the intact HIV reservoir and for achieving HIV remission in children.
Murine typhus, caused by infection with Rickettsia typhi, is a neglected disease contributing to infectious disease burden in south- and southeast Asia. Despite its importance, we have minimal knowledge of the genomics of R. typhi, with only four complete genomes being sequenced prior to this work. We sequenced a further 25 genomes including historical strains collected before 1976 from both human and rat hosts, and recent genomes isolated from patients at a single hospital in Laos. Whole genome SNP analysis reveals extremely low levels of genetic diversity across the 29 genomes, with overall nucleotide diversity (π) of 1.27e-05 and evidence of purifying selection, and a minimal pan-genome. Phylogenetic analysis shows clustering of the genome by historic or modern origin, with the exception of one modern strain which is most closely related to historic strains from Thailand, and no clustering by host origin. The highly conserved genome of R. typhi suggests strong constraints on genome evolution in this obligate intracellular parasite, and has implications for the design of future murine typhus diagnostic tools and vaccines.
We integrated 18O-stable isotope probing and viromics (SIP-viromics) to investigate soil viral activity after rewetting. Among 354 detected viral populations, only 22% incorporated 18O, indicating most virions persisted through this induction event without replicating, forming a genetic reservoir of viral diversity. Actinomycetota were dominant predicted hosts of lytic infections, supporting Cull-the-Winner dynamics. As the first SIP-viromics application, this study reveals viral persistence and turnover during microbial responses to environmental perturbation.
Structures of nucleoprotein (N)–RNA complexes of the Bornaviridae, a virus family in the order Mononegavirales, have remained unknown. Using cryo-electron microscopy (cryo-EM), we characterized N structures of Borna disease virus 1 (BoDV-1), the type species of the Bornaviridae, which causes fatal human encephalitis. N forms complexes in both RNA-free and RNA-bound states, revealing conserved features throughout the order, as well as BoDV-1-specific stoichiometry, thereby offering insights into viral evolution. We redefine assembly principles governing N–RNA complexes with the discovery of a previously unrecognized, RNA–independent mechanism involving domain-swapping and truncated subunits. Mutational analyses identified residues essential for nucleocapsid formation and RNA synthesis. Cryo-EM of mutant complexes captured RNA-free assemblies, suggesting that initial N oligomerization precedes RNA binding. These findings explain several unknowns in N–RNA complex structure and suggest an alternative to canonical RNA-driven assembly models, offering a new conceptual framework for nucleocapsid formation.
Vaccinium is a genus of shrubs known for their antioxidant and anti-inflammatory compounds. While the commercially cultivated blueberry (V. corymbosum) originates from North America, European species like V. myrtillus (bilberry) and V. uliginosum (bog bilberry) remain uncultivated due to poor adaptability to conventional growing media. At present, bilberries, which are more valued for fresh consumption, are exclusively harvested from wild populations, particularly in subalpine and mountain environments like the Italian Alps and Apennines, where unique soil and climate conditions prevail. This study explores the bacterial and fungal communities associated with V. myrtillus and V. uliginosum in wild populations of the Central Italian Apennines, using 16S rRNA and ITS amplicon sequencing. Microbiota from roots, leaves, rhizosphere, and bulk soil were analyzed, revealing distinct microbial community compositions based on species and plant compartments. Bacterial diversity was highest in bulk soil, while fungal diversity dominated plant tissues. Co-occurrence network analysis showed greater connectivity in V. uliginosum microbiota, suggesting higher resilience. Functional predictions indicated roles in nitrogen cycling, cellulose degradation, and plant-microbe interactions. These findings offer insights into the native microbiota of wild Vaccinium species and could inform future conservation and cultivation efforts.
Genome sequences provide fundamental information for both basic and applied life sciences. Whole-genome sequencing is now requested for describing novel prokaryotic species and designating their type strains, which serve as representative and well-characterized strains of the species. Indeed, the number of sequenced prokaryotic genomes has been rapidly increasing. However, a considerable number of isolated strains, particularly ″fastidious″ type strains such as strict anaerobes and slow growers, remain without genome sequence information. Here we report the whole-genome sequencing of 290 bacterial and 61 archaeal strains, including fastidious type strains, obtained from Japan Collection of Microorganisms (JCM) using a combination of short- and long-read sequencing technologies. The dataset includes 284 type strain genomes and 235 complete genomes. Notably, in the dataset, genomes of over 200 strains, including over 150 type strains, had not been made publicly available. Comparative genomic analysis suggests that some strains need to be assigned to novel taxa or reclassified. Functional gene survey indicates that some strains possess previously unrecognized potential for carbon fixation or bioactive secondary metabolite production. Our dataset will contribute to more accurate taxonomic classification, fill gaps in the phylogeny of prokaryotes, and provide insights into their physiology and ecology.