GSR-ST: A generalized spatial-temporal framework for genomic signals and regions prediction using multi-scale feature fusion.

Variation in surface charge of the cell envelope across bacterial species is not well understood. The absence of systematic investigations comparing taxonomically diverse bacterial species has led to confusion about the influence of cell wall type (i.e. gram-negative or gram-positive bacteria) on surface charge. In this study, dynamic light scattering (DLS) was used to determine surface charge via zeta potential of 12 phylogenetically diverse bacterial species spanning four bacterial phyla. Surface charge of six gram-positive species (Bacillus subtilis, Streptococcus thermophilus, Paenibacillus polymyxa, Kocuria rosea, Mycobacterium smegmatis, and Streptomyces albus) and six gram-negative species (Chryseobacterium shigense, Sphingobacterium multivorum, Flavobacterium pectinovorum, Escherichia coli, Stenotrophomonas maltophilia, and Pseudomonas putida) were characterized. Results show that surface charge is not dependent on cell wall type but instead varies across diverse species, as there is significant overlap between the surface charge means of the gram-positive and gram-negative bacterial groups. The methodology used in this study provides a consistent, high-throughput technique for assessing surface charge. Data presented herein suggest that routine surface charge measurements are informative to investigations of interactions between bacteria and other systems, such as antimicrobial therapeutics and water purification systems.

ObjectivesCarbapenemase-producing Gram-negative bacteria represent a major clinical challenge. Rapid detection and characterization of carbapenemase group is essential for guiding therapy and for infection control. We evaluated the performance of Certest ResisCheck® Carbapenemases, a novel lateral flow immunoassay (LFIA), for detecting the main carbapenemase groups from bacterial culture.Materials and methodsA multicentre collection of 351 well-characterized clinical Gram-negative isolates, studied by whole-genome sequencing, was tested using this assay. 57 KPC, 53 NDM, 63 VIM, 68 OXA-48-like, 64 IMP and 100 carbapenem resistant non-carbapenemase producers were included.ResultsThe LFIA showed an overall sensitivity of 96.4% and specificity of 100%. The positive predictive value (PPV) was 100%, and the negative predictive value (NPV) was 91.7%. No false-positives or cross-reactions with extended-spectrum β-lactamases (ESBLs) or cephalosporinases were observed either with isolates harbouring multiple carbapenemases. Detection rates for IMP and NDM variants reached 100%. Sensitivity for KPC, OXA-like and VIM was slightly lower (93.0%, 95.6% and 96.8%, respectively). Nine false-negative results were observed: KPC-31 (n = 4), VIM-63 (n = 2) and OXA-1054-producing (n = 3) isolates. Increasing the bacterial inoculum managed to obtain a positive result for KPC-31 and VIM-63, but OXA-1054 remained undetected.ConclusionsCertest ResisCheck® Carbapenemases is a rapid, accurate, and reliable tool for detecting carbapenemases in clinical settings. While some rare variants may escape detection, its strong performance across diverse bacterial species supports its use as part of routine diagnostics to guide timely antimicrobial therapy and control the spread of resistance.

The study objective was to assess occupational exposure to bacteria and antimicrobial resistance genes (ARGs) present on air filters of utility vehicles used in the working environment of mechanical-biological treatment (MBT) facilities, in the context of workers health risks. The study was conducted in summer 2024 in 9 air filters from utility vehicles used in MBT plants in Poland. External filters were removed from the vehicle's ventilation system, packed and shipped according to instructions. From the duplicate filters samples DNA was isolated and high-throughput next-generation sequencing (NGS) was carried out. Bioinformatic data analysis was conducted to detect bacteria and ARGs in air filters' surfaces. Totally, 34 bacterial taxa were detected in relative abundance ≥0.5%. The genera most frequently present at the highest relative abundances: Saccharomonospora, Thermobifida, Nocardiopsis, Pectobacterium, Aerococcus, Thermoactinomyces, Novibacillus and Streptomyces. Across all bacteria isolated from the analyzed filters, regardless of their relative abundance, a total of 91 taxa were classified into risk groups 2 or 3 (86 and 5 taxa, respectively). The most frequently detected ARGs were those encoding resistance to a single class of antibiotics (AAC(3)-VIIa, aadA2, ANT(6)-Ia, APH(3'')-Ia, APH(3'')-Ib, APH(6)-Id, cml, cmx, lnuA, lnuD, novA, parY, sul2, vanHF, vanJ, vanRA, vanRI, vanRO - each at least on 4 air filters). Antimicrobial resistance genes encoding multi antibiotic resistance were also detected: CRP, emtA, erm(34), erm(36), ermA, ermC, ermF, ermG, ermT, ermX, ernB, H-NS, mel, msrA, msrE, mtrA, optrA, ramA, ykkD - each at least on 1 air filter. Despite the limited number of analyzed filters, the study demonstrated a high bacterial species diversity in the MBT plant environment. The MBT workers are exposed to bacteria with high pathogenic potential and to ARGs encoding resistance to antibiotics used exclusively in human medicine, used in human and veterinary medicine, and not intended for human use. Int J Occup Med Environ Health. 2026;39(2).

Antarctic marine food webs are expected to be significantly impacted by future climate change. In particular, the recent rapid regional warming in the West Antarctic Peninsula has, and will continue to have, a negative impact on endemic marine biodiversity. However, despite the growing recognition of the role microbial symbionts play in mediating responses to environmental change, microbiome characterisation has been conducted for only a small fraction of the marine invertebrates in the Southern Ocean. Our study examined the effects of feeding guild, seasonality, and experimental warming (6 months at + 2°C) on the gut microbiome of six species of near-shore marine Antarctic echinoderms sampled from waters off Rothera Research Station, Antarctica. Our study used 16S rRNA amplicon sequencing of the V3-V4 region, with analyses including measurements of alpha and beta-diversity alongside co-occurrence network analyses. Of the six invertebrate species sampled in winter, peak species diversity values in gut microbiomes were observed in the omnivores, Ophionotus victoriae and Sterechinus neumayeri, with lower values in the scavenger/predator, Odontaster validus, and the suspension feeders, Cucumaria georgiana, Echinopsolus charcoti, and Heterocucumis steineni. In the seasonal experiment, H. steineni bacterial gut species diversity doubled from winter to early summer yet decreased by a similar magnitude during the same period in O. victoriae. Despite these opposing diversity trends, both species displayed similar increases in the relative abundances of Bacteroidota and Bacillota in winter and early summer in their gut microbiomes. Bacterial diversity in the gut microbiome of the sea cucumbers E. charcoti and H. steineni, was not impacted by six-months at + 2 ˚C above ambient, although C. georgiana displayed a decrease in observed ASVs following this treatment. These results suggest a strong influence of feeding guild and seasonality on the gut microbiomes of these invertebrates. There appeared to be little effect of warming (+ 2°C) on the taxonomic composition of the gut microbiomes of the three holothurians. This highlights the need to examine the functional significance of experimental warming treatments using metabolomics and transcriptomics alongside microbial species diversity analyses to understand whether gut microbiomes can aid resilience under future climate change.

Engineered Bacteria as living detectors of tumor DNA: A new diagnostic frontier.

Genetic code reprogramming allows for ribosomal incorporation of exotic amino acids, such as d-α- and d-β-amino acids, into peptides. However, their incorporation efficiency remains much lower than that of canonical l-amino acids, making their multiple/consecutive incorporations difficult. The side chain of d-α-amino acids clashes with U2506 of 23S rRNA, hindering the incorporation of d-α-amino acids with bulky side chains; therefore, consecutive incorporation has been limited to small d-α-amino acids. To overcome this limitation, we screened ribosomes from phylogenetically diverse bacterial species to identify variants that enable consecutive incorporation of bulky d-amino acids. The Alteromonas macleodii (AM) ribosome is capable of incorporating d-α-, d-β-, and α,α-disubstituted amino acids with efficiencies superior to the Escherichia coli (EC) ribosome. It elongates six consecutive d-Ser and two consecutive 1-aminocyclobutane-1-carboxylic acid with yields 6.1- and 7.3-fold higher than the EC ribosome. Moreover, consecutive incorporation of nine types of bulky d-α-amino acids (d-Asn, d-Asp, d-Gln, d-Met, d-Phe, d-Thr, d-Trp, d-Tyr, d-Val) was achieved for the first time. A model macrocyclic peptide containing four d-amino acids and one α,α-disubstituted amino acid was also synthesized by the AM ribosome. These results expand the potential of ribosomal synthesis for peptide libraries containing structurally diverse, bulky d-amino acids.

ABSTRACTThe global rise of antimicrobial resistance (AMR) demands innovative strategies to limit the spread of multidrug-resistant bacteria. Conjugative plasmids, particularly those in the incompatibility group P (IncP), play a central role in disseminating resistance genes across diverse bacterial species via their encoded Type IV secretion systems (T4SS). Here, we characterize the single-stranded RNA bacteriophage (ssRNA phage) PRR1, which selectively targets AMR ESKAPEE pathogens carrying the IncP plasmid RP4, and assess its ability to inhibit conjugation. Using cryo–electron microscopy, we first resolved the mature PRR1 virion at 3.45 Å resolution revealing two phage maturation protein (Mat)-RNA interactions within the 3’ untranslated region (UTR) – a conserved interaction (Mat-U1) and a novel interaction (Mat-V1) for ssRNA phages. To characterize the PRR1-RP4 pilus interaction, we performed alanine-scanning mutagenesis and pinpointed four critical TrbC pilin residues (S12, W13, S72, and R77) for infection. Computational modeling revealed that these residues are located near the termini of the pilin at the phage-pilus interface. Notably, native and non-infectious, UV-crosslinked PRR1 were sufficient to block RP4 transfer, indicating conjugation inhibition does not require a complete infection cycle. Finally, combining PRR1 and antibiotic treatment yielded nine unique phage-resistant mutants within T4SS-associated genes on the RP4 plasmid. Eight of these mutants nearly abolished conjugation, while thetrbEframeshift mutant retained ∼30% of wild-type efficiency, which is pivotal to clarifying the relationship between phage infection and pilus function. Collectively, these results establish ssRNA phages as specific T4SS plasmid targeting agents and underscore their potential to limit horizontal gene transfer in AMR pathogens.IMPORTANCEAntimicrobial resistance (AMR) spreads rapidly through horizontal gene transfer, largely driven by conjugative plasmids. Despite their central role, few strategies exist to directly block plasmid transfer. Here, we show that the IncP plasmid-dependent ssRNA phage PRR1 can inhibit the spread of antibiotic resistance genes by targeting the RP4 T4SS pilus. Structural and mutational analyses reveal previously unrecognized RNA packaging interactions and identify four pilin residues critical for infection. Remarkably, non-infectious PRR1 particles alone are sufficient to block conjugation, offering inhibition without the selective pressure from phage replication. Almost all PRR1-resistant RP4 mutants lost or had severely reduced plasmid transfer, while the remaining mutant is critical for studying the link between T4SS function and phage infection. These results highlight ssRNA phages as precise agents for limiting AMR gene dissemination.

Transport of proteins and small molecules across cellular membrane is crucial for bacterial interaction with the environment and survival against antibiotics. In Gram-negative bacteria that possess two layers of membranes, specialized macromolecular machines are required to transport substrates across the cell envelope, often via an indirect stepwise process. The major facilitator superfamily (MFS)-type tripartite efflux pumps use proton electrochemical gradient to extrude drugs in diverse bacterial species, but the architecture of the assembly and structural mechanisms remain elusive. A representative MFS-type tripartite efflux pump, EmrAB-TolC, mediates resistance to multiple antimicrobial drugs through proton-coupled EmrB, a member of the DHA2 transporter family. Here, we report the high-resolution (3.13 Å) structure of the EmrAB-TolC pump, revealing a distinct, asymmetric architecture emerging from the assembly of TolC:EmrA:EmrB with a ratio of 3:6:1 and contacts that are essential for the pump assembly. Key residues involved in drug transport are identified and corroborated by mutagenesis and antibiotic sensitivity assays. The structural and functional data support a model for one-step drug transport bythe MFS pump across the entire envelope of Gram-negative bacteria.

We present a living, synthetic bacterial cell made by transplanting a complete genome into a dead cell. After killing Mycoplasma capricolum cells by chemically crosslinking their genome with Mitomycin C (MMC), we installed synthetic Mycoplasma mycoides genomes into the resulting dead cells using Whole Genome Transplantation (WGT)1,2. During WGT, a synthetic donor genome is placed into a recipient cell, thereby reprogramming that cell to adopt a new genetic identity3. WGT has only been demonstrated using species within one phylogenetic clade of Mollicutes bacteria4. A major barrier to expanding WGT to diverse bacterial species has been the inability to inactivate the recipient genome, leading to false positive transplants due to homologous recombination of antibiotic resistance markers from the donor genome into the recipient cell genome. Here, we address this key limitation by removing reliance on an antibiotic resistance marker to select for transplants; recipient cells are dead unless revived by the installation of a new genome. Our work demonstrates a general approach to fully inactivate the recipient cell genome, reports the first living synthetic bacterial cell constructed from non-living parts, and advances WGT for building engineered or synthetic cells for diverse applications.

Conventional approaches to heterologous gene expression rely on codon optimization, which is limited to swapping synonymous codons and fails to capture deeper adaptive changes. In contrast, naturally evolved orthologous genes include non-synonymous mutations, insertions, and deletions that confer functional adaptation to different host contexts. Here we present OrthologTransformer, a Transformer-based deep learning model that converts orthologous genes between species by learning from large-scale orthologous gene datasets. The model recapitulates evolutionary differences-from synonymous codon swaps to amino acid-changing mutations and indels-to predict coding sequences optimized for target species while preserving protein function. In extensive tests across diverse bacterial species pairs, the model's context-aware gene designs more closely resembled native host orthologs, preserved protein functionality, and achieved superior expression yields compared to codon-optimized sequences. As proof of concept, an OrthologTransformer-redesigned PETase expressed in Bacillus subtilis showed robust activity, producing approximately 10-fold more reaction product than the codon-optimized enzyme, and achieving higher expression levels, thereby demonstrating improved enzyme performance via AI-guided gene design.

Background: Increasing evidence identifies intratumoral bacteria as key modulators of tumor progression, chemoresistance, and immunosuppression, presenting major obstacles to conventional cancer therapies. Recent advances in nanotechnology have enabled new strategies for selective targeting bacteria within the tumor microenvironment, potentially improving anticancer efficacy. Methods: A scoping review was conducted to outline the current landscape of nano-based therapeutic approaches aimed at the simultaneous elimination of intratumoral bacteria and cancer. Preclinical research publications involving in vivo antitumor efficacy evaluations were retrieved from three databases, Web of Science, PubMed, and Scopus, using the key words "(kill* OR eradicate* OR eliminate*) AND intratumoral AND (bacteria OR infection)". Key information from the eligible studies was extracted and analyzed. Results: The diversity of bacterial species, cancer models, and evaluation methodologies employed in these preclinical studies were summarized, followed by critical examination of the design principles, therapeutic outcomes, and translational challenges of various nanomedicine platforms, including passive and active targeting drug delivery systems, phototherapy, phage therapy, and emerging modalities. Nano-based therapeutics functionalized with both antibacterial and anticancer properties were shown to effectively overcome bacteria-induced treatment resistance. Conclusions: Targeting intratumoral bacteria may significantly enhance the efficacy of existing treatments and contribute to the evolution of precision oncology. The insights gained from this review are expected to guide future systematic reviews and inform research directions in the development of dual-functional nanomedicines for cancer therapy.

Electrochemically engineered plasmonic nanostructuring on universally captured bacterial surfaces for label-free and ultrasensitive SERS pathogen detection.

The role of molecular chirality in shaping interactions among organisms remains unclear. We discovered that amino acid stereochemistry provides a social avoidance mechanism for Bacillus subtilis spores. Germination of spores is triggered by L-alanine and competitively inhibited by its enantiomer D-alanine, produced almost exclusively by bacteria. The biological role of this enantiomer sensing remains unclear. We quantified the L- and D-alanine concentrations secreted by over 20 diverse bacterial species. We find that enantiomer ratios secreted by these species are located just beyond the germination response range for B. subtilis spores. Spores thus avoid germination when sensing other species. By forcing germination in pairwise co-cultures, we show that the presence of another bacterial species is often detrimental to germinating spores. Spores thus appear to exploit the L-/D-alanine ratio as a social avoidance mechanism, revealing a benefit for enantiomer sensing and potential importance of amino acid handedness in ecosystems.

Healthcare-associated infections (HAIs) remain a major cause of morbidity in hospitalized patients and residents of long-term care facilities. Conventional chemical disinfectants have limitations such as corrosion, toxicity, and economic burden. This study investigated the antibacterial properties of nanobubble water (NBW) as a novel physicochemical disinfection method that does not require chemical additives. NBW was generated using four gases-air, nitrogen (N2), oxygen (O2), and carbon dioxide (CO2)- and tested against Escherichia coli (E. coli), Extended-spectrum beta-lactamase (ESBL)-producing E. coli, Staphylococcus aureus, and Methicillin-resistant S. aureus (MRSA). Nanobubble size and stability were analyzed, and time-dependent antibacterial activity was evaluated by colony-forming unit (CFU) assays. Transmission electron microscopy (TEM) was used to assess bacterial ultrastructural changes following NBW exposure. Generated nanobubbles measured 50-100 nm in diameter and were stable over time. NBW exhibited intrinsic, time-dependent antibacterial effects that were independent of the solvent or dissolved gas itself. Antibacterial activity was more pronounced at lower bacterial loads and differed by gas type and bacterial species: N2-NBW was particularly effective against MRSA, whereas CO2-NBW and Air-NBW showed activity against ESBL-producing E. coli. Several NBW types demonstrated antibacterial effects against drug-resistant strains, although the magnitude and duration varied. Importantly, short-term exposure (≤60 min) did not reduce bacterial counts, indicating that measurable effects require prolonged immersion. NBW generated using relatively safe and inexpensive gases (excluding ozone, which is known to be cytotoxic) exerts reproducible antibacterial activity against diverse bacterial species, including resistant strains. However, its lack of rapid disinfectant action suggests that NBW is better suited for long-term immersion rather than short-contact disinfection. These findings support NBW as a potential safer and cost-effective strategy for immersion-based infection control and mitigation of antimicrobial resistance.

Histidine kinases are an integral component of bacterial two-component systems (TCSs), playing a pivotal role in signal transduction pathways, resulting in both resistance and virulence. However, their inherent membrane-bound nature often results in poor solubility, making them difficult to isolate and rendering them incompatible with most in vitro biochemical techniques. Consequently, much of the research on two-component systems has centered on response regulators, limiting both drug discovery efforts and our broader understanding of key signal transduction mechanisms. To address these challenges, we sought to straightforwardly generate cytosolic truncation mutants of histidine kinases that retain their autophosphorylation and phosphotransfer capabilities. Previously, we successfully developed a cytosolic truncation mutant of PmrB (PmrBc) that maintained these critical functions, demonstrating its suitability as a viable surrogate for in vitro investigations, including inhibitor compound screening. Building upon this foundation, we have refined our methods and here demonstrate these improvements by producing functional histidine kinase truncation mutants from the following diverse bacterial species: Escherichia coli; PhoQ, BasS and Klebsiella pneumoniae; and PmrB and PhoQ.

Male genitourinary (mGU) malignancies, including prostate, bladder, kidney, testicular, and penile cancers, represent a clinically and epidemiologically significant subset of global cancer burden. Although well-established etiological factors such as genetic mutations, androgen signaling, and environmental exposures contribute to tumorigenesis, the underlying mechanisms remain ill-defined. Recent advances in next-generation sequencing and metagenomics technologies have facilitated a deeper understanding of the human microbiome, revealing its potential role in carcinogenesis. While the gut microbiome has been extensively studied, emerging evidence indicates that site-specific microbial communities within the genitourinary (GU) tract may significantly influence cancer susceptibility, progression, and therapeutic outcomes. Accordingly, this review aims to comprehensively summarize the current evidence examining the relationship between the GU microbiome and the development, progression, and treatment of mGU cancers. To provide the specific context, relevant publications were collected from Google Scholar, PubMed, Science Direct, Dimension AI, and EBSCO Host using specific keywords such as “bladder cancer”, “dysbiosis”, “genitourinary”, “genitourinary cancer”, “microbiome”, “pathogens”, “penile cancer”, “prostate cancer”, “renal cancer”, “testicular cancer”, “urogenital microbiome”. We did not add any limits to the publication date during the inclusion of papers. However, it is noteworthy that the initial reports, including the aforementioned keywords, have been published since 2015. Emerging evidence highlights a significant association between the dysbiosis of the GU microbiome and the development of mGU cancers. Notably, an increase in bacterial richness and species diversity has been correlated with a rapid progression of these cancers, suggesting that such features may be explored as potential candidate biomarkers. Advanced sequencing and meta-omics technologies have enabled the identification of distinct microbial signatures with emerging diagnostic, prognostic, and therapeutic potential. Despite these advancements, the understanding of the functional and mechanistic roles of microbiota, particularly within the penile and seminal environments, remains limited.

Universal stress proteins (USPs) have remained an enigma since their first description by Nystrom and Neidhardt in 1992. Despite being upregulated under diverse stresses and found across a range of bacterial species, decades of studies suggested only general and potentially redundant protective functions for USPs. Recent studies have uncovered that USPs are critical regulators of bacterial survival processes in Actinobacteria, most notably in Mycobacterium tuberculosis, one of the most prolific and lethal of human pathogens. This brief review places these recent studies in the context of earlier publications and discusses their importance for future USP research, our understanding of these regulatory proteins, and novel therapeutic options that these proteins present in Mycobacterium tuberculosis, related Actinobacteria, and across diverse bacterial species. Impact Statement Universal stress proteins (USPs) have recently been directly implicated in survival processes in Mycobacteria, related Actinobacteria, and multiple bacterial pathogens. This new understanding identifies these stress-responsive proteins as important targets for mechanistic studies in bacterial survival and promising targets for novel antimicrobial therapeutics.

Dynamic succession patterns, nitrogen cycling potential, and multi-scale assembly mechanisms of cross-habitat bacterial communities in lakes driven by seasonal frozen conditions.

BmpA is a putative substrate-binding protein from Borrelia burgdorferi , the causative agent of Lyme disease, an organism with limited metabolic capacity that relies on salvage pathways rather than de novo nucleotide biosynthesis. Here, we determine the crystal structure of BmpA to a resolution of 2.6 Å, revealing a conserved substrate-binding protein fold with a deeply buried nucleoside-binding pocket. Using microscale thermophoresis, we show that BmpA binds thymidine with high affinity followed by cytidine and adenosine, whereas binding to ribose, guanosine, inosine, and uridine was not detected. Structure-guided mutagenesis further demonstrates that two conserved aromatic residues (Phe27 and Phe176) are essential for thymidine recognition, as alanine substitution at either position abolishes detectable binding. Additionally, a Foldseek-based structural homology search identified related proteins across diverse bacterial and archaeal species that share a conserved overall fold and binding-site architecture despite low sequence similarity, consistent with an evolutionarily conserved scaffold that can accommodate distinct nucleoside ligands. Together, our work illustrates how conserved binding protein architectures enable selective nucleoside acquisition and provides a foundation for understanding nutrient uptake strategies in organisms with reduced genomes.