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Bacterial conjugation, wherein DNA is transferred between cells through direct contact, is highly prevalent in complex microbial communities and is responsible for spreading myriad genes related to human and environmental health. Despite their importance, much remains unknown regarding the mechanisms driving the spread and persistence of these plasmids in situ. Studies have demonstrated that transferring, acquiring, and maintaining a plasmid imposes a significant metabolic burden on the host. Simultaneously, emerging evidence suggests that the presence of a conjugative plasmid can also provide both obvious and unexpected benefits to their host and local community. Combined, this highlights a continuous cost-benefit tradeoff at the population level, likely contributing to overall plasmid abundance and long-term persistence. Yet, while the metabolic burdens of plasmid conjugation, and their causes, are widely studied, their attendant potential advantages are less clear. Here, we summarize current perspectives on conjugative plasmids' metabolic burden and then highlight the lesser-appreciated yet critical benefits that plasmid-mediated metabolic burdens may provide. We argue that this largely unexplored tradeoff is critical to both a fundamental theory of microbial populations and engineering applications and therefore warrants further detailed study., (© 2024 Wiley Periodicals LLC.)

Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by progressive muscle degeneration, primarily affecting young males. In this study, we investigated arginine-modified hydroxyapatite nanoparticles (R-HAp) as a novel non-viral vector for DMD gene therapy, particularly for delivering the large 18.8 kb dystrophin gene. Addressing the limitations of traditional adeno-associated viral vectors, R-HAp demonstrated efficient binding and delivery of the dystrophin plasmid to DMD patient-derived skeletal muscle cells. Using confocal imaging and RT-PCR analysis, our results showed effective gene delivery and expression in both mouse myotubes and patient-derived cells, with sustained expression evident up to 5 days post transfection. The patient-derived myotubes also showed dystrophin protein production 7 days post transfection. These findings suggest R-HAp nanoparticles as a promising and cost-effective alternative for DMD treatment, highlighting their potential for overcoming current gene therapy challenges.

Gene II Protein (Gp2/P2) is a nicking enzyme of the M13 bacteriophage that plays a role in the DNA replication of the viral genome. P2 recognizes a specific sequence at the f1 replication origin and nicks one of the strands and starts replication. This study was conducted to address the limitations of previous experiments, improve methodologies, and precisely determine the biochemical activity conditions of the P2 enzyme in vitro. For these purposes, the gene encoding P2 was cloned in Escherichia coli and expressed as a hybrid protein together with a green fluorescent protein (P2-GFP). P2-GFP was purified via metal affinity chromatography, and its nicking activity was determined by conversion of supercoiled DNA to open circular or linear forms. We discovered that, among the two loops of the f1 origin defined previously, P2 can recognize just the A1 loop. When a supercoiled plasmid containing the f1 origin was treated with P2-GFP, the plasmid was present in an open circular form, indicating that a nick was created on only one of the strands. However, when the A1 loop sequence was inserted into the 3' ends of both strands by cloning a PCR product obtained by primers with the A1 loop sequence, the plasmid was linearized by treatment with P2-GFP, indicating that nicks were created on both strands. Certain infectious diseases are caused by single-stranded DNA viruses, and some of them have specific nicking enzymes that enable strand displacement and free 3' end of a single strand that works as a primer for their replication mechanisms like M13 bacteriophages, such as parvovirus B19. Despite there being different host viruses such as bacteria and humans, their DNA replication mechanisms are very similar in this concept. Investigating the features of the P2-nicking enzyme may deepen the understanding of human pathogenic single-stranded viruses and facilitate the development of drugs that inhibit viral replication.

Parasitic elements often spread to hosts through the delivery of their nucleic acids to the recipient. This is particularly true for the primary parasites of bacteria, bacteriophages (phages) and plasmids. Although bacterial immune systems can sense a diverse set of infection signals, such as a protein unique to the invader or the disruption of natural host processes, phage and plasmid nucleic acids represent some of the most common molecules that are recognized as foreign to initiate defense. In this review, we will discuss the various elements of invader nucleic acids that can be distinguished by bacterial host immune systems as "non-self" and how this signal is relayed to activate an immune response., Competing Interests: Declaration of interests L.A.M. is a cofounder and Scientific Advisory Board member of Intellia Therapeutics, Scientific Advisory Board member of Ancilia Biosciences, and a cofounder of Eligo Biosciences., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metal-organic frameworks (MOFs) provide diverse applications across a wide range of scientific disciplines, including drug/nucleic acid (NA) delivery. In the subclass of MOFs, zeolitic imidazolate framework-8 (ZIF-8) is well regarded due to its exceptional physicochemical properties. Biomolecules can be encapsulated and released under precise conditions within ZIF, making it an important material for materials science and biomedical applications. Different solvents and synthesis methods influence the ZIF's topologies and framework structures. The physicochemical properties of plasmid-encapsulated ZIF (plasmid@ZIF) can be controlled by tuning the precursors and biomolecular concentration. Using plasmid@ZIF, this study demonstrated that nucleic acids can be loaded precisely and released with a controlled bioactivity within cells. It was found that the ZIF phases substantially influenced both NA delivery into the cell and physicochemical properties. As a result of this study, we better understand MOFs' potential in NA delivery, and it emphasizes the importance of precisely controlling their physicochemical properties.

Inducible systems are crucial to metabolic engineering and synthetic biology, enabling organisms that function as biosensors and produce valuable compounds. However, almost all inducible systems are strain-specific, limiting comparative analyses and applications across strains rapidly. This study designed and presented a robust workflow for developing the cross-species inducible system. By applying this approach, two reconstructed inducible systems (a 2,4-diacetylphloroglucinol-inducible system PphlF3R1 and an anhydrotetracycline-inducible system Ptet2R2*) were successfully developed and demonstrated to function in three model microorganisms, including Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. To enhance their practicality, both inducible systems were subsequently placed on the plasmid and genome for detailed characterization to determine the optimal expression conditions. Furthermore, the more efficient inducible system Ptet2R2* was employed to express various reporter proteins and gene clusters in these three strains. Moreover, the aTc-inducible system Ptet2R2*, combined with T7 RNA polymerase and dCas12a, was utilized to develop a single-input genetic circuit that enables the simultaneous activation and repression of gene expression. Overall, the cross-species inducible system serves as a stringent, controllable and effective tool for protein expression and metabolic pathway control in different bacteria., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Current plasmid propagation in E. coli compromises large inverted repeats, such as inverted terminal repeats (ITRs) of adeno-associated virus (AAV). Direct long-read sequencing analyses upon varying strains and culture conditions revealed ITR instability caused by a slipped misalignment mechanism, although other mechanism probably contribute. ITRs stabilized in absence of SbcC, which is part of the SbcCD nuclease complex, a human Mre11-Rad50 homolog, or at elevated growth temperatures (e.g. 42°C), with a combination being optimal. Resulting full ITR transgene plasmids improved rAAV yield and purity in HEK-293 productions. The findings advance plasmid biology, cloneable sequences and therapeutic AAV manufacturing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Small RNAs (sRNAs) play a crucial role in modulating target gene expression through short base-pairing interactions and serve as integral components of many stress response pathways and regulatory circuits in bacteria. Transcriptome analyses have facilitated the annotation of dozens of sRNA candidates in the ubiquitous environmental model bacterium Caulobacter crescentus, but their physiological functions have not been systematically investigated so far. To address this gap, we have established CauloSOEP, a multi-copy plasmid library of C. crescentus sRNAs, which can be studied in a chosen genetic background and under select conditions. Demonstrating the power of CauloSOEP, we identified sRNA AbnZ to impair cell viability and morphology. AbnZ is processed from the 3' end of the polycistronic abn mRNA encoding the tripartite envelope-spanning efflux pump AcrAB-NodT. A combinatorial approach revealed the essential membrane translocation module TamAB as a target of AbnZ, implying that growth inhibition by AbnZ is linked to repression of this system., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Hydroxytyrosol, a fine chemical, is widely utilized in food and pharmaceutical industries. In this study, we constructed a pathway to produce hydroxytyrosol by co-expressing tyrosin-phenol lyase (TPL), L-amino acid dehydrogenase (aadL), α-keto acid decarboxylase (KAD), aldehyde reductase (yahK) and glucose dehydrogenase (gdh). We changed combinations between plasmids with different copy numbers and target genes, resulting in 84% increase in hydroxytyrosol production. The yield of hydroxytyrosol was further increased 89.3% by optimizing the temperature and pH. Finally, 55.3 mM hydroxytyrosol was produced within 14 h by fed-batch biotransformation. This study provides a novel approach for hydroxytyrosol production., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Structural maintenance of chromosomes (SMC) complexes play pivotal roles in genome organization and maintenance across all domains of life. In prokaryotes, SMC-family Wadjet complexes structurally resemble the widespread MukBEF but serve a defensive role by inhibiting plasmid transformation. We previously showed that Wadjet specifically cleaves plasmid DNA; however, the molecular mechanism underlying plasmid recognition remains unclear. Here, we use in vitro single-molecule imaging to directly visualize DNA loop extrusion and plasmid cleavage by Wadjet. We find that Wadjet is a symmetric loop extruder that simultaneously reels in DNA from both sides of a loop and that this activity requires a dimeric JetABC supercomplex. On surface-anchored plasmid DNAs, Wadjet extrudes the full length of a 44-kb-pair plasmid, stalls, and cleaves DNA. Our findings reveal the role of loop extrusion in the specific recognition and elimination of plasmids by Wadjet and establish loop extrusion as an evolutionarily conserved mechanism among SMC complexes across all kingdoms of life., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Conjugation-mediated DNA delivery is the primary mode for antibiotic resistance spread in bacteria; yet, molecular mechanisms regulating the conjugation process remain largely unexplored. While conjugative plasmids typically require bacterial attachment to solid surfaces for facilitation of donor-to-recipient proximity, the pLS20 conjugative plasmid, prevalent among Gram-positive Bacillus spp., uniquely requires fluid environments to enhance its transfer. Here, we show that pLS20, carried by Bacillus subtilis, induces multicellular clustering, which can accommodate various species, hence offering a stable platform for DNA delivery in a liquid milieu. We further discovered that induction of pLS20 promoters, governing crucial conjugative genes, is dependent on the presence of donor cell flagella, the major bacterial motility organelle. Moreover, the pLS20 regulatory circuit is controlled by a mechanosensing signal transduction pathway responsive to flagella rotation, thus activating conjugation gene expression exclusively during the host motile phase. This flagella-conjugation coupling strategy may allow the dissemination of the plasmid to remote destinations, allowing infiltration into new niches., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Studies on radiosensitization of biological damage by O 2 began about a century ago and it remains one of the most significant subjects in radiobiology. It has been related to increased production of oxygen radicals and other reactive metabolites, but only recently to the action of the numerous low-energy electrons (LEEs: 0-30 eV) produced by ionizing radiation. We provide the first complete set of G-values (yields of specific products per energy deposited) for all conformational damages induced to plasmid DNA by LEEs (G LEE (O 2 )) and 1.5 keV X-rays (G X (O 2 )) under oxygen at atmospheric pressure. The experiments are performed in a chamber, under humidity levels ranging from 2.5 to 33 water molecules/base. Photoelectrons from 0 to 30 eV are produced by X-rays incident on a tantalum substrate covered with DNA. Damage yields are measured by electrophoresis as a function of X-ray fluence. The oxygen enhancement ratio G LEE (O 2 )/G LEE (N 2 ), which lies around 2 for potentially lethal cluster lesions, is similar to that found with cells. The average ratio, G LEE (O 2 )/G X (O 2 ), of 12 for cluster lesions and crosslinks strongly suggest that DNA damages that harm cells are much more likely to be created by LEEs than any other initial species generated by X-rays in the presence of O 2 .

We introduce a biomolecular circuit for precise control of gene expression in mammalian cells. The circuit leverages the stochiometric interaction between the artificial transcription factor VPR-dCas9 and the anti-CRISPR protein AcrIIA4, enhanced with synthetic coiled-coil domains to boost their interaction, to maintain the expression of a reporter protein constant across diverse experimental conditions, including fluctuations in protein degradation rates and plasmid concentrations, by automatically adjusting its mRNA level. This capability, known as robust perfect adaptation (RPA), is crucial for the stable functioning of biological systems and has wide-ranging implications for biotechnological applications. This system belongs to a class of biomolecular circuits named antithetic integral controllers, and it can be easily adapted to regulate any endogenous transcription factor thanks to the versatility of the CRISPR-Cas system. Finally, we show that RPA also holds in cells genomically integrated with the circuit, thus paving the way for practical applications in biotechnology that require stable cell lines.

Cationic polymers that bind with the plasmids to form polyplexes protect the DNA from enzymatic degradation and improve cellular and tissue uptake. Complete or near complete gel retardation of the polyplex is an important assay to determine the optimal polymer: plasmid ratio for in vitro and in vivo studies. Nevertheless, despite minimal to moderate gel retardation of histidine-lysine (HK) polyplexes formed with low peptide: plasmid DNA ratios (1:2 and 1:4; w:w), the polyplexes effectively targeted the tumor in vivo. To understand the lack of predictability of the initial gel mobility shift assays, we revisited the retardation and stability of polyplexes with these electrophoresis assays. Because the histidine component with a pKa of about 6.0 will have a greater positive charge and may bind plasmids with a higher affinity at lower pHs, we compared the retardation of the two HK polyplexes when the pH of the running buffer of the gel mobility shift assay was altered. Both HK polyplexes were retarded significantly more when the running buffer had a pH of 7.3 instead of the standard pH of 8.3. Indeed, the HK polyplexes formed at the 1:2 ratio showed complete retardation at pH 7.3. Consequently, while both HK polyplexes formed at these low ratios targeted the tumor, the polyplex formed with the 1:2 ratio had reduced tumor gene expression variability and lower lung and liver values. Thus, the selection of the optimal ratios for the linear HK and plasmid for transfection studies in vivo was improved with a running buffer pH of 7.3., Competing Interests: Declaration of competing interest The authors of this manuscript declare that there are no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In the past two decades, 25 different species of the genus Pantoea within the Enterobacteriaceae family, have been isolated from different environmental niches. These species have a wide range of biological roles. Versatility in functions and hosts indicate that this genus has undergone extensive genetic diversification, which can be attributed to the different extra-chromosomal genetic elements or plasmids found across this genus. We have analyzed the functions of these plasmids and categorized them into four major groups for a better understanding of their future applications. The first and second group includes plasmids that contribute to genetic diversification and pathogenicity, respectively. The third group comprises cryptic plasmids of Pantoea. The last group includes plasmids that play a role in the metabolic versatility of the genus Pantoea. We have analyzed the data available up to May 2023 from two databases (viz; NCBI and PLSDB). In our analysis we have found a vast gap in knowledge. Complete gene annotations are available for only a few of the plasmids. This review highlights these challenges as an avenue for future research., Competing Interests: Declarations Conflict of interest The authors declare no conflict of interest., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

UCMSCs were reprogrammed to iPSCs using Yamanaka factor bearing episomal plasmids. SCIKFi001-B exhibited pluripotency, had typical iPSC morphology and didn't retain any residual episomal plasmid. Although karyotyping showed chromosomal translocation, this abnormality seemed to have little impact on the functionality of SCIKFi001-B since it retained its ability to differentiate to three-germ layer. While karyotypic abnormality might negate use in therapeutic and clinical settings, this line remained a valuable educational tool for iPS cell culture techniques. Finally, our study highlighted the importance of routine karyotyping on iPSC lines as abnormal karyotypes oftentimes bear no discernible effect on cell morphology nor functionality., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Ultrasonic-mediated plasmid transformation is a promising microbial transformation strategy with broad application prospects that has attracted interest across various fields. Limited research exists on developing a quantitative model to understand the relationship between transformation efficiency and ultrasonic power. Within the ultrasonic range that did not damage plasmids, the maximum transformation efficiency reached at 4.84 × 10 5 CFU/μg DNA. A kinetic model based on changes in membrane permeability was utilized to determine the membrane permeability at different power levels. The results indicated a linear correlation between ultrasonic power, transformation efficiency, and membrane permeability within a specific range. A quantitative relationship model was established based on ultrasonic power and transformation efficiency in E. coli. Electron microscopy revealed that E. coli cells subjected to ultrasonic treatment exhibited pore formation and cellular expansion. Furthermore, the integrity of the bacterial membrane was compromised as ultrasonic power increased. Nine genes associated with the functional terms of cell membrane components and transmembrane transport were identified in E. coli DH5α. According to qRT-PCR results, genes with these functions (including cusC, uidC, tolQ, tolA, ompC, yaiY) play crucial roles in ultrasound-mediated transformation of E. coli DH5α. This study suggested that ultrasound-mediated transformation in E. coli DH5α is not a simple physical-chemical process but rather involves the regulation of responsive membrane-related genes. This research establishes the groundwork for future comprehensive investigations into the molecular mechanism of ultrasound-mediated transformation and provides insights for the application of ultrasound technology in genetic engineering and related fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Strong promoters and stable mRNAs are essential for the overproduction of heterologous proteins in Bacillus subtilis. To improve the strength of natural promoters and ensure robust protein output, promoter and genetic insulator engineering have been used. A series of plasmids containing single and dual promoters and genetic insulators to express alt3796 were engineered, which encoded an unsaturated glucuronyl hydrolase (UGL). As a first step, we screened the host and deleted the signal peptide (SP ALT ) of alt3796, successfully expressed secreted ALT3796 from B. subtilis WB800. Subsequently, to improve expression, we screened the dual promoter P Hag-spoVG from a collection of 22 promoters, which yielded higher enzymatic activity. Finally, using a recombinant strain carrying a plasmid with the P Hag-spoVG dual promoter and a genetic insulator, we obtained 40.9 U/mL of activity. Purified recombinant ALT3796 exhibited good stability and specifically degraded ulvan. In conclusion, a system for the heterologous expression of ALT3796 was constructed, and the obtained protein exhibited favorable properties, suggesting its potential for preparing novel ulvan oligosaccharides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Gamma-aminobutyric acid (GABA), which is synthesized from l-glutamic acid via glutamate decarboxylase (Gad), is used as food, supplements, and biodegradable plastics. Our previous study demonstrated an Escherichia coli mutant (ΔΔ) strain, lacking type I NADH dehydrogenase (NDH-I) and cytochrome bo 3 oxidase (Cytbo 3 ), produced 7 g/L glutamic acid on MS1 glucose-minimal medium. In this study, the ΔΔ strain was used for improving GABA production. A plasmid (pMBL19-gadB') expressing a mutated E. coli GadB (Glu89Gln/Δ452-466), retaining activity at neutral pH, was introduced into the ΔΔ strain and its parent strain (W1485). The ΔΔ strain carrying pMBL19-gadB' exhibited a twofold increase in GABA production compared to the W1485 strain carrying pMBL19-gadB'. Deleting the C-terminal (Δ471-511) of GadC antiporter in the ΔΔ strain further improved GABA yield to 1.5 g/L when cultured in MS1 glucose-minimal medium. On the other hand, a large amount of glutamic acid produced by the ΔΔ strain was not fully converted to GABA, likely due to the inhibition of GadB activity by the accumulation of acetic acid. Although there is room for improvement, these results indicate the efficacy of the ΔNDH-IΔCytbo 3 double mutation in augmenting GABA production., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Plastic pollution arising from single-use plastic bottles (SUPBs) and containers leads to the formation of micro/nanoplastics (NPs). These NPs raise concerns due to their potential toxicity and interactions with microorganisms. In various environments, including our digestive system, both microorganisms and plastics coexist. The interactions between these NPs and microorganisms can have far-reaching consequences, potentially affecting the ecosystems and human health. Therefore, understanding these interactions is crucial for addressing the challenges posed by plastic pollution. This study investigated the role of NPs in propagating antibiotic resistance (AR), specifically through outer membrane vesicles (OMVs), which is a mechanism that has not been fully explored to date in terms of NPs' effects. To explore this, NPs were synthesized using polyethylene terephthalate (PET) SUPBs, mimicking the natural chemical composition of environmental nanoplastics, unlike previous studies, which used pure PET, polystyrene (PS) or other pure plastic materials. The resulting PET bottle-derived nanoplastics (PBNPs), which exhibited diverse shapes and sizes (50-850 nm), were found to facilitate horizontal gene transfer (HGT) through transformation and outer membrane vesicles (OMVs), enabling the transport of plasmids among bacteria. In transformation, PBNPs physically carried plasmids across the bacterial membrane. In another scenario, PBNPs induced oxidative stress and bacterial surface damage, which led to the upregulation of stress response-associated genes and the escalation of OMV secretion in E. coli . This novel pathway highlights how PBNPs contribute to AR gene dissemination, potentially exacerbating the global antibiotic resistance crisis. Furthermore, PBNPs mediate cross-species gene transfer from E. coli to Lactobacillus acidophilus , underscoring their impact on diverse microorganisms, including those in the human gut. Our findings suggest that nanoplastics may be an unrecognized contributor to the rising tide of antibiotic resistance, with significant consequences for human health and the environment. Molecular analyses revealed the upregulated expression of genes associated with stress response and OMV secretion, offering deeper insights into the biological mechanisms affected by PBNPs. This study offers crucial insights into the interactions of NPs and microorganisms for developing strategies to address the ecological and health implications of nanoplastic contamination.

Background: Plasmids play a major role in the transfer of antimicrobial resistance (AMR) genes among bacteria via horizontal gene transfer. The identification of plasmids in short-read assemblies is a challenging problem and a very active research area. Plasmid binning aims at detecting, in a draft genome assembly, groups (bins) of contigs likely to originate from the same plasmid. Several methods for plasmid binning have been developed recently, such as PlasBin-flow, HyAsP, gplas, MOB-suite, and plasmidSPAdes. This motivates the problem of evaluating the performances of plasmid binning methods, either against a given ground truth or between them., Results: We describe PlasEval, a novel method aimed at comparing the results of plasmid binning tools. PlasEval computes a dissimilarity measure between two sets of plasmid bins, that can originate either from two plasmid binning tools, or from a plasmid binning tool and a ground truth set of plasmid bins. The PlasEval dissimilarity accounts for the contig content of plasmid bins, the length of contigs and is repeat-aware. Moreover, the dissimilarity score computed by PlasEval is broken down into several parts, that allows to understand qualitative differences between the compared sets of plasmid bins. We illustrate the use of PlasEval by benchmarking four recently developed plasmid binning tools-PlasBin-flow, HyAsP, gplas, and MOB-recon-on a data set of 53 E. coli bacterial genomes., Conclusion: Analysis of the results of plasmid binning methods using PlasEval shows that their behaviour varies significantly. PlasEval can be used to decide which specific plasmid binning method should be used for a specific dataset. The disagreement between different methods also suggests that the problem of plasmid binning on short-read contigs requires further research. We believe that PlasEval can prove to be an effective tool in this regard. PlasEval is publicly available at https://github.com/acme92/PlasEval., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

The methylotrophic yeast Komagataella phaffii is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant "dead" MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research. CRISPRi utilizing dMAD7 does not introduce double-strand breaks but only binds to the DNA to regulate gene expression. Here, we report the first use of dMAD7 in K. phaffii to regulate the expression of the enhanced green fluorescent protein (eGFP). A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. Integration loci/events of investigated strains were assessed through whole genome sequencing. Additionally, RNA-sequencing experiments corroborated the whole genome sequencing results and showed a significantly reduced expression of eGFP in strains containing a dMAD7 plasmid, among others. Our findings conclusively demonstrate the utility of dMAD7 in K. phaffii through successfully regulating eGFP expression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

l-Methionine, a valuable sulfur-containing amino acid, holds great significance as a feed additive, nutraceutical, pharmaceutical, or even in the cosmetic industry. However, achieving efficient microbial production of l-methionine remains challenging due to its complex biosynthetic pathway and plasmid loss during fermentation. Herein, l-methionine biosynthesis was improved by enhancing succinyl-CoA supply, introducing a direct-sulfurylation pathway, and weakening the l-threonine branched pathway. The engineered strain produced 21.55 g/L l-methionine with a yield of 0.14 g/g glucose in a 5 L bioreactor. To eliminate the need for antibiotics and minimize plasmid loss, the hok / sok system was incorporated into the plasmid. The resulting plasmid pAm10 enabled strain M2 thrB A1G to produce 20.39 g/L of l-methionine without antibiotics in 5 L of fed-batch cultivation, a 42.58% increase compared to the control. This study highlights the potential of plasmid-based antibiotic-free fermentation for efficient and cost-effective production of l-methionine, as well as other amino acids or chemicals.

Protein synthesis is influenced by the chemical and structural properties of the amino acids incorporated into the polypeptide chain. Motifs containing consecutive prolines can slow the translation speed and cause ribosome stalling. Translation elongation factor P (EF-P) facilitates peptide bond formation in these motifs, thereby alleviating stalled ribosomes and restoring the regular translational speed. Ribosome pausing at various polyproline motifs has been intensively studied using a range of sophisticated techniques, including ribosome profiling, proteomics, and in vivo screening, with reporters incorporated into the chromosome. However, the full spectrum of motifs that cause translational pausing in Escherichia coli has not yet been identified. Here, we describe a plasmid-based dual reporter for rapid assessment of pausing motifs. This reporter contains two coupled genes encoding mScarlet-I and chloramphenicol acetyltransferase to screen motif libraries based on both bacterial fluorescence and survival. In combination with a diprolyl motif library, we used this reporter to reveal motifs of different pausing strengths in an E. coli strain lacking efp . Subsequently, we used the reporter for a high-throughput screen of four motif libraries, with and without prolines at different positions, sorted by fluorescence-associated cell sorting (FACS) and identify new motifs that influence the translational efficiency of the fluorophore. Our study provides an in vivo platform for rapid screening of amino acid motifs that affect translational efficiencies.

Enzymes that produce volatile metabolites can be coded into genetic circuits to report nondisruptively on microbial behaviors in hard-to-image soils. However, these enzyme reporters remain challenging to apply in gene transfer studies due to leaky off states that can lead to false positives. To overcome this problem, we designed a reporter that uses ribozyme-mediated gene-fragment complementation of a methyl halide transferase (MHT) to regulate the synthesis of methyl halide gases. We split the mht gene into two nonfunctional fragments and attached these to a pair of splicing ribozyme fragments. While the individual mht -ribozyme fragments did not produce methyl halides when transcribed alone in Escherichia coli , coexpression resulted in a spliced transcript that translated the MHT reporter. When cells containing one mht -ribozyme fragment transcribed from a mobile plasmid were mixed with cells that transcribed the second mht -ribozyme fragment, methyl halides were only detected following rare conjugation events. When conjugation was performed in soil, it led to a 16-fold increase in methyl halides in the soil headspace. These findings show how ribozyme-mediated gene-fragment complementation can achieve tight control of protein reporter production, a level of control that will be critical for monitoring the effects of soil conditions on gene transfer and the fidelity of biocontainment measures developed for environmental applications.

The interaction of low energy electrons (LEEs; 1-30 eV) with genomic material can induce multiple types of damage that may cause the loss of genetic information, mutations, genome instability, and cell death. For all damages measurable by electrophoresis, we provide the first complete set of G -values (yield of a specific product per energy deposited) induced in plasmid DNA by the direct and indirect effects of LEEs ( G LEE ) and 1.5 keV X-rays ( G X ) under identical conditions. Low energy photoelectrons are produced via X-rays incident on a tantalum (Ta) substrate covered with DNA and placed in a chamber filled with nitrogen at atmospheric pressure, under four different humidity levels, ranging from dry conditions to full hydration (Γ = 2.5 to Γ = 33, where Γ is the number of water molecules/nucleotide). Damage yields are measured as a function of X-ray fluence and humidity. G LEE values are between 2 and 27 times larger than those for X-rays. At Γ = 2.5 and 33, G LEE values for double strand breaks are 27 and 16 times larger than G X , respectively. The indirect effect contributes ∼50% to the total damage. These G -values allow quantification of potentially lethal lesions composed of strand breaks and/or base damages in the presence of varying amounts of water, i.e., closer to cellular conditions.

Semiconductor nanowires are known for their unusual geometry, providing unique electronic and optical properties. Substrates with vertical nanowires have highly non-uniform surfaces, which are attractive in terms of the study of live cells that can interact and be labeled with the wires. Despite several previous works studying cells cultivated over nanowires, questions regarding cell rupture and interaction with the wires remain open. Here, we demonstrate that nanowires can not only penetrate the cell membrane, but even be broken by a cell and trapped inside it. Even after mechanical poration of the membrane manifested by the efficient transfection and delivery of a fluorescent protein encoding plasmid, the cells are found to be viable for 7 days of incubation. The endocytosed wires are then aligned along the nucleus periphery and ousted to pseudopodia with the formation of nanowire-rich fibrils as a result of complex intracellular processes. We demonstrate that endocytosis of the wires may lead to their chemical modification manifested by the red shift of the luminescence spectra. Analysis of the wires' breakdown reveals that the cells can generate forces as high as several hundreds of nN. Using this work, we demonstrate several phenomena with the potential to be used in intriguing methods for intracellular visualization and the development of biointerfaces.

In many bacteria, the essential factors Rho and NusG mediate termination of synthesis of nascent transcripts (including antisense RNAs) that are not being simultaneously translated. It has been proposed that in Rho's absence toxic RNA-DNA hybrids (R-loops) may be generated from nascent untranslated transcripts, and genome-wide mapping studies in Escherichia coli have identified putative loci of R-loop formation from more than 100 endogenous antisense transcripts that are synthesized only in a Rho-deficient strain. Here we provide evidence that engineered expression in wild-type E. coli of several such individual antisense regions on a plasmid or the chromosome generates R-loops that, in an RNase H-modulated manner, serve to disrupt genome integrity. Rho inhibition was associated with increased prevalence of antisense R-loops also in Xanthomonas oryzae pv. oryzae and Caulobacter crescentus. Our results confirm the essential role of Rho in several bacterial genera for prevention of toxic R-loops from pervasive yet cryptic endogenous antisense transcripts. Engineered antisense R-looped regions may be useful for studies on both site-specific impediments to bacterial chromosomal replication and the mechanisms of their resolution., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Circular RNA (circRNA) exhibits a higher stability and intracellular half-life than linear RNA and has better potential in the fields of RNA vaccines and RNAi drugs. The current strategies for circRNA preparation have low efficiency, high costs, and high complexity, which significantly limits their applications. In this paper, we propose a one-step synthesis of circRNA based on E. coli. The four RNA sequence lengths of 1700, 1400, 500, and 64 nt were connected to group II intron elements from the surface protein region of Clostridium tetani and then inserted downstream of the T7 promoter in the pET28a plasmid to assist in cyclization. Then, circRNA was produced in HT115, where the yields of pET28-1700, pET28-1400, pET28-500, and pET28-64 were improved to 820, 783, 691, and 460 ng/1 mL, respectively. Consequently, this system could achieve the mass production of circRNA using only a simple E. coli culture and inducible expression. Meanwhile, the overexpressed circRNA and small circular interference RNA (sciRNA) maintained their biological functions in the protein translation and RNAi. Therefore, this simple and efficient one-step synthesis system can be applied to the functional study and preparation of circRNA in the future.

Enhanced production stability and efficiency along with a decrease in production costs are required to build efficient microbial cell factories. Target genes can be integrated into the genome to enhance genetic stability, reduce reliance on antibiotics, and alleviate the metabolic burden. However, selecting the optimal insertion site for the desired gene expression levels remains challenging. Therefore, 18 commonly used Escherichia coli integration sites were systematically characterized in this study. Promoters of different strengths were combined with integration sites, yielding a differential intensity range of up to 93-fold. This indicated the versatility and precision of this approach for controlling gene expression levels. Referring to the library, pathway genes were strategically integrated into the E. coli genome based on their respective expression levels. Genetically stable and highly efficient engineered strains that could biosynthesize arbutin and p -aminobenzoic acid were constructed.

The trade-off between rapid growth and other important physiological traits (e.g., survival and adaptability) poses a fundamental challenge for microbes to achieve fitness maximization. Studies on Bacillus subtilis biology often use strains derived after a process of lab 'domestication' from an ancestral strain known as Marburg strain. The domestication process led to loss of a large plasmid (pBS32) encoding a phosphatase (RapP) that dephosphorylates the Spo0F protein and thus regulates biofilm formation and sporulation. Here, we show that plasmid pBS32, and more specifically rapP, enhance growth rates by preventing premature expression of the Spo0F-Spo0A-mediated adaptive response during exponential phase. This results in reallocation of proteome resources towards biosynthetic, growth-promoting pathways without compromising long-term fitness during stationary phase. Thus, RapP helps B. subtilis to constrain physiological trade-offs and economize cellular resources for fitness improvement., (© 2024. The Author(s).)

The ability to manipulate gene expression is valuable for elucidating gene function. In the fission yeast Schizosaccharomyces pombe, the most widely used regulatable expression system is the nmt1 promoter and its two attenuated variants. However, these promoters have limitations, including a long lag, incompatibility with rich media and unsuitability for non-dividing cells. Here, we present a tetracycline-inducible system free of these shortcomings. Our system features the enotetS promoter, which achieves a similar induced level and a higher induction ratio compared to the nmt1 promoter, without exhibiting a lag. Additionally, our system includes four weakened enotetS variants, offering an expression range similar to that of the nmt1 series promoters but with more intermediate levels. To enhance usability, each promoter is combined with a Tet-repressor-expressing cassette in an integration plasmid. Importantly, our system can be used in non-dividing cells, enabling the development of a synchronous meiosis induction method with high spore viability. Moreover, our system allows for the shutdown of gene expression and the generation of conditional loss-of-function mutants. This system provides a versatile and powerful tool for manipulating gene expression in fission yeast., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Conjugative dissemination of mobile genetic elements (MGEs) among bacteria is initiated by assembly of the relaxosome at the MGE's origin-of-transfer (oriT) sequence. A critical but poorly defined step of relaxosome assembly involves recruitment of the catalytic relaxase to its DNA strand-specific nicking site within oriT. Here, we present evidence by AlphaFold modeling, affinity pulldowns, and in vivo site-directed photocrosslinking that the TraK Ribbon-Helix-Helix DNA-binding protein recruits TraI to oriT through a dynamic interaction in which TraI's C-terminal unstructured domain (TraI CTD ) wraps around TraK's C-proximal tetramerization domain. Upon relaxosome assembly, conformational changes disrupt this contact, and TraI CTD instead self-associates as a prerequisite for relaxase catalytic functions or substrate engagement with the transfer channel. These findings delineate key early-stage processing reactions required for conjugative dissemination of a model MGE., (© 2024 Federation of European Biochemical Societies.)

Environmental pollution is growing every day in terms of the increase in population, industrialization, and urbanization. Shewanella azerbaijanica is introduced as a highly potent bacterium in metal bioremediation. The mtrC gene was selected as a cloning target to improve electron flux chains in the EET (extracellular electron transfer) pathway. Using the SDM (site-directed mutagenesis) technique, the unique gene assembly featured the mtrC gene sandwiched between two napD/B genes to disrupt the nitrate reduction pathway, which serves as the primary metal reduction competitor. Shew-mtrC gene construction was transferred to expression plasmid pET28a (+) in the expression host bacteria (E. coli BL21 and S. azerbaijanica), in pUC57, cloning plasmid, which was transferred to the cloning host bacteria E. coli Top10 and S. azerbaijanica. All cloning procedures (i.e., synthesis, insertion, transformation, cloning, and protein expression) were verified and confirmed by precise tests. ATR-FTIR analysis, CD, western blotting, affinity chromatography, SDS-PAGE, and other techniques were used to confirm the expression and structure of the MtrC protein. The genome sequence and primers were designed according to the submitted Shewanella oneidensis MR-1 genome, the most similar bacteria to this native species. The performance of recombinant S. azerbaijanica bacterium in metal bioremediation, as sustainable strategy, has to be verified by more research., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Dynamically unstable polymers capture and move cellular cargos in bacteria and eukaryotes, but regulation of their assembly remains poorly understood. Here we describe polymerization of Alp7A, a bacterial actin-like protein (ALP) that distributes copies of plasmid pLS20 among daughter cells in Bacillus subtilis . Purified ATP-Alp7A forms dynamically unstable polymers with a high critical concentration for net assembly (cc N = 10.3 µM), but a much lower critical concentration for filament elongation (cc E = 0.6 µM). Rapid nucleation and stabilization of Alp7A polymers by the accessory factor, Alp7R, decrease cc N into the physiological range. Stable populations of Alp7A filaments appear under two conditions: (i) when Alp7R slows catastrophe rates or (ii) when Alp7A concentrations are high enough to promote filament bundling. These results reveal how dynamic instability maintains high steady-state concentrations of monomeric Alp7A, and how accessory factors regulate Alp7A assembly by modulating cc N independently of cc E ., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

The LitR/CarH family comprises adenosyl B 12 -based photosensory transcriptional regulators that control light-inducible carotenoid production in nonphototrophic bacteria. In this study, we established a blue-green light-inducible hyperexpression system using LitR and its partner ECF-type sigma factor LitS in streptomycin-producing Streptomyces griseus NBRC 13350. The constructed multiple-copy number plasmid, pLit19, carried five genetic elements: pIJ101rep, the thiostrepton resistance gene, litR, litS, and σ LitS -recognized light-inducible crtE promoter. Streptomyces griseus transformants harboring pLit19 exhibited a light-dependent hyper-production of intracellular reporter enzymes including catechol-2,3-dioxygenase and β-glucuronidase, extracellular secreted enzymes including laccase and transglutaminase, and secondary metabolites including melanin, flaviolin, and indigoidine. Cephamycin-producing Streptomyces sp. NBRC 13304, carrying an entire actinorhodin gene cluster, exhibited light-dependent actinorhodin production after the introduction of the pLit19 shuttle-type plasmid with the pathway-specific activator actII-ORF4. Insertion of sti fragment derived from Streptomyces phaeochromogenes pJV1 plasmid into pLit19 increased its light sensitivity, allowing gene expression under weak light irradiation. The two constructed Escherichia coli-Streptomyces shuttle-type pLit19 plasmids were found to have abilities similar to those of pLit19. We successfully established an optogenetically controlled hyperproduction system for S. griseus NBRC 13350 and Streptomyces sp. NBRC 13304., (© 2024. The Author(s).)

Background: The high-value aryl tetralin lignan (+)-pinoresinol is the main precursor of many plant lignans including (-)-podophyllotoxin, which is used for the synthesis of chemotherapeutics. As (-)-podophyllotoxin is traditionally isolated from endangered and therefore limited natural sources, there is a particular need for biotechnological production. Recently, we developed a reconstituted biosynthetic pathway from (+)-pinoresinol to (-)-deoxypodophyllotoxin, the direct precursor of (-)-podophyllotoxin, in the recombinant host Escherichia coli. However, the use of the expensive substrate (+)-pinoresinol limits its application from the economic viewpoint. In addition, the simultaneous expression of multiple heterologous genes from different plasmids for a multi-enzyme cascade can be challenging and limits large-scale use., Results: In this study, recombinant plasmid-free E. coli strains for the multi-step synthesis of pinoresinol from ferulic acid were constructed. To this end, a simple and versatile plasmid toolbox for CRISPR/Cas9-assisted chromosomal integration has been developed, which allows the easy transfer of genes from the pET vector series into the E. coli chromosome. Two versions of the developed toolbox enable the efficient integration of either one or two genes into intergenic high expression loci in both E. coli K-12 and B strains. After evaluation of this toolbox using the fluorescent reporter mCherry, genes from Petroselinum crispum and Zea mays for the synthesis of the monolignol coniferyl alcohol were integrated into different E. coli strains. The product titers achieved with plasmid-free E. coli W3110(T7) were comparable to those of the plasmid-based expression system. For the subsequent oxidative coupling of coniferyl alcohol to pinoresinol, a laccase from Corynebacterium glutamicum was selected. Testing of different culture media as well as optimization of gene copy number and copper availability for laccase activity resulted in the synthesis of 100 mg/L pinoresinol using growing E. coli cells., Conclusions: For efficient and simple transfer of genes from pET vectors into the E. coli chromosome, an easy-to-handle molecular toolbox was developed and successfully tested on several E. coli strains. By combining heterologous and endogenous enzymes of the host, a plasmid-free recombinant E. coli growing cell system has been established that enables the synthesis of the key lignan pinoresinol., (© 2024. The Author(s).)

Background: Conjugation-based episome delivery is a highly efficient method used to transfer DNA into the diatom Phaeodactylum tricornutum, facilitating the production of recombinant proteins and high-value metabolites. However, previous reports have indicated phenotypic heterogeneity among individual cells from clonally propagated exconjugant cell lines, potentially affecting the stability of recombinant protein production in the diatom., Results: Here, we characterized the differences between subpopulations with distinct fluorescence intensity phenotypes derived from a single exconjugant colony of P. tricornutum expressing the enhanced green fluorescent protein (eGFP). We analyzed the expression cassette sequence integrity, plasmid copy number, and global gene expression. Our findings reveal that lower copy numbers and the deletion of the expression cassette in part of the population contributed to low transgene expression. Gene co-expression analysis identified a set of genes with similar expression pattern to eGFP including a gene encoding a putative Flp recombinase, which may be related to variations in fluorescence intensity. These genes thus present themselves as potential candidates for increasing recombinant proteins production in P. tricornutum episomal expression system., Conclusions: Overall, our study elucidates genetic and transcriptomic differences between distinct subpopulations in a clonally propagated culture, contributes to a better understanding of heterogeneity in diatom expression systems for synthetic biology applications., (© 2024. The Author(s).)

Currently, pJP4 is one of the best-known plasmids for the biodegradation of xenobiotics that mediate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), which is associated with serious health and environmental risks. Although the sequencing and proposed theory of pJP4 formation occurred almost 20 years ago (2004), pJP4 is still the model object of many studies focused on the biodegradation of 2,4-D. The uniqueness of this plasmid is due to the presence of two evolutionarily distinct gene clusters, tfd I and tfd II , controlling the degradation of 2,4-D. Recent advances in plasmid biology, especially those concerning the characterization of new IncP-1 plasmids and the systematization of tfd gene cluster findings, serve as a basis for proposing new insights into the formation of the clusters' architecture of the canonical plasmid, pJP4, and their distribution among other plasmids. In the present work, a comparative genomic and phylogenetic in silico study of plasmids with tfd I and tfd II clusters was carried out. The possible initial distribution patterns of tfd I clusters among plasmids of different incompatibility groups (non-IncP-1) and tfd II clusters among IncP-1 plasmids using the IS 1071 -based composite transposon were revealed. A new theory on the formation of the architecture of the tfd I and tfd II clusters of pJP4 through sequential internal rearrangements, recombination, and IS JP4 insertion, is proposed. In addition, small gene clusters resulting from internal rearrangements of pJP4 ( tfd II SA and ORF31/32) served as fingerprints for exploring the distribution of tfd I and tfd II clusters. The revealed patterns and formulated theory extend the frontiers of plasmid biology and will be beneficial for understanding the role of plasmids in bacterial adaptation to xenobiotic-contaminated environments.

Drug-resistant pathogens significantly threaten human health and life. Simply killing drug-resistant pathogens cannot effectively eliminate their threat since the drug-resistant genes (DRGs) released from dead drug-resistant pathogens are difficult to eliminate and can further spread via horizontal gene transfer, leading to the spread of drug resistance. The development of antibacterial materials with sterilization and DRGs cleavage activities is highly crucial. Herein, a living system, Ce-PEA@Bdello, is fabricated with bacterial killing and DRGs cleavage activities for blocking bacterial drug resistance dissemination by engineered Bdellovibrio bacteriovorus (Bdello). Ce-PEA@Bdello is obtained by engineering Bdello with dopamine and a multinuclear cerium (IV) complex. Ce-PEA@Bdello can penetrate and eliminate kanamycin-resistant P. aeruginosa (Kan R ) biofilms via the synergistic effect of predatory Bdello and photothermal polydopamine under near-infrared light. Additionally, the DNase-mimicking ability of Ce-PEA@Bdello endows it with genome and plasmid DNA cleavage ability. An in vivo study reveals that Ce-PEA@Bdello can eliminate P. aeruginosa (Kan R ) and cleave DRGs in scald/burn infected wounds to block the spread of drug resistance and accelerate wound healing. This bioactive system constructed from natural living materials offers a promising means for blocking the spread of drug resistance., (© 2024 Wiley‐VCH GmbH.)

Surface-associated microbial systems are hotspots for the spread of plasmid-encoded antibiotic resistance, but how surface association affects plasmid transfer and proliferation remains unclear. Surface association enables prolonged spatial proximities between different populations, which promotes plasmid transfer between them. However, surface association also fosters strong metabolic interactions between different populations, which can direct their spatial self-organization with consequences for plasmid transfer and proliferation. Here, we hypothesize that metabolic interactions direct the spatial self-organization of different populations and, in turn, regulate the spread of plasmid-encoded antibiotic resistance. We show that resource competition causes populations to spatially segregate, which represses plasmid transfer. In contrast, resource cross-feeding causes populations to spatially intermix, which promotes plasmid transfer. We further show that the spatial positionings that emerge from metabolic interactions determine the proliferation of plasmid recipients. Our results demonstrate that metabolic interactions are important regulators of both the transfer and proliferation of plasmid-encoded antibiotic resistance., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In this issue of Cell Reports, Ma et al. 1 identify causative regulatory links between self-organization in surface-attached bacterial colonies and the rate of horizontal gene transfers (conjugations) and subsequent selection of the newly arising population of recipient bacteria (transconjugants)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

DdmDE is a novel plasmid defense system that was discovered in the seventh pandemic Vibrio cholerae strain of the biotype O1 EI Tor. In this issue of Cell, Yang and coworkers reveal the mechanisms underlying the assembly and activation of the DdmDE defense system., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgos) and the DNA defense module DdmDE system. Through biochemical analysis, structural determination, and in vivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study investigates the synthesis and optimization of nanobots (NBs) loaded with pDNA using the layer-by-layer (LBL) method and explores the impact of their collective motion on the transfection efficiency. NBs consist of biocompatible and biodegradable poly(lactic- co -glycolic acid) (PLGA) nanoparticles and are powered by the urease enzyme, enabling autonomous movement and collective swarming behavior. In vitro experiments were conducted to validate the delivery efficiency of fluorescently labeled NBs, using two-dimensional (2D) and three-dimensional (3D) cell models: murine urothelial carcinoma cell line (MB49) and spheroids from human urothelial bladder cancer cells (RT4). Swarms of pDNA-loaded NBs showed enhancements of 2.2- to 2.6-fold in delivery efficiency and 6.8- to 8.1-fold in material delivered compared to inhibited particles (inhibited enzyme) and the absence of fuel in a 2D cell culture. Additionally, efficient intracellular delivery of pDNA was demonstrated in both cell models by quantifying and visualizing the expression of eGFP. Swarms of NBs exhibited a >5-fold enhancement in transfection efficiency compared to the absence of fuel in a 2D culture, even surpassing the Lipofectamine 3000 commercial transfection agent (cationic lipid-mediated transfection). Swarms also demonstrated up to a 3.2-fold enhancement in the amount of material delivered in 3D spheroids compared to the absence of fuel. The successful transfection of 2D and 3D cell cultures using swarms of LBL PLGA NBs holds great potential for nucleic acid delivery in the context of bladder treatments.

Bacterial conjugation is a process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It is the main means by which antibiotic resistance genes spread among bacterial populations. It is crucially dependent upon the elaboration of an extracellular appendage, termed "pilus," by a large double-membrane-spanning secretion system termed conjugative "type IV secretion system." Here we present the structure of the conjugative pilus encoded by the R388 plasmid. We demonstrate that, as opposed to all conjugative pili produced so far for cryoelectron microscopy (cryo-EM) structure determination, the conjugative pilus encoded by the R388 plasmid is greatly stimulated by the presence of recipient cells. Comparison of its cryo-EM structure with existing conjugative pilus structures highlights a number of important differences between the R388 pilus structure and that of its homologs, the most prominent being the highly distinctive conformation of its bound lipid., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Synonymous codons were originally viewed as interchangeable, with no phenotypic consequences. However, substantial evidence has now demonstrated that synonymous substitutions can perturb a variety of gene expression and protein homeostasis mechanisms, including translational efficiency, translational fidelity, and cotranslational folding of the encoded protein. To date, most studies of synonymous codon-derived perturbations have focused on effects within a single gene. Here, we show that synonymous codon substitutions made far within the coding sequence of Escherichia coli plasmid-encoded chloramphenicol acetyltransferase ( cat ) can significantly increase expression of the divergent upstream tetracycline resistance gene, tetR . In four out of nine synonymously recoded cat sequences tested, expression of the upstream tetR gene was significantly elevated due to transcription of a long antisense RNA (asRNA) originating from a transcription start site within cat . Surprisingly, transcription of this asRNA readily bypassed the native tet transcriptional repression mechanism. Even more surprisingly, accumulation of the TetR protein correlated with the level of asRNA, rather than total tetR RNA. These effects of synonymous codon substitutions on transcription and translation of a neighboring gene suggest that synonymous codon usage in bacteria may be under selection to both preserve the amino acid sequence of the encoded gene and avoid DNA sequence elements that can significantly perturb expression of neighboring genes. Avoiding such sequences may be especially important in plasmids and prokaryotic genomes, where genes and regulatory elements are often densely packed. Similar considerations may apply to the design of genetic circuits for synthetic biology applications., Competing Interests: Competing interests statement:The authors declare no competing interest.

Cell communication coordinates developmental processes, maintains homeostasis, and contributes to disease. Therefore, understanding the relationship between cells in a shared environment is crucial. Here we introduce Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a cell neighbour-labelling system based upon secretion and uptake of positively supercharged fluorescent protein s36GFP. We fused s36GFP to mNeonGreen or to a HaloTag, facilitating ultra-bright, sensitive, colour-of-choice labelling. Secretor cells transfer PUFFFIN to neighbours while retaining nuclear mCherry, making identification, isolation, and investigation of live neighbours straightforward. PUFFFIN can be delivered to cells, tissues, or embryos on a customisable single-plasmid construct composed of interchangeable components with the option to incorporate any transgene. This versatility enables the manipulation of cell properties, while simultaneously labelling surrounding cells, in cell culture or in vivo. We use PUFFFIN to ask whether pluripotent cells adjust the pace of differentiation to synchronise with their neighbours during exit from naïve pluripotency. PUFFFIN offers a simple, sensitive, customisable approach to profile non-cell-autonomous responses to natural or induced changes in cell identity or behaviour., (© 2024. The Author(s).)

Gene overexpression by transient transfection of in vitro cultured model cell lines with plasmid DNA is a commonly used method for studying molecular aspects of human biology and pathobiology. However, there is accumulating evidence suggesting that human cells may actively secrete fragments of DNA and the implications of this phenomenon for in vitro cultured cells transiently transfected with foreign nucleic acids has been overlooked. Therefore, in the current study we investigated whether a cell-to-cell transmission of acquired plasmid DNA takes place in a commonly used human cell line model. We transiently transfected HEK293 cells with EGFP encoding plasmids to serve as donor cells and either co-cultured these with stably mCherry expressing recipient cells in different set-ups or transferred their culture medium to the recipient cells. We found that recipient cells produced EGFP after being co-cultured with donor cells but not when they were exposed to their culture medium. The employment of different co-culture set-ups excluded that the observed effect stemmed from technical artefacts and provided evidence that an intercellular plasmid transfer takes place requiring physical proximity between living cells. This phenomenon could represent a significant biological artefact for certain studies such as those addressing protein transmissions in prion diseases., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier Inc.)

Core-shell tecto dendrimers (CSTDs) with excellent physicochemical properties and good tumor penetration and gene transfection efficiency have been demonstrated to have the potential to replace high-generation dendrimers in biomedical applications. However, their characterization and related biological properties of CSTDs for enhanced tumor penetration and gene delivery still lack in-depth investigation. Herein, three types of dual-responsive CSTDs are designed for thorough physicochemical characterization and investigation of their tumor penetration and gene delivery efficiency. Three types of CSTDs are prepared through phenylborate ester bonds of phenylboronic acid (PBA)-decorated generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers as cores and monose (galactose, glucose, or mannose)-conjugated G3 PAMAM dendrimers as shells and thoroughly characterized via NMR and other techniques. It is shown that the produced CSTDs display strong correlation signals between the PBA and monose protons, similar hydrodynamic diameters, and dual reactive oxygen species- and pH-responsivenesses. The dual-responsive CSTDs are proven to have structure-dependent tumor penetration property and gene delivery efficiency in terms of small interference RNA for gene silencing and plasmid DNA for gene editing, thus revealing a great potential for different biomedical applications., (© 2024 Wiley‐VCH GmbH.)