The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a significant risk to global health, warranting the formulation of efficient preventive and therapeutic measures to tackle its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein of coronaviruses plays a pivotal role in viral attachment and entry into host cells. The receptor-binding domain (RBD) of the SARS-CoV-2 S protein has demonstrated a robust binding affinity to ACE2 receptors in humans. Consequently, it has become a prime target for therapeutic interventions using antibodies, vaccines, or other designed inhibitors. We engineered an RBD sequence with refined ORF boundaries guided by structural insights, which enabled efficient in vitro refolding. This highlights the critical role of precise expression cassette design in a plasmid, extending beyond conventional parameters such as promoter or fusion tag selection. Using customized refolding procedures, we obtained 10-12 mg of active protein from a one-liter LB culture. The biological activity of the refolded RBD was confirmed by monitoring its interaction with the designed LCB1 miniprotein ligand by surface plasmon resonance, wherein they exhibited significant affinity levels as reflected by their dissociation constants (K values <10 nM). The resulting RBD could be an ideal target for designing potent COVID-19 antivirals.

Little is known about the role of mobile genetic elements in natural ecosystems such as the infant gut microbiome. Here, we conduct the most comprehensive longitudinal study of the infant plasmidome to date by analyzing monthly fecal samples from 12 infants from birth to one year of age. We employ an assembly-based bioinformatic pipeline for the reconstruction and identification of full-length plasmids, including a novel approach for assigning putative plasmid hosts. We then investigated plasmid content and dynamics in the infant gut microbiome. After assembly and identification, we identified 620 unique circular plasmids in the infant cohort, including a number of novel sequences. Independent assembly of the same plasmids in several samples and infants helped corroborate the authenticity of the plasmids. Among the observed plasmids was the recently described ubiquitous and abundant Bacteroides plasmid pBI143. Overall, the genus Bacteroides had the highest plasmid carriage, while the highest plasmid diversity was observed in Clostridium, including 5 previously unknown widespread plasmids. Lastly, we leveraged the longitudinal nature of our dataset to investigate contemporaneous correlations between temporal variations in plasmid abundances and species dynamics. This enabled us to link co-residing plasmids and tightly linked plasmid-taxon pairs within each infant. These insights into plasmid ecology help us understand determinants driving plasmid distribution in complex microbial communities.

Microbial genomes are continuously being rearranged by mobile genetic elements (MGEs), leading to configurations that may confer novel phenotypic traits such as antibiotic resistance, degradation of compounds, or metabolic features. Standard genomic sequencing provides a snapshot of a genome in one configuration, but this static image does not give insight into the dynamics of genomic evolution and whether MGEs are actively changing a genome. We applied single-strain mobilome sequencing to Escherichia coli K-12 MG1655 under various stress conditions: UV, SDS, nalidixic acid, tetracycline, cetrimide, and copper. Under these conditions, we quantified the activity of a range of genetic elements, including extrachromosomal circular DNA (eccDNA) from IS elements, RNA genes, the UV-inducible e14 prophage, and intergenic repetitive sites (REP). Of the stressors, copper and SDS are among the largest inducers of eccDNA formation from some IS elements, while elevated levels of hypothetical RNA/DNA heteroduplexes of ribosomal and transfer RNAs, and Rhs-nuclease proteins are induced under various stressors, especially copper and SDS. This approach holds promise for quantifying the genetic response to environmental stress and implications for genome plasticity. The mobilization of IS elements upon copper and other stressors helps to explain co-selection of heavy metals with antibiotic resistance genes and MGEs.

Replicon typing identifies sequences similar to known DNA replication initiators and is widely used to detect specific plasmid groups (e.g., IncP-1) in genome and metagenome sequencing data. However, the characteristics of these homologous sequences in public databases have not been systematically assessed, making it difficult to determine whether detecting a specific replicon type reliably indicates the presence of a particular plasmid group. Here, we conducted amino acid sequence alignments to identify sequences similar to the replication initiation protein TrfA of the IncP-1 plasmid RK2 in the NCBI non-redundant (nr) database. In the nr nucleotide database, TrfA-matched nucleotide sequences were found across diverse taxonomic groups and replicons, including complete and partial plasmids and chromosomes. In total, 76 protein sequences from the reference plasmid RK2 were screened against the nucleotide sequences of the trfA-harboring plasmids to identify candidate IncP-1 plasmids. TrfA-related proteins, originating from bacterial chromosomes, plasmids, and phages, were selected from the nr amino acid database and used to infer phylogenetic trees. Our phylogenetic analyses reveal that TrfA homologs have diverged through vertical inheritance within IncP-1 and horizontal gene transfer across replicons and taxa. These findings caution against overreliance on single-gene replicon typing to infer plasmid group identity from sequence data.

Bacterial adaptation to environmental stress involves stringent regulation of DNA replication. While the mechanisms controlling chromosomal replication under adverse conditions, such as amino acid starvation, are relatively well characterized, the molecular basis for stress-induced inhibition of plasmid replication remains largely unknown. In this study, we investigated how amino acid starvation affects the replication of the broad-host-range RK2 plasmid in Escherichia coli, focusing on the plasmid-encoded replication initiator TrfA and host-encoded initiator DnaA. We found that the RK2 plasmid origin of replication (oriV) occupation by TrfA and DnaA is prevented in stress conditions. We also did not detect increase of plasmid DNA level showing that new rounds of RK2 replication are not initiated. The replication-inactive state persisted even in cells expressing a hyperactive monomeric TrfA variant that is incapable of handcuffing, indicating that other regulatory mechanisms, beyond handcuffing, contribute to the prevention of the plasmid replication. The reduction of initiators binding to the plasmid origin during stress coincided with a substantial decrease in the intracellular levels of TrfA, as shown in this study, and of DnaA, as reported previously. Given that cell division is arrested during stress, the only explanation for the observed gradual decrease of TrfA levels is proteolysis. Our findings demonstrate that during amino acids starvation, RK2 plasmid replication in E. coli is likely prevented by a significant drop in initiator proteins concentrations. This uncovers a previously underappreciated layer of plasmid replication control under stress conditions.

Conjugative plasmids are key drivers of horizontal gene transfer and the spread of antimicrobial resistance. Their successful establishment in new hosts requires overcoming diverse bacterial defence mechanisms, such as restriction-modification systems, CRISPR-Cas systems, and the SOS response. Plasmids achieve this through a leading region-encoded zygotic program of anti-defence genes expressed early in conjugation. This program employs diverse strategies, including single-stranded promoters, repressed double-stranded promoters, and protein translocation. This review explores the diversity of these zygotic programs, the mechanisms underlying their timely regulation, and the array of anti-defence functions they encode. Further investigation of leading region genes is crucial for discovering novel counter-defence strategies and understanding their tailored regulation across diverse plasmid and bacterial species, ultimately enabling us to better understand and potentially manipulate plasmid transfer.

Members of the marine bacterial genus Tenacibaculum cause disease in finfish and outbreaks result in significant animal harm and losses in aquaculture around the globe. Plasmids have not been previously identified in Tenacibaculum, but long-read DNA sequencing of genomes from disease-associated Tenacibaculum isolates collected between 2017 and 2020 in British Columbia, Canada, revealed circular putative plasmids in three Tenacibaculum species. In addition to high-quality circular assembly, the putative plasmids contained genes encoding plasmid replication, mobility, and partitioning proteins. Genes for type B conjugation machinery and type 6 secretion system components were also identified on each of the two largest plasmid sequences. Several protocols were tested to visualize and enrich Tenacibaculum plasmid DNA. Rolling-circle replication with Phi29 DNA polymerase amplified putative plasmids smaller than 100 kb. Alkaline lysis extraction provided weak enrichment of putative plasmid DNA, but plasmids could not be confidently resolved by Eckhardt extraction and electrophoresis in agarose gels. The newly assembled plasmids matched previously sequenced Tenacibaculum contigs, suggesting that publicly available Tenacibaculum genomes contain unrecognized plasmids. The discovery of putative plasmids in Tenacibaculum is significant because plasmids often confer important functions to host cells and serve as vehicles for horizontal gene transfer within and beyond the host bacterial species.

We previously reported the development of a Cre/lox-based gene disruption system for multiple markerless gene disruption in Thermus thermophilus; however, it was a time-consuming method because it functioned at 50 °C, the minimum growth temperature of T. thermophilus HB27. In the present study, we improved this system by introducing random mutations into the cre-expressing plasmid, pSH-Cre. One of the resulting mutant plasmids, pSH-CreFM allowed us to remove selection marker genes by Cre-mediated recombination at temperatures up to 70 °C. By using the thermostable Cre/lox system with pSH-CreFM, we successfully constructed two valuable pTT27 megaplasmid mutant strains, a plasmid-free strain and β-galactosidase gene deletion strain, which were produced by different methods. The thermostable Cre/lox system improved the time-consuming nature of the original Cre/lox system, but it was not suitable for multiple markerless gene disruption in T. thermophilus because of its highly efficient induction of Cre-mediated recombination even at 70 °C. However, in vivo megaplasmid manipulations performed at 65 °C were faster and easier than with the original Cre/lox system. Collectively, these results indicate that this system is a powerful tool for engineering T. thermophilus megaplasmids.

The plasmid pAnox1, isolated from Anoxybacillus gonensis 05S15, was sequenced and characterized as a circular, double-stranded DNA molecule of 1592 base pairs with a GC content of 40.01 %. Despite its cryptic nature and small genome, bioinformatic analyses identified conserved motifs associated with replication-related proteins, though BLAST searches revealed no significant homology with other plasmids. The plasmid genome contains five putative Open Reading Frames (ORFs), four palindromic sequences, and two direct repeats on both strands, suggesting regulatory roles. Electron microscopy and Southern hybridization studies confirmed that pAnox1 follows a Rolling Circle (RC) replication mode. The study further demonstrated that the plasmid encodes three distinct transcripts: ORF-1 and ORF-3 are oriented in the same direction, while ORF-5 is on the opposite strand. RACE and LACE analyses revealed transcript lengths of 903 bp for ORF1, 499 bp for ORF3, and 211 bp for ORF5. Quantitative real-time PCR estimated the relative copy number of pAnox1 at 127 ± 2 copies per chromosomal equivalent. This novel RC-type plasmid in the Anoxybacillus genome holds promise as a cloning and expression vector for biotechnological applications and in vivo protein engineering.

Bacteria, the primary microorganisms used for industrial molecule production, do not naturally generate miRNAs. This study aims to systematically review current literature on miRNA expression systems in bacteria and address three key questions: (1) Which microorganism is most efficient for heterologous miRNA production? (2) What essential elements should be included in a plasmid construction to optimize miRNA expression? (3) Which commercial plasmid is most used for miRNA expression? Initially, 832 studies were identified across three databases, with fifteen included in this review. Three species-Escherichia coli, Salmonella typhimurium, and Rhodovulum sulfidophilum-were found as host organisms for recombinant miRNA expression. A total of 78 miRNAs were identified, out of which 75 were produced in E. coli, one in R. sulfidophilum (miR-29b), and two in S. typhimurium (mi-INHA and miRNA CCL22). Among gram-negative bacteria, R. sulfidophilum emerged as an efficient platform for heterologous production, thanks to features like nucleic acid secretion, RNase non-secretion, and seawater cultivation capability. However, E. coli remains the widely recognized model for large-scale miRNA production in biotechnology market. Regarding plasmids for miRNA expression in bacteria, those with an lpp promoter and multiple cloning sites appear to be the most suitable due to their robust expression cassette. The reengineering of recombinant constructs holds potential, as improvements in construct characteristics maximize the expression of desired molecules. The utilization of recombinant bacteria as platforms for producing new molecules is a widely used approach, with a focus on miRNAs expression for therapeutic contexts.

Plasmids can impact the evolution of their hosts, e.g. due to carriage of mutagenic genes, through cross-talk with host genes or as result of SOS induction during transfer. Here we demonstrate that plasmids can affect the level of microindel mutations in the host genome. These mutations are driven by the production of single-stranded DNA molecules that invade replication forks at microhomologies and subsequently get integrated into the genome. Using the gammaproteobacterial model organism Acinetobacter baylyi, we show that carriage of broad host range plasmids from different incompatibility groups can cause microindel mutations directly or indirectly. The plasmid vector pQLICE belonging to the incompatibility group Q (IncQ) and replicating by a characteristic strand displacement mechanism can generate chromosomal microindel mutations directly with short stretches of DNA originating from pQLICE. In addition, results with the IncP plasmid vector pRK415 (theta replication mechanism) show that the presence of plasmids can increase microindel mutation frequencies indirectly (i.e., with chromosomal ectopic DNA), presumably through plasmid-chromosome interactions that lead to DNA damages. These results provide new mechanistic insights into the microindel mutation mechanism, suggesting that single-stranded DNA repair intermediates are the causing agents. By contrast, the IncN plasmid RN3 appears to suppress host microindel mutations. The suppression mechanism remains unknown. Other plasmids in this study (belonging to IncA/C2, IncW, pBBR incompatibility groups) confer ambiguous or no quantifiable mutagenic effects.

In this work, we report the construction of four bacterial luciferase-based promoter probe vectors with an expanded set of selectable markers, designed to facilitate their use in antibiotic-resistant bacteria. These vectors contain the low-copy-number, broad-host-range pBBR origin of replication and an origin of transfer, allowing efficient conjugative transformation into various bacterial genera. The broad host range origin also enables their use in bacterial strains that harbor other plasmids, as the pBBR origin is compatible with a wide variety of other plasmid replication systems. The utility of these vectors was demonstrated by quantifying capsule gene expression in both classical and hypervirulent Klebsiella pneumoniae strains lacking tolC, which encodes the outer membrane pore protein for tripartite transport systems. Our results revealed that the tolC mutation reduced capsule gene expression, highlighting a critical role for tolC in K. pneumoniae pathobiology and the utility of bioluminescence for studying gene expression in real time. These new vectors provide a flexible platform for circumventing antibiotic resistance phenotypes and studying gene expression across diverse bacterial species, including strains containing additional plasmids.

SGI1 and its many variant forms are integrative mobilizable elements that rely on IncA or IncC plasmids for transfer functions. However, the coexistence of SGI1 with the plasmid is unstable in the longer term. Here, we have investigated the effect of SGI1 type integrative elements on the initial entry of these plasmids. Using two transfer proficient IncC plasmids and the IncA plasmid RA1, exclusion indices were 40-100-fold for SGI1-I or SGI1-D which have a complete backbone. Using the SGI1-K and SGI1-LK1 variants that lack backbone segments, loss of a region of 5793 bp that includes the traHG transfer genes and the downstream open reading frame S010 was found to abolish exclusion. S010 was shown to be co-transcribed with traHG and hence also under the control of an AcaDC inducible promoter. However, complementation with a 5.2 kbp fragment that included the traHG-S010 operon did not restore exclusion activity to SGI1-LK1. Part of S013 that encodes a small polypeptide of unknown function, was also lost from SGI1-LK1. S013 and the adjacent S014 gene were also co-transcribed. However complementation with S013-S014 did not restore exclusion activity to SGI1-LK1. Hence, the precise cause of the SGI1-mediated plasmid exclusion remains elusive.

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.

The length of a plasmid is a key property which is linked to many aspects of plasmid biology. When distributions of plasmid lengths are shown in the literature, they are usually plotted with length on a logarithmic scale. However, a quantity and its logarithm have distinct distributions which may differ considerably in shape. Mistaking the distribution of log-lengths for the distribution of lengths can therefore lead to distorted conclusions about the distribution; in particular, the distribution of log-lengths may be bimodal when the distribution of lengths is only unimodal. This particular confusion has arisen in the literature where the length distribution is often claimed to be bimodal based on examination of what is in fact the log-length distribution. While the length distribution is indeed bimodal within many bacterial families, it is not across the ensemble of all plasmids. We suggest that authors should be careful to show the plasmid length distribution, or to distinguish the two distributions, to avoid misleading inferences.

The predominant type of plasmids found in Acinetobacter species encode a Rep_3 initiation protein and many of these carry their accessory genes in dif modules. Here, available sequences of the 14 members of the group of Rep_3 plasmids typed as R3-T33, using a threshold of 95% identity in the repA gene, were compiled and compared. These plasmids were from various Acinetobacter species. The pdif sites were identified allowing the backbone and dif modules to be defined. As for other Rep_3 plasmids carrying dif modules, orfX encoding a protein of unknown function was found downstream of repA followed by a pdif site in the orientation XerC binding site-spacer-XerD binding site. Most backbones (n = 12) also included mobA and mobC genes but the two plasmids with the most diverged repA and orfX genes had different backbone contents. Although the gene content of the plasmid backbone was largely conserved, extensive recombinational exchange was detected and only two small groups carried identical or nearly identical backbones. Individual plasmids were associated with 1 to 13 dif modules. Many different dif modules were identified, including ones containing antibiotic or chromate resistance genes and several toxin/antitoxin gene pairs. In some cases, modules carrying the same genes were significantly diverged. Generally, the orientation of the pdif sites alternated such that C modules (XerC binding sites internal) alternated with D modules (XerD binding sites internal). However, fusions of two dif modules via mutational inactivation or loss of a pdif site were also detected.

Plasmids found in Acinetobacter species are not found in other Gram-negative species. There are many distinct plasmid types and the majority encode a Rep_3 family RepA replication initiation protein. Among these, a number were known to carry dif modules. Here, the representative plasmid for each of the 78 reported R3 types was examined to identity features of the plasmid backbone that are associated with the carriage of dif modules. A conserved open reading frame designated orfX (IPR047783) was found downstream of repA in 35 of them, and the backbones of those 35 plasmids were bounded by recombination sites recognised by XerC and XerD, known as pdif sites. These plasmid backbones are all equivalent to a C-type dif module as the pdif sites are in the orientation D/C at one end and C/D at the other end, i.e. the XerC binding sites are internal. Hence, to provide the XerD binding sites and generate a complete plasmid at least one D-type dif module is needed. Phylogenies of the RepA and OrfX proteins revealed that plasmids with closely-related RepA proteins are not always associated with closely-related OrfX proteins and vice-versa indicating extensive backbone recombination. Folded structures of diverse OrfX proteins predicted using AlphaFold 3 revealed an N-terminal HTH domain followed by a long α-helix that is predicted to promote dimerization and a disordered C-terminus. Given the correlation between the presence of orfX and one or more dif modules, the possibility that OrfX is involved in dif module movement deserves to be investigated.

The majority of large multiresistance plasmids of Staphylococcus aureus utilise a RepA_N-type replication initiation protein, the expression of which is regulated by a small antisense RNA (RNAI) that overlaps the rep mRNA leader. The pSK41/pGO1-family of conjugative plasmids additionally possess a small (86 codon) divergently transcribed ORF (orf86) located upstream of the rep locus. The product of pSK41 orf86 was predicted to have a helix-turn-helix motif suggestive of a likely function in transcriptional repression. In this study, we investigated the effect of Orf86 on transcription of thirteen pSK41 backbone promoters. We found that Orf86 only repressed transcription from the rep promoter, and hence now redesignate the product as Cop. Over-expression of Cop in trans reduced the copy number of pSK41 mini-replicons, both in the presence and absence of rnaI. in vitro protein-DNA binding experiments with purified 6 × His-Cop demonstrated specific DNA binding, adjacent to, and partially overlapping the -35 hexamer of the rep promoter. The crystal structure of Cop revealed a dimeric structure similar to other known transcriptional regulators. Cop mRNA was found to result from "read-through" transcription from the strong RNAI promoter that escapes the rnaI terminator. Thus, P is responsible for transcription of two distinct negative regulators of plasmid copy number; the antisense RNAI that primarily represses Rep translation, and Cop protein that can repress rep transcription. Deletion of cop in a native plasmid did not appear to impact copy number, indicating a cryptic auxiliary role.