Conjugative plasmids in combination with translocatable elements play a key role in the spread of antibiotic resistance genes. The IncZ plasmid pIE545 is one of the group of plasmids that have been used since the 1980s to examine and compare the properties of conjugative plasmids in the I-complex and is the only member of the group for which a complete sequence was not available. pIE545 was found in a Klebsiella pneumoniae in Germany in the 1970s and was known to confer resistance to kanamycin, neomycin and sulphonamides. Here, the complete pIE545 sequence was determined and its structure examined. The plasmid is 109,697 bp, made up of an 86.7-kb backbone that includes determinants for replication, maintenance and conjugation functions, together with 23 kb of accessory regions. A 10.2-kb integrative element, IE-545, that interrupts the backbone impB gene is itself interrupted by an IS102-bounded transposon, TnaphA1-pIE545, that includes the aphA1 kanamycin/neomycin resistance gene and an ISEc52-like element. The sulphonamide resistance gene sul2 is in a fragment of GIsul2 inserted between nikB and trbC. An IS2 interrupts pilL, and a second copy of ISEc52-like is in one of the two additional alternate ends for the pilV gene found in the shufflon. The pIE545 shufflon is found in other backbones that contain a Z-type replicon but is distinct from the shufflons of I1- and I2-type plasmids. The uninterrupted version of IE-545 is found in both plasmids and chromosomes interrupting impB or umuC genes.
Carbapenem-resistant Klebsiella pneumoniae ST11 is a high-risk lineage predominantly associated with healthcare settings. Here, we report the phenotypic and genomic characterization of a KPC-producing K. pneumoniae ST11 isolate recovered from a free-living, yellow-chevroned parakeet (Brotogeris chiriri) in Brazil. Antimicrobial susceptibility testing revealed resistance to multiple drug classes, including carbapenems. Whole-genome sequencing using a hybrid short- and long-read approach resolved a 5.5 Mb chromosome and five plasmids belonging to the IncFIB/HI1B, IncFIB/FII, IncN15, IncX3, and ColRNAI groups. The blaKPC-2 gene was located within a Tn4401-like element on an IncN15 plasmid, while additional resistance determinants were embedded in mosaic Tn402-derived regions and class 1 integrons. The genome displayed a complex mobilome comprising insertion sequences, transposon derivatives, and nine intact chromosomal prophages. Multiple heavy-metal tolerance operons and efflux systems were also identified. In vivo infection assays using Galleria mellonella demonstrated increased virulence compared with a reference strain. These findings document the occurrence of a clinically relevant carbapenem-resistant K. pneumoniae ST11 isolate in a free-living bird and support the consideration of wildlife-associated samples in integrated One Health surveillance of high-risk antimicrobial-resistant clones.
T vectors are core tools for TA cloning and are widely used for the direct cloning of PCR products. In the present study, a novel versatile T vector, designated pZBT01C, was developed based on a modified lysis gene E (mE) from bacteriophage PhiX174, which allows only positive tranformants to grow. Ten unique cloning sites were introduced into the mE gene, flanked by two XcmI sites. Universal M13-48/M13-47 primer binding sites were incorporated 35 bp upstream and downstream of the TA cloning site, and the enhancer T7g10 and Shine-Dalgarno box were placed upstream to stabilize mE expression. With a compact size of 3029 bp, pZBT01C is prepared by XcmI digestion to generate 3'-T overhangs. At the induction temperature, the plasmid pZBT01C exhibited strong host-killing activity. In cloning tests, all randomly selected 20 colonies were confirmed to be positive by colony PCR. The potential of pZBT01C as an expression system was further demonstrated by temperature-inducible expression of the reporter gene GFP. Owing to its near-zero-background performance and versatile design, pZBT01C is expected to have broad application prospects in PCR product cloning and related molecular biology applications.
Antibiotic resistance genes associated with IS26-family mobile genetic elements are globally distributed in the Gram-negative plasmidome. In many cases, antibiotic resistance genes are co-located with genes of unknown function. Two particular IS26-associated gene clusters, mph(A)-mrx-mphR(A) and cysH-eamA-tet(A)-tetR(A), are prevalent on plasmids and chromosomes of common Gram-negative pathogens. While mph(A) encodes a macrolide phosphotransferase and tet(A) encodes a tetracycline efflux pump, the roles, if any, of mrx, cysH, and eamA remain poorly understood. To test the hypothesis that these genes consistently co-located with mph(A) and tet(A) act cooperatively to enhance antibiotic resistance, recombinant E. coli strains carrying various genetic combinations from each cluster were subjected to antimicrobial susceptibility testing. While the mph(A) gene alone provided minimal protection from azithromycin, inclusion of mrx increased resistance by at least 48-fold. Disruption of mrx by the insertion of a nonsense mutation or the addition of an efflux pump inhibitor abrogated this effect, indicating that the Mrx protein is a drug exporter that is required for maximal effect. In the absence of mph(A), mrx had no effect on azithromycin resistance, suggesting that Mrx only effluxes the phosphorylated form of azithromycin. Similarly, inclusion of cysH-eamA with tet(A) increased resistance to tetracycline antibiotics by at least 2-fold over tet(A) alone. Disruption of either cysH or eamA had no effect on this enhancement, but disruption of both genes eliminated it entirely. A more complete understanding of antibiotic resistance among Gram-negative bacteria requires elucidation of the roles of modulator genes co-located with primary resistance genes on mobile genetic elements.
The accurate determination of protein-protein interactions (PPIs) within living cells is essential for our understanding of cellular processes. Molecular interactions can be determined through complementation systems, in which the spatial proximity of two molecular fragments reconstitutes their activity. Among these, NanoLuc Binary Technology (NanoBiT®) utilizes the functional complementation of a large (LgBiT) and a small (SmBiT) luciferase fragment, but its reliability and biological interpretability depends critically on balanced expression of the interacting proteins. To ensure a highly reproducible and finely controlled expression of the interaction partners, we developed a novel plasmid system allowing stable and biallelic integration of NanoLuc plasmids into the safe harbor locus on human chromosome 19. To avoid overexpression artifacts the expression of both interaction partners is precisely regulated by the Tet-On system. We used this inducible binary technology (iBiT) system to investigate a largely unexplored interaction between the full-length isoform 1 and the N-terminally truncated isoform 2 of p62/Sequestosome 1. These experiments demonstrate that cell lines stably expressing iBiT constructs enable highly reproducible, longitudinal, and time-resolved analyses of protein-protein interactions, providing a robust platform for assessing both protein-protein interaction dynamics and their pharmacological modulation in intact cells.
Plasmids play a critical role in bacterial evolution and represent major drivers of the emergence and dissemination of antimicrobial resistance. As primary mobile genetic elements (MGEs), plasmids facilitate the horizontal transfer of resistance determinants alongside genes associated with virulence, metabolic functions, and broader adaptive advantages. Recent studies have further highlighted the importance of conjugative plasmids, such as IncI1-like elements, in mediating the spread of extended-spectrum β-lactamase (ESBL) genes and other clinically relevant traits across diverse bacterial populations. Whether the recurrent detection of these plasmids is coincidental or reflects unique genetic features that enhance their capacity for transmission remains an important question in microbial genomics. In this context, the present study analyses complete genome sequences and whole-genome maps of Escherichia coli O157:H7 strains to characterize their antimicrobial resistance genes, virulence-associated loci, prophage content, and plasmid profiles. Publicly available sequences from the NCBI GenBank repository were examined using comparative genomic tools, including BRIG, VirulenceFinder, ResFinder, PlasmidFinder, and PHASTEST. This work also underscores the limited availability of whole-genome data for E. coli O157:H7 and O157:H7NM in developing regions, particularly within African countries, highlighting the need for expanded genomic surveillance. Comparative analyses revealed that most strains displayed high genomic similarity to the reference Sakai strain, with relatively few missing regions, although a subset exhibited reduced homology marked by numerous gaps. Prophages, bacteriophages integrated into the bacterial genome, were found to contribute substantially to genomic diversity, influencing virulence potential, antimicrobial resistance, and patterns of horizontal gene transfer. These findings emphasize the complex role of mobile genetic elements in shaping the evolution of E. coli O157:H7 and reinforce the importance of continued genomic sequencing to further elucidate the pathogen's diversity and adaptive mechanisms.
Vibrio (V.) parahaemolyticus is a marine-associated bacterium that has previously been linked to foodborne illness associated with seafood consumption. Various plasmids harbouring antimicrobial resistance and virulence genes have been described for V. parahaemolyticus. By whole genome sequencing, we found two V. parahaemolyticus strains harbouring a large additional circular genomic element of 0.882 Mbp. NCBI database search revealed that this element represents a rare but globally distributed megaplasmid detected in four additional Vibrio strains spanning distinct species (V. parahaemolyticus, V. cholerae and V. vulnificus), geographical origins, and hosts. In addition to the two megaplasmid-harbouring V. parahaemolyticus strains from our study, we also identified two megaplasmid-free isogenic strains in our strain collection, confirming that the megaplasmid is indeed a plasmid encoding non-obligatory functional traits. The divergent GC content and codon usage of the megaplasmid suggest a non-Vibrio origin. The genetic diversity found in the six investigated megaplasmid sequences indicates adaptation within the different Vibrio hosts. Although many of the megaplasmid genes could not be categorised through Cluster of Orthologous Genes (COG) classification, genes coding for partitioning systems, type IV secretion systems, defence systems, and toxin-antitoxin modules were identified. These contribute to a high plasmid stability, as we demonstrated by the curing experiments performed with our strains. Consequently, the megaplasmid described here represents a highly stable but adaptive genetic element that is characterised by as yet unexplored genetic traits.
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.
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 (KD values <10 nM). The resulting RBD could be an ideal target for designing potent COVID-19 antivirals.
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 6iii 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.
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.
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.
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.
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.
