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Transposable genetic elements and plasmid evolution.

Transposable elements of DNA that are structurally defined and genetically discrete units seem to have an important role in the evolution of bacterial plasmids. Recombination occurring at the termini of such elements can result in the joining together of unrelated DNA segments that lack extensive nucleotide sequence homology. In addition, transposable elements serve as novel biological switches capable of turning on and off the expression of nearby genes as a consequence of their insertion into or excision from plasmid genomes.

Alleles

Evolution of Pseudomonas R-plasmids: consequences of Tn1 insertion and resultant partial diploidy to chromosome and Tra- R-plasmid mobilization.

Tn1 transposes from pRO161, a Tra- derivative of RP1, to Pseudomonas aeruginosa sex factor FP2. The acquisition of Tn1 by FP2 results in its ability to mobilize pRO161 to other bacteria. Genetic evidence presented here suggests two sequential mechanisms. Initially, transposition of Tn1 results in trans-diploidy for the Tra+ and Tra- plasmids. This subsequently allows mobilization of the Tra- R-plasmid dependent on a host recombination mechanism. Transconjugants from this mating contain either stable cointegrate R-plasmids or aggregates resulting from dissociation of the cointegrates into a Tra+ and Tra- plasmid. These aggregates have lost at least part of Tn1 from their parent FP2:Tn1 component, but now they mobilize the tra- R-plasmid from a recombination-deficient (Rec-) genetic background as well as from Rec+ donor strains. Transconjugants from these retransfer matings are aggregates. These results suggest a contribution of transposons to R-plasmid evolution and dissemination beyond the mere acquisition of resistance to a given antibiotic.

Chromosomes, Bacterial

Translocatable elements in Staphylococcus aureus.

The properties of the first translocatable element in Gram-positive bacteria, a 5.2 kb segment encoding erythromycin resistance in S. aureus, are described. This element translocates from plasmid to multiple chromosomal sites and from chromosome to multiple plasmid sites, sometimes causing insertional inactivation and deletion. The genetic control of translocation and its role in natural plasmid evolution are discussed and preliminary evidence for translocation of penicillin and chloramphenicol resistance is presented. In the latter case, translocation involves in intact plasmid.

Chloramphenicol

Mesorhizobium bavaricum sp. nov. and Mesorhizobium monacense sp. nov., two novel Lotus-associated species harbouring symbiotic plasmids.

Legumes establish a mutualistic interaction with nitrogen-fixing rhizobia. Lotus japonicus is a model for studying this symbiosis; however, only a limited number of rhizobial species nodulating this host have been taxonomically described. Here, we characterise four Mesorhizobium strains (DC-1.1T, Qj1B1, DC-1.5T, and Qj2B2) isolated from root nodules of Lotus japonicus and Lotus burttii. Multi-locus phylogeny and phylogenomic analyses resolved these isolates into two well-supported monophyletic clades. Genome-based comparisons supported their classification as distinct taxa, with strains DC-1.1T and Qj1B1 showing 95.2% average nucleotide identity (ANI) and 62.9-63.5% digital DNA-DNA hybridisation (dDDH) values relative to Mesorhizobium newzealandense ICMP 19545T, whereas DC-1.5T and Qj2B2 exhibited 92.5-92.8% ANI and 49.9-50.5% dDDH compared with Mesorhizobium waimense ICMP 19557T. Together with chemotaxonomic and physiological traits, these data support the proposal of two novel species, Mesorhizobium bavaricum sp. nov. (DC-1.1T and Qj1B1) and Mesorhizobium monacense sp. nov. (DC-1.5T and Qj2B2). Metagenomic analyses predicted high environmental prevalence for these novel taxa, particularly within soil habitats. Isolates DC-1.1T, Qj1B1, and DC-1.5T effectively nodulated Lotus burttii and significantly promoted plant growth, whereas Qj2B2 neither nodulated nor enhanced growth. Comparative genomic analysis revealed that the nodulating isolates harbour symbiotic genes (nod, fix, and nif) on symbiotic plasmids, a rare feature in Mesorhizobium strains, whereas Qj2B2 lacks essential nod and nif genes. Consistent with these genomic features, symbiotaxonomic analysis assigned the nodulating isolates to symbiovar loti. These results highlight the potential of these isolates as models for comparative analyses of symbiotic plasmid evolution and horizontal gene transfer.

Mesorhizobium

Genomic characterization of blaIMP-harboring plasmids in Klebsiella spp.

UNLABELLED: The spread of carbapenem-resistant Klebsiella spp. poses a significant public health threat, partly due to the acquisition of the blaIMP genes, which encode IMP-type metallo-β-lactamases. These enzymes confer resistance to a broad spectrum of β-lactam antibiotics, including carbapenems, thereby complicating treatment options. This study aims to provide a comprehensive genomic characterization of blaIMP-harboring plasmids across different species within the genus Klebsiella, based on the genomic characteristics of the plasmid pT117-2 of Klebsiella variicola strain T117 isolated from clinical settings in China, along with all available blaIMP-harboring plasmids of Klebsiella spp. fromthe GenBank database until 26 April 2025. Among the 123 blaIMP-harboring plasmids of Klebsiella spp., nine variants were identified, with blaIMP-4 (carried by 69 plasmids) and blaIMP-1 (carried by 37 plasmids) being the most prevalent. The blaIMP-4 gene was associated with IncN type (~50 kb, conjugative) and untypeable (~300 kb, non-mobilizable) plasmids in China, whereas in Australia, it was linked to IncC (~200 kb) and IncM2 (~80 kb) type conjugative plasmids. Meanwhile, blaIMP-1 was found to be associated with IncN (~50 kb), IncM (~80 kb), and IncFII (80 ~200 kb) type conjugative plasmids mainly in Japan. Notably, our results highlight the prevalence of IncN-type conjugative plasmids, including the plasmid pT117-2 identified in this study, as key vehicles for the dissemination of blaIMP genes. This study provides critical insights into the genetic mechanisms of blaIMP-harboring plasmids persistence and spread in Klebsiella spp., advancing our understanding of their dissemination. IMPORTANCE: Carbapenem-resistant Enterobacterales (CRE) mediated by metallo-β-lactamases (MBLs) pose a major global public health threat that challenges clinical antimicrobial therapy; based on our study, blaIMP-4 in China is predominantly associated with IncN plasmids (forming the "IncN-blaIMP-4-qnrS1" axis), while blaIMP-1 in Japan links to IncN/IncM/IncFII plasmids, with these regional differences highlighting the need for geographically targeted surveillance, and notably, the high-risk ST146 Klebsiella variicola carrying blaIMP-4 on a conjugative IncN plasmid serves as an underrecognized reservoir for resistance genes, extending surveillance beyond common pathogenic Enterobacterales; limitations of this study include restricted sample size and geographic scope, and future research should validate these patterns via multi-center studies, explore plasmid evolution mechanisms, and integrate findings into routine surveillance to optimize antibiotic stewardship and infection control, thereby mitigating the global spread of MBL-mediated CRE.

Plasmids

Genomic characterization of KPC-2 and NDM coproducing carbapenem-resistant Klebsiella pneumoniae in a hospital: discovery of ST1869 clone and a novel hybrid plasmid.

UNLABELLED: To characterize the plasmid architecture and molecular background of KPC-NDM coproducing carbapenem-resistant Klebsiella pneumoniae (KN-CRKP) in a South China hospital. Five KN-CRKP isolates were collected, including three from one patient. All underwent Illumina sequencing; two (ST11 and ST1869) additionally had Nanopore sequencing. Antimicrobial susceptibility testing strain sequence types, conjugation assays, resistance gene profiling, plasmid typing, genetic structure comparison, core-genome single nucleotide polymorphisms (SNPs) analysis, and plasmid clustering were performed. All isolates exhibited an imipenem minimum inhibitory concentration (MIC) of ≥128 µg/mL and harbored multiple resistance genes. One isolate (1/5) belonged to ST1869 and co-harbored blaKPC-2 and blaNDM-5. The blaNDM-5-carrying plasmid was a novel IncI1/X3 fusion plasmid that also carried blaCMY-42. Unlike several IncX3 plasmids carrying blaNDM in publicly available KN-CRKP genomes from South China, this IncI1/X3 hybrid lacked a complete conjugative transfer system. ST11 was the predominant clone (4/5), co-harboring blaKPC-2 and blaNDM-1. A rare genetic structure, ΔISKpn6-blaKPC-2-ISKpn28, was identified on IncFII plasmids carrying blaKPC-2. Plasmid clustering analysis of 126 comparative KN-CRKP genomes showed diverse sequence types and plasmid backgrounds associated with the KPC/NDM co-production pattern. The observed plasmid diversity and structural variation in KN-CRKP support continued genomic surveillance, with particular attention to the ST1869 clone, the novel IncI1/X3 hybrid plasmid harboring blaNDM-5 and blaCMY-42, and the rare "ΔISKpn6-blaKPC-2-ISKpn28" genetic structure. Expanded genomic data on KN-CRKP are needed to further elucidate its resistance mechanisms and plasmid evolutionary trajectories. IMPORTANCE: The co-production of KPC and NDM carbapenemases in Klebsiella pneumoniae poses a formidable threat to clinical antimicrobial therapy, as these enzymes confer resistance to virtually all β-lactam agents, including carbapenems. Here, we report novel genomic features of KN-CRKP in South China, including the emergence of the ST1869 clone, a unique IncI1/X3 hybrid plasmid harboring blaNDM-5 and blaCMY-42, and the rare ΔISKpn6-blaKPC-2-ISKpn28 genetic structure. These findings substantially expand current understanding of plasmid evolution and resistance gene dissemination in this region. The identification of diverse resistance mechanisms and clonal backgrounds supports enhanced genomic surveillance and infection-control awareness for pan-resistant Enterobacterales.

Plasmids

Tn125-borne blaNDM-1 is decoupled from clonal background in a transcontinental Acinetobacter baumannii ST126/KL14 lineage.

BACKGROUND/OBJECTIVES: Carbapenem-resistant A. baumannii (CRAB) is a WHO Critical Priority pathogen. The blaNDM-1-carrying ST126/KL14 lineage has been independently reported from Vietnam (2015), Malaysia (2016), the USA (2023-2026), and Costa Rica (2024). Whether these geographically distinct reports represent a single transcontinental clone and through what mechanism blaNDM-1 disseminates has not been formally tested. METHODS: We performed comprehensive whole-genome reanalysis of the Vietnamese sentinel isolate DMS06669_L1 using three nested panels (n = 19, n = 138, and n = 609 Vietnamese A. baumannii genomes) and surveyed 429 plasmids extracted from 99 NDM-1 A. baumannii genomes retrieved from NCBI Pathogen Detection. RESULTS: Four ST126/KL14 isolates share high inter-regional average nucleotide identity (ANI; 99.77-99.92%) but wide intra-clade core-SNP distances (41-731 SNPs, well above the ∼20-40-SNP range typical of single-outbreak transmission clusters) and lack a significant molecular clock, consistent with a related transcontinental lineage rather than a single recent clone. NDM-1 plasmid evolution is statistically uncorrelated with chromosomal sequence type (Spearman ρ = 0.131, P = 0.573). At 609-strain population scale, under a fragmentation-aware detection criterion, all 41 blaNDM-1-carrying Vietnamese strains also carry ISAba125, with none carrying blaNDM-1 without it (Fisher exact test; Haldane-Anscombe-corrected OR ≈2.0 × 10³, 95% CI 1.2 × 10² to 3.4 × 10⁴; P = 4.74×10⁻⁴⁸; φ = 0.79). CONCLUSIONS: The blaNDM-1 dissemination pattern in this ST126/KL14 lineage is primarily consistent with Tn125 transposition acting alongside plasmid-borne spread. Standard MLST-based surveillance is insufficient; multi-level genomic monitoring - including chromosomal and plasmid-level detection of the Tn125/ISAba125 unit - is required to track this resistance threat.

A. baumannii

Structural evolution of bacterial plasmids: role of translocating genetic elements and DNA sequence insertions.

Recent evidence suggests that plasmids have evolved by site-specific recombinational events involving translocation and insertion of discretely defined DNA segments. The role of translocating genetic elements and repeated DNA sequences in the formation and structural evolution of bacterial plasmids, and in the control of plasmid gene expression, is the subject of this brief review. Insertion sequence (IS) regions are discrete segments of DNA that are known to cause strongly polar mutations in the genes of Escherichia coli and several bacteriophages as a consequence of their insertion into bacterial or phage genomes. Recent investigations have identified three separate kinds of IS segments on plasmids, and have indicated that such regions may have a role in 1) site-specific reversible dissociation of antibiotic resistance plasmids into their component segments, 2) recombination of certain plasmids with the bacterial chromosome, and 3) translocation of segments of plasmid DNA onto other replicons, or onto different sites of the same replicon. In addition, such DNA sequences, which may be repeated on plasmid genomes in either direct or reverse orientation, are involved in the control of plasmid gene expression. Inverted repeats other than the genetically characterized IS segments also appear to be involved in recA-independent, recombination and translocation of plasmid DNA segments. These inverted repeats contain palindromic nucleotide sequences on each strand of DNA and are detectable as hairpin-loop structures by electron microscope heteroduplex analysis. Such palindromes resemble the recognition sites for restriction endonucleases, some of which are encoded by plasmids, suggesting that similar endonucleolytic enzymes may be involved in the translocation of plasmid DNA segments.

Base Sequence

Instability of plasmid DNA sequences: macro and micro evolution of the antibiotic resistance plasmid R6-5.

Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possible other replicons occur more frequently than has hitherto been appreciated. The sequences changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existing in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous, and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.

Anti-Bacterial Agents

Evolution of linear plasmids.

Linear plasmids are genetic elements commonly found in yeast, filamentous fungi, and higher plants. In contrast to all other plasmids they possess terminal inverted repeats and terminal bound proteins and encode their own DNA and RNA polymerases. Here we present alignments of conserved amino acid sequences of both the DNA and RNA polymerases encoded by those linear plasmids for which DNA sequence data are available. Additionally these sequences are compared to a number of polymerases encoded by related viral and cellular entities. Phylogenetic trees have been established for both types of polymerases. These trees appear to exhibit very similar subgroupings, proving the accuracy of the method employed.

Amino Acid Sequence

Putative evolution of Myxococcus fulvus 124B02 plasmid pMF1 from a chromosomal segment in another Myxococcus species.

Myxobacteria or order Myxococcales (old nomenclature) or phylum Myxococcota (new terminology) are fascinating organisms well known for their diverse peculiar physiological, taxonomic, and genomic properties. Researchers have long sought to identify plasmids within these organisms, yet thus far, only two organisms from different families have been found to harbor a plasmid. This study delves into the putative evolution of one of these plasmids, i.e., pMF1 present in Myxococcus fulvus 124B02 in the suborder Cystobacterineae and family Myxococcaceae. Here, we first reannotated the pMF1 plasmid genome sequence and identified two additional open reading frames or putative genes which were not annotated until now. We further reported that all pMF1 plasmid genes depict homology with Myxococcus stipitatus CYD1 draft genome (contig 28) and a chromosomal segment of M. stipitatus DSM14675 in a syntenic manner, implying the presence of plasmid-like structure in M. stipitatus CYD1, integrated into its chromosome. To comprehend the relationship among these three species, we conducted phylogenetic analyses using 16S and concatenated housekeeping genes and genome-to-genome distance calculator (GGDC) analysis, which confirmed that M. stipitatus CYD1 is a distinct and novel species within the genus Myxococcus. Overall, this comparative genomic study sheds light on the putative emergence of the pMF1 plasmid from a common ancestor of closely related yet distinct species, M. stipitatus CYD1, possibly through the partition from its chromosome as a segment.IMPORTANCEMyxobacteria are not well known to have plasmids. Until now, only two organisms have been shown to have plasmids, raising a pertinent question about how these plasmids evolved randomly within the phylum Myxococcota. The study presented in this manuscript delves into the emergence of the pMF1 plasmid found in Myxococcus fulvus 124B02, a member of the suborder Cystobacterineae and family Myxococcaceae. Our research addresses this intriguing topic of plasmid identification and evolution within myxobacteria, which are a group of fascinating organisms that have garnered significant interest due to their diverse physiological, taxonomic, and genomic properties.

Plasmids

Ecological and evolutionary implications of a mobile genetic element-rich haloarchaeon with unique osmotic resilience.

We isolated a novel halophilic archaeon, strain DSL9, representing the proposed new species Haloliberatus hailidukes gen. nov., sp. nov., from Dishui Lake, China. Unlike most obligate halophiles, DSL9 survives in low salinity, even distilled water, without lysis. Genomic analysis revealed dual salinity adaptation strategies: salt-in and compatible solutes, including a complete trehalose biosynthesis pathway. The strain harbors multiple plasmids, notably a 111,311 bp large plasmid (pHdsl9-3) encoding replication (Orc1/Cdc6, SSB), transcription (TFIIB), transmission (T4SS cluster, ArdC-like protein), and recombination (XerA) modules. pHdsl9-3 provides auxiliary functions such as defense, genome diversification, ion detoxification, and suggests active horizontal gene transfer. Similar elements are widespread in Halobacteriales, highlighting their role in haloarchaeal genetic diversity and plasticity. The encoded XerA hinted at a function beyond DNA dimer resolution, suggesting it may have been adapted by other archaeal mobile genetic elements. These findings underscore the need to investigate plasmid-driven evolution and environmental adaptation mechanisms in haloarchaea.IMPORTANCEThis study reports the isolation and characterization of DSL9, a novel halophilic archaeon from a freshwater lake. Remarkably, DSL9 defies the typical obligate halophilic lifestyle by surviving in low-salinity environments, including distilled water, without cell lysis. A key discovery is the identification of a 111,311 bp large plasmid harboring essential modules for replication, transcription, transmission, and integration. Widespread distribution of similar elements across Halobacteriales suggests their crucial role in haloarchaeal genetic diversity and plasticity, warranting further study of plasmid-mediated evolution and adaptation strategies.

Lakes

DNA inversion regions Min of plasmid p15B and Cin of bacteriophage P1: evolution of bacteriophage tail fiber genes.

Plasmid p15B and the genome of bacteriophage P1 are closely related, but their site-specific DNA inversion systems, Min and Cin, respectively, do not have strict structural homology. Rather, the complex Min system represents a substitution of a Cin-like system into an ancestral p15B genome. The substituting sequences of both the min recombinase gene and the multiple invertible DNA segments of p15B are, respectively, homologous to the pin recombinase gene and to part of the invertible DNA of the Pin system on the defective viral element e14 of Escherichia coli K-12. To map the sites of this substitution, the DNA sequence of a segment adjacent to the invertible segment in the P1 genome was determined. This, together with already available sequence data, indicated that both P1 and p15B had suffered various sequence acquisitions or deletions and sequence amplifications giving rise to mosaics of partially related repeated elements. Data base searches revealed segments of homology in the DNA inversion regions of p15B, e14, and P1 and in tail fiber genes of phages Mu, T4, P2, and lambda. This result suggest that the evolution of phage tail fiber genes involves horizontal gene transfer and that the Min and Pin regions encode tail fiber genes. A functional test proved that the p15B Min region carries a tail fiber operon and suggests that the alternative expression of six different gene variants by Min inversion offers extensive host range variation.

Amino Acid Sequence

Evolution of multiple-antibiotic-resistance plasmids mediated by transposable plasmid deoxyribonucleic acid sequences.

Two plasmid deoxyribonucleic acid sequences mediating multiple antibiotic resistance transposed in vivo between coexisting plasmids in clinical isolates of Serratia marcescens. This event resulted in the evolution of a transferable multiresistance plasmid. Both sequences, designated in Tn1699 and Tn1700, were flanked by inverted deoxyribonucleic acid repetitions and could transpose between replicons independently of the Excherichia coli recA gene function. Tn1699 and Tn1700 mediated ampicillin, carbenicillin, kanamycin, and gentamicin resistance but differed in the type of gentamicin-acetyltransferase enzymes that they encoded. The structural genes for these enzymes share a great deal of polynucleotide sequence similarity despite their phenotypic differences. The transposition of Tn1699 and Tn1700 to coresident transferable plasmids has contributed to the dissemination of antibiotic resistance among other gram-negative bacteria. These organisms have recently caused nosocomial infections in epidemic proportions.

Anti-Bacterial Agents

SegMantX: A Novel Tool for Detecting DNA Duplications Uncovers Prevalent Duplications in Plasmids.

Segmental duplications play an important role in genome evolution via their contribution to copy-number variation, gene-family diversification, and the emergence of novel functions. The detection of segmental duplications is challenging due to heterogeneous amelioration of sequence similarity among duplicates, which hinders the reconstruction of continuous sequence alignment. Here we introduce SegMantX, a novel approach for the identification of diverged segmental duplications in prokaryote genomes using local alignment chaining. In this approach, local alignments resulting from a preliminary sequence similarity search (e.g. BLASTn) are chained into continuous segments. Evaluating the performance of SegMantX using simulated sequences shows that the tool can detect diverged duplications beyond the sensitivity limits of standard alignment-based methods. Applying SegMantX to 6,784 enterobacterial plasmids, we find that 65% plasmids contain duplicated regions and gene duplications, most of which correspond either to dispersed, noncoding regions or duplicated mobile genetic elements (MGEs; e.g. transposons and insertion sequences). Furthermore, we demonstrate the applicability of SegMantX for the identification of diverged gene transfers between replicons and plasmid hybridization events. Our findings highlight MGEs as drivers of segmental duplications in plasmid evolution, leading to the amplification of their cargo genes, including antibiotic resistance genes. SegMantX provides a powerful framework for reconstructing diverged segmental duplications and other alignment problems.

Plasmids