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Chlormequat (CCC) is widely used in agricultural production to increase the crop yield. However, the effects of CCC on transfer of ARGs in agricultural system are still unclear. In this study, using E.coli DH5α (carrying RP4 plasmid with Amp R , Tet R , Kan R ) as the donor bacterium, E.coli HB101, endophytic Pseudomonas sp. Ph6 or rhizosphere Pseudomonas putida KT2440 as the recipient strain, three conjugative systems were designed to investigate the effects of CCC on ARG transfer. Meanwhile, hydroponics experiments were designed to study the ARG spread in the rice-nutrient solution system after CCC application. The results showed that CCC significantly promoted the RP4 conjugation by expanding cell membrane permeability and improving the relative transcription levels of trfAp, trbBp, traA and traL genes in RP4. Furthermore, the conjugation frequency between E. coli and Pseudomonas was much higher than that between E. coli cells. Compared with spraying foliage with 2500 mg·L -1 of CCC, soaking seeds with 250 mg·L -1 of CCC was more beneficial to the colonization of ARB in rice, and also increased the abundance of ARGs in rice cultivation system. These results remind that the use of CCC in agricultural production might promote the ARG transmission in agro-ecosystems; however, foliage spraying with 2500 mg·L -1 of CCC could control its spread., 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.)

Over the past few decades, the global spread of antimicrobial resistance has underscored the urgent need to develop innovative non-antibiotic antibacterial strategies and to reduce antibiotic use worldwide. In response to this challenge, several methods have been developed that rely on gene transfer by conjugation to deliver toxic compounds or CRISPR systems specifically designed to kill or resensitize target bacterial strains to antibiotics. This review explores the design, potential, and limitations of these conjugation-based antibacterial strategies, focusing on the recent advances in the delivery of CRISPR systems as antibacterial effectors., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Streptomyces are ubiquitous soil-dwelling bacteria with large, linear genomes that are of special importance as a source of metabolites used in human and veterinary medicine, agronomy, and industry. Conjugative elements (actinomycetes integrative and conjugative elements, AICEs) are the main drivers of Streptomyces Horizontal Gene Transfer. AICE transfer has long been known to be accompanied by mobilization of chromosomal DNA. However, the magnitude of DNA transfer, or the localization of acquired DNA across their linear chromosome, has remained undetermined. We here show that conjugative crossings in sympatric strains of Streptomyces result in the large-scale, genome-wide distributed replacement of up to one-third of the recipient chromosome, a phenomenon for which we propose the name "Streptomyces Chromosomal Transfer" (SCT). Such chromosome blending results in the acquisition, loss, and hybridization of Specialized Metabolite Biosynthetic Gene Clusters, leading to a novel metabolic arsenal in exconjugant offspring. Harnessing conjugation-mediated specialized metabolite biosynthesis gene cluster diversification holds great promise in the discovery of new bioactive compounds including antibiotics., Competing Interests: Conflict of Interest The authors declare that there are no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Plasmids are major drivers of gene mobilization by means of horizontal gene transfer and play a key role in spreading antimicrobial resistance among pathogens 1,2 . Despite various bacterial defence mechanisms such as CRISPR-Cas, restriction-modification systems and SOS-response genes that prevent the invasion of mobile genetic elements 3 , plasmids robustly transfer within bacterial populations through conjugation 4,5 . Here we show that the leading region of plasmids, the first to enter recipient cells, is a hotspot for an extensive repertoire of anti-defence systems, encoding anti-CRISPR, anti-restriction, anti-SOS and other counter-defence proteins. We further identified in the leading region a prevalence of promoters known to allow expression from single-stranded DNA 6 , potentially facilitating rapid protection against bacterial immunity during the early stages of plasmid establishment. We demonstrated experimentally the importance of anti-defence gene localization in the leading region for efficient conjugation. These results indicate that focusing on the leading region of plasmids could lead to the discovery of diverse anti-defence genes. Combined, our findings show a new facet of plasmid dissemination and provide theoretical foundations for developing efficient conjugative delivery systems for natural microbial communities., (© 2024. The Author(s).)

We report the creation of 17 Escherichia coli strains harboring the conjugative plasmid pLCasCureT with a CRISPR-Cas9 system to surgically "cure" the most common plasmids among Enterobacterales species. This approach can create isogenic pairs of strains to study host-plasmid interactions, correlate plasmid genotype and phenotype, and create plasmid-free cloning strains., 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.)

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.)

Aims: To investigate the molecular events associated with acquiring macrolide resistance genes [mefE/mel (Mega) or ermB] in Streptococcus pneumoniae (Spn) during nasopharyngeal colonization., Methods and Results: Genomic analysis of 128 macrolide-resistant Spn isolates revealed recombination events in genes of the conjugation apparatus, or the competence system, in strains carrying Tn916-related elements. Studies using confocal and electron microscopy demonstrated that during the transfer of Tn916-related elements in nasopharyngeal cell biofilms, pneumococcal strains formed clusters facilitating their acquisition of resistance determinants at a high recombination frequency (rF). Remarkably, these aggregates comprise both encapsulated and nonencapsulated pneumococci that span extracellular and intracellular compartments. rF assessments showed similar rates regardless Mega was associated with large integrative and conjugative elements (ICEs) (>23 kb) or not (∼5.4 kb). The rF for Mega Class IV(c) insertion region (∼53 kb) was three orders of magnitude higher than the transformation of the capsule locus. Metabolomics studies of the microenvironment created by colonization of human nasopharyngeal cells revealed a link between the acquisition of ICEs and the pathways involving nicotinic acid and sucrose., Conclusions: Pneumococcal clusters, both extracellular and intracellular, facilitate macrolide resistance acquisition, and ICEs were acquired at a higher frequency than the capsule locus. Metabolic changes could serve as intervention targets., 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 Ltd.. All rights reserved.)

Horizontal gene transfer plays a key role in the global dissemination of antimicrobial resistance (AMR). AMR genes are often carried on self-transmissible plasmids, which are shared amongst bacteria primarily by conjugation. Antibiotic use has been a well-established driver of the emergence and spread of AMR. However, the impact of commonly used non-antibiotic compounds and environmental pollutants on AMR spread has been largely overlooked. Recent studies found common prescription and over-the-counter drugs, artificial sweeteners, food preservatives, and environmental pollutants, can increase the conjugative transfer of AMR plasmids. The potential mechanisms by which these compounds promote plasmid transmission include increased membrane permeability, upregulation of plasmid transfer genes, formation of reactive oxygen species, and SOS response gene induction. Many questions remain around the impact of most non-antibiotic compounds on AMR plasmid conjugation in clinical isolates and the long-term impact on AMR dissemination. By elucidating the role of routinely used pharmaceuticals, food additives, and pollutants in the dissemination of AMR, action can be taken to mitigate their impact by closely monitoring use and disposal. This review will discuss recent progress on understanding the influence of non-antibiotic compounds on plasmid transmission, the mechanisms by which they promote transfer, and the level of risk they pose.

The emergence and spread of antibiotic resistance in the environment pose a serious threat to global public health. It is acknowledged that non-antibiotic stresses, including disinfectants, pharmaceuticals and organic pollutants, play a crucial role in horizontal transmission of antibiotic resistance genes (ARGs). Despite the widespread presence of non-steroidal anti-inflammatory drugs (NSAIDs), notably in surface water, their contributions to the transfer of ARGs have not been systematically explored. Furthermore, previous studies have primarily concentrated on model strains to investigate whether contaminants promote the conjugative transfer of ARGs, leaving the mechanisms of ARG transmission among antibiotic resistant bacteria in natural aqueous environments under the selective pressures of non-antibiotic contaminants remains unclear. In this study, the Escherichia coli (E. coli) K12 carrying RP4 plasmid was used as the donor strain, indigenous strain Aeromonas veronii containing rifampicin resistance genes in Taihu Lake, and E. coli HB101 were used as receptor strains to establish inter-genus and intra-genus conjugative transfer systems, examining the conjugative transfer frequency under the stress of ketoprofen. The results indicated that ketoprofen accelerated the environmental spread of ARGs through several mechanisms. Ketoprofen promoted cell-to-cell contact by increasing cell surface hydrophobicity and reducing cell surface charge, thereby mitigating cell-to-cell repulsion. Furthermore, ketoprofen induced increased levels of reactive oxygen species (ROS) production, activated the DNA damage-induced response (SOS), and enhanced cell membrane permeability, facilitating ARG transmission in intra-genus and inter-genus systems. The upregulation of outer membrane proteins, oxidative stress, SOS response, mating pair formation (Mpf) system, and DNA transfer and replication (Dtr) system related genes, as well as the inhibition of global regulatory genes, all contributed to higher transfer efficiency under ketoprofen treatment. These findings served as an early warning for a comprehensive assessment of the roles of NSAIDs in the spread of antibiotic resistance in natural aqueous environments., 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 Ltd. All rights reserved.)

DNA transfer is ubiquitous in the human gut microbiota, especially among species of the order Bacteroidales. In silico analyses have revealed hundreds of mobile genetic elements shared between these species, yet little is known about the phenotypes they encode, their effects on fitness, or pleiotropic consequences for the recipient's genome. In this work, we show that acquisition of a ubiquitous integrative conjugative element (ICE) encoding a type VI secretion system (T6SS) shuts down the native T6SS of Bacteroides fragilis . Despite inactivating this T6SS, ICE acquisition increases the fitness of the B. fragilis transconjugant over its progenitor by arming it with the new T6SS. DNA transfer causes the strain to change allegiances so that it no longer targets ecosystem members with the same element yet is armed for communal defense.

The increase in antibiotic resistance in bacteria has prompted the efforts in developing new alternative strategies for pathogenic bacteria. We explored the feasibility of targeting Vibrio cholerae by neutralizing bacterial cellular processes rather than outright killing the pathogen. We investigated the efficacy of delivering engineered regulatory small RNAs (sRNAs) to modulate gene expression through DNA conjugation. As a proof of concept, we engineered several sRNAs targeting the type VI secretion system (T6SS), several of which were able to successfully knockdown the T6SS activity at different degrees. Using the same strategy, we modulated exopolysaccharide production and motility. Lastly, we delivered an sRNA targeting T6SS into V. cholerae via conjugation and observed a rapid knockdown of the T6SS activity. Coupling conjugation with engineered sRNAs represents a novel way of modulating gene expression in V. cholerae opening the door for the development of novel prophylactic and therapeutic applications., Importance: Given the prevalence of antibiotic resistance, there is an increasing need to develop alternative approaches to managing pathogenic bacteria. In this work, we explore the feasibility of modulating the expression of various cellular systems in Vibrio cholerae using engineered regulatory sRNAs delivered into cells via DNA conjugation. These sRNAs are based on regulatory sRNAs found in V. cholerae and exploit its native regulatory machinery. By delivering these sRNAs conjugatively along with a real-time marker for DNA transfer, we found that complete knockdown of a targeted cellular system could be achieved within one cell division cycle after sRNA gene delivery. These results indicate that conjugative delivery of engineered regulatory sRNAs is a rapid and robust way of precisely targeting V. cholerae ., Competing Interests: The authors declare no conflict of interest.

Humans use dietary supplements for several intended effects, such as supplementing malnutrition. While these compounds have been developed for host end benefits, their ancillary impact on the gut microbiota remains unclear. The human gut has been proposed as a reservoir for the prevalent lateral transfer of antimicrobial resistance and virulence genes in bacteria through plasmid conjugation. Here, we studied the effect of dietary zinc supplements on the incidence of plasmid conjugation in vitro . Supplement effects were analyzed through standardized broth conjugation assays. The avian pathogenic Escherichia coli (APEC) strain APEC-O2-211 was a donor of the multidrug resistance plasmid pAPEC-O2-211A-ColV, and the human commensal isolate E. coli HS-4 was the plasmid-free recipient. Bacterial strains were standardized and mixed 1:1 and supplemented 1:10 with water, or zinc derived from either commercial zinc supplements or zinc gluconate reagent at varying concentrations. We observed a significant reduction in donors, recipients, and transconjugant populations in conjugations supplemented with zinc, with a dose-dependent relationship. Additionally, we observed a significant reduction ( P < 0.05) in log conjugation efficiency in zinc-treated reactions. Upregulation of the mRNA for the plasmid replication initiation gene repA and the subset of transfer genes M , J , E , K , B , P , C , W , U , N , F , Q , D , I , and X was observed. Furthermore, we observed a downregulation of the conjugal propilin gene traA and the entry exclusion gene traS . This study demonstrates the effect of dietary zinc supplements on the conjugal transfer of a multidrug resistance plasmid between pathogenic and commensal bacteria during in vitro conditions.IMPORTANCEThis study identifies dietary zinc supplementation as a potential novel intervention for mitigating the emergence of multidrug resistance in bacteria, thus preventing antibiotic treatment failure and death in patients and animals. Further studies are required to determine the applicability of this approach in an in vivo model., Competing Interests: The authors declare no conflict of interest.

This study aimed to investigate the diversity of conjugative and chromosomally integrated mobile genetic elements (cciMGEs) within six oral streptococci species. cciMGEs, including integrative and conjugative elements (ICEs) and integrative and mobilizable elements (IMEs), are stably maintained on the host cell chromosome; however, under certain conditions, they are able to excise, form extrachromosomal circles, and transfer via a conjugation apparatus. Many cciMGEs encode "cargo" functions that aid survival in new niches and evolve new antimicrobial resistance or virulence properties, whereas others have been shown to influence host bacterial physiology. Here, using a workflow employing preexisting bioinformatics tools, we analyzed 551 genomes for the presence of cciMGEs across six common health- and disease-associated oral streptococci. We identified 486 cciMGEs, 173 of which were ICEs and 233 of which were IMEs. The cciMGEs were diverse in size, cargo genes, and relaxase types. We identified several novel relaxase proteins and a widespread IME carrying a small multidrug resistance transporter. Additionally, we provide evidence that several of the bioinformatically predicted cciMGEs encoded within various Streptococcus mutans strains are capable of excision and circularization, a critical step for cciMGE conjugative transfer. These findings highlight the significance and potential impact of MGEs in shaping the genetic landscape, pathogenicity, and antimicrobial resistance profiles of the oral microbiota.IMPORTANCEOral streptococci are important players in the oral microbiome, influencing both health and disease states within dental bacterial communities. Evolutionary adaptation, shaped in a major part by the horizontal transfer of genes, is essential for their survival in the oral cavity and within new environments. Conjugation is a significant driver of horizontal gene transfer; however, there is limited information regarding this process in oral bacteria. This study utilizes publicly available genome sequences to identify conjugative and chromosomally integrated mobile genetic elements (cciMGEs) across several species of oral streptococci and presents the preliminary characterization of these elements. Our findings significantly enhance our understanding of the mobile genomic landscape of oral streptococci critical for human health, with valuable insights into how cciMGEs might influence the survival and pathogenesis of these bacteria in the oral microbiome., Competing Interests: The authors declare no conflict of interest.

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria. Conjugative relaxases are translocated through a type IV secretion system into the recipient cell, covalently attached to the transferred DNA strand. We fused relaxase R388-TrwC with the endonuclease Cas12a and confirmed that it can be transported through a T4SS. The fusion protein maintained its activity upon translocation by conjugation into the recipient cell, as evidenced by the induction of the SOS signal resulting from DNA breaks produced by the endonuclease in the recipient cell, and the detection of mutations at the target position. We further show how a template DNA provided on the transferred DNA can be used to introduce specific mutations. The guide RNA can also be encoded by the transferred DNA, enabling its production in the recipient cells where it can form a complex with the Cas nuclease transferred as a protein. This self-contained setup enables to target wild-type bacterial cells. Finally, we extended this strategy to the delivery of relaxases fused to base editors. Using TrwC and MobA relaxases as drivers, we achieved precise editing of transconjugants. Thus, conjugation provides a delivery system for Cas-derived editing tools, bypassing the need to deliver and express a cas gene in the target cells., Competing Interests: Competing interests statement:D.B. is a shareholder and advisor of Eligo Bioscience. D.L.G.-H., D.B., and M.L. are listed as inventors on a patent related to the technology discussed in this article: Proteína quimérica relaxasa-cas, ES2897017B2, 2022.

We have recently described a clinical isolate of Providencia rustigianii strain JH-1 carrying the genes for cytolethal distending toxin (CDT) in a conjugative plasmid. A cdtB mutant of strain JH-1, which lost CDT activity, was still found to retain invasiveness and diarrheagenicity. The strain was subjected to phenotypic and genetic analyses including whole genome sequencing (WGS) to explore the genetic determinants of the observed invasiveness and diarrheagenic properties. Analysis and annotation of WGS data revealed the presence of two distinct type III secretion systems (T3SS) in strain JH-1, one of which was located on the chromosome designated as cT3SS (3,992,833 bp) and the other on a mega-plasmid designated as pT3SS (168,819 bp). Comparative genomic analysis revealed that cT3SS is generally conserved in Providencia spp. but pT3SS was limited to a subset of Providencia spp., carrying cdt genes. Strain JH-1 was found to invade HeLa cells and induce fluid accumulation with characteristic pathological lesions in rabbit ileal loops. Remarkably, these phenomena were associated with the pT3SS but not cT3SS. The plasmid could be transferred by conjugation from strain JH-1 to other strains of P. rustigianii , Providencia rettgeri , and Escherichia coli with concomitant transfer of these virulence properties. This is the first report of a functional and mobile T3SS in P. rustigianii and its association with invasiveness and diarrheagenicity of this bacterium. These data suggest that P. rustigianii and other CDT-producing Providencia strains might carry T3SS and exert their diarrheagenic effect by exploiting the T3SS nano-machinery.IMPORTANCEThe precise mechanism of virulence of Providencia rustigianii is unclear, although some strains produce cytolethal distending toxin as a putative virulence factor. We have detected the presence of a type III secretion system (T3SS) for the first time on a plasmid in a P. rustigianii strain. Plasmid-mediated T3SS seems to be directly involved in virulence of P. rustigianii and may serve as a means of horizontal transfer of T3SS genes. Our results may have implication in understanding the mechanism of emergence of new pathogenic strains of P. rustigianii ., Competing Interests: The authors declare no conflict of interest.

Horizontal gene transfer (HGT) is a pivotal mechanism driving bacterial evolution, conferring adaptability within dynamic marine ecosystems. Among HGT mechanisms, conjugation mediated by type IV secretion systems (T4SSs) plays a central role in the ecological success of marine bacteria. However, the conditions promoting conjugation events in the marine environment are not well-understood. Roseobacters, abundant marine bacteria commonly associated with algae, possess a multitude of T4SSs. Many Roseobacters are heterotrophic bacteria that rely on algal secreted compounds to support their growth. These compounds attract bacteria, facilitating colonization and attachment to algal cells. Algae and their metabolites bring bacteria into close proximity, potentially promoting bacterial HGT. Investigation across various Roseobacters revealed that algal exudates indeed enhance plasmid transfer through conjugation. While algal exudates do not influence the transcription of bacterial conjugative machinery genes, they promote bacterial attachment, potentially stabilizing proximity and facilitating HGT. Notably, under conditions where attachment is less advantageous, the impact of algal exudates on conjugation is reduced. These findings suggest that algae enhance bacterial conjugation primarily by fostering attachment and highlight the importance of studying bacterial HGT within the context of algal-bacterial interactions., Importance: This study explores how algal-bacterial interactions influence horizontal gene transfer (HGT) among marine bacteria. HGT, a key driver of bacterial evolution, is facilitated by conjugation mediated by type IV secretion systems (T4SSs). Through investigating Roseobacters, abundant marine bacteria often found to be associated with algae, the study reveals that algal exudates enhance plasmid transfer via conjugation. This enhancement is attributed to the promotion of bacterial attachment by algal compounds, emphasizing the role of algal-bacterial interactions in shaping genetic exchange within dynamic marine ecosystems. Understanding these mechanisms is crucial for elucidating bacterial adaptability and evolution in the marine environment., Competing Interests: The authors declare no conflict of interest.

Cyanobacterial blooms are expanding world-wide in freshwater and marine environments, and can cause serious ecological and environmental issues, which also contribute to the spread of antibiotic resistance genes (ARGs). However, the mechanistic understanding of cyanobacteria-mediated resistance dynamics is not fully elucidated yet. We selected Microcystis aeruginosa as a model cyanobacteria to illustrate how cyanobacteria mediate the evolution and transfer processes of bacterial antibiotic resistance. The results show that the presence of cyanobacteria significantly decreased the abundance of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) by 3%-99% and 2%-18%, respectively. In addition, it clearly altered bacterial community structure, with the dominant genera evolving from Acinetobacter (27%) and Enterobacter (42%) to Porphyrobacter (59%). The abundance of ARGs positively correlated with Proteobacteria and Firmicutes, rather than Cyanobacteria, and Bacteroidetes. In the presence of cyanobacteria, the transfer events of bacterial resistance genes via conjugation were found to decrease by 10%-89% (p < 0.05). Surprisingly, we found an extradentary high transfer frequency (about 0.1) for the ARGs via plasmid conjugation from the bacteria into M. aeruginosa population. It confirmed the role of cyanobacterial population as the competent hosts to facilitate ARGs spreading. Our findings provide valuable information on the risk evaluation of ARGs caused by cyanobacterial blooms in aquatic environments, key for the protection and assessment of aquatic environmental quality., 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 Ltd. All rights reserved.)

The phenomenon of exclusion allows conjugative plasmids to selectively impede the entry of identical or related elements into their host cell to prevent the resulting instability. Entry exclusion blocks DNA translocation into the recipient cell, whereas surface exclusion destabilizes the mating pair. IncC conjugative plasmids largely contribute to the dissemination of antibiotic-resistance genes in Gammaproteobacteria. IncC plasmids are known to exert exclusion against their relatives, including IncC and IncA plasmids, yet the entry exclusion factor eexC alone does not account for the totality of the exclusion phenotype. In this study, a transposon-directed insertion sequencing approach identified sfx as necessary and sufficient for the remaining exclusion phenotype. Sfx is an exclusion factor unrelated to the ones described to date. A cell fractionation assay localized Sfx in the outer membrane. Reverse transcription PCR and beta-galactosidase experiments showed that sfx is expressed constitutively at a higher level than eexC. A search in Gammaproteobacteria genomes identified Sfx homologs encoded by IncC, IncA and related, untyped conjugative plasmids and an uncharacterized family of integrative and mobilizable elements that likely rely on IncC plasmids for their mobility. Mating assays demonstrated that sfx is not required in the donor for exclusion, ruling out Sfx as the exclusion target. Instead, complementation assays revealed that the putative adhesin TraN in the donor mediates the specificity of surface exclusion. Mating assays with TraN homologs from related untyped plasmids from Aeromonas spp. and Photobacterium damselae identified two surface exclusion groups, with each Sfx being specific of TraN homologs from the same group. Together, these results allow us to better understand the apparent incompatibility between IncA and IncC plasmids and to propose a mechanistic model for surface exclusion mediated by Sfx in IncC plasmids and related elements, with implications for the rampant dissemination of antibiotic resistance., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: V.B. has served as guest editor in 2021/2022 for PLoS Genetics. All other authors have no competing interest., (Copyright: © 2024 Rivard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

It has been shown that an evolutionary tradeoff between vertical (host growth rate) and horizontal (plasmid conjugation) transmissions contributes to global plasmid fitness. As conjugative IncC plasmids are important for the spread of multidrug resistance (MDR), in a broad range of bacterial hosts, we investigated vertical and horizontal transmissions of two multidrug-resistant IncC plasmids according to their backbones and MDR-region rearrangements, upon plasmid entry into a new host. We observed plasmid genome deletions after conjugation in three diverse natural Escherichia coli clinical strains, varying from null to high number depending on the plasmid, all occurring in the MDR region. The plasmid burden on bacterial fitness depended more on the strain background than on the structure of the MDR region, with deletions appearing to have no impact. Besides, we observed an increase in plasmid transfer rate, from ancestral host to new clinical recipient strains, when the IncC plasmid was rearranged. Finally, using a second set of conjugation experiments, we investigated the evolutionary tradeoff of the IncC plasmid during the critical period of plasmid establishment in E. coli K-12, by correlating the transfer rates of deleted or non-deleted IncC plasmids and their costs on the recipient strain. Plasmid deletions strongly improved conjugation efficiency with no negative growth effect. Our findings indicate that the flexibility of the MDR-region of the IncC plasmids can promote their dissemination, and provide diverse opportunities to capture new resistance genes. In a broader view, they suggest that the vertical-horizontal transmission tradeoff can be manipulated by the plasmid to improve its fitness., Competing Interests: The authors declare no conflict of interest.

Plasmids are ubiquitous mobile genetic elements, that can be either costly or beneficial for their bacterial host. In response to constant viral threat, bacteria have evolved various immune systems, such as the prevalent restriction modification (innate immunity) and CRISPR-Cas systems (adaptive immunity). At the molecular level, both systems also target plasmids, but the consequences of these interactions for plasmid spread are unclear. Using a modeling approach, we show that restriction modification and CRISPR-Cas are effective as barriers against the spread of costly plasmids, but not against beneficial ones. Consequently, bacteria can profit from the selective advantages that beneficial plasmids confer even in the presence of bacterial immunity. While plasmids that are costly for bacteria may persist in the bacterial population for a certain period, restriction modification and CRISPR-Cas can eventually drive them to extinction. Finally, we demonstrate that the selection pressure imposed by bacterial immunity on costly plasmids can be circumvented through a diversity of escape mechanisms and highlight how plasmid carriage might be common despite bacterial immunity. In summary, the population-level outcome of interactions between plasmids and defense systems in a bacterial population is closely tied to plasmid cost: Beneficial plasmids can persist at high prevalence in bacterial populations despite defense systems, while costly plasmids may face extinction., Competing Interests: Conflict of Interest None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

ICE KKS102 Tn 4677 carries a bph operon for the mineralization of polychlorinated biphenyls (PCBs)/biphenyl and belongs to the Tn 4371 ICE (integrative and conjugative element) family. In this study, we investigated the role of the traR gene in ICE transfer. The traR gene encodes a LysR-type transcriptional regulator, which is conserved in sequence, positioning, and directional orientation among Tn 4371 family ICEs. The traR belongs to the bph operon, and its overexpression on solid medium resulted in modest upregulation of traG (threefold), marked upregulation of xis (80-fold), enhanced ICE excision and, most notably, ICE transfer frequency. We propose the evolutional roles of traR , which upon insertion to its current position, might have connected the cargo gene activation and ICE transfer. This property of ICE, i.e., undergoing transfer under environmental conditions that lead to cargo gene activation, would instantly confer fitness advantages to bacteria newly acquiring this ICE, thereby resulting in efficient dissemination of the Tn 4371 family ICEs.IMPORTANCEOnly ICE KKS102 Tn 4677 is proven to transfer among the widely disseminating Tn 4371 family integrative and conjugative elements (ICEs) from β and γ-proteobacteria. We showed that the traR gene in ICE KKS102 Tn 4677 , which is conserved in the ICE family with fixed location and direction, is co-transcribed with the cargo gene and activates ICE transfer. We propose that capturing of traR by an ancestral ICE to the current position established the Tn 4371 family of ICEs. Our findings provide insights into the evolutionary processes that led to the widespread distribution of the Tn 4371 family of ICEs across bacterial species., Competing Interests: The authors declare no conflict of interest.

Paracoccus denitrificans has been identified as a representative strain with heterotrophic nitrification-aerobic denitrification capabilities (HN-AD), and demonstrates strong denitrification proficiency. Previously, we isolated the DYTN-1 strain from activated sludge, and it has showcased remarkable nitrogen removal abilities and genetic editability, which positions P. denitrificans DYTN-1 as a promising chassis cell for synthetic biology engineering, with versatile pollutant degradation capabilities. However, the strain's low stability in plasmid conjugation transfer efficiency (PCTE) hampers gene editing efficacy, and is attributed to its restriction modification system (R-M system). To overcome this limitation, we characterized the R-M system in P. denitrificans DYTN-1 and identified a DNA endonuclease and 13 DNA methylases, with the DNA endonuclease identified as HNH endonuclease. Subsequently, we developed a plasmid artificial modification approach to enhance conjugation transfer efficiency, which resulted in a remarkable 44-fold improvement in single colony production. This was accompanied by an increase in the frequency of positive colonies from 33.3% to 100%. Simultaneously, we cloned, expressed, and characterized the speculative HNH endonuclease capable of degrading unmethylated DNA at 30°C without specific cutting site preference. Notably, the impact of DNA methylase M9 modification on the plasmid was discovered, significantly impeding the cutting efficiency of the HNH endonuclease. This revelation unveils a novel R-M system in P. denitrificans and sheds light on protective mechanisms employed against exogenous DNA invasion. These findings pave the way for future engineering endeavors aimed at enhancing the DNA editability of P. denitrificans .

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.)

Bacterial conjugation, a commonly used method to horizontally transfer functional genes from donor to recipient strains, plays an important role in the genetic manipulation of bacteria for basic research and industrial production. Successful conjugation depends on the donor-recipient cell recognition and a tight mating junction formation. However, the efficiency of conjugative transfer is usually very low. In this work, we developed a new technique that employed DNA molecule "glue" to increase the match frequency and the interaction stability between the donor and recipient cells. We used two E. coli strains, ETZ and BL21, as a model system, and modified them with the complementary ssDNA oligonucleotides by click chemistry. The conjugation efficiency of the modified bacteria was improved more than 4 times from 10 %-46 %. This technique is simple and generalizable as it only relies on the active amino groups on the bacterial surface. It is expected to have broad applications in constructing engineered bacteria., (© 2024 Wiley-VCH GmbH.)

The aim of this work was to provide a theoretical and scientific basis for the treatment, prevention, and control of clinical drug-resistant bacterial infections by studying the molecular epidemiology and horizontal transfer mechanism of optrA -carrying linezolid-resistant Enterococcus faecalis strains (LREfs) that were clinically isolated in a tertiary hospital in Kunming, China. Non-repetitive LREfs retained in a tertiary A hospital in Kunming, China. The strains were identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The transferability and horizontal transfer mechanism of optrA gene were analyzed using polymerase chain reaction (PCR), whole-genome sequencing (WGS), and conjugation experiments. A total of 39 LREfs strains were collected, and all of them were multi-drug resistant. There were 30 LREfs strains (76.9%) carrying the optrA gene, The cfr, poxtA genes and mutations in the 23S rRNA gene were not detected. The conjugation experiments showed that only three of 10 randomly selected optrA -carrying LREfs were successfully conjugated with JH2-2. Further analysis of one successfully conjugated strain revealed that the optrA gene, located in the donor bacterium, formed the IS1216E-erm(A)-optrA-fexA-IS1216E transferable fragment under the mediation of the mobile genetic element (MGE) IS1216E , which was then transferred to the recipient bacterium via horizontal plasmid transfer. Carrying the optrA gene is the primary resistance mechanism of LREfs strains. The optrA gene could carry the erm(A) and fexA genes to co-transfer among E. faecalis . MGEs such as insertion sequence IS1216E play an important role in the horizontal transfer of the optrA gene., (© 2024 Peini Yang et al., published by Sciendo.)

The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies., (© 2024. The Author(s).)

The problem of bacterial resistance caused by antibiotic abuse is seriously detrimental to global human health and ecosystem security. The two-dimensional nanomaterial (2D) such as black phosphorus (BP) is recently expected to become a new bacterial inhibitor and has been widely used in the antibacterial field due to its specific physicochemical properties. Nevertheless, the effects of 2D-BP on the propagation of antibiotic resistance genes (ARGs) in environments and the relevant mechanisms are not clear. Herein, we observed that the sub-inhibitory concentrations of 2D-BP dramatically increased the conjugative transfer of ARGs mediated by the RP4 plasmid up to 2.6-fold at the 125 mg/L exposure level compared with the untreated bacterial cells. Nevertheless, 2D-BP with the inhibitory concentration caused a dramatic decrease in the conjugative frequency. The phenotypic changes revealed that the increase of the conjugative transfer caused by 2D-BP exposure were attributed to the excessive reactive oxygen species and oxidative stress, and increased bacterial cell membrane permeability. The genotypic evidence demonstrated that 2D-BP affecting the horizontal gene transfer of ARGs was probably through the upregulation of mating pair formation genes (trbBp and traF) and DNA transfer and replication genes (trfAp and traJ), as well as the downregulation of global regulatory gene expression (korA, korB, and trbA). In summary, the changes in the functional and regulatory genes in the conjugative transfer contributed to the stimulation of conjugative transfer. This research aims to broaden our comprehension of how nanomaterials influence the dissemination of ARGs by elucidating their effects and mechanisms., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.)

Dissemination of antibiotic resistance genes (ARGs) has become a critical threat to public health. Activated sludge, rich in extracellular polymeric substances (EPS), is an important pool of ARGs. In this study, mechanisms of conjugation transfer of ARGs induced by EPS, including tightly bound EPS (TBEPS), soluble EPS (SEPS), and loosely bound EPS (LBEPS), were explored in terms of molecular diversities and electron transfer properties of EPS. Conjugation transfer frequency was increased by 9.98-folds (SEPS), 4.21-folds (LBEPS), and 15.75-folds (TBEPS) versus the control, respectively. Conjugation-related core genes involving SOS responses (9 genes), membrane permeability (18 genes), intercellular contact (17 genes), and energy metabolism pathways (13 genes) were all upregulated, especially in the presence of TBEPS. Carbohydrates and aliphatic substances in SEPS and LBEPS were contributors to ARG transfer, via influencing reactive oxygen species (ROS) formation (SEPS) and ROS and adenosine triphosphate (ATP) production (LBEPS). TBEPS had the highest redox potential and greatest lability and facilitated electron transfer and alternated respiration between cells, thus promoting ARG transfer by producing ATP. Generally, the chemical molecular characteristics and redox properties of EPS facilitated ARG transfer mainly by influencing lipid peroxidation and ATP, respectively., 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.)

Wastewater treatment plants are one of the major pathways for microplastics to enter the environment. In general, microplastics are contaminants of global concern that pose risks to ecosystems and human health. Here, we present a proof-of-concept for reduction of microplastic pollution emitted from wastewater treatment plants: delivery of recombinant DNA to bacteria in wastewater to enable degradation of polyethylene terephthalate (PET). Using a broad-host-range conjugative plasmid, we enabled various bacterial species from a municipal wastewater sample to express FAST-PETase, which was released into the extracellular environment. We found that FAST-PETase purified from some transconjugant isolates could degrade about 40% of a 0.25 mm thick commercial PET film within 4 days at 50°C. We then demonstrated partial degradation of a post-consumer PET product over 5-7 days by exposure to conditioned media from isolates. These results have broad implications for addressing the global plastic pollution problem by enabling environmental bacteria to degrade PET., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Objectives: To investigate the tet(X) gene, a determinant of tigecycline resistance, in the emerging pathogen Elizabethkingia meningoseptica and its association with an integrative and conjugative element (ICE)., Methods: All E. meningoseptica genomes from the National Center for Biotechnology Information (n = 87) were retrieved and annotated for resistome searches using the CARD database. A phylogenic analysis was performed based on the E. meningoseptica core genome. The ICE was identified through comparative genomics with other ICEs occurring in Elizabethkingia spp., Results: Phylogenetic analysis revealed E. meningoseptica genomes from six countries distributed across different lineages, some of which persisted for years. The common resistome of these genomes included bla BlaB , bla CME , bla GOB , ranA/B, aadS, and catB (genes associated with resistance to β-lactams, aminoglycosides, and chloramphenicol). Some genomes also presented additional resistance genes (dfrA, ereD, bla VEB , aadS, and tet(X)). Interestingly, tet(X) and aadS were located in an ICE of 49 769 bp (ICEEmSQ101), which was fully obtained from the E. meningoseptica SQ101 genome. We also showed evidence that the other 27 genomes harboured this ICE. The distribution of ICEEmSQ101, carrying tet(X), was restricted to a single Chinese lineage., Conclusions: The tet(X) gene is not prevalent in the species E. meningoseptica, as previously stated for the genus Elizabethkingia, since it is present only in a single Chinese lineage. We identified that several E. meningoseptica genomes harboured an ICE that mobilized the Elizabethkingia tet(X) gene and exhibited characteristics similar to the ICEs of other Flavobacteria, which would favour their transmission in this bacterial family., Competing Interests: Competing interests None declared., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: The aim of this study was to characterize an NDM-1-producing Acinetobacter seifertii isolates from a patient in South Korea., Methods: Antibiotic susceptibility testing and genotyping using multigene sequencing were performed and whole plasmid sequences were determined., Results: The genotype of A. seifertii was ST1899 and was resistant to ceftazidime, trimethoprim-sulfamethoxazole, and piperacillin-tazobactam, in addition to carbapenem. bla NDM-1 was surrounded by the ISAba125 insertion sequence within the structure of Tn125 in the 47 kb-sized plasmid. The plasmid exhibited a structure similar to that of other plasmids of diverse Acinetobacter sp. found worldwide. Transconjugation and the growth curve indicated that the plasmid was adapted to A. seifertii rather than other closely related Acinetobacter sp., Conclusions: Acquisition of carbapenem resistance by horizontal transfer of the bla NDM-1 -carrying plasmid from another Acinetobacter species was found with no growth defect., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: To investigate the clinical and molecular epidemiological characteristics of bla IMP-4 -carrying Klebsiella pneumoniae in a tertiary hospital in China., Methods: Ten carbapenem-resistant K. pneumoniae (CRKP) isolates carrying the bla IMP-4 gene were collected. Molecular characteristics were analysed using whole-genome sequencing. Plasmid conjugation experiments were used to analyse conjugation of the plasmids. We compared and analysed K. pneumoniae-carrying bla IMP-4 genomic datasets obtained from the National Center for Biotechnology Information (NCBI) with the strains in this study., Results: All 10 CRKP isolates carrying bla IMP-4 were collected from 10 adult patients in the respiratory intensive care unit. These strains were only sensitive to polymyxins and tigecycline due to them simultaneously carrying multiple resistance genes, namely bla OKP-A-5 , fosA, oqxA, and oqxB. Notably, R29 harboured two carbapenemase genes (bla NDM-1 and bla IMP-4 ). These strains had similar drug-resistant phenotypes and genes, all belonging to sequence type (ST)196. Additionally, the patients had experienced spatiotemporal intersection during hospitalization, suggesting that these strains underwent clonal transmission, but they belonged to different clonal clusters from the bla IMP-4 -positive K. pneumoniae currently published in the NCBI. Among the 10 strains, bla IMP-4 was located on the IncN plasmid, and six strains had successfully transferred the plasmid to the recipient strain EC600 through plasmid conjugation., Conclusions: The bla IMP-4 -positive ST196 CRKP isolate showed clonal distribution in the respiratory intensive care unit, which was mediated by the IncN plasmid. Consequently, there should be increased monitoring of carbapenem-resistant strains in clinical settings to prevent and control its transmission., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Plasmids orchestrate bacterial adaptation across diverse environments and facilitate lateral gene transfer within bacterial communities. Their presence can perturb host metabolism, creating a competitive advantage for plasmid-free cells. Plasmid stability hinges on efficient replication and partition mechanisms. While plasmids commonly encode histone-like nucleoid-structuring (H-NS) family proteins, the precise influence of plasmid-encoded H-NS proteins on stability remains elusive. In this study, we examined the conjugative plasmid pMBL6842, harboring the hns gene, and observed its positive regulation of parAB transcription, critical for plasmid segregation. Deletion of hns led to rapid plasmid loss, which was remedied by hns complementation. Further investigations unveiled adverse effects of hns overexpression on the bacterial host. Transcriptome analysis revealed hns's role in regulating numerous bacterial genes, impacting both host growth and swimming motility in the presence of the hns gene. Therefore, our study unveils the multifaceted roles of H-NS in both plasmid stability and host physiology, underscoring its biological significance and paving the way for future inquiries into the involvement of H-NS in horizontal gene transfer events., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Objectives: Carbapenem-resistant Klebsiella pneumoniae (CRKP), a superbug that can be difficult or impossible to treat, has become a worldwide problem. This study presents the first report of a CRKP strain carrying a plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A) and the subsequent analysis of its genomic features., Methods: Isolation and identification of bacteria, antimicrobial susceptibility test, whole genome sequencing, and conjugation experiments assay were conducted in clinical epidemiological investigations and plasmid genetic characterisation analysis., Results: A total of 116 strains of bacteria were isolated from patients with bloodstream infections (BSI) between 2018 and 2023. A total of 89.66% of the isolates were carbapenem-resistant Enterobacteriaceae (CRE), with the majority (75/116) being CRKP. Among these, a novel plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A) simultaneously was found in CRKP46, and the three genes mediated conjugation by IS26, ISAba125, and IS26, respectively. This plasmid conferred carbapenem resistance to E. coli J53 after conjugative transfer, which was 2 times greater than that of CRKP46., Conclusion: The present study identified the occurrence of a rare plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A), and the spread of these genes was mediated by the corresponding mobile elements. The increased carbapenem resistance created by this novel plasmid challenges public health security and poses a potential threat to human health; therefore, it deserves attention., (Copyright © 2024 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

In recent decades, there has been a rapid increase in the prevalence of multidrug-resistant pathogens, posing a challenge to modern antibiotic-based medicine. This has highlighted the need for novel treatments that can specifically affect the target microorganism without disturbing other co-inhabiting species, thus preventing the development of dysbiosis in treated patients. Moreover, there is a pressing demand for tools to effectively manipulate complex microbial populations. One of the approaches suggested to address both issues was to use conjugation as a tool to modify the microbiome by either editing the genome of specific bacterial species and/or the removal of certain taxonomic groups. Conjugation involves the transfer of DNA from one bacterium to another, which opens up the possibility of introducing, modifying or deleting specific genes in the recipient. In response to this proposal, there has been a significant increase in the number of studies using this method for gene delivery in bacterial populations. This MicroReview aims to provide a detailed overview on the use of conjugation for microbiome engineering, and at the same time, to initiate a discussion on the potential, limitations and possible future directions of this approach., (© 2023 John Wiley & Sons Ltd.)

Klebsiella pneumoniae (Kp) is increasingly recognized as a reservoir for a range of antibiotic resistance genes and a pathogen that frequently causes severe infections in both hospital and community settings. In this study, we have identified a novel mechanism of conjugative transfer of a non-conjugative virulence plasmid through the formation of a fusion plasmid between the virulence plasmid and a novel 59,162 bp IncN- plasmid. This plasmid was found to be a multidrug-resistance (MDR) plasmid and carried a T4SS cluster, which greatly facilitated the efficient horizontal transfer of the fusion plasmid between Kp strains. The fused virulence plasmid conferred the resistance of serum killing and macrophage phagocytosis to the transconjugants. Importantly, this plasmid was shown to be essential for Kp virulence in a mouse model. Mechanistic analysis revealed that the virulence factors encoded by this virulence plasmid contributed to resistance to in vivo clearance and induced a high level of proinflammatory cytokine IL-1β, which acts as an inducer for more neutrophil recruitment. The transmission of the fusion plasmid in Kp has the potential to convert it into both MDR and hypervirulent Kp, accelerating its evolution, and posing a serious threat to human health. The findings of this study provide new insights into the rapid evolution of MDR and hypervirulent Kp in recent years., 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 GmbH. All rights reserved.)

Pasteurella multocida ( P. multocida ) is a bacterial pathogen responsible for a range of infections in humans and various animal hosts, causing significant economic losses in farming. Integrative and conjugative elements (ICEs) are important horizontal gene transfer elements, potentially enabling host bacteria to enhance adaptability by acquiring multiple functional genes. However, the understanding of ICEs in P. multocida and their impact on the transmission of this pathogen remains limited. In this study, 42 poultry-sourced P. multocida genomes obtained by high-throughput sequencing together with 393 publicly available P. multocida genomes were used to analyse the horizontal transfer of ICEs. Eighty-two ICEs were identified in P. multocida , including SXT/R391 and Tn916 subtypes, as well as three subtypes of ICE Hin 1056 family, with the latter being widely prevalent in P. multocida and carrying multiple resistance genes. The correlations between insertion sequences and resistant genes in ICEs were also identified, and some ICEs introduced the carbapenem gene bla OXA-2 and the bleomycin gene bleO to P. multocida . Phylogenetic and collinearity analyses of these bioinformatics found that ICEs in P. multocida were transmitted vertically and horizontally and have evolved with host specialization. These findings provide insight into the transmission and evolution mode of ICEs in P. multocida and highlight the importance of understanding these elements for controlling the spread of antibiotic resistance.

This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral strain ECNX52 was constructed by introducing the bla NDM-5 -carrying IncX3 plasmid into E. coli C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the repA mutation on plasmid copy number was determined. Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 μg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein ompF . Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 μg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by repA D140Y (GAT→TAT) mutation. Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.

Mating assays are common laboratory experiments for measuring the conjugation frequency, i.e. efficiency at which a plasmid transfers from a population of donor cells to a population of recipient cells. Selective plating remains a widely used quantification method to enumerate transconjugants at the end of such assays. However, conjugation frequencies may be inaccurately estimated because plasmid transfer can occur on transconjugant-selective plates rather than only during the intended mating duration. We investigated the influence of cell density on this phenomenon. We conducted mating experiments with IncPα plasmid RP4 harbored in Escherichia coli at a fixed cell density and mating conditions, inoculated a serial dilution of the mating mixture on transconjugant-selective plates or in transconjugant-selective broth, and compared the results to a model of cell-to-cell distance distribution. Our findings suggest that irrespective of the mating mode (liquid vs solid), the enumeration of transconjugants becomes significantly biased if the plated cell density exceeds 28 Colony Forming Unit (CFU)/mm 2 (or 1.68•10 5  CFU/standard 9 cm Petri dish). This threshold is determined with a 95% confidence interval of ± 4 CFU/mm 2 (± 2.46•10 4  CFU/standard 9 cm Petri dish). Liquid mating assays were more sensitive to this bias because the conjugation frequency of RP4 is several orders of magnitude lower in suspension compared to surface mating. Therefore, if selective plating is used, we recommend to plate at this density threshold and that negative controls are performed where donors and recipients are briefly mixed before plating at the same dilutions as for the actual mating assay. As an alternative, a liquid enumeration method can be utilized to increase the signal-to-noise ratio and allow for more accurate enumeration of transconjugants., (© 2024. The Author(s).)

Nanocolloids (Nc) are widespread in natural water environment, whereas the potential effects of Nc on dissemination of antibiotic resistance remain largely unknown. In this study, Nc collected from the Yellow River in Henan province was tested for its ability to influence the conjugative transfer of resistant plasmid in aqueous environment. The results revealed that the conjugative transfer of RP4 plasmid between Escherichia coli was down-regulated by 52%-91% upon exposure to 1-10 mg/L Nc and the reduction became constant when the dose became higher (20-200 mg/L). Despite the exposure of Nc activated the anti-oxidation and SOS response in bacteria through up-regulating genes involved in glutathione biosynthesis and DNA recombination, the inhibition on the synthesis and secretion of extracellular polysaccharide induced the prevention of cell-cell contact, leading to the reduction of plasmid transfer. This was evidenced by the decreased bacterial adhesion and lowered levels of genes and metabolites relevant to transmembrane transport and D-glucose phosphorylation, as clarified in phenotypic, transcriptomics and metabolomics analysis of E. coli. The significant down-regulation of glycolysis/gluconeogenesis and TCA cycle was associated with the shortage of ATP induced by Nc. The up-regulation of global regulatory genes (korA and trbA) and the reduction of plasmid genes (trfAp, trbBp, and traG) expression also contributed to the suppressed conjugation of RP4 plasmid. The obtained findings remind that the role of ubiquitous colloidal particles is nonnegligible when practically and comprehensively assessing the risk of antibiotic resistance in the environment., 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 Ltd. All rights reserved.)

Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has three predicted extracellular hydrolase domains: metallo-peptidase (LytM), soluble lytic transglycosylase (SLT), and cysteine, histidine-dependent amidohydrolases/peptidases (CHAP). Here, we report the structure of the LytM domain and show that its active site is degenerate and lacks the active site metal. Furthermore, we show that only the predicted SLT domain is functional in vitro and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Furthermore, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information for understanding the function of Gram-positive T4SSs.IMPORTANCEAntibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from Enterococcus faecalis . This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in E. faecalis as well as other medically relevant Gram-positive bacteria., Competing Interests: The authors declare no conflict of interest.

Toxin-antitoxin systems (TAs) are abundant in bacterial chromosomes and can arrest growth under stress, but usually remain inactive. TAs have been increasingly implicated in halting the growth of infected bacteria from bacteriophages or foreign genetic elements 1,2 to protect the population (abortive infection, Abi). The vast diversity and abundance of TAs and other Abi systems 3 suggest they play an important immunity role, yet what allows them to sense attack remains largely enigmatic. Here, we describe a method called toxin activation-inhibition conjugation (TAC-TIC), which we used to identify gene products that trigger or block the toxicity of phage-defending tripartite retron-TAs 4 . TAC-TIC employs high-density arrayed mobilizable gene-overexpression libraries, which are transferred into cells carrying the full TA system or only its toxic component, on inducible vectors. The double-plasmid transconjugants are then pinned on inducer-containing agar plates and their colony fitness is quantified to identify gene products that trigger a TA to inhibit growth (TAC), or that block it from acting (TIC). TAC-TIC is optimized for the Singer ROTOR pinning robot, but can also be used with other robots or manual pinners, and allows screening tens of thousands of genes against any TA or Abi (with toxicity) within a week. Finally, we present a dual conjugation donor/cloning strain (Escherichia coli DATC), which accelerates the construction of TAC-TIC gene-donor libraries from phages, enabling the use of TAC-TIC for identifying TA triggers and antidefense mechanisms in phage genomes., (© 2024. European Molecular Biology Laboratory, under exclusive licence to Springer Nature Limited.)

Plasmid-mediated conjugative transfer facilitates the dissemination of antibiotic resistance, yet the comprehensive regulatory mechanisms governing this process remain elusive. Herein, we established pure bacteria and activated sludge conjugation system to investigate the regulatory mechanisms of conjugative transfer, leveraging metformin as an exogenous agent. Transcriptomic analysis unveiled that substantial upregulation of genes associated with the two-component system (e.g., AcrB/AcrA, EnvZ/Omp, and CpxA/CpxR) upon exposure to metformin. Furthermore, downstream regulators of the two-component system, including reactive oxygen species (ROS), cytoplasmic membrane permeability, and adenosine triphosphate (ATP) production, were enhanced by 1.7, 1.4 and 1.1 times, respectively, compared to the control group under 0.1 mg/L metformin exposure. Moreover, flow sorting and high-throughput sequencing revealed increased microbial community diversity among transconjugants in activated sludge systems. Notably, the antibacterial potential of human pathogenic bacteria (e.g., Bacteroides, Escherichia-Shigella, and Lactobacillus) was augmented, posing a potential threat to human health. Our findings shed light on the spread of antibiotic resistance bacteria and assess the ecological risks associated with plasmid-mediated conjugative transfer in wastewater treatment systems., 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 Ltd. All rights reserved.)

Antimicrobial resistance (AMR) poses a significant threat to global public health. Notably, resistance to carbapenem and extended-spectrum β-lactam antibiotics in Gram-negative bacteria is a major impediment to treating infections. Genes responsible for antibiotic resistance are frequently carried on plasmids, which can transfer between bacteria. Therefore, exploring strategies to prevent this transfer and the prevalence of AMR plasmids is timely and pertinent. Here, we show that certain natural product extracts and associated pure compounds can reduce the conjugation of AMR plasmids into new bacterial hosts. Using our established high-throughput fluorescence-based flow cytometry assay, we found that the natural products were more active in reducing transmission of the IncK extended-spectrum β-lactamase-encoding plasmid pCT in Escherichia coli EC958c, compared to Klebsiella pneumoniae Ecl8 carrying the IncFII carbapenemase-encoding plasmid pKpQIL. The exception was the natural product rottlerin, also active in K. pneumoniae . In classical conjugation assays, rottlerin also reduced the conjugation frequency of the IncFII bla NDM-1 carrying plasmid pCPE16&#95;3 from a clinical K. pneumoniae isolate. Our data indicate that the natural products tested here, in their current molecular structure, reduced conjugation by a small amount, which is unlikely to achieve a large-scale reduction in AMR in bacterial populations. However, certain natural products like rottlerin could provide a foundation for further research into compounds with effective anti-plasmid activity.

Ammonia availability has a crucial role in agriculture as it ensures healthy plant growth and increased crop yields. Since diazotrophs are the only organisms capable of reducing dinitrogen to ammonia, they have great ecological importance and potential to mitigate the environmental and economic costs of synthetic fertilizer use. Rhizobia are especially valuable being that they can engage in nitrogen-fixing symbiotic relationships with legumes, and they demonstrate great diversity and plasticity in genomic and phenotypic traits. However, few rhizobial species have sufficient genetic tractability for synthetic biology applications. This study established a basic genetic toolbox with antibiotic resistance markers, multi-host shuttle plasmids and a streamlined protocol for biparental conjugation with Mesorhizobium and Bradyrhizobium species. We identified two repABC origins of replication from Sinorhizobium meliloti (pSymB) and Rhizobium etli (p42d) that were stable across all three strains of interest. Furthermore, the NZP2235 genome was sequenced and phylogenetic analysis determined its reclassification to Mesorhizobium huakuii . These tools will enable the use of plasmid-based strategies for more advanced genetic engineering projects and ultimately contribute towards the development of more sustainable agriculture practices by means of novel nitrogen-fixing organelles, elite bioinoculants, or symbiotic association with nonlegumes., Competing Interests: The authors declare there are no competing interests.

The use of antimicrobials in poultry leaves residues in the litter, favoring the emergence of antimicrobial-resistant pathogens and making it a source of contamination. An in vitro 4 × 4 factorial trial was performed to investigate the influence of four treatments, consisting of antimicrobial sub-concentrations, on the transference of IncB/O-plasmid through conjugation in four groups. Each group was composed of one plasmid donor bacterium (Escherichia coli H2332) and a recipient bacterium (Escherichia coli J62 or Salmonella enterica serovars, Enteritidis, Typhimurium, or Heidelberg). Our results showed a little decrease in the conjugation frequency in almost all treatments between the two bacterial species, which varied according to each strain. The MIC test revealed an increase of up to 4096-fold in resistance to beta-lactams in Salmonella serovars after plasmid acquisition. This finding suggests that some genetic apparatus may be involved in increased antimicrobial resistance in Salmonella serovars after the acquisition of primary resistance determinants., 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 Ltd. All rights reserved.)

Objectives: To characterize blaNDM-carrying Salmonella recovered from a pig slaughterhouse., Methods: In this study, 46 environment samples were collected from a slaughterhouse in China, and screened for carbapenem-resistant Enterobacterales. WGS, antimicrobial susceptibility testing and conjugation experiments were carried out to identify the isolates' resistance phenotypes and genetic characteristics. The phylogenetic relatedness of the Salmonella isolates obtained in this study and Salmonella (ST34 and ST29) in GenBank was determined., Results: Two ST34 Salmonella Typhimurium and one ST29 Salmonella Stanley, recovered from three environmental samples (6.52%), were positive for blaNDM-1 and blaNDM-5, respectively. The two ST34 S. Typhimurium strains exhibited a close relationship (10-36 SNPs) with two human-derived blaNDM-1-bearing isolates from China (Hong Kong and Guangxi Province) and two blaNDM-negative ST34 Salmonella strains from the UK. The blaNDM-1 genes were located on IncHI2/ST3 plasmids. The capture of blaNDM-1 by the IncHI2/ST3 plasmid seems to be due to homologous recombination mediated by circular structures, as the genetic arrangements of the blaNDM-1 gene contain two IS26 elements of the same orientation. The blaNDM-5 gene was also carried by the IncHI2/ST3 plasmid, which shares highly similar structures with other blaNDM-5-bearing IncHI2/ST3 plasmids from other sources (fish, chicken, duck, human)., Conclusions: This is the first report of a blaNDM-5-carrying IncHI2/ST3 plasmid in Salmonella. The clonal spread of NDM-1-producing ST34 S. Typhimurium across human and animal-associated environments, and the widespread dissemination of epidemic blaNDM-5-carrying IncHI2/ST3 plasmids among Enterobacteriaceae in China indicate the potential of further dissemination of blaNDM among Salmonella, which poses a threat to public health., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Objective: To investigate the effect of low concentration of Wenyang Tonglin Decoction (WTD) on the binding conditions of R45 plasmid conjugative transfer under liquid phase conjugation and its mechanism., Methods: Escherichia coli CP9 (R45) and Staphylococcus aureus RN450RF were cultured in medium containing WTD, and their minimum inhibitory concentration (MIC) values were obtained. Using promoter fusion technology, E. coli CP9 (R45) containing a promoter fusion was obtained. β-Galactosidase activity of TrfAp and TrbBp was tested, and the mRNA expression of regulatory factors (TrbA, KorA, and KorB) was detected by real-time fluorescent quantitative polymerase chain reaction., Results: The MIC of E. coli CP9 (R45) was 400 g/L and that of S. aureus RN450RF was 200 g/L. When the drug concentration in the culture medium was 200 g/L, the highest number of conjugants was (3.47 ±0.20) × 10 7 CFU/mL At 90 h of conjugation, the maximum number of conjugants was (1.15 ±0.06) × 10 8 CFU/mL When the initial bacterial concentration was 10 8 CFU/mL, the maximum number of conjugants was (3.47 ± 0.20) × 10 7 CFU/mL. When the drug concentration was 200 g/L, the β-galactosidase activity of TrfAp and TrbBp significantly increased; the relative quantification of TrbA, KorA and KorB were significantly inhibited., Conclusion: Low concentration of WTD promoted the development of bacterial resistance by affecting promoters and inhibiting the expression of regulatory factors., (© 2024. The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a recipient bacterial cell. Bacterial conjugation is the primary means by which antibiotic resistance genes spread among bacterial populations (Barlow 2009; Virolle et al, 2020). Conjugative T4SSs form pili: long extracellular filaments that connect with recipient cells. Previously, we solved the cryo-electron microscopy (cryo-EM) structure of a conjugative T4SS. In this article, based on additional data, we present a more complete T4SS cryo-EM structure than that published earlier. Novel structural features include details of the mismatch symmetry within the OMCC, the presence of a fourth VirB8 subunit in the asymmetric unit of both the arches and the inner membrane complex (IMC), and a hydrophobic VirB5 tip in the distal end of the stalk. Additionally, we provide previously undescribed structural insights into the protein VirB10 and identify a novel regulation mechanism of T4SS-mediated pilus biogenesis by this protein, that we believe is a key checkpoint for this process., (© 2024. The Author(s).)