A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii.

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

Modular and integrative activity reporters enhance biochemical studies in the yeast ER.

The yeast endoplasmic reticulum sequestration and screening (YESS) system is a broadly applicable platform to perform high-throughput biochemical studies of post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.

Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes.

Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.

DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity.

Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.

Variation in the plasmid backbone and dif module content of R3-T33 Acinetobacter plasmids.

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

Plasmid partitioning driven by collective migration of ParA between nucleoid lobes.

The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy-number plasmids. However, despite extensive research, the spatiotemporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo have remained unclear. In this study, we utilize high-throughput imaging, quantitative data analysis, and computational modeling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid. As previously observed, we find that F-plasmid ParA undergoes collective migrations ("flips") between cell halves multiple times per cell cycle. We reveal that a constricting nucleoid is required for these migrations and that they are triggered by a plasmid crossing into the cell half with greater ParA. Using simulations, we show that these dynamics can be explained by the combination of nucleoid constriction and cooperative ParA binding to the DNA, in line with the behavior of other ParA proteins. We further show that these ParA flips act to equally partition plasmids between the two lobes of the constricted nucleoid and are therefore important for plasmid stability, especially in fast growth conditions for which the nucleoid constricts early in the cell cycle. Overall, our work identifies a second mode of action of the ParABS system and deepens our understanding of how this important segregation system functions.

Is the distribution of plasmid lengths bimodal?

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

Improved Manufacturing Methods of Extracellular Vesicles Pseudotyped with the Vesicular Stomatitis Virus Glycoprotein.

Extracellular vesicles (EV), which expose the vesicular stomatitis virus glycoprotein (VSVG) on their surface, are used for delivery of nucleic acids and proteins in human cell lines. These particles are biomanufactured using methods that are difficult to scale up. Here, we describe the development of the first EV-VSVG production process in serum-free media using polyethylenimine (PEI)-based transient transfection of HEK293 suspension cells, as well as the first EV-VSVG purification process to utilize both ultracentrifugation and chromatography. Three parameters were investigated for EV-VSVG production: cell density, DNA concentration, and DNA:PEI ratio. The best production titer was obtained with 3 × 106 cells/mL, a plasmid concentration of 2 µg/mL, and a DNA:PEI ratio of 1:4. The production kinetics of VSVG was performed and showed that the highest amount of VSVG was obtained 3 days after transfection. Addition of cell culture supplements during the transfection resulted in an increase in VSVG production, with a maximum yield obtained with 2 mM of sodium butyrate added 18 h after transfection. Moreover, the absence of EV-VSVG during cell transfection with a GFP-coding plasmid revealed to be ineffective, with no fluorescent cells. An efficient EV-VSVG purification procedure consisting of a two-step concentration by low-speed centrifugation and sucrose cushion ultracentrifugation followed by a heparin affinity chromatography purification was also developed. Purified bioactive EV-VSVG preparations were characterized and revealed that EV-VSVG are spherical particles of 176.4 ± 88.32 nm with 91.4% of protein similarity to exosomes.

The Epstein-Barr Virus Enhancer Interaction Landscapes in Virus-Associated Cancer Cell Lines.

Epstein-Barr virus (EBV) persists in human cells as episomes. EBV episomes are chromatinized and their 3D conformation varies greatly in cells expressing different latency genes. We used HiChIP, an assay which combines genome-wide chromatin conformation capture followed by deep sequencing (Hi-C) and chromatin immunoprecipitation (ChIP), to interrogate the EBV episome 3D conformation in different cancer cell lines. In an EBV-transformed lymphoblastoid cell line (LCL) GM12878 expressing type III EBV latency genes, abundant genomic interactions were identified by H3K27ac HiChIP. A strong enhancer was located near the BILF2 gene and looped to multiple genes around BALFs loci. Perturbation of the BILF2 enhancer by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) altered the expression of BILF2 enhancer-linked genes, including BARF0 and BALF2, suggesting that this enhancer regulates the expression of linked genes. H3K27ac ChIP followed by deep sequencing (ChIP-seq) identified several strong EBV enhancers in T/NK (natural killer) lymphoma cells that express type II EBV latency genes. Extensive intragenomic interactions were also found which linked enhancers to target genes. A strong enhancer at BILF2 also looped to the BALF loci. CRISPRi also validated the functional connection between BILF2 enhancer and BARF1 gene. In contrast, H3K27ac HiChIP found significantly fewer intragenomic interactions in type I EBV latency gene-expressing primary effusion lymphoma (PEL) cell lines. These data provided new insight into the regulation of EBV latency gene expression in different EBV-associated tumors. IMPORTANCE EBV is the first human DNA tumor virus identified, discovered over 50 years ago. EBV causes ~200,000 cases of various cancers each year. EBV-encoded oncogenes, noncoding RNAs, and microRNAs (miRNAs) can promote cell growth and survival and suppress senescence. Regulation of EBV gene expression is very complex. The viral C promoter regulates the expression of all EBV nuclear antigens (EBNAs), some of which are very far away from the C promoter. Another way by which the virus activates remote gene expression is through DNA looping. In this study, we describe the viral genome looping patterns in various EBV-associated cancer cell lines and identify important EBV enhancers in these cells. This study also identified novel opportunities to perturb and eventually control EBV gene expression in these cancer cells.

Plasmid carriage and the natural complexity of bacterial populations contributes to plasmid persistence / El transporte de plásmidos y la complejidad natural de las poblaciones bacterianas contribuyen a la persistencia del plásmido

Introduction: Plasmids carry and transport genes that assist their hosts to survive in many environments. Many studies have examined the conditions for plasmid persistence in bacterial populations. A limitation includes that a majority of the mathematical models for examining plasmid persistence only included bacteria from similar colonies. However, most bacterial cells inhabit complex communities where plasmids disseminate between varied bacterial host cells. Thus, there is a gap in knowledge concerning the persistence of plasmids in natural bacterial populations. To address a few of these gaps in knowledge, the present study attempted to examine the effects of plasmid carriage on intrinsic stages of bacterial populations in Bacillus subtilis co-cultures. Material and methods: B. subtilis cells were transformed with CRISPR-hCas-9 plasmid vectors where the natural phases of bacterial growth, biofilm production, and antibiotic resistance were examined in relation to plasmid carriage. These three natural phases were measured in relation to plasmid carriage through in vitro co-culture assays. Results: After calculating the CFU/mL, bacterial growth in the B. subtilis-Carrier with Escherichia coli (B. sub-C-E. coli) and Vibrio harveyi (B. sub-C-VH) co-cultures significantly decreased with a paired-t-test two-tailed P=0. The WT B. subtilis-V.H samples, the B. subtilis Carrier-V.H co-cultures, and the controls each scored a total of 40, 47, and 46 of crystal violet (CV) intensity of biofilm, respectively. Biofilm formation decreased after co-culturing E. coli with the B. subtilis-Carrier, yielding a P<0.001. The antibiotic resistance levels of the co-cultures increased by 3% for the B. sub-C-V.H samples while the B. sub-C-E. coli co-cultures decreased in antibiotic sensitivity by approximately 1.5%. Conclusions: Plasmid carriage contributes to plasmid persistence via altering the natural phases of bacterial populations (AU)

Introducción: Los plásmidos portan y transportan genes que ayudan a sus huéspedes a sobrevivir en muchos entornos. Muchos estudios han examinado las condiciones para la persistencia de plásmidos en poblaciones bacterianas. Una limitación incluye que la mayoría de los modelos matemáticos para examinar la persistencia de plásmidos solo incluyeron bacterias de colonias similares. Sin embargo, la mayoría de las células bacterianas habitan en comunidades complejas donde los plásmidos se diseminan entre diversas células huésped bacterianas. Por lo tanto, existe un vacío en el conocimiento sobre la persistencia de plásmidos en poblaciones bacterianas naturales. Para abordar algunas de estas lagunas en el conocimiento, el presente estudio intentó examinar los efectos del transporte de plásmidos en las etapas intrínsecas de las poblaciones bacterianas en cocultivos de Bacillus subtilis. Material y métodos: Células de B. subtilis se transformaron con vectores plasmídicos CRISPR-hCas-9 donde se examinaron las fases naturales de crecimiento bacteriano, producción de biopelículas y resistencia a los antibióticos en relación con el transporte del plásmido. Estas tres fases naturales se midieron en relación con el transporte de plásmidos a través de ensayos de cocultivo in vitro. Resultados: Después de calcular las UFC/mL, el crecimiento bacteriano en los cocultivos de B. subtilis-Carrier con Escherichia coli (B. sub-C-E. coli) y Vibrio harveyi (B. sub-C-VH) disminuyó significativamente con un -t-test de dos colas P=0. Las muestras WT B. subtilis-V.H, los cocultivos B. subtilis Carrier-V.H y los controles obtuvieron cada uno un total de 40, 47 y 46 de intensidad de biopelícula cristal violeta (CV), respectivamente. La formación de biopelículas disminuyó después de cocultivar E. coli con B. subtilis-Carrier, lo que arrojó un P<0,001 (AU)

Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers.

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.

Rapid, Efficient, and Cost-Effective Gene Editing of Enterococcus faecium with CRISPR-Cas12a.

Considered a serious threat by the Centers for Disease Control and Prevention, multidrug-resistant Enterococcus faecium is an increasing cause of hospital-acquired infection. Here, we provide details on a single-plasmid CRISPR-Cas12a system for generating clean deletions and insertions. Single manipulations were carried out in under 2 weeks, with successful deletions/insertions present in >80% of the clones tested. Using this method, we generated three individual clean deletion mutations in the acpH, treA, and lacL genes and inserted codon-optimized unaG, enabling green fluorescent protein (GFP)-like fluorescence under the control of the trehalase operon. The use of in vivo recombination for plasmid construction kept costs to a minimum. IMPORTANCE Enterococcus faecium is increasingly associated with hard-to-treat antibiotic-resistant infections. The ability to generate clean genomic alterations is the first step in generating a complete mechanistic understanding of how E. faecium acquires pathogenic traits and causes disease. Here, we show that CRISPR-Cas12a can be used to quickly (under 2 weeks) and cheaply delete or insert genes into the E. faecium genome. This substantial improvement over current methods should speed up research on this important opportunistic pathogen.

Escherichia fergusonii, an Underrated Repository for Antimicrobial Resistance in Food Animals.

A total of 1,400 samples of food animals (pigs, chickens, and ducks) were collected between July and September 2019 in China to uncover the prevalence of E. fergusonii and its potential role in the evolution of antimicrobial resistance (AMR). An isolation of E. fergusonii was performed and pulsed-field gel electrophoresis (PFGE) was used to uncover the genetic relationship. The AMR of E. fergusonii isolates was comprehensively characterized using broth microdilution-based antimicrobial susceptibility testing, S1-PFGE, southern hybridization, whole-genome sequencing, and in-depth bioinformatics analysis. As a result, a total of 133 E. fergusonii isolates were obtained. These isolates could be grouped into 41 PFGE subclades, suggesting a diverse genetic relationship. The resistance phenotypes of sulfafurazole (97.74%) and tetracycline (94.74%) were the most frequently found. Of the E. fergusonii isolates, 51.88% were extended spectrum beta-lactamase (ESBL)-positive. Forty-three different AMR genes were revealed based on 25 genome sequences harboring mcr-1. Briefly, aph(6)-Id, aph(3'')-Ib and tet(A) genes were the most frequently observed, with the highest rate being 76.00% (19/25). Three mcr-1-harboring plasmids were identified after Nanopore sequencing, including pTB31P1 (IncHI2-IncHI2A, 184,652 bp), pTB44P3 (IncI2, 62,882 bp), and pTB91P1 (IncHI2-IncHI2A, 255,882 bp). Additionally, 25 E. fergusonii isolates harboring mcr-1 were clustered together with other E. fergusonii isolates from different regions and sources available in GenBank, suggesting a possible random process of mcr-1 transmission in E. fergusonii. In conclusion, E. fergusonii is widespread in food animals in China and might be an important reservoir of AMR genes, especially mcr-1, and facilitate the evolution of AMR. IMPORTANCEE. fergusonii, a member of the genus Escherichia, has been reported to transmit via the food chain and cause diseases in humans. However, the prevalence of multidrug-resistant E. fergusonii, especially mcr-1-positive E. fergusonii isolates, has rarely been reported. Here, we collected 1,400 samples from food animals in three provinces of China and obtained 133 E. fergusonii isolates (9.5%). We found that the prevalence of E. fergusonii isolates was diverse, with high levels of antimicrobial resistance. Among them, 18.8% E. fergusonii isolates carried the colistin resistance gene mcr-1. Thus, E. fergusonii may facilitate the evolution of colistin resistance as a reservoir of mcr-1. As far as we know, the prevalence and AMR of E. fergusonii in the food animals in this study was first reported in China. These findings increase our understanding of the role of E. fergusonii in public health and the evolution of antibiotic resistance.

Characterization of Genetic Elements Carrying mcr-1 Gene in Escherichia coli from the Community and Hospital Settings in Vietnam.

Colistin is widely used in agriculture and aquaculture as prophylaxis, particularly in Asia. Recently, mcr-1 and other mobilizable genes conferring colistin resistance have spread globally in community and hospital populations. Characterizing mcr-1 mobile genetic elements and host genetic background is important to understand the transmission of this resistance mechanism. We conducted whole-genome sequencing of 94 mcr-1-positive Escherichia coli isolates (Mcr1-Ec isolates) from human and animal feces, food, and water in a community cohort (N = 87) and from clinical specimens from a referral hospital (N = 7) in northern Vietnam. mcr-1 was plasmid-borne in 71 and chromosomally carried in 25 (2 isolates contain one copy on chromosome and one copy on a plasmid) of 94 E. coli isolates from the community and hospital settings. All seven clinical isolates carried mcr-1 on plasmids. Replicon types of mcr-1-carrying plasmids included IncI2, IncP, IncX4, and IncFIA single replicons and combinations of IncHI2, IncN, and IncX1 multireplicons. Alignment of a long-read sequence of an IncI2 plasmid from animal feces with short-read sequences of IncI2 plasmids from a healthy human, water, and hospitalized patients showed highly similar structures (query cover from 90% to 98%, overall identity of >81%). We detected the potential existence of multireplicon plasmids harboring mcr-1 regardless of sample setting, confirming 10/71 with long-read sequencing. An intact/conserved Tn6330 transposon sequence or its genetic context variants were found in 6/25 Mcr1-Ec isolates with chromosomally carried mcr-1. The dissemination of mcr-1 is facilitated by a high diversity of plasmid replicon types and a high prevalence of the chromosomal Tn6330 transposon. IMPORTANCE The article presented advances our understanding of genetic elements carrying mcr-1 in Escherichia coli in both community and hospital settings. We provide evidence to suggest that diverse plasmid types, including multireplicon plasmids, have facilitated the successful transmission of mcr-1 in different reservoirs. The widespread use of colistin in agriculture, where a high diversity of bacteria are exposed, has allowed the selection and evolution of various transmission mechanisms that will make it a challenge to get rid of. Colocalization of mcr-1 and other antibiotic resistance genes (ARGs) on multireplicon plasmids adds another layer of complexity to the rapid dissemination of mcr-1 genes among community and hospital bacterial populations and to the slow pandemic of antimicrobial resistance (AMR) in general.

Carbapenem Resistance in Acinetobacter nosocomialis and Acinetobacter junii Conferred by Acquisition of blaOXA-24/40 and Genetic Characterization of the Transmission Mechanism between Acinetobacter Genomic Species.

Carbapenem resistance is increasing among Gram-negative bacteria, including the genus Acinetobacter. This study aimed to characterize, for the first time, the development of carbapenem resistance in clinical isolates of Acinetobacter junii and Acinetobacter nosocomialis conferred by the acquisition of a plasmid-borne blaOXA-24/40 gene and also to characterize the dissemination of this gene between species of Acinetobacter. Carbapenem-resistant A. nosocomialis HUAV-AN66 and A. junii HUAV-AJ77 strains were isolated in the Arnau de Vilanova Hospital (Spain). The genomes were sequenced, and in silico analysis were performed to characterize the genetic environment and the OXA-24/40 transmission mechanism. Antibiotic MICs were determined, and horizontal transfer assays were conducted to evaluate interspecies transmission of OXA-24/40. Carbapenems MICs obtained were ≥64 mg/L for HUAV-AN66 and HUAV-AJ77. Genome analysis revealed the presence in both strains of a new plasmid, designated pHUAV/OXA-24/40, harboring the carbapenem-resistance gene blaOXA-24/40 and flanked by sequences XerC/XerD. pHUAV/OXA-24/40 was successfully transferred from A. nosocomialis and A. junii to a carbapenem-susceptible A. baumannii strain, thus conferring carbapenem resistance. A second plasmid (pHUAV/AMG-R) was identified in both clinical isolates for the successful horizontal transfer of pHUAV/OXA-24/40. blaOXA-24/40-carrying plasmids of the GR12 group and showing high identity with pHUAV/OXA-24/40 were identified in at least 8 Acinetobacter species. In conclusion the carbapenemase OXA-24/40 is described for the first time in A. nosocomialis and A. junii. In both isolates the blaOXA-24/40 gene was located in the GR12 pHUAV/OXA-24/40 plasmid. GR12 plasmids are implicated in the dissemination and spread of carbapenem resistance among Acinetobacter species. IMPORTANCE Acinetobacter baumannii is one of the most relevant pathogens in terms of antibiotic resistance. The main resistance mechanisms are the carbapenem-hydrolyzing class D ß-lactamases (CHDLs), especially OXA-23 and OXA-24/40. In addition to A. baumannii, there are other species within the genus Acinetobacter, which in general exhibit much lower resistance rates. In this work we characterize for the first time two clinical isolates of Acinetobacter nosocomialis and Acinetobacter junii, isolated in the same hospital, carrying the carbapenemase OXA-24/40 and displaying high resistance rates to carbapenems. By means of bioinformatics analysis we have also been able to characterize the mechanism by which this carbapenemase is horizontally transferred interspecies of Acinetobacter spp. The dissemination of carbapenemase OXA-24/40 between non-baumannii Acinetobacter species is concerning since it prevents the use of most ß-lactam antibiotics in the fight against these resistant isolates.

Metabolic engineering of Escherichia coli BL21 strain using simplified CRISPR-Cas9 and asymmetric homology arms recombineering.

BACKGROUND: The recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA with homology arms for precise genome editing. However, generation of homology arms is a time-consuming, costly and inefficient process that is often overlooked. RESULTS: In this study, we first optimized a CRISPR-Cas genome engineering protocol in the Escherichia coli (E. coli) BL21 strain and successfully deleted 10 kb of DNA from the genome in one round of editing. To further simplify the protocol, asymmetric homology arms were produced by PCR in a single step with two primers and then purified using a desalting column. Unlike conventional homology arms that are prepared through overlapping PCR, cloning into a plasmid or annealing synthetic DNA fragments, our method significantly both shortened the time taken and reduced the cost of homology arm preparation. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across the BL21 genome. Noteworthy, gRNA design is important for the CRISPR-Cas system and a general heuristic gRNA design has been proposed in this study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene yield by ~ threefold. CONCLUSIONS: Our work has optimized the homology arms design for gene deletion in BL21. The protocol efficiently edited BL21 to improve lycopene production. The same workflow is applicable to any E. coli strain in which genome engineering would be useful to further increase metabolite production.

EGF domain peptide of Developmentally regulated endothelial locus1 facilitates gene expression of extracellularly applied plasmid DNA.

BACKGROUND: The successful development of messenger RNA vaccines for SARS-CoV-2 opened up venues for clinical nucleotide-based vaccinations. For development of DNA vaccines, we tested whether the EGF domain peptide of Developmentally regulated endothelial locus1 (E3 peptide) enhances uptake of extracellularly applied plasmid DNA. METHODS: DNA plasmid encoding lacZ or GFP was applied with a conditioned culture medium containing E3 peptide to cell lines in vitro or mouse soleus muscles in vivo, respectively. After 48 h incubation, gene expression was examined by ß-galactosidase (ß-gal) assay and fluorescent microscope, respectively. RESULTS: Application of E3 peptide-containing medium to cultured cell lines induced intense ß-gal activity in a dose-dependent manner. Intra-gastrocnemius injection of E3 peptide-containing medium to mouse soleus muscle succeeded in the induction of GFP fluorescence in many cells around the injection site. CONCLUSIONS: The administration of E3 peptide facilitates transmembrane uptake of extracellular DNA plasmid which induces sufficient extrinsic gene expression.

Plasmid Fusion and Recombination Events That Occurred during Conjugation of poxtA-Carrying Plasmids in Enterococci.

Linezolid plays a crucial role in the treatment of infections caused by multiresistant Gram-positive bacteria. The poxtA gene not only confers oxazolidinone and phenicol resistance but also decreases susceptibility to tetracycline. In this study, we investigated structural changes in mobilizable poxtA-carrying plasmids in enterococci which occurred during conjugation experiments using S1-PFGE (pulsed-field gel electrophoresis), Southern blot hybridization, and whole-genome sequencing (WGS) analysis. Two poxtA-carrying strains were identified in Enterococcus faecalis E006 and Enterococcus lactis E843, respectively. E. faecalis E006 contains the 121,520-bp conjugative plasmid pE006-121 and the 19,832-bp mobilizable poxtA-carrying plasmid pE006-19, while E. lactis E843 contains the 171,930-bp conjugative plasmid pE843-171 and the 27,847-bp mobilizable poxtA-carrying plasmid pE843-27. Moreover, both poxtA-carrying plasmids were mobilized by their respective conjugative plasmid in enterococci by plasmid fusion; one was generated by homologous recombination in E. faecalis through an identical 864-bp homologous region in the plasmids of the parental strain, while another was generated by an IS1216E-mediated plasmid integration in E. lactis, involving a replicative transposition. IMPORTANCE Until now, all the poxtA genes described in enterococci, including E. faecalis, E. faecium, and E. hirae, are plasmid-borne, suggesting that plasmids play an important role in the dissemination of the poxtA gene among enterococci. This study showed that the mobilizable poxtA-carrying plasmid could transfer with the help of conjugative plasmid in enterococci via plasmid fusion, with one generated by homologous recombination in E. faecalis, and another by replicative transposition in E. lactis. During both the fusion events, the poxtA-carrying plasmids changed from nonconjugative to conjugative, leading to the generation and enhanced dissemination of the larger phenicol-oxazolidinone-tetracycline resistance-encoding plasmids in enterococci.

Intracellular Transposition and Capture of Mobile Genetic Elements following Intercellular Conjugation of Multidrug Resistance Conjugative Plasmids from Clinical Enterobacteriaceae Isolates.

Mobile genetic elements (MGEs) are often associated with antimicrobial resistance genes (ARGs). They are responsible for intracellular transposition between different replicons and intercellular conjugation and are therefore important agents of ARG dissemination. Detection and characterization of functional MGEs, especially in clinical isolates, would increase our understanding of the underlying pathways of transposition and recombination and allow us to determine interventional strategies to interrupt this process. Entrapment vectors can be used to capture active MGEs, as they contain a positive selection genetic system conferring a selectable phenotype upon the insertion of an MGE within certain regions of that system. Previously, we developed the pBACpAK entrapment vector that results in a tetracycline-resistant phenotype when MGEs translocate and disrupt the cI repressor gene. We have previously used pBACpAK to capture MGEs in clinical Escherichia coli isolates following transformation with pBACpAK. In this study, we aimed to extend the utilization of pBACpAK to other bacterial taxa. We utilized an MGE-free recipient E. coli strain containing pBACpAK to capture MGEs on conjugative, ARG-containing plasmids following conjugation from clinical Enterobacteriaceae donors. Following the conjugative transfer of multiple conjugative plasmids and screening for tetracycline resistance in these transconjugants, we captured several insertion sequence (IS) elements and novel transposons (Tn7350 and Tn7351) and detected the de novo formation of novel putative composite transposons where the pBACpAK-located tet(A) is flanked by ISKpn25 from the transferred conjugative plasmid, as well as the ISKpn14-mediated integration of an entire 119-kb, blaNDM-1-containing conjugative plasmid from Klebsiella pneumoniae. IMPORTANCE By analyzing transposition activity within our MGE-free recipient, we can gain insights into the interaction and evolution of multidrug resistance-conferring MGEs following conjugation, including the movement of multiple ISs, the formation of composite transposons, and cointegration and/or recombination between different replicons in the same cell. This combination of recipient and entrapment vector will allow fine-scale experimental studies of factors affecting intracellular transposition and MGE formation in and from ARG-encoding MGEs from multiple species of clinically relevant Enterobacteriaceae.