Plasmid Assembly Research Articles

Gene expression screening and cell factory engineering for enhancing echinocandin B production in Aspergillus nidulans NRRL8112.

Echinocandin B (ECB) is a key precursor of the antifungal drug anidulafungin and its biosynthesis occurs via ani gene cluster in Aspergillus nidulans NRRL8112. Strain improvement for industrial ECB production has mainly relied on mutation breeding due to the lack of genetic tools. Here, a CRISPR-base-editing tool was developed in A. nidulans NRRL8112 for simultaneous inactivation of the nkuA gene and two marker genes, pryoA and riboB, which enabled efficient genetic manipulation. Then, in-vivo plasmid assembly was harnessed for ani gene expression screening, identifying the rate-limiting enzyme AniA and a pathway-specific transcription factor AniJ. Stepwise titer enhancement was achieved by overexpressing aniA and/or aniJ, and ECB production reached 1.5g/L during 5-L fed-batch fermentation, an increase of ~ 30-fold compared with the parent strain. This study, for the first time, revealed the regulatory mechanism of ECB biosynthesis and harnessed genetic engineering for the development of an efficient ECB-producing strain.

Sequencing Strategy to Ensure Accurate Plasmid Assembly.

Despite the wide use of plasmids in research and clinical production, the need to verify plasmid sequences is a bottleneck that is too often underestimated in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for de novo assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads) de novo assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most accurate, consistent assembly generation and identified mutations relative to the reference sequence. Although Sanger sequencing can be used to verify specific regions, some GC-rich and repetitive regions were difficult to resolve using any method, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs.

A modular toolkit for environmental Rhodococcus, Gordonia, and Nocardia enables complex metabolic manipulation.

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome but largely lack genetic tools comparable to those available in model species such as Escherichia coli or Pseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework, along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate Rhodococcus ruber C208, and demonstrate conserved functionality in 11 additional environmental isolates of Rhodococcus, Nocardia, and Gordonia. This toolkit encompasses five Mycobacteriale origins of replication, five broad-host-range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway in Rhodococcus erythropolis N9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux toward the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species.IMPORTANCESoil-dwelling Actinomycetes, particularly the Mycobacteriales, include both diverse new hosts for sustainable biomanufacturing and emerging opportunistic pathogens. Rhodococcus, Gordonia, and Nocardia are three abundant genera with particularly flexible metabolisms and untapped potential for natural product discovery. Among these, Rhodococcus ruber C208 was shown to degrade polyethylene; Gordonia paraffinivorans can assimilate carbon from solid hydrocarbons; and Nocardia neocaledoniensis (and many other Nocardia spp.) possesses dual isoprenoid biosynthesis pathways. Many species accumulate high levels of carotenoid pigments, indicative of highly active isoprenoid biosynthesis pathways which may be harnessed for fermentation of terpenes and other commodity isoprenoids. Modular genetic toolkits have proven valuable for both fundamental and applied research in model organisms, but such tools are lacking for most Actinomycetes. Our suite of genetic tools and DNA assembly framework were developed for broad functionality and to facilitate rapid prototyping of genetic constructs in these organisms.

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field.

Sequencing Strategy to Ensure Accurate Plasmid Assembly.

Despite the wide use of plasmids in research and clinical production, the need to verify plasmid sequences is a bottleneck that is too often underestimated in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for de novo assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads) de novo assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most accurate, consistent assembly generation and identified mutations relative to the reference sequence. Although Sanger sequencing can be used to verify specific regions, some GC-rich and repetitive regions were difficult to resolve using any method, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs.

Bacterial dynamics of the plastisphere microbiome exposed to sub-lethal antibiotic pollution

BackgroundAntibiotics and microplastics are two major aquatic pollutants that have been associated to antibiotic resistance selection in the environment and are considered a risk to human health. However, little is known about the interaction of these pollutants at environmental concentrations and the response of the microbial communities in the plastisphere to sub-lethal antibiotic pollution. Here, we describe the bacterial dynamics underlying this response in surface water bacteria at the community, resistome and mobilome level using a combination of methods (next-generation sequencing and qPCR), sequencing targets (16S rRNA gene, pre-clinical and clinical class 1 integron cassettes and metagenomes), technologies (short and long read sequencing), and assembly approaches (non-assembled reads, genome assembly, bacteriophage and plasmid assembly).ResultsOur results show a shift in the microbial community response to antibiotics in the plastisphere microbiome compared to surface water communities and describe the bacterial subpopulations that respond differently to antibiotic and microplastic pollution. The plastisphere showed an increased tolerance to antibiotics and selected different antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs). Several metagenome assembled genomes (MAGs) derived from the antibiotic-exposed plastisphere contained ARGs, virulence factors, and genes involved in plasmid conjugation. These include Comamonas, Chryseobacterium, the opportunistic pathogen Stenotrophomonas maltophilia, and other MAGs belonging to genera that have been associated to human infections, such as Achromobacter. The abundance of the integron-associated ciprofloxacin resistance gene aac(6’)-Ib-cr increased under ciprofloxacin exposure in both freshwater microbial communities and in the plastisphere. Regarding the antibiotic mobilome, although no significant changes in ARG load in class 1 integrons and plasmids were observed in polluted samples, we identified three ARG-containing viral contigs that were integrated into MAGs as prophages.ConclusionsThis study illustrates how the selective nature of the plastisphere influences bacterial response to antibiotics at sub-lethal selective pressure. The microbial changes identified here help define the selective role of the plastisphere and its impact on the maintenance of environmental antibiotic resistance in combination with other anthropogenic pollutants. This research highlights the need to evaluate the impact of aquatic pollutants in environmental microbial communities using complex scenarios with combined stresses.3sW_WiGiki6wh61j1XWikeVideo

The plasmidome associated with Gram-negative bloodstream infections: A large-scale observational study using complete plasmid assemblies

Plasmids carry genes conferring antimicrobial resistance and other clinically important traits, and contribute to the rapid dissemination of such genes. Previous studies using complete plasmid assemblies, which are essential for reliable inference, have been small and/or limited to plasmids carrying antimicrobial resistance genes (ARGs). In this study, we sequenced 1,880 complete plasmids from 738 isolates from bloodstream infections in Oxfordshire, UK. The bacteria had been originally isolated in 2009 (194 isolates) and 2018 (368 isolates), plus a stratified selection from intervening years (176 isolates). We demonstrate that plasmids are largely, but not entirely, constrained to a single host species, although there is substantial overlap between species of plasmid gene-repertoire. Most ARGs are carried by a relatively small number of plasmid groups with biological features that are predictable. Plasmids carrying ARGs (including those encoding carbapenemases) share a putative ‘backbone’ of core genes with those carrying no such genes. These findings suggest that future surveillance should, in addition to tracking plasmids currently associated with clinically important genes, focus on identifying and monitoring the dissemination of high-risk plasmid groups with the potential to rapidly acquire and disseminate these genes.

Nanopore adaptive sampling effectively enriches bacterial plasmids.

Antimicrobial resistance causes millions of deaths every year. Mobile genetic elements like bacterial plasmids are key drivers for the dissemination of antimicrobial resistance genes. This makes the characterization of plasmids via DNA sequencing an important tool for clinical microbiologists. Since plasmids are often underrepresented in bacterial samples, plasmid sequencing can be challenging and laborious. To accelerate the sequencing process, we evaluate nanopore adaptive sampling as an in silico method for the enrichment of low-abundant plasmids. Our results show the potential of this cost-efficient method for future plasmid research but also indicate issues that arise from using reference sequences.

Yeast recombinational cloning for heterologous biosynthesis of polyketides: a molecular microbiology laboratory module for undergraduate students.

Recombinant plasmids are essential tools in molecular biotechnology, and reliable plasmid assembly methods have, therefore, become a prerequisite for the successful cloning and transfer of genes. Among the multitude of available plasmid assembly strategies, in vivo homologous recombinational cloning in yeast has emerged as a cost-effective and relatively simple method. Since we use this method routinely in our group for assembling large plasmids with secondary metabolite gene clusters and for direct heterologous production of polyketides in Saccharomyces cerevisiae, we developed an exercise module for undergraduate students where they would get hands-on experience with these molecular practices. The exercises target several molecular techniques, including PCR, restriction enzyme digestion, and yeast recombinational cloning. The students will learn about plasmid assembly and yeast transformation methods by performing these experiments while inherently acquiring new skills valuable for their subsequent laboratory work or projects.

PlayBack cloning: simple, reversible, cost-effective cloning for the combinatorial assembly of complex expression constructs.

With advancements in multicomponent molecular biological tools, the need for versatile, rapid and cost-effective cloning that enables successful combinatorial assembly of DNA plasmids of interest is becoming increasingly important. Unfortunately, current cloning platforms fall short regarding affordability, ease of combinatorial assembly and, above all, the ability to iteratively remove individual cassettes at will. Herein we construct, implement and make available a broad set of cloning vectors, called PlayBack vectors, that allow for the expression of several different constructs simultaneously under separate promoters. Overall, this system is substantially cheaper than other multicomponent cloning systems, has usability for a wide breadth of experimental paradigms and includes the novel feature of being able to selectively remove components of interest at will at any stage of the cloning platform.

Characterization of the Environmental Plasmidome of the Red Sea.

Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments. However, while the number of marine microbiome studies is constantly increasing, very little is known about marine plasmids, and they are very poorly represented in public databases. To extend the repertoire of environmental marine plasmids, we established a pipeline for the de novo assembly of plasmids in the marine environment by analyzing available microbiome metagenomic sequencing data. By applying the pipeline to data from the Red Sea, we identified 362 plasmid candidates. We showed that the distribution of plasmids corresponds to environmental conditions, particularly, depth, temperature, and physical location. At least 7 of the 362 candidates are most probably real plasmids, based on a functional analysis of their open reading frames (ORFs). Only one of the seven has been described previously. Three plasmids were identified in other public marine metagenomic data from different locations all over the world; these plasmids contained different cassettes of functional genes at each location. Analysis of antibiotic and metal resistance genes revealed that the same positions that were enriched with genes encoding resistance to antibiotics were also enriched with resistance to metals, suggesting that plasmids contribute site-dependent phenotypic modules to their ecological niches. Finally, half of the ORFs (50.8%) could not be assigned to a function, emphasizing the untapped potential of the unique marine plasmids to provide proteins with multiple novel functions. IMPORTANCE Marine plasmids are understudied and hence underrepresented in databases. Plasmid functional annotation and characterization is complicated but, if successful, may provide a pool of novel genes and unknown functions. Newly discovered plasmids and their functional repertoire are potentially valuable tools for predicting the dissemination of antimicrobial resistance, providing vectors for molecular cloning and an understanding of plasmid-bacterial interactions in various environments.

Genomic characterisation of Campylobacter jejuni Cj26: A high-level ciprofloxacin/erythromycin-resistant strain isolated from a poultry carcass in southern Brazil

The draft genome sequence of Campylobacter jejuni (Cj26) was analysed to investigate genetic mechanisms of antimicrobial resistance, virulence-associated genes, and phylogenetic context. Antimicrobial resistance was assessed by agar dilution and disk diffusion. Cj26 was sequenced using NovaSeq 6000 technology. The genome was assembled and annotated. Resistance genes and chromosomal mutations were analysed using the Center for Genomic Epidemiology services, and the multilocus sequence type, SVR-flaA, and porA were determined. The virulome was determined using the Virulence Factor Database. Plasmid detection and assembly were performed using Unicycler v0.5.0 software. To infer the core genome phylogeny, prokka v1.14.5 was employed in conjunction with IQtree v2.0.3. The Cj26 strain showed a high level of resistance to ciprofloxacin (32 µg/mL) and erythromycin (>128 µg/mL) and resistance to tetracycline and ampicillin. Multilocus sequence typing revealed that the strain belonged to sequence type 353. The substitutions Tre-86-Ile in gyrA and A2075G in 23s RNA were detected, along with the genes tetO, aph(3')-III, ant(6)-Ia, and blaOXA 460. A consistent relationship among accessory and core genes was identified. When compared to other sequence type 353 genomes from Brazil, Cj26 clustered with strains that had more antimicrobial resistance genes than the other clusters. This report provides insight into the antimicrobial resistance determinants found in a C. jejuni strain and offers a valuable resource for further studies on Campylobacter genomics and antimicrobial resistance.

Large-scale analysis of putative plasmids in clinical multidrug-resistant Escherichia coli isolates from Vietnamese patients.

In the past decades, extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant (CR) Escherichia coli isolates have been detected in Vietnamese hospitals. The transfer of antimicrobial resistance (AMR) genes carried on plasmids is mainly responsible for the emergence of multidrug-resistant E. coli strains and the spread of AMR genes through horizontal gene transfer. Therefore, it is important to thoroughly study the characteristics of AMR gene-harboring plasmids in clinical multidrug-resistant bacterial isolates. The profiles of plasmid assemblies were determined by analyzing previously published whole-genome sequencing data of 751 multidrug-resistant E. coli isolates from Vietnamese hospitals in order to identify the risk of AMR gene horizontal transfer and dissemination. The number of putative plasmids in isolates was independent of the sequencing coverage. These putative plasmids originated from various bacterial species, but mostly from the Escherichia genus, particularly E. coli species. Many different AMR genes were detected in plasmid contigs of the studied isolates, and their number was higher in CR isolates than in ESBL-producing isolates. Similarly, the blaKPC-2, blaNDM-5, blaOXA-1, blaOXA-48, and blaOXA-181 β-lactamase genes, associated with resistance to carbapenems, were more frequent in CR strains. Sequence similarity network and genome annotation analyses revealed high conservation of the β-lactamase gene clusters in plasmid contigs that carried the same AMR genes. Our study provides evidence of horizontal gene transfer in multidrug-resistant E. coli isolates via conjugative plasmids, thus rapidly accelerating the emergence of resistant bacteria. Besides reducing antibiotic misuse, prevention of plasmid transmission also is essential to limit antibiotic resistance.

Multiplexed long-read plasmid validation and analysis using OnRamp.

Recombinant plasmid vectors are versatile tools that have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation; however, this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids owing to read-length limits. Although high-throughput sequencing does provide full-plasmid sequencing at scale, it is impractical and costly when used outside of library-scale validation. Here, we present Oxford nanopore-based rapid analysis of multiplexed plasmids (OnRamp), an alternative method for routine plasmid validation that combines the advantages of high-throughput sequencing's full-plasmid coverage and scalability with Sanger's affordability and accessibility by leveraging nanopore's long-read sequencing technology. We include customized wet-laboratory protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is deployed on the OnRamp web app, which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views. OnRamp is designed to be broadly accessible regardless of programming experience to facilitate more widespread adoption of long-read sequencing for routine plasmid validation. Here we describe the OnRamp protocols and pipeline and show our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure at less than half the cost of equivalent Sanger sequencing.

A modular cloning (MoClo) toolkit for reliable intracellular protein targeting in the yeast Saccharomyces cerevisiae

Modular Cloning (MoClo) allows the combinatorial assembly of plasmids from standardized genetic parts without the need of error-prone PCR reactions. It is a very powerful strategy which enables highly flexible expression patterns without the need of repetitive cloning procedures. In this study, we describe an advanced MoClo toolkit that is designed for the baker's yeast Saccharomyces cerevisiae and optimized for the targeting of proteins of interest to specific cellular compartments. Comparing different targeting sequences, we developed signals to direct proteins with high specificity to the different mitochondrial subcompartments, such as the matrix and the intermembrane space (IMS). Furthermore, we optimized the subcellular targeting by controlling expression levels using a collection of different promoter cassettes; the MoClo strategy allows it to generate arrays of expression plasmids in parallel to optimize gene expression levels and reliable targeting for each given protein and cellular compartment. Thus, the MoClo strategy enables the generation of protein-expressing yeast plasmids that accurately target proteins of interest to various cellular compartments.

Retraction for Kim et al., "Complete Sequence of blaKPC-2-Harboring Plasmid with a Mosaic of IncN1- and IncN3-Type Plasmids in a Klebsiella pneumoniae Isolate from South Korea".

Volume 60, no. 2, p. 1167–1169, 2016, https://doi.org/10.1128/aac.02532-15. We hereby retract the above-mentioned article published in Antimicrobial Agents and Chemotherapy because of the presence of some errors in the sequencing analysis for the published plasmid. Following the publication of the Expression of Concern at https://doi.org/10.1128/aac.02220-21, we recently reexamined the primary sequence data of pKPC-DK05 and found that there are some inaccuracies in the plasmid assembly and errors in the presentation of primers. Thus, we retract the paper and apologize to the readers and regret any inconvenience that this causes.

Retraction for Shin et al., “ bla NDM-5 -Bearing IncFII-Type Plasmids of Klebsiella pneumoniae Sequence Type 147 Transmitted by Cross-Border Transfer of a Patient”

Volume 60, no. 3, p. 1932–1934, 2016, https://doi.org/10.1128/aac.02722-15. We hereby retract this article published in Antimicrobial Agents and Chemotherapy because of the presence of some errors in sequencing analysis for the published plasmids. Following the publication of the Expression of Concern (https://doi.org/10.1128/aac.02221-21), we recently reexamined the primary sequence data of pCC1409-1 and pCC1410-1 and found that there are some inaccuracies in the plasmid assembly and errors in the presentation of primers. Thus, we retract the paper, and we apologize to the readers and regret any inconvenience that this causes.

Serine Integrase-based Recombination Enables Direct Plasmid Assembly In Vivo

Serine Integrase-based Recombination Enables Direct Plasmid Assembly In Vivo

Nuclear transformation of a dinoflagellate symbiont of corals

Dinoflagellates are a diverse and ecologically important group of single-celled eukaryotes. Many are photosynthetic autotrophs while others are predatory, parasitic, or symbiotic. One major group — the Symbiodiniaceae — is well known for its role as coral symbionts that provide the coral host with vital nutrients. While genetic transformation protocols have been published for some non-symbiotic dinoflagellate species, robust methods for genetic manipulation of coral symbionts are lacking, hindering a detailed molecular understanding of this critical symbiotic interaction. Here, we describe the successful transformation of coral symbiont Breviolum minutum (strain SSB01). Using Golden Gate modular plasmid assembly and electroporation, we drove transient NLS-GFP expression from an endogenous dinoflagellate virus nuclear protein (DVNP) promoter and successfully targeted GFP to the dinoflagellate nucleus. We further determined that puromycin can efficiently select transformed cells using the puromycin N-acetyltransferase (pac) resistance gene. Transformed cells could be maintained under antibiotic selection for at least 12 months without losing resistance, albeit with slowly attenuating fluorescence signal. We thus tested the expression of hybrid GFP-2A-PAC polypeptides under the control of a single promoter sequence to overcome loss of fluorescence, but lack of efficient 2A cleavage seemingly hindered antibiotic selection interfering GFP function. Despite this, our transformation approach now allows unanswered questions of dinoflagellate biology to be addressed, as well as fundamental aspects of dinoflagellate-coral symbiosis.

Wide distribution of Escherichia coli carrying IncF plasmids containing bla NDM-5 and rmtB resistance genes from hospitalized patients in England.

Introduction. The New Delhi metallo-β-lactamase (NDM) variant NDM-5 was first described in 2011 in an isolate of Escherichia coli. We noted that a high proportion of isolates of E. coli positive for bla NDM carbapenemase genes submitted to the UK Health Security Agency (formerly Public Health England) between 2019 and mid-2021 carried the bla NDM-5 allele, with many co-harbouring rmtB, rendering them highly resistant to aminoglycosides as well as to most β-lactams.Hypothesis/Gap Statement. This observation suggested that a common plasmid may be circulating.Aim. To compare these isolates and describe the plasmids carrying these resistance elements.Methodology. All isolates were sequenced on an Illumina platform, with five also subjected to long-read nanopore sequencing to provide complete assemblies. The locations of bla NDM-5, rmtB and other associated genetic elements were identified. Susceptibility testing to a wide range of antibiotics was carried out on representative isolates.Results. The 34 isolates co-harbouring bla NDM-5 and rmtB were from 14 hospital groups and six different regions across England and consisted of 11 distinct sequence types. All carried IncF plasmids. Assembly of the NDM plasmids in five isolates revealed that they carried rmtB and bla NDM-5 in an IncF conjugative plasmid ranging in size from 85.5 to 161 kb. All carried a highly conserved region, previously described in E. coli plasmid pHC105-NDM, that included bla TEM-1B and rmtB followed by sequence bounded by two IS26 elements containing ΔISAba125, bla NDM-5, ble, trpF and tat followed by ISCR1 and an integron with sul1, aadA2 and dfrA12 cassettes. This arrangement has been described in isolates from other countries and continents, suggesting that such plasmids are widely distributed, at least in E. coli, with similar plasmids also found in Klebsiella pneumoniae. Tested isolates were resistant to most antibiotics except colistin, fosfomycin and tigecycline.Conclusion. These observations suggest that conjugative plasmids carrying a highly conserved resistance gene segment have become widespread in England and elsewhere. This study highlights the value of routine whole-genome sequencing in identifying genetic elements responsible for resistance dissemination.