Plasmid Copy Number Research Articles

Development of a Real-Time PCR Assay for Noninvasive Detection of Miamiensis avidus in Olive Flounder Aquaculture.

Miamiensis avidus (syn. Philasterides dicentrarchi) leads to high mortality and economic losses in olive flounder (Paralichthys olivaceus) aquaculture. In this study, we developed a real-time PCR assay targeting the cytochrome c oxidase subunit I (cox1) gene of M. avidus to sensitively detect and quantify the parasite in seawater. The assay showed a strong linear correlation between the log copy number of the standard plasmid and Ct values (R2 = 0.9985), achieving an amplification efficiency of 96.26%. The cox1 gene copy number was estimated at 6017 ± 2794 copies per cell. The assay specifically detected M. avidus (cox1 genotypes 1-4) without cross-reacting with Cryptocaryon irritans or Uronema elegans, identifying as few as two M. avidus cells in 1 L of seawater with 100% accuracy (Ct value: 31.95 ± 0.78). The noninvasive seawater sampling demonstrates higher sensitivity than invasive tissue sampling. A high density of M. avidus (57.81 cells/10 L) was detected in tanks housing olive flounder with clinical signs of infection, and sporadic detection occurred in both inflow and effluent seawater. The developed real-time PCR assay provides a sensitive, specific and noninvasive method for detecting and quantifying M. avidus, enabling improved disease prevention and management in fish farms.

Development of a Real-Time Enzymatic Recombinase Amplification Assay (RT-ERA) and an ERA Combined with a Lateral Flow Dipstick (LFD) Assay (ERA-LFD) for Enteric Microsporidian (Enterospora epinepheli) in Grouper Fishes

Enterospora epinepheli poses a severe threat to grouper aquaculture due to the absence of effective prevention and treatment strategies. To address this challenge, we developed and validated two isothermal diagnostic tools, the real-time enzymatic recombinase amplification (RT-ERA) assay and the enzymatic recombinase amplification combined with a lateral flow dipstick (ERA-LFD) assay, targeting the 18S rDNA gene of the parasite. These assays operate under isothermal conditions at ≤40 °C and offer rapid detection, with RT-ERA yielding results in 14~20 min and ERA-LFD in approximately 10 min. The RT-ERA assay demonstrated a strong linear relationship between plasmid copy numbers and cycle threshold (Ct) values (y = −2.1226x + 19.562, R2 = 0.9915), enabling accurate quantification. Both methods displayed a detection limit of 2 × 100 copies/μL and no cross-reactivity with other aquaculture pathogens. Validation using grouper tissue and water samples from Hainan, China, demonstrated 100% concordance rates with basic ERA and outperformed compared to conventional PCR. These assays provide sensitive, specific, and rapid detection tools for effective monitoring and pathogen load assessment of E. epinepheli, with broad applicability to pathogen detection in aquaculture systems.

Preventing Multimer Formation in Commonly Used Synthetic Biology Plasmids.

Plasmids are an essential tool for basic research and biotechnology applications. To optimize plasmid-based circuits, it is crucial to control plasmid integrity, including the formation of plasmid multimers. Multimers are tandem repeats of entire plasmids formed by failed dimer resolution during replication. Multimers can affect the behavior of synthetic circuits, especially ones that include DNA-editing enzymes. However, occurrence of multimers is not commonly assayed. Here we survey four commonly used plasmid backbones for occurrence of multimers in cloning (JM109) and wild-type (MG1655) strains of Escherichia coli. We find that multimers occur appreciably only in MG1655, with the fraction of plasmids existing as multimers increasing with both plasmid copy number and culture passaging. In contrast, transforming multimers into JM109 can yield strains that contain no singlet plasmids. We present an MG1655 ΔrecA single-locus knockout that avoids multimer production. These results can aid synthetic biologists in improving design and reliability of plasmid-based circuits.

The insertion of the inverted repeat of an insertion sequence (IS) element from Lacticaseibacillus rhamnosus changes the host range and stability of pGK12, a shuttle vector for lactic acid bacteria.

This study highlights the significant impact of insertion sequence (IS) elements on plasmid replication in lactobacilli. The spontaneous integration of an IS element from the Laticaseibacillus rhamnosus genome into plasmid pGK12 not only expands its host range in previously incompatible strains but also changes plasmid stability and copy number. This expansion of the plasmid's host range is crucial for developing versatile genetic tools across diverse lactobacilli species. Additionally, the stable plasmid variant of pGK12 with the IS element insertion offers a valuable tool for cloning and gene expression in lactobacilli. These findings enhance our understanding of plasmid-IS element interactions and may provide insight into a new method to expand the host range of existing plasmids.

Deletion of pcnB affects antibiotic susceptibility in resistant Escherichia coli by reducing copy number of ColE1-family plasmids

Plasmids play a major role in the spread of antibiotic resistance genes in bacteria. Plasmid copy number (PCN) is often tightly regulated. In plasmids of the ColE1-type, this regulation happens by a negative feedback mechanism using an antisense RNA. Here, we employed a sequencing-based method for determining PCN to demonstrate that copy number of different ColE1-family plasmids harboring antibiotic resistance genes increases during antibiotic treatment. Further, we show that deletion of the gene pcnB reduces the copy number of ColE1-family plasmids in E. coli MG1655, which in turn results in a reduced resistance to antimicrobials of the classes aminoglycosides, β-lactams and tetracyclines. In the absence of antibiotic selection, the deletion of pcnB also decreased the number of ColE1-type plasmids in a bacterial population. Hence, PcnB, which polyadenylates RNA, marking it for decay, represents a potential drug and helper-drug target that could be used to reduce PCN to re-sensitize bacteria with multi-copy-number resistance-plasmids to treatment with different antimicrobials.

Machine learning detection of heteroresistance in Escherichiacoli.

Machine learning detection of heteroresistance in Escherichiacoli.

Construction of a Plasmid-Free Escherichia coli Strain with Enhanced Heme Supply to Produce Active Hemoglobins.

Heme is an important cofactor and plays crucial roles in the correct folding of hemoproteins. The synthesis of heme can be enhanced by the plasmid-based expression of heme biosynthetic genes. However, plasmid-based expression is genetically unstable and requires the utilization of antibiotics to maintain high copy numbers of plasmids. The rate-limiting steps in heme biosynthesis were first analyzed based on previous studies and the accumulation of heme intermediates was achieved by adding heme precursor (5-aminolevulinic acid, ALA). Next, the intracellular accumulation of porphyrin was increased by deleting the porphyrin transporter TolC. Finally, the heme synthetic genes were modified by integrating the hemA and hemL genes into the cheW and yciQ locus, assembling the rate-limiting enzymes HemC and HemD with RIAD-RIDD tags, replacing the promoters of hemE/hemH genes with the constitutive promoter PJ23100, and deleting the heme degradation gene yfeX. An enhanced heme supply HEME2 strain was obtained with a heme titer of 0.14 mg/L, which was 4.60-fold higher than that of the C41(DE3) strain. The HEME2 strain was applied to produce human hemoglobin and leghemoglobin. The titer and peroxidase activity of human hemoglobin were 1.29-fold and 42.4% higher in the HEME2-hHb strain than the values in the control strain C41-hHb. In addition, the peroxidase activity and heme content of leghemoglobin were increased by 39.2% and 53.4% in the HEME2-sHb strain compared to the values in the control strain C41-sHb. A plasmid-free Escherichia coli C41(DE3) strain capable of efficient and stable heme supply was constructed and can be used for the production of high-active hemoglobins.

YmoA functions as a molecular stress sensor in Yersinia

Pathogenic bacteria sense and respond to environmental fluctuations, a capability essential for establishing successful infections. The YmoA/Hha protein family are conserved transcription regulators in Enterobacteriaceae, playing a critical role in these responses. Specifically, YmoA in Yersinia adjusts the expression of virulence-associated traits upon temperature shift. Still, the molecular mechanisms transducing environmental signals through YmoA remain elusive. Our study employs nuclear magnetic resonance spectroscopy, biological assays and RNA-seq analysis to elucidate these mechanisms. We demonstrate that YmoA undergoes structural fluctuations and conformational dynamics in response to temperature and osmolarity changes, correlating with changes in plasmid copy number, bacterial fitness and virulence function. Notably, chemical shift analysis identifies key roles of a few specific residues and of the C-terminus region in sensing both temperature and salt-driven switch. These findings demonstrate that YmoA acts as a central stress sensor in Yersinia, fine-tuning virulence gene expression and balancing metabolic trade-offs.

ARRDC3, a novel α-arrestin, modulates WSSV replication and AHPND pathogenesis in Litopeneaus vannamei

ARRDC3, a novel α-arrestin, modulates WSSV replication and AHPND pathogenesis in Litopeneaus vannamei

Dissemination of IncQ1 Plasmids Harboring NTEKPC-IId in a Brazilian Hospital.

KPC is a clinically significant serine carbapenemase in most countries, and its rapid spread threatens global public health. blaKPC transmission is commonly mediated by Tn4401 transposons. The blaKPC gene has also been found in non-Tn4401 elements (NTEKPC). To fill the gap in the understanding of the stability and dissemination of NTEKPC-carrying plasmids, we selected and characterized carbapenem-resistant bacteria isolated between 2009 and 2016 from a hospital for a retrospective study of their plasmids conjugation capacity, impact on fitness, and replication in different species. Different clones were selected using PFGE, and their genomes were sequenced using Illumina and Oxford Nanopore methods. Minimum inhibitory concentrations (MICs) were determined by broth microdilution. Plasmid copy numbers (PCNs) were determined using qPCR. Doubling time was used to analyze fitness change. Most isolates (67%, 33/49) carried blaKPC, of which 85% presented blaKPC in a NTEKPC. The 25 isolates selected presented the blaKPC gene in NTEKPC-IId in IncQ1-type plasmids, showing multispecies dissemination. IncQ1 plasmids were mobilizable and PCN seemed to be directly linked to the species, presenting a high-copy number, mainly in K. pneumoniae. No relationship was observed between IncQ1 PCN and carbapenems MIC values. IncQ1 and a conjugative plasmid from K. pneumoniae BHKPC10 were transferred to E. coli J53 without fitness changes, and MIC values were maintained for carbapenems despite the low transconjugant PCN. In addition to IncQ1 with NTEKPC, Enterobacter cloacae BHKPC28 contained the mcr-9 gene in an IncHI2/IncHI2A conjugative plasmid, which may help the mobility of IncQ1 and the dissemination of two resistance determinants to last-resort antibiotics. Understanding the interaction between plasmids and high-risk lineages can help develop new therapies to prevent the dissemination of resistance traits.

Construction and Characterization of MoClo-Compatible Vectors for Modular Protein Expression in E. coli.

Cloning methods are fundamental to synthetic biology research. The capability to generate custom DNA constructs exhibiting predictable protein expression levels is crucial to the engineering of biology. Golden Gate cloning, a modular cloning (MoClo) technique, enables rapid and reliable one-pot assembly of genetic parts. In this study, we expand on the existing MoClo toolkits by constructing and characterizing compatible low- (p15A) and medium-copy (pBR322) destination vectors. Together with existing high-copy vectors, these backbones enable a protein expression range covering a 500-fold difference in normalized fluorescence output. We further characterize the expression- and burden profiles of each vector and demonstrate their use for the optimization of growth-coupled enzyme expression. The optimal expression of adhE (encoding alcohol dehydrogenase) for ethanol-dependent growth of Escherichia coli is determined using randomized Golden Gate Assembly, creating a diverse library of constructs with varying expression strengths and plasmid copy numbers. Through selective growth experiments, we show that relatively low expression levels of adhE facilitated optimal growth using ethanol as the sole carbon source, demonstrating the importance of adding low-copy vectors to the MoClo vector repertoire. This study emphasizes the importance of varying vector copy numbers in selection experiments to balance expression levels and burden, ensuring accurate identification of optimal conditions for growth. The vectors developed in this work are publicly available via Addgene (catalog #217582-217609).

Optimization of Conditions for Production of Soluble E. coli Poly(A)-Polymerase for Biotechnological Applications.

Poly(A) polymerase (PAP 1) from Escherichia coli is the primary enzyme responsible for synthesizing poly(A) tails on RNA molecules, signaling RNA degradation in bacterial cells. In vitro, PAP 1 is used to prepare libraries for RNAseq and to produce mRNA vaccines. However, E. coli PAP 1's toxicity and instability in low-salt buffers complicate its expression and purification. Here, we optimized the conditions for the production of recombinant PAP 1. For that, E. coli PAP 1 was expressed in seven E. coli strains with different origins and genetic backgrounds, followed by assessment of the overall protein yield, solubility, and enzymatic activity. Among the tested strains, BL21 (DE3) pLysS achieved the best balance of cell density, total PAP 1 yield, solubility, and specific activity. Rosetta 2 (DE3) and Rosetta Blue (DE3) hosting the pRARE plasmid exhibited the lowest solubility, likely due to excessive translation efficiency. Higher induction temperatures (>18 °C) exacerbated PAP 1's insolubility. Interestingly, PAP 1 accumulation correlated with an increase in the plasmid copy number encoding the enzyme, indicating its potential utility as a surrogate marker of PAP 1 activity. These findings provide insights into optimizing E. coli PAP 1 production for biotechnological applications.

Metabolic Engineering of Escherichia coli for Efficient Production of Ectoine.

Ectoine is a valuable compatible solute with extensive applications in bioengineering, cosmetics, medicine, and the food industry. While certain halophilic bacteria can naturally produce ectoine, as a model organism for biomanufacturing, Escherichia coli offers significant advantages to be engineered for potentially high-level ectoine production. However, complex metabolic flux distributions and byproduct formation present bottlenecks that limit ectoine production in E. coli. In this study, we aimed to enhance ectoine production in E. coli BL21(DE3) through systematic metabolic engineering strategies. We investigated the effects of the ectABC gene cluster sequence, plasmid copy number, and key gene copy number on ectoine synthesis. Using the original ectABC sequence with the high-copy-number plasmid pRSFDuet-1 resulted in the highest level of ectoine production. Knocking out genes encoding homoserine dehydrogenase and diaminopimelate decarboxylase reduced competing pathways, further increasing ectoine yield. Overexpression of aspartate semialdehyde dehydrogenase, aspartate kinase I (thrA*), aspartate aminotransferase, and aspartate ammonia-lyase (aspA) was performed, and optimal gene copy numbers were determined. When the copy numbers of thrA* and aspA were both three, ectoine synthesis improved, reaching 1.91 g/L. Enhancing the oxaloacetate pool by overexpressing phosphoenolpyruvate carboxylase (ppc) or introducing pyruvate carboxylase (pyc) from Corynebacterium glutamicum further increased ectoine production to 4.99 g/L. Balancing NADPH and ATP levels through cofactor engineering contributed to additional production improvements. Combining these strain engineering strategies, we ultimately constructed strain C24, which produced 35.33 g/L ectoine in a 5 L fermenter with a glucose conversion rate of 0.21 g/g. These results demonstrate that targeted metabolic engineering can significantly enhance ectoine production in E. coli, providing a foundation for industrial-scale production.

Dynamic control of the plasmid copy number maintained without antibiotics in Escherichia coli

BackgroundManipulating the gene expression is the key strategy to optimize the metabolic flux. Not only transcription, translation, and post-translation level control, but also the dynamic plasmid copy number (PCN) control has been studied. The dynamic PCN control systems that have been developed to date are based on the understanding of origin replication mechanisms, which limits their application to specific origins of replication and requires the use of antibiotics for plasmid maintenance. In this study, we developed a dynamic PCN control system for Escherichia coli that is maintained without antibiotics. This is achieved by regulating the transcription level of the translation initiation factor IF-1 (infA), an essential gene encoded on the plasmid, while deleting it from the plasmid-bearing host cell.ResultsWhen validated using GFP as a reporter protein, our system demonstrated a 22-fold dynamic range in PCN within the CloDF13 origin. The system was employed to determine the optimal copy number of the plasmid carrying the cad gene, which converts an intermediate of the tricarboxylic acid cycle (TCA cycle) to itaconic acid. By optimizing the PCN, we could achieve an itaconic acid titer of 3 g/L, which is 5.3-fold higher than the control strain.ConclusionsOur system offers a strategy to identify the optimal expression level of genes that have a competitive relationship with metabolic pathways crucial for the growth of the host organism. This approach can potentially be applied to other bacterial hosts by substituting the sensing module or the essential gene.

Regulation of Metabolic Pathways to Enhance Difucosyllactose Biosynthesis in Escherichia coli.

Difucosyllactose (DFL), an important kind of fucosylated human milk oligosaccharides (HMOs), has garnered considerable attention due to its excellent physiological activities in infants. Previously, we achieved de novo biosynthesis of DFL; however, substantial residual intermediates of fucosyllactoses (FL) were detected. In this study, DFL biosynthesis was optimized, and residual FL were reduced by regulating metabolic pathways. Different plasmid combinations were used to regulate gene expression, achieving an optimal flux balance between 2'-FL and DFL. The expression level of key enzyme α-1,3-fucosyltransferase (α-1,3-FT, FucTa) was then enhanced by increasing plasmid copy number and integrating fucTa gene into the chromosome. Exocytosis of 2'-FL was reduced by deleting the sugar efflux transporter setA gene, thereby minimizing residual FL. Finally, strain BSF41 produced 55.3 g/L of DFL with only 2.59 g/L of residual FL in a 5 L fermentor, representing the highest reported titer to date. This study provides an important foundation for advancing the biosynthesis of fucosylated HMOs.

Phenotypic Plasticity During Organofluorine Degradation Revealed by Adaptive Evolution.

A major factor limiting the biodegradation of organofluorine compounds has been highlighted as fluoride anion toxicity produced by defluorinating enzymes. Here, two highly active defluorinases with different activities were constitutively expressed in Pseudomonas putida ATCC 12633 to examine adaption to fluoride stress. Each strain was grown on α-fluorophenylacetic acid as the sole carbon source via defluorination to mandelic acid, and each showed immediate fluoride release and delayed growth. Adaptive evolution was performed for each recombinant strain by serial transfer. Both strains adapted to show a much shorter lag and a higher growth yield. The observed adaptation occurred rapidly and reproducibly, within 50 generations each time. After adaption, growth with 50-70 mM α-fluorophenylacetic acid was significantly faster with more fluoride release than a preadapted culture due to larger cell populations. Genomic sequencing of both pre- and postadapted strain pairs revealed decreases in the defluorinase gene content. With both defluorinases, adaption produced a 56%-57% decrease in the plasmid copy number. Additionally, during adaption of the strain expressing the faster defluorinase, two plasmids were present: the original and a derivative in which the defluorinase gene was deleted. An examination of the enzyme rates in the pathway suggested that the defluorinase rate was concurrently optimised for pathway flux and minimising fluoride toxicity. The rapid alteration of plasmid copy number and mutation was consistent with other studies on microbial responses to stresses such as antibiotics. The data presented here support the idea that fluoride stress is significant during the biodegradation of organofluorine compounds and suggest engineered strains will be under strong selective pressure to decrease fluoride stress.

Influence of Surface Material and Nutrient Conditions on Green Fluorescent Protein Production in Escherichia coli Biofilms

Escherichia coli biofilms have been investigated as a platform for producing recombinant proteins. This study aimed to assess the effect of different surface materials and culture media on E. coli biofilm formation and enhanced Green Fluorescent Protein (eGFP) production. Three culture media with different carbon and nitrogen sources (Lysogeny broth, Terrific broth, and M9ZB broth) were tested in combination with three materials with distinct surface properties (stainless steel, polyvinyl chloride, and silicone rubber). Biofilm formation, specific eGFP production, and plasmid copy number were monitored in microtiter plates for 9 days. Microscopy and culturability results indicated that biofilm formation was highest in Terrific broth, regardless of the surface material. Additionally, polyvinyl chloride surfaces exposed to Terrific broth provided the most advantageous conditions for achieving the highest specific eGFP production and plasmid maintenance in biofilms. These findings are relevant for establishing operational conditions for producing recombinant proteins and other high-value-added compounds on larger-scale biofilm platforms.

Binary vector copy number engineering improves Agrobacterium-mediated transformation.

The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60-100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.

Survival of antimicrobial resistant Salmonella Heidelberg inoculated into microcosms of fresh pine wood shavings for broiler litter.

S. Heidelberg survived up to 21 days in PWS which is often used as broiler bedding. S. Heidelberg abundance and survival was correlated with the water activity of PWS. S. Heidelberg strains that carried higher copy numbers of small Col plasmids were the dominant strains isolated from PWS at later time points. S. Heidelberg strains harboring transmissible plasmid carrying AmpC-like beta-lactamase gene persisted longer in PWS without antibiotic pressures for AMR.

No two clones are alike: characterization of heterologous subpopulations in a transgenic cell line of the model diatom Phaeodactylum tricornutum

BackgroundConjugation-based episome delivery is a highly efficient method used to transfer DNA into the diatom Phaeodactylum tricornutum, facilitating the production of recombinant proteins and high-value metabolites. However, previous reports have indicated phenotypic heterogeneity among individual cells from clonally propagated exconjugant cell lines, potentially affecting the stability of recombinant protein production in the diatom.ResultsHere, we characterized the differences between subpopulations with distinct fluorescence intensity phenotypes derived from a single exconjugant colony of P. tricornutum expressing the enhanced green fluorescent protein (eGFP). We analyzed the expression cassette sequence integrity, plasmid copy number, and global gene expression. Our findings reveal that lower copy numbers and the deletion of the expression cassette in part of the population contributed to low transgene expression. Gene co-expression analysis identified a set of genes with similar expression pattern to eGFP including a gene encoding a putative Flp recombinase, which may be related to variations in fluorescence intensity. These genes thus present themselves as potential candidates for increasing recombinant proteins production in P. tricornutum episomal expression system.ConclusionsOverall, our study elucidates genetic and transcriptomic differences between distinct subpopulations in a clonally propagated culture, contributes to a better understanding of heterogeneity in diatom expression systems for synthetic biology applications.