Increase In Plasmid Copy Number Research Articles

Plasmid-Stabilizing Strains for Antibiotic-Free Chemical Fermentation.

Plasmid-mediated antibiotic-free fermentation holds significant industrial potential. However, the requirements for host elements and energy during plasmid inheritance often cause cell burden, leading to plasmid loss and reduced production. The stable maintenance of plasmids is primarily achieved through a complex mechanism, making it challenging to rationally design plasmid-stabilizing strains and characterize the associated genetic factors. In this study, we introduced a fluorescence-based high-throughput method and successfully screened plasmid-stabilizing strains from the genomic fragment-deletion strains of Escherichia coli MG1655 and Bacillus subtilis 168. The application of EcΔ50 in antibiotic-free fermentation increased the alanine titer 2.9 times. The enhanced plasmid stability in EcΔ50 was attributed to the coordinated deletion of genes involved in plasmid segregation and replication control, leading to improved plasmid maintenance and increased plasmid copy number. The increased plasmid stability of BsΔ44 was due to the deletion of the phage SPP1 surface receptor gene yueB, resulting in minimized sporulation, improved plasmid segregational stability and host adaptation. Antibiotic-free fermentation results showed that strain BsΔyueB exhibited a 61.99% higher acetoin titer compared to strain Bs168, reaching 3.96 g/L. When used for the fermentation of the downstream product, 2,3-butanediol, strain BsΔyueB achieved an 80.63% higher titer than Bs168, reaching 14.94 g/L using rich carbon and nitrogen feedstocks. Overall, our work provided a plasmid-stabilizing chassis for E. coli and B. subtilis, highlighting their potential for antibiotic-free fermentation of valuable products and metabolic engineering applications.

Temperatures above 37°C increase virulence of a convergent Klebsiella pneumoniae sequence type 307 strain.

Convergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307. Hypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics. Our results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways. This study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.

Three concurrent mechanisms generate gene copy number variation and transient antibiotic heteroresistance

Heteroresistance is a medically relevant phenotype where small antibiotic-resistant subpopulations coexist within predominantly susceptible bacterial populations. Heteroresistance reduces treatment efficacy across diverse bacterial species and antibiotic classes, yet its genetic and physiological mechanisms remain poorly understood. Here, we investigated a multi-resistant Klebsiella pneumoniae isolate and identified three primary drivers of gene dosage-dependent heteroresistance for several antibiotic classes: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms imposed fitness costs and were genetically unstable, leading to fast reversion to susceptibility in the absence of antibiotics. We used a mouse gut colonization model to show that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. Importantly, we observed that the three mechanisms are prevalent among Escherichia coli bloodstream isolates. Our findings underscore the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations.

Calcium-responsive plasmid copy number regulation is dependent on discrete YopD domains in Yersinia pseudotuberculosis

Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.

Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol.

The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L-1 in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanol-based overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains.

Central carbon metabolite profiling reveals vector-associated differences in the recombinant protein production host Escherichia coli BL21

The Gram-negative bacterium Escherichia coli is the most widely used host for recombinant protein production, both as an industrial expression platform and as a model system at laboratory scale. The recombinant protein production industry generates proteins with direct applications as biopharmaceuticals and in technological processes central to a plethora of fields. Despite the increasing economic significance of recombinant protein production, and the importance of E. coli as an expression platform and model organism, only few studies have focused on the central carbon metabolic landscape of E. coli during high-level recombinant protein production. In the present work, we applied four targeted CapIC- and LC-MS/MS methods, covering over 60 metabolites, to perform an in-depth metabolite profiling of the effects of high-level recombinant protein production in strains derived from E. coli BL21, carrying XylS/Pm vectors with different characteristics. The mass-spectrometric central carbon metabolite profiling was complemented with the study of growth kinetics and protein production in batch bioreactors. Our work shows the robustness in E. coli central carbon metabolism when introducing increased plasmid copy number, as well as the greater importance of induction of recombinant protein production as a metabolic challenge, especially when strong promoters are used.

Cells with stochastically increased methyltransferase to restriction endonuclease ratio provide an entry for bacteriophage into protected cell population.

The action of Type II restriction-modification (RM) systems depends on restriction endonuclease (REase), which cleaves foreign DNA at specific sites, and methyltransferase (MTase), which protects host genome from restriction by methylating the same sites. Wehere show that protection from phage infection increases as the copy number of plasmids carrying the Type II RM Esp1396I system is increased. However, since increased plasmid copy number leads to both increased absolute intracellular RM enzyme levels and to a decreased MTase/REase ratio, it is impossible to determine which factor determines resistance/susceptibility to infection. By controlled expression of individual Esp1396I MTase or REase genes in cells carrying the Esp1396I system, we show that a shift in the MTase to REase ratio caused by overproduction of MTase or REase leads, respectively, to decreased or increased protection from infection. Consistently, due to stochastic variation of MTase and REase amount in individual cells, bacterial cells that are productively infected by bacteriophage have significantly higher MTase to REase ratios than cells that ward off the infection. Our results suggest that cells with transiently increased MTase to REase ratio at the time of infection serve as entry points for unmodified phage DNA into protected bacterial populations.

A Flp-SUMO hybrid recombinase reveals multi-layered copy number control of a selfish DNA element through post-translational modification.

Mechanisms for highly efficient chromosome-associated equal segregation, and for maintenance of steady state copy number, are at the heart of the evolutionary success of the 2-micron plasmid as a stable multi-copy extra-chromosomal selfish DNA element present in the yeast nucleus. The Flp site-specific recombination system housed by the plasmid, which is central to plasmid copy number maintenance, is regulated at multiple levels. Transcription of the FLP gene is fine-tuned by the repressor function of the plasmid-coded partitioning proteins Rep1 and Rep2 and their antagonist Raf1, which is also plasmid-coded. In addition, the Flp protein is regulated by the host’s post-translational modification machinery. Utilizing a Flp-SUMO fusion protein, which functionally mimics naturally sumoylated Flp, we demonstrate that the modification signals ubiquitination of Flp, followed by its proteasome-mediated degradation. Furthermore, reduced binding affinity and cooperativity of the modified Flp decrease its association with the plasmid FRT (Flp recombination target) sites, and/or increase its dissociation from them. The resulting attenuation of strand cleavage and recombination events safeguards against runaway increase in plasmid copy number, which is deleterious to the host—and indirectly—to the plasmid. These results have broader relevance to potential mechanisms by which selfish genomes minimize fitness conflicts with host genomes by holding in check the extra genetic load they pose.

Plasmid copy number and qnr gene expression in selection of fluoroquinolone-resistant Escherichia coli.

Fluoroquinolone resistance in Enterobacteriales is developed by chromosomal and plasmid-mediated mechanisms. Plasmids play an important role in dissemination of resistant genes and they carry genes that protect bacteria in different stress-induced situations. In this study, we studied Escherichia coli strains, each carried one plasmid-mediated quinolone resistance determinant namely, qnrA1, qnrB1, qnrC1, and qnrD1. We exposed 0.5 McFarland density of each strain to 0.5 mg/L ciprofloxacin from the period of 30, 60, 90, and 120min over 24h. All treated strains were further exposed to a constantly increasing 1, 2, 4, and 8 mg/L ciprofloxacin solution through 24, 48, and 120h. In given timepoints, RNA was extracted from all treated strains. Expression of qnrA1, qnrB1, qnrC1, and qnrD1 was investigated by quantitative PCR. Mutations in gyrA and parC genes were analyzed by PCR and nucleic acid sequencing. In this study, during 0.5 mg/L ciprofloxacin exposition, the following expression levels were detected: 1.2 for qnrA1, 1.47 for qnrD1, 12.44 for qnrC1, and 80.63 for qnrB1. In case of long-term study, we selected a resistant strain in qnrB1-positive E. coli, and its expression increased from 105.91 to 212.31. On the contrary, plasmid copy number increased in time from 1 to 4.13. No mutations in gyrA or in parC chromosomal genes of treated strains were detected. Our results show that qnrB1-positive E. coli strain was able to develop fluoroquinolone resistance by upregulated qnrB1 expression that was linked to a minor increase in plasmid copy number but no mutations occurred in gyrA or parC.

Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation.

ColE1-like plasmid vectors are widely used for expression of recombinant genes in E. coli. For these vectors, segregation of individual plasmids into daughter cells during cell division appears to be random, making them susceptible to loss over time when no mechanisms ensuring their maintenance are present. Here we use the plasmid pGFPuv in a recA relA strain as a sensitized model to study factors affecting plasmid stability in the context of recombinant gene expression. We find that in this model, plasmid stability can be restored by two types of genetic modifications to the plasmid origin of replication (ori) sequence: point mutations and a novel 269nt duplication at the 5' end of the plasmid ori, which we named DAS (duplicated anti-sense) ori. Combinations of these modifications produce a range of copy numbers and of levels of recombinant expression. In direct contradiction with the classic random distribution model, we find no correlation between increased plasmid copy number and increased plasmid stability. Increased stability cannot be explained by reduced levels of recombinant gene expression either. Our observations would be more compatible with a hybrid clustered and free-distribution model, which has been recently proposed based on detection of individual plasmids in vivo using super-resolution fluorescence microscopy. This work suggests a role for the plasmid ori in the control of segregation of ColE1 plasmids that is distinct from replication initiation, opening the door for the genetic regulation of plasmid stability as a strategy aimed at enhancing large-scale recombinant gene expression or bioremediation.

Acquisition of Carbapenem Resistance by Plasmid-Encoded-AmpC-Expressing Escherichia coli.

Although AmpC β-lactamases can barely degrade carbapenems, if at all, they can sequester them and prevent them from reaching their targets. Thus, carbapenem resistance in Escherichia coli and other Enterobacteriaceae can result from AmpC production and simultaneous reduction of antibiotic influx into the periplasm by mutations in the porin genes. Here we investigated the route and genetic mechanisms of acquisition of carbapenem resistance in a clinical E. coli isolate carrying blaCMY-2 on a plasmid by selecting for mutants that are resistant to increasing concentrations of meropenem. In the first step, the expression of OmpC, the only porin produced in the strain under laboratory conditions, was lost, leading to reduced susceptibility to meropenem. In the second step, the expression of the CMY-2 β-lactamase was upregulated, leading to resistance to meropenem. The loss of OmpC was due to the insertion of an IS1 element into the ompC gene or to frameshift mutations and premature stop codons in this gene. The blaCMY-2 gene was found to be located on an IncIγ plasmid, and overproduction of the CMY-2 enzyme resulted from an increased plasmid copy number due to a nucleotide substitution in the inc gene. The clinical relevance of these genetic mechanisms became evident from the analysis of previously isolated carbapenem-resistant clinical isolates, which appeared to carry similar mutations.

DNA-Binding Proteins Regulating pIP501 Transfer and Replication.

pIP501 is a Gram-positive broad-host-range model plasmid intensively used for studying plasmid replication and conjugative transfer. It is a multiple antibiotic resistance plasmid frequently detected in clinical Enterococcus faecalis and Enterococcus faecium strains. Replication of pIP501 proceeds unidirectionally by a theta mechanism. The minimal replicon of pIP501 is composed of the repR gene encoding the essential rate-limiting replication initiator protein RepR and the origin of replication, oriR, located downstream of repR. RepR is similar to RepE of related streptococcal plasmid pAMβ1, which has been shown to possess RNase activity cleaving free RNA molecules in close proximity of the initiation site of DNA synthesis. Replication of pIP501 is controlled by the concerted action of a small protein, CopR, and an antisense RNA, RNAIII. CopR has a dual function: It acts as transcriptional repressor at the repR promoter and, in addition, prevents convergent transcription of RNAIII and repR mRNA (RNAII), which indirectly increases RNAIII synthesis. CopR binds asymmetrically as a dimer at two consecutive binding sites upstream of and overlapping with the repR promoter. RNAIII induces transcriptional attenuation within the leader region of the repR mRNA (RNAII). Deletion of either control component causes a 10- to 20-fold increase of plasmid copy number, while simultaneous deletions have no additional effect. Conjugative transfer of pIP501 depends on a type IV secretion system (T4SS) encoded in a single operon. Its transfer host-range is considerably broad, as it has been transferred to virtually all Gram-positive bacteria including Streptomyces and even the Gram-negative Escherichia coli. Expression of the 15 genes encoding the T4SS is tightly controlled by binding of the relaxase TraA, the transfer initiator protein, to the operon promoter overlapping with the origin of transfer (oriT). The T4SS operon encodes the DNA-binding proteins TraJ (VirD4-like coupling protein) and the VirB4-like ATPase, TraE. Both proteins are actively involved in conjugative DNA transport. Moreover, the operon encodes TraN, a small cytoplasmic protein, whose specific binding to a sequence upstream of the oriT nic-site was demonstrated. TraN seems to be an effective repressor of pIP501 transfer, as conjugative transfer rates were significantly increased in an E. faecalis pIP501ΔtraN mutant.

Increased plasmid copy number is essential for Yersinia T3SS function and virulence.

Pathogenic bacteria have evolved numerous virulence mechanisms that are essential for establishing infections. The enterobacterium Yersinia uses a type III secretion system (T3SS) encoded by a 70-kilobase, low-copy, IncFII-class virulence plasmid. We report a novel virulence strategy in Y. pseudotuberculosis in which this pathogen up-regulates the plasmid copy number during infection. We found that an increased dose of plasmid-encoded genes is indispensable for virulence and substantially elevates the expression and function of the T3SS. Remarkably, we observed direct, tight coupling between plasmid replication and T3SS function. This regulatory pathway provides a framework for further exploration of the environmental sensing mechanisms of pathogenic bacteria.

Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum.

BackgroundLeuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc.ResultsUsing an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α–amylase.ConclusionsThe high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.

248. A Bona Fide Mammalian Replicator Enhances All Aspects of Episomal Gene Transfer Into Human Hematopoietic Progenitor Cells

Episomal gene transfer for gene therapy applications is limited at present mainly because of luck of long term persistence of episomes in the nucleus of cells. However, episomal vectors may offer a valid alternative to viral vectors as well as to gene editing strategies. To expand the potential of episomal vectors, we used the Initiation of Replication region, (IR), from β-globin gene locus, which is capable to promote DNA replication ectopically (Wang L et al 2004) and can rescue DNA replication within the context of Scaffold /Matrix Attachment Region (S/MAR) based episomal vector (Giannakoppulos A. et al 2009). We constructed two episomal vectors, derived from the protopype S/MAR based episomal vector pEPI-eGFP (6.7Kb), namely, pEP-IR and its control pEPI-SFFV, by replacing CMV promoter of eGFP by the Spleen Focus forming Virus (SFFV) promoter, while vector pEP-IR carries the IR element just upstream the SFFV promoter. Stress Induced Duplex Destabilization (SIDD) analysis of the novel plasmids indicated that these changes would not disrupt their function as episomal vectors and could be beneficial. All three vectors (pEP-IR and control vectors pEPI-SFFV and pEPI-eGFP) showed excellent episomal properties upon transfer into K562 cells. Additionally, they showed transfection efficiency around 30% upon transfer into CD34+ cells from peripheral blood, from adult, mobilised, healthy donors. CD34+/eGFP+ cells sorted by FACS were placed in semisolid cultures and produced differentiated cell-colonies within 14 days. Non-fluorescent colonies do not contain plasmids, as documented previously too (Papapetrou E. et al 2006). Vector pEP-IR shows an enhancement of all aspects of vector performance: 100% fluorescent colonies against (0%) for pEPI-eGFP as found previously (Papapetrou E. et al 2006) and 56% for pEPI-SFFV, in Fluorescent microscopy; increase of plasmid copy number per fluorescent cell, which was 1.78 for pEP-IR against 1.12 for pEPI-SFFV, as estimated by Real Time PCR. The ratio of the number of fluorescent, stably transfected colonies to the number of transfected, seeded cells presenting the rate of establishment in semisolid cultures of transfected CD34+ cells, was 5.5% for pEPI-SFFV and 15% for pEP-IR. It is thus determined that the relative capacity for establishment for pEP-IR is 3-fold higher than that of control vector pEPI-SFFV. Finally, estimation of eGFP mRNA showed that pEP-IR supports 3 times higher eGFP expression per plasmid copy, compared to pEPI-SFFV. Vector pEPI-IR in a series of over five transfections of CD34+ cells, routinely produced fluorescent cells in the area of 99.8%. Practically, vector pEP-IR is expected to be established in all cells that give rise to the differentiated cell-progeny of CD34+ cells, that is in 10% to 20% of the total sorted CD34+/eGFP+ cells. These estimates for vector pEPI-IR can be of clinical importance.

Pharmacokinetic (PK) and Pharmacodynamic (PD) Results of a Novel, First-in-Class Modulator of Eukaryotic Translation Initiation Factor 5A (eIF5A) in Patients (pts) with Relapsed or Refractory B-Cell Malignancies: Data from a Phase 1-2 Study

IntroductioneIF5A is the only known protein to be modified by hypusination and is highly conserved across species. Hypusinated eIF5A, the predominant form in normal and cancer cells, is involved in cell survival and inflammatory pathway activation. siRNAs targeting eIF5A inhibit NF-kB activation and reduce pro-inflammatory cytokine production. Accumulation of the unhypusinated lysine form of eIF5A is associated with apoptosis. Mutants of eIF5A that cannot be hypusinated (e.g. eIF5AK50R) are pro-apoptotic in vitro and have anti-tumoral activity in vivo in multiple cancer types including melanoma and lung cancer. SNS01-T is a novel therapeutic with a dual mechanism of eIF5A modulation: inducing cell death via siRNA-mediated inhibition of hypusinated eIF5A while simultaneously causing over-expression of pro-apoptotic eIF5AK50R via a DNA plasmid with a B-cell promoter to induce tumor cell death. SNS01-T significantly inhibited tumor growth and increased survival in mouse models of myeloma (MM), mantle cell and diffuse large B-cell lymphoma. The phase 1-2 study of SNS01-T has completed 4 planned dosing cohorts 0.0125, 0.05, 0.2 and 0.375 mg/kg twice weekly IV for 6 weeks in pts with refractory B-cell cancers.MethodsPK and PD secondary endpoints included characterization of PK by measuring pExp5A plasmid DNA and eIF5A siRNA in blood and bone marrow (BM), assessing potential immunogenicity of SNS01-T by measuring serum concentrations of antibodies against SNS01-T nanoparticles, and measuring serum concentrations of select proinflammatory cytokines by enzyme-linked immunosorbent assay in serum and plasma samples. Blood PK timepoints were 30 minutes before the first infusion and at 30 minutes, 2, 6, and 24 hours after the first infusions on Week 1, Week 3, and Week 6 and at the 4, 8, and 12 week visits after the last infusion; BM samples were collected 1 day after the final infusion. Serum and plasma PD sampling timepoints were 30 minutes before and at 2, 6, and 24 hours after the first infusion on Week 1, Week 3, and Week 6, and at 4 weeks after the final infusion. Cytokines assayed included TNF-α, IFN-α, IFN-ß, IFN-g, CXCL1, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12.ResultsTable 1 shows data available for interpretation as of August 2014. The remaining samples are under analysis and will be presented. Abstract 2121. Table 1:Data available for interpretation (number of patients)SNS01-T DoseOverall(n=18)0.0125 mg/kg(n=6)0.05 mg/kg(n=4)0.2 mg/kg(n=4)0.375 mg/kg(n=4)Blood PK DNA and RNA156441Bone marrow PK82321Serum antibodies to SNS01-T nanoparticles186444Serum and plasma cytokines156441Plasmid and siRNA blood levels generally peaked 30 minutes post-dosing at weeks 1, 3 and 6 of dosing. Both plasmid and siRNA exhibited rapid clearance from the blood, with levels dropping to near pre-dosing levels within 24 hours of administration. pExp5A plasmid DNA was detectable in the bone marrow of 2 pts at cohort 1, 2 at cohort 2, 1 at cohort 3 and 1 at cohort 4. eIF5A siRNA was not detectable in bone marrow. No antibodies to SNS01-T nanoparticles were detected at any timepoint at any dose level. Cytokines remained within the expected range of inter-patient variability, similar to baseline across all timepoints at the first 2 dose levels. At dose level 3, levels of IL-6, IL-8 and TNF-α in particular increased at the 2 and 6 hour timepoints but had recovered to baseline levels 24-hours post dosing. This effect was more pronounced at the first infusion.ConclusionsPCR analysis demonstrated the presence of both plasmid DNA and siRNA components of SNS01-T in blood at all dose levels, with a dose-dependent increase in plasmid copy number. Plasmid DNA was also detected in bone marrow collected 24 hours after the final infusion of SNS01-T. Pro-inflammatory cytokines did increase within hours of infusion but returned to baseline within 24 hours, synchronous with the clinical infusion reactions (see Abstract 70148). No evidence of an anti-SNS01-T antibody response was observed in any subject. Phase 2 trials are planned. DisclosuresBexon:Senesco: Consultancy. Craig:Senesco: PI Other. Siegel:Senesco: PI Other. Bensinger:Senesco: PI Other. Novitzky:Senesco: PI Other. McDonald:Senesco: PI Other. Gutierrez:Senesco: PI Other. Libby:Senesco: PI Other. van Rhee:Senesco: PI Other. Heidel:Senesco: Consultancy. Thompson:Senesco: Consultancy, Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Barranco:Senesco: Consultancy. Taylor:Senesco: Research Funding. Browne:Senesco: Employment. Kurman:Senesco: Consultancy. Lust:Senesco: PI Other. Dondero:Senesco: Employment.

Analysis of a DNA region from low-copy-number plasmid pYAN-1 ofSphingobium yanoikuyaeresponsible for plasmid stability

We identified and analyzed a DNA region that is required for the stable maintenance of plasmids in the genus Sphingomonas. This DNA fragment, a 244 bp, is localized in the upstream region of the repA gene of low-copy-number small plasmid pYAN-1 (4896 bp) of Sphingobium yanoikuyae. It has four inverted repeats and one direct repeat for possible secondary structures. We were able to stabilize not only another unstable plasmid, pYAN-2, in the genus Sphingomonas, but also the unstable plasmid pSC101 without par locus in Escherichia coli. The copy-number levels between the unstable plasmid and the parental plasmid were similar, and these results suggest that the stabilization of unstable plasmids by this DNA region of pYAN-1 was not due to an increase in plasmid copy number. We concluded that the stabilization of the plasmid was due to a plasmid partition mechanism encoded by a DNA fragment of pYAN-1.

Construction of a prophage-free variant of Corynebacterium glutamicum ATCC 13032 for use as a platform strain for basic research and industrial biotechnology.

The activity of bacteriophages and phage-related mobile elements is a major source for genome rearrangements and genetic instability of their bacterial hosts. The genome of the industrial amino acid producer Corynebacterium glutamicum ATCC 13032 contains three prophages (CGP1, CGP2, and CGP3) of so far unknown functionality. Several phage genes are regularly expressed, and the large prophage CGP3 (∼190 kbp) has recently been shown to be induced under certain stress conditions. Here, we present the construction of MB001, a prophage-free variant of C. glutamicum ATCC 13032 with a 6% reduced genome. This strain does not show any unfavorable properties during extensive phenotypic characterization under various standard and stress conditions. As expected, we observed improved growth and fitness of MB001 under SOS-response-inducing conditions that trigger CGP3 induction in the wild-type strain. Further studies revealed that MB001 has a significantly increased transformation efficiency and produced about 30% more of the heterologous model protein enhanced yellow fluorescent protein (eYFP), presumably as a consequence of an increased plasmid copy number. These effects were attributed to the loss of the restriction-modification system (cg1996-cg1998) located within CGP3. The deletion of the prophages without any negative effect results in a novel platform strain for metabolic engineering and represents a useful step toward the construction of a C. glutamicum chassis genome of strain ATCC 13032 for biotechnological applications and synthetic biology.

Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis

Biofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity in Enterococcus faecalis cells carrying plasmid pCF10. In this study, we examined whether biofilm growth affected the copy number and expression of antibiotic resistance determinants for several plasmids with diverse replication systems. Four different E. faecalis plasmids, unrelated to pCF10, demonstrated increased copy number in biofilm cells. In biofilm cells, we also observed increased transcription of antibiotic resistance genes present on these plasmids. The increase in plasmid copy number correlated with increased plating efficiency on high concentrations of antibiotics. Single-cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells. Regrowth of harvested biofilm cells in liquid culture resulted in a rapid reduction of plasmid copy number to that observed in the planktonic state. These results suggest a possible mechanism by which biofilm growth could reduce susceptibility to antibiotics in clinical settings.

New multi-purpose high copy number vector with greater mitotic stability for diverse applications in fission yeast Schizosaccharomyces pombe

We have constructed a pUC19-based multipurpose ATG vector in Schizosaccharomyces pombe with higher copy number and mitotic stability possible with commonly used vectors. The vector, having an NdeI site in its polylinker to provide ATG site for expression, carries a greatly truncated version of URA3 gene, URA3m, of Saccharomyces cerevisiae as a selection marker. In addition, it contains the mat2P-right flank region (mat2P-RF) of S. pombe as an autonomous replicating sequence (ARS) and a polylinker with wider choice of restriction sites. While URA3m confers an increase in plasmid copy number up to 200copies/cell, mat2P-RF imparts greater mitotic stability than the standard ars1 element of S. pombe. Finally, the vector also includes the transcription termination signal of the nmt1 gene (Tnmt1). This basic vector should serve as a versatile tool for studies of gene function in S. pombe.