The replication initiator of the cholera pathogen's second chromosome shows structural similarity to plasmid initiators
Explore the source record for details and available documents.
99 GENERAL AND MISCELLANEOUS↗
SEARCH · Engineering Papers
Results for “plasmids”
Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
A novel conjugative transposon carrying an autonomously amplified plasmid
Tetracyclines serve as broad-spectrum antibiotics to treat bacterial infections. The discovery of new tetracycline resistance genes has led to new questions about the underlying mechanisms of resistance, gene transfer, and their relevance to human health. We tracked changes in the abundance of a 55-kbp conjugative transposon (CTn214) carrying tetQ, a tetracycline resistance gene, within a Bacteroides fragilis metagenome-assembled genome derived from shotgun sequencing of microbial DNA extracted from the ileal pouch of a patient with ulcerative colitis. The mapping of metagenomic reads to CTn214 revealed the multi-copy nature of a 17,044-nt region containing tetQ in samples collected during inflammation and uninflamed visits. B. fragilis cultivars isolated from the same patient during periods of inflammation harbored CTn214 integrated into the chromosome or both a circular, multi-copy, extrachromosomal region of the CTn214 containing tetQ and the corresponding integrated form. The tetracycline-dependent mechanism for the transmission of CTn214 is nearly identical to a common conjugative transposon found in the genome of B. fragilis (CTnDOT), but the autonomously amplified nature of a circular 17,044-nt region of CTn214 that codes for tetQ and the integration of the same sequence in the linear chromosome within the same cell is a novel observation. Genome and transcriptome sequencing of B. fragilis cultivars grown under different concentrations of tetracycline and ciprofloxacin indicates that tetQ in strains containing the circular form remains actively expressed regardless of treatment, while the expression of tetQ in strains containing the linear form increases only in the presence of tetracycline.
59 BASIC BIOLOGICAL SCIENCES↗
Genetic Complexity of CC5 Staphylococcus aureus Isolates Associated with Sternal Bursitis in Chickens: Antimicrobial Resistance, Virulence, Plasmids, and Biofilm Formation
Sternal bursitis, a common inflammatory condition in poultry, poses significant challenges to both animal welfare and public health. This study aimed to investigate the prevalence, antimicrobial resistance, and genetic characteristics of Staphylococcus aureus isolates associated with sternal bursitis in chickens. Ninety-eight samples were collected from affected chickens, and 24 S. aureus isolates were identified. Antimicrobial susceptibility testing revealed resistance to multiple agents, with a notable prevalence of aminoglycoside resistance genes. Whole genome sequencing elucidated the genetic diversity and virulence profiles of the isolates, highlighting the predominance of clonal complex 5 (CC5) strains. Additionally, biofilm formation assays demonstrated moderate biofilm production capacity among the isolates. These findings underscore the importance of vigilant monitoring and targeted interventions to mitigate the impact of sternal bursitis in poultry production systems.
Silva, Vanessa (ORCID:0000000194068433)↗
Genotypic and Phenotypic Characterization of Incompatibility Group FIB Positive Salmonella enterica Serovar Typhimurium Isolates from Food Animal Sources
Salmonella enterica is one of the most common bacterial foodborne pathogens in the United States, causing illnesses that range from self-limiting gastroenteritis to more severe, life threatening invasive disease. Many Salmonella strains contain plasmids that carry virulence, antimicrobial resistance, and/or transfer genes which allow them to adapt to diverse environments, and these can include incompatibility group (Inc) FIB plasmids. This study was undertaken to evaluate the genomic and phenotypic characteristics of IncFIB-positive Salmonella enterica serovar Typhimurium isolates from food animal sources, to identify their plasmid content, assess antimicrobial resistance and virulence properties, and compare their genotypic isolates with more recently isolated S. Typhimurium isolates from food animal sources. Methods: We identified 71 S. Typhimurium isolates that carried IncFIB plasmids. These isolates were subjected to whole genome sequencing and evaluated for bacteriocin production, antimicrobial susceptibility, the ability to transfer resistance plasmids, and a subset was evaluated for their ability to invade and persist in intestinal human epithelial cells. Results: Approximately 30% of isolates (n = 21) displayed bacteriocin inhibition of Escherichia coli strain J53. Bioinformatic analyses using PlasmidFinder software confirmed that all isolates contained IncFIB plasmids along with multiple other plasmid replicon types. Comparative analyses showed that all strains carried multiple antimicrobial resistance genes and virulence factors including iron acquisition genes, such as iucABCD (75%), iutA (94%), sitABCD (76%) and sitAB (100%). In 17 cases (71%), IncFIB plasmids, along with other plasmid replicon types, were able to conjugally transfer antimicrobial resistance and virulence genes to the susceptible recipient strain. For ten strains, persistence cell counts (27%) were noted to be significantly higher than invasion bacterial cell counts. When the genome sequences of the study isolates collected from 1998–2003 were compared to those published from subsequent years (2005–2018), overlapping genotypes were found, indicating the perseverance of IncFIB positive strains in food animal populations. This study confirms that IncFIB plasmids can play a potential role in disseminating antimicrobial resistance and virulence genes amongst bacteria from several food animal species.
59 BASIC BIOLOGICAL SCIENCES↗
Occurrence of NDM-1-producing Morganella morganii and Proteus mirabilis in a single patient in Portugal: probable in vivo transfer by conjugation
Abstract Objectives To decipher the genetics of acquisition of carbapenemase-encoding genes identified in two carbapenem-resistant Enterobacteriaceae recovered from a single patient in Portugal. Methods Carbapenemase genes were searched by PCR assays and mating-out assays were performed to further characterize the plasmid support of the carbapenemase genes. Genetic characterization of the plasmid supports was performed by whole-plasmid sequencing using the Illumina technology. Results We identified here two NDM-1-producing isolates, namely a Morganella morganii and a Proteus mirabilis, sharing the same blaNDM-1-positive plasmid. This 154 kb plasmid belonged to the IncA/C2 type, recently renamed IncC, and co-harboured two AmpC β-lactamase genes, namely blaCMY-4 and blaDHA-1, in addition to the 16S rRNA methylase gene armA encoding high-level resistance to aminoglycosides. In addition, the M. morganii isolate produced the CTX-M-33 extended-spectrum β-lactamase possessing weak carbapenemase activity, encoded by another plasmid. Conclusions We showed here that, in addition to KPC-type and OXA-181 carbapenemases, which have been identified as widespread in this country, another concern is the emergence of NDM-1-producing enterobacterial isolates in Portugal. We demonstrated here the in vivo plasmid transfer of a blaNDM-1-positive plasmid leading to dissemination of this carbapenemase gene within different enterobacterial species in a single patient.
Aires-de-Sousa, Marta↗
Assessing horizontal gene transfer in the rhizosphere of Brachypodium distachyon using fabricated ecosystems (EcoFABs)
ABSTRACT Horizontal gene transfer (HGT) is a major process by which genes are transferred between microbes in the rhizosphere. However, examining HGT remains challenging due to the complexity of mimicking conditions within the rhizosphere. Fabricated ecosystems (EcoFABs) have been used to investigate several complex processes in plant-associated environments. Here we show that EcoFABs are efficient tools to examine and measure HGT frequency in the rhizosphere. We provide the first demonstration of gene transfer via a triparental conjugation system in the Brachypodium distachyon rhizosphere in an EcoFAB using Pseudomonas putida KT2440 as both donor and recipient bacterial strain with the donor containing a mobilizable and non-self-transmissible plasmid. We observed that the frequency of plasmid transfer in the rhizosphere is potentially dependent on the plant developmental stage and the composition and amount of root exudates. The frequency of plasmid transfer also increased with higher numbers of donor cells. We demonstrate the transfer of plasmid from P. putida to another B. distachyon root colonizer, Burkholderia sp. OAS925, showing HGT within a rhizosphere microbial community. Environmental stresses also influenced the rate and efficiency of HGT in the rhizosphere between different species and genera. This study provides a robust workflow to evaluate transfer of engineered plasmids in the rhizosphere when such plasmids are potentially introduced in a field or other plant-associated environments. IMPORTANCE We report the use of EcoFABs to investigate the HGT process in a rhizosphere environment. It highlights the potential of EcoFABs in recapitulating the dynamic rhizosphere conditions as well as their versatility in studying plant-microbe interactions. This study also emphasizes the importance of studying the parameters impacting the HGT frequency. Several factors such as plant developmental stages, nutrient conditions, number of donor cells, and environmental stresses influence gene transfer within the rhizosphere microbial community. This study paves the way for future investigations into understanding the fate and movement of engineered plasmids in a field environment.
Priya, Shweta↗
Revisiting the unique structure of autonomously replicating sequences in Yarrowia lipolytica and its role in pathway engineering
Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Here, our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.
59 BASIC BIOLOGICAL SCIENCES↗
Evaluation of DNA Vaccine Candidates against Foot-and-Mouth Disease Virus in Cattle
We evaluated four DNA vaccine candidates for their ability to produce virus-like particles (VLPs) and elicit a protective immune response against Foot-and-mouth disease virus (FMDV) in cattle. Two traditional DNA plasmids and two DNA minicircle constructs were evaluated. Both the pTarget O1P1-3C plasmid and O1P1-3C minicircle encoded a wild-type FMDV 3C protease to process the P1-2A polypeptide, whereas the O1P1-HIV-3CT minicircle used an HIV-1 ribosomal frameshift to down-regulate expression of a mutant 3C protease. A modified pTarget plasmid with a reduced backbone size, mpTarget O1P1-3C LT , used a 3C protease containing two mutations reported to enhance expression. All constructs produced mature FMDV P1 cleavage products in transfected cells, as seen by western blot analysis. Three constructs, O1P1-3C minicircles, pTarget O1P1-3C, and mpTarget O1P1-3C LT plasmids, produced intracellular VLP crystalline arrays detected by electron microscopy. Despite VLP formation in vitro, none of the DNA vaccine candidates elicited protection from clinical disease when administered independently. Administration of pTarget O1P1-3C plasmid enhanced neutralizing antibody titers when used as a priming dose prior to administration of a conditionally licensed adenovirus-vectored FMD vaccine. Further work is needed to develop these DNA plasmid-based constructs into standalone FMD vaccines in cattle.
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
Engineered Escherichia coli platforms for tyrosine-derivative production from phenylalanine using phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system
Aromatic compounds derived from tyrosine are important and diverse chemicals that have industrial and commercial applications. Although these aromatic compounds can be obtained by extraction from natural producers, their growth is slow, and their content is low. To overcome these problems, many of them have been chemically synthesized from petroleum-based feedstocks. However, because of the environmental burden and depleting availability of feedstock, microbial cell factories are attracting much attention as sustainable and environmentally friendly processes. To facilitate development of microbial cell factories for producing tyrosine derivatives, we developed simple and convenient tyrosine-producing Escherichia coli platforms with a bacterial phenylalanine hydroxylase, which converted phenylalanine to tyrosine with tetrahydromonapterin as a cofactor, using a synthetic biology approach. By introducing a tetrahydrobiopterin-regeneration system, the tyrosine titer of the plasmid-based engineered strain was 4.63 g/L in a medium supplemented with 5.00 g/L phenylalanine with a test tube. The strains were successfully used to produce industrially attractive compounds, such as tyrosol with a yield of 1.58 g/L by installing a tyrosol-producing module consisting of genes encoding tyrosine decarboxylase and tyramine oxidase on a plasmid. Gene integration into E. coli chromosomes has an advantage over the use of plasmids because it increases genetic stability without antibiotic feeding to the culture media and enables more flexible pathway engineering by accepting more plasmids with artificial pathway genes. Therefore, we constructed a plasmid-free tyrosine-producing platform by integrating five modules, comprising genes encoding the phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system, into the chromosome. The platform strain could produce 1.04 g/L of 3,4-dihydroxyphenylalanine, a drug medicine, by installing a gene encoding tyrosine hydroxylase and the tetrahydrobiopterin-regeneration system on a plasmid. Moreover, by installing the tyrosol-producing module, tyrosol was produced with a yield of 1.28 g/L. We developed novel E. coli platforms for producing tyrosine from phenylalanine at multi-gram-per-liter levels in test-tube cultivation. The platforms allowed development and evaluation of microbial cell factories installing various designed tyrosine-derivative biosynthetic pathways at multi-grams-per-liter levels in test tubes.
09 BIOMASS FUELS↗
Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii
Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4 . The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas , land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.
59 BASIC BIOLOGICAL SCIENCES↗
Next-Generation Sequencing Data from a CUT&RUN Study of R. toruloides IFO0880 Cse4 and Orc1 Binding Sites
Rhodotorula toruloides has been increasingly explored as a host for bioproduction of lipids, fatty acid derivatives and terpenoids. Various genetic tools have been developed, but neither a centromere nor an autonomously replicating sequence (ARS), both necessary elements for stable episomal plasmid maintenance, has yet been reported. In this study, cleavage under targets and release using nuclease (CUT&RUN), a method used for genome-wide mapping of DNA–protein interactions, was used to identify R. toruloides IFO0880 genomic regions associated with the centromeric histone H3 protein Cse4, a marker of centromeric DNA. Fifteen putative centromeres ranging from 8 to 19 kb in length were identified and analyzed, and four were tested for, but did not show, ARS activity. These centromeric sequences contained below average GC content, corresponded to transcriptional cold spots, were primarily nonrepetitive and shared some vestigial transposon-related sequences but otherwise did not show significant sequence conservation. Future efforts to identify an ARS in this yeast can utilize these centromeric DNA sequences to improve the stability of episomal plasmids derived from putative ARS elements.
Genome Engineering↗
Comparative Genomic Analysis of Labrenzia aggregata (Alphaproteobacteria) Strains Isolated From the Mariana Trench: Insights Into the Metabolic Potentials and Biogeochemical Functions
Hadal zones are marine environments deeper than 6,000 m, most of which comprise oceanic trenches. Microbes thriving at such depth experience high hydrostatic pressure and low temperature. The genomic potentials of these microbes to such extreme environments are largely unknown. Here, we compare five complete genomes of bacterial strains belonging to Labrenzia aggregata ( Alphaproteobacteria ), including four from the Mariana Trench at depths up to 9,600 m and one reference from surface seawater of the East China Sea, to uncover the genomic potentials of this species. Genomic investigation suggests all the five strains of L. aggregata as participants in nitrogen and sulfur cycles, including denitrification, dissimilatory nitrate reduction to ammonium (DNRA), thiosulfate oxidation, and dimethylsulfoniopropionate (DMSP) biosynthesis and degradation. Further comparisons show that, among the five strains, 85% gene functions are similar with 96.7% of them encoded on the chromosomes, whereas the numbers of functional specific genes related to osmoregulation, antibiotic resistance, viral infection, and secondary metabolite biosynthesis are majorly contributed by the differential plasmids. A following analysis suggests the plasmidic gene numbers increase along with isolation depth and most plasmids are dissimilar among the five strains. These findings provide a better understanding of genomic potentials in the same species throughout a deep-sea water column and address the importance of externally originated plasmidic genes putatively shaped by deep-sea environment.
Zhong, Haohui↗
Influence of Space Flight Factors on the Genetic Properties of Streptomyces Lividans 66 (PIJ702)
Gram-positive Streptomyces bacteria display genetic instability in response to external factors. Strain S. lividans 66 harbors the multicopy plasmid pIJ702 with selective and differential marker genes for antibiotic thiostrepton resistance and melanin production. Culture plates of modified ISP agar medium with and without thiostrepton were flown on Foton-M2. Suboptimal flight temperatures, which were simulated for asynchronous ground controls, resulted in slow growth and failure to differentiate and sporulate. Flight samples and asynchronous controls showed a high frequency of failing to express plasmid markers compared to laboratory controls. This was associated with loss of plasmid DNA and likely resulted from suboptimal temperatures for flight cultures and controls. Neither restriction fragment length polymorphism, nor polymerase chain reaction amplification coupled with denaturing gradient gel electrophoresis, revealed differences between pIJ702 DNA from flight vs. control clones. Mutations of the plasmid marker genes resulting from specific spaceflight factors, e.g., microgravity and radiation, were not detected.
Tabakov, V. Yu.↗
CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) for Near-Perfect Selective Transformation
The CRISPR (Clustered, Regularly Interspaced, Short Palindromic Repeats)/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be also applied in vitro to fundamental cloning steps to facilitate efficient plasmid selection for transformation and selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts with a single-guide RNA (sgRNA)-Cas9 nuclease complex. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes multiple plasmids as well as unwanted ligation byproducts resulting in an unprecedented increase in the transformation success rate from approximately 20% to nearly 100%. Thus, this CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient application to conventional molecular cloning to achieve near-perfect selective transformation.
molecular cloning↗
Toxin-Antitoxin Systems Reflect Community Interactions Through Horizontal Gene Transfer
Bacterial evolution through horizontal gene transfer (HGT) reflects their community interactions. In this way, HGT networks do well at mapping community interactions, but offer little toward controlling them—an important step in the translation of synthetic strains into natural contexts. Toxin–antitoxin (TA) systems serve as ubiquitous and diverse agents of selection; however, their utility is limited by their erratic distribution in hosts. Here we examine the heterogeneous distribution of TAs as a consequence of their mobility. By systematically mapping TA systems across a 10,000 plasmid network, we find HGT communities have unique and predictable TA signatures. We propose these TA signatures arise from plasmid competition and have further potential to signal the degree to which plasmids, hosts, and phage interact. To emphasize these relationships, we construct an HGT network based solely on TA similarity, framing specific selection markers in the broader context of bacterial communities. This work both clarifies the evolution of TA systems and unlocks a common framework for manipulating community interactions through TA compatibility.
59 BASIC BIOLOGICAL SCIENCES↗
Enhancement of anion-exchange chromatography of DNA using compaction agents
The use of adsorptive chromatography for preparative nucleic acid separations is often limited by low capacity. The possibility that the adsorbent surface area sterically accessible to nucleic acid molecules could be increased by reducing their radius of gyration with compaction agents has been investigated. The equilibrium adsorption capacity of Q Sepharose anion-exchange matrix for plasmid DNA at 600 mM NaCl was enhanced by up to ca. 40% in the presence of 2.5 mM spermine. In addition, compaction agent selectivity has been demonstrated. Spermine, for example, enhances the adsorption of both plasmid and genomic DNA, spermidine enhances binding only of plasmid, and hexamine cobalt enhances only the binding of genomic DNA. Compaction may be generally useful for enhancing adsorptive separations of nucleic acids.
NASA Discipline Environmental Health↗
Restoration and Modification of Magnetosome Biosynthesis by Internal Gene Acquisition in a Magnetotactic Bacterium
We report that the Integration of a large-sized DNA fragment into a chromosome is an important strategy for characterization of cellular functions in microorganisms. Magnetotactic bacteria synthesize intracellular organelles comprising membrane-bound single crystalline magnetite, also referred to as magnetosomes. Magnetosomes have gained interest in both scientific and engineering sectors as they can be utilized as a material for biomedical and nanotechnological applications. Although genetic engineering of magnetosome biosynthesis mechanism has been investigated, the current method requires cumbersome gene preparation processes. Here, the chromosomal integration of a plasmid containing ≈27 magnetosome genes (≈26 kbp region) in a non-magnetic mutant of Magnetospirillum magneticum AMB-1 using a broad-host-range plasmid is shown. The genome sequencing of gene-complemented strains reveals the chromosomal integration of the plasmid with magnetosome genes at a specific site, most likely by catalysis of an endogenous transposase. Magnetosome production is successfully enhanced by integrating a variation of magnetosome gene operons in the chromosome. This chromosomal integration mechanism will allow the design of functional magnetosomes de novo and M. magneticum AMB-1 may be used as a chassis for the designed magnetosome production.
59 BASIC BIOLOGICAL SCIENCES↗
Origin of replication discovery for environmentally isolated Pantoea strain enables expression of heterologous proteins, pathways and products
Leveraging predicted origin sequences from a previously characterized groundwater plasmidome, we constructed a barcoded plasmid library to screen for previously unknown origins. Testing this library against a panel of representative bacterial strains led to the identification of 3 previously unknown origins that replicate in gram-negative bacteria not previously associated with these origin sequences. Experimental validation confirmed that a plasmid bearing origin 6911 as the sole origin could replicate with a copy number of 9 (±2) in Pantoea sp. MT58, a fast growing and metal tolerant, environmentally important bacterium. Plasmids based on this new origin were used to express the reporter protein GFP, and non-native metabolite pathways for the natural product indigoidine and the terpenoid compound isoprenol. Functional previously unknown origins of replication in such non-model organisms can expand the toolkit for genetic manipulations of both model and less-studied bacteria.
molecular biology↗