Genetic transformation of Rhodopseudomonas sphaeroides by plasmid DNA

1982 ◽  
Vol 152 (1) ◽  
pp. 89-97
Author(s):  
C S Fornari ◽  
S Kaplan
Keyword(s):  
Plasmid Dna ◽  
Recombinant Dna ◽  
Effective Means ◽  
Viable Cell ◽  
Broad Host Range ◽  
Dna Uptake ◽  
Rhodopseudomonas Sphaeroides ◽  
Open Circular ◽  
Polyethylene Glycol 6000

A broad-host-range cloning vector, pUI81, was constructed in vitro from plasmids RSF1010 and pSL25 (a pBR322 derivative) and used to assay for transformation in Rhodopseudomonas sphaeroides. Washing cells with 500 mM Tris was an effective means of inducing competence for DNA uptake. Transformation frequencies as high as 10(-5) (transformants per viable cell) have been achieved by incubating Tris-treated cells with plasmid DNA, 100 mM CaCl2, and 20% polyethylene glycol 6000. Maximum frequencies were obtained when recipient cells were spread onto selective media after a 6.5-h outgrowth period in antibiotic-free medium. The structure (open circular versus closed, covalent circular), size, and concentration of plasmid DNA all significantly affected the transformation frequency. Four different plasmids, all small and suitable as cloning vectors, have been introduced by transformation into several different R. sphaeroides strains. Recombinant DNA carried on small, nonconjugative plasmids with broad host ranges can now be directly transferred to R. sphaeroides by this method.

scholarly journals Marker Rescue Studies of the Transfer of Recombinant DNA to Streptococcus gordonii In Vitro, in Foods and Gnotobiotic Rats

2003 ◽  
Vol 69 (10) ◽  
pp. 6121-6127 ◽  
Author(s):  
Mitra Kharazmi ◽  
Silke Sczesny ◽  
Michael Blaut ◽  
Walter P. Hammes ◽  
Christian Hertel
Keyword(s):  
Plasmid Dna ◽  
Recombinant Dna ◽  
Serum Proteins ◽  
Horse Serum ◽  
Streptococcus Gordonii ◽  
Chromosomal Dna ◽  
Marker Rescue ◽  
Transgenic Potatoes

ABSTRACT A plasmid marker rescue system based on restoration of the nptII gene was established in Streptococcus gordonii to study the transfer of bacterial and transgenic plant DNA by transformation. In vitro studies revealed that the marker rescue efficiency depends on the type of donor DNA. Plasmid and chromosomal DNA of bacteria as well as DNA of transgenic potatoes were transferred with efficiencies ranging from 8.1 × 10−6 to 5.8 × 10−7 transformants per nptII gene. Using a 792-bp amplification product of nptII the efficiency was strongly decreased (9.8 × 10−9). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 × 10−10), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 × 10−11. No marker rescue was detected in extracts of transgenic potatoes despite addition of HHS. In vivo transformation of S. gordonii LTH 5597 was studied in monoassociated rats by using plasmid DNA. No marker rescue could be detected in vivo, although transformation was detected in the presence of saliva and fecal samples supplemented with HHS. It was also shown that plasmid DNA persists in rat saliva permitting transformation for up to 6 h of incubation. It is suggested that the lack of marker rescue is due to the absence of competence-stimulating factors such as serum proteins in rat saliva.

scholarly journals Construction and Application of aluxABCDEReporter System for Real-Time Monitoring of Enterococcus faecalis Gene Expression and Growth

2012 ◽  
Vol 78 (19) ◽  
pp. 7003-7011 ◽  
Author(s):  
Sabina Leanti La Rosa ◽  
Dzung B. Diep ◽  
Ingolf F. Nes ◽  
Dag Anders Brede
Keyword(s):  
Real Time ◽  
Enterococcus Faecalis ◽  
Galleria Mellonella ◽  
Viable Cell ◽  
Model Systems ◽  
Infection Model ◽  
Broad Host Range ◽  
Reporter System ◽  
Content Type

ABSTRACTThe present work describes the construction of a novel molecular tool for luciferase-based bioluminescence (BL) tagging ofEnterococcus faecalis. To this end, a vector (pSL101) and its derivatives conferring a genetically encoded bioluminescent phenotype on all tested strains ofE. faecaliswere constructed. pSL101 harbors theluxABCDEoperon from pPL2luxand the pREG696 broad-host-range replicon andaxe-txetoxin-antitoxin cassette, providing segregational stability for long-term plasmid persistence in the absence of antibiotic selection. The bioluminescent signals obtained from three highly expressed promoters correlated linearly (R2> 0.98) with the viable-cell count. We employedlux-taggedE. faecalisstrains to monitor growth in real time in milk and urinein vitro. Furthermore, bioluminescence imaging (BLI) was used to visualize the magnitude of the bacterial burden during infection in theGalleria mellonellamodel system. To our knowledge, pSL101 is the first substrate addition-independent reporter system developed for BLI ofE. faecalisand an efficient tool for spatiotemporal tracking of bacterial growth and quantitative determination of promoter activity in real time, noninvasively, in infection model systems.

scholarly journals Comparative studies on in vitro expression and in vivo immunogenicity of supercoiled and open circular forms of plasmid DNA vaccines

Vaccine ◽  
2008 ◽  
Vol 26 (8) ◽  
pp. 1136-1141 ◽  
Author(s):  
Vinod Bhaskara Pillai ◽  
Michael Hellerstein ◽  
Tianwei Yu ◽  
Rama Rao Amara ◽  
Harriet L. Robinson
Keyword(s):  
Plasmid Dna ◽  
Comparative Studies ◽  
Dna Vaccines ◽  
In Vitro Expression ◽  
Open Circular

Evaluation of biocompatibility and efficiency of plasmid DNA delivery by gene-activated hydrogels in vitro

2019 ◽  
Vol XIV (2) ◽  
Author(s):  
I.Y. Bozo ◽  
A.A. Titova ◽  
M.N. Zhuravleva ◽  
A.I. Bilyalov ◽  
M.O. Mavlikeev ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Dna Delivery

scholarly journals Cyclooxygenase Inhibition Alters Proliferative, Migratory, and Invasive Properties of Human Glioblastoma Cells In Vitro

2021 ◽  
Vol 22 (9) ◽  
pp. 4297
Author(s):  
Matthew Thomas Ferreira ◽  
Juliano Andreoli Miyake ◽  
Renata Nascimento Gomes ◽  
Fábio Feitoza ◽  
Pollyana Bulgarelli Stevannato ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Biology ◽  
Gelatin Zymography ◽  
Viable Cell ◽  
Cell Counting ◽  
Gelatinolytic Activity ◽  
Ep4 Receptor ◽  
And Migration ◽  
Proliferation And Migration

Prostaglandin E2 (PGE2) is known to increase glioblastoma (GBM) cell proliferation and migration while cyclooxygenase (COX) inhibition decreases proliferation and migration. The present study investigated the effects of COX inhibitors and PGE2 receptor antagonists on GBM cell biology. Cells were grown with inhibitors and dose response, viable cell counting, flow cytometry, cell migration, gene expression, Western blotting, and gelatin zymography studies were performed. The stimulatory effects of PGE2 and the inhibitory effects of ibuprofen (IBP) were confirmed in GBM cells. The EP2 and EP4 receptors were identified as important mediators of the actions of PGE2 in GBM cells. The concomitant inhibition of EP2 and EP4 caused a significant decrease in cell migration which was not reverted by exogenous PGE2. In T98G cells exogenous PGE2 increased latent MMP2 gelatinolytic activity. The inhibition of COX1 or COX2 caused significant alterations in MMP2 expression and gelatinolytic activity in GBM cells. These findings provide further evidence for the importance of PGE2 signalling through the EP2 and the EP4 receptor in the control of GBM cell biology. They also support the hypothesis that a relationship exists between COX1 and MMP2 in GBM cells which merits further investigation as a novel therapeutic target for drug development.

scholarly journals 1250. Novel Boronic Acid Transition State Analogs (BATSI) with in vitro inhibitory activity against class A, B and C β-lactamases

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S643-S643
Author(s):  
Maria F Mojica ◽  
Christopher Bethel ◽  
Emilia Caselli ◽  
Magdalena A Taracila ◽  
Fabio Prati ◽  
...  
Keyword(s):  
Active Site ◽  
Inhibitory Activity ◽  
Covalent Bond ◽  
Thiol Group ◽  
Viable Cell ◽  
Free Thiol ◽  
Transition State Analogs ◽  
Cell Counts ◽  
Free Thiol Group

Abstract Background Catalytic mechanisms of serine β-lactamases (SBL; classes A, C and D) and metallo-β-lactamases (MBLs) have directed divergent strategies towards inhibitor design. SBL inhibitors act as high affinity substrates that -as in BATSIs- form a reversible, dative covalent bond with the conserved active site Ser. MBL inhibitors bind the active-site Zn2+ ions and displace the nucleophilic OH-. Herein, we explore the efficacy of a series of BATSI compounds with a free-thiol group at inhibiting both SBL and MBL. Methods Exploratory compounds were synthesized using stereoselective homologation of (+) pinandiol boronates to introduce the amino group on the boron-bearing carbon atom, which was subsequently acylated with mercaptopropanoic acid. Representative SBL (KPC-2, ADC-7, PDC-3 and OXA-23) and MBL (IMP-1, NDM-1 and VIM-2) were purified and used for the kinetic characterization of the BATSIs. In vitro activity was evaluated by a modified time-kill curve assay, using SBL and MBL-producing strains. Results Kinetic assays revealed that IC50 values ranged from 1.3 µM to >100 µM for this series. The best compound, s08033, demonstrated inhibitory activity against KPC-2, VIM-2, ADC-7 and PDC-3, with IC50 in the low μM range. Reduction of at least 1.5 log10-fold of viable cell counts upon exposure to sub-lethal concentrations of antibiotics (AB) + s08033, compared to the cells exposed to AB alone, demonstrated the microbiological activity of this novel compound against SBL- and MBL-producing E. coli (Table 1). Table 1 Conclusion Addition of a free-thiol group to the BATSI scaffold increases the range of these compounds resulting in a broad-spectrum inhibitor toward clinically important carbapenemases and cephalosporinases. Disclosures Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

scholarly journals Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
M. Adamczyk ◽  
E. Lewicka ◽  
R. Szatkowska ◽  
H. Nieznanska ◽  
J. Ludwiczak ◽  
...  
Keyword(s):  
Dna Binding ◽  
Host Range ◽  
Coiled Coil ◽  
Amino Acid Sequences ◽  
Broad Host Range ◽  
Biophysical Properties ◽  
Dna Binding Sites ◽  
In Silico Studies ◽  
Wide Range

Abstract Background DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. Results This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions. Conclusion KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

Multifunctional self-assembled cationic peptide nanostructures efficiently carry plasmid DNA in vitro and exhibit antimicrobial activity with minimal toxicity

2014 ◽  
Vol 2 (30) ◽  
pp. 4848-4861 ◽  
Author(s):  
Santosh Yadav ◽  
Manohar Mahato ◽  
Rajiv Pathak ◽  
Diksha Jha ◽  
Bipul Kumar ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Plasmid Dna ◽  
Cationic Peptide ◽  
Minimal Toxicity ◽  
Amphiphilic Peptide ◽  
Self Assembled ◽  
Multifunctional Properties

An amphiphilic peptide–aminoglycoside (Pep–Neo) conjugate has been synthesized, self-assembled into nanostructures and evaluated for its multifunctional properties.

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