scholarly journals Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu

2002 ◽  
Vol 68 (2) ◽  
pp. 705-712 ◽  
Author(s):  
Arja Lamberg ◽  
Sari Nieminen ◽  
Mingqiang Qiao ◽  
Harri Savilahti
Keyword(s):  
Divalent Cations ◽  
Erwinia Carotovora ◽  
Insertion Mutagenesis ◽  
Bacterial Cells ◽  
Divalent Metal Ions ◽  
Bacterial Genomes ◽  
Dna Transposition ◽  
Bacterial Chromosomes ◽  
Transposition Reaction

ABSTRACT An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 104 to 106 CFU/μg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.

scholarly journals A Direct Transposon Insertion Tool for Modification and Functional Analysis of Viral Genomes

2003 ◽  
Vol 77 (1) ◽  
pp. 123-134 ◽  
Author(s):  
Heikki Vilen ◽  
Juha-Matti Aalto ◽  
Anna Kassinen ◽  
Lars Paulin ◽  
Harri Savilahti
Keyword(s):  
Functional Characterization ◽  
General Nature ◽  
Insertion Mutagenesis ◽  
Bacterial Cells ◽  
Virus Propagation ◽  
Recombinant Phage ◽  
Dna Transposition ◽  
Viral Genomes ◽  
Genomic Regions

ABSTRACT Advances in DNA transposition technology have recently generated efficient tools for various types of functional genetic analyses. We demonstrate here the power of the bacteriophage Mu-derived in vitro DNA transposition system for modification and functional characterization of a complete bacterial virus genome. The linear double-stranded DNA genome of Escherichia coli bacteriophage PRD1 was studied by insertion mutagenesis with reporter mini-Mu transposons that were integrated in vitro into isolated genomic DNA. After introduction into bacterial cells by electroporation, recombinant transposon-containing virus clones were identified by autoradiography or visual blue-white screening employing α-complementation of E. coli β-galactosidase. Additionally, a modified transposon with engineered NotI sites at both ends was used to introduce novel restriction sites into the phage genome. Analysis of the transposon integration sites in the genomes of viable recombinant phage generated a functional map, collectively indicating genes and genomic regions essential and nonessential for virus propagation. Moreover, promoterless transposons defined the direction of transcription within several insert-tolerant genomic regions. These strategies for the analysis of viral genomes are of a general nature and therefore may be applied to functional genomics studies in all prokaryotic and eukaryotic cell viruses.

scholarly journals Effects of Mn and Fe Levels onBacillus subtilisSpore Resistance and Effects of Mn2+, Other Divalent Cations, Orthophosphate, and Dipicolinic Acid on Protein Resistance to Ionizing Radiation

2010 ◽  
Vol 77 (1) ◽  
pp. 32-40 ◽  
Author(s):  
Amanda C. Granger ◽  
Elena K. Gaidamakova ◽  
Vera Y. Matrosova ◽  
Michael J. Daly ◽  
Peter Setlow
Keyword(s):  
Ionizing Radiation ◽  
Deinococcus Radiodurans ◽  
Dipicolinic Acid ◽  
Divalent Cations ◽  
Wild Type ◽  
Divalent Metal Ions ◽  
Protein Resistance ◽  
Wet Heat ◽  
Resistance To Ionizing Radiation

ABSTRACTSpores ofBacillus subtilisstrains with (wild type) or without (α−β−) most DNA-binding α/β-type small, acid-soluble proteins (SASP) were prepared in medium with additional MnCl2concentrations of 0.3 μM to 1 mM. These haploid spores had Mn levels that varied up to 180-fold and Mn/Fe ratios that varied up to 300-fold. However, the resistance of these spores to desiccation, wet heat, dry heat, and in particular ionizing radiation was unaffected by their level of Mn or their Mn/Fe ratio; this was also the case for wild-type spore resistance to hydrogen peroxide (H2O2). However, α−β−spores were more sensitive to H2O2when they had high Mn levels and a high Mn/Fe ratio. These results suggest that Mn levels alone are not essential for wild-type bacterial spores' extreme resistance properties, in particular ionizing radiation, although high Mn levels sensitize α−β−spores to H2O2, probably by repressing expression of the auxiliary DNA-protective protein MrgA. Notably, Mn2+complexed with the abundant spore molecule dipicolinic acid (DPA) with or without inorganic phosphate was very effective at protecting a restriction enzyme against ionizing radiationin vitro, and Ca2+complexed with DPA and phosphate was also very effective in this regard. These latter data suggest that protein protection in spores against treatments such as ionizing radiation that generate reactive oxygen species may be due in part to the spores' high levels of DPA conjugated to divalent metal ions, predominantly Ca2+, much like high levels of Mn2+complexed with small molecules protect the bacteriumDeinococcus radioduransagainst ionizing radiation.

Some factors affecting the adherence of probiotic Propionibacterium acidipropionici CRL 1198 to intestinal epithelial cells

2002 ◽  
Vol 48 (5) ◽  
pp. 449-457 ◽  
Author(s):  
Gabriela Zárate ◽  
Vilma Morata De Ambrosini ◽  
Adriana Perez Chaia ◽  
Silvia González
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Divalent Cations ◽  
Metabolic Effects ◽  
Bacterial Cells ◽  
Intestinal Epithelial ◽  
Propionibacterium Acidipropionici ◽  
Sodium Metaperiodate

Adhesion to the intestinal mucosa is generally considered an important property of probiotic microorganisms and has been related to many of their health benefits. This study investigated some factors that could affect or be involved in the adherence of Propionibacterium acidipropionici CRL 1198, a dairy strain with suggested probiotic effects and high adherence in vitro and in vivo to intestinal epithelial cells. In vitro adhesion of propionibacteria was decreased by gastric digestion but not affected by bile and pancreatic enzymes. Adherence was also decreased by pretreatment of bacterial cells with protease, sodium metaperiodate, and trichloroacetic acid, revealing that different features of the cell surface, like protein factors, carbohydrates, and teichoic acids, are involved in the process. Adherence to intestinal epithelial cells was enhanced by calcium and was dependent on other divalent cations. Adhesion to intestinal mucus was also demonstrated. The results should explain the metabolic effects in the host previously obtained with this strain and support the potential of Propionibacterium for development of new probiotics.Key words: propionibacteria, adhesion, probiotics.

scholarly journals An Efficient DNA Sequencing Strategy Based on the Bacteriophage Mu in Vitro DNA Transposition Reaction

1999 ◽  
Vol 9 (3) ◽  
pp. 308-315 ◽  
Author(s):  
Saija Haapa ◽  
Sini Suomalainen ◽  
Simo Eerikäinen ◽  
Matti Airaksinen ◽  
Lars Paulin ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Sequence Data ◽  
Sequence Information ◽  
Bacteriophage Mu ◽  
Reaction Products ◽  
Dna Transposition ◽  
Sequencing Strategy ◽  
Transposon Insertions ◽  
Transposition Reaction

A highly efficient DNA sequencing strategy was developed on the basis of the bacteriophage Mu in vitro DNA transposition reaction. In the reaction, an artificial transposon with a chloramphenicol acetyltransferase (cat) gene as a selectable marker integrated into the target plasmid DNA containing a 10.3-kb mouse genomic insert to be sequenced. Bacterial clones carrying plasmids with the transposon insertions in different positions were produced by transforming transposition reaction products into Escherichia coli cells that were then selected on appropriate selection plates. Plasmids from individual clones were isolated and used as templates for DNA sequencing, each with two primers specific for the transposon sequence but reading the sequence into opposite directions, thus creating a minicontig. By combining the information from overlapping minicontigs, the sequence of the entire 10,288-bp region of mouse genome including six exons of mouse Kcc2 gene was obtained. The results indicated that the described methodology is extremely well suited for DNA sequencing projects in which considerable sequence information is on demand. In addition, massive DNA sequencing projects, including those of full genomes, are expected to benefit substantially from the Mu strategy.[The sequence data reported in this paper have been submitted to the GenBank data library under accession no. AJ011033.]

Influence of monovalent metal ions on metal binding and catalytic activity of the 10–23 DNAzyme

2020 ◽  
Vol 402 (1) ◽  
pp. 99-111
Author(s):  
Hannah Rosenbach ◽  
Jan Borggräfe ◽  
Julian Victor ◽  
Christine Wuebben ◽  
Olav Schiemann ◽  
...  
Keyword(s):  
Metal Ions ◽  
Metal Binding ◽  
Divalent Cations ◽  
Reaction Rates ◽  
Divalent Metal ◽  
Divalent Metal Ions ◽  
Monovalent Metal ◽  
Biophysical Techniques ◽  
Target Rna

AbstractDeoxyribozymes (DNAzymes) are single-stranded DNA molecules that catalyze a broad range of chemical reactions. The 10–23 DNAzyme catalyzes the cleavage of RNA strands and can be designed to cleave essentially any target RNA, which makes it particularly interesting for therapeutic and biosensing applications. The activity of this DNAzyme in vitro is considerably higher than in cells, which was suggested to be a result of the low intracellular concentration of bioavailable divalent cations. While the interaction of the 10–23 DNAzyme with divalent metal ions was studied extensively, the influence of monovalent metal ions on its activity remains poorly understood. Here, we characterize the influence of monovalent and divalent cations on the 10–23 DNAzyme utilizing functional and biophysical techniques. Our results show that Na+ and K+ affect the binding of divalent metal ions to the DNAzyme:RNA complex and considerably modulate the reaction rates of RNA cleavage. We observe an opposite effect of high levels of Na+ and K+ concentrations on Mg2+- and Mn2+-induced reactions, revealing a different interplay of these metals in catalysis. Based on these findings, we propose a model for the interaction of metal ions with the DNAzyme:RNA complex.

Mechanisms involved in effects of antimicrobial peptides, bactenectins ChBac3.4, ChBac5, and mini-ChBac7.5Nb, on bacterial cells

2017 ◽  
pp. 43-50
Author(s):  
О.В. Шамова ◽  
М.С. Жаркова ◽  
П.М. Копейкин ◽  
Д.С. Орлов ◽  
Е.А. Корнева
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Innate Immunity ◽  
Infectious Diseases ◽  
Metabolic Activity ◽  
Capra Hircus ◽  
Bacterial Cells ◽  
Antibiotic Resistant ◽  
Resistant Strains

Антимикробные пептиды (АМП) системы врожденного иммунитета - соединения, играющие важную роль в патогенезе инфекционных заболеваний, так как обладают свойством инактивировать широкий спектр патогенных бактерий, обеспечивая противомикробную защиту живых организмов. В настоящее время АМП рассматриваются как потенциальные соединения-корректоры инфекционной патологии, вызываемой антибиотикорезистентными бактериями (АБР). Цель данной работы состояла в изученим механизмов антибактериального действия трех пептидов, принадлежащих к семейству бактенецинов - ChBac3.4, ChBac5 и mini-ChBac7.5Nb. Эти химически синтезированные пептиды являются аналогами природных пролин-богатых АМП, обнаруженных в лейкоцитах домашней козы Capra hircus и проявляющих высокую антимикробную активность, в том числе и в отношении грамотрицательных АБР. Методы. Минимальные ингибирующие и минимальные бактерицидные концентрации пептидов (МИК и МБК) определяли методом серийных разведений в жидкой питательной среде с последующим высевом на плотную питательную среду. Эффекты пептидов на проницаемость цитоплазматической мембраны бактерий для хромогенного маркера исследовали с использованием генетически модифицированного штамма Escherichia coli ML35p. Действие бактенецинов на метаболическую активность бактерий изучали с применением маркера резазурина. Результаты. Показано, что все исследованные пептиды проявляют высокую антимикробную активность в отношении Escherichia coli ML35p и антибиотикоустойчивых штаммов Escherichia coli ESBL и Acinetobacter baumannii in vitro, но их действие на бактериальные клетки разное. Использован комплекс методик, позволяющих наблюдать в режиме реального времени динамику действия бактенецинов в различных концентрациях (включая их МИК и МБК) на барьерную функцию цитоплазматической мембраны и на интенсивность метаболизма бактериальных клеток, что дало возможность выявить различия в характере воздействия бактенецинов, отличающихся по структуре молекулы, на исследуемые микроорганизмы. Установлено, что действие каждого из трех исследованных бактенецинов в бактерицидных концентрациях отличается по эффективности нарушения целостности бактериальных мембран и в скорости подавления метаболизма клеток. Заключение. Полученная информация дополнит существующие фундаментальные представления о механизмах действия пролин-богатых пептидов врожденного иммунитета, а также послужит основой для биотехнологических исследований, направленных на разработку на базе этих соединений новых антибиотических препаратов для коррекции инфекционных заболеваний, вызываемых АБР и являющимися причинами тяжелых внутрибольничных инфекций. Antimicrobial peptides (AMPs) of the innate immunity are compounds that play an important role in pathogenesis of infectious diseases due to their ability to inactivate a broad array of pathogenic bacteria, thereby providing anti-microbial host defense. AMPs are currently considered promising compounds for treatment of infectious diseases caused by antibiotic-resistant bacteria. The aim of this study was to investigate molecular mechanisms of the antibacterial action of three peptides from the bactenecin family, ChBac3.4, ChBac5, and mini-ChBac7.5Nb. These chemically synthesized peptides are analogues of natural proline-rich AMPs previously discovered by the authors of the present study in leukocytes of the domestic goat, Capra hircus. These peptides exhibit a high antimicrobial activity, in particular, against antibiotic-resistant gram-negative bacteria. Methods. Minimum inhibitory and minimum bactericidal concentrations of the peptides (MIC and MBC) were determined using the broth microdilution assay followed by subculturing on agar plates. Effects of the AMPs on bacterial cytoplasmic membrane permeability for a chromogenic marker were explored using a genetically modified strain, Escherichia coli ML35p. The effect of bactenecins on bacterial metabolic activity was studied using a resazurin marker. Results. All the studied peptides showed a high in vitro antimicrobial activity against Escherichia coli ML35p and antibiotic-resistant strains, Escherichia coli ESBL and Acinetobacter baumannii, but differed in features of their action on bacterial cells. The used combination of techniques allowed the real-time monitoring of effects of bactenecin at different concentrations (including their MIC and MBC) on the cell membrane barrier function and metabolic activity of bacteria. The differences in effects of these three structurally different bactenecins on the studied microorganisms implied that these peptides at bactericidal concentrations differed in their capability for disintegrating bacterial cell membranes and rate of inhibiting bacterial metabolism. Conclusion. The obtained information will supplement the existing basic concepts on mechanisms involved in effects of proline-rich peptides of the innate immunity. This information will also stimulate biotechnological research aimed at development of new antibiotics for treatment of infectious diseases, such as severe in-hospital infections, caused by antibiotic-resistant strains.

scholarly journals Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Leeann Klassen ◽  
Greta Reintjes ◽  
Jeffrey P. Tingley ◽  
Darryl R. Jones ◽  
Jan-Hendrik Hehemann ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Ex Vivo ◽  
Rapid Assessment ◽  
Vital Role ◽  
Strain Level ◽  
Metabolic Phenotype ◽  
Specific Cell ◽  
Bacterial Cells ◽  
Carbohydrate Utilization

AbstractGut microbiomes, such as the microbial community that colonizes the rumen, have vast catabolic potential and play a vital role in host health and nutrition. By expanding our understanding of metabolic pathways in these ecosystems, we will garner foundational information for manipulating microbiome structure and function to influence host physiology. Currently, our knowledge of metabolic pathways relies heavily on inferences derived from metagenomics or culturing bacteria in vitro. However, novel approaches targeting specific cell physiologies can illuminate the functional potential encoded within microbial (meta)genomes to provide accurate assessments of metabolic abilities. Using fluorescently labeled polysaccharides, we visualized carbohydrate metabolism performed by single bacterial cells in a complex rumen sample, enabling a rapid assessment of their metabolic phenotype. Specifically, we identified bovine-adapted strains of Bacteroides thetaiotaomicron that metabolized yeast mannan in the rumen microbiome ex vivo and discerned the mechanistic differences between two distinct carbohydrate foraging behaviors, referred to as “medium grower” and “high grower.” Using comparative whole-genome sequencing, RNA-seq, and carbohydrate-active enzyme fingerprinting, we could elucidate the strain-level variability in carbohydrate utilization systems of the two foraging behaviors to help predict individual strategies of nutrient acquisition. Here, we present a multi-faceted study using complimentary next-generation physiology and “omics” approaches to characterize microbial adaptation to a prebiotic in the rumen ecosystem.

scholarly journals Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Pia Montanucci ◽  
Silvia Terenzi ◽  
Claudio Santi ◽  
Ilaria Pennoni ◽  
Vittorio Bini ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Chemical Properties ◽  
Live Cells ◽  
High Performing ◽  
Physical Chemical ◽  
Degenerative Disorders ◽  
Selection Of ◽  
Better Than

Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation,in vitroandin vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules’ basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others.

Sign in / Sign up

Export Citation Format

Share Document