Uptake of exogenous DNA by plant protoplasts

1972 ◽  
Vol 50 (10) ◽  
pp. 2077-2080 ◽  
Author(s):  
Kanji Ohyama ◽  
Oluf L. Gamborg ◽  
Raymond A. Miller
Keyword(s):  
Escherichia Coli ◽  
Time Course ◽  
Cultured Cells ◽  
Incubation Temperature ◽  
Plant Protoplasts ◽  
Exogenous Dna ◽  
E Coli ◽  
Ammi Visnaga

Uptake by plant protoplasts isolated from cultured cells was demonstrated using Escherichia coli14C-DNA and Ammi visnaga protoplasts. About 0.6 to 2.8% of exogenous E. coli14C-DNA was taken up into the protoplasts, of which about 20% appeared to be acid-precipitable. The time course of uptake, the effect of incubation temperature, and the effects of DNA and protoplast concentration were studied. DEAE-dextran, poly-L-lysine, and poly-L-ornithine were found to enhance the uptake markedly.

scholarly journals Escherichia coli Competence Gene Homologs Are Essential for Competitive Fitness and the Use of DNA as a Nutrient

2006 ◽  
Vol 188 (11) ◽  
pp. 3902-3910 ◽  
Author(s):  
Vyacheslav Palchevskiy ◽  
Steven E. Finkel
Keyword(s):  
Escherichia Coli ◽  
Sole Source ◽  
Extracellular Dna ◽  
Nutrient Source ◽  
Genetic Competence ◽  
Exogenous Dna ◽  
Competitive Fitness ◽  
E Coli ◽  
Widespread Phenomenon

ABSTRACT Natural genetic competence is the ability of cells to take up extracellular DNA and is an important mechanism for horizontal gene transfer. Another potential benefit of natural competence is that exogenous DNA can serve as a nutrient source for starving bacteria because the ability to “eat” DNA is necessary for competitive survival in environments containing limited nutrients. We show here that eight Escherichia coli genes, identified as homologs of com genes in Haemophilus influenzae and Neisseria gonorrhoeae, are necessary for the use of extracellular DNA as the sole source of carbon and energy. These genes also confer a competitive advantage to E. coli during long-term stationary-phase incubation. We also show that homologs of these genes are found throughout the proteobacteria, suggesting that the use of DNA as a nutrient may be a widespread phenomenon.

scholarly journals Moringa oleifera Seeds Extract Activity on Enteropathogenic Escherichia coli and Aeromonas hydrophyla Cells in Aquatic Microcosm

2019 ◽  
Vol 7 (2) ◽  
pp. 13 ◽  
Author(s):  
Claire Stephane Metsopkeng ◽  
Chretien Lontsi Djimeli ◽  
Olive Vivien Noah Ewoti ◽  
Lucienne Marlyse Moungang ◽  
Paul Alain Nana ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Moringa Oleifera ◽  
Potable Water ◽  
Incubation Temperature ◽  
Enteropathogenic Escherichia Coli ◽  
Incubation Condition ◽  
E Coli ◽  
Extract Concentration ◽  
Cell Incubation ◽  
Potential Exploitation

This study aimed to evaluate in microcosm condition, the survival of Aeromonas hydrophila and Enteropathogenic Escherichia coli (EPEC), in the presence of M. oleifera aqueous seeds extract at concentrations varying from 1 to 40 g/L, and under 4 °C and 23 °C incubation temperature. It has been noted that cell abundances decrease gradually with the increasing in the seeds extract concentration. However, a marked cells regrowth was sometimes noted. In monospecies cell incubation condition, under 4 °C, the EPEC cells inhibition percentages (CIP) values varied from 52.12 to 99.84%. Those of A. hydrophila varied from 13.2 to 96%. The lowest CIPs were noted at the extract concentration 1g/L for EPEC and A. hydrophila. The highest CIP value was registered at 10 and 40 g/L for EPEC and at 15 g/L for A. hydrophila. Under 23 °C incubation, the EPEC CIPs values varied from 74.04 to 99.9% and those of A. hydrophila varied from 21.2 to 97.8%. For E. coli, the lowest and the highest CIP were recorded at the extract concentration 1g/L and 30 g/L, respectively. In bispecies cells incubation condition, the CIPs were relatively different. These results show the potential exploitation of M. oleifera extracts in the microbiological treatment of potable water.

scholarly journals Characterization of Fluorescent Chimeras of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxins Produced by Use of the Twin Arginine Translocation System

2005 ◽  
Vol 73 (6) ◽  
pp. 3627-3635 ◽  
Author(s):  
Juliette K. Tinker ◽  
Jarrod L. Erbe ◽  
Randall K. Holmes
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Fusion Proteins ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Secretory Diarrhea ◽  
E Coli ◽  
Twin Arginine Translocation ◽  
Heat Labile

ABSTRACT Cholera toxin (CT) is an AB5 toxin responsible for the profuse secretory diarrhea resulting from Vibrio cholerae infection. CT consists of a pentameric, receptor-binding B subunit (CTB) and a monomeric A subunit (CTA) that has latent enzymatic activity. In addition to its enterotoxicity, CT has potent mucosal adjuvant activity and can also function as a carrier molecule with many potential applications in cell biology. In earlier studies, the toxic CTA1 domain was replaced by several other antigenic protein domains to produce holotoxin-like chimeras for use as potential mucosal vaccines. In the present study we utilized the twin arginine translocation (tat) system to produce fluorescent CT chimeras, as well as fluorescent chimeras of Escherichia coli heat-labile toxins LTI and LTIIb. Fusion proteins containing either green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP) and the A2 domain of CT, LTI, or LTIIb were transported to the periplasm of E. coli by the tat system, and the corresponding B polypeptides of CT, LTI, and LTIIb were transported to the periplasm by the sec system. The fluorescent fusion proteins were shown to assemble spontaneously and efficiently with the corresponding B polypeptides in the periplasm to form chimeric holotoxin-like molecules, and these chimeras bound to and entered cultured cells in a manner similar to native CT, LTI, or LTIIb. The GFP and mRFP derivatives of CT, LT, and LTIIb developed here are useful tools for studies on the cell biology of trafficking of the CT/LT family of bacterial enterotoxins. In addition, these constructs provide proof in principle for the development of novel chimeric CT-like or LT-like vaccine candidates containing CTA2 fusion proteins that cannot be delivered to the periplasm of E. coli by use of the sec secretion pathway.

scholarly journals Glutathione-Dependent Conversion ofN-Ethylmaleimide to the Maleamic Acid by Escherichia coli: an Intracellular Detoxification Process

2000 ◽  
Vol 66 (4) ◽  
pp. 1393-1399 ◽  
Author(s):  
D. McLaggan ◽  
H. Rufino ◽  
M. Jaspars ◽  
I. R. Booth
Keyword(s):  
Escherichia Coli ◽  
Time Course ◽  
E Coli ◽  
Transient Activation ◽  
Low Concentrations ◽  
Maleamic Acid ◽  
High Concentrations ◽  
Hydrolysis Of ◽  
Strong Activator ◽  
Detoxification Process

ABSTRACT The electrophile N-ethylmaleimide (NEM) elicits rapid K+ efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K+ efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM andN-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-Ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD+-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K+ efflux systems.

scholarly journals The Escherichia coli Common Pilus and the Bundle-Forming Pilus Act in Concert during the Formation of Localized Adherence by Enteropathogenic E. coli

2009 ◽  
Vol 191 (11) ◽  
pp. 3451-3461 ◽  
Author(s):  
Zeus Saldaña ◽  
Ayşen L. Erdem ◽  
Stephanie Schüller ◽  
Iruka N. Okeke ◽  
Mark Lucas ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Triple Mutant ◽  
E Coli ◽  
Accessory Factor ◽  
Isogenic Mutants ◽  
Dose Dependent ◽  
Background Expression

ABSTRACT Although the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) mediates microcolony formation on epithelial cells, the adherence of BFP-deficient mutants is significantly abrogated, but the mutants are still adherent due to the presence of intimin and possibly other adhesins. In this study we investigated the contribution of the recently described E. coli common pilus (ECP) to the overall adherence properties of EPEC. We found that ECP and BFP structures can be simultaneously observed in the course (between zero time and 7 h during infection) of formation of localized adherence on cultured epithelial cells. These two pilus types colocalized at different levels of the microcolony topology, tethering the adhering bacteria. No evidence of BFP disappearance was found after prolonged infection. When expressed from a plasmid present in nonadherent E. coli HB101, ECP rendered this organism highly adherent at levels comparable to those of HB101 expressing the BFP. Purified ECP bound in a dose-dependent manner to epithelial cells, and the binding was blocked with anti-ECP antibodies, confirming that the pili possess adhesin properties. An ECP mutant showed only a modest reduction in adherence to cultured cells due to background expression levels of BFP and intimin. However, isogenic mutants not expressing EspA or BFP were significantly less adherent when the ecpA gene was also deleted. Furthermore, a ΔespA ΔecpA double mutant (unable to translocate Tir and to establish intimate adhesion) was at least 10-fold less adherent than the ΔespA and ΔecpA single mutants, even in the presence of BFP. A Δbfp ΔespA ΔecpA triple mutant showed the least adherence compared to the wild type and all the isogenic mutant strains tested, suggesting that ECP plays a synergistic role in adherence. Our data indicate that ECP is an accessory factor that, in association with BFP and other adhesins, contributes to the multifactorial complex interaction of EPEC with host epithelial cells.

TOXICITY OF COPPER TO ESCHERICHIA COLI IN RELATION TO INCUBATION TEMPERATURE AND METHOD OF STERILIZATION OF MEDIA

1967 ◽  
Vol 13 (10) ◽  
pp. 1299-1309 ◽  
Author(s):  
Carmen Melanie Wells Burke ◽  
Ilda McVeigh
Keyword(s):  
Experimental Data ◽  
Escherichia Coli ◽  
Growth Rate ◽  
Optical Density ◽  
Incubation Temperature ◽  
Synthetic Medium ◽  
E Coli ◽  
Viable Cells ◽  
Sterilization Process

The growth of Escherichia coli ATCC 9637 in a basal synthetic medium was very similar in cultures incubated at 35, 37, 40, and 42 °C. The inclusion in the medium of copper at concentrations of 0.1 and 0.2 μg/ml resulted in a decrease in the growth rate at each of these temperatures; the higher the temperature, the greater the decrease. Copper caused a decrease in the growth rate of E. coli in the medium sterilized by filtration as well as in that sterilized by autoclaving. However, in the medium sterilized by heat there were alternate periods in which the optical density (O.D.) of culture increased and periods during which no increase in O.D. occurred. Decreases of 50% or more in the numbers of viable cells occurred in conjunction with the plateaus in the O.D. of the cultures. A greater delay in the initiation of growth occurred in both the copper-free and the copper-containing media sterilized by heat than in the corresponding medium sterilized by filtration; the longer the exposure of the medium to heat during the sterilization process, the greater the delay in the initiation of growth. Several theories are presented and discussed as possible explanations of the experimental data obtained.

Bacterial overgrowth by indigenous microflora in the phytohemagglutinin-fed rat

1988 ◽  
Vol 34 (8) ◽  
pp. 1009-1013 ◽  
Author(s):  
J. G. Banwell ◽  
R. Howard ◽  
I. Kabir ◽  
J. W. Costerton
Keyword(s):  
Escherichia Coli ◽  
Time Course ◽  
Control Diet ◽  
Bacterial Overgrowth ◽  
Microbial Colonization ◽  
Facultative Anaerobe ◽  
E Coli ◽  
Heat Stable ◽  
Red Kidney Bean ◽  
Indigenous Microflora

Phytohemagglutinin lectin (PHA) derived from red kidney bean (Phaseolus vulgaris) causes bacterial and protozoal colonization of the rat small intestine. To provide additional insights into this phenomenon we have studied the time course and population dynamics of microbial colonization of the major aerobe – facultative anaerobe groups which characterize this microflora. Compared with controls, PHA caused proliferation of a consistent adherent microbial flora in the jejunum (P < 0.01). The predominant bacteria identified were Escherichia coli. a Streptococcal sp., and Lactobacillus. Escherichia coli isolates expressed no predominant serotype or fimbriae; none elaborated heat-labile or heat-stable toxin. Both E. coli and Streptococcal sp. populations increased within 24 h of PHA feeding and were sustained during further exposure to PHA (P < 0.05). On reversion to a control diet, coliform counts fell progressively within 24–48 h and continued to decline, whereas gram-positive rod and coccus flora became the more prominent colonizers through days 1 to 4 of the reversion.

scholarly journals Microgravity Alters the Physiological Characteristics of Escherichia coli O157:H7 ATCC 35150, ATCC 43889, and ATCC 43895 under Different Nutrient Conditions

2014 ◽  
Vol 80 (7) ◽  
pp. 2270-2278 ◽  
Author(s):  
H. W. Kim ◽  
A. Matin ◽  
M. S. Rhee
Keyword(s):  
Escherichia Coli ◽  
Optical Density ◽  
Minimal Medium ◽  
Cultured Cells ◽  
Physiological Characteristics ◽  
Nutrient Metabolism ◽  
Content Type ◽  
E Coli ◽  
Food Borne ◽  
Space Conditions

ABSTRACTThe aim of this study is to provide understanding of microgravity effects on important food-borne bacteria,Escherichia coliO157:H7 ATCC 35150, ATCC 43889, and ATCC 43895, cultured in nutrient-rich or minimal medium. Physiological characteristics, such as growth (measured by optical density and plating), cell morphology, and pH, were monitored under low-shear modeled microgravity (LSMMG; space conditions) and normal gravity (NG; Earth conditions). In nutrient-rich medium, all strains except ATCC 35150 showed significantly higher optical density after 6 h of culture under LSMMG conditions than under NG conditions (P< 0.05). LSMMG-cultured cells were approximately 1.8 times larger than NG-cultured cells at 24 h; therefore, it was assumed that the increase in optical density was due to the size of individual cells rather than an increase in the cell population. The higher pH of the NG cultures relative to that of the LSMMG cultures suggests that nitrogen metabolism was slower in the latter. After 24 h of culturing in minimal media, LSMMG-cultured cells had an optical density 1.3 times higher than that of NG-cultured cells; thus, the higher optical density in the LSMMG cultures may be due to an increase in both cell size and number. Since bacteria actively grew under LSMMG conditions in minimal medium despite the lower pH, it is of some concern that LSMMG-culturedE. coliO157:H7 may be able to adapt well to acidic environments. These changes may be caused by changes in nutrient metabolism under LSMMG conditions, although this needs to be demonstrated in future studies.

scholarly journals Contribution of the Ler- and H-NS-Regulated Long Polar Fimbriae of Escherichia coli O157:H7 during Binding to Tissue-Cultured Cells

2008 ◽  
Vol 76 (11) ◽  
pp. 5062-5071 ◽  
Author(s):  
Alfredo G. Torres ◽  
Terry M. Slater ◽  
Shilpa D. Patel ◽  
Vsevolod L. Popov ◽  
Margarita M. P. Arenas-Hernández
Keyword(s):  
Escherichia Coli ◽  
Cultured Cells ◽  
Immunogold Labeling ◽  
Regulatory Sequence ◽  
Escherichia Coli O157 ◽  
Wild Type ◽  
E Coli ◽  
First Time ◽  
Better Than

ABSTRACT The expression of the long polar fimbriae (LPF) of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by a tightly regulated process, and, therefore, the role of these fimbriae during binding to epithelial cells has been difficult to establish. We recently found that histone-like nucleoid-structuring protein (H-NS) binds to the regulatory sequence of the E. coli O157:H7 lpf1 operon and “silences” its transcription, while Ler inhibits the action of the H-NS protein and allows lpf1 to be expressed. In the present study, we determined how the deregulated expression of LPF affects binding of EHEC O157:H7 to tissue-cultured cells, correlating the adherence phenotype with lpf1 expression. We tested the adherence properties of EHEC hns mutant and found that this strain adhered 2.8-fold better than the wild type. In contrast, the EHEC ler mutant adhered 2.1-fold less than the wild type. The EHEC hns ler mutant constitutively expressed the lpf genes, and, therefore, we observed that the double mutant adhered 5.6-fold times better than the wild type. Disruption of lpfA in the EHEC hns and hns ler mutants or the addition of anti-LpfA serum caused a reduction in adhesion, demonstrating that the increased adherence was due to the expression of LPF. Immunogold-labeling electron microscopy showed that LPF is present on the surface of EHEC lpfA + strains. Furthermore, we showed that EHEC expressing LPF agglutinates red blood cells from different species and that the agglutination was blocked by the addition of anti-LpfA serum. Overall, our data confirmed that expression of LPF is a tightly regulated process and, for the first time, demonstrated that these fimbriae are associated with adherence and hemagglutination phenotypes in EHEC O157:H7.

scholarly journals Fluidity of Structure and Swiveling of Helices in the Subunit c Ring of Escherichia coli ATP Synthase as Revealed by Cysteine-Cysteine Cross-Linking

2007 ◽  
Vol 282 (46) ◽  
pp. 33788-33794 ◽  
Author(s):  
Owen D. Vincent ◽  
Brian E. Schwem ◽  
P. Ryan Steed ◽  
Warren Jiang ◽  
Robert H. Fillingame
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Time Course ◽  
Structural Model ◽  
Ring Structure ◽  
High Yield ◽  
Cross Linking ◽  
E Coli ◽  
The Cross ◽  
Ilyobacter Tartaricus

Subunit c in the membrane-traversing F0 sector of Escherichia coli ATP synthase is known to fold with two transmembrane helices and form an oligomeric ring of 10 or more subunits in the membrane. Models for the E. coli ring structure have been proposed based upon NMR solution structures and intersubunit cross-linking of Cys residues in the membrane. The E. coli models differ from the recent x-ray diffraction structure of the isolated Ilyobacter tartaricus c-ring. Furthermore, key cross-linking results supporting the E. coli model prove to be incompatible with the I. tartaricus structure. To test the applicability of the I. tartaricus model to the E. coli c-ring, we compared the cross-linking of a pair of doubly Cys substituted c-subunits, each of which was compatible with one model but not the other. The key finding of this study is that both A21C/M65C and A21C/I66C doubly substituted c-subunits form high yield oligomeric structures, c2, c3... c10, via intersubunit disulfide bond formation. The results indicate that helical swiveling, with resultant interconversion of the two conformers predicted by the E. coli and I. tartaricus models, must be occurring over the time course of the cross-linking experiment. In the additional experiments reported here, we tried to ascertain the preferred conformation in the membrane to help define the most likely structural model. We conclude that both structures must be able to form in the membrane, but that the helical swiveling that promotes their interconversion may not be necessary during rotary function.

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