scholarly journals Making All Parts of the 16S rRNA of Escherichia coli Accessible In Situ to Single DNA Oligonucleotides

2006 ◽  
Vol 72 (1) ◽  
pp. 733-744 ◽  
Author(s):  
L. Şafak Yilmaz ◽  
Hatice E. Ökten ◽  
Daniel R. Noguera
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Fluorescence Intensity ◽  
Rational Design ◽  
Mechanistic Model ◽  
Mechanistic Modeling ◽  
E Coli ◽  
Dna Oligonucleotides ◽  
Nucleic Acid Interactions

ABSTRACT rRNA accessibility is a major sensitivity issue limiting the design of working probes for fluorescence in situ hybridization (FISH). Previous studies empirically highlighted the accessibility of target sites on rRNA maps by grouping probes into six classes according to their brightness levels. In this study, a recently proposed mechanistic model of FISH, based on the thermodynamics of secondary nucleic acid interactions, was used to evaluate the accessibility of the 16S rRNA of Escherichia coli to fluorescein-labeled oligonucleotides when thermodynamic and kinetic barriers were eliminated. To cover the entire 16S rRNA, 109 probes were designed with an average thermodynamic affinity (ΔG o overall) of −13.5 kcal/mol. Fluorescence intensity was measured by flow cytometry, and a brightness threshold between classes 3 and 4 was used as the requirement for proof of accessibility. While 46% of the probes were above this threshold with conventional 3-h hybridizations, extending the incubation period to 96 h dramatically increased the fraction of bright probes to 86%. Insufficient thermodynamic affinity and/or fluorophore quenching was demonstrated to cause the low fluorescence intensity of the remaining 14% of the probes. In the end, it was proven that every nucleotide in the 16S rRNA of E. coli could be targeted with a bright probe and, therefore, that there were no truly inaccessible target regions in the 16S rRNA. Based on our findings and mechanistic modeling, a rational design strategy involving ΔG o overall, hybridization kinetics, and fluorophore quenching is recommended for the development of bright probes.

scholarly journals Is the In Situ Accessibility of the 16S rRNA of Escherichia coli for Cy3-Labeled Oligonucleotide Probes Predicted by a Three-Dimensional Structure Model of the 30S Ribosomal Subunit?

2003 ◽  
Vol 69 (8) ◽  
pp. 4935-4941 ◽  
Author(s):  
Sebastian Behrens ◽  
Bernhard M. Fuchs ◽  
Florian Mueller ◽  
Rudolf Amann
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Rational Design ◽  
Tertiary Structure ◽  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Order Structure ◽  
Structure Model

ABSTRACT Systematic studies on the hybridization of fluorescently labeled, rRNA-targeted oligonucleotides have shown strong variations in in situ accessibility. Reliable predictions of target site accessibility would contribute to more-rational design of probes for the identification of individual microbial cells in their natural environments. During the past 3 years, numerous studies of the higher-order structure of the ribosome have advanced our understanding of its spatial conformation. These studies range from the identification of rRNA-rRNA interactions based on covariation analyses to physical imaging of the ribosome for the identification of protein-rRNA interactions. Here we reevaluate our Escherichia coli 16S rRNA in situ accessibility data with regard to a tertiary-structure model of the small subunit of the ribosome. We localized target sequences of 176 oligonucleotides on a 3.0-Å-resolution three-dimensional (3D) model of the 30S ribosomal subunit. Little correlation was found between probe hybridization efficiency and the proximity of the probe target region to the surface of the 30S ribosomal subunit model. We attribute this to the fact that fluorescence in situ hybridization is performed on fixed cells containing denatured ribosomes, whereas 3D models of the ribosome are based on its native conformation. The effects of different fixation and hybridization protocols on the fluorescence signals conferred by a set of 10 representative probes were tested. The presence or absence of the strongly denaturing detergent sodium dodecyl sulfate had a much more pronounced effect than a change of fixative from paraformaldehyde to ethanol.

scholarly journals Rapid Detection, Identification, and Enumeration ofEscherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

2001 ◽  
Vol 67 (1) ◽  
pp. 142-147 ◽  
Author(s):  
Henrik Stender ◽  
Adam J. Broomer ◽  
Kenneth Oliveira ◽  
Heather Perry-O'Keefe ◽  
Jens J. Hyldig-Nielsen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Sensitivity And Specificity ◽  
Bacterial Species ◽  
Rdna Sequence ◽  
Sequence Information ◽  
Sequence Alignments ◽  
E Coli ◽  
Municipal Water

ABSTRACT A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturableEscherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coliwere detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targetingP. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other thanE. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.

scholarly journals In situ delivery of biobutyrate by probiotic Escherichia coli for cancer therapy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chung-Jen Chiang ◽  
Yan-Hong Hong
Keyword(s):  
Escherichia Coli ◽  
Cancer Therapy ◽  
Rational Design ◽  
Hypoxic Condition ◽  
Human Colorectal Cancer ◽  
Apoptosis Pathway ◽  
Mitochondrial Apoptosis Pathway ◽  
Cycle Arrest ◽  
Human Colorectal Cancer Cell

AbstractButyrate has a bioactive function to reduce carcinogenesis. To achieve targeted cancer therapy, this study developed bacterial cancer therapy (BCT) with butyrate as a payload. By metabolic engineering, Escherichia coli Nissle 1917 (EcN) was reprogrammed to synthesize butyrate (referred to as biobutyrate) and designated EcN-BUT. The adopted strategy includes construction of a synthetic pathway for biobutyrate and the rational design of central metabolism to increase the production of biobutyrate at the expense of acetate. With glucose, EcN-BUT produced primarily biobutyrate under the hypoxic condition. Furthermore, human colorectal cancer cell was administrated with the produced biobutyrate. It caused the cell cycle arrest at the G1 phase and induced the mitochondrial apoptosis pathway independent of p53. In the tumor-bearing mice, the injected EcN-BUT exhibited tumor-specific colonization and significantly reduced the tumor volume by 70%. Overall, this study opens a new avenue for BCT based on biobutyrate.

scholarly journals High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

2005 ◽  
Vol 71 (12) ◽  
pp. 8157-8164 ◽  
Author(s):  
Ying Zhong Tang ◽  
Karina Yew Hoong Gin ◽  
Tok Hoon Lim
Keyword(s):  
Escherichia Coli ◽  
Signal Intensity ◽  
Fluorescence Signal ◽  
Standard Protocol ◽  
Oligonucleotide Probes ◽  
E Coli ◽  
Hybridization Efficiency ◽  
Hybridization Time ◽  
Seawater Samples

ABSTRACT Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).

scholarly journals Dissection of 16S rRNA Methyltransferase (KsgA) Function in Escherichia coli

2007 ◽  
Vol 189 (23) ◽  
pp. 8510-8518 ◽  
Author(s):  
Koichi Inoue ◽  
Soumit Basu ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Growth Defect ◽  
Methyltransferase Activity ◽  
Additional Function ◽  
Multicopy Suppressor ◽  
E Coli ◽  
Acid Shock ◽  
Cold Sensitive ◽  
Increased Sensitivity

ABSTRACT A 16S rRNA methyltransferase, KsgA, identified originally in Escherichia coli is highly conserved in all living cells, from bacteria to humans. KsgA orthologs in eukaryotes possess functions in addition to their rRNA methyltransferase activity. E. coli Era is an essential GTP-binding protein. We recently observed that KsgA functions as a multicopy suppressor for the cold-sensitive cell growth of an era mutant [Era(E200K)] strain (Q. Lu and M. Inouye, J. Bacteriol. 180:5243-5246, 1998). Here we observed that although KsgA(E43A), KsgA(G47A), and KsgA(E66A) mutations located in the S-adenosylmethionine-binding motifs severely reduced its methyltransferase activity, these mutations retained the ability to suppress the growth defect of the Era(E200K) strain at a low temperature. On the other hand, a KsgA(R248A) mutation at the C-terminal domain that does not affect the methyltransferase activity failed to suppress the growth defect. Surprisingly, E. coli cells overexpressing wild-type KsgA, but not KsgA(R248A), were found to be highly sensitive to acetate even at neutral pH. Such growth inhibition also was observed in the presence of other weak organic acids, such as propionate and benzoate. These chemicals are known to be highly toxic at acidic pH by lowering the intracellular pH. We found that KsgA-induced cells had increased sensitivity to extreme acid conditions (pH 3.0) compared to that of noninduced cells. These results suggest that E. coli KsgA, in addition to its methyltransferase activity, has another unidentified function that plays a role in the suppression of the cold-sensitive phenotype of the Era(E200K) strain and that the additional function may be involved in the acid shock response. We discuss a possible mechanism of the KsgA-induced acid-sensitive phenotype.

scholarly journals 16S rRNA Mutations That Confer Tetracycline Resistance in Helicobacter pylori Decrease Drug Binding in Escherichia coli Ribosomes

2005 ◽  
Vol 187 (11) ◽  
pp. 3708-3712 ◽  
Author(s):  
Lisa Nonaka ◽  
Sean R. Connell ◽  
Diane E. Taylor
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
16S Rrna ◽  
Binding Site ◽  
Tetracycline Resistance ◽  
Drug Binding ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
E Coli ◽  
H Pylori

ABSTRACT Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

scholarly journals Effect of gamma irradiation on viability and DNA of Staphylococcus epidermidis and Escherichia coli

2006 ◽  
Vol 55 (9) ◽  
pp. 1271-1275 ◽  
Author(s):  
Andrej Trampuz ◽  
Kerryl E. Piper ◽  
James M. Steckelberg ◽  
Robin Patel
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Gamma Irradiation ◽  
Staphylococcus Epidermidis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Techniques ◽  
Bacterial Cells ◽  
E Coli

Gamma irradiation is widely used for sterilization; however, its effect on elimination of amplifiable DNA, an issue of relevance to molecular diagnostic approaches, has not been well studied. The effect of gamma irradiation on the viability of Staphylococcus epidermidis and Escherichia coli (using quantitative cultures) and on their DNA (using quantitative 16S rRNA gene PCR) was evaluated. Viability was abrogated at 2.8 and 3.6 kGy for S. epidermidis and E. coli, respectively. The radiation dose required to reduce viable bacteria by one log10 (D 10 value) was 0.31 and 0.35 kGy for S. epidermidis and E. coli, respectively. D 10 values for amplifiable DNA extracted from bacteria were 2.58 and 3.09 kGy for S. epidermidis and E. coli, respectively, whereas D 10 values for amplifiable DNA were significantly higher for DNA extracted from irradiated viable bacterial cells (22.9 and 52.6 kGy for S. epidermidis and E. coli, respectively; P<0.001). This study showed that gamma irradiation of DNA in viable bacterial cells has little effect on amplifiable DNA, was not able to eliminate amplifiable 16S rRNA genes at a dose of up to 12 kGy and cannot therefore be used for elimination of DNA contamination of PCR reaction components or laboratory equipment when this DNA is present in microbial cells. This finding has practical implications for those using molecular diagnostic techniques in microbiology.

scholarly journals Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

2004 ◽  
Vol 186 (20) ◽  
pp. 6845-6854 ◽  
Author(s):  
Koichi Mori ◽  
Reiko Bando ◽  
Naoki Hieda ◽  
Tetsuo Toraya
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Protein S ◽  
Amino Acid Residues ◽  
Permeabilized Cells ◽  
E Coli ◽  
Cell Extracts ◽  
Auxiliary Protein

ABSTRACT The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5′ end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.

Sign in / Sign up

Export Citation Format

Share Document