scholarly journals A systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria

2017 ◽  
Author(s):  
Nathan M. Belliveau ◽  
Stephanie L. Barnes ◽  
William T. Ireland ◽  
Daniel L. Jones ◽  
Mike J. Sweredoski ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Regulatory Sequences ◽  
Bacterial Genomes ◽  
Information Theoretic ◽  
E Coli ◽  
Biophysical Models ◽  
Wide Range ◽  
Reporter Assays ◽  
Nucleotide Resolution ◽  
Almost All

Gene regulation is one of the most ubiquitous processes in biology. But while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. At present, characterizing the molecular mechanisms by which individual regulatory sequences operate requires focused efforts using low-throughput methods. Here we show how a combination of massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used to dissect bacterial promoters in a systematic and scalable way. We demonstrate this method on both well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli. In all cases we recover nucleotide-resolution models of promoter mechanism. For some promoters, including previously unannotated ones, the approach allowed us to further extract quantitative biophysical models describing input-output relationships. This method opens up the possibility of exhaustively dissecting the mechanisms of promoter function in E. coli and a wide range of other bacteria.

scholarly journals Systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria

2018 ◽  
Vol 115 (21) ◽  
pp. E4796-E4805 ◽  
Author(s):  
Nathan M. Belliveau ◽  
Stephanie L. Barnes ◽  
William T. Ireland ◽  
Daniel L. Jones ◽  
Michael J. Sweredoski ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Regulatory Sequences ◽  
Bacterial Genomes ◽  
Information Theoretic ◽  
E Coli ◽  
Biophysical Models ◽  
Wide Range ◽  
Reporter Assays ◽  
Nucleotide Resolution ◽  
Almost All

Gene regulation is one of the most ubiquitous processes in biology. However, while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. At present, characterizing the molecular mechanisms by which individual regulatory sequences operate requires focused efforts using low-throughput methods. Here, we take a first step toward multipromoter dissection and show how a combination of massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used to dissect multiple bacterial promoters in a systematic way. We show this approach on both well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli. In all cases, we recover nucleotide-resolution models of promoter mechanism. For some promoters, including previously unannotated ones, the approach allowed us to further extract quantitative biophysical models describing input–output relationships. Given the generality of the approach presented here, it opens up the possibility of quantitatively dissecting the mechanisms of promoter function in E. coli and a wide range of other bacteria.

scholarly journals Additional Og-Typing PCR Techniques Targeting Escherichia coli-Novel and Shigella-Unique O-Antigen Biosynthesis Gene Clusters

2020 ◽  
Vol 58 (11) ◽  
Author(s):  
Atsushi Iguchi ◽  
Hironobu Nishii ◽  
Kazuko Seto ◽  
Jiro Mitobe ◽  
Kenichi Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Strong Association ◽  
Gene Clusters ◽  
Epidemiological Studies ◽  
Content Type ◽  
E Coli ◽  
O Antigen ◽  
Wide Range ◽  
Biosynthesis Gene ◽  
Almost All

ABSTRACT The O-serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and controls. O-serogroup diversification shows a strong association with the genetic diversity in some O-antigen biosynthesis gene clusters. Through genomic studies, in addition to the types of O-antigen biosynthesis gene clusters (Og-types) from conventional O-serogroup strains, a number of novel Og-types have been found in E. coli isolates. To assist outbreak investigations and surveillance of pathogenic E. coli at inspection institutes, in previous studies, we developed PCR methods that could determine almost all conventional O-serogroups and some novel Og-types. However, there are still many Og-types that may not be determined by simple genetic methods such as PCR. Thus, in the present study, we aimed to develop an additional Og-typing PCR system. Based on the novel Og-types, including OgN32, OgN33, and OgN34, presented in this study, we designed an additional 24 PCR primer pairs targeting 14 novel and 2 diversified E. coli Og-types and 8 Shigella-unique Og-types. Subsequently, we developed 5 new multiplex PCR sets consisting of 33 primers, including the aforementioned 24 primers and 9 primers reported in previous studies. The accuracy and specificity of the PCR system was validated using approximately 260 E. coli and Shigella O-serogroup and Og-type reference strains. The Og-typing PCR system reported here can determine a wide range of Og-types of E. coli and may help epidemiological studies, in addition to the surveillance of pathogenic E. coli.

scholarly journals Measuring cis-regulatory energetics in living cells using allelic manifolds

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Talitha L Forcier ◽  
Andalus Ayaz ◽  
Manraj S Gill ◽  
Daniel Jones ◽  
Rob Phillips ◽  
...  
Keyword(s):  
Living Cells ◽  
Massively Parallel ◽  
Editorial Note ◽  
Regulatory Sequences ◽  
Allelic Series ◽  
Cooperative Interactions ◽  
Biophysical Models ◽  
Prior Literature ◽  
Reporter Assays ◽  
Classical Genetics

Gene expression in all organisms is controlled by cooperative interactions between DNA-bound transcription factors (TFs), but quantitatively measuring TF-DNA and TF-TF interactions remains difficult. Here we introduce a strategy for precisely measuring the Gibbs free energy of such interactions in living cells. This strategy centers on the measurement and modeling of ‘allelic manifolds’, a multidimensional generalization of the classical genetics concept of allelic series. Allelic manifolds are measured using reporter assays performed on strategically designed cis-regulatory sequences. Quantitative biophysical models are then fit to the resulting data. We used this strategy to study regulation by two Escherichia coli TFs, CRP andσ70RNA polymerase. Doing so, we consistently obtained energetic measurements precise to∼0.1kcal/mol. We also obtained multiple results that deviate from the prior literature. Our strategy is compatible with massively parallel reporter assays in both prokaryotes and eukaryotes, and should therefore be highly scalable and broadly applicable.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that minor issues remain unresolved (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

scholarly journals Measuring cis-regulatory energetics in living cells using allelic manifolds

2018 ◽  
Author(s):  
Talitha Forcier ◽  
Andalus Ayaz ◽  
Manraj S. Gill ◽  
Daniel Jones ◽  
Rob Phillips ◽  
...  
Keyword(s):  
Gene Expression ◽  
Living Cells ◽  
Massively Parallel ◽  
Regulatory Sequences ◽  
Allelic Series ◽  
Cooperative Interactions ◽  
Biophysical Models ◽  
Prior Literature ◽  
Reporter Assays ◽  
Classical Genetics

AbstractGene expression in all organisms is controlled by cooperative interactions between DNA-bound transcription factors (TFs), but quantitatively measuring TF-DNA and TF-TF interactions remains difficult. Here we introduce a strategy for precisely measuring the Gibbs free energy of such interactions in living cells. This strategy centers on the measurement and modeling of “allelic manifolds”, a multidimensional generalization of the classical genetics concept of allelic series. Allelic manifolds are measured using reporter assays performed on strategically designed cis-regulatory sequences. Quantitative biophysical models are then fit to the resulting data. We used this strategy to study regulation by twoEscherichia coliTFs, CRP and σ70RNA polymerase. Doing so, we consistently obtained energetic measurements precise to ~ 0.1 kcal/mol. We also obtained multiple results that deviate from the prior literature. Our strategy is compatible with massively parallel reporter assays in both prokaryotes and eukaryotes, and should therefore be highly scalable and broadly applicable.

scholarly journals Recording mobile DNA in the gut microbiota using an Escherichia coli CRISPR-Cas spacer acquisition platform

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christian Munck ◽  
Ravi U. Sheth ◽  
Daniel E. Freedberg ◽  
Harris H. Wang
Keyword(s):  
Escherichia Coli ◽  
Horizontal Gene Transfer ◽  
Genetic Material ◽  
Dna Transfer ◽  
Experimental Measurements ◽  
Mobile Dna ◽  
Bacterial Genomes ◽  
E Coli ◽  
Nucleotide Resolution ◽  
Dna Acquisition

AbstractThe flow of genetic material between bacteria is central to the adaptation and evolution of bacterial genomes. However, our knowledge about DNA transfer within complex microbiomes is lacking, with most studies of horizontal gene transfer (HGT) relying on bioinformatic analyses of genetic elements maintained on evolutionary timescales or experimental measurements of phenotypically trackable markers. Here, we utilize the CRISPR-Cas spacer acquisition process to detect DNA acquisition events from complex microbiota in real-time and at nucleotide resolution. In this system, an E. coli recording strain is exposed to a microbial sample and spacers are acquired from transferred plasmids and permanently stored in genomic CRISPR arrays. Sequencing and analysis of acquired spacers enables identification of the transferred plasmids. This approach allowed us to identify individual mobile elements without relying on phenotypic markers or post-transfer replication. We found that HGT into the recording strain in human clinical fecal samples can be extensive and is driven by different plasmid types, with the IncX type being the most actively transferred.

scholarly journals Biophysical models of cis-regulation as interpretable neural networks

2019 ◽  
Author(s):  
Ammar Tareen ◽  
Justin B. Kinney
Keyword(s):  
Neural Networks ◽  
Gene Regulation ◽  
Deep Learning ◽  
Large Classes ◽  
Biophysical Models ◽  
Learning Techniques ◽  
Wide Range ◽  
Reporter Assays ◽  
Post Hoc ◽  
Learning Frameworks

AbstractThe adoption of deep learning techniques in genomics has been hindered by the difficulty of mechanistically interpreting the models that these techniques produce. In recent years, a variety of post-hoc attribution methods have been proposed for addressing this neural network interpretability problem in the context of gene regulation. Here we describe a complementary way of approaching this problem. Our strategy is based on the observation that two large classes of biophysical models of cis-regulatory mechanisms can be expressed as deep neural networks in which nodes and weights have explicit physiochemical interpretations. We also demonstrate how such biophysical networks can be rapidly inferred, using modern deep learning frameworks, from the data produced by certain types of massively parallel reporter assays (MPRAs). These results suggest a scalable strategy for using MPRAs to systematically characterize the biophysical basis of gene regulation in a wide range of biological contexts. They also highlight gene regulation as a promising venue for the development of scientifically interpretable approaches to deep learning.

scholarly journals Antibacterial Activity And Genotoxicity Effect of Ethanolic Leaves Extract of Rosmarinus Officinalis

2020 ◽  
Vol 14 (3) ◽  
pp. 2165-2171
Author(s):  
Fayez Althobaiti
Keyword(s):  
Pathogenic Bacteria ◽  
Ethanolic Extract ◽  
Secondary Compounds ◽  
Rosmarinus Officinalis ◽  
Bacterial Genomes ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Wide Range ◽  
High Site ◽  
Resistant Strains

The recent increasing use of artificial antibiotics has prompted an expansion in resistant strains and high site reactions. Medicinal plants have for quite some time been utilized as traditional medicine to treat pathogenic bacteria. In such manner, consistently numerous scientists are sending a range of plant’s secondary compounds to the customer advertise for the treatment of human illnesses. Accordingly, the distinguishing proof of plant spices with antimicrobial impacts can assist with delivering new medications with a wide range of impacts. The aim of the present research was to examine the ability of ethanolic leaves extracts of Rosmarinus officinalis plant as antibacterial agent against the Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli. The zone of inhibition increased with increase in concentration of the test solution. Higher activity of ethanolic extract was found against S. aureus (2.4 cm) than E. coli (1.8 cm). In addition, the repetitive element PCR (Rep-PCR) significantly showed that several genetic numbers of polymorphic bands were observed in S. aureus and E. coli treated bacteria with leaves extracts and not observed in the control. These results indicate that these extracts have a genotoxicity effect on the two bacterial genomes. The obtained results demonstrate that R. officinalis can be used as a potential source of antibacterial and genotoxicity factors.

Small Molecule Permeation Across Membrane Channels: Chemical Modification to Quantify Transport Across OmpF

2018 ◽  
Author(s):  
Jiajun Wang ◽  
Jayesh Arun Bafna ◽  
Satya Prathyusha Bhamidimarri ◽  
Mathias Winterhalter
Keyword(s):  
Dwell Time ◽  
Covalent Binding ◽  
Channel Structure ◽  
Ion Current ◽  
E Coli ◽  
Current Fluctuation ◽  
Wide Range ◽  
Outer Membrane Channel ◽  
Biological Channels ◽  
Constriction Region

Biological channels facilitate the exchange of small molecules across membranes, but surprisingly there is a lack of general tools for the identification and quantification of transport (i.e., translocation and binding). Analyzing the ion current fluctuation of a typical channel with its constriction region in the middle does not allow a direct conclusion on successful transport. For this, we created an additional barrier acting as a molecular counter at the exit of the channel. To identify permeation, we mainly read the molecule residence time in the channel lumen as the indicator whether the molecule reached the exit of the channel. As an example, here we use the well-studied porin, OmpF, an outer membrane channel from <i>E. coli</i>. Inspection of the channel structure suggests that aspartic acid at position 181 is located below the constriction region (CR) and we subsequently mutated this residue to cysteine, where else cysteine free and functionalized it by covalent binding with 2-sulfonatoethyl methanethiosulfonate (MTSES) or the larger glutathione (GLT) blockers. Using the dwell time as the signal for transport, we found that both mono-arginine and tri-arginine permeation process is prolonged by 20% and 50% respectively through OmpF<sub>E181C</sub>MTSES, while the larger sized blocker modification OmpF<sub>E181C</sub>GLT drastically decreased the permeation of mono-arginine by 9-fold and even blocked the pathway of the tri-arginine. In case of the hepta-arginine as substrate, both chemical modifications led to an identical ‘blocked’ pattern observed by the dwell time of ion current fluctuation of the OmpF<sub>wt</sub>. As an instance for antibiotic permeation, we analyzed norfloxacin, a fluoroquinolone antimicrobial agent. The modulation of the interaction dwell time suggests possible successful permeation of norfloxacin across OmpF<sub>wt</sub>. This approach may discriminate blockages from translocation events for a wide range of substrates. A potential application could be screening for scaffolds to improve the permeability of antibiotics.

The Characterization of Antibiotic Resistance of Bacterial Isolates from Intensive Care Unit Patient Samples in a University Affiliated Hospital in Romania

2019 ◽  
Vol 70 (5) ◽  
pp. 1778-1783
Author(s):  
Andreea-Loredana Golli ◽  
Floarea Mimi Nitu ◽  
Maria Balasoiu ◽  
Marina Alina Lungu ◽  
Cristiana Cerasella Dragomirescu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Bacterial Pathogens ◽  
Resistance Rate ◽  
Resistance Pattern ◽  
Bacterial Isolates ◽  
E Coli ◽  
Acinetobacter Spp ◽  
Almost All ◽  
Klebsiella Spp

To determine the resistance pattern of bacterial pathogens involved in infections of the patients aged between 18-64 years, admitted in a ICU from a 1518-bed university-affiliated hospital. A retrospective study of bacterial pathogens was carried out on 351 patients aged between 18-64 years admitted to the ICU, from January to December 2017. In this study there were analysed 469 samples from 351 patients (18-64 years). A total of 566 bacterial isolates were obtained, of which 120 strains of Klebsiella spp. (35.39%%), followed by Nonfermenting Gram negative bacilli, other than Pseudomonas and Acinetobacter (NFB) (75- 22.12%), Acinetobacter spp. (53 - 15.63%), Pseudomonas aeruginosa and Proteus (51 - 15.04%), and Escherichia coli (49 - 14.45%). The most common isolates were from respiratory tract (394 isolates � 69.61%). High rates of MDR were found for Pseudomonas aeruginosa (64.70%), MRSA (62.65%) and Klebsiella spp. (53.33%), while almost all of the isolated NFB strains were MDR (97.33%). There was statistic difference between the drug resistance rate of Klebsiella and E. coli strains to ceftazidime and ceftriaxone (p[0.001), cefuroxime (p[0.01) and to cefepime (p[0.01). The study revealed an alarming pattern of antibiotic resistance in the majority of ICU isolates.

An Improved Membrane Filtration Method for Enumeration of Faecal Coliforms and E. Coli by a Fluorogenic β-Glucuronidase Assay

1993 ◽  
Vol 27 (3-4) ◽  
pp. 267-270 ◽  
Author(s):  
M. T. Augoustinos ◽  
N. A. Grabow ◽  
B. Genthe ◽  
R. Kfir
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Membrane Filtration ◽  
Faecal Coliforms ◽  
Environmental Waters ◽  
Filtration Method ◽  
E Coli ◽  
Wide Range ◽  
Pure Cultures ◽  
Glucuronidase Assay

A fluorogenic β-glucuronidase assay comprising membrane filtration followed by selective enumeration on m-FC agar at 44.5°C and further confirmation using tlie 4-metliylumbelliferyl-β-D-glucuronide (MUG) containing medium was evaluated for the detection of Escherichia coli in water. A total of 200 typical blue and non-typical blue colonies were isolated from sea and fresh water samples using initial selective enumeration on m-FC agar. Pure cultures of the selected colonies were further tested using the MUG assay and identified using the API 20E method. Of the colonies tested which were shown to be positive using the MUG assay 99.4% were Escherichia coli. The results of this study indicate the combination of the m-FC method followed by the MUG assay to be highly efficient for the selection and confirmation of E. coli from a wide range of environmental waters.

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