scholarly journals Metabolic Regulation of a Bacterial Cell System with Emphasis on Escherichia coli Metabolism

2013 ◽  
Vol 2013 ◽  
pp. 1-47 ◽  
Author(s):  
Kazuyuki Shimizu
Keyword(s):  
Escherichia Coli ◽  
Metabolic Regulation ◽  
Cell System ◽  
Regulation Mechanism ◽  
Bacterial Cells ◽  
Gene Expressions ◽  
Global Regulators ◽  
Acid Shock ◽  
Carbon Catabolite Regulation ◽  
Catabolite Regulation

It is quite important to understand the overall metabolic regulation mechanism of bacterial cells such as Escherichia coli from both science (such as biochemistry) and engineering (such as metabolic engineering) points of view. Here, an attempt was made to clarify the overall metabolic regulation mechanism by focusing on the roles of global regulators which detect the culture or growth condition and manipulate a set of metabolic pathways by modulating the related gene expressions. For this, it was considered how the cell responds to a variety of culture environments such as carbon (catabolite regulation), nitrogen, and phosphate limitations, as well as the effects of oxygen level, pH (acid shock), temperature (heat shock), and nutrient starvation.

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 A novel application of bubble-eye strain of Carassius auratus for ex vivo fish immunological studies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hiroto Nakajima ◽  
Atsushi Miyashita ◽  
Hiroshi Hamamoto ◽  
Kazuhisa Sekimizu
Keyword(s):  
Inflammatory Cytokines ◽  
Immune Cells ◽  
Ex Vivo ◽  
Large Bubble ◽  
Suitable Model ◽  
Bacterial Cells ◽  
Functional Impairments ◽  
Gene Expressions ◽  
Pro Inflammatory Cytokines

AbstractIn this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution

2021 ◽  
Vol 2021 (11) ◽  
pp. pdb.prot101212 ◽  
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Escherichia Coli ◽  
Convenient Method ◽  
Plasmid Dna ◽  
Transformation Efficiency ◽  
Bacterial Cells ◽  
E Coli ◽  
One Step ◽  
And Storage

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli. The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

scholarly journals Bacterial Secretions of Nonpathogenic Escherichia coli Elicit Inflammatory Pathways: a Closer Investigation of Interkingdom Signaling

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Amin Zargar ◽  
David N. Quan ◽  
Karen K. Carter ◽  
Min Guo ◽  
Herman O. Sintim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Negative Feedback ◽  
Cell Contact ◽  
Bacterial Cells ◽  
Content Type ◽  
Phenotypic Differences ◽  
E Coli ◽  
Autoinducer 2 ◽  
Human Epithelial Cells

ABSTRACTThere have been many studies on the relationship between nonpathogenic bacteria and human epithelial cells; however, the bidirectional effects of the secretomes (secreted substances in which there is no direct bacterium-cell contact) have yet to be fully investigated. In this study, we use a transwell model to explore the transcriptomic effects of bacterial secretions from two different nonpathogenicEscherichia colistrains on the human colonic cell line HCT-8 using next-generation transcriptome sequencing (RNA-Seq).E. coliBL21 and W3110, while genetically very similar (99.1% homology), exhibit key phenotypic differences, including differences in their production of macromolecular structures (e.g., flagella and lipopolysaccharide) and in their secretion of metabolic byproducts (e.g., acetate) and signaling molecules (e.g., quorum-sensing autoinducer 2 [AI-2]). After analysis of differential epithelial responses to the respective secretomes, this study shows for the first time that a nonpathogenic bacterial secretome activates the NF-κB-mediated cytokine-cytokine receptor pathways while also upregulating negative-feedback components, including the NOD-like signaling pathway. Because of AI-2's relevance as a bacterium-bacterium signaling molecule and the differences in its secretion rates between these strains, we investigated its role in HCT-8 cells. We found that the expression of the inflammatory cytokine interleukin 8 (IL-8) responded to AI-2 with a pattern of rapid upregulation before subsequent downregulation after 24 h. Collectively, these data demonstrate that secreted products from nonpathogenic bacteria stimulate the transcription of immune-related biological pathways, followed by the upregulation of negative-feedback elements that may serve to temper the inflammatory response.IMPORTANCEThe symbiotic relationship between the microbiome and the host is important in the maintenance of human health. There is a growing need to further understand the nature of these relationships to aid in the development of homeostatic probiotics and also in the design of novel antimicrobial therapeutics. To our knowledge, this is the first global-transcriptome study of bacteria cocultured with human epithelial cells in a model to determine the transcriptional effects of epithelial cells in which epithelial and bacterial cells are allowed to “communicate” with each other only through diffusible small molecules and proteins. By beginning to demarcate the direct and indirect effects of bacteria on the gastrointestinal (GI) tract, two-way interkingdom communication can potentially be mediated between host and microbe.

scholarly journals Escherichia coli Harboring a Natural IncF Conjugative F Plasmid Develops Complex Mature Biofilms by Stimulating Synthesis of Colanic Acid and Curli

2008 ◽  
Vol 190 (22) ◽  
pp. 7479-7490 ◽  
Author(s):  
Thithiwat May ◽  
Satoshi Okabe
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Three Dimensional ◽  
F Plasmid ◽  
Form Complex ◽  
Global Regulators ◽  
E Coli ◽  
Colanic Acid ◽  
Regulatory Systems ◽  
Initial Deposition

ABSTRACT It has been shown that Escherichia coli harboring the derepressed IncFI and IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore, in this study we investigated the contribution of a natural IncF conjugative F plasmid to the formation of E. coli biofilms. Unlike the presence of a derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm formation by generating the cell-to-cell mating F pili between pairs of F+ cells (approximately two to four pili per cell) and by stimulating the formation of colanic acid and curli meshwork. Formation of colanic acid and curli was required after the initial deposition of F-pilus connections to generate a three-dimensional mushroom-type biofilm. In addition, we demonstrated that the conjugative factor of F plasmid, rather than a pilus synthesis function, was involved in curli production during biofilm formation, which promoted cell-surface interactions. Curli played an important role in the maturation process. Microarray experiments were performed to identify the genes involved in curli biosynthesis and regulation. The results suggested that a natural F plasmid was more likely an external activator that indirectly promoted curli production via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the RpoS and HN-S global regulators). These data provided new insights into the role of a natural F plasmid during the development of E. coli biofilms.

scholarly journals Cooperative Roles of Nitric Oxide-Metabolizing Enzymes To Counteract Nitrosative Stress in Enterohemorrhagic Escherichia coli

2019 ◽  
Vol 87 (9) ◽  
Author(s):  
Takeshi Shimizu ◽  
Akio Matsumoto ◽  
Masatoshi Noda
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Stationary Phase ◽  
Nitrosative Stress ◽  
Enterohemorrhagic Escherichia Coli ◽  
The Other ◽  
Anaerobic Growth ◽  
Bacterial Cells ◽  
Metabolizing Enzymes ◽  
Microaerobic Conditions

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) has at least three enzymes, NorV, Hmp, and Hcp, that act independently to lower the toxicity of nitric oxide (NO), a potent antimicrobial molecule. This study aimed to reveal the cooperative roles of these defensive enzymes in EHEC against nitrosative stress. Under anaerobic conditions, combined deletion of all three enzymes significantly increased the NO sensitivity of EHEC determined by the growth at late stationary phase; however, the expression of norV restored the NO resistance of EHEC. On the other hand, the growth of Δhmp mutant EHEC was inhibited after early stationary phase, indicating that NorV and Hmp play a cooperative role in anaerobic growth. Under microaerobic conditions, the growth of Δhmp mutant EHEC was inhibited by NO, indicating that Hmp is the enzyme that protects cells from NO stress under microaerobic conditions. When EHEC cells were exposed to a lower concentration of NO, the NO level in bacterial cells of Δhcp mutant EHEC was higher than those of the other EHEC mutants, suggesting that Hcp is effective at regulating NO levels only at a low concentration. These findings of a low level of NO in bacterial cells with hcp indicate that the NO consumption activity of Hcp was suppressed by Hmp at a low range of NO concentrations. Taken together, these results show that the cooperative effects of NO-metabolizing enzymes are regulated by the range of NO concentrations to which the EHEC cells are exposed.

Genetic engineering: possibilities and prospects for its application in industrial microbiology

1980 ◽  
Vol 290 (1040) ◽  
pp. 369-386 ◽  
Keyword(s):  
Escherichia Coli ◽  
Genetic Engineering ◽  
Detailed Analysis ◽  
Bacterial Cells ◽  
Bacterial Enzymes ◽  
Distant Future ◽  
Cells In Culture ◽  
Wide Range ◽  
Hybrid Dna

A wide range of techniques is now available for the construction of hybrid DNA molecules comprising components from disparate species. Transfer of segments of DNA from other organisms, and especially eukaryotes, to Escherichia coli permits their preparation in quantities sufficient for detailed analysis of their structure and mechanism of expression. This information could be exploited to enhance the quantity or quality of polypeptide products from bacterial cells. Greatly increased yields of bacterial enzymes have been obtained in this way in several instances. The approaches that have been pioneered with bacteria are currently being applied to higher organisms. Much work is in progress with yeasts, in which transformation has been successfully demonstrated, with animal viruses and cells in culture and with some plant systems and offers the promise of wider applications of genetic engineering in the not too distant future.

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