scholarly journals THE EFFECT ON THE GENETIC APPARATUS OF THE MICROBIAL CELL OF COMPOUNDS BASED ON SUBSTITUTED 1H-INDOL-4-, 5-, 6-, 7-YLAMINES

2019 ◽  
Author(s):  
A. Maseykina ◽  
I. Stepanenko ◽  
S. Yamashkin ◽  
E. Slastnikov
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Microbial Cell ◽  
Transcriptional Analysis ◽  
Antimicrobial Compounds ◽  
Bacterial Cells ◽  
Response Curves ◽  
Sos Induction ◽  
Sos Chromotest ◽  
Wide Range

The study of new antimicrobial compounds includes determining the mechanism of their effect on the microbial cell. As a rule, the effect of most modern synthetic antimicrobials is associated either with the suppression of DNA synthesis, or with the suppression of bacterial protein synthesis at the level of translation or transcription.There are sensitive and simple methods for screening and monitoring the potential genotoxic activity of a wide range of natural and synthetic compounds. To date, the Ames test has been widely used, based on the sensitivity of Salmonella strains to carcinogenic chemicals, although some compounds that cause Ames negative reactions could actually be carcinogenic to animals.Similarly, the SOS chromotest is an SOS transcriptional analysis that can evaluate DNA damage caused by chemical and physical mutagens. It measures the expression of a reporter gene (β-galactosidase). The β-galactosidase enzyme processes ortho-nitrophenyl galactopyranoside to form a yellow compound detected at 420 nm. Then, the induction of β-galactosidase normalizes the activity of alkaline phosphatase, an enzyme expressed constitutively by Escherichia coli. SOS chromotest is also widely used for genotoxicological studies. The answer is quick (several hours) and does not require the survival of the test strain. Dose response curves for various chemicals include a linear region. The slope of this area is taken as a measure of SOS induction.Therefore, an SOS chromotest was selected for the study, which allows one to identify the DNA-mediated effect of the studied compounds.The aim of the work was to evaluate the SOS-inducing activity of antimicrobial compounds based on substituted 1H-indol-4-, 5-, 6-, 7-ylamines.The strain Escherichia coli PQ 37 with the genotype F-thr leu his-4 pyrD thi galE lacΔU169 srl300 :: Th10 rpoB rpsL uvrA rfa trp :: Mis + sfi A :: Mud (Ar, lac) cts, Due to the presence of sfi A genes :: lac Z, lacZ β-galactosidase gene expression in strain PQ 37 is controlled by the promoter of the sfiA gene, one of the components of the E. coli SOS regulon. The indicator of the SOS-inducing activity of the studied compounds in the SOS chromotest is the activity of β-galactosidase, which evaluates the activity of active microorganisms - alkaline phosphatase, which also allows you to control the toxic effect of the studied compounds on bacterial cells.The results showed that 4,4,4-trifluoro-N-(6-methoxy-1,2,3-trimethyl-1H-indol-5-yl)-3-oxobutanamide (1), 4,4,4-trifluoro-N-(6-methyl-2-phenyl-1H-indol-5-yl)-3-oxobutanamide (2) and N-(1,5-dimethyl-2-phenyl-1H-indol-6-yl)-4,4,4-trifluoro-3-oxobutanamide (3) does not possess SOS-inducing activity in the studied concentrations. 4-Hydroxy-8-phenyl-4-(trifluoromethyl)-1,3,4,7-tetrahydro-2H-pyrrolo [2,3-h] -quinolin-2-one (4), 9-hydroxy-5-methyl-2-phenyl-9-(trifluoromethyl)-1,6,8,9-tetrahydro-7Н-pyrrolo-[2,3-f]quinolin-7-one (5), 6-hydroxy-2,3-dimethyl-6-(trifluoromethyl)-1,6,7,9-tetrahydro-8H-pyrrolo[3,2-h]quinolin-8-one (6) and 1,2,3,9-tetramethyl-6-(trifluoromethyl)-1,9-dihydro-8H-pyrrolo [3,2-h]quinolin-8-one (7) showed dose-dependent SOS-inducing activity in bactericidal concentrations. The obtained research results allowed us to identify compounds 4, 5, 6, 7, the mechanism of action of which includes exposure to DNA of a microbial cell.

SOS induction of selected naturally occurring substances in Escherichia coli (SOS chromotest)

1999 ◽  
Vol 445 (1) ◽  
pp. 81-91 ◽  
Author(s):  
Sebastian Kevekordes ◽  
Volker Mersch-Sundermann ◽  
Christian M Burghaus ◽  
Jan Spielberger ◽  
Heinz H Schmeiser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sos Induction ◽  
Naturally Occurring ◽  
Sos Chromotest

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.

scholarly journals Plasticity in Escherichia coli cell wall metabolism promotes fitness and mediates intrinsic antibiotic resistance across environmental conditions

2018 ◽  
Author(s):  
Elizabeth A Mueller ◽  
Petra Anne Levin
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Growth Parameter ◽  
Culture Conditions ◽  
Cell Wall Integrity ◽  
Bacterial Cells ◽  
Intrinsic Resistance ◽  
Wide Range ◽  
Peptidoglycan Cell Wall ◽  
Wide Ph Range

ABSTRACTAlthough the peptidoglycan cell wall is an essential structural and morphological feature of most bacterial cells, the extracytoplasmic enzymes involved in its synthesis are frequently dispensable under standard culture conditions. By modulating a single growth parameter—extracellular pH—we discovered a subset of these so-called “redundant” enzymes in Escherichia coli are required for maximal fitness across pH environments. Among these pH specialists are the class A penicillin binding proteins PBP1 a and PBP1 b; defects in these enzymes attenuate growth in alkaline and acidic conditions, respectively. Genetic, biochemical, and cytological studies demonstrate that synthase activity is required for cell wall integrity across a wide pH range, and differential activity across pH environments significantly alters intrinsic resistance to cell wall active antibiotics. Together, our findings reveal previously thought to be redundant enzymes are instead specialized for distinct environmental niches, thereby ensuring robust growth and cell wall integrity in a wide range of conditions.

scholarly journals Plasticity of Escherichia coli cell wall metabolism promotes fitness and antibiotic resistance across environmental conditions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Elizabeth A Mueller ◽  
Alexander JF Egan ◽  
Eefjan Breukink ◽  
Waldemar Vollmer ◽  
Petra Anne Levin
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Growth Parameter ◽  
Culture Conditions ◽  
Cell Wall Integrity ◽  
Editorial Note ◽  
Bacterial Cells ◽  
Wide Range ◽  
Peptidoglycan Cell Wall ◽  
Wide Ph Range

Although the peptidoglycan cell wall is an essential structural and morphological feature of most bacterial cells, the extracytoplasmic enzymes involved in its synthesis are frequently dispensable under standard culture conditions. By modulating a single growth parameter—extracellular pH—we discovered a subset of these so-called ‘redundant’ enzymes in Escherichia coli are required for maximal fitness across pH environments. Among these pH specialists are the class A penicillin binding proteins PBP1a and PBP1b; defects in these enzymes attenuate growth in alkaline and acidic conditions, respectively. Genetic, biochemical, and cytological studies demonstrate that synthase activity is required for cell wall integrity across a wide pH range and influences pH-dependent changes in resistance to cell wall active antibiotics. Altogether, our findings reveal previously thought to be redundant enzymes are instead specialized for distinct environmental niches. This specialization may ensure robust growth and cell wall integrity in a wide range of conditions.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 all the issues have been addressed (see decision letter).

scholarly journals Assessment of Photodynamic Destruction of Escherichia coli O157:H7 and Listeria monocytogenes by Using ATP Bioluminescence

2003 ◽  
Vol 69 (11) ◽  
pp. 6393-6398 ◽  
Author(s):  
N. A. Romanova ◽  
L. Y. Brovko ◽  
L. Moore ◽  
E. Pometun ◽  
A. P. Savitsky ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Yeast Cells ◽  
Bacterial Cells ◽  
E Coli ◽  
Intracellular Atp ◽  
Microdilution Method ◽  
Wide Range ◽  
Atp Bioluminescence ◽  
Time Courses

ABSTRACT Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.

scholarly journals Persister Cells Resuscitate via Ribosome Modification by 23S rRNA Pseudouridine Synthase RluD

2019 ◽  
Author(s):  
Sooyeon Song ◽  
Thomas K. Wood
Keyword(s):  
Escherichia Coli ◽  
Small Rna ◽  
23S Rrna ◽  
Bacterial Cells ◽  
Persister Cells ◽  
E Coli ◽  
Pseudouridine Synthase ◽  
Dormant Cells ◽  
Wide Range ◽  
Persister Cell

ABSTRACTUpon a wide range of stress conditions (e.g., nutrient, antibiotic, oxidative), a subpopulation of bacterial cells known as persisters survive by halting metabolism. These cells resuscitate rapidly to reconstitute infections once the stress is removed and nutrients are provided. However, how these dormant cells resuscitate is not understood well but involves reactivating ribosomes. By screening 10,000 compounds directly for stimulating Escherichia coli persister cell resuscitation, we identified that 2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (BPOET) stimulates resuscitation. Critically, by screening 4,267 E. coli proteins, we determined that BPOET activates hibernating ribosomes via 23S rRNA pseudouridine synthase RluD, which increases ribosome activity. Corroborating the increased waking with RluD, production of RluD increased the number of active ribosomes in persister cells. Also, inactivating the small RNA RybB which represses rluD led to faster persister resuscitation. Hence, persister cells resuscitate via activation of RluD.

scholarly journals Properties of AdeABC and AdeIJK Efflux Systems of Acinetobacter baumannii Compared with Those of the AcrAB-TolC System of Escherichia coli

2014 ◽  
Vol 58 (12) ◽  
pp. 7250-7257 ◽  
Author(s):  
Etsuko Sugawara ◽  
Hiroshi Nikaido
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Ethidium Bromide ◽  
Expression Level ◽  
Antimicrobial Compounds ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Quantitative Basis

ABSTRACTAcinetobacter baumanniicontains RND-family efflux systems AdeABC and AdeIJK, which pump out a wide range of antimicrobial compounds, as judged from the MIC changes occurring upon deletion of the responsible genes. However, these studies may miss changes because of the high backgrounds generated by the remaining pumps and by β-lactamases, and it is unclear how the activities of these pumps compare quantitatively with those of the well-studied AcrAB-TolC system ofEscherichia coli. We expressedadeABCandadeIJKofA. baumannii, as well asE. coliacrAB, in anE. colihost from whichacrABwas deleted. TheA. baumanniipumps were functional inE. coli, and the MIC changes that were observed largely confirmed the substrate range already reported, with important differences. Thus, the AdeABC system pumped out all β-lactams, an activity that was often missed in deletion studies. When the expression level of the pump genes was adjusted to a similar level for a comparison with AcrAB-TolC, we found that bothA. baumanniiefflux systems pumped out a wide range of compounds, but AdeABC was less effective than AcrAB-TolC in the extrusion of lipophilic β-lactams, novobiocin, and ethidium bromide, although it was more effective at tetracycline efflux. AdeIJK was remarkably more effective than a similar level of AcrAB-TolC in the efflux of β-lactams, novobiocin, and ethidium bromide, although it was less so in the efflux of erythromycin. These results thus allow us to compare these efflux systems on a quantitative basis, if we can assume that the heterologous systems are fully functional in theE. colihost.

scholarly journals A Luminescent Escherichia coli Biosensor for the High Throughput Detection of β-Lactams

2002 ◽  
Vol 7 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Satu J. Valtonen ◽  
Jussi S. Kurittu ◽  
Matti T. Karp
Keyword(s):  
Escherichia Coli ◽  
Model Organism ◽  
Coli Strain ◽  
Bacterial Cells ◽  
Response Curves ◽  
Light Production ◽  
High Throughput Detection ◽  
High Concentrations ◽  
Luciferase Enzyme ◽  
Responsive Element

A group-specific bioluminescent Escherichia coli strain for studying the action of β-lactam antibiotics is described. The strain contains a plasmid, pBlaLux1, in which the luciferase genes from Photorhabdus luminescent are inserted under the control of the β-lactam-responsive element ampR/ampC from Citrobacter freundii. In the presence of β-lactams, the bacterial cells are induced to express the luciferase enzyme and three additional enzymes generating the substrate for the luciferase reaction. This biosensor for β-lactams does not need any substrate or cofactor additions, and the bioluminescence can be measured very sensitively in real time by using a luminometer. Basic parameters affecting the light production and induction in the gram-negative model organism E. coli SNO301/pBlaLux1 by various β-lactams were studied. The dose-response curves were bell shaped, indicating toxic effects for the sensor strain at high concentrations of β-lactams. Various β-lactams had fairly different assay ranges: ampicillin, 0.05-1.0 μg/ml; piperacillin, 0.0025-25 μg/ml; imipenem, 0.0025-0.25 μg/ml; cephapirin, 0.025-2.5 μg/ml; cefoxitin, 0.0025-1.5 μg/ml; and oxacillin, 25-500 μg/ml. Also, the induction coefficients (signal over background noninduced control) varied considerably from 3 to 158 in a 2-hour assay. Different non-β-lactam antibiotics did not cause induction. Because the assay can be automated using microplate technologies, the approach may be suitable for higher throughput analysis of β-lactam action.

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.

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