scholarly journals Chromogenic Escherichia coli reporter strain for screening DNA damaging agents

AMB Express ◽  
2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Josué Daniel Mora-Garduño ◽  
Jessica Tamayo-Nuñez ◽  
Felipe Padilla-Vaca ◽  
Fátima Berenice Ramírez-Montiel ◽  
Ángeles Rangel-Serrano ◽  
...  

AbstractThe presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.

1999 ◽  
Vol 181 (15) ◽  
pp. 4549-4553 ◽  
Author(s):  
Teruhito Yamashita ◽  
Katsuhiro Hanada ◽  
Mihoko Iwasaki ◽  
Hirotaka Yamaguchi ◽  
Hideo Ikeda

ABSTRACT Illegitimate recombination that usually takes place at a low frequency is greatly enhanced by treatment with DNA-damaging agents. It is thought that DNA double-strand breaks induced by this DNA damage are important for initiation of illegitimate recombination. Here we show that illegitimate recombination is enhanced by overexpression of the DnaB protein in Escherichia coli. The recombination enhanced by DnaB overexpression occurred between short regions of homology. We propose a model for the initiation of illegitimate recombination in which DnaB overexpression may excessively unwind DNA at replication forks and induce double-strand breaks, resulting in illegitimate recombination. The defect in RecQ has a synergistic effect on the increased illegitimate recombination in cells containing the overproduced DnaB protein, implying that DnaB works in the same pathway as RecQ does but that they work at different steps.


2009 ◽  
Vol 191 (15) ◽  
pp. 4959-4965 ◽  
Author(s):  
Katherine R. Ona ◽  
Charmain T. Courcelle ◽  
Justin Courcelle

ABSTRACT Nitrofurazone is reduced by cellular nitroreductases to form N 2-deoxyguanine (N 2-dG) adducts that are associated with mutagenesis and lethality. Much attention recently has been given to the role that the highly conserved polymerase IV (Pol IV) family of polymerases plays in tolerating adducts induced by nitrofurazone and other N 2-dG-generating agents, yet little is known about how nitrofurazone-induced DNA damage is processed by the cell. In this study, we characterized the genetic repair pathways that contribute to survival and mutagenesis in Escherichia coli cultures grown in the presence of nitrofurazone. We find that nucleotide excision repair is a primary mechanism for processing damage induced by nitrofurazone. The contribution of translesion synthesis to survival was minor compared to that of nucleotide excision repair and depended upon Pol IV. In addition, survival also depended on both the RecF and RecBCD pathways. We also found that nitrofurazone acts as a direct inhibitor of DNA replication at higher concentrations. We show that the direct inhibition of replication by nitrofurazone occurs independently of DNA damage and is reversible once the nitrofurazone is removed. Previous studies that reported nucleotide excision repair mutants that were fully resistant to nitrofurazone used high concentrations of the drug (200 μM) and short exposure times. We demonstrate here that these conditions inhibit replication but are insufficient in duration to induce significant levels of DNA damage.


Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3181-3195 ◽  
Author(s):  
Joe J. Harrison ◽  
Howard Ceri ◽  
Nicole J. Roper ◽  
Erin A. Badry ◽  
Kimberley M. Sproule ◽  
...  

Bacterial cultures produce subpopulations of cells termed ‘persisters’, reputedly known for high tolerance to killing by antibiotics. Ecologically, antibiotics produced by competing microflora are only one potential stress encountered by bacteria. Another pressure in the environment is toxic metals that are distributed ubiquitously by human pollution, volcanic activity and the weathering of minerals. This study evaluated the time- and concentration-dependent killing of Escherichia coli planktonic and biofilm cultures by the water-soluble metal(loid) oxyanions chromate (), arsenate (), arsenite (), selenite (), tellurate () and tellurite (). Correlative to previous reports in the literature, control antibiotic assays indicated that a small proportion of E. coli biofilm populations remained recalcitrant to killing by antibiotics (even with 24 h exposure). In contrast, metal oxyanions presented a slow, bactericidal action that eradicated biofilms. When exposed for 2 h, biofilms were up to 310 times more tolerant to killing by metal oxyanions than corresponding planktonic cultures. However, by 24 h, planktonic cells and biofilms were eradicated at approximately the same concentration in all instances. Coloured complexes of metals and chelators could not be generated in biofilms exposed to or , suggesting that the extracellular polymeric matrix of E. coli may have a low binding affinity for metal oxyanions. Viable cell counts at 2 and 24 h exposure revealed that, at high concentrations, all of the metal oxyanions had killed 99 % (or a greater proportion) of the bacterial cells in biofilm populations. It is suggested here that the short-term survival of <1 % of the bacterial population corresponds well with the hypothesis that a small population of persister cells may be responsible for the time-dependent tolerance of E. coli biofilms to high concentrations of metal oxyanions.


2008 ◽  
Vol 74 (5) ◽  
pp. 1502-1507 ◽  
Author(s):  
Yanming Liu ◽  
Ainslie Gilchrist ◽  
Jing Zhang ◽  
Xing-Fang Li

ABSTRACT A sensitive method for specific detection of viable Escherichia coli O157:H7 cells, including viable but nonculturable (VBNC) cells, in water samples was developed. This method involved capture of the bacterial cells on a low-protein-binding membrane and direct extraction and purification of RNA followed by reverse transcription-PCR and electronic microarray detection of the rfbE and fliC genes of E. coli O157:H7. It detected as few as 1 CFU of E. coli O157:H7 in diluted cultures, 3 to 4 CFU/liter in tap water, 7 CFU/liter in river water, and 50 VBNC cells in 1 liter of river water, demonstrating the best limit of detection reported to date for VBNC cells in environmental water samples.


2021 ◽  
Vol 9 ◽  
Author(s):  
Julie K. Buick ◽  
Andrew Williams ◽  
Matthew J. Meier ◽  
Carol D. Swartz ◽  
Leslie Recio ◽  
...  

Higher-throughput, mode-of-action-based assays provide a valuable approach to expedite chemical evaluation for human health risk assessment. In this study, we combined the high-throughput alkaline DNA damage-sensing CometChip® assay with the TGx-DDI transcriptomic biomarker (DDI = DNA damage-inducing) using high-throughput TempO-Seq®, as an integrated genotoxicity testing approach. We used metabolically competent differentiated human HepaRG™ cell cultures to enable the identification of chemicals that require bioactivation to cause genotoxicity. We studied 12 chemicals (nine DDI, three non-DDI) in increasing concentrations to measure and classify chemicals based on their ability to damage DNA. The CometChip® classified 10/12 test chemicals correctly, missing a positive DDI call for aflatoxin B1 and propyl gallate. The poor detection of aflatoxin B1 adducts is consistent with the insensitivity of the standard alkaline comet assay to bulky lesions (a shortcoming that can be overcome by trapping repair intermediates). The TGx-DDI biomarker accurately classified 10/12 agents. TGx-DDI correctly identified aflatoxin B1 as DDI, demonstrating efficacy for combined used of these complementary methodologies. Zidovudine, a known DDI chemical, was misclassified as it inhibits transcription, which prevents measurable changes in gene expression. Eugenol, a non-DDI chemical known to render misleading positive results at high concentrations, was classified as DDI at the highest concentration tested. When combined, the CometChip® assay and the TGx-DDI biomarker were 100% accurate in identifying chemicals that induce DNA damage. Quantitative benchmark concentration (BMC) modeling was applied to evaluate chemical potencies for both assays. The BMCs for the CometChip® assay and the TGx-DDI biomarker were highly concordant (within 4-fold) and resulted in identical potency rankings. These results demonstrate that these two assays can be integrated for efficient identification and potency ranking of DNA damaging agents in HepaRG™ cell cultures.


2011 ◽  
Vol 77 (15) ◽  
pp. 5141-5148 ◽  
Author(s):  
Morteza Abbaszadegan ◽  
Absar Alum ◽  
Hamed Abbaszadegan ◽  
Valerie Stout

ABSTRACTThe lack of efficient methods for concentrating viruses in water samples leads to underreporting of viral contamination in source water. A novel strategy for viral concentration was developed using the expression of target virus receptors on bacterial cells. Poliovirus type 1, the most studied enterovirus, was used as a surrogate for enteric viruses. The human poliovirus receptor (hPVR) gene was expressed on the surface ofEscherichia colicells by using the ice nucleation protein (INP) gene. ThehPVRgene was ligated to the 3′ end of theINPgene after the removal of the stop codon. The resulting open reading frame (ORF) was used for the projection of hPVR onto the outer membrane ofE. coli. Gene expression was tested by SDS-PAGE, Western blot, and dot blot analyses, and virion capture ability was confirmed by transmission electron microscopy. The application of engineeredE. colicells for capturing viruses in 1-liter samples of source and drinking water resulted in 75 to 99% procedural recovery efficiency. Cell surface display of viral receptors on bacterial cells opens a new prospect for an efficient and inexpensive alternative tool for capturing and concentrating waterborne viruses in water samples.


2002 ◽  
Vol 7 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Satu J. Valtonen ◽  
Jussi S. Kurittu ◽  
Matti T. Karp

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.


2016 ◽  
Vol 198 (11) ◽  
pp. 1576-1584 ◽  
Author(s):  
Ge Ren ◽  
Zhou Wang ◽  
Ye Li ◽  
Xiaoqing Hu ◽  
Xiaoyuan Wang

ABSTRACTWhen 10Escherichia colimutant strains with defects in lipopolysaccharide (LPS) core biosynthesis were grown on agar medium at 30°C, four of them, the ΔwaaF, ΔwaaG, ΔwaaP, and ΔwaaBstrains, formed mucoid colonies, while the other six, the ΔwaaU, ΔwaaR, ΔwaaO, ΔwaaC, ΔwaaQ, and ΔwaaYstrains, did not. Using light microscopy with tannin mordant staining, the presence of exopolysaccharide around the cells of the mutants that formed mucoid colonies could be discerned. The ΔwaaFmutant produced the largest amounts of exopolysaccharide, regardless of whether it was grown on agar or in liquid medium. The exopolysaccharide was isolated from the liquid growth medium of ΔwaaFcells, hydrolyzed, and analyzed by high-performance liquid chromatography with an ion-exchange column, and the results indicated that the exopolysaccharide was consistent with colanic acid. When the key genes related to the biosynthesis of colanic acid, i.e.,wza,wzb,wzc, andwcaA, were deleted in the ΔwaaFbackground, the exopolysaccharide could not be produced any more, further confirming that it was colanic acid. Colanic acid could not be produced in strains in whichrcsA,rcsB,rcsD, orrcsFwas deleted in the ΔwaaFbackground, but a reduced level of colanic acid production was detected when thercsCgene was deleted, suggesting that a change of lipopolysaccharide structure in ΔwaaFcells might be sensed by the RcsCDB phosphorelay system, leading to the production of colanic acid. The results demonstrate thatE. colicells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural deficiency of lipopolysaccharide.IMPORTANCELipopolysaccharide and colanic acid are important forms of exopolysaccharide forEscherichia colicells. Their metabolism and biological significance have been investigated, but their interrelation with the cell stress response process is not understood. This study demonstrates, for the first time, thatE. colicells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural change of lipopolysaccharide, suggesting that bacterial cells can monitor the outer membrane integrity, which is essential for cell survival and damage repair.


2016 ◽  
Vol 60 (11) ◽  
pp. 6867-6871 ◽  
Author(s):  
Peng Cui ◽  
Hongxia Niu ◽  
Wanliang Shi ◽  
Shuo Zhang ◽  
Hao Zhang ◽  
...  

ABSTRACTPersisters are small populations of quiescent bacterial cells that survive exposure to bactericidal antibiotics and are responsible for many persistent infections and posttreatment relapses. However, little is known about how to effectively kill persister bacteria. In the work presented here, we found that colistin, a membrane-active antibiotic, was highly active againstEscherichia colipersisters at high concentrations (25 or 50 μg/ml). At a clinically relevant lower concentration (10 μg/ml), colistin alone had no apparent effect onE. colipersisters. In combination with other drugs, this concentration of colistin enhanced the antipersister activity of gentamicin and ofloxacin but not that of ampicillin, nitrofurans, and sulfa drugsin vitro. The colistin enhancement effect was most likely due to increased uptake of the other antibiotics, as demonstrated by increased accumulation of fluorescence-labeled gentamicin. Interestingly, colistin significantly enhanced the activity of ofloxacin and nitrofurantoin but not that of gentamicin or sulfa drugs in the murine model of urinary tract infection. Our findings suggest that targeting bacterial membranes is a valuable approach to eradicating persisters and should have implications for more effective treatment of persistent bacterial infections.


Author(s):  
О.В. Шамова ◽  
М.С. Жаркова ◽  
П.М. Копейкин ◽  
Д.С. Орлов ◽  
Е.А. Корнева

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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