scholarly journals Staphylococcus aureus MazF Specifically Cleaves a Pentad Sequence, UACAU, Which Is Unusually Abundant in the mRNA for Pathogenic Adhesive Factor SraP

2009 ◽  
Vol 191 (10) ◽  
pp. 3248-3255 ◽  
Author(s):  
Ling Zhu ◽  
Koichi Inoue ◽  
Satoshi Yoshizumi ◽  
Hiroshi Kobayashi ◽  
Yonglong Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Cell Growth ◽  
Bioinformatics Analysis ◽  
Human Tissues ◽  
Regulatory Relationship ◽  
E Coli ◽  
Pathogenic Factors ◽  
Inhibit Cell Growth ◽  
Adhesive Factor

ABSTRACT Escherichia coli mRNA interferases, such as MazF and ChpBK, are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems present in its genome. A MazF homologue in Staphylococcus aureus (MazFSa) has been shown to inhibit cell growth when induced in E. coli. Here, we determined the cleavage site for MazFSa with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. MazFSa specifically cleaves the RNA at a pentad sequence, U↓ACAU. Bioinformatics analysis revealed that this pentad sequence is significantly abundant in several genes, including the sraP gene in the S. aureus N315 strain. This gene encodes a serine-rich protein, which is known to play an important role in adhesion of the pathogen to human tissues and thus in endovascular infection. We demonstrated that the sraP mRNA became extremely unstable in comparison with the ompA mRNA only when MazFSa was induced in E. coli. Further bioinformatics analysis indicated that the pentad sequence is also significantly abundant in the mRNAs for all the pathogenic factors in S. aureus. This observation suggests a possible regulatory relationship between the MazEFSa TA module and the pathogenicity in S. aureus.

EFECTO INHIBITORIO DE EXTRACTOS DE Vitex mollis Kunth CONTRA BACTERIAS Y ESPECIES DE Fusarium DE IMPORTANCIA HUMANA Y AGRÍCOLA

2018 ◽  
Vol 41 (4) ◽  
pp. 353-363
Author(s):  
Alberto J. Valencia-Botin ◽  
Melesio Gutiérrez-Lomelí ◽  
Juan A. Morales-Del-Río ◽  
Pedro J. Guerrero-Medina ◽  
Miguel A. Robles-García ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Salmonella Enterica ◽  
Micrococcus Luteus ◽  
E Coli

Actualmente existe la necesidad de hacer frente al problema de la resistencia a los antibióticos y al uso indiscriminado de fungicidas químicos en la agricultura. El objetivo de este trabajo fue evaluar el efecto inhibitorio de extractos acuosos, metanólicos, acetónicos y hexánicos de hoja y tallo de Vitex mollis Kunth (Lamiaceae) contra diferentes bacterias (Escherichia coli, Micrococcus luteus, Salmonella enterica y Staphylococcus aureus) y especies del hongo Fusarium (F. verticillioides, F. oxysporum, F. tapsinum y F. oxysporum f.sp. lycopersici) de importancia en la salud y en la agricultura, así como determinar su composición química general. Se determinaron las concentraciones inhibitorias mínimas (CIM) de todos los extractos por la técnica de microdilución, excepto del hexánico, que no presentó inhibición en las bacterias estudiadas. S. enterica fue la bacteria que mostró mayor sensibilidad al extracto metanólico de tallo (CIM = 28 μg mL-1), le siguieron M. luteus (CIM = 32 μg mL-1), S. aureus (CIM = 75 μg mL-1) y E. coli (CIM = 80 μg mL- 1). Los extractos metanólicos y acuosos de tallo presentaron mayor porcentaje de inhibición contra los diferentes tipos de Fusarium evaluados por el método de dilución en agar. Los extractos de V. mollis inhibieron a F. verticillioides entre 62 y 91 % con 120 μg mL-1 de extracto. El orden de las especies de hongos inhibidas por los extractos fue: F. verticillioides > F. oxysporum > F. tapsinum > F. oxysporum f.sp. lycopersici. La composición química de las especies se determinó mediante pruebas para fenoles, taninos, flavonoides, triterpenos, alcaloides, cumarinas y saponinas. Ninguno de los extractos presentó alcaloides y saponinas. Los fenoles (37.1 mg EAG/g muestra seca) y flavonoides (26.8 mg EQ/g muestra seca) fueron los compuestos mayoritarios en los extractos metanólicos y acuosos. En conclusión, se requieren cantidades muy pequeñas de extracto para la inhibición de bacterias y de Fusarium; por lo tanto, V. mollis puede ser considerada una fuente de metabolitos para este fin y en la agricultura como control alternativo dentro de un manejo integrado de enfermedades.

Substituted 4-Formyl-2H-chromen-2-ones: Their Reaction with N-(2,3,4,6-Tetra-Oacetyl- β-D-galactopyranosyl)thiosemicarbazide, Antibacterial and Antifungal Activity of Their Thiosemicarbazone Products

2020 ◽  
Vol 24 (19) ◽  
pp. 2272-2282
Author(s):  
Vu Ngoc Toan ◽  
Nguyen Minh Tri ◽  
Nguyen Dinh Thanh
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Selenium Dioxide ◽  
Antibacterial And Antifungal Activity ◽  
E Coli ◽  
Antibacterial And Antifungal Activities ◽  
Alkyl Ethers ◽  
Antibacterial And Antifungal

Several 6- and 7-alkoxy-2-oxo-2H-chromene-4-carbaldehydes were prepared from corresponding alkyl ethers of 6- and 7-hydroxy-4-methyl-2-oxo-2H-chromen-2-ones by oxidation using selenium dioxide. 6- and 7-Alkoxy-4-methyl-2H-chromenes were obtained with yields of 57-85%. Corresponding 4-carbaldehyde derivatives were prepared with yields of 41-67%. Thiosemicarbazones of these aldehydes with D-galactose moiety were synthesized by reaction of these aldehydes with N-(2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosyl) thiosemicarbazide with yields of 62-74%. These thiosemicarbazones were screened for their antibacterial and antifungal activities in vitro against bacteria, such as Staphylococcus aureus, Escherichia coli, and fungi, such as Aspergillus niger, Candida albicans. Several compounds exhibited strong inhibitory activity with MIC values of 0.78- 1.56 μM, including 8a (against S. aureus, E. coli, and C. albicans), 8d (against E. coli and A. niger), 9a (against S. aureus), and 9c (against S. aureus and C. albicans).

Synthesis of Chalcones and Nucleosides Incorporating [1, 3, 4]Oxadiazolenone Core and Evaluation of their Antifungal and Antibacterial Activities

2020 ◽  
Vol 15 (6) ◽  
pp. 665-679
Author(s):  
Alok K. Srivastava ◽  
Lokesh K. Pandey
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Aspergillus Niger ◽  
Gram Negative ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Good Antibacterial Activity

Background: [1, 3, 4]oxadiazolenone core containing chalcones and nucleosides were synthesized by Claisen-Schmidt condensation of a variety of benzaldehyde derivatives, obtained from oxidation of substituted 5-(3/6 substituted-4-Methylphenyl)-1, 3, 4-oxadiazole-2(3H)-one and various substituted acetophenone. The resultant chalcones were coupled with penta-O-acetylglucopyranose followed by deacetylation to get [1, 3, 4] oxadiazolenone core containing chalcones and nucleosides. Various analytical techniques viz IR, NMR, LC-MS and elemental analysis were used to confirm the structure of the synthesised compounds.The compounds were targeted against Bacillus subtilis, Staphylococcus aureus and Escherichia coli for antibacterial activity and Aspergillus flavus, Aspergillus niger and Fusarium oxysporum for antifungal activity. Methods: A mixture of Acid hydrazides (3.0 mmol) and N, Nʹ- carbonyl diimidazole (3.3 mmol) in 15 mL of dioxane was refluxed to afford substituted [1, 3, 4]-oxadiazole-2(3H)-one. The resulted [1, 3, 4]- oxadiazole-2(3H)-one (1.42 mmol) was oxidized with Chromyl chloride (1.5 mL) in 20 mL of carbon tetra chloride and condensed with acetophenones (1.42 mmol) to get chalcones 4. The equimolar ratio of obtained chalcones 4 and β -D-1,2,3,4,6- penta-O-acetylglucopyranose in presence of iodine was refluxed to get nucleosides 5. The [1, 3, 4] oxadiazolenone core containing chalcones 4 and nucleosides 5 were tested to determined minimum inhibitory concentration (MIC) value with the experimental procedure of Benson using disc-diffusion method. All compounds were tested at concentration of 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.62 mg/mL, 0.31 mg/mL and 0.15 mg/mL for antifungal activity against three strains of pathogenic fungi Aspergillus flavus (A. flavus), Aspergillus niger (A. niger) and Fusarium oxysporum (F. oxysporum) and for antibacterial activity against Gram-negative bacterium: Escherichia coli (E. coli), and two Gram-positive bacteria: Staphylococcus aureus (S. aureus) and Bacillus subtilis(B. subtilis). Result: The chalcones 4 and nucleosides 5 were screened for antibacterial activity against E. coli, S. aureus and B. subtilis whereas antifungal activity against A. flavus, A. niger and F. oxysporum. Compounds 4a-t showed good antibacterial activity whereas compounds 5a-t containing glucose moiety showed better activity against fungi. The glucose moiety of compounds 5 helps to enter into the cell wall of fungi and control the cell growth. Conclusion: Chalcones 4 and nucleosides 5 incorporating [1, 3, 4] oxadiazolenone core were synthesized and characterized by various spectral techniques and elemental analysis. These compounds were evaluated for their antifungal activity against three fungi; viz. A. flavus, A. niger and F. oxysporum. In addition to this, synthesized compounds were evaluated for their antibacterial activity against gram negative bacteria E. Coli and gram positive bacteria S. aureus, B. subtilis. Compounds 4a-t showed good antibacterial activity whereas 5a-t showed better activity against fungi.

Comparison of the Effects of Silver in Nanostructured and Ultrahigh Diluted Form on Growth and Volatile Compounds Produced by Escherichia coli and Staphylococcus aureus

2020 ◽  
Vol 10 (3) ◽  
pp. 316-329
Author(s):  
Fateme Mirzajani ◽  
Amin Hamidi
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Life Cycles ◽  
Gas Chromatography Mass Spectrometry ◽  
Untreated Sample ◽  
E Coli ◽  
Volatile Metabolites ◽  
Octyl Acetate ◽  
Volatile Organic ◽  
And Staphylococcus Aureus

Introduction: In this project, the growth and volatile metabolites profiles of Escherichia coli (E. coli ) and Staphylococcus aureus were monitored under the influence of silver base chemical, nanoparticle and ultra-highly diluted compounds. Materials & Methods: The treatments were done for 12000 life cycles using silver nanoparticles (AgNPs) as well as ultra-highly diluted Argentum nitricum (Arg-n). Volatile organic metabolites analysis was performed using gas chromatography mass spectrometry (GC-MS). The results indicated that AgNPs treatment made the bacteria resistant and adapted to growth in the nanoparticle condition. The use of ultra-highly diluted Arg-n initially increased growth but it decreased later. Also, with the continuous usage of these materials, no more bacterial growth was observed. Results: The most important compounds produced by E. coli are Acetophenone, Octyl acetate, Styrene, 1,8-cineole, 4-t-butyl-2-(1-methyl-2-nitroethyl)cyclohexane, hexadecane and 2-Undecanol. The main compounds derived from S. aureus are Acetophenone,1,8-cineole, Benzaldehyde, 2-Hexan-1-ol, Tridecanol, Dimethyl Octenal and tetradecane. Acetophenone and 1,8-cineole were common and produced by both organisms. Conclusion: Based on the origin of the produced volatiles, main volatiles percentage of untreated sample is hydrocarbon (>50%), while bacteria treatments convert the ratio in to aldehydes, ketones and alcohols in the case of AgNPs, (>80%) and aldehydes, ketones and terpenes in the case of Arg-n (>70%).

TiO2 antifouling coating based on epoxy-modified tung oil waterborne resin

2021 ◽  
pp. 096739112110111
Author(s):  
Hailiang Hu ◽  
Minmin Chen ◽  
Mengye Cao
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Survival Rate ◽  
Light Irradiation ◽  
Fouling Resistance ◽  
Tung Oil ◽  
E Coli ◽  
Antifouling Coating ◽  
Waterborne Epoxy

The eco-friendly functionalized TiO2/polymer antifouling (AF) coating was successfully synthesized by dispersing TiO2 nanoparticles in waterborne epoxy-modified tung oil resin. The AF effectiveness of coating was evaluated toward Staphylococcus aureus ( S. aureus, ATCC6538), Escherichia coli ( E. coli, ATCC8739) and diatom ( Cyclotella sp., FACHB-1635). The nanoTiO2/polymer AF coating showed good antimicrobial activity both under the light and dark conditions by comparison with the pristine TiO2 nanoparticles and bulk polymer. Under light irradiation for 50 min, the AF coating showed only 8.4% and 8% survival rate for S. aureus and E. coli. In addition, The AF coatings exhibited favorable inhibition efficacy toward the growth and adhesion of Cyclotella sp., and the efficacy was enhanced with the increase of TiO2 content. It can be concluded that TiO2 nanoparticles endow the AF coatings with promoted fouling resistance properties.

scholarly journals Fabrication of a Novel AgBr/Ag2MoO4@InVO4 Composite with Excellent Visible Light Photocatalytic Property for Antibacterial Use

Nanomaterials ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1541
Author(s):  
Jie Zhang ◽  
Jia Wang ◽  
Qingjun Zhu ◽  
Binbin Zhang ◽  
Huihui Xu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Degradation Rate ◽  
Photocatalytic Property ◽  
Photocatalytic Reaction ◽  
Composite Photocatalyst ◽  
E Coli ◽  
Radical Trapping ◽  
Visible Light Photocatalytic

A novel AgBr/Ag2MoO4@InVO4 composite photocatalyst with different heterojunction structures was successfully constructed by compounding InVO4 with Ag2MoO4 and AgBr. According to the degradation, antibacterial and free radical trapping data, the photocatalytic antibacterial and antifouling activities of AgBr/Ag2MoO4@InVO4 composite were evaluated, and the corresponding photocatalytic reaction mechanism was proposed. Adding AgBr/Ag2MoO4@InVO4 composite, the degradation rate of ciprofloxacin (CIP) achieved 95.5% within 120 min. At the same time, the antibacterial rates of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) achieved 99.99%. The AgBr/Ag2MoO4@InVO4 composite photocatalyst showed promising usage in photocatalytic antibacterial and purification areas.

scholarly journals ATIVIDADE ANTIMICROBIANA DE MICRORGANISMOS ISOLADOS DE GRÃOS DE KEFIR

2018 ◽  
Vol 19 (0) ◽  
Author(s):  
Priscila Alves Dias ◽  
Daiani Teixeira Silva ◽  
Cláudio Dias Timm
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Listeria Monocytogenes ◽  
Salmonella Typhimurium ◽  
Salmonella Enterica ◽  
Escherichia Coli O157 ◽  
E Coli

Resumo Kefir é o produto da fermentação do leite pelos grãos de kefir. Esses grãos contêm uma mistura simbiótica de bactérias e leveduras imersas em uma matriz composta de polissacarídeos e proteínas. Muitos benefícios à saúde humana têm sido atribuídos ao kefir, incluindo atividade antimicrobiana contra bactérias Gram positivas e Gram negativas. A atividade antimicrobiana de 60 microrganismos isolados de grãos de kefir, frente à Escherichia coli O157:H7, Salmonella enterica subsp. enterica sorotipos Typhimurium e Enteritidis, Staphylococcus aureus e Listeria monocytogenes, foi estudada através do teste do antagonismo. A ação antimicrobiana dos sobrenadantes das bactérias ácido-lácticas que apresentaram atividade no teste do antagonismo foi testada. O experimento foi repetido usando sobrenadantes com pH neutralizado. Salmonella Typhimurium e Enteritidis sobreviveram por 24 horas no kefir em fermentação. E. coli O157:H7, S. aureus e L. monocytogenes foram recuperados até 72 horas após o início da fermentação. Todos os isolados apresentaram atividade antimicrobiana contra pelo menos um dos patógenos usados no teste do antagonismo. Sobrenadantes de 25 isolados apresentaram atividade inibitória e três mantiveram essa atividade com pH neutralizado. As bactérias patogênicas estudadas sobreviveram por tempo superior àquele normalmente utilizado para a fermentação do kefir artesanal, o que caracteriza perigo em potencial para o consumidor quando a matéria-prima não apresentar segurança sanitária. Lactobacillus isolados de grãos de kefir apresentam atividade antimicrobiana contra cepas de E. coli O157:H7, Salmonella sorotipos Typhimurium e Enteritidis, S. aureus e L. monocytogenes além daquela exercida pela diminuição do pH.

SEPSIS MARKERS AND SENSITIVITY OF STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLI ISOLATES IN A GENERAL HOSPITAL SETTING

Vestnik ◽  
2021 ◽  
pp. 68-74
Author(s):  
М.Е. Рамазанов ◽  
В.Н. Сон ◽  
М.Р. Рысулы ◽  
С.Т. Турсуналиев ◽  
Е.Б. Еспенбетов
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Hospital Setting ◽  
Bloodstream Infections ◽  
High Sensitivity ◽  
Clinical Isolates ◽  
Empiric Antibiotic Therapy ◽  
E Coli ◽  
Number Of Patients ◽  
Empiric Antibiotic

Представлены результаты проспективного обследования 80 больных ГКБ №7 с бактериемией с октября 2019 года по февраль 2021 года из различных отделений госпиталя. Производилась оценки показателей маркеров сепсиса - пресепсина, прокальцитонина и С-реактивного белка (СРБ) в крови больных в динамике эмпирической терапии антимикробными препаратами (АМП). Наибольшее число больных с выявленной бактериемией находилось в отделении ОАРИТ - 39 пациентов, у 25 из них был диагностирован сепсис по шкале СЕПСИС III, вызванный известными патогенами Staphylococcus aureus (46,6%) и Escherichia coli (36,6%). Для эмпирического лечения применялись различные антибиотики: ампенициллин, амикацин, меропенем, цефотаксим, метрид, ципрофлоксацин, ципрокс, цефлокс, цефазолин, цефтриаксон, левофлоксацин. Уровни прокальцитонина составляют для больных с клиническими изолятами E. coli 20,8±3,1нг/мл, а для изолятов St. aureus 15,7±1,8 нг/мл. После терапии АМП наблюдается значительное снижение показателей до 1,43±0,6 и 2,3±0,9 нг/мл., что позволяет признать эффективность эмпирической антибиотикотерапии при инфекциях кровотока. Высокая чувствительность клинических изолятов Escherichia coli отмечена к препаратам группы карбапенемов - имипенему и меропенему (90,9%), низкая к эртапенему (72,7%). 100% чувствительность все изоляты показали по отношению к АМП из группы глицилциклинов - тигециклину, который структурно сходен с тетрациклинами. Высокой резистеностью клинические изоляты Staphylococcus aureus обладают к пенициллину (92,9%), липопептиду природного происхождения даптомицину (85,8%) и препарату из группы линкозамидов - клиндамицину (64,3%). The results of a prospective examination of 80 patients with bacteremia from October 2019 to February 2021 from various departments of the hospital are presented. The largest number of patients with detected bacteremia were in the OARIT department - 39 patients, 25 of them were diagnosed with sepsis according to the SEPSIS III scale, caused by known pathogens Staphylococcus aureus (46.6%) and Escherichia coli (36.6%). For empirical treatment, various antibiotics were used: ampenicillin, amikacin, meropenem, cefotaxime, metrid, ciprofloxacin, ciprox, ceflox, cefazolin, ceftriaxone, levofloxacin. Procalcitonin levels for patients with clinical E. coli isolates are 20.8 ± 3.1 ng / ml, and for St. aureus 15.7 ± 1.8 ng / ml. After AMP therapy, there is a significant decrease in indicators to 1.43 ± 0.6 and 2.3 ± 0.9 ng / ml, which makes it possible to recognize the effectiveness of empiric antibiotic therapy for bloodstream infections. High sensitivity of clinical isolates of Escherichia coli was noted to drugs of the carbapenem group - imipenem and meropenem (90.9%), low to ertapenem (72.7%). All isolates showed 100% sensitivity to AMPs from the glycylcycline group - tigecycline, which is structurally similar to tetracyclines. Clinical isolates of Staphylococcus aureus are highly resistant to penicillin (92.9%), natural lipopeptide daptomycin (85.8%), and a drug from the lincosamide group - clindamycin (64.3%).

Antimicrobial Effects of Novel H2O2-Ag+ Complex on Membrane Damage to Staphylococcus aureus,Escherichia coli and Salmonella typhimurium

2021 ◽  
Author(s):  
Bing Han ◽  
Xiaoyu Han ◽  
Mengmeng Ren ◽  
Yilin You ◽  
Jicheng Zhan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Staphylococcus Aureus ◽  
Salmonella Typhimurium ◽  
Membrane Damage ◽  
Bactericidal Effect ◽  
E Coli ◽  
Bacterial Cell Membrane ◽  
Time Kill ◽  
Environmental Disinfection

Diseases caused by harmful microorganisms pose a serious threat to human health. Safe and environment-friendly disinfectants are, therefore, essential in preventing and controlling such pathogens. This study aimed to investigate the antimicrobial activity and mechanism of a novel hydrogen peroxide and silver (H 2 O 2 -Ag + ) complex (HSC) in combatting Staphylococcus aureus ATCC 29213, Escherichia coli O157:H7 NCTC 12900 and Salmonella typhimurium SL 1344. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against S. aureus were found to be 0.014 % H 2 O 2 -3.125 mg/L Ag + , while 0.028 % H 2 O 2 -6.25 mg/L Ag + for both E. coli and S. typhimurium . Results of the growth curve assay and time-kill trial suggest that the HSC could inhibit the growth of the tested bacteria, as 99.9 % of viable cells were killed following treatment at the 1 MIC for 3 h. Compared with Oxytech D10 disinfectant (0.25 % H 2 O 2 -5 mg/L Ag + ), the HSC exhibited better antibacterial efficacy at a lower concentration (0.045 % H 2 O 2 -10 mg/L Ag + ). The mechanism of antibacterial action of HSC was found including the disruption of the bacterial cell membrane, followed by entry into the bacteria cell to reduce intracellular adenosine triphosphate (ATP) concentration, and inhibit the activity of antioxidases, superoxide dismutase (SOD) and catalase (CAT). The enhanced bactericidal effect of hydrogen peroxide combined with silver indicates a potential for its application in environmental disinfection, particularly in the food industry.

scholarly journals Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases.

1996 ◽  
Vol 40 (12) ◽  
pp. 2714-2720 ◽  
Author(s):  
F Blanche ◽  
B Cameron ◽  
F X Bernard ◽  
L Maton ◽  
B Manse ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Strong Support ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Type Ii ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Dna Relaxation

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

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