Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed

Macromolecular Biosynthesis Assay for Evaluation of Influence of an Antimicrobial on the Synthesis of Macromolecules

采用大分子生物合成法评估抗菌剂对大分子合成的影响

JN Justyna Nowakowska
NK Nina Khanna
RL Regine Landmann

发布: 2013年06月20日第3卷第12期 DOI: 10.21769/BioProtoc.798 浏览次数: 11551

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Antimicrobial Agents and Chemotherapy, featuring study using the protocol.

Jan 2012

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

吉西他滨化疗细菌降解的功能测定

吉西他滨化疗细菌降解的功能测定

Serkan Sayin and Amir Mitchell

2023年09月05日 1133 阅读

基于高效液相色谱法的史氏分枝杆菌DisA环二腺苷酸（C-di-AMP）合成酶活性研究

基于高效液相色谱法的史氏分枝杆菌DisA环二腺苷酸（C-di-AMP）合成酶活性研究

Avisek Mahapa [...] Dipankar Chatterji

2024年12月20日 1017 阅读

靶向纳米孔测序技术在结核分枝杆菌及其耐药性检测中的应用

靶向纳米孔测序技术在结核分枝杆菌及其耐药性检测中的应用

Chen Tang [...] Guangxin Xiang

2025年02月05日 1098 阅读

Abstract

One of the most compelling approaches in the discovery of novel antimicrobials is screening of natural sources. In our publication we report on the activity of a compound 8-hydroxyserrulat-14-en-19-oic acid (EN4), a diterpene isolated from the Australian plant Eremophila neglecta. We evaluate its applicability for treatment of implant-associated infections. A comprehensive analysis of the mechanism of action of EN4 against staphylococci revealed its membranolytic properties and a general inhibition of macromolecular biosynthesis, which was confirmed in a macromolecular biosynthesis assay and suggested a multitarget activity. The method used to investigate an influence of EN4 on the synthesis of peptidoglycan, RNA, DNA and proteins is based on precipitation of macromolecules with trichloroacetic acid. These macromolecules are synthesised from respective [3H]-labelled precursors. The incorporated radioactivity with and without an antimicrobial is measured and it reflects the mode of action of the tested compound. Antibiotics with known mechanisms of action are used as controls.

Keywords: Macromolecular synthesis (大分子合成) search Mechanism of action (作用机制) search Antibiotic (抗生素) search Antimicrobial (抗菌) search Bacterial biosynthesis (细菌的生物合成) search

Materials and Reagents

  1. [3H] N-acetylglucosamine (Hartmann Analytic, catalog number: ART 0101 )
  2. [3H] Uridine (Hartmann Analytic, catalog number: MT-602 )
  3. [3H] Thymidine (PerkinElmer, catalog number: NET35500 )
  4. [3H] Leucine (Hartmann Analytic, catalog number: MT-672 )
  5. Triptic soy broth (TSB) (Becton Dickinson, catalog number: 211825 )
  6. Control antimicrobials for the influence on synthesis of:
    1. Peptidoglycan: vancomycin (Vancocin 500 mg) (Teva Pharma)
    2. RNA: actinomycin D (Sigma-Aldrich, catalog number: A1410 ) 
    3. DNA: ciprofloxacin (Ciproxin Infusion 0.2 g) (Bayer) 
    4. Proteins: chloramphenicol (Applichem, catalog number: A1806 )
    5. All macromolecules (antiseptic activity): chlorhexidine dihydrochloride (Sigma-Aldrich, catalog number: C8527 ) 
      Note: All substances prepared according to the manufacturer’s instructions; minimal inhibitory concentration (MIC) of each antimicrobial can be determined according to Clinical and Laboratory Standards Institute guidelines (see Reference 1) (The following MICs were determined for S. aureus WSPPA: MICvancomycin = 2 μg/ml, MICactinomycin D = 6.25 μg/ml, MICciprofloxacin = 2 μg/ml, MICchloramphenicol = 8 μg/ml, MICchlorhexidine = 0.78 μg/ml)
  7. Scintillation cocktail (Ultima Gold, catalog number: 6013321 ).
  8. Phosphate-buffered saline (PBS) (Reagens, catalog number: 9007695 ).
  9. Na2HPO4 (Sigma-Aldrich, catalog number: S3264 )
  10. KH2PO4 (Sigma-Aldrich, catalog number: P8416 )
  11. MgSO4.7H2O (Fluka, catalog number: 63138 )
  12. NH4Cl (Sigma-Aldrich, catalog number: A9434 )
  13. NaCl (Sigma-Aldrich, catalog number: S3014 )
  14. Sodium citrate tribasic dihydrate (Sigma-Aldrich, catalog number: S4641 )
  15. Glucose (B. Braun Medical, catalog number: 395176 )
  16. Various amino acids (i.e., L-alanine (Sigma-Aldrich, catalog number: A7469 )
  17. L-Valine (Sigma-Aldrich, catalog number: V0513 )
  18. L-Isoleucine (Sigma-Aldrich, catalog number: I7403 )
  19. L-Aspartic acid (Sigma-Aldrich, catalog number: A7219 )
  20. L-Glutamic acid (Sigma-Aldrich, catalog number: G8415 )
  21. L-Serine (Sigma-Aldrich, catalog number: S4311 )
  22. L-Threonine (Sigma-Aldrich, catalog number: T8441 )
  23. L-Cysteine hydrochloride (Sigma-Aldrich, catalog number: C6852 )
  24. L-Arginine (Sigma-Aldrich, catalog number: A8094 )
  25. L-Leucine (Sigma-Aldrich, catalog number: L8912 )
  26. L-Lysine (Sigma-Aldrich, catalog number: L9037 )
  27. L-Proline (Sigma-Aldrich, catalog number: P5607 )
  28. L-Phenylalanine (Sigma-Aldrich, catalog number: P5482 )
  29. L-Tryptophan (Sigma-Aldrich, catalog number: T8941 )
  30. L-Histidine monohydrochloride (Sigma-Aldrich, catalog number: H5659
  31. Glycine (Sigma-Aldrich, catalog number: G7126 )
  32. Cyanocobalamine (Sigma-Aldrich, catalog number: V2876 )
  33. p-Aminobenzoate (Fluka, catalog number: 06940 )
  34. Biotin (Sigma-Aldrich, catalog number: B3399 )
  35. Nicotinic acid (Sigma-Aldrich, catalog number: N0761 )
  36. D-pantothenic acid hemicalcium salt (Sigma-Aldrich, catalog number: P5155 )
  37. Pyridoxine hydrochloride (Sigma-Aldrich, catalog number: P6280 )
  38. Thiamine hydrochloride (Sigma-Aldrich, catalog number: T1270 )
  39. Riboflavin (Sigma-Aldrich, catalog number: R9504 )
  40. ZnCl2 (Sigma-Aldrich, catalog number: 208086
  41. MnCl2.4H2O (Sigma-Aldrich, catalog number: M5005 )
  42. BH3O3 (Fluka, catalog number: 15665 )
  43. CoCl2.6H2O (Sigma-Aldrich, catalog number: C8661 )
  44. CuCl2.2H2O (Sigma-Aldrich, catalog number: C3279 )
  45. NiCl2.6H2O (Sigma-Aldrich, catalog number: 223387 )
  46. Na2MoO4.2H2O (Sigma-Aldrich, catalog number: M1003 )
  47. FeCl2.4H2O (Fluka, catalog number: 44939 )
  48. NaOH (Sigma-Aldrich, catalog number: S5881 )
  49. Uracil (Sigma-Aldrich, catalog number: U0750 )
  50. Cytosine (Sigma-Aldrich, catalog number: C3506 )
  51. Adenine (Sigma-Aldrich, catalog number: A2786 )
  52. Guanine (Sigma-Aldrich, catalog number: G11950 )
  53. Sodium dodecyl sulphate (SDS) (Sigma-Aldrich, catalog number: L4390 )
  54. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T9159 )
  55. 15 ml TPP centrifuge tubes (TPP 91015 )
  56. 10% TCA, 5% TCA, 5% TCA/1.5 M NaCl (see Recipes)
  57. Completely defined medium (CDM) (see Recipes)
    Note: This medium has been optimised for Staphylococcus (S.) aureus and may require further optimisation if other bacteria are investigated. 

Equipment

  1. 37 °C bacterial culture incubator
  2. Sterile capped glass tubes (to reduce bacterial adherence) (GlasKeller, catalog number: 2613111 )
  3. Ultracentrifuge tubes (2 ml) (Eppendorf, catalog number: 0030120.094 )
  4. Scintillation tubes (PerkinElmer, catalog number: 6000288 )
  5. Benchtop centrifuge
  6. Vacuum pump
  7. Liquid scintillation analyser (Tri-CARB, catalog number: 1900TR )

Procedure

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Nowakowska, J., Khanna, N. and Landmann, R. (2013). Macromolecular Biosynthesis Assay for Evaluation of Influence of an Antimicrobial on the Synthesis of Macromolecules. Bio-protocol 3(12): e798. DOI: 10.21769/BioProtoc.798.

ris ris Download Citation in RIS Format

分类

微生物学 > 微生物生物化学 > 其它化合物

微生物学 > 抗微生物试验 > 抗细菌试验

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件