Genetic Manipulation of Nocardia Species

Dipesh Dhakal1, Amit Kumar Jha1, Anaya Pokhrel1, Anil Shrestha1, Jae Kyung Sohng1

1 Institute of Biomolecule Reconstruction, Department of BT‐Convergent Pharmaceutical Engineering, Sun Moon University, Chungnam, Republic of Korea
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10F.2
DOI:  10.1002/9780471729259.mc10f02s40
Online Posting Date:  February, 2016
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Abstract

Nocardia spp. are aerobic, Gram‐positive, catalase positive, and non‐motile actinomycetes. They are associated with human infections. However, some species produce important natural products, degrade toxic chemicals, and are involved in biotransformation of valuable products. The lack of robust genetic tools has hindered detailed studies and advanced research. This unit describes the major genetic engineering approaches using Nocardia sp. CS682 as a prototype. These methods will certainly help in understanding the basis of their pathogenicity as well as biosynthetic and biotransforming abilities. It can be expected that knowledge of the biochemistry behind their pathogenicity will be crucial in developing effective treatment strategies. These genetic tools can be utilized to develop rational metabolic engineering approaches for crafting host strains with higher production or biotransformation ability. © 2016 by John Wiley & Sons, Inc.

Keywords: Nocardia spp; genomic DNA; transformation; RNA isolation; protein extraction

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: DNA Transfer Via Electroporation in Nocardia spp.
  • Support Protocol 1: Making Electrocompetent Nocardia sp. CS682
  • Basic Protocol 2: Extraction of Genomic DNA from Nocardia spp.
  • Basic Protocol 3: Direct Colony PCR of Nocardia spp.
  • Basic Protocol 4: Extraction of RNA from Nocardia spp.
  • Basic Protocol 5: Reverse Transcription PCR
  • Basic Protocol 6: Extraction of Total Protein from Nocardia spp.
  • Reagents and Solutions
  • Commentary
  • Literatures cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: DNA Transfer Via Electroporation in Nocardia spp.

  Materials
  • Electrocompetent Nocardia sp. CS682 cells (see protocol 2Support Protocol)
  • DNA to be electroporated
  • Brain‐heart infusion (BHI) medium (see recipe)
  • BHI/agar plate containing appropriate antibiotics
  • 1.5‐ml microcentrifuge tubes
  • 1‐mm electroporation cuvettes
  • Electroporator
  • Ice
  • 14‐ml plastic round bottom tubes
  • 37°C incubator with variable shaking ability
  • L‐shaped plate spreader or glass rods

Support Protocol 1: Making Electrocompetent Nocardia sp. CS682

  Materials
  • Nocardia sp. CS682 culture (see unit 10.1)
  • Brain‐heart infusion (BHI) medium (see recipe)
  • 10× glycine/Tween 80 (GT) solution (see recipe)
  • 10% glycerol (see recipe)
  • 500‐ml Erlenmeyer flask
  • 37°C shaking incubator
  • 50‐ml conical centrifuge tubes
  • Ice
  • Centrifuge (Hanil Union 32R)

Basic Protocol 2: Extraction of Genomic DNA from Nocardia spp.

  Materials
  • Brain‐heart infusion (BHI) medium (see recipe)
  • Mycelium or spore stock of Nocardia sp. CS682
  • 10× glycine/Tween 80 (GT) solution (see recipe)
  • Lysis buffer (see recipe)
  • 200 mg/ml lysozyme (Sigma, cat. no. L6876) in 10 mM Tris·Cl, pH 7.5 (see appendix 2A), make fresh
  • 10% SDS (see appendix 2A)
  • 0.5 M EDTA, pH 8.0 (see appendix 2A)
  • 20 mg/ml pronase E (Sigma, cat. no. P6911)
  • 5 M potassium acetate, pH 7.5 (store in aliquots at −20°C; see appendix 2A)
  • Phenol
  • Chloroform
  • RNase solution (see recipe)
  • 70% and 100% ethanol
  • Isopropanol
  • Tris/EDTA (TE) buffer (see appendix 2A)
  • 500‐ml baffled Erlenmeyer flasks
  • 37°C shaking incubator
  • 50‐ml screw cap conical centrifuge tubes (Fisher, cat. no. 05‐539‐13)
  • Centrifuge (Hanil Union 32R)
  • −70°C freezer
  • 37°C and 72°C water bath
  • Microscope slides
  • Ice
  • Blunted glass Pasteur pipets
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge (Hanil Smart R17)
  • Spectrophotometer

Basic Protocol 3: Direct Colony PCR of Nocardia spp.

  Materials
  • DMSO
  • Mycelium or spore stock of Nocardia sp. CS682
  • 2× PrimeSTAR GC Buffer containing Mg2+ (Clonetech Laboratories)
  • 2.5 mM (each) dNTP mix (see appendix 2A)
  • 20 μM primer 1
  • 20 μM primer 2
  • 2.5 U/μl PrimeSTAR HS DNA Polymerase (Clonetech Laboratories)
  • PCR tubes
  • Toothpicks, sterile
  • Blunted glass Pasteur pipet
  • Thermal cycler

Basic Protocol 4: Extraction of RNA from Nocardia spp.

  Materials
  • Brain‐heart infusion (BHI) medium (see recipe)
  • Mycelium or spore stock of Nocardia sp. CS682
  • 10× glycine/Tween 80 (GT) solution (see recipe)
  • RNAprotect Bacteria Reagent (Qiagen, cat. no. 76506)
  • 200 mg/ml lysozyme (Sigma, cat. no. L6876) in 50 mM Tris·Cl, pH 7.5 (see appendix 2A), make fresh
  • Tris/EDTA (TE) buffer (see appendix 2A)
  • 10 mg/ml proteinase K (Sigma, cat. no. P4914) dissolved in water
  • 2‐Mercaptoethanol (Sigma, cat. no. M3148)
  • RNeasy Mini Kit (Qiagen, cat. no. 74104) containing:
    • RLT buffer
    • RW1 buffer
    • RPE buffer
    • RNase‐free water
    • RNeasy Mini Spin Columns with collection tubes
  • 80% ethanol
  • RNase‐Free DNase Set (Qiagen, cat. no 79254) containing:
    • DNase 1
    • RDD buffer
  • 500‐ml baffled Erlenmeyer flask
  • 37°C shaking incubator
  • Spectrophotometer
  • 1.5‐ml and 2‐ml microcentrifuge tubes
  • Centrifuge (Hanil Smart R17)
  • Glass beads, acid washed (150 to 212 μm; Sigma, cat. no. 04K5305)
  • Mini‐Beadbeater (Biospec Products)

Basic Protocol 5: Reverse Transcription PCR

  Materials
  • RNA extracted from Nocardia spp. (see protocol 5)
  • RNase‐free deionized H 2O
  • QuantiTect SYBR Green PCR Kit (Qiagen, cat. no 204143)
  • QuantiTect Reverse Transcription Kit (Qiagen, cat. no 205311)
  • 20 μM primer 1
  • 20 μM primer 2
  • Spectrophotometer

Basic Protocol 6: Extraction of Total Protein from Nocardia spp.

  Materials
  • Brain‐heart infusion (BHI) medium (see recipe)
  • Mycelium or spore stock of Nocardia sp. CS682
  • 10× glycine/Tween 80 (GT) solution (see recipe)
  • Protein extraction (PE) buffer (see recipe)
  • 50× proteinase inhibitor cocktail (PIC; see recipe)
  • Protein loading (PL) buffer (see recipe)
  • 500‐ml baffled Erlenmeyer flask
  • 37°C shaking incubator
  • 50‐ml screw cap conical centrifuge tube
  • Centrifuge (Hanil Union 32R)
  • Vortex
  • Ice
  • –70°C freezer
  • Sonicator
  • 1.5‐ml microcentrifuge tube
  • Microcentrifuge (Hanil Smart R17)
  • 95°C water bath
  • Additional reagents and equipment for quantifying protein by Bradford assay (see appendix 3A) and for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE; see appendix 3M)
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Figures

Videos

Literature Cited

Literatures cited
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  Luo, Q., Hiessl, S., Poehlein, A., and Steinbüchel, A. 2013. Microbial gutta‐percha degradation shares common steps with rubber degradation by Nocardia nova SH22a. Appl. Environ. Microbiol. 79:1140‐1149. doi: 10.1128/AEM.03016‐12.
  Maharjan, S., Koju, D., Lee, H.C., Yoo, J.C., and Sohng, J.K. 2012. Metabolic engineering of Nocardia sp. CS682 for enhanced production of nargenicin A1. Appl. Biochem. Biotechnol. 166:805‐817. doi: 10.1007/s12010‐011‐9470‐1.
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