Azospirillum brasilense: Laboratory Maintenance and Genetic Manipulation

Jessica Gullett1, Lindsey O'Neal1, Tanmoy Mukherjee1, Gladys Alexandre1

1 Biochemistry & Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 3E.2
DOI:  10.1002/cpmc.39
Online Posting Date:  November, 2017
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Abstract

Bacteria of the genus Azospirillum, including the most comprehensively studied Azospirillum brasilense, are non‐pathogenic soil bacteria that promote the growth of diverse plants, making them an attractive model to understand non‐symbiotic, beneficial plant‐bacteria associations. Research into the physiology and genetics of these organisms spans decades and a range of molecular tools and protocols have been developed for allelic exchange mutagenesis, in trans expression of genes, and fusions to reporter genes. © 2017 by John Wiley & Sons, Inc.

Keywords: Azospirillum; functional complementation; mutagenesis; plasmids; reporter genes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Growth of A. brasilense From a Frozen Stock
  • Basic Protocol 2: Growth of A. brasilense in Liquid Medium
  • Basic Protocol 3: Bacterial Conjugation in A. brasilense
  • Support Protocol 1: Preparation of E. coli Donor and Helper Strains for Bacterial Conjugation
  • Basic Protocol 4: gusA as a Reporter of Promoter Activity: Analysis in Liquid Cultures
  • Basic Protocol 5: gusA as a Reporter of Promoter Activity: Analysis on Plant Surfaces
  • Basic Protocol 6: Insertional Mutagenesis of A. brasilense
  • Basic Protocol 7: Markerless Deletion Mutagenesis in A. brasilense
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Growth of A. brasilense From a Frozen Stock

  Materials
  • Tryptone yeast (TY) or minimal medium for A. brasilense (MMAB) medium solid plates
  • A. brasilense frozen stock (see unit 3.1)
  • Disposable sterile pipet tips
  • Inoculating loop
  • 28°C incubator shaker
  • Low temperature freezer (−80°C)

Basic Protocol 2: Growth of A. brasilense in Liquid Medium

  Materials
  • Tryptone yeast (TY) or minimal medium for A. brasilense with nitrogen source (MMAB + N; see recipe)
  • Ampicillin (200 µg/ml)
  • A. brasilense colonies on a solid plate
  • Sterile glass tubes
  • Sterilized pipet tips
  • 28°C incubator with shaker at 200 rpm

Basic Protocol 3: Bacterial Conjugation in A. brasilense

  Materials
  • A. brasilense recipient colonies freshly grown on tryptone yeast (TY) or minimal medium for A. brasilense (MMAB and MMAB‐N) plates
  • Tryptone yeast (TY) or minimal medium for A. brasilense (MMAB) liquid and solid media
  • LB liquid medium
  • Antibiotic stock solutions
  • E. coli donor colonies on fresh LB plates supplemented with appropriate antibiotics
  • E. coli helper colonies on fresh LB plates with appropriate antibiotics (if performing a triparental mating; see protocol 4)
  • Sterilized 0.8% KCl
  • Sterile glass tubes
  • 28°C and 37°C incubators
  • 2‐ml sterilized Eppendorf tubes
  • Tabletop centrifuge
  • D‐medium plates
  • Styrofoam box with lid
  • Sterile inoculating loop

Support Protocol 1: Preparation of E. coli Donor and Helper Strains for Bacterial Conjugation

  Materials
  • Plasmid DNA to be transformed (donor and/or helper)
  • Chemically competent cells; S17‐1 for biparental (Thermo Fisher Scientific) or Top10 for triparental (Thermo Fisher Scientific)
  • Solid agar plates with appropriate antibiotics (dependent on plasmid resistance)
  • 70% ethanol
  • LB broth
  • Pipets and sterile tips
  • 2‐ml Eppendorf tubes
  • Tabletop centrifuge
  • Incubator at 37°C
  • L‐shaped cell spreader

Basic Protocol 4: gusA as a Reporter of Promoter Activity: Analysis in Liquid Cultures

  Materials
  • Liquid culture of A. brasilense expressing gusA from a plasmid in which the promoter region of the gene of interest has been cloned in front of a promoterless gusA gene (e.g., pFUS1 vector) or constitutively expressing gusA (e.g., pJBA21Tc; prepared as described in protocol 3)
  • MUG assay solution (see recipe)
  • Spectrophotometer
  • Flat‐bottomed clear 96‐well microplates (select plates with low autofluorescence and/or absorbance at 365 nm and 445 nm)
  • Multichannel pipets (12 or 8 channel; to dispense volumes of 10 μl and 100 μl)
  • Low temperature freezer (−80°C)
  • 37°C incubator
  • Fluorescence microplate reader (e.g., Biotek Synergy 2)

Basic Protocol 5: gusA as a Reporter of Promoter Activity: Analysis on Plant Surfaces

  Materials
  • Liquid culture of A. brasilense expressing gusA from a plasmid in which the promoter region of the gene of interest has been cloned in front of a promoterless gusA gene (e.g., pFUS1 vector) or constitutively expressing gusA (e.g., pJBA21Tc) and prepared as described in protocol 3
  • Antibiotics to be used for plasmid maintenance
  • Surface‐sterilized and germinated seedlings, 3 to 5 days (depending on plants being inoculated)
  • Sterilized 0.8% KCl (see recipe)
  • 4% (w/v) Farhaeus semisoft agar (distributed into sterile glass tubes; see recipe)
  • 50 mM sodium phosphate buffer supplemented with 0.1 g/ml X‐Gluc (see recipe)
  • Spectrophotometer
  • 2‐ml sterilized Eppendorf tubes
  • Tabletop centrifuge
  • Sterile glass tubes
  • Whatman 3 MM filter paper
  • Dessicator
  • Compound light microscope

Basic Protocol 6: Insertional Mutagenesis of A. brasilense

  Additional Materials (also see protocol 3)
  • Suicide vector in which the mutated gene of interest (GOI; insertion mutagenesis or markerless deletion) is cloned
  • A. brasilense strain in which gene of interest (GOI) will be mutated
  • 0.8% sterile KCl
  • Minimal medium for A. brasilense without nitrogen source (MMAB – N; see recipe) without antibiotic (markerless deletion) and with antibiotics (insertion mutagenesis with antibiotic added corresponding to inserted cassette [deletion‐insertion mutagenesis] or suicide vector [insertion mutagenesis])
  • Tryptone yeast (TY) liquid medium (see recipe)
  • Tryptone yeast (TY) solid plates (see recipe)
  • Antibiotic stock solutions (for selection of double homologous recombinants)
  • 28°C incubator
  • Thermocycler

Basic Protocol 7: Markerless Deletion Mutagenesis in A. brasilense

  Additional Materials (also see protocol 7)
  • Tryptone yeast (TY) solid plates supplemented with 10% sucrose
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Figures

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Literature Cited

Literature Cited
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