Genetic Manipulation of Stenotrophomonas maltophilia

Elliott Welker1, Yayra Domfeh1, Deepti Tyagi1, Sanjivni Sinha1, Nathan Fisher1

1 North Dakota State University, Department of Veterinary and Microbiological Sciences, Fargo, North Dakota
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6F.2
DOI:  10.1002/9780471729259.mc06f02s37
Online Posting Date:  May, 2015
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Stenotrophomonas maltophilia is a Gram‐negative, aerobic, motile, environmental bacterium that is emerging as an important nosocomial pathogen with high rates of attributable mortality in severely ill patients. S. maltophilia is of particular concern to patients suffering from cystic fibrosis (CF) as it has been shown to colonize airway epithelial and establish a chronic infection. Here we describe several molecular techniques for the genetic manipulation of this bacterium, including DNA extraction, RNA extraction, conjugation of plasmids from Escherichia coli and allelic exchange. © 2015 by John Wiley & Sons, Inc.

Keywords: Stenotrophomonas maltophilia; genetic manipulation; nosocomial; hospital acquired infection

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation of S. maltophilia Genomic DNA
  • Alternate Protocol 1: Quick Extraction of S. maltophilia DNA for PCR
  • Basic Protocol 2: Extraction of S. maltophilia RNA
  • Basic Protocol 3: Transfer of Plasmid DNA to S. maltophilia Recipient from E. coli Donor
  • Basic Protocol 4: Allelic Exchange in S. maltophilia Using pEX18Tc
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Isolation of S. maltophilia Genomic DNA

  • Viable S. maltophilia on a Petri plate (see unit 6.1, Mahdi et al., )
  • Luria‐Bertani (LB) broth ( appendix 4A)
  • 0.1× SSC (see recipe)
  • 50 mg/ml stock lysozyme solution (see recipe)
  • 20 mg/ml stock proteinase K solution (see recipe)
  • Tris‐saturated phenol (pH 7.5; molecular biology grade)
  • Chloroform (molecular biology grade)
  • 70% ethanol in nuclease‐free double‐distilled water (ddH 2O), ice‐cold (see recipe)
  • Absolute ethanol (≥99.8%), ice‐cold
  • Buffer 1 (see recipe)
  • Lysis buffer (see recipe)
  • TE buffer, pH 8.0 ( appendix 2A)
  • 5 M sodium chloride (NaCl) solution (see recipe)
  • Microcentrifuge tubes, nuclease free
  • Orbital shaker set to 37°C, 225 rpm
  • Inoculating loop or needle, sterile
  • 15‐ and 50‐ml conical tubes, sterile
  • 100‐ml culture flask, sterile

Alternate Protocol 1: Quick Extraction of S. maltophilia DNA for PCR

  • Viable S. maltophilia on a Petri plate (see unit 6.1; Mahdi et al., ).
  • TE buffer, pH 8.0 ( appendix 2A)
  • Inoculating loop or needle, sterile
  • Sterile 200‐μl PCR tubes
  • Microcentrifuge fitted with 200‐μl tube adapter
  • Two water baths, set to 55°C and 80°C, or a thermocycler

Basic Protocol 2: Extraction of S. maltophilia RNA

  • Viable S. maltophilia on a Petri plate (see unit 6.1; Mahdi et al., )
  • Luria‐Bertani (LB) broth ( appendix 4A)
  • Acid phenol:chloroform (pH 4.3 to 4.7)
  • Chloroform
  • Double‐distilled water (ddH 2O), nuclease free
  • 70% ethanol in nuclease‐free ddH 2O, ice‐cold (see recipe)
  • Absolute ethanol (≥99.8%), ice‐cold
  • Isoamyl alcohol
  • RNasin Plus RNase Inhibitor (Promega)
  • 0.1 M dithiothreitol (DTT)
  • RQ1 RNase‐Free DNase
  • 10× RQ1 RNase‐Free DNase buffer
  • Sample buffer (see recipe)
  • Stop solution (see recipe)
  • 3 M sodium acetate, pH 5.2 (see recipe)
  • 20% sodium dodecyl sulfate (SDS; see recipe)
  • TE buffer, pH 8.0 ( appendix 2A)
  • 0.1‐mm zirconium beads, acid‐washed (see recipe)
  • 15‐ and 50‐ml conical tube, sterile
  • Beadbeater (Biospec Products)
  • Microcentrifuge tubes, sterile
  • Swinging bucket centrifuge, pre‐chilled to 4°C
  • Orbital shaker set to 37°C, 225 rpm
  • 8‐ml bead‐beating tubes
  • Water bath set to 65°C
  • Ice bath

Basic Protocol 3: Transfer of Plasmid DNA to S. maltophilia Recipient from E. coli Donor

  • Viable S. maltophilia on a Petri plate (recipient; unit 6.1, Mahdi et al., )
  • Viable E. coli on a Petri plate (donor)
  • Broad‐host range shuttle vector
  • LB broth ( appendix 4A)
  • LB agar plates, dry ( appendix 4A)
  • Primers SM1 and SM2 (see recipe)
  • 15‐ml centrifuge tube, sterile
  • Orbital shaker set to 37°C, 225 rpm
  • Inoculating loop or needle, sterile

Basic Protocol 4: Allelic Exchange in S. maltophilia Using pEX18Tc

  • pEX18Tc (gift from Dr. H. P. Schweizer, Colorado State University; Hoang et al., )
  • LB broth ( appendix 4A)
  • LB agar ( appendix 4A)
  • Sucrose
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Literature Cited

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