Laboratory Maintenance of Bdellovibrio

Carey Lambert1, R. Elizabeth Sockett1

1 Nottingham University, Institute of Genetics, Queen's Medical Centre, Nottingham, United Kingdom
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 7B.2
DOI:  10.1002/9780471729259.mc07b02s9
Online Posting Date:  May, 2008
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Abstract

Bdellovibrio bacteriovorus is a Gram‐negative bacterium that preys upon other Gram‐negative bacteria. It does this by penetrating the outer membrane and peptidoglycan to establish itself in the periplasm where it grows at the expense of the contents of the prey cell before ultimately lysing the prey. Wild‐type Bdellovibrio are prey‐(or host‐) dependent and the protocols described in this unit deal with the techniques required to grow this bacterium on its prey cells. Protocols are also presented to generate and culture host‐independent mutants as well as novel protocols to evaluate Bdellovibrio predation. Curr. Protoc. Microbiol. 9:7B.2.1‐7B.2.13. © 2008 by John Wiley & Sons, Inc.

Keywords: Bdellovibrio bacteriovorus; predator‐prey interactions; luminescence assay; plaque assay; host‐independent growth; storage of cultures

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Routine Culturing of Prey Lysates
  • Basic Protocol 2: Reviving Bdellovibrio from Frozen Stocks
  • Basic Protocol 3: Growth of Bdellovibrio as Single Plaques for Enumeration
  • Basic Protocol 4: Generation of Host‐Independent Bdellovibrio
  • Basic Protocol 5: Routine Maintenance of HI Cultures
  • Basic Protocol 6: Preparing Frozen Glycerol Stocks for Long‐Term Storage
  • Basic Protocol 7: Bdellovibrio Predation Efficiency Assay on Luminescent Prey
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Routine Culturing of Prey Lysates

  Materials
  • Prey cells (e.g., E. coli K12; refer to appendix 4A for methods of culturing E. coli K12)
  • 1% YT agar plates (see recipe for YT agar)
  • YT broth (see recipe)
  • HEPES buffer supplemented with CaCl 2 (see recipe)
  • Overnight culture of prey lysate
  • 37°C static incubator
  • 250‐ml conical flasks or sterile universal tubes (Sterilin)
  • 29° and 37°C shaking incubator
  • Phase contrast microscope with a 100× oil immersion lens

Basic Protocol 2: Reviving Bdellovibrio from Frozen Stocks

  Materials
  • Prey cells (e.g., E. coli K12; refer to appendix 4A for methods of culturing E. coli K12)
  • 1% YT agar plates (see recipe for YT broth)
  • YT broth (see recipe)
  • 1% YPSC bottom agar (see recipe for YPSC broth)
  • 25 g/liter CaCl 2·2H 2O solution, sterile
  • 0.6% YPSC top agar (see recipe for YPSC broth)
  • Bdellovibrio frozen stock (made from a previous prey lysate, see protocol 6)
  • HEPES buffer supplemented with CaCl 2 (see recipe)
  • 29° and 37°C static incubators
  • 37°C shaking incubator
  • Test tubes
  • 55°C water bath
  • Phase contrast microscope

Basic Protocol 3: Growth of Bdellovibrio as Single Plaques for Enumeration

  Materials
  • Prey cells (e.g., E. coli K12; refer to appendix 4A for methods of culturing E. coli K12)
  • 1% YT agar plates (see recipe for YT broth)
  • YT broth (see recipe)
  • 1% YPSC bottom agar (see recipe for YPSC broth)
  • 25 g/liter CaCl 2·2H 2O solution, sterile
  • HEPES buffer supplemented with CaCl 2 (see recipe)
  • Sample to be enumerated
  • 0.6% YPSC top agar (see recipe for YPSC broth)
  • 29° and 37°C static incubators
  • 37°C shaking incubator
  • Test tubes
  • 55°C water bath

Basic Protocol 4: Generation of Host‐Independent Bdellovibrio

  Materials
  • Overnight prey lysate of host‐dependent Bdellovibrio
  • PY agar (see recipe for PY broth)
  • PY broth (see recipe)
  • Phase contrast microscope
  • Petri dishes
  • 0.45‐µm syringe filters
  • 10‐ml syringe
  • 15‐ml tubes (Falcon)
  • Benchtop centrifuge
  • Plate spreaders
  • 29°C static incubator
  • Bijou universal tubes (7‐ml screw‐top plastic tubes; Sterilin cat. no. 129B)
  • 29°C shaking incubator

Basic Protocol 5: Routine Maintenance of HI Cultures

  Materials
  • PY agar (see recipe for PY broth)
  • Frozen stock of HI strain
  • PY broth (see recipe)
  • Petri dishes
  • 29°C static incubator
  • Bijou universal tubes
  • Conical flasks
  • 29°C shaking incubator

Basic Protocol 6: Preparing Frozen Glycerol Stocks for Long‐Term Storage

  Materials
  • Culture of host‐dependent Bdellovibrio
  • Culture of host‐independent Bdellovibrio
  • Sterile 80% glycerol
  • Liquid nitrogen in suitable Dewar container
  • Phase contrast microscope
  • 1.5‐ml plastic freezer tubes (Nunc)
  • Perforated ladle/slotted spoon
  • −80°C freezer containing freezer box

Basic Protocol 7: Bdellovibrio Predation Efficiency Assay on Luminescent Prey

  Materials
  • Bdellovibrio mutant strains to be tested (usually kanamycin resistant due to insertional inactivation of a gene under study)
  • 109JK control strain
  • Luminescent E. coli S17‐1 (pCL100, kanamycin resistant) (Lambert et al., )
  • E. coli S17‐1 (pZMR100) (Rogers et al., )
  • YT agar (see recipe for YT broth)
  • YPSC agar (see recipes for both top and bottom agar)
  • HEPES buffer supplemented with CaCl 2 (see recipe)
  • YT broth (see recipe)
  • PY broth (see recipe)
  • 50 mg/ml kanamycin sulfate
  • Petri dishes
  • 55°C and boiling water baths
  • 1.5‐ml microcentrifuge tubes
  • Multichannel pipettor
  • 96‐well microtiter plates for luminometer
  • Photoluminometer capable of reading a kinetic assay of luminescence over time in a 96‐well microtiter well and with temperature control and shaking (e.g., Anthos Lucy, Perkin‐Elmer Instruments Victor, BMG Labtechnology Fluostar Optima, Tecan Genios)
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Figures

Videos

Literature Cited

   Koval, S.F. and Hynes, S.H. 1991. Effect of paracrystalline protein surface layers on predation by Bdellovibrio bacteriovorus. J. Bacteriol. 173: 2244‐2249.
   Lambert, C., Smith, M.C.M., and Sockett, R.E. 2003. A novel assay to monitor predator‐prey interactions for Bdellovibrio bacteriovorus 109J reveals a role for methyl‐accepting chemotaxis proteins in predation. Environ. Microbiol. 5: 127‐132.
   Martin, M.O. 2002. Predatory prokaryotes: an emerging research opportunity. J. Mol. Microbiol. Biotechnol. 4: 467‐477.
   Rittenberg, S. 1982. Bdellovibrios‐intraperiplasmic growth. In Experimental Microbial Ecology. (R.G. Burns and J.H. Slater, eds.) Blackwell Scientific Publications, Oxford, UK.
   Rogers, M., Ekaterinaki, N., Nimmo, E., and Sherrat, D. 1986. Analysis of Tn7 transposition. J. Mol. Gen. Genet. 205: 550‐556.
   Seidler, R.J. and Starr, M.P. 1969a. Isolation and characterisation of host‐independent bdellovibrios. J. Bacteriol. 100: 769‐785.
   Seidler, R.J. and Starr, M.P. 1969b. Factors affecting the growth of Bdellovibrio bacteriovorus developing within E. coli. J. Bacteriol. 97: 912‐923.
   Stolp, H. and Starr, M.P. 1963. Bdellovibrio bacteriovorus gen. et sp. n., a predatory, ectoparasitic, and bacteriolytic microorganism. Antonie Van Leeuwenhoek 29: 217‐248.
   Tudor, J.J., McCann, M.P., and Acrich, I.A. 1990. A new model for the penetration of prey cells by bdellovibrios. J. Bacteriol. 172: 2421‐2426.
   Varon, M. and Shilo, M. 1980. Ecology of aquatic bdellovibrios. In Advances in Aquatic Microbiology, vol. 2. (M.R. Droop and H.W. Jannesch, eds.) pp. 1‐41. Academic Press, London.
Key References
   Beck, S., Schwudke, D., Strauch, E., Appel, B., and Linscheid, M. 2004. Bdellovibrio bacteriovorus strains produce a novel major outer membrane protein during predacious growth in the periplasm of prey bacteria. J. Bacteriol. 186: 2766‐2773.
  Key paper that refutes earlier work on outer‐membrane protein transfer from predator to prey.
   Cotter, T.W. and Thomashow, M.F. 1992. Identification of a Bdellovibrio bacteriovorus genetic locus, hit, associated with the host‐independent phenotype. J. Bacteriol. 174: 6018‐6024.
  Key paper describing molecular genetics of some HI strains.
   Martin, 2002. See above.
  Review of widespread potential applications of Bdellovibrio.
   Rendulic, S., Jagtap, P., Rosinus, A., Eppinger, M., Baar, C., Christa, L., Keller, H., Lambert, C., Evans, K.J., Goesmann, A., Meyer, F., Sockett, R.E., and Schuster, S.C. 2004. A predator unmasked: Life cycle of Bdellovibrio bacteriovorus from a genomic perspective. Science 303: 689‐692.
  First examination of genomic contents of a Bdellovibrio.
   Shemesh, Y. and Jurkevitch, E. 2004. Plastic phenotypic resistance to predation by Bdellovibrio and like organisms in bacterial prey. Environ. Microbiol. 6: 12‐18.
  Describes the non‐inherited nature of transient prey resistance to Bdellovibrio in cultures.
   Snyder, A.R., Williams, H.N., Baer, M.L., Walker, K.E., and Stine, O.C. 2002. 16S rDNA sequence analysis of environmental Bdellovibrio‐and‐like organisms (BALO) reveals extensive diversity. Int. J. Syst. Evol. Microbiol. 52: 2089‐2094.
  Describes the wide diversity of predatory bacteria in environmental samples.
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