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Chapter 16

Measurement of the Bacteriophage Inactivation Kineticswith Purified Receptors

Andrew M. Kropinski

Abstract

Practical methods are described for studying the interaction between bacterial viruses and their surfacereceptors.

Key words:  Adsorption, neutralization, inactivation, receptor, T4, M13, lipopolysaccharide, outermembrane protein, flagella, teichoic acids, pili.

1 Introduction

Bacteriophages bind to all possible cell surface receptors includ-ing pili [M13, D3112, F116]   ( 1, 2 ), flagella [χ, SP3, PBP1](3 , 4 , 5 ), lipopolysaccharide (LPS) [T7, P22], surface proteins[T1,T5,  λ, AR1]   ( 6 ), teichoic acids [SP50,  ϕ25]   ( 7 ), and cap-sules [K29, K1F, H4489A]  ( 8 , 9,10,11). In certain cases, such asT4, two receptors are used ( 12 ). These observations make phagesextremely useful tools for selecting receptor-deficient mutants,and for characterizing strains for specific receptors. An exampleof the latter would be the use of phages in typing systems.

Rather than using viable cells, receptor studies have been alsocarried out with cell extracts ( 13 ), cell walls preparations ( 14,15 ),

purified lipopolysaccharide ( 16,17 ), and complexes of outer mem-brane proteins with LPS  ( 12,15 ). In many cases the phages bindirreversibly to their isolated receptor resulting in inactivation. Thiscan be tested in the following manner which is optimized fromour studies of phage–LPS interactions  ( 17,18 , 19 , 20 ).

Martha R. J. Clokie, Andrew M. Kropinski (eds.), Bacteriophages: Methods and Protocols, Volume 1: Isolation,Characterization, and Interactions, vol. 501,   C 2009 Humana Press, a part of Springer Science+Business MediaDOI 10.1007/978-1-60327-164-6 16 Springerprotocols.com

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2 Materials

1. 1.0, 0.1, and 0.01 ml micropipetters such as Finnpipette or

Eppendorf pipetters (Fisher Scientific). These should be peri-odically recalibrated.

2. Sterile tips.3. Sterile capped 13× 100mm test tubes.4. Two sterile 125 ml Erlenmeyer flasks.5. Heating block or waterbath set at 48◦C.6. Waterbath shaker set at the growth temperature of your bac-

terium.7. Visible spectrophotometer set at 650 nm.8. Overnight culture of your bacterium grown in medium of 

choice supplemented with 1–10 mM CaCl2, subcultured and

grown to mid-log phase (Note 1).9. Phage diluted in growth medium (plus  Ca2+) to a titer of 

1–3× 105. Prewarm to the assay temperature just prior tothe experiment.

10. Bucket or styrofoam box containing crushed ice.11. Agar plates and overlays (Chapter   7).

3 Methods

1. Set up a rack containing 12, 13× 100mm  glass test tubes,and add carefully 1.6 ml of distilled water or buffer to thefirst tube (Note 2).

2. Add 0.9 ml of water or buffer to remainder of the tubes.3. Number the tubes 1 through 11, and the last tube “C.”4. Add 0.2 ml of LPS to the first tube so as to achieve a final

concentration of 200µg/ml. The stock solution of LPS istherefore 1.7 mg/ml.

5. Mix, and using a new pipette tip transfer 0.9 ml from tube“1” to tube “2.” Mix and continue to make doubling dilu-tions to tube number “11.”

6. Discard 0.9 ml from tube “11.”7. Add 0.1 ml of phage preparation, diluted in broth or buffer,

to each tube so as to achieve a final titer of 3 × 103 pfu/ml(Note 3).

8. Place the tubes in a waterbath or heating block at the desiredtemperature.

9. After an incubation period of 1 h remove 0.1 ml from eachtube to molten overlay medium, seed with host cells and pouronto plates.

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Phage neutralization 159

10. After appropriate incubation count and record the plaguenumbers, and calculate the percentage of phage neutralizedat each concentration of LPS.

11. Plot the data on two or three cycle semilog paper (or in asoftware package using a log scale) with the final concentra-

tion of LPS on the log scale and the number of plaques onthe linear scale. From this you can easily calculate the  PhI50i.e., the concentration of LPS which inactivates 50% of thephage.

4 Notes

1. The medium that which you use to grow your bacterium andpropagate your phage. If you are starting a new project Irecommend that you use the medium recommended for the

propagating the host bacterium (see   for example AmericanType Culture Collection (ATCC at http://www.atcc.org/) orDeutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ at http://www.dsmz.de/). N.B. many phages require1–10 mM divalent ions (such as  Ca2+ or  Mg2+) for optimaladsorption and the medium should be supplemented accord-ingly  ( 21,22 ).

2. We noted that dilutions of LPS in broth had an inhibitory affect on the PhI50  value.

3. This assay is optimized for phages which produce small plaquesin which accurate counts of 300 plaques per plate can be easily made. It is far more difficult to accurately count the numberof phages, such as T7, which produce large plaques.

References

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1990.   Pseudomonas aeruginosa   transposablebacteriophages D3112 and B3 require pili andsurface growth for adsorption. Journal of Bac-teriology  172 :1899–1904.

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4. Samuel,A.D., T.P.Pitta, W.S.Ryu, P.N.Danese,E.C.Leung, and H.C.Berg. 1999. Flagellardeterminants of bacterial sensitivity to chi-phage. Proceedings of the National Academy of Sciences of the United States of America96 :9863–9866.

5. Lovett,P.S. 1972. PBPI: a flagella specific bac-teriophage mediating transduction in  Bacillus  pumilus . Virology  47 :743–752.

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9. Stummeyer,K., A.Dickmanns, M.Muhlenhoff,R.Gerardy-Schahn, and R.Ficner. 2005. Crys-tal structure of the polysialic acid-degradingendosialidase of bacteriophage K1F. NatureStructural & Molecular Biology  12 :90–96.

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12. Hantke,K. 1978. Major outer membrane pro-teins of  E. coli  K12 serve as receptors for thephages T2 (protein Ia) and 434 (protein Ib).Molecular & General Genetics 164 :131–135.

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14. Valyasevi,R., W.E.Sandine, and B.L.Geller.1990. The bacteriophage kh receptor of  Lac- tococcus lactis   subsp. cremoris KH is therhamnose of the extracellular wall polysaccha-ride. Applied & Environmental Microbiology 56 :1882–1889.

15. Yu,F. and S.Mizushima. 1982. Roles of lipopolysaccharide and outer membrane pro-tein OmpC of   Escherichia coli   K-12 in the

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