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We are attempting to isolate bacteriophages from environmental soil samples and most protocols recommend adding CaCl2 to the media to aid bacteriophage infection, even though they all recommend different concentrations.

This paper claims:

Addition of divalent ions (CaCl2 and MgCl2) may also facilitate plaque formation through aiding phage adsorption to the bacterial receptor

But how does CaCl2 aid phage adsorption exactly? And by extension, is there an ideal concentration of CaCl2 that maximizes the adsorption when growing bacteria in liquid media?

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    $\begingroup$ Not knowing anything about phage infection itself in this context, I would guess that the ideal concentration varies depending on phage, host, and environment. There may not be a universal ("You should always use 10 mM") ideal concentration. If the papers you're reading are all dealing with the same context you are, and the recommended concentrations aren't too far away from each other, i.e., over an order of magnitude or so, then averaging them might not be a bad idea. Or, pick one paper and use its conditions as your guide so you can replicate their results. $\endgroup$
    – MattDMo
    Commented Nov 19, 2022 at 22:44
  • $\begingroup$ @MattDMo we are going to test with two concentrations since the concentration varied so much in the various papers. 1 mM/liter and 50 mM/liter. We did read one paper that seems to use 3 M/liter which seems high. None of the papers detail what mechanism makes CaCl work however. $\endgroup$ Commented Nov 20, 2022 at 16:09
  • $\begingroup$ @JohanWikström doing a range of concentrations would be a good idea - you'll likely select different populations of phage. 3M may have been a typo as I don't think there are any bacteria beyond extreme halophiles that will tolerate that sort of salt concentration. I would guess cofactor - divalent cations are commonly cofactors for proteins. $\endgroup$
    – bob1
    Commented Nov 20, 2022 at 20:02

1 Answer 1

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The relationship between calcium ion concentration and efficiency of phage infection has been observed since Delbrück's work classifying phage mutants.1 A short letter in Nature authored by H. Williams Smith in 1948 is the first source I can find recommending CaCl2 supplementation of media for bacteriophage propagation.2

Stassano and de Beaufort, Asheshov, Bordet, Burnet, and others have reported on the inhibitory effect of sodium citrate when added to media used for the demonstration of phage action. This suggested that a deficiency of calcium in the medium might be responsible for the inhibition of phage action referred to above. On the suggestion of Dr. G. P. Gladstone of the Lister Institute, calcium chloride in concentrations of $M$/100 were added to two of the batches of inhibitory media; the third was unfortunately discarded. The effect was striking. The media were now found to be completely satisfactory for the demonstration of bacteriophage activity.
As a result of this experience, it is considered advisable to add calcium chloride to solid culture media that are to be used for the typing of staphylococci by the bacteriophage method.


To answer your first question ...

But how does CaCl2 aid phage adsorption exactly?

... structural and biochemical studies have shown that calcium ions are important cofactors for many adhesion-related phage proteins. Some examples:

  • p2, a phage infecting Lactococcus lactis, requires Ca2+ to induce the "open" conformation of the receptor binding component of its baseplate.3
  • Bacillus phage SPP1 requires Ca2+ to stabilize the infective conformation of its tail tip.4
  • Escherichia phage Mu needs Ca2+ for efficient binding of its central spike to the host membrane.5

Beyond adsorption, Ca2+ has a role as a counterion during phage DNA injection 6-8 and as a stabilizing factor during capsid assembly.9,10


To address your second question ...

is there an ideal concentration of CaCl2 that maximizes the adsorption when growing bacteria in liquid media?

... that will depend on the phage-bacteria system and other details of your culture environment.


References

  1. Delbrück M. Biochemical Mutants of Bacterial Viruses. J Bacteriol. 1948 Jul; 56(1):1-16.
  2. Smith HW. Calcium-deficient media; their effect on phage action. Nature. 1948 Mar 13; 161(4089):397.
  3. Sciara G, Bebeacua C, Bron P, Tremblay D, Ortiz-Lombardia M, Lichière J, van Heel M, Campanacci V, Moineau S, Cambillau C. Structure of lactococcal phage p2 baseplate and its mechanism of activation. Proc Natl Acad Sci USA. 2010 Apr 13;107(15):6852-7.
  4. Goulet A, Lai-Kee-Him J, Veesler D, Auzat I, Robin G, Shepherd DA, Ashcroft AE, Richard E, Lichière J, Tavares P, Cambillau C, Bron P. The opening of the SPP1 bacteriophage tail, a prevalent mechanism in Gram-positive-infecting siphophages. J Biol Chem. 2011 Jul 15;286(28):25397-405.
  5. Harada K, Yamashita E, Nakagawa A, Miyafusa T, Tsumoto K, Ueno T, Toyama Y, Takeda S. Crystal structure of the C-terminal domain of Mu phage central spike and functions of bound calcium ion. Biochim Biophys Acta. 2013 Jan;1834(1):284-91.
  6. Fernandes S, Labarde A, Baptista C, Jakutytè L, Tavares P, São-José C. A non-invasive method for studying viral DNA delivery to bacteria reveals key requirements for phage SPP1 DNA entry in Bacillus subtilis cells. Virology. 2016 Aug;495:79-91.
  7. Alatossava T, Jütte H, Seiler H. Transmembrane cation movements during infection of Lactobacillus lactis by bacteriophage LL-H. J Gen Virol. 1987 Jun;68 (Pt 6):1525-32.
  8. Cvirkaite-Krupovic V, Krupovic M, Daugelavicius R, Bamford DH. Calcium ion-dependent entry of the membrane-containing bacteriophage PM2 into its Pseudoalteromonas host. Virology. 2010 Sep 15;405(1):120-8.
  9. McKenna R, Bowman BR, Ilag LL, Rossmann MG, Fane BA. Atomic structure of the degraded procapsid particle of the bacteriophage G4: induced structural changes in the presence of calcium ions and functional implications. J Mol Biol. 1996 Mar 8;256(4):736-50.
  10. Plevka P, Kazaks A, Voronkova T, Kotelovica S, Dishlers A, Liljas L, Tars K. The structure of bacteriophage phiCb5 reveals a role of the RNA genome and metal ions in particle stability and assembly. J Mol Biol. 2009 Aug 21;391(3):635-47.
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