I need to start some work that involves infecting Escherichia coli with bacteriophage Chi. The phages were propagated on Salmonella. Will they be able to grow immediately on the usual Escherichia K12 derivatives, or will I encounter difficulties with differential restriction systems or maybe with somewhat different surface/flagella properties? What do I need to consider while planning the experiments?
Thanks a lot for bothering.
I don't know about chi specifically, but in general bacteriophages will face host restriction. You can easily test this by titering the phage stock on both E. coli and Salmonella and seeing if the plating efficiency is the same on both hosts or is significantly different. If different, just select one plaque from the E. coli plate and use to propagate a new phage stock in E. coli, this should then work fine.
Michael Benedik is correct; in addition and better still: determine efficiency of plating with various strains of E. coli, lysogenic and 'innocent'. Consult pp 514-522 in "The Genetics of Bacteria and their Viruses" by William Hayes, Blackwell Sci Publ, 2nd Edition, 1968...
You indeed need to address a few questions. I don't know the Chi phage. But I can think of the following general questions:
- Is the phage receptor present in the new host?
- Does the new host have one or several restriction systems? Does the phage have some sort of 'anti-restriction' function?
- What about CRISPR sequences present in the new host? And anti-CRiSPR proteins in the phages (thought I don't think any of those proteins has been identified in Salmonella phages yet, but I'm not sure).
That may help to have some sort of preliminary evaluation of the number of phages that are likekly to make it to the new host.
If the receptor is absent, and there are restriction systems/CRISPR in the new host it is unlikely you'll get plaques. If the receptor is absent and you can manage to get rid of the restriction systems you may have a chance to isolate a mutant of the phage with a broader host range, but that will most likely require to plate a high titre lysate as this type of mutant may be rare. Than you can switch to the strain with restriction systems.
If you have the sequences of the phage and the host genomes you may be able to pick up the restriction system specificity and the restriction sites on the phage, which would then provide an idea of the factor of restriction (one site, around 1 in a 100 phages will grow if I remember correctly). I suppose you could also check for CRISPR systems and look for spacers corresponding to your phage sequence.
Good luck.
Dear Ariane, Dear Michael,
presence of the phage receptor is not a problem. Chi binds to flagella of several enterobacteria, including Salmonella typhimurium and Escherichia coli, and delivers its DNA at the flagellar baisis. So far my text book (and lecture) knowledge. I will find out about CRISPR motives. It's funny, but I do not find data on plating efficiencies on different hosts. Maybe, this is one of those experiments, that it is easier to do it than to find the literature.
Thank you anyhow, I will let you know what comes out of it.
If you wish, Johannes, I can send you a short pps with these pages that I was using for teaching before retiring.
- Let me have your email address where to send it to; mine is [email protected]
Johannes, yes I had meant for you to just do the plating efficiency experiment. You can have the answer in 24 hours.
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