Bacterial 2′,3′-cGAMP activates a SAVED effector to form membrane-disrupting filaments and restrict phage replication
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Bacterial cGAS-like enzymes produce 2′,3′-cGAMP to activate an ion channel that restricts phage replication
Really interesting paper! I appreciated the mixes of methodologies used in this study - it was an enjoyable read!
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These experiments suggest that active BtCap14 transports Cl- from the cis to trans chambers.
its not immediately clear to me why chloride transport on its own would block phage replication - I'm quite curious to know what is happening in infected cells. There are some methodologies for measuring ion flux during phage infection in living cells. It could be really useful to apply them to your system, to 1) confirm that chloride efflux is happening in vivo and 2) to maybe measure flux of other types of ions to see if this is part of a large system of changing the concentration gradients of different ions across the cell membrane in a way that might be more mechanistically connected to antiphage defense.
You could also consider staining the membranes of cells that have been recently infected with phage to see if the membrane potential is …
These experiments suggest that active BtCap14 transports Cl- from the cis to trans chambers.
its not immediately clear to me why chloride transport on its own would block phage replication - I'm quite curious to know what is happening in infected cells. There are some methodologies for measuring ion flux during phage infection in living cells. It could be really useful to apply them to your system, to 1) confirm that chloride efflux is happening in vivo and 2) to maybe measure flux of other types of ions to see if this is part of a large system of changing the concentration gradients of different ions across the cell membrane in a way that might be more mechanistically connected to antiphage defense.
You could also consider staining the membranes of cells that have been recently infected with phage to see if the membrane potential is disrupted.
Lastly, Im curious if this system is sensitve to extracellular (or extra-vesicualar) chloride/anion concentrations. Different media have different chloride levels, it could be really informative to grow the bacterial in a higher concentration of NaCl for instance and see if the activity of the system is affected
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SPβ or Φ29
Can you briefly comment on how similar or different all these B. subtilus phages are from one another? Are these different classes of phages (myo vs podo vs sipho) or different life styes (lytic vs temperate) or having any other notable features of their biology that could be driving this (genome modification, shell formation, etc) ?
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104-fold protection against phages SPP1 and phiB002
I'm curious about the survivors that are plaquing here...are these escapers? if you pick of these plaques, are they sensitive or resistant to your system?
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OD600 of cultures expressing wild-type and catalytically inactive BtCBASS both collapsed.
Its difficult to visualize this collapse with the way that the graphs are plotted on what looks like a log10 scale (2F). It would be easier to see this point with a linear scale, and you could also potentially consider extending the time frame of the growth period for longer to help the reader get a sense of the growth dynamics.
I have done a lot of experiments like this varying phage MOI and measuring both bacterial OD over time in a plate reader and phage output at the end of 24 hours - often you can get pretty clear binary results at the end of a long time course.
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