Temperate Phages

One class of bacteriophages are the “virulent” phages. Virulent viruses carry out lytic, productive growth in an infected host cell.  Bacteriophages can also be temperate. Temperate phages have an additional option of forming a repressed prophage in the host cell.  The prophage turns off lytic genes and ensures that the prophage genome is replicated and distributed to progeny cells as the host cell divides.  Temperate phages make a decision which path to go down, depending on whether the infected cell’s metabolic state is robust.  Under appropriate conditions, the prophage can become induced to lytic growth [See Ptashne’s “A Genetic Switch”, incidentally one of the very best books ever about a creature and its biology.].  A rule-of-thumb view is that lytic phages are by far most likely to be found as free viruses, and thus likely to be found in the above enrichment scheme.  A working view of temperate phages is that they mostly exist as prophages, and make the leap to a new host only occasionally.  A search for a temperate phage should therefore be focused on gut bacteria as discussed below [under construction].
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Isolating a temperate phage Method 1

When bacterial genomes are sequenced, prophages are often present in the sequence; these are prophages of temperate bacteriophages.  Prophages are generally integrated in the bacterial genome, though the prophage of some bacteriophages are in a plasmid state.  The typical enteric isolate will harbor one or more prophages.   A culture of a bacterium harboring a prophage generally has a small fraction of induced cells that release mature virus particles.  For example, a culture of an E. coli stain carrying a lambda prophage will have about 1 free phage for every 1000 cells.  That is, an overnight broth culture with a cell titer of 109 cells/ml will have a phage titer of about 106 phages/ml.  One way to determine the free phages is to treat the bacterial culture with a few drops of chloroform to kill the bacteria, spin to clarify, and then plate the supernate out with cells of a non-lysogen.  Finding the non-lysogen is the trick.  Lambda was in the lab for 30 years or so before Esther Lederberg happened to use a strain spontaneously cured of its lambda prophage plus a sample containing free lambda virions.  An easier approach is to choose a non-lysogenic host and look for bacteria that release temperate phages able to form plaques on it.  In principle, one can take 25 colonies from a streak plate like the one shown in Picture 2, grow small cultures and do the test just described above.  Another even easier way is to take advantage of induction by UV light.  

Isolating a temperate phage Method 2


Induction is when a prophage transitions to lytic, productive growth.  Lambda and many other temperate phages are very efficiently induced to lytic growth by UV light.  See Ptashne’s book for how that works.  In fact, the genetic switch of the book’s title is the switch from repressed prophage to lytic viral development.  In our lab we have a homemade UV box, which consists of a germicidal lamp in a box plugged into the power strip so it can be easily switched on and off.  The lamp is a two pronged (T5) bulb, about 18” long.  It is positioned on the roof of the box, and it is 19 cm away from the surface where we put plates to irradiate them.  We have calibrated the lamp’s dosage using a strain of E. coli carrying a lambda prophage.  We put E. coli (λ+) cells in a UV transparent buffer (necessary as broth media have organics that absorb UV.  We irradiated the culture for a  few seconds, took a sample, UVed some more, and took another sample, etc.  These aliquots were diluted into broth and after 70 min (a time sufficient for the lytic cycle to complete), we determined the phage titer of the cultures. The optimal dose, 12 seconds in our case, was the dose that yielded the highest phage titer.   Using UV, one can find out if a cultured bacterium is a lysogen carrying a UV-inducible prophage.  We picked 25 colonies of a MacConkey plate streaked with the fecal slurry, and gridded them on a lawn of E. coli C that had been poured in soft agar on a broth plate.   We have a sterile grid system but the spotting can easily be done with loopfuls.  The plate was irradiated for 12 seconds, and incubated overnight at 37C.  The results are in Picture 5. 

Picture 5

Results of spotting 25 cultures of enteric bacteria from a MacConkey plate.  Top row from L to R = isolates 1-5.  Next row is 6-10, and so on.  It looks like 8 isolates, namely 2, 5, 7, 8, 10, 12, 17 and  25 are non-lysogens by this assay.  The remaining spots are surrounded by lysis halos suggesting phage growth.  See text for further commentary. 

The UV plate indicates that about 13 isolates give good lysis spots, consistent with them being lysogens.  There are other possible explanations for the lysis haloes, a major one being that a toxin, called a colicin, is being released , not a phage.  Colicins are protein toxins, not viruses, and do not replicate.  A confirmation that a strain is a lysogen is to grow up a culture , sterilize it with chloroform, and spot the culture supernate on a lawn of the host to see if the supernatant contains phages that can produce plaques. 

More comments about the print plate in Picture 5.
1. The cultures may not be pure.  For example, isolates 16 and 22 and a few others, have halos and yet show heavy bacterial growth, and some pigmentation.  These might be mixtures of two or more bacteria. 


2. Look at isolate 17 – its print has spread far from the original spot location – this guy is very motile.
To study some of these cultures further, isolates 1, 6, 11, 16, and 21 were streaked out on a broth plate: these are the ones in the leftmost column in Picture 5.
At left, broth plate streaked with isolates used in Picture 5.  Counterclockwise from the top they are: isos 1, 6, 11, 16 and 21.  Note isos 16 and 21, at the bottom and next right, are fuzzy, indicating high mobility.  It is not clear that these cultures are single bacterial strains – they still may be mixtures of 2 or more strains.