More Bacterial Chromosomes and PFGE

[Brian_D Lanoil]

This is a follow-up to a post I made before.  I feel that a little more 
detail in my protocols and what I observed might be helpful for people to 
better understand my problem.  Thank you to those who have already responded.

I have been using CHEF to separate and observe chromosomes from the 
marine bacterium Alteromonas haloplanktis.  As a control on these gels, I 
have been running undigested whole E. coli chromosomes.  I prepared the 
plugs by treating the log phase cells with chloramphenicol (0.2 mg/mL) for 
1 hr (approximately 3 doubling times) to allow completion of replication but 
not reinitiation.  I then sealed the cells in agarose plugs and digested 
for 16 hours at 37 C with a lysis buffer containing lysozyme, EDTA, 
and salts.  Then I digested further with a solution of 0.5 M EDTA, 1% 
Sarkosyl, and 1 mg/mL proteinase K for 24 hours.  I then washed 
extensively with 0.5 M EDTA.

Using a switching protocol optimized to separate in the range of ~350 kb 
to 5 Mb, I then separated the chromosomes out without any linearization 
or digestion.  I observed multiple bands in the E. coli lane, the 
smallest of which was the predicted size.  A similar pattern was seen in 
A. haloplanktis, although the size of this genome is not known.  The E. 
coli strain contains no plasmids, and is, to my knowledge, a complete 
chromosome.  Since large circular chromosomes do not enter the gel during 
PFGE (?) unless fortuitously nicked during preparation (in which case 
they should run at the correct size for the linear version), I am asking 
what could be causing these extra bands.

Several people have responded that the extra bands are concatemers of the 
chromosome which are formed due to the inability of the chloramphenicol 
to affect the cells fully, either due to concentration or time.  I have 
tried several different concentrations, and I have seen no effect, 
although the lowest that I have gone is 0.1 mg/mL (it is difficult to do 
these experiments since these gels take 5 days to run).  I have not 
attempted different times, but I think that it is unlikely that this is a 
factor since one hour is approximately 3 doubling times for E. coli and 
almost 2 doubling times for A. haloplanktis.

I appreciate the response I have gotten already, and I thank you in 
advance for any advice you might give.

thanks,

Brian Lanoil