>cbystrom at darkwing.uoregon.edu (Cory Bystrom) writes:
>I've been trying to find an answer regarding some characteristics of
>bacteria that grow on MacConkeys agar. Microbiology texts haven't been
>much help and I'd like to hear some opinions.
>I've prepared two mutants of a sugar kinase and added the respective sugar
>to the agar to select for the correct clones.
I hope you used MacConkey base (without lactose), right?
>When I sequence each of the clones picked from the MacConkeys agar I
confirm that they both carry the mutation. However, the color of the
colonies from the two mutants is strikingly different. One gives the
classic deep red colonies with bile precipitate under overgrown areas.
You don't say what bug and what sugars do you use. Anyway, in
enteric bacteria most sugars would be transported (and phosphorylated)
through PTS, sometimes with poor affinity (your 1% sugar in MacConkey
should be enough, though). Curtis and Epstein (J.Bact., 1975) got white
colonies of E. coli on MacConkey-glucose only when they eliminated ptsG
and ptsM and glucokinase.
>The second gives weakly pink colonies and the agar slowly becomes yellow
>after two days of incubation. I expect that both of these mutations to have
>no effect on the activity of the>kinase that I've mutated.
>This being said, is it possible that the mutations *have* changed the
>activity of the kinase and that this is reflected in the ability for the
>bacteria to ferment the selected sugar? Is it possible that the level of
>expression is different?
Yes, all that is possible. Why don't you just measure the kinase
activity in extracts? You could do it
1) using radioactive sugar and collecting sugar-phosphate formed on an
anion-exchange resin;
2) using pyruvate kinase and lactate dehydrogenase coupling system to
measure sugar-dependent ADP formation:
Sugar + ATP ------> Sugar-P + ADP
ADP + PEP ------> ATP + Pyruvate
Pyruvate + NADH ------> Lactate + NAD
You just have to measure the decrease of absorbance at 340 nm caused by
addition of your sugar.
Hope this helps. I could dig out references if needed.
Michael Galperin galperin at biotek.mcb.uconn.edu
Molecular and Cell Biology 75304.226 at compuserve.com
Univ. of Connecticut U-125 (860) 486-1898 (lab)
Storrs, CT 06269-3125 (860) 486-4331 (fax)
