The non-bold was spoken. All the figures and tables were on transparencies.
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Control of beta-Galactosidase Activity
Beyond Those of the Lac-Operon
Mireille A. Captieux, Univ. of Edinburgh, Scotland
Taylor E. Swain, Jamestown High School, Wmsbg, VA
Alicia A. Mills, Messiah College, PA
Lauren M. Mills, Messiah College, PA
Carl W. Vermeulen, PhD, Science-Projects.Com, Wmsbg, VA.
Of course, we must first start with the Lac-Operon. Allow me to acquaint you with my symbolism:
It has been said that you know you have made a truly great discovery if your work shows up in the textbooks of grade schoolers. Well, I don't know if the lac-operon has trickled down that far, but it is taught in the high schools across this country. Besides it is a part of most college genetics and microbiology lab curricula, and one of the most used subjects in graduate degree qualifying exams. So I suppose that Jacob, Monod, Cuzin and Brenner do warrant a Nobel (smirk).
Anyway, the gist of it is to produce an enzyme to breakdown the sugar lactose.
Now you can see just how audacious we were to think that we could build upon this foundation, but then, my far flung group were young enough not to know just how audacious they were.
It thus occurred to us that Jacob and his friends hadn't finished the story about the control of beta-galactosidase - or "lactase", as I shall continue to refer to it. Afterall, the lac-operon is only an on/off switch, and as any production line manager knows - one doesn't merely turn on the conveyer belt. One must also manage the speed. Thus the operon level is a form of very coarse control, and we knew that there must be some sort of fine control also.
Thus by assimilating another lesson with our knowledge of the operon, we reasoned that the most likely candidate for fine-tuning the activity of lactase would be by some sort of feedback mechanism. What might this be?
Again, following the footsteps of chemists associated with the Pasteur and Cambridge groups, we noted that galactose moiety shows up in several of the analytically important analogs of lactose. One of these is ONPG, which you see lactase hydrolyzes to yield a colored product allowing the reaction to be followed photometrically.
Another analog is IPTG, which is able to induce, or turn on, the operon, but it itself cannot be hydrolyzed by lactase. Thus the induction of the gene and the performance of the gene's function could be separated.
Well, this emboldened us to suggest the perhaps the product galactose itself might be chemical instrument of this feedback control.
So we set out to test this first in a qualitative fashion:
And this is what happened:
With our reasoning confirmed,
WHAT NEXT?
In order to determine the type of inhibition, which is, in essence, telling us where the galactose affixes to the lactase to inhibit its breakdown of lactose, we would have to perform a series of simple quantitative runs with various concentrations of the inhibitor (galactose) with various concentrations of substrate, ONPG.
Note that where the lines cross will give us the type of inhibition. Being that ONPG were IPTG analogs of lactose, we took the not-so-wild guess that galactose was also an analog of lactose, and that it probably would fit into the same reaction site as does lactose itself, such as shown here:
Afer going through all the preliminary plots for finding initial velocities, this is what we found:
Thus galactose is a competitive inhibitor. Of course, it was surprising to me that the intersection was at or very close to (0,0) - something I had never seen before in analyses of other enzymes using the Lineweaver-Burk plotting method.
But all this in vitro work is fine and dandy, but means little if it does not work in vivo.
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But with our luck this will happen: it will "eat" the galactose and shun the lactose altogether! We were concerned about this because, looking back at the lac-operon, any movement of galactose into glycolysis would yield ATP, which, in turn, would inhibit the expression of the lac-operon.
To prevent that possibility, we needed to use a strain that was incapable of using galactose. And we would compare this gal- strain to a gal+ strain.
| Syndey Brenner's Accession Number | Yale's Accession Number | Genotype | Relevant Phenotype |
|---|---|---|---|
| MO | 4995 | F- λ- e14- relA1 rspL150(strR), spoT1 (see note) | lac+ gal+ |
| M26 | 4996 | same as above except it is also galE15 | lac+ gal- |
| Note: "F- " indicates this is a 'female' cell (i.e.: without an F fertility factor) "λ-" indicates that this cell is not lysogenized with phage λ "e14- relA1 rspL150(strR), spoT1" all indicate that these cells have various abnormal ribosome functions | |||
Thus we had a strain with this altered pathway:
Before we continue, there are a few in vivo phenomena we needed to know about.
(((For "Flickering" I will NOT show the diagram to the right, but rather have the real gadget in front of me to demonstrate to the crowd. If the reostat is turned full on as if there were no feed back on the enzyme, then the light will visibly flicker. However, if the reostat is turned down, the light dims to a point that it will not activate the photoelectric cell to turn off the circuit. Thus the "operon" is on, but the production of light ("lactase activity") is dimmed.)))
The second thing to know was how to make use of the anaerobic conversion of sugars to that mixure of acids. Shown here is a plate of what is called MacConkey Agar upon which the two strains above were streaked. The plate contains a rich medium of amino acids, a pH indicator AND galactose. On the left side, acid is being produced and turns the methyl-red red, while on the right side, where galactose cannot be used, the bacteria now use the amino acids exclusively for their food and energy and release a lot of ammonia making the pH go basic leaving the pH indicator pale.
Now let's see how our two strains work on various concoctions of sugars in this type of agar. We hope that you don't mind looking at halves of plates for awhile: they fit better on the screen. In this first panel, we are looking at the "wild" - unmutated strain:
So we see that sugars are necessary for the production of acid. Now let's see how the gal- deals with these media.
Just as expected - but look closely! On lactose it doesn't make as much acid as did the non-mutant. Could this be due to a buildup of feedback-inhibiting galactose that cannot be forwarded into glycolysis? Ah, ha! This could be a hint of good things to come! So let's see what happens to these two strains on a medium containing lactose and an excess of galactose. But first let's try to imagine what would happen. Look at the middle plate (above). If the reason for the diminished amount of red is because some galactose was formed, what would it be like if we had previously added even more? It should be pale! So let's see:
Viola! It looks like we are on to something. But we need to know one more thing for a real pièce de résistance. We need to know if there is indeed the predicted lactase in those mutant cells, because if there is, that lactase must be severely inhibited within the cell by all the added galactose. So what we did was wash the cells free of the excess galactose (and lactose), break them open using a mixture of detergent (SDS) and chloroform, and see if they can breakdown ONPG.
Not only does the mutant have lactase in them, but they also seem to have even more than the normal cells. Why?
We should be able to answer that if we assimilate our newfound information into the overall scheme of lactase control:
Now for the whole picture:
If the galactose so overwhelms lactase activity, the cell runs low on ATP, which is just the condition adenyl cyclase needs to make more cAMP, which along with the constant bath of de-repressing lactose enforces the expression of the operon.
Thus we have extended knowledge about this most commonly studied of genes.
In summary, there is easily detectible fine control of lactase activity beyond the coarse control of the lac-operon!
What is more is that we have also laid out several extensions which we feel should be amenable to a number of instructors of laboratories in microbiology and genetics. Of particular interest in these days of safety - both personal and environmental - is that this is one of very few enzyme inhibition labs that involves no heavy-weight toxins such as heavy metals or carcinogens. If you are interested, the protocol can be found within the site index of my website:
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