Mating Experiments using E. coli

(Reference manual: For specific techniques, reference will be made to pages in Pierce and Leboffe: Exercises for the Microbiology Laboratory (1999); Morton Publishing Co. (ISBN 0-89582-462-0))

Somatic Chromosome Recombination
(Usually called "Conjugation" or "Sexduction."

.....Many years ago, William B. Hayes, who was working in the laboratory of Edward A. Adelberg (Nobel of bacterial genetics), discovered a very strange thing. Unlike the case of F-lac transfer, in the previous exercise, Hayes found that his strain of E. coli could transfer not only the lac-operon, but also any other gene on the somatic chromosome. ..Adelberg was so incredulous that he refused to add his name to the research paper. ..Thus Hayes got all the credit for discovering the tool that rapidly led to the determination of where many nutritional genes lay on a "chromosome." ..Within only five years, more than 1,000 genes had been mapped. ..This made little E. coli the best genetically mapped organism in the world, and established it as the top research micro-organism.

.....Rather quickly the mystery of how this strain could transfer its genes was elucidated (dictionary anyone?). ..It was found that F could lead another existence: ..not only could it get along freely in the cytoplasm, but it could also insert itself into the somatic chromosome by a type of recombination. ..The result of this was that F became a super F-prime (F'). .. While F-lac contained all the normal F-genes plus the lac-operon from the somatic chromosome, the new super-F' contained the F-genes plus ALL the somatic genes. By the way, the official name of a super-F' is "Hfr" (for high frequency recombination). .. As it turned out, there are many different types of Hfr - all depending on where the F "integrated" into the somatic genome. .. And, to complicate things, F is "polar" and thus can integrate in either of two different directions relative to neighboring somatic genes AT THE SAME PLACE.

.....But how can such a cell mate? .. Here's how you might picture it - and don't worry about getting lost with the technical jargon, the last paragraph before the Exercise will be an analogy that will help you to completely understand what transpires. .. F, like all plasmid DNA is duplicated by its own special DNA-polymerase. .. This enzyme sits down at one end of the inserted F-DNA, but instead of reading INTO the F-genes, it reads the opposite direction - that is, into the beginning of the somatic DNA, and keeps reading until it reaches its own special "STOP" sign, which is all the way around the huge piece of somatic DNA and through the F-DNA itself, right up to the place where it originally sat down and began reading - more than an hour and a half ago!

.....BUT The little "loose" end of DNA, that was made as the enzyme started its reading, was directed into the pilus to be transferred to the recipient cell. ..Thus the longer the enzyme kept at its job of replicating the donor's DNA, the more DNA was sent through the pilus. .. Thus, eventually, all genes would go through the pilus - SO LONG AS brownian motion didn't rip the two cells apart (ouch!) and break the DNA. ..Thus it is rare that the whole chromosome is transferred, and very common for the first genes to go over to the F^- recipient cell. ..For sake of easier discussion lets term those genes near the beginning as "early" and those all the way around at the other end as "late", with the F-genes themselves being the "last."

.....THUS the "later" the gene is located the less chance it has of being transferred because it becomes ever more probable that brownian motion will interrupt the mating. .. (Of course, Hayes helped out by taking periodic samples from mating cultures and putting them in a blender!)

.....Your exercise will be to mate a strain of F^- with two different of the many types of Hfr. ..You will do this by a very simple method called 'cross-streaking:' .. on each of your different plates you will make a vertical swab of you F^-, and then you will make horizontal swabs of each of your two Hfr's - each at a different place, of course. .. What is especially important is that your Hfr swabbing go only across the plate ONCE. .. Picture in your mind what is happening: ..as the Hfr swab hits the vertical F^--line, some of the F^- bacteria are picked up and dragged along to be dropped off next to any Hfr cells that also happen to fall off the swab at that point. ..Thus the two bacteria will be next to each other allowing mating to occur. ..Growth will occur IF the medium in the plate allows it.

.....ANALOGY. If at first you are befuddled by the above explanation for how an Hfr sends its DNA through a pilus into the F^- cell, "pretend" it is this way. ..With a pencil, draw the DNA of the Hfr as a large circle. .. On that circle place the F as a big dot. .. Then erase a tiny bit of the circle right next to the dot. .. Convert the new end of the LINE into an arrow-head. Now straighten the whole thing out: ..you have an arrow with a point at one end and the big dot as its tail. .. Now imagine that the HFR duplicates this and you have a second arrow. ..The Hfr sends this arrow through the pilus. .. The point goes first (it is officially called the "origin" or "O"). .. The arrow slowly slips through the pilus into the F^- cell taking about 90 to 100 minutes of time to do so (at 37°C). .. The last thing to go into the recipent cell is the "F" - converting the recipient into an F⁺, at least, and maybe an HFR. ..Thus the genes near the arrowhead are transferred sooner than those near the end where the "F" is located. ..One thing that made Dr. Hayes so famous was that he could map the genes on the E. coli chromosome by merely watching the clock, because the DNA slips through the pilus at a constant rate.

..... You drew your first circle and put the dot on it and then erased the bit of line on one side of the dot. .. Suppose that you had done your erasing on the other side of the dot. You'd have an Hfr that was completely the opposite type of donor than the first. .. The early genes on the first, would be very late ones on the second.

..... And consider that when you drew your first circle and put the dot on it, you could draw other circles and put dots on them in different places. ..And erase the bits of line on either side of those dots. ..Yes, indeed! ..There are many different Hfr types of donors.

..... Because the very first Hfr was found by Hayes, it is called "HfrH", and is the depicted in books as having the "conventional" orientation... Draw a circle and put the dot at the top. Erase the bit just to the right of the dot. ..The earliest gene on that picture is the one that codes for the making of the amino acid threonine. ..Now let us liken our circle to a clock's face.

..Here are the rough positions of a few genes on that face:

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