Historical Background
The search for bacterial conjugation stemmed directly from the desire to study genetics in bacterial systems.
EDWARD TATUM & HIS MUTANTS
George Beadle and Edward Tatum, working with the fungus Neurospora had shown that mutants could be blocked in different steps of the same biochemical pathway. This led to their famous "one gene - one enzyme" hypothesis.
Beadle, writing in 1945, recognized the dilemma of establishing this hypothesis in bacteria:
| The genetic definition of a gene implies sexual reproduction. It is only through segregation and recombination of genes during meiosis and fusion of gametes that the gene exhibits its unitary property. In bacteria, for example, in which cell reproduction is vegetative, there are presumably units functionally homologous with the genes of higher organisms, but there is no means by which these can be identified by the techniques of classical genetics. |
Beadle |
Tatum |
We should also note that Beadle and Tatum's work was revolutionary for their use of nutritional mutants of Neurospora as conditional mutants. They were awarded the The Nobel Prize in Physiology or Medicine in 1958 "for their discovery that genes act by regulating definite chemical events"
Tatum, nevertheless, tried to see if he could extend his nutritional studies to E. coli K12. He isolated a large number of double mutant strains. This was a significant advance because he demonstrated the first real proof of heredity in bacteria -- the mutant phenotype persisted over generations.
Some of the strains he obtained were:
Strain Auxotroph for: 58-161 methionine & biotin 58-278 phenylalanine & biotin 58-309 cysteine & biotin 58-336 isoleucine & biotin 58-580 thiamine & biotin 58-741 histidine & biotin 58-2651 proline & biotin 679-183 proline & threonine 679-662 glutamic acid & threonine 679-680 leucine & threonine
Unknown to him, the real importance in these mutants lay in the fact that mutant 679-680 was genetically different from all of the others. We will see later that it plays a critical role in Lederberg's discovery of conjugation.
JOSHUA LEDERBERG & THE DISCOVERY OF CONJUGATION
In 1945, Joshua Lederberg decided to use nutritional mutants to look for evidence of mating in bacteria:
| He conceived the use of nutritional mutants as a means of searching for mating in bacteria. With the prototrophic recovery approach in mind, he devised a possible experiment. "The basic protocol ... entailed the use of a pair of nutritional mutants, say A+B- and A-B+. If crossing occurred, one could plate out billions of cells in a selective medium ... one should be able to find even a single A+B+ recombinant. |  |
| Text from "The Emergence of Bacterial Genetics" by T.D. Brock, p81. |
He isolated 2 E. coli mutants: one was met-pro+; the other was met+pro-. He tried a mating experiment but he found no evidence for mating.
When Lederberg heard that Tatum had isolated double mutant strains of E. coli, he wanted to try these:
Such double mutants were of special value since their reversion to prototrophy should be extremely low. Another value perceived by Lederberg for obtaining Tatum's strains was the following: In the event that mating types or sterility factors were present in Escherichia coli, mating might not occur. Self-incompatibility was common in fungi, and one of the best ways of obtaining distinct mating types was to use independent isolates. this idea was expressed in Lederberg's first letter to Tatum: "It should ... be advantageous to use stocks of heterogeneous origin in the event that there exist mating types, sterility factors, etc." This idea, of course, proved to be true. However, Lederberg had no way of knowing that Tatum's Escherichia coli K-12 would be one of the rare strains containing an F-plasmid and that this plasmid had been lost in the threonine- leucine- double mutant (679-680). Text from "The Emergence of Bacterial Genetics" by T.D. Brock, p81. The basic experiment to demonstrate conjugation was as follows:
[G10-4]
- Start with 3 cultures of bacteria:
- strain A (thr-leu-) alone
- strain B (bio-met-thi-) alone
- a mixture of strain A and strain B
- Grow all 3 cultures in minimal media supplemented with the 5 nutrients (threonine, leucine, biotin, methionine and thiamine)
- Collect the cells, wash them in minimal media, and plate them on minimal media plates without any supplements.
This protocol selects for prototrophs. The only cells which can grow are cells that no longer have any nutrient requirements.
The results were as follows:
- plate 1 -- strain A alone -- no colonies were observed
- plate 2 -- strain B alone -- no colonies were observed
- plate 3 -- mixture of strain A and strain B -- 1 colony per 107 cells plated was observed.
Since no colonies grew on plates 1 or 2, the colonies on plate 3 could not have arisen as a result of reversion. However, the frequency with which colonies were seen on plate 3 explains why Lederberg's initial experiment was unsuccessful. This frequency is the same as the natural mutation rate. Thus any experiments with single mutants is unable to distinguish between prototroph recovery due to reversion and prototroph recovery due to any other reason.
Although the experiment seems convincing, a number of alternative explanations need to be excluded:
Could the cells have been transformed?
Might cells of one strain have lysed allowing their DNA to be taken up by cells of the other strain?
This possibility was ruled out by using the FILTRATES of each culture. No recovery or prototrophs was found indicating that free DNA was not involved.
Could cross-feeding have been responsible?
Might cells of one strain have secreted metabolic intermediates that were used for growth by the other?
This possibility was ruled out by the U-TUBE EXPERIMENT. This experiment demonstrated that physical contact between the cells was required.
Tom Brock in "The Emergence of Bacterial Genetics" lists three reasons why Lederberg's "simple experiment was considered so brilliant":
- It represented the first use of conditional mutants to select against the parental type.
- The mutants were double mutants so reversion artefacts were avoided.
- The prototrophic recovery technique had enormous sensistivity.
Once he had demonstrated the existence of genetic mating in bacteria (E. coli), Lederberg thought that he would be able to use the technique to construct a complete genetic map of E. coli. He soon ran into difficulties interpreting the results. Fundamentally, the problem was that he did not really know or understand the real mechanism underlying mating in bacteria. That understanding was provided by the work of Bill Hayes.
BILL HAYES & THE DISCOVERY OF FERTILITY
Hayes studied the kinetics of bacterial mating not just the end results of mating to develop an insight into the process. Just like Lederberg, he used a streptomycin selection procedure.
He did a series of mating experiments, all of which pointed to the conclusion that a successful mating depended on the continued viability of one parent only and that mating was unidirectional.
EXPT #1A.
Consider a mating experiment with following strains:
- strain A is met-strS
- strain B is thr-leu-strR
- grow cultures of both strains, mix the cultures, and plate on minimal media plates containing streptomycin.
In this experiment, prototrophic recombinants are recovered at a normal frequency.
EXPT #1B.
Consider the same experiment with the same two strains EXCEPT that the streptomycin resistances are reversed:
- strain A is met-strR
- strain B is thr-leu-strS
- grow cultures of both strains, mix the cultures, and plate on minimal media plates containing streptomycin
This time, no prototrophs are recovered -- this is a sterile cross.
Comparing the results of these two experiments, it seems that the continued viability of strain B is essential for successful recovery of prototrophs.
EXPT #2.
In this experiment, both strain A and strain B are strS.
Streptomycin is used to kill one of the two strains. Treatment with streptomycin does not cause the cell to lyse. Streptomycin treated cells are able to carry out conjugation for a short while after treatment.
- If strain A is treated with streptomycin prior to mixing the two cultures, then normal prototrophic recovery is observed.
- If strain B is treated with streptomycin prior to mixing the two cultures, then a sterile cross results.
Once again, the continued viability of strain B is essential.
These experimental observations led to the suggestion that strain A was a DONOR strain and that strain B was a RECIPIENT strain.
Fertility
It was soon shown that FERTILITY, the ability to act as a donor, was a genetic trait.
We denote donor strains as F+ and recipient strains as F-. We can summarize the results of bacterial crosses between F+ and F- strains as follows:
F+ x F- This cross is FERTILE.
Progeny are F+
Transfer of fertility occurs at a much higher frequency than the transfer of bacterial markers which occurs at a low frequency.
F- x F- This cross is STERILE. F+ x F+ This cross is FERTILE and it occurs at a very low efficiency. The discovery of fertility in bacteria revolutionized the field of bacterial genetics and provided the tools that were necessary to move genes from one bacterial cell to another. Jim Watson heard about Hayes work at a small meeting on microbial genetics in Pallanza in 1952. In his book "The Double Helix", Watson describes the impact of Hayes' announcement of his discovery:
Bill's appearance was the sleeper of the three-day gathering: before his talk no one except Cavalli-Sforza knew he existed. As soon as he had finished his unassuming report, however, everyone in the audience knew that a bombshell had exploded in the world of Joshua Lederberg. In 1946, Joshua, then only twenty, burst upon the biological world by announcing that bacteria mated and showed recombination. Since then he had carried out such a prodigious number of pretty experiments that virtually no one except Cavalli dared work in the same field. Text from "The Double Helix" by J.D. Watson, chapter 20.
The Fertility Factor -- The F Plasmid
We now know that the fertility factor or F factor is a very large (94,500 bp) circular dsDNA plasmid; it is generally independent of the host chromosome.
The F factor controls its own replication. It has two origins of replication: oriV is the origin for bidirectional replication; oriS is the origin for unidirectional replication. The F factor also has genes that regulate DNA synthesis so that its copy number is kept at a low level; and, genes that regulate the partition into the daughter cells after E. coli divides.
The F factor is self-mobilizable -- it can transfer itself to other cells. This requires the functions coded by a large number of transfer (tra and trb) genes. Transfer initiates at a special origin of transfer, oriT, and proceeds via a rolling circle mechanism of replication. DNA is transfered through pili which form on the surface of a donor cell and can attach to the surface of a recipient cell. Pili cannot attach to other donor cells due to the presence of the proteins coded by the traS and traT genes -- this phenomenon is called surface exclusion.
The transfer genes are all located within a 33.3 Kbp transfer region. The following table summarizes these genes and their functions:
FUNCTION GENES synthesis of pili traA, traL, traE, traK, traB, traV, traC, traW, traU, traF, traQ, traH, traG surface exclusion traS, traT stabilization of mating pairs traN, traG DNA transfer traM, traY (exonuclease),traD, traI (helicase), traZ (exonuclease) regulation finP, finO, traJ (positive regulator) The F factor also contains a number of Insertion Sequences (IS2, IS3a and IS3b) and the transposon Tn1000 (also known as gamma-delta).
View the Genbank entry for Plasmid F, complete sequence
Transfer of F-factors
F factors transfer themselves from one cell to another. oriT is nicked by TraY and one of the 2 strands of the F plasmid is displaced as the TraI helicase unwinds it permitting it to be transferred to the recipient cell through the F-pilus. The transferred strand is covalently linked to the TraD protein during transfer. Once it has been transferred, the strand circularizes and second strand synthesis takes place.
Transfer of chromosomal DNA -- I
The progeny of an F+ x F- mating receive a complete copy of F with a very high frequency. Hence the progeny are also F+ or FERTILE. However, this does not explain how chromosomal markers can be transferred during a bacterial mating.
Chromosomal markers are transferred at a relatively low frequency -- much lower than the frequency with which fertility is transferred. How are chromosomal markers transferred?
The answer comes from another discovery made both by Bill Hayes and by Luigi Luca Cavalli-Sforza.
Hfr strains
Hayes and Cavalli-Sforza both discovered strains of E. coli that transferred chromosomal markers at a much higher frequency than normal. They called these HIGH FREQUENCY of RECOMBINATION strains or Hfr strains.
We can compare the behaviour of an Hfr strain with an F+ strain during a bacterial mating:
F+ x F- Progeny are F+
Low frequency of recombinants.
Hfr x F- Progeny are F-
High frequency of recombinants.
Understanding this behaviour of Hfr strains in constrast to that of F+ strains came from:
- the interrupted mating experiment -- by Elie Wollman & François Jacob (see below)
- an insight of Allan Campbell
Campbell's suggestion was that both E. coli and the F plasmid were circular molecules and that recombination between them would generate a single larger circular molecule.
Now, if conjugational transfer were to start while the F plasmid was integrated, not only would the F-DNA be transferred but the chromosomal DNA into which it had integrated would also be transferred.
Transfer of such a large molecule would take a long time. The probability of an entire chromosome of E. coli being transferred would be extremely low. Furthermore, since oriT is in the middle of the integrated F-DNA, and since transfer is a unidirectional process, transfer of the F-DNA would not be complete until the entire bacterial chromosome had been transferred. Thus, the progeny of an Hfr x F- cross would almost always be F-.
However, since a large amount of bacterial DNA would be transferred during an Hfr x F- cross, there would be a high probability that recombination between the transferred chromosomal DNA and the recipient chromosomal DNA would occur. If the donor and recipient strains could be distinguished genetically, then the results of this recombination could be observed.
Transfer of chromosomal DNA -- II
We can distinguish between the Hfr state and an Hfr strain. Once recombination between the F plasmid and the host chromosome occurs, the cell is in an Hfr state. A plasmid that can reversibly integrate into the host chromosome is called an episome.
Integration of F plasmids can occur at many places. The F plasmid contains mobile genetic elements (3 Insertion Sequences and a Transposon) which also occur in the E. coli chromsome. Recombination between a chromosomal element and an F-plasmid element generates a strain in the Hfr state. Bacterial markers that are close to the site of F plasmid integration are transferred at a very high frequency.
Recombination between an F plasmid and the bacterial chromosome is readily reversible. Under normal circumstances, the F plasmid will excise itself before conjugational transfer occurs. Hfr strains were identified because, in essence, the F plasmid got stuck in the integrated Hfr state.
Transfer of bacterial markers in a typical conjugation experiment occurs because in any population of F+ cells, a small percentage will be in the integrated state. If conjugation occurs while the cell is in this state, there will be a high frequency of recombinants but the progeny will mostly be F-.
The Interrupted Mating Experiment
The mechanism of transfer of bacterial markers was demonstrated by the interrupted mating experiment of Elie Wollman and François Jacob.
Wollman adopted the idea of using a kitchen blender to disrupt the cell pairs that form during conjugation from the experiment of Hershey and Chase. The experimental design was simple:
- Mix donor and recipient cells.
- Incubate to allow conjugation to get started
- At time t, blend the culture in the kitchen blender. This disrupts the cell pairs but does not break the individual cells.
- Plate recipient cells (use streptomycin selection).
- Screen for recombinant markers.
The actual cross used in this experiment was the following:
HfrH(aziRtonRlac+gal+strS)
x
F-(aziStonSlac-gal-strR)
HfrH was the Hayes Hfr strain. The results of this experiment showed that the bacterial markers were transferred in the order :
- azide resistance (actually, a mutation in the secA gene - normally involved in protein secretion)
- bacteriophage T1 resistance (a mutation in the fhuA gene - codes for the outer membrane receptor for ferrichrome, colicin M, and phages T1, T5 and phi80)
- lactose metabolism
- galactose metabolism
By using different Hfr strains -- in which F has integrated in different places -- it became both conceptually and practically possible to map genes in E. coli simply by measuring and comparing time of transfer.
Transfer of chromosomal DNA -- III
As we have noted, recombination between an F plasmid and the bacterial chromosome is readily reversible. Normally this process is quite precise, i.e. the F plasmid excises correctly.
Occasionally, though, this does not happen. Sometimes the F plasmid will excise incorrectly and, in doing so, it will take a small section of bacterial chromosome with it. The resulting F plasmid carries all of the genetic functions that it needs for conjugation but in addition it now carries a very specific bacterial marker. These type of modified F plasmids are denoted F' (F prime) factors.
[G10-3d]
F' factors are FERTILE. They transfer fertility at a normal frequency and they transfer some chromosomal markers or loci -- those that are now contained on the F plasmid -- at a similar high frequency. They transfer most chromosomal markers at the usual low frequency.
F' factors were discovered by Edward Adelberg. Their discovery was described by Elie Wollman to Tom Brock in "The Emergence of Bacterial Genetics" (p104):
"Adelberg had brought back to Berkeley some of our Hfr strains. I spent the year 1958-59 in Berkeley -- finishing the writing of our book. Once Ed Adelberg came to me telling me that one of the Hfr strains had changed: the frequency of recombinants was less than expected, but all were donors of intermediate frequency. I suggested that, by comparison with HFT (Hfr) phage the sex factor had left its site accompanied by neighbouring genetic fragments. This was verified experimentally. Lwoff, who had come to visit, brought the news back to François Jacob who immediately used it for making partial Lac diploids. This is the history of F prime factors" F' factors were extremely important in the early days of molecular biology. They were, in fact, the first cloning vectors -- the conceptual groundwork for all modern cloning was provided by the use of F' factors.
F' factors have also been used to map the E. coli chromosome.
The following two methods can be used for isolating F' factors.
- Select for early transfer of a marker which is normally transferred late during normal conjugation.
- Select for stable transfer of a marker using a recA- recipient strain which is defective in recombination.
In both cases, a second mating will confirm that the marker must reside on an F' factor.
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