(chapter 16)

A bacterium contains about 100 times more DNA than most of its bacteriophages. The DNA in a bacterium is found in the single main circular chromosome and also as small covalently closed circles (ccc) of DNA known as plasmids. One kind of plasmid is known as the sex element or F plasmid (F for 'fertility'), or  F factor or F element.

CONJUGATION

E. coli cells that carry an F element are known as F⁺ and such cells are found at low frequency among natural populations of E. coli. The F plasmid contains at least 25 tra genes, about 12 of which specify formation of the sex pili (singular pilus) or fimbriae. These long appendages extending from the surface of F⁺ cells are hollow cylinders with a central hole of about 2 nm.

Fig of F⁺ cell

Cells that possess F pili are sensitive to infection by single stranded RNA (ssRNA) phages. F^- cells lack pili and are insensitive to such RNA phages.

An F⁺ cell will ignore another F⁺ cell but will readily establish contact with an  F^- cell via an F pilus (only cells of opposite 'mating types' can 'mate'). Once contact is made the pilus is believed to become modified and to serve as a protoplasmic channel between the two cells and it is often referred to as a conjugation tube.

Usually only the genetic element to be transferred is the  F plasmid. The F plasmid is thought to replicate by the rolling circle mechanism. One strand of the  F factor is nicked at the origin and the 5' end of one strand is drawn into the recipient F^- cell where it is copied. Meanwhile the second strand remains in the donor and serves as a rolling template for its own replication. This process takes about 1 minute. (Fig 16.10)

Fig: F⁺ x F^- conjugation

In the transfer process the origin is always transferred first, followed by the remainder of the F factor. When the complete F factor has been transferred, the F^- cell becomes an F⁺ cell as a result of transfer of the F factor genes. Repeated conjugation between F⁺ and F^- cells leads to an increase in the proportion of F⁺ cells in the population. In this way virtually every cell in a mixed population will become F⁺. Cells can sometimes be induced to lose the F factor by chemical treatment or repeated subculturing.

Summary

Some E. coli cells possess a plasmid, called the F factor, that is required for mating. E. coli cells carrying the F factor are designated F⁺ and those without it are F^- . The F⁺ cells (donors) can mate with F^- cells (recipients) in a process called conjugation, which leads to the one-way transfer of a copy of the F factor from donor to recipient during replication of the F factor. As a result, both donor and recipient are F⁺. None of the bacterial chromosome is transferred during F⁺ x F^- conjugation.

High-Frequency Recombination Strains

In about 1/10⁵ F⁺ cells, the F plasmid becomes integrated into the main bacterial chromosome by a single crossover event. An F⁺ cell which has its F plasmid integrated in this way is known as an Hfr (for high-frequency recombination) strain. The integrated F plasmid is replicated along with the main chromosome and the integration process is similar to that for l prophages. The Hfr strains also possess F-pili and undergo conjugation with F^- cells. (Fig 16.11)

In Hfr x F^- matings, the 5' leading strand starts at a nick in the DNA at the replication origin of the F element. Part of the F factor first moves into the recipient cell where it is copied. The attached bacterial chromosome also begins to be transferred to the recipient. Depending on the position of the replication origin at the time of integration, this usually means that only some of the F element is transferred as the remainder of the F element is at the other end of the bacterial chromosome. For all of the F element to be transferred, the entire bacterial chromosome would have to be transferred as well. This happens only rarely as transfer normally breaks before completion. Therefore the recipient usually remains F^-, but is diploid for some of its genome. This organism is known as a partial diploid, merodiploid or merozygote.

If there are differences between genes on the donor chromosome and those of the recipient, recombination in such partial diploids can lead to the formation of recombinant classes. In a double crossover event, a segment of donor DNA is exchanged for the homologous segment of host DNA. (As the bacterial chromosome is circular, a single crossover would result in a non-viable linear molecule). The remaining fragments of DNA are lost during subsequent cell division and all daughter cells are haploid. This high frequency of recombinants produced as a result of Hfr x F^- conjugations that gave these integrated strains their names long before the molecular basis of conjugation was understood.

These early experiments were carried out by Hayes in London and Jacob and Wollman in Paris in the 1950s. The early results were puzzling, but the realisation that conjugation involved transfer of a linear Hfr chromosome to the F^- cell was established genetically by Jacob & Wollman in their interrupted mating experiments.

Interrupted Mating

Cells of Hayes= first Hfr strain called Hfr H were mixed with cells of an F^- strain carrying a number of mutations:

threonine requirement (thr^- )

leucine requirement (leu^- )

azide resistance (azi^R)

resistance to bacteriophage T1 (ton^R)

inability to utilise lactose as a carbon source (lac^- )

inability to utilise galactose as a carbon source (gal^- )

streptomycin resistance (str^R)

Hfr                    x                      F^-

thr⁺  leu⁺  azi^Ston^R             X                   thr^-  leu^-  azi^Rton^R

lac⁺ gal⁺ str^S                                             lac^- gal^- str^R

The separated cells were then plated on MM + streptomycin. This medium selected for thr⁺, leu⁺, str^R recombinants. The thr⁺ and leu⁺ genes would come from the Hfr parent, while str^R would come from the F^- parent. No such recombinants were found when conjugation was interrupted under 8 minutes. Beginning at about 8 min after mixing, a few thr⁺, leu⁺, str^R colonies grew up on the plates after incubation. Samples taken after longer time intervals produced progressively more recombinants and these were tested out on different media to assess the other markers.

Up until 9 min after mixing, no other Hfr markers appeared. At 9 min the azide sensitivity (azi^S) appeared and increased in frequency in subsequent samples until it peaked at about 90% of the progeny cells. At about 10 min the bacteriophage T1 sensitivity marker (ton^S) appeared among recombinants. lac⁺ followed at about 16 min and gal⁺ at about 25 min. These results could be interpreted in terms of the order of genes being transferred through the conjugation tube. (Figs 16.14, 16.15)

Fig: Frequency of Hfr markers among recombinants

Fig: Transfer of Hfr markers

As transfer appears to be at an approximate constant rate we have a new way of mapping. The bacterial chromosome can be mapped by recombination frequencies with respect to time. Hence mapping is in minutes rather than map units. The E. coli map is normally presented in terms of minutes.

Different Hfr strains were subsequently isolated which had different properties, e.g. Hfr AB311 which transferred the markers in the reverse order:

thr⁺ leu⁺ azi^S ton^S lac⁺ gal⁺
 
 

Other Hfr strains transferred different regions of the bacterial chromosome, so it appeared that the F plasmid could integrate at a number of sites around the chromosome and in different orientations. By using these different Hfr strains a complete genetical map of the E. coli chromosome was constructed, which was found to be circular and is now 100 min long. That is to say it would take 100 min for the entire chromosome to be transferred through conjugation. (Rate of transfer would be 38600 bp min^-1 or ~ 600 bp sec^-1 on average, but the rate of transfer from mating couple to mating couple is not constant.) As you may know, E. coli cells can divide after 20 min, so Hayes remarked that in E. coli the sex act takes 4.5 times as long as the life cycle.

In most conjugation events, only about 1/5^th of the genome is transferred before the DNA being transferred breaks, so only rarely do F^- cells convert to Hfr or F⁺ types as only rarely is the entire bacterial chromosome transferred.

FN factors

Integration of the F element into the bacterial chromosome involves specific sequences of DNA about 1 kb long known as insertion sequences (IS). There are about 4 such sequences on the F plasmid and more than 20 on the E. coli chromosome. Pairing between homologous IS elements on the plasmid and the E. coli chromosome allows recombination to occur and a single cross over will allow integration of the plasmid to form an Hfr strain.

Fig: F plasmid insertion

Occasionally the F element excises to become a free F plasmid. The F plasmid is an example of an episome (a genetic element in E. coli that may behave as an autonomous unit replicating independently of the host chromosome, or as an integrated unit attached to the host chromosome and replicating with it e.g. phage l and sex factor F).

Excision may be precise - at the same IS sites as insertion - or may involve a different IS element and the F element may take some of the host chromosomal DNA with it. Since the F factor may integrate at one of many IS sites on the bacterial chromosome, different chromosomal segments may be picked up by incorrect excision of the plasmid. This chromosomal DNA may carry with it a marker, e.g. lac. The result is an F factor that also carries the lac genes of the host chromosome, and is known as an FN (F prime) plasmid - in this case FN (lac).

Fig: Formation of F' (lac)

The FN behaves as F⁺ in matings with F^- cells, except that recipients as well as being FN, will also be partially diploid for the lac region of the host chromosome, forming again a merodiploid. These merodiploids are reasonably stable and allow dominance relationships to be determined for alleles of E. coli genes. Merodiploids can be used for complementation studies (as trans-heterozygotes).

Recombination between FN and the main chromosome can also occur and this is called F-duction or sexduction. If more than 1 marker is transferred (sexduced) at the same time, then mapping can be performed.

To summarise conjugation:

The circular F factor can integrate into the circular bacterial chromosome by a single crossover event. Strains in which this has happened can conjugate with F^- strains, and transfer of the bacterial chromosome occurs. Strains carrying the integrated F factor are called Hfr (high-frequency recombination) strains. In Hfr x F^- matings, the chromosome is transferred in a one-way fashion from the Hfr cell to the F^- cell, beginning at a specific site called the origin (O). The further away a gene is from O, the later it is transferred to the F^-, and this is the basis for mapping genes by their times of entry into the F^- cell. Conjugation and interrupted mating allow mapping of large chromosome segments.
 
 

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