1. Bacterial conjugation is the transfer of genetic material (conjugative plasmid) between bacteria through direct cell to cell contact, or through a bridge-like connection between the two cells. conjugation is a mechanism of horizontal gene transfer.

2.  The prototype for conjugative plasmids is the F-plasmid, also called the F-factor.  The F-plasmid is an episome (a plasmid that can integrate itself into the bacterial chromosome by homologous recombination) of about 100 kb length.

3.  It carries its own origin of replication, the oriV, as well as an origin of transfer, or oriT.  There can only be one copy of the F-plasmid in a given bacterium, either free or integrated.  The host bacterium is called F-positive or F-plus (denoted F+). Strains that lack F plasmids are called F-negative or F-minus (F-) (received F).

4.  Among other genetic information, the F-plasmid carries a tra and a trb locus, which together are about 33 kb long and consist of about 40 genes.  The tra locus includes the pilin gene and regulatory genes, which together form pili on the cell surface, polymeric proteins that can attach themselves to the surface of F- bacteria and initiate the conjugation.

6.  When conjugation is initiated, a relaxase enzyme creates a nick in one plasmid DNA strand at the origin of transfer, or oriT.nick  The relaxase may work alone or in a complex of over a dozen proteins, known collectively as a relaxosome.  In the F-plasmid system, the relaxase enzyme is called TraI and the relaxosome consists of TraI, TraY, TraM, and the integrated host factor, IHF.

7.  The transferred, or T-strand, is unwound from the duplex plasmid and transferred into the recipient bacterium in a 5'-terminus to 3'-terminus direction.  The remaining strand is replicated, either independent of conjugative action beginning at the oriV) or in concert with conjugation (conjugative replication similar to the rolling circle).rolling circle

9.  If the F-plasmid becomes integrated into the host genome, donor chromosomal DNA may be transferred along with plasmid DNA and the cell is called Hfr.  The certain amount of chromosomal DNA that is transferred depends on how long the bacteria remain in contact; for common laboratory strains of E. coli the transfer of the entire bacterial chromosome takes about 100 minutes. The transferred DNA can be integrated into the recipient genome via homologous recombination.

10.  Some strains of bacteria with an integrated F- plasmid can be isolated and grown in pure culture. Because such strains transfer chromosomal genes very efficiently, they are called Hfr (high frequency of recombination). 10

11.  F plasmid recombine to the chromosome by homologous recombination. (How?)  F plasmid carry two copies of IS3, one copy of IS2 and one copy of Tn1000.  The chromosome has about six copies of IS2, five copies of IS3 and multiple copies of Tn1000.  These homologous sites can be used as a source of homologous recombination. 11

12.  F´ are formed from Hfr strains.  The F plasmid that is integrated into the chromosome can come back.  The F factor can come back out with only F plasmid reforming F+.  The F factor can come back out and carry chromosomal DNA either from left or right of the original insertion these are known as type I F prime. 12

13.  If the F´ carry genes that were transferred early by the starting Hfr strain then it’s a Type IA F´,  If the F´ carry genes that were transferred late by the starting Hfr strain then it’s a Type IB F´,  Its possible for the F factor to carry genes from both sides of the original chromosome and these are called Type IIF´ 13

14.  Merodiploid ( two copies of the gene into the same cell) 14

15.  Mapping genes 15 A B C D

17. Transduction:  Is the process of moving a piece of chromosome (DNA ) from one cell to another using a bacteriophage to carry DNA.  First described by Zinder and Lederburg in 1952.

18.  There are two types of transducing phage 1- Generalized: capable of moving any piece of DNA from one cell to another. 2- specialized: move the same piece of DNA every time.

20.  P1 has a double stranded DNA (90kb).  P1 infect E.coli and package DNA into a phage head from a specific site on the P1 DNA known as pac site, once the DNA is inserted into the phage head, a p1tail is added to make a complete phage that is capable of infecting another E.coli cell.

21.  Specialized transducing phage carrying a defined region of the chromosome.  Unlike generalized transduing phage specialized transducing phage carry chromsomal DNA in addition to phage DNA.  Example is λ.

22.  λ recombine with the bacterial chromosome using attB site by site specific recombination.

23. Identifying transduced bacteria  Screening: examining each individual bacterium is known as screening.  Selection: bacteria with specific characteristics or phenotype can be selected by growth or non growth on specified media. A- Lethal selection B- non lethal selection

24. 1- Mapping the orders of the gene. 3- strain construction 4- Localized mutagenesis. 5- Making merodiploides using specialized transduction. 6- Moving mutations from plasmids to specialized transducing phage to the chromosome.