1. Chapter 9 Genetics of Bacteria and Their Viruses Jones and Bartlett Publishers © 2005

2. Mobile DNA Plasmids Insertion sequences and transposons Integrons R plasmids

3. Mobile DNA Plasmids – can be circular or linear. Not essential for bacterial cells Can be 1-2 copies or up to 50 copies per host cell. Range in size from a few kb to a few hundred kb. Plasmids depend on host DNA replication enzymes.

4. F plasmid: Conjugation In E. coli, a large plasmid can be transferred between cells (distinct from fission and replication). F factor (F for fertility) Low copy number, 100 kb F + vs F - Pilus (pl., pili), a tube-like structure that connects two cells undergoing conjugation. 20 genes necessary for pilus assembly and DNA transfer.

5. Conjugation in E. coli between a male (F + ) and a female (F - ) cell

6. Insertion sequences and transposons Transposable elements are responsible for gene mobilization. How they work to move genes, and act as mutagens will be discussed later. In bacteria, the smallest transposable elements are insertion sequences, or IS elements. IS elements are 1-3 kb and encode a transposase protein and a few related proteins.

7. Tn5 (a composite transposon) has IS elements at each end (IS50 left and IS50 right) and drug resistance genes in the middle IS elements are the simplest known fully functional transposons (they have one gene flanked by inverted repeats). Neo, ble and str refer to the antibiotics neomycin, bleomycin and streptomycin.

8. Mobilization of non-conjugative plasmids Transposons can move between host DNA of a bacterium and plasmids in the cell. E. coli contains 1-6 genomic copies of each of 6 naturally occurring IS elements. In cells with a particular IS element, 20- 60% contain the IS element in plasmids. These IS elements in plasmids can serve as recombination sites (homology).

9. Recombination between 2 transposons leads to the fusion of 2 plasmid circles creating a larger circle Cointegrate = a composite plasmid. In this way, nonconjugative plasmids can catch a ride with a conjugative plasmid.

10. Site-specific recombination involves 2 short target sequences and an enzyme capable of recombining them Cre recombinase recognizes the loxP site.

11. Site-specific recombination and antibiotic resistance Multiple-antibiotic resistance units can be assembled to form integron. An integron is a DNA element which encodes a site-specific recombinase as well as a recognition sequence that allows other similar elements to join the integron. A cassette is a circular antibiotic-resistance- coding region flanked by a recognition region.

12. One or more short circular DNA molecules may be inserted in a DNA target by site-specific recombination

13. R plasmids Some conjugative plasmids in nature have accumulated different transposons containing multiple antibiotic-resistance genes (or transposons with integrons with the resistance genes). These plasmids with multiple resistance genes are called R plasmids, and cause clinical trouble.

14. Bacterial Traits Bacteria are small: about 1/1000 th the volume of eukaryotic cells. Escherichia coli are about 1  m long by 0.5  m in diameter. With favorable temperature and nutrients, bacteria can divide every 20-60 minutes. They are haploid, with only one allele at each locus. They make clones in culture.

15. Bacterial Traits E. coli can grow either on solid media on a Petri plate, or in liquid culture. A single cell of E. coli that doubles every hour can make over 10 million cells in 24 hours. The colony will be visible on a plate, and will be a clone

16. Bacterial genetics Mutants of bacteria exist: –Unable to use certain nutrients –Auxotrophic strains (wild type are prototrophs and can grow on a minimal medium) –Sensitivity or resistance to drugs or phage. –Conditional mutants (temperature sensitivity). –Carbon source mutants

17. Types of genetic transfer in bacteria Transformation – does not require contact; one cell lyses, another takes up the DNA from the lysed cell. Conjugation – requires actual physical contact between the donor cell and the recipient. Transduction – involves a bacteriophage.

18. Genetic transformation can be used to create genetic maps in bacteria