1. Bacterial Physiology (Micr430)
Lecture 12
Bacterial Genetics
(Based on Snyder and Champness Book)

2. Definitions you should know
Conjugation
Transformation
Transduction
Merodiploid
Plasmid
cis-acting vs trans-acting

3. PLASMIDS
A plasmid that can mediate its own transfer to a new strain - conjugative plasmid; otherwise it’s nonconjugative.
Plasmid partitioning - a process that ensures proper distribution of replicated plasmids into daughter cells.
Two plasmids belonging to the same incompatibility group cannot be maintained stably.

4. PLASMIDS
Many plasmids harbor resistance (R) factors that confer resistance to antibiotics.
Most plasmids are circular; Streptomyces have linear plasmids
Plasmid replication:
Theta type
rolling circle

5. CONJUGATION F factor is the prototype conjugative plasmid.
F factor has genes coding for sex pili formation;
Presence of sex pili is correlated with the ability of the cell to serve as a donor of genetic material
oriT is origin of transfer

8. Hfr formation
F factor can exist autonomously in a cell as a plasmid or can integrate into the bacterial chromosome.
A cell that contains an integrated F factor is called Hfr cell (high frequency of recombination)
An integrated F factor still has transfer functions; a conjugation with an F- strain will transfer host DNA
Host genetic map can be obtained

10. TRANSFORMATION
Natural Transformation occurs in many bacterial genera including Streptococcus, Haemophilus, Neisseria, Xanthomonas, Rhizobium, Bacillus and Staphylococcus.
Competence is defined as a physiological state that permits a cell to take up transforming DNA and be genetically changed by it.

11. Natural transformation
Natural competence discovery - the Griffith experiment

12. Gram positive transformation
In Streptococcus pneumoniae, the competence state is transient and persists for only a short period of time during late exponential phase.
It is induced by competence-stimulating peptide (CSP); binding of this activator protein to receptors on the plasma membrane triggers synthesis of many new proteins
Cells develop capacity to bind DNA molecules

13. Gram negative transformation
Neisseria gonorrheae and Haemophilus influenzae are naturally competent, but only during stationary phase.
In Neisseria gonorrheae, DNA first binds nonspecifically to cell surface. Then an unknown protein or protein complex recognizes a specific sequence (GCCGTCTGAA) within the bound DNA
Transport of the tagged DNA across the outer membrane involves a type IV pilus structure made up by PilE protein.

14. Gram negative transformation
Once in the periplasm, donor DNA needs help from ComL and Tpc to get through murein layer.
An inner membrane protein ComA helps to transport donor DNA across the inner membrane

15. Forced Competence
Organisms not considered naturally transformable (E. coli and S. typhimurium) can be transformed under special laboratory conditions
These include CaCl2 treatment of cells or electrical shock (electroporation) to transfer plasmid DNA into the cells

16. TRANSDUCTION There are two types of transduction:
Generalized: phage-mediated transfer of any portion of a donor cell’s genome into a second host.
Specialized transduction is mediated by bacteriophage that integrate into a specific site on the bacterial chromosome; this specificity limits transfer of genetic material to host markers that lie within immediate vicinity of this site.

17. RECOMBINATION There are two types of recombinations:
General or RecA-dependent recombination requires a large region of homology between donor and recipient DNAs.
Occurs more frequent
RecA-independent or nonhomologous recombination requires very little sequence homology.
Occurs rarely
Requires special proteins

18. General Recombination: requirements
Identical or very similar sequences in the cross-over region
Complementary base pairing between double-stranded DNA molecules
Recombination enzymes
Heteroduplex formation

19. Models of recombination
Holliday double-strand invasion model

20. Migration of Holliday junctions

21. Single-strand
Invasion
Model

22. Transposable elements
Transposable elements are discrete sequences of DNA that encode functions to catalyze the movement of the transposable element from one DNA site to a second.
Two types of transposition:
Replicative: involving both replication and recombination with a copy of the element remaining at the original site
Conservative: no replication involved; the element is simply moved to a new location

23. Transposable elements classes
Insertion sequences (IS), encoding no function other than transposition, with inverted repeats at either of its ends
Composite transposons – formed by two IS elements of the same type, bracketing other genes such as drug resistance

24. Composite transposons