1. Chapter 14 The Prokaryotic Chromosome: Genetic Analysis in Bacteria

2. Outline of Chapter 14 General overview of bacteria
Range of sizes
Metabolic activity
How to grow them for study
The bacterial genome
Structure
Organization
Transcription
Replication
Evolution of large, circular chromosomes
Structure and function of small circular plasmids
Gene transfer in bacteria
Transformation
Conjugation
Transduction
A comprehensive example
Genetic tools to dissect bacterial chemotaxis

3. General overview of bacteria
One of the three major lineages of life
Eukaryotes – organisms whose cells have encased nuclei
Prokaryotes – lack a nuclear membrane
Archea
1996 complete genome of Methanococcus jannaschii sequenced
More than 50% of genes completely different than bacteria and eukaryotes
Of those that are similar, genes for replication, transcription, and translation are same as eukaryotes
Genes for survival in unusual habitats similar to some bacteria
Bacteria
Similar genome structure, morphology, and mechanisms of gene transfer to archea
Evolutionary biologist believe earliest single celled organism, probably prokaryote existed 3.5 billion years ago

4. A family tree of living organisms
Figure 14.1
Fig. 14.1

5. Diversity of bacteria Outnumber all other organisms on Earth
10,000 species identified
Smallest – 200 nanometers in diameter
Largest – 500 micrometers in length (10 billion times larger than the smallest bacteria)
Habitats range from land, aquatic, to parasitic
Remarkable metabolic diversity allows them to live almost anywhere

6. Common features of bacteria
Lack defined nuclear membrane
Lack membrane bound organelles
Chromosomes fold to form a nucleoid body
Membrane encloses cells with mesosome which serves as a source of new membranes during cell division
Most have a cell wall
Mucus like coating called a capsule
Many move by flagella

7. Table 14.1

8. Power of bacterial genetics is the potential to study rare events
Bacteria multiply rapidly
Liquid media – E. coli grow to concentration of 109 cells per milliliter within a day
Agar media – single bacteria will multiply to 107 – 108 cells in less than a day
Most studies focus on E. coli
Inhabitant of intestines in warm blooded animals
Grows without oxygen
Strains in laboratory are not pathogenic
Prototrphic – makes all the enzymes it needs for amino acid and nucleotide synthesis
Grows on minimal media containing glucose as the only carbon source
Divides about once every hour in minimal media and every 20 minutes in enriched media
Rapid multiplication make it possible to observe very rare genetic events

9. The bacterial genome is composed of one circular chromosome
4-5 Mb long
Condenses by supercoiling and looping into a densely packed nucleoid body
Chromosomes replicate inside cell and cell divides by binary fission
Figure 14.4 b
Fig b

10. E. coli lysed to release chromosome
Figure 14.4 a
Fig a

11. How to find mutations in bacterial genes
Mutations affecting colony morphology
Mutations conferring resistance to antibiotics or bacteriophages
Mutations that create auxotrophs
Mutations affecting the ability of cells to break down and use complicated chemicals in the environment
Mutations in essential genes whose protein products are required under all conditions of growth

12. How to identify mutations by a genetic screen
Genetic screens provide a way to observe mutations that occur very rarely such as spontaneous mutations (1 in 106 to 1 in 108 cells)
Replica plating – simultaneous transfer of thousands of colonies from one plate to another
Treatments with mutagens – increase frequency of mutations
Enrichment procedures – increase the proportion of mutant cells by killing wild-type cells
Testing for visible mutants on a petri plate

13. Bacteria nomenclature
wild-type – ‘+’
mutant gene – ‘-’
three lower case, italicized letters – a gene (e.g., leu+ is wild type leucine gene)
The phenotype for a bacteria at a specific gene is written with a capital letter and no italics (e.g., Leu+ is a bacteria with that does not need leucine to grow, and Leu- is a bacteria that does need leucine to grow.)

14. Structure and organization of E. coli chromosome
4.6 million base pairs
open reading frames (ORFs)
90% of genome encodes protein (compare that to humans!)
4288 genes, 40% of which we do not know what they do.
almost no repeated DNA
427 genes have a transport function, other classes also identified
bacteriophage sequences found in 8 places (must have been invaded by viruses at least 8 times during history.

15. Insertion sequences dot the E. coli chromosome
Transposable elements place DNA sequences at various locations in the genome.
Geneticists use transposable elements to insert DNA at various locations in bacterial genomes.
If you were to insert a piece of DNA into a bacterial genome using a transposable element, can you think of a molecular method that you could use to find out which gene you inserted the DNA into?

16. Transposable elements in bacteria
Figure 14.6 a, b,c
Fig. 14.6

17. Transcription in bacteria
Transcription machinery moves clockwise
Different strands code for different genes
Several genes may be transcribed in one segment
RNA polymerase may transcribe adjacent genes at the same time in a counterclockwise direction
Highly transcribed genes generally oriented in direction of replication fork movement

18. DNA replication in E. coli
Figure 14.7
Fig. 14.7

19. Plasmids: smaller circles of DNA that do not carry essential genes
Plasmids vary in size ranging from 1kb – 3 Mb.
Plasmids can carry genes that confer resistance to antibiotics and toxic substances.
Plasmids are not needed for reproduction or normal growth, but they can be beneficial.
Plasmids can carry genes from one bacteria to another. Bacteria can thus become resistant to a drug, put the resistance gene in the plasmid, and transfer it to other bacteria. This transfer of plasmid DNA can even occur across species.

20. Some plasmids contain multiple antibiotic resistance genes
Figure 14.8 b

21. Gene Transfer in Bacteria
Figure 14.9
Fig. 14.9

22. Transformation
Fragments of donor DNA enter the recipient and alter its genotype
Natural transformation – recipient cell has enzymatic machinery for DNA import
Artificial transformation – damage to recipient cell walls allows donor DNA to enter cells
Treat cells by suspending in calcium at cold temperatures
Electroporation – mix donor DNA with recipient bacteria and subject to very brief high-voltage shock

23. Mechanism of natural transformation
Figure 14.10
Fig

24. Conjugation – A type of gene transfer requiring cell-to-cell contact
Figure 14.11
Fig

25. The F plasmid and conjugation
Figure a
Fig a

26. The process of conjugation
Figure b

27. The F plasmid occasionally integrates into the E. coli chromosome
Hfr cells have integrated part of chromosome
Episomes – plasmids that can integrate into host chromosome
Exconjugate – recipient cell with integrated DNA
Integrated plasmid can initiate DNA transfer by conjugation, but may take some of bacterial chromosome as well
Figure 14.13
Fig

28. Gene transfer in a mating between Hfr donor and F- recipient
Figure 14.14
Fig

29. Mapping genes in Hfr and F- crosses by interrupted mating experiments
Figure 14.15

30. Interrupted mating studies confirm bacterial chromosome is a circle
Cross between Hfr and F-
The F plasmid integrates into different locations in different orientations into the circular donor chromosome
Figure a, b
Fig a, b

31. Partial genetic map of the E. coli chromosome
Figure c
Fig c

32. Recombination analysis improves accuracy of map
Interupted mating experiments accurate to only 2 minutes
Frequency of recombination between genes is more accurate
Start by considering only exconjugates that have all of the genes to be mapped (select for the last gene transferred)
Living cells must have even number of crossovers
Consider as a three-point cross

33. Mapping genes using a three-point cross
Figure a, b
Fig

34. Different classes of crossovers: quadruple crossover is least frequent
Figure c
Fig c

35. F’ plasmids can be used for complementation studies
F’ plasmids replicate as discrete circles of DNA inside host cells.
Transferred in same manner as F plasmids
A few chromosomal genes will always be transferred as part of the F’ plasmid
Can create partial diploids
Merozygotes – partial diploids in which two gene copies are identical
Heterogenotes – partial dipoids carrying different alleles of the same gene

36. F’ plasmid formation and transfer
Figure a, b
Fig a, b

37. Complementation testing using F’ plasmids
Creation of a heterogenote
Phenotype of partial diploid establishes whether mutations complement each other or not
Figure c
Fig c

38. Transduction: Gene transfer via bactgeriophages
Bacteriophages
Widely distributed in nature
Infect, multiply, and kill bacterial host cells
Transduction - may incorporate some of bacterial chromosome into its own chromosome and transfer it to other cells
Bacteriophage particles are produced by the lytic cycle
Phage inject DNA into cell
Phage DNA expresses its genes in host cell and replicate
Reassemble into new phage particles
Cells lyse and phage infect other cells
Lysate is population of phage after lytic cyle is complete

39. Generalized transduction
Figure 14.19
Fig

40. Mapping genes by generalized transduction
Frequency of recombination between genes
P1 bacteriophage often used for mapping
90kb can be constransduced corresponding to about 2% recombination or 2 minutes
First find approximate location of gene by mating mutant strain to different Hfr strains
P1 transduction then used to map to specific location

41. Figure 14.20
Fig

42. Temperate phage can integrate into bacterial genome through lysogenic cycle creating a prophage
Figure 14.21
Fig

43. Recombination between att sites on the phage and bacterial chromosomes allows integration of the prophage
Figure b
Fig b

44. Errors in prophage excision produce specialized transducing phage
Adjacent genes are included in circular phage DNA that forms after excision
Figure c
Fig c