Chapter 14 The Prokaryotic Chromosome: Genetic Analysis in Bacteria

1. Chapter 14The 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 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

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 Fig. 14.4 b

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

21. Gene Transfer in Bacteria 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 Fig. 14.10

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

25. The F plasmid and conjugation Fig. 14.12 a

26. The process of conjugation

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 Fig. 14.13

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

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

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 Fig. 14.16 a, b

31. Partial genetic map of the E. coli chromosome Fig. 14.16 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 Fig. 14.17

34. Different classes of crossovers: quadruple crossover is least frequent Fig. 14.17 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 Fig. 14.18 a, b

37. Complementation testing using F’ plasmids • Creation of a heterogenote • Phenotype of partial diploid establishes whether mutations complement each other or not Fig. 14.18 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 100-200 new phage particles • Cells lyse and phage infect other cells • Lysate is population of phage after lytic cyle is complete

39. Generalized transduction Fig. 14.19

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. Fig. 14.20

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

43. Recombination between att sites on the phage and bacterial chromosomes allows integration of the prophage Fig. 14.22 b

44. Errors in prophage excision produce specialized transducing phage • Adjacent genes are included in circular phage DNA that forms after excision Fig. 14.22 c