1. 1 Bacterial Genomes Remember no nucleus!! Bacterial chromosome - Large ds circular DNA molecule = haploid - E. coli has about 4,300 genes (~4.2 Mb) 100x more DNA than the average virus 1000x less DNA than eukaryotic cell - Chromosome is tightly coiled into dense body = nucleoid

2. 2 Prok Genome Size Bacteria Size (Mbp) Escherichia coli4.64 Bacillus subtilis4.20 Streptococcus pyrogenes1.85 Mycobacterium genitalium0.58 ArchaeaSize (Mbp) Methanococcus jannaschii 1.66 Sulfolobus solfactaricus2.25 Pyrococcus furiosus1.75 Mega = 10 6

3. 3 Euk Genome Size Organism Mbp Homo sapiens3,000 Drosophilia melanogaster 165 Plasmodium falciparum 23 Saccharomyces cerevisiae 12.07 Eukaryotes also have Mitochondrial DNA Chloroplast DNA

4. 4 - Bacteria divide by simple division = binary fission - Division proceeded by chromosome replication from single origin of replication - E. coli cells can divide every 20 min under optimal conditions - DNA molecules are identical except for mutations - Mutation rate ~1 mutation/chromosome/generation - With short generation time = lots of mutations ~ 10 7 -10 8 mutations/12 hours

5. 5 Extrachromosomal DNA - Many bacteria have extrachromosomal molecules of DNA = plasmids - Plasmids contain an average of ~10-50 genes - Cells can contain 1-100 plasmids Resistance (R) plasmids - Usually carry genes that detoxify antibiotics - Allows bacteria to be resistant (R) to drugs that would normally kill them - Also often contain genes for sex pilus = can be transferred by conjugation (F plasmids)

6. 6 Recombination in Bacteria - Bacteria are haploid, have only 1 copy of each gene on circular chromosome - There are mechanisms to introduce pieces of DNA from one cell to another to produce a partial diploid - Partial diploids, because usually only small pieces of DNA with only a few genes are transferred - The foreign DNA in a partial diploid can replace endogenous DNA in the chromosome by homologous recombination

7. 7 Genetic recombination - exchange of genes between two related chromosomes, forms new combinations of genes Involves crossover event between chromosomes Results in hybrid chromosomes Each now has properties of both original chromosomes In eukaryotes, occurs during meiosis Increases genetic diversity

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9. 9 Transformation - Bacteria take up naked foreign DNA from the environment - Consequences can be that mutant alleles are replaced with wildtype alleles or vice versa by homologous recombination=crossing over - Not all bacteria can be “naturally” transformed - Competence - Can create “competent cells” in the lab Can generate partial diploids in 3 different ways

10. 10 Types of Transfer of Genetic Material Genes can be passed from parental cell to progeny cell – vertical gene transfer Only method of transfer in higher eukaryotes (yeasts may be an exception) Also used by bacteria Bacteria can also undergo horizontal gene transfer Transfer of genetic material from one cell to another Can result in a recombination event Generates recombinant bacteria

11. 11 Transforming Principle Experiment 1928 Fred Griffith Streptococcus pneumoniae Smooth Virulent Rough Avirulent α-hemolysis of RBCs Blood Agar Plate No capsule!

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13. 13 The Transforming Principle R cells were “transformed” Something in the S cells transformed the R cells The standard assumption was that proteins were responsible

14. 14 How were the Bacteria Transformed? Rough colonies lacked functional gene for capsule production DNA containing functional gene from heat killed smooth bacteria taken up by rough bacteria Recombination event replaced defective capsule production gene with functional gene Once rough bacteria can now make capsule and are transformed to smooth colony virulent phenotype

15. 15 Conjugation General features - Transfer of genetic material between 2 bacteria that are temporally joined - The donor cell transfer DNA to the recipient cell - A sex pilus from the male initially joins the 2 cells via cytoplasmic bridge - “Maleness” is the ability to form a sex pilus and donate DNA - Maleness requires an F factor found either on the bacterial chromosome or on a plasmid

16. 16 Conjugation Bacteria can exchange genetic information through conjugation Requires presence of fertility plasmid (F plasmid) Contains genes required for production of sex pilus Can connect two bacteria with pilus, one with plasmid (F+) one without (F-) F plasmid transferred Converts F- to F+

17. 17 How conjugation works - F factor is an episome = can exist as an autonomous or integrated (into bacterial chromosome) plasmid - The F factor contains ~25 genes mostly used to make the sex pilus - Cells with the F factor = F + = conjugation donors - Cells without the F factor = F - = conjugation recipients - When F + and F - meet, F + donates the F factor to F - cell and converts it to F +

18. 18 F factor on plasmid - the plasmid is only transferred during mating F factor integrated into the bacteria chromosome - occurs at a specific site - the resulting cell is Hfr (High frequency of recombination).

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20. 20 Transduction - Occurs when phage picks up piece of degraded bacterial chromosome by mistake - The bacterial DNA is transferred from one host to another by the phage during infection

21. 21 More on phage during the virus lectures

22. 22 In general, prokaryotic genes are organized (and Expressed) as operons An operon consists of: Several genes that encode enzymes under the control of a single promoter - usually all enzymes needed for a specific activity - all transcribed as one long mRNA - polycistronic mRNA - mRNA that contains that codes for more than one gene within the same mRNA transcript Regulation of Genes in Prokaryotes

23. 23 promoter region - site where RNA polymerase binds - binding to promoter is necessary for transcription of the mRNA that encodes the enzymes operator region - binding site between the promoter and first structural gene - acts as an “on-off” switch repressor protein - binds to the operator region - prevents transcription inducer molecule - binds to repressor & allows transcription

24. 24 Lac Operon Escherichia coli Transcriptional Control in Prokaryotes

25. 25 Disaccharide Monosaccharide  galactosidase Genes in the lac operon are designed to breakdown and import lactose

26. 26 Three lactose metabolizing enzymes are under the control of one promoter

27. 27 Repressor protein binds to operator No transcription No need to make  -gal & other enzymes when lactose is not present Repressor is produced constitutively Negative Control

28. 28 allolactose A small amount is converted to allolactose

29. 29 Positive Control of the Lac Operon - Activator protein CRP cAMP Receptor Protein -Concentration of glucose is low -cAMP accumulates -CRP/cAMP binds to the promoter -There’s a special sequence / binding location -Maximal rates of transcription occur -Synthesize a lot of  -gal & other enzymes

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31. 31 Positive Control – High Glucose - There is little cAMP - CRP can not be activated - E. coli prefers glucose If there’s plenty of glucose No reason for produce  -gal The lac operon is shut down in the presence of glucose

32. 32 [glucose]  [cAMP]  Reduced transcription