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

Prok Genome SizeBacteria Size (Mbp)Escherichia coli 4.64Bacillus subtilis 4.20Streptococcus pyrogenes 1.85Mycobacterium genitalium 0.58

Archaea Size (Mbp)Methanococcus jannaschii 1.66Sulfolobus solfactaricus 2.25Pyrococcus furiosus 1.75

Mega = 106

3

Euk Genome SizeOrganism MbpHomo sapiens 3,000Drosophilia melanogaster 165Plasmodium falciparum 23Saccharomyces cerevisiae 12.07

Eukaryotes also have Mitochondrial DNAChloroplast DNA

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 ~ 107-108 mutations/12 hours

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

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

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

8

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

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

Transforming Principle Experiment 1928

Fred Griffith Streptococcus pneumoniae

SmoothVirulent

RoughAvirulent

α-hemolysisof RBCs

Blood Agar Plate

No capsule!

12

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

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

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

ConjugationBacteria 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

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

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).

19

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

More on phage during the virus lectures

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

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

Lac Operon

Escherichia coli

Transcriptional Control in Prokaryotes

25

Disaccharide

Monosaccharide galactosidase

Genes in the lac operon are designed to breakdown and import lactose

26

Three lactose metabolizing enzymes are underthe control of one promoter

27

Repressor protein binds to operator

No transcription

No need to make -gal & other enzymeswhen lactose is not present

Repressor is producedconstitutively

Negative Control

28

allolactoseA small amount isconverted to allolactose

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

30

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

[glucose] [cAMP] Reduced transcription