2.9: bacteria

Question 1

what is a bacterium?

Answer 1

• small unicellular prokaryotes
• lacks a true nucleus and membrane-bound organelles

Question 2

what kind of DNA do bacteria have?

Answer 2

circular DNA

Question 3

what type of ribosomes do bacteria have?

Answer 3

70S

Question 4

how many oriR do bacteria have

Answer 4

single

Question 5

what are plasmids?

Answer 5

• extrachromosomal DNA
• replicate independently of chromosomal DNA
• involved in conjugation & antibiotic resistance

Question 6

describe the process of DNA replication (before binary fission)

Answer 6

1. DNA double helix unwinds and unzips @ oriR
2. replicates via semi-conservative model
3. bidirectional replication
4. circular DNA -> interlocking structure -> topoisomerase cuts, reseals 2 DNA molecules

Question 7

describe binary fission

Answer 7

1. bacterial chromosome is attached to plasma membrane @ oriC
2. replicates , 2nd oriC attaches to membrane adjacently
3. cell elongates by growing membrane, depositing cell wall material btwn 2 chromos
4. after growing, septum forms in btwn
5. septum fuses -> 2 identical daughter cells

Question 8

what is the significance of genetic variation?

Answer 8

• rapidly changing environment
• natural selection
• occur due to point mutations/genetic transfer

Question 9

what is conjugation?

Answer 9

• unidirectional transfer of F plasmid
• via direct contact
• from F+ -> F-

Question 10

what does the F plasmid contain

Answer 10

fertility factor
- enables sex pilus production

surface protein gene
- surface exclusion
- sex pilus will not attach

endonuclease gene
- cut strand of dsDNA @ oriT

regulatory gene

origin of replication
- rolling circle mechanism

Question 11

describe the process of conjugation

Answer 11

1. F+ produces sex pilus that binds to specific binding site @ F- cell
2. sex pilus retracts, pulling cells tgt
3. temporary cytoplasmic bridge built
4. 1 strand of F plasmid dsDNA cleaved by endonuclease @ oriT, transferred to recipient cell
5. F plasmid replicated via rolling circle mechanism
6. free 3’ end of cleaved DNA extended by DNA pol -> new strand
7. new strand displaces cleaved strand via 5’ end
8. ss F plasmid transferred acts as template for 2nd DNA strand
9. ds plasmid in recipient circularises

F- -> F+ !!

Question 12

what is transformation

Answer 12

uptake of naked, foreign DNA in environment by competent bacterial cells

incorporated into genome

change in genotype and phenotype of cell

Question 13

describe the process of transformation

Answer 13

1. dsDNA of dead recognised
2. bound by competence factors on CSM of recipient
3. dsDNA fragment enters
4. one strand degraded by endonuclease/exonuclease
5. other dsDNA aligns w homologous regions of bacteria
6. integrated via homologous recombination
7. needs 2 crossover events

recombinant cell !!

Question 14

how to induce competence

Answer 14

heat shock
- cold CaCl2
- brief heat
- transient pores

electroporation
- electric shock
- transient pores

induce DNA uptake !!

Question 15

what is generalised transduction?

Answer 15

• lytic cycle bacteriophages (virulent: T4 phage)
• random transfer of donor bacterial genome -> recipient

Question 16

describe generalised transduction

Answer 16

1. bacteriophage binds to host cell receptor
2. injects viral genome
3. phage degrades bacterial chromosome into small fragments using phage nuclease
4. random fragments of lysed bacterial genome may be wrongly packaged into phage capsid
   transducting (defective) phage !!
5. post lysis of host cell: transducing phage attaches to new host
6. injects viral DNA + DNA from old host
7. homologous recombination
   recombinant bacterial cell !!

Question 17

what is specialised transduction?

Answer 17

only lysogenic cycle bacteriophages (temperate: lambda)

transfer of specific bacterial DNA

Question 18

describe the process of specialised transduction

Answer 18

1. bacteriophage attaches to host
2. injects viral genome -> forms prophage
3. enters lysogenic cycle / spontaneously induced: lytic cycle
4. prophage excised

incorrect excision: host genes excised accidentally

5. both phage DNA and bacterial genes assembled into capsid.
6. old host cell DNA + viral DNA into 2nd cell

recombinant genome !!

Question 19

what is an operon?

Answer 19

cluster of genes w related functions

all turned on/off tgt

promoter + operator + structural genes = single polycistronic mRNA

Question 20

what is a promoter?

Answer 20

RNA pol binding site

upstream of structural genes

Question 21

what is an operator?

Answer 21

repressor protein binding site

prevents RNA pol from binding to promoter and initiating transcription

Question 22

what is the purpose of regulation in bacteria?

Answer 22

• economical use of energy and resources
• prevents wastage
• genes expressed only when necessary
• bacteria can respond rapidly to changes in env
• selective advantage to bacteria

Question 23

what is a repressible operon?

Answer 23

• controls anabolic pathways
• usually switched on
• repressor protein synthesised in inactive form

eg trp operon

Question 24

what is the structure of a trp operon

Answer 24

1. promoter
2. operator
3. structural genes

trpE, trpD, trpC, trpB, trpA

*outside operon: regulatory gene trpR (codes for inactive repressor protein)

Question 25

when tryptophan is ABSENT

Answer 25

1. trpR synthesised in inactive form 2. RNA pol binds to promoter 3. synthesise structural genes -> polycistronic mRNA formed 4. translation of polycistronic mRNA -> 5 enzymes responsible for tryptophan synthesis formed 5. trp operon on : bacterium synthesises tryptophan as it contains 5 enzymes for trp anabolism

Question 26

when tryptophan is PRESENT IN EXCESS

Answer 26

1. repressor protein in inactive form 2. tryptophan accumulates -> acts as corepressor -> bind to allosteric site of inactive trp repressor 3. repressor undergoes conformational change 4. active repressor protein binds to operator at DNA binding site 5. RNA pol cannot bind to promoter 6. transcription cannot proceed 7. trp operon off 8. no polycistronic mRNA 9. no 5 enzymes in tryptophan synthesis 10. no synthesis of trp

Question 27

what is an inducible operon?

Answer 27

- catabolic pathways - usually switched off - deactivated when inducer binds to it

Question 28

what is the structure of a lac operon?

Answer 28

1. promoter 2. operator 3. structural genes: lacZ, lacY, lacA *outside operon: lacI repressor

Question 29

what does lacZ code for?

Answer 29

beta-galactosidase breaks down lactose into glucose and galactose converts lactose -> allolactose

Question 30

what does lacY code for?

Answer 30

lactose permease transport of lactose into bacterium from external environment

Question 31

what does the lacA gene code for?

Answer 31

transacetylase may be involved in toxic by-product removal

Question 32

regulation of lac operon

Answer 32

dual regulation negative: through lac repressor positive: catabolite activator protein (CAP)

Question 33

(negative regulation) when LACTOSE and GLUCOSE ABSENT

Answer 33

1. lacI gene constitutively transcribed 2. continuous production of lac repressor protein 3. active lac repressor protein binds to operator via DNA binding site 4. absence of lactose: bound repressor prevents RNA pol from binding to promoter 5. transcription of structural genes blocked lac operon is switched OFF

Question 34

(negative regulation) when LACTOSE PRESENT

Answer 34

1. lactose enters cell via permease 2. converted to allolactose by beta-galactosidase 3. allolactose acts as inducer + binds to allosteric site of active lac repressor 4. inactivates repressor by altering conformation 5. repressor inactive and detaches from operator 6. RNA pol binds to promoter to initiate transcription of structural genes lac operon SWITCHED ON 7. single polycistronic mRNA 8. produce 3 enzymes involved in lactose metabolism

Question 35

catabolite activator protein

Answer 35

DNA binding site: allows to bind to activator/CAP binding site in promoter allosteric site specific for binding of cAMP - CAP naturally inactive - activated when bound to cAMP -> CAP-cAMPcomplex

Question 36

(positive regulation) GLUCOSE and LACTOSE both PRESENT

Answer 36

1. inactive repressor bound to inducer 2. lac operon switches on 3. high glucose level INHIBITS adenylyl cyclase 4. low levels of cAMP 5. no binding of cAMP to CAP 6. low levels of cAMP-CAP complex 7. RNA pol binds to promoter less stably 8. structural genes transcribed at lower rate 9. enzymes synthesised at lower rate 10. bacterium may use lactose BUT GLUCOSE PREFERRED RAHHH

Question 37

(positive regulation) GLUCOSE ABSENT, LACTOSE PRESENT

Answer 37

1. absence of glucose: high adenlyl cyclase activity 2. cAMP increase 3. cAMP binds to allosteric site of CAP -> cAMP-CAP complex 4. activating CAP which binds to CAP binding site in promoter binding of cAMP-CAP complex to CAP binding site INCREASES AFFINITY OF PROMOTER FOR RNA POL - increases rate of transcription initiation of lac operon - structural genes transcribed at high rate - enzymes synthesised at high rate - bacterium take up and break down lactose at faster rate - lactose is now main substrate for respiration