2.9: bacteria Flashcards
what is a bacterium?
- small unicellular prokaryotes
- lacks a true nucleus and membrane-bound organelles
what kind of DNA do bacteria have?
circular DNA
what type of ribosomes do bacteria have?
70S
how many oriR do bacteria have
single
what are plasmids?
- extrachromosomal DNA
- replicate independently of chromosomal DNA
- involved in conjugation & antibiotic resistance
describe the process of DNA replication (before binary fission)
- DNA double helix unwinds and unzips @ oriR
- replicates via semi-conservative model
- bidirectional replication
- circular DNA -> interlocking structure -> topoisomerase cuts, reseals 2 DNA molecules
describe binary fission
- bacterial chromosome is attached to plasma membrane @ oriC
- replicates , 2nd oriC attaches to membrane adjacently
- cell elongates by growing membrane, depositing cell wall material btwn 2 chromos
- after growing, septum forms in btwn
- septum fuses -> 2 identical daughter cells
what is the significance of genetic variation?
- rapidly changing environment
- natural selection
- occur due to point mutations/genetic transfer
what is conjugation?
- unidirectional transfer of F plasmid
- via direct contact
- from F+ -> F-
what does the F plasmid contain
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
describe the process of conjugation
- F+ produces sex pilus that binds to specific binding site @ F- cell
- sex pilus retracts, pulling cells tgt
- temporary cytoplasmic bridge built
- 1 strand of F plasmid dsDNA cleaved by endonuclease @ oriT, transferred to recipient cell
- F plasmid replicated via rolling circle mechanism
- free 3’ end of cleaved DNA extended by DNA pol -> new strand
- new strand displaces cleaved strand via 5’ end
- ss F plasmid transferred acts as template for 2nd DNA strand
- ds plasmid in recipient circularises
F- -> F+ !!
what is transformation
uptake of naked, foreign DNA in environment by competent bacterial cells
incorporated into genome
change in genotype and phenotype of cell
describe the process of transformation
- dsDNA of dead recognised
- bound by competence factors on CSM of recipient
- dsDNA fragment enters
- one strand degraded by endonuclease/exonuclease
- other dsDNA aligns w homologous regions of bacteria
- integrated via homologous recombination
- needs 2 crossover events
recombinant cell !!
how to induce competence
heat shock
- cold CaCl2
- brief heat
- transient pores
electroporation
- electric shock
- transient pores
induce DNA uptake !!
what is generalised transduction?
- lytic cycle bacteriophages (virulent: T4 phage)
- random transfer of donor bacterial genome -> recipient
describe generalised transduction
- bacteriophage binds to host cell receptor
- injects viral genome
- phage degrades bacterial chromosome into small fragments using phage nuclease
- random fragments of lysed bacterial genome may be wrongly packaged into phage capsid
transducting (defective) phage !! - post lysis of host cell: transducing phage attaches to new host
- injects viral DNA + DNA from old host
- homologous recombination
recombinant bacterial cell !!
what is specialised transduction?
only lysogenic cycle bacteriophages (temperate: lambda)
transfer of specific bacterial DNA
describe the process of specialised transduction
- bacteriophage attaches to host
- injects viral genome -> forms prophage
- enters lysogenic cycle / spontaneously induced: lytic cycle
- prophage excised
incorrect excision: host genes excised accidentally
- both phage DNA and bacterial genes assembled into capsid.
- old host cell DNA + viral DNA into 2nd cell
recombinant genome !!
what is an operon?
cluster of genes w related functions
all turned on/off tgt
promoter + operator + structural genes = single polycistronic mRNA
what is a promoter?
RNA pol binding site
upstream of structural genes
what is an operator?
repressor protein binding site
prevents RNA pol from binding to promoter and initiating transcription
what is the purpose of regulation in bacteria?
- 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
what is a repressible operon?
- controls anabolic pathways
- usually switched on
- repressor protein synthesised in inactive form
eg trp operon
what is the structure of a trp operon
- promoter
- operator
- structural genes
trpE, trpD, trpC, trpB, trpA
*outside operon: regulatory gene trpR (codes for inactive repressor protein)
when tryptophan is ABSENT
- trpR synthesised in inactive form
- RNA pol binds to promoter
- synthesise structural genes -> polycistronic mRNA formed
- translation of polycistronic mRNA -> 5 enzymes responsible for tryptophan synthesis formed
- trp operon on : bacterium synthesises tryptophan as it contains 5 enzymes for trp anabolism
when tryptophan is PRESENT IN EXCESS
- repressor protein in inactive form
- tryptophan accumulates -> acts as corepressor -> bind to allosteric site of inactive trp repressor
- repressor undergoes conformational change
- active repressor protein binds to operator at DNA binding site
- RNA pol cannot bind to promoter
- transcription cannot proceed
- trp operon off
- no polycistronic mRNA
- no 5 enzymes in tryptophan synthesis
- no synthesis of trp
what is an inducible operon?
- catabolic pathways
- usually switched off
- deactivated when inducer binds to it
what is the structure of a lac operon?
- promoter
- operator
- structural genes: lacZ, lacY, lacA
*outside operon: lacI repressor
what does lacZ code for?
beta-galactosidase
breaks down lactose into glucose and galactose
converts lactose -> allolactose
what does lacY code for?
lactose permease
transport of lactose into bacterium from external environment
what does the lacA gene code for?
transacetylase
may be involved in toxic by-product removal
regulation of lac operon
dual regulation
negative: through lac repressor
positive: catabolite activator protein (CAP)
(negative regulation) when LACTOSE and GLUCOSE ABSENT
- lacI gene constitutively transcribed
- continuous production of lac repressor protein
- active lac repressor protein binds to operator via DNA binding site
- absence of lactose: bound repressor prevents RNA pol from binding to promoter
- transcription of structural genes blocked
lac operon is switched OFF
(negative regulation) when LACTOSE PRESENT
- lactose enters cell via permease
- converted to allolactose by beta-galactosidase
- allolactose acts as inducer + binds to allosteric site of active lac repressor
- inactivates repressor by altering conformation
- repressor inactive and detaches from operator
- RNA pol binds to promoter to initiate transcription of structural genes
lac operon SWITCHED ON
- single polycistronic mRNA
- produce 3 enzymes involved in lactose metabolism
catabolite activator protein
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
(positive regulation) GLUCOSE and LACTOSE both PRESENT
- inactive repressor bound to inducer
- lac operon switches on
- high glucose level INHIBITS adenylyl cyclase
- low levels of cAMP
- no binding of cAMP to CAP
- low levels of cAMP-CAP complex
- RNA pol binds to promoter less stably
- structural genes transcribed at lower rate
- enzymes synthesised at lower rate
- bacterium may use lactose BUT GLUCOSE PREFERRED RAHHH
(positive regulation) GLUCOSE ABSENT, LACTOSE PRESENT
- absence of glucose: high adenlyl cyclase activity
- cAMP increase
- cAMP binds to allosteric site of CAP -> cAMP-CAP complex
- 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
