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DNA BERPINDAH-PINDAH (MOBILE DNA)

DNA yg mampu berpindah dari satu tempat ke tempat lain,


misal plasmid, phage dan transposon; dpt bertindak sebagai
vehicle pd proses strains improvement

Phage
Plasmi
d
Transposo

1.PLASMID

Plasmid
Extrakromosom, benang ganda,
biasanya supercoiled
Ditemukan pada bakt G+ dan G- ;
satu sel individu sering harbors
membebaskan sejumlah plasmid
DNA adalah berada di luar e
kromosom; replikon plasmid
terpisah dari anakan sel dan
dipelihara di dalam sel anakannya
Plasmid manunjukkan sedikit
homologi (bila ada) thd sequen
kromosom DNA inang

Plasmids are not usually essential


Plasmid biasanya tdk mengkode gen-gen esensial
; wl dmkn, gen-gen plasmid bisa memberi suatu
keuntungan selektif bagi bakteri, misal:

Produksi bacteriocin
Degradasi seny2 toksik
Simbiosis dg jasad eukariot (fiksasi nitrogen)
Produksi antibiotik
Resisten thd antibiotik
Enterotoxins (E. coli)
Hemolysins
Mhslk Faktor adherence & colonization (pili)
Mhslk proteins serum resistensi
Mhslk patwai katabolik dan anabolik

Types of Plasmids
Fertility plasmids--F plasmids
(mating/conjugation).
Resistance plasmids--R plasmids
(antibiotic resistance--see handout)
Col plasmids--encode colicins
(bacteroicins)
Virulence plasmids--encode determinants
required for pathogenesis
Metabolic plasmids-mediate degradation
of toxic compounds

Insertionsequences(IS)assistinthe
integrationoftransposons(Tn)into
homologussitesofrecipientsgenome.
DifferentHfrstrainsareproducedasa
result

Genesforconjugativetransfer

Originoftransfer
Replication&segregationgenesofFplasmid

99kbF(Fertility)PlasmidGeneticMap(E.coli)

Understanding Plasmids
4. Types of plasmids & their biological significance
Remember- essential host functions not part of plasmid
genes
Pseudomonas- entire metabolic degradation pathways of
unique compounds camphor, napthalene.
Cryptic plasmids we know little of the plasmid functions

Understanding Plasmids
5. Resistance Plasmids (R plasmids)
R100 carries resistance for sulfonamdes, tetracyline,
chloramphenicol, spectinomycin, mercury. Broad enteric host
range- Escherichia, Proteus, Klebsiella, Salmonella, Shigella,

Understanding Plasmids
6. Toxins & other virulence characteristics
Ability to attach and colonise a host
o E. coli Colonisation Factor Antigen (CFA) assists in
attaching to intestinal epithelia
production of toxins, enzymes that damage the host
o E coli hemolysin- lyse RBS & E. coli enterotoxin- induce
salt and water secretion into the bowel

Understanding Plasmids
7. Bacteriocins
Bacterocins inhibit or kill related species or different strains of
the same species (limited inhibitory spectrum to antibiotics)
E. coli plasmids- Col

Insertion Sequences, Transposons & Integrons


Mobilised via plasmids.
Promote changes to the host DNA- Rearrange and or delete
genes
Integrons are IS elements or transposons which create and
move large gene clusters as a single unit - PAI

Plasmids: Examples

R plasmid

pBR322 (ColEI replicon)

Analysis and Classification


Replikon diklasifikasikan oleh gugus "incompatibilitas"
Kompatibel plasmid memp sistem replicasi & segregasi
berbeda. Incompatibel plasmid mengg sistem replikasi &
segrgasi yg sama. Sepertinya mrk tdk dpt dibedakan satu dg yg
lain selama proses-proses ini, keduanya tdk dpt dipertahankan
Jumlah kopi plasmid/sel bs tinggi (30 or lbh) or rendah (1-2).
Replicasinya adl stringent or relaxed

Stringent: mereplikasi sejalan dg khromosom (low copy #)


Relaxed: mereplicasi scr tdk terpengaaruh kromosom (high copy #)

Panel A: coexistence of 2
plasmids from different
incompatibility (Inc) groups
-both maintained
Panel B: curing of one
plasmid. In the absence of
selection, asymmetric
partitioning results in the
loss of one of each plasmid
-ultimately cells
contain one
plasmids of the
other
but not both

Analisis dan Klasifikasi (cont.)


Isolated as covalently closed circular (ccc) DNA; sized
by agarose gel electrophoresis.
Restriction enzymes used to obtain a fragment profile
useful in epidemiology.
DNA hybridization used to detect sequence
relationships.
Many plasmids remain cryptic; confer an undefined
phenotype.

ReplicasiPlasmid
Mengikuti aturan replikasi DNA kromosom, bila
replikasi mrpk stringent (low copy # plasmids;
e.g., F plasmids).
High copy # plasmids (i.e., relaxed plasmids)
Mengikuti aturannya sdr; mekanisme replikasi
distinct amplification of plasmid DNA

Replication: theta or sigma


Unidirectional

Bidirectional

Rolling circle

Plasmid transmission

Sequen DNA Plasmid dpt ditransmisikan diantr bakteri dg cara


transformasi, transduksi, or konjugasi. Krn plasmids berreplikasi scr
independen, rekombinasi tdk dibutuhkan unt menurunkan sifat-sifat
Konjugatif (self-transmissable) plasmids encode a conjugation system.
"Mobilizable" plasmids transfer through the cell contacts established by
conjugative plasmids. Typically, the frequency of transfer is low. (The F
plasmid transfer system is depressed).
"Wide host range" conjugative plasmids are able to replicate in many
organisms; "narrow host range" plasmids, in only a few. However, DNA
can be delivered to many organisms in either case; such DNA can then
be maintained in the recipient through integration with other replicons
(transposition; cointegrate formation).
Properties disseminated by plasmids: As plasmids move from host to
host they interact with other host replicons (including the chromosome)
& acquire genes by recombination or transposition.

Plasmids:Pathogenesis
A

Examples:Shigella(A)and
Yersinia(B)bothhavevirulence
plasmidsthatarerequiredfor
optimalvirulenceofthese
bacteria.Otherfactorsalso
contributetopathogenesisthat
arenotplasmidencoded(i.e.,
shigatoxin).Strainscuredof
theseplasmidshaveanattenuated
virulencephebnotype.

2. PHAGE

Sifat umum Phage


Phages adalah parasit bakteri.
Tdk dpt berreplikasi di luar sel inang
Mengg mesin inang (ribosomes,
enzymes) unt mensintesis komponen virus
Mengg sistem enersi sel inang ( karenanya
hanya dpt mengreplikasi pd bakt hdp)
Ribuan tipe beda diketh namun hanya
sedikit species/strain yg spesifik

T4 dan T2 BAKTERIOFAG

T2 PHAGE MENGINFEKSI BAKTERI

Struktur Phage
1. Genome
single or double- benang DNA, sirkuler or linier.
linier, benang tunggal RNA.
2. Kapsid
protein shell ("coat").
pelindung asam nukleat genome.
Bentuknya icosohedral or filamentous
3. Ekor
Struktur protein yg nempel pd kapsid.
Struktur fisiknya sgt kompleks.
Terlibat dlm penempelan phage pd sel inang dan
pemindahan DNAphage ke sel inang
Tidak semua phages memiliki ekor

Kehidupan siklus litik


Fase reproduktif yg mhslk anakan phage
baru
Biasanya (tdk selalu) mhslk kematian & lisis
sel inang
Phage yg hanya mampu tumbuh liti dikenal
sbg virulen

Tingkatan dlm siklus litik

Adsorpsi: penempelan phage ke sel inang via reseptor spesifik

Penetrasi: introduktsi DNA phage ke dlm sel inang; mekanisme


sering tdk jelas

Ekspresi: transkripsi/translasi genom phage ke protein spesifik


penghasil virus; two temporal stages, early and late. protein awal
biasanya unt replikasi & transkripsi, protein akhir unt perakitan

Replikasi: sintesis genome phage baru

Perakitan: pengorganisasian anakan genom phage ke dlm kapsid

Pelepasan: anakan hsl rakitan ninggalkan sel, dg melisiskan sel


inang ("burst").

Generalised transduction

Lisogeni
Only observed for phage with double stranded
DNA genomes (phages capable of lysogeny are
called temperate).
Phage genome becomes part of host cell
chromosome (integrates into host DNA, e.g.
Lambda) or is maintained as a low copy number
plasmid (e.g. P1 phage).
Integrated phage genome called a prophage.
Bacterium with prophage called a lysogen.

Stages in Lysogeny
Adsorption
Introduction of phage DNA into host chromosome
(recombination-dependent event).
Expression: only of genes required for lysogeny.
Maintenance: prophage replicates along with bacterial
genome.
Induction: disruption of the lysogenic state and initiation of
the lytic phase.
Replication, Assembly and Release: as in the lytic cycle

Lytic Phages
RNA Phages (f2, MS2, R17, Q)

Simplest of phages.
Genome is single-stranded, linear RNA (3000-4000
bases), (+) strand.
Encode three proteins; CP (coat protein), A (attachment
protein), and Rep (RNA replicase).
All utilize F-pili as receptors for attachment.
Rep copies (+) strand into (-) strands then uses (-)
strands to make new (+) strands for packaging.
Lyse cells and release 10-20,000 phage per cell;
mechanism of lysis unknown.

Phages Litik
Single Stranded DNA Phages

Icosohedral capsid ( X174) or filamentous (f1,


M13, fd).
Adsorb to F-pili.
Replication proceeds via double stranded (RF)
intermediate; RF form produces new progeny
DNA by rolling circle replication.
> X174 lyses cell to release progeny

> filamentous phages "bud" from host cell and do not


cause lysis or death.

T4

2. Genome Structure

Double stranded (ds), linear (170Kb,


200 genes).
DNA is replicated bidirectionally and
forms long concatemers.
Concatemers are cut and packaged to
form a "headful" unit.
Resultant DNA in phage is terminally
redundant and circularly permuted.
Concatemer:

T4
3. Life Cycle
-Infection with T4 causes shut-off of host
cell protein, DNA, and RNA synthesis.
-Progeny phage assembled within 15-20
min.
-Cells lyse within 20-25 min releasing
300 progeny phage.

Lambda ( )
Immunity:
Lysogenic strains of bacteria cannot be
infected with a phage of the same type as the
prophage.
Resistance to superinfection
In the lysogen, the cI repressor is dominant,
so an incoming phage is immediately
repressed and cannot replicate.

MAP GENOM T4-FAG

3.TRANSPOSON

Transposons

Mobile Genetic Elements

Transposons or Transposable elements


(TEs)

move around the genome

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Transposable elements in
prokaryotes

Insertion sequence (IS) elements

Transposons (Tn)

Bacteriophage Mu
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Insertion sequence (IS)


elements

Simplest type of transposable element found in


bacterial chromosomes and plasmids

Encode only genes for mobilization and insertion

Range in size from 768 bp to 5 kb

IS1 first identified in E. colis glactose operon is


768 bp long and is present with 4-19 copies in the
E. coli chromosome

Ends of all known IS elements show inverted


terminal repeats (ITRs)
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Integrasi elemen IS dlm DNA


kromosom

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Three different mechanisms


for transposition

Conservative transposition

Replicative transposition

Retrotransposition

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Three different mechanisms


for transposition

Conservative transposition: elemen itu sdr


berpindah dari TK donor ke dalam TK target

Replicative transposition: element moves a


copy itself a copy of itself to a new site via a DNA
intermediate (Element memindahkan satu kopiny
sdr ke TK yg baru via DNA antara)

Retrotransposition: The element makes an RNA


copy of itself which is reversed-transcribed
into a
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DNA copy which is then inserted (cDNA)

Conservative transposition

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Replicative transposition

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Retrotransposition

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common feature of mobile elements

Generation of short direct repeats flanking the


newly inserted element

This results for a staggered cut being made in


the DNA strands at the site of insertion

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Transposons (Tn)

Similar to IS elements but are more


complex structurally and carry additional
genes

2 types of transposons:

Composite transposons

Noncomposite transposons
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Composite
transposons

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IS10R is an autonomous element, while IS10L is non-autonomous


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Insertionsequences(IS)assistinthe
integrationoftransposons(Tn)into
homologussitesofrecipientsgenome.
DifferentHfrstrainsareproducedasa
result

Genesforconjugativetransfer

Originoftransfer
Replication&segregationgenesofFplasmid

99kbF(Fertility)PlasmidGeneticMap(E.coli)

Composite Transposons

Tetracycline resistance is carried by a


transposable element
The transposon is a composite transposon,
composed of IS-elements flanking an included
sequence, in this case containing an antibiotic
resistance gene
IS10R is an autonomous element
while IS10L is non-autonomous
Composite transposons probably evolved from IS
elements by the chance location of a pair in close
proximity to one another. Inactivation of one
element by mutation would not harm ability to
transpose and would assure continued
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transposition of the entire transposon

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