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8/17/2019 Bakul Dna Mobile-2016

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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

Plasmid

Transposo

Phage


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1PLA!MID


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Plasmid – Extrakromosom, benang ganda,biasanya supercoiled

– Ditemukan pada bakt G+ dan G- ;

satu sel individu sering harborsmembebaskan sejumlah plasmid

– DNA adalah berada di luar ekromosom; replikon plasmid

terpisah dari anakan sel dandipelihara di dalam sel anakannya

– Plasmid manunjukkan sedikithomologi (bila ada) thd sequenkromosom DNA inang


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Plasmids are not usually essential

• Plasmid biasanya tdk mengkode gen-gen esensial; wl dmkn, gen-gen plasmid bisa memberi suatukeuntungan 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


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• Fertility plasmids--F plasmids(mating/conjugation).

• Resistance plasmids--R plasmids

(antibiotic resistance--see handout)• Col plasmids--encode colicins(bacteroicins)

•

Virulence plasmids--encode determinantsrequired for pathogenesis

• Metabolic plasmids-mediate degradationof toxic compounds

Types of Plasmids


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99kb F (Fertility) Plasmid Genetic Map ( E. coli)

Insertion sequences (IS) assist in the

integration of transposons (Tn) into

homologus sites of recipients genome.

Different Hfr strains are produced as aresult

Replication & segregation genes of F plasmid

Genes for conjugative transfer

Origin of transfer


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Understanding Plasmids

4. Types of plasmids & their biological significance

Remember- essential host functions not part of plasmidgenes

Pseudomonas- entire metabolic degradation pathways ofunique compounds – camphor napthalene.

!ryptic plasmids – we "now little of the plasmid functions


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#. Resistance Plasmids $R plasmids%

R'' carries resistance for sulfonamdes tetracylinechloramphenicol spectinomycin mercury. (road enteric hostrange- )scherichia Proteus *lebsiella +almonella +higella


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,. Toins & other irulence characteristics

• /bility to attach and colonise a hosto ). coli !olonisation 0actor /ntigen $!0/% assists inattaching to intestinal epithelia

• production of toins en1ymes that damage the host

o ) coli hemolysin- lyse R(+ & ). coli enterotoin- inducesalt and water secretion into the bowel


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2. (acteriocins

(acterocins inhibit or "ill related species or different strains ofthe same species $limited inhibitory spectrum to antibiotics%

). coli plasmids- !ol


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3nsertion +equences Transposons & 3ntegrons

obilised ia plasmids.

Promote changes to the host 56/- Rearrange and or delete

genes3ntegrons are 3+ elements or transposons which create andmoe large gene clusters as a single unit - P/3


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R plasmid

Plasmids: Examples


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pBR322 (ColEI replicon)


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Analysis and Classification

– Replikon diklasifikasikan oleh gugus "incompatibilitas"Kompatibel plasmid memp sistem replicasi & segregasiberbeda. Incompatibel plasmid mengg sistem replikasi &

segrgasi yg sama. Sepertinya mrk tdk dpt dibedakan satu dg yglain 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 #)


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Panel A: coexistence of 2plasmids from differentincompatibility (Inc) groups-both maintained

Panel B: “curing” of oneplasmid. In the absence ofselection, asymmetricpartitioning results in theloss of one of each plasmid

-ultimately cellscontain oneplasmids of the otherbut not both


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– Isolated ascovalently closed circular(ccc) DNA; sizedby agarose gel electrophoresis.

– Restriction enzymes used to obtain a fragment profile

useful in epidemiology.

– DNA hybridization used to detect sequencerelationships.

–

Many plasmids remaincryptic; confer an undefinedphenotype.

Analisis dan Klasifikasi (cont.)


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ReplicasiPlasmid

Mengikuti aturan replikasi DNA kromosom, bilareplikasi mrpk stringent (low copy # plasmids;e.g., F plasmids).

High copy # plasmids (i.e., “relaxed” plasmids)Mengikuti aturannya sdr; mekanisme replikasidistinct“amplification” of plasmid DNA


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Replication: “theta” or “sigma”

Unidirectional

Bidirectional

Rolling circle


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Plasmid transmission

• Sequen DNA Plasmid dpt ditransmisikan diantr bakteri dg caratransformasi, transduksi, or konjugasi. Krn plasmids berreplikasi scrindependen, rekombinasi tdk dibutuhkan unt menurunkan sifat-sifat

• Konjugatif (self-transmissable) plasmids encode a conjugation system.

"Mobilizable" plasmids transfer through the cell contacts established byconjugative plasmids. Typically, the frequency of transfer is low. (The Fplasmid transfer system is depressed).

• "Wide host range" conjugative plasmids are able to replicate in manyorganisms; "narrow host range" plasmids, in only a few. However, DNAcan 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 tohost they interact with other host replicons (including the chromosome)& acquire genes by recombination or transposition.


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Plasmids: Pathogenesis

Examples: Shigella (A) and

Yersinia (B) both have virulence

plasmids that are required for

optimal virulence of these

bacteria. Other factors also

contribute to pathogenesis that

are not plasmid-encoded (i.e.,

shiga toxin). Strains “cured” ofthese plasmids have an attenuated

virulence phebnotype.

A

B


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" PHA#E


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Sifat umumPhage

• 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 ( karenanyahanya dpt mengreplikasi pd bakt hdp)

• Ribuan tipe beda diketh namun hanyasedikit species/strain yg spesifik


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T$ %dan T" BA&TERIO'A#


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T2 PA!" #"N!$N%"&'$ (A&T")$


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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 danpemindahan DNAphage ke sel inang

•Tidak semua phages memiliki ekor


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Kehidupan siklus litik

• Fase reproduktif yg mhslk anakan phagebaru

• Biasanya (tdk selalu) mhslk kematian & lisissel inang

•Phage yg hanya mampu tumbuh liti dikenalsbg virulen


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Tingkatan dlm siklus litik

• Adsorpsi: penempelan phage ke sel inang via reseptor spesifik

• Penetrasi: introduktsi DNA phage ke dlm sel inang; mekanismesering tdk jelas

• Ekspresi: transkripsi/translasi genom phage ke protein spesifikpenghasil virus; two temporal stages, early and late. protein awalbiasanya 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 selinang ("burst").


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Lisogeni

• Only observed for phage with double strandedDNA genomes (phages capable of lysogeny arecalled temperate).

• Phage genome becomes part of host cellchromosome (integrates into host DNA, e.g.Lambda) or is maintained as a low copy numberplasmid (e.g. P1 phage).

• Integrated phage genome called a prophage.

• Bacterium with prophage called a lysogen.


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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 bacterialgenome.

• Induction: disruption of the lysogenic state and initiation ofthe lytic phase.

• Replication, Assembly and Release: as in the lytic cycle


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Lytic Phages

• RNA Phages (f2, MS2, R17, Qß) – Simplest of phages.

– Genome is single-stranded, linear RNA (3000-4000bases), (+) strand.

– Encode three proteins; CP (coat protein), A (attachmentprotein), 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.


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T4• 2. Genome Structure

–Double stranded (ds), linear (≈170Kb,200 genes).

–DNA is replicated bidirectionally andforms long concatemers.

–Concatemers are cut and packaged to

form a "headful" unit. –Resultant DNA in phage is terminallyredundant and circularly permuted.

–Concatemer:


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3. Life Cycle

-Infection with T4 causes shut-off of hostcell protein, DNA, and RNA synthesis.

-Progeny phage assembled within 15-20min.

-Cells lyse within 20-25 min releasing

≈300 progeny phage.

T4


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Lambda( )

–Immunity:

• Lysogenic strains of bacteria cannot be

infected with a phage of the same type as theprophage.

–Resistance tosuperinfection

• In the lysogen, the cI repressor is dominant,so an incoming phage is immediatelyrepressed and cannot replicate.

MAP #ENOM T$ 'A#


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MAP #ENOM T$-'A#


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T


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Transposons


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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 inbacterial chromosomes and plasmids

Encode only genes for mobiliation and insertion

!ange in sie from "#$ bp to % kb

IS& first identified in E. coli 's glactose operon is"#$ bp long and is present ith *&+ copies in the

E. coli chromosome

Ends of all knon IS elements sho in,ertedterminal repeats (IT!s)


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Three different mechanisms

for transposition

0onser,ati,e transposition

!eplicati,e transposition

!etrotransposition


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Three different mechanisms

for transposition 0onser,ati,e transposition: elemen itu sdr

berpindah dari TK donor ke dalam TK target

!eplicati,e transposition: element moves acopy itself a copy of itself to a new site via a DNA

intermediate (lement memindahkan satu kopiny

sdr ke TK yg baru via DNA !antara")

!etrotransposition: The element makes an #NA

copy of itself which is re,ersed*transcribed into a

DNA co which is then inserted cDNA


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0onser,ati,e transposition


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!eplicati,e transposition


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!etrotransposition


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$eneration 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

common feature of mobile elements


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Transposons (Tn)

Similar to IS elements but are morecomple1 structurally and carry additionalgenes

2 types of transposons3

0omposite transposons

.oncomposite transposons


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0omposite

transposons


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%&'# is an autonomous element while %&'* is non+autonomous

http://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.html


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99kb F (Fertility) Plasmid Genetic Map ( E. coli)

Insertion sequences (IS) assist in the

integration of transposons (Tn) into

homologus sites of recipients genome.

Different Hfr strains are produced as aresult

Replication & segregation genes of F plasmid

Genes for conjugative transfer

Origin of transfer


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0omposite Transposons

Tetracycline resistance is carried by atransposable element

The transposon is a composite transposoncomposed of %&+elements flanking an includedse,uence in this case containing an antibioticresistance gene

%&'# is an autonomous element while %&'* is non+autonomous

-omposite transposons probably evolved from %&elements by the chance location of a pair in closepro.imity to one another/ %nactivation of oneelement by mutation would not harm ability totranspose and would assure continued
http://opbs.okstate.edu/~melcher/mg/MGW3/MG32221.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32211.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://www.ndsu.nodak.edu/instruct/mcclean/plsc431/transelem/trans5.htmhttp://www.ndsu.nodak.edu/instruct/mcclean/plsc431/transelem/trans5.htmhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32214.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32211.htmlhttp://opbs.okstate.edu/~melcher/mg/MGW3/MG32221.html

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