fismik basic microbial genetics

8/2/2019 FISMIK Basic Microbial Genetics

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Basic Microbial Geneticspart 1

Bahan kuliah Genetika Mikrobia

Prof. Dr. A. Endang Sutariningsih Soetarto, MSc

Fakultas Biologi UGM


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Genetika dan regulasi metabolik dayadan formasi enzyme

a. Biosintesis mikromolekul dan regulasi


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A. DNA (in prokaryotes)

Every living organism has DNA = cell database.Bacteria have single chromosome (circular in all except Borrelia burgdorferi, cause ofLyme disease), no nucleus.

View TEM of bacterium, illustrating DNA (marked n) in cell cytoplasm. Cell is adividing Neisseria gonorrhoeae, cause of gonorrhea.

View electron micrograph of isolated bacterial DNA Central dogma: information is encoded in DNA.

To express this information, RNA is transcribed with same coding, thentranslated into amino acid sequence which folds to form active proteins.

View animation of central dogma (Note: shockwave plug-in required)

DNA encodes two types of molecules: database for protein structure (access by sequential transcription & translation) database for needed t-RNA, r-RNA molecules (access by transcription alone)

Several bacterial genomes have been completely sequenced.Some chromosome sizes are listed in the table, along with sizes of yeast and human DNAfor comparison.
http://www.sp.uconn.edu/~terry/images/other/NgonorTEM.jpghttp://www.ai.mit.edu/people/tk/ce/ecoli-dna.gifhttp://instruct1.cit.cornell.edu/Courses/biomi290/MOVIES/CENTRALDOG.HTMLhttp://instruct1.cit.cornell.edu/Courses/biomi290/MOVIES/CENTRALDOG.HTMLhttp://www.ai.mit.edu/people/tk/ce/ecoli-dna.gifhttp://www.sp.uconn.edu/~terry/images/other/NgonorTEM.jpghttp://www.sp.uconn.edu/~terry/images/other/NgonorTEM.jpg


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Some chromosome sizes are listed in the table, alongwith sizes of yeast and human DNA for comparison

Organism Domain Chromosome size (base pairs) Predicted polypeptide coding regions

Mycoplasma genitalium Bacteria 0.58 Million bp 470 proteins

Hemophilus influenzae Bacteria 1.83 Million bp 1740 proteins

Methanococcus jannaschii Archaea 1.66 Million bp 1682 proteins

Escherichia coli Bacteria 4.64 Million bp 4288 proteins

Largest yeast chromosome

now mappedEukarya 1.55 Million bp ?

Entire yeast genome Eukarya 15 Million bp ?

Smallest human

chromosome (Y) Eukarya 50 Million bp ?

Largest human

chromosome (1)Eukarya 250 Million bp ?

Entire human genome Eukarya 3 Billion ?
http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=gmghttp://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=ghihttp://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=arghttp://www.genome.wisc.edu/http://genome-www.stanford.edu/Saccharomyces/MAP/GENOMICVIEW/GenomicView.htmlhttp://genome-www.stanford.edu/Saccharomyces/MAP/GENOMICVIEW/GenomicView.htmlhttp://www.genome.wisc.edu/http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=arghttp://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=ghihttp://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=gmg


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E. colicell is often used as "modelorganism".

Cell ~ 0.2-2 um in diameter,2 m in length;

has single circularDNA chromosome.

DNA can be measured invarious ways:

~ 1400 m in length~ 4700 kilobase pairs

~ 2 x 109 daltons in mass


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DNA prompts many intriguing questions.For example

how does cell manage a giant ball of string, which

must be identically copied prior to replication,without generating hopeless tangle? Many genes are not expressed most of the time,

only under certain circumstances. How does cellregulate this expression?

Any damage to DNA is likely to be lethal, sincethere is no "backup" copy of the database. Howdoes cell detect and repair damage?

E. colicell is often used as "model organism".


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DNA structure

DNA made from subunits called nucleotides. Each nucleotide contains:

Purine (Adenine or A, Guanine or G) or

Pyrimidine (Cytosine or C, Thymine or T) bases.

deoxyribose sugar. View pentose sugars. 1, 2, or 3 phosphate groups. View phosphate groups.
http://www.sp.uconn.edu/~terry/images/mols/Nucacids08.GIFhttp://www.sp.uconn.edu/~terry/images/mols/Nucacids09.GIFhttp://www.sp.uconn.edu/~terry/images/mols/Nucacids09.GIFhttp://www.sp.uconn.edu/~terry/images/mols/Nucacids08.GIFhttp://info.bio.cmu.edu/Courses/BiochemMols/BuildBlocks/Nucs.html


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Nucleotidesare named accordingto # of phosphates:e.g., dATP = deoxy adenosine triphosphate

dAMP = deoxy adenosine monophosphateNote: nucleotides in RNA don't have

deoxyribose, don't have prefix "d";names like ATP, ADP, AMP refer toRNA nucleotides containing ribosesugar

Watson & Crick discoveredstructure of DNA by analyzing X-ray data from Franklin & Wilkins.DNA = double helix,"backbone" consists of alternating

units:-- deoxyribose -- phosphate --deoxyribose -- phosphate --


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MICROBIAL GENETICS

A. Polypeptides, Proteins, and EnzymesB. Deoxyribonucleic Acid (DNA)C. DNA Replication

D. Ribonucleic Acid (RNA)E. Polypeptide and Protein SynthesisF. Changes in structure of DNA ( genetic code )G. Transfer of Genetic Material and Genetic

Recombination in Bacteria

H. Bacterial Resistance to Chemotherapeutic AgentI. Genetics of Viruses


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Two purposes of the genome


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DNA Replication

Deoxyribonucleic Acid(DNA)

DNA Replication Ribonucleic Acid (RNA)
http://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNArep/dnarep.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNA/dna.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNArep/dnarep.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNA/dna.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNA/dna.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNArep/dnarep.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNArep/dnarep.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/RNA/rna.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/RNA/rna.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/RNA/rna.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/RNA/rna.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNArep/dnarep.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNArep/dnarep.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNArep/dnarep.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNArep/dnarep.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNA/dna.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNA/dna.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNArep/dnarep.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNArep/dnarep.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/DNA/DNArep/dnarep.html


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Polypeptide and Protein Synthesis

1. An Overview

2. Transcription

3. Translation4. Enzyme Regulation
http://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/overview/overview.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/overview/overview.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/transcription/transcription.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/transcription/transcription.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/translation/translation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/translation/translation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/regulation/regulation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/regulation/regulation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/regulation/regulation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/regulation/regulation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/translation/translation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/translation/translation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/transcription/transcription.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/transcription/transcription.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/overview/overview.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/protsyn/overview/overview.html


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Gene expression - the making of a trait

A. Transcription mRNA

B. Translation -tRNA

C. Product =protein


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DNA the blueprint

A. Instructions for aHuman Being

B. Protecting the

blueprintC. Reading the blueprint= Transcription (readby mRNA
http://www.ucmp.berkeley.edu/glossary/gloss3/DNA2.gif


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Polypeptide and Protein Synthesis

D. Translating to thenecessary buildingmaterials = Translation

E.The building process =Protein Synthesis

F.The final process

tRNA translates the message (or theblueprint) into the actual physicalneeds. (amino acids for proteins)


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Instructions Instructions copied Instructions translated Construction Product

DNA mRNA tRNA rRNA Protein

Blueprint Architect Construction Manager Crew Building


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Regulation of gene expression inprokaryotes

Many genes occur in operons

several structural genes (for protein) controlled bya single promoter

mRNA is polycistronic: has multiple start & stopsignals, codes for multiple polypeptides

In some cases, transcription depends onspecific DNA region, the operator, which is

near or overlapping the promoter. Operator site can bind repressor proteins,

controlled by some effector molecule


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Regulation of gene expression in prokaryotes


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Transcription & Translation are coupled

Prot. synthesis begins before transcription ends

multiple ribosomes bind to each mRNA

All control at level of transcription

once m-RNA is made, it is translated.


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Some genes are expressed constitutively

Always translated when cells synthesizingprotein

Examples

ribosomal genes genes for replication & transcription

genes for central metabolism enzymes

Note: some proteins made from constitutivegenes occur at much higher levels thanothers. Why? Strong vs. weak promoters


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Some genes are regulated by repressorproteins

Classic example: the lac operon

gene is regulated by negative control;

in absence of specific repressor, gene istranscribed just like constitutive gene. Inorder to regulate, must add specific block.

Must say "no"; otherwise gene is not down-regulated


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Changes in structure of DNA(genetic code

MutationsChanges in bacteria (phenotype)

1. Colony morphology :

a. Loss of pigmentationb. Smooth rough ( loss of capsule)2. Biochemical activity

Change in enzyme production

3. VirulenceChange in ability to produce disease

4. Drug resistance


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MUTATIONSA. Definition :

1. Permanent change in the sequence of nucleotides in DNA

2. Passed to all daughter cells (inherited)

B. Types of mutations:1. Spontaneous mutations - due to mistakes occurring during DNA replication2. Induced mutations - due to mutation causing agents (mutagens - chemicals,UV,

C. Changes in DNA mutation1. Point mutation - base substitution

a. Substitution 1 base

b. Inversion 1 base pair

2. Frameshift mutation

a. Addition base pairs

b. Deletion base pairs

c. Alters all triplets beyond the point of addition or deletion3. Nonsense mutation

a. Creates a stop codon in m-RNA (may be due to a point mutation)

b. Incomplete protein (nonsense protein) produced

4. Dimer

a. Presence UV light

b. Covalent bonds formed between adjacent thymines


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Transfer of Genetic Material andGenetic Recombination in Bacteria

1. An Overview

2. Transformation

3. Transduction4. Conjugation

5. Recombinant DNA
http://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/overview/overview.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/transformation/transformation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/transformation/transformation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/transduction/transduction.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/transduction/transduction.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/conjugation/conjugation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/conjugation/conjugation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/recombinant/recombinant.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/recombinant/recombinant.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/recombinant/recombinant.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/recombinant/recombinant.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/recombinant/recombinant.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/recombinant/recombinant.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/conjugation/conjugation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/conjugation/conjugation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/transduction/transduction.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/transduction/transduction.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/transformation/transformation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/transformation/transformation.htmlhttp://student.ccbcmd.edu/courses/bio141/lecguide/unit6/genetics/recombination/overview/overview.html


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TRANSFER OF GENETIC MATERIAL

A. Common properties:

1. Unidirectional - donor cell recipient cell2. Fragment DNA transferred

B. Three methods of transfer:

1. TransformationViable bacteria absorb naked fragments of DNA released from dead

bacteria

2. TransductionBacterial virus (bacteriophage) transfers DNA fragments from onebacterial cell to another

3. Conjugation

a. Occurs amongst bacteria with sex pilib. Bridge formed between cells by pilus

c. Fragment of DNA (plasmid) transferred from one bacterialcell (donor) to another bacterial cell (recipient)


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Conjugation in Gram Negative Bacteria

I. Characteristics

II. Mechanism

Cell-cell interactions DNA metabolism

DNA transfer

III. Host range


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DNA transfer requires cell-cell contact

conjugation is prevented by filter that allows mixing of mediumbut prevents contact between donor and recipient cells

DNA transfer occurs via a conjugal pore -

resistance to DNase DNA transfer occurs in one direction

from donor to recipient not vice versa DNA transfer does not require protein

Synthesis In donor cell - Str sensitivedonors can mate with Str resistant

recipients in the presence of streptomycin DNA transfer requires energy in donor cell

primarily ATP

Distinguishing characteristics of conjugation


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Mechanism

Conjugation conferredby certain types of genetic elements, particularlyplasmids. Transfer of plasmid from donor to recipient does not require plasmid DNAto recombine with recipient DNA.

Conjugation is conservative -- donor retains a copy of the plasmid aftertransfer, indicating that plasmid replication occurs during conjugation.

Plasmid replicationrequires a "mating bridge" between the donor andrecipient cells.

The mating bridgeis a region of contact between the donor and recipientcells where the DNA is presumably transferred via a pore (although thepore has not yet been realiably visualized). Before the mating bridge canform:

donor must recognize recipient celldonor must make contact with recipient cell

These two events do not necessarily occur simultaneously.


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Mechanism of Conjugation


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Molecular events that occur duringplasmid transfer


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Molecular events that occur during plasmidtransfer

C j ti


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Conjugation

Multiple genes are required for conjugation.For example, for the TiC58 plasmid the trb operons encode the gene products

required for recognition of the recipient and mating pair formation.

Genes in this cluster encode the sex pilus.The sex pilus is essential for conjugation.

very long (e.g. the pilus produced by F-plasmid) orvery short (e.g. the pilus produced by plasmid RP4).DNA is not transferred via the sex pilus.

Although the receptor is yet not known for any conjugal plasmid,

required for recognition of the recipient cell. Plasmids like F that code for long flexous pili mate well in liquid media, but

plasmids that code for short stiff pili usually mate well only on solid surfaces.Following contact, the long flexous pilus of F acts as a retractile motor -- the donor and

recipient cells are pulled together as the pilus subunits depolymerize into thecytoplasmic membrane of the donor cells. In contrast, the retractile nature of other pilihas not been demonstrated.

The tra operons encode gene products required for theDNA replication functions.


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The locations of some of the IS1 IS2 and IS3


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The locations of some of the IS1, IS2, and IS3insertions on the E. coliK-12 chromosome

and the orientation of the insertions

Hfr's can be isolated at manysites in E. coli and in differentorientations relative to thechromosome.

A few examples of Hfr insertionsthat have been isolated in E. coli

Note that the E. coli chromosomeis shown linearized in the figure.

The numbers at the top of thefigure represent minutes (or"centisomes") on the E. coligenetic map.


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Formation of Hfr's in Salmonella typhimurium

in contrast, S typhimuriumlacks many of the IS elementspresent in E. coli.

Hence, Hfr insertions caused byhomologous recombinationbetween IS elements on the F-

plasmid and chromosome arerare in S. typhimurium.


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fismik basic microbial genetics

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