Bacteriology Chapter 10 Part i i

8/13/2019 Bacteriology Chapter 10 Part i i

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Chapter 10 (continued)

Bacterial Genetics


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Transformation vs. Transduction vs.

Conjugation


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Genetic TransformationGenetic Transformation: process by which free DNA is

incorporated into a recipient cells and brings about

genetic change.A number of prok. are naturally transformable, including

gram-pos. and gram-neg. Bacteriaand someArchaea.

Only a small number of genes from one cell can be

transferred to another by a single transformation event.


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Competent CellsCompetent cells: able to take up a molecule of DNA via transformation.

Only certain strains are competent and this ability is geneticallydetermined.

Competence is regulated with special proteins playing a role in DNAuptake and processing.

ssDNA or dsDNA may taken up by cells, though it must be in ssDNAform to be incorporated into the genome by recombination.

Competent cells bind up to 1000X more DNA than noncompetent cells

(dsDNA binds better to cells).DNA fragments compete with each other for uptake.

While the max. frequency of transformation = 20% of the population,actual values = 0.1-1.0%.

Min. conc. of DNA yielding detectable transformants = 0.00001 g/ml.


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Integration of Transforming DNADNA is either taken up single-stranded or dsDNA is takenup and one strand is degradedssDNA.

Next, the ssDNA associates with competence-specificprotein that remains attached to the DNA to protect it fromnuclease attack until it reaches the chromosome, whereRecA takes over.

DNA is then integrated into the genome of the recipient by

recombination.During replication of this heteroduplex DNA, one parentaland one recombinant DNA molecule are formed. Onsegregation at cell division, the recombinant DNA molecule

is present in the transformed cell.


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TransfectionTransfection: Bacteria transformed by

bacteriophage DNA instead of DNA from another

bacterium.Transfection by a lytic bacteriophage leads to

normal virus production.


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Artificially Induced

Competence/ElectroporationOnly a few bacteria exhibit natural transformation. Otherbacteria can be made competent through artificially inducedcompetence.

High conc. of cold Ca2+ions causes E. coli to becomecompetent at low efficiency.

Electroporation: a technique in which cells are exposed topulsed electric fields to open small pores in their

membranes. DNA present outside the cells can enterthrough these pores. This method works for a variety ofprok. and euk. Plasmids can be transferred directly fromone cell to another because DNA can exit as well as enter

through these pores.


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TransductionTransduction: DNA is transferred from cell to cell via viruses.

A variety of prok. can undergo transduction and a variety ofphages can transduce.

2 types of transductionvirus ends up defective andhomologous recomb. can occur in either case:(1) generalized transduction: host DNA derived from virtuallyany portion of the host genome becomes a part of the DNA ofthe mature virus particle in place of the virus genome.(2) specialized transduction: occurs only in some temperateviruses; DNA from a specific region of the host chromosome isintegrated directly into the virus genomeusually replacingsome of the virus genes.


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Generalized Transduction


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Specialized Transduction


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Phage ConversionPhage conversion: phenotypic alterations made

in a lysogenized cell, can be acquisition of

immunity to further infection by the same type ofphage or can be some other change.

ex. toxin production in Corynebacterium

diphtheriae.


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PlasmidsPlasmids: genetic elements that replicate independently ofthe host chromosome.

Thousands of different types of plasmids are known, almostall of which are dsDNA, most of which are supercoiled andcircular, are vary in size from 1-1000 kbp.

Different plasmids are present in cells in a particular numberof plasmid molecules per cell = copy number, which can

vary from 1-100.Most gram-neg. plasmids replicate similar to the chrom.,although some replicate unidirectionally. Most gram-pos.plasmids replicate by the rolling circle mechanism similar to

a phage.


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Plasmids (cont.)Cells can contain different types of plasmids. A cell in whichtwo plasmids cannot be maintained together are said to beincompatible.

Curing: elimination of a plasmid from a cell. Curing canoccur spontaneously or with the help of chemicals orelectroporation.

Plasmids lack extracellular form.

The main mechanism of cell-to-cell plasmid transfer =conjugation.

Plasmids that govern their own transfer by cell-to-cellcontact = conjugative.


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Types of PlasmidsWhile all plasmids carry genes that ensure their own replication, somecarry genes for conjugation, as well as other unique propertiesconferred upon the cell.

Resistance (R) plasmids: confer resistance to antibiotics and otherinhibitors of growth. These plasmids often transfer resistance to othercells via cell-to-cell contact, resulting in antibiotic resistant populations.R plasmids with genes for resistance to most antibiotics are known.

The following virulence factors of pathogenic bacteria can be encoded

on plasmids:(1) ability of microorganisms to attach and colonize specific sites in thehost(2) formation of substances (ex. toxins, etc.) that cause damage to thehost. What is this similar to that we just discussed?


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BacteriocinsBacteriocins: agents produced by bacteria that inhibit

or kill closely related species or different strains of the

same species. They are different from antibiotics,which have a wider spectrum of activity.

Bacteriocins are often plasmid-encoded.

Bacteriocins are named according to the organism that

produces them.

They can interfere with another cells proton motive

force and, thus, have practical uses such as food

preservatives.


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Conjugation and Chromosome

MobilizationWhat is bacterial conjugation also known as?

Conjugation is a plasmid-encoded mechanism, but canmobilize host chromosome as well.

The F plasmid of E. coli first confirmed the occurrence ofconjugation.

Conjugation involves a donor cell containing a conjugativeplasmid and a recipient cell, which does not. What are

these cells also known as?

Sex pilus: may be specified by the plasmid, allowing forspecific pairing between donor and recipient. The pilusformed by the F plasmid is called the F pilus.


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DNA Transfer During ConjugationDNA synthesis is necessary for DNA transfer to occur.

Rolling circle replication: model best explains DNA

transfer during conjugation. This process is triggeredby cell-to-cell contact.

At the end of the process, both donor and recipient

possess plasmids and the recipient can become a

donor, spreading the plasmids between populations like

infectious agents.


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Transfer of Plasmid DNA by

Conjugation


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Hfr (High Frequency of

Recombination) StrainsF plasmid: conjugative, can integrate into host chromosome (=

episome), and can also mobilize chromosome transfer.

Cells with an unintegrated F plasmid = F+, while those having a

chromosome-integrated F plasmid = Hfr, and cells without and F

plasmid = F-.

Conjugation with Hfr donor transfer of host chromosome.

After transfer, an Hfr strain remains Hfr since it retains a copy of

the F plasmid in the chromosome.

Note: ori= origin, ex. of replication or of transfer


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F PlasmidCells having an F plasmid are able to synthesize and F pilus, mobilize DNA for

transfer to another cell, and alter surface receptors so that the cell can no

longer serve as a recipient.

The F plasmid can integrate into the host chromosome at sites called insertionsequences (IS) . Once integrated, the F plasmid no longer controls its own

replication.

Usually, because of breakage of the DNA strand during transfer, only part of

the donor chromosome is transferred. Although Hfr strains transmit

chromosomal genes at high frequency, they usually do not convert F- strainsto F+ or Hfr because the entire F plasmid is rarely transferred. However, F+

strains can convert F- strains to F+ because the entire F plasmid is

transferred.


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Interrupted MatingRecombinants from conjugation can be selected for.

In an Hfr strain, the transfer of chromosomal genes will always occur inthe same order from a fixed site on a given Hfr strain.

Interrupted mating: interrupt mating pairs by agitation after a certaintime that conjugation has occurred. Genes present closer to the originenter the F- cell first.

This technique leads to genetic mapping since you can determine theorder in which the genes occur by the order in which they are

transferred. Genes at certain points can be referred to as positioned atso many minutes.

Genetic recombination is dependent on the occurrence of homologousrecombination and is not a result of genetic transfer alone.

F = cells in which the F plasmid has been excised from the

chromosome and takes some chromosomal DNA with it.


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Interrupted Mating Experiment


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Other Conjugation SystemsConjugative transposons can be transferred from

the chromosome of a donor to a recipient and

can mobilize other genetic elements.Conjugative transposons are common to gram-

pos. cells.


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ComplementationComplementation is used to determine whether or not two mutations are in the

same gene by restoring function of a gene by complementing the defective

(mutant) gene with a normal (nonmutant) copy of that gene. Homologous

recombination can restore gene function (unless both of the mutations include

changes in exactly the same base pairs) but cannot reveal whether or not the

mutations were in the same gene.

The two mutations are said to complement each other.

Bacterial gene transfer must be done in order to conduct this test.

Complementation does notinvolve recombination.Cistron= genetwo mutations in the same cistron cannot complement each

other.

In diploid organisms: Cis= 2 mutations from the same parent, Trans= 2

mutations from different parent.


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Complementation (cont.)


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Transposons and Insertion

SequencesSome genes are capable of moving under certain conditions. The process by

which a gene moves from one place to another in the genome = transposition.

Transposition is relatively rare.

Not all genes are capable of transposition. Transposition of genes is linked tothe presence of special genetic elements called transposable elements.

There are 3 types of transposable elements in Bacteria:

(1) insertion sequences

(2) transposons

(3) some special viruses (ex. Mu)Transposable elements have 2 features in common:

(1) transposaseenzyme necessary for transposition

(2) inverted terminal repeats and the ends of their DNA.


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Mechanisms of TranspositionTwo mechanisms of transposition:(1) Conservative: the transposable element is excised from onelocation in the chromosome and becomes reinserted at a second

location. The copy number of a conservative transposon remainsat one. Direct repeats are formed in the target site at the ends ofthe transposon.(2) Replicative (ex. bacteriophage Mu): transposons areduplicated and a new copy is inserted at another location. A

composition structure called a cointegrateis formed.Transposition is essentially a recombination event, but one thatdoes not occur between homologous sequences or use thegeneral recombination system of the cell. It is called site-specificrecombinationand involves transposase instead of RecA.


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Mechanisms of Transposition (cont.)


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Mutagenesis with Transposable

ElementsIf the insertion site for a transposable element is

within a gene, insertion of the transposon will

result in loss of linear continuity of the gene,leading to mutation = transposon mutagenesis =

means of creating mutants throughout the

chromosome.


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IntegronsIntegrons = genetic elements that can capture and express

genes from other sources.

Integrons code for integrase, which catalyzes a type of site-specific recombination.

Integrase can integrate gene cassettesand a promoter that

can then express the newly integrated gene cassette.

The genes in the gene cassette that are captured are notcaptured randomly, but have specific DNA sequences

recognized by the integrase and genes that are not

expressed until they become part of an integron.


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Restriction EnzymesProtect prok. from foreign DNA, ex. viruses.

Restriction enzymes recognize certain sequences of DNA and cut the DNA.

Palindrome: sequence of bases that reads the same when read from either

right or left. Palindromes are often the target of REs.Introduce double stranded breaks.

In a random DNA molecule, one would expect any 4-bp sequence to occur ~

once every 256 bps based on the probability of 1/4 X 1/4 x 14 x 1/4.

A 6 bp sequence would appear every 4096 bps in random DNA and a 8 bp

sequence would appear once every 1000 bps, so NotI cuts the E. coli genome(4600 bps) 21x, therefor the recognition sequence for NotI occurs more often

than predicted.

There are over 2000 REs known with over 200 specificities.


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Protection from RestrictionCells protect their own DNA from their REs by

methods such as methylation of their own

sequences that would be targeted by their REs.


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RE Analysis of DNARE analysis is done by gel electrophoresis - whats the

procedure for this?

Can be used to generate a physical map of DNA.Nucleic acids can be purified from gels and used to

transform cells or for nucleic acid hybridization as

nucleic acid probes to find similar sequences from

different genetic elements = Southern blot (RNAhybridization = Northern blot, protein hybridization =

Western blot).


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Sequencing and Synthesizing DNA2 procedures: (1) Maxim and Gilbert method (2) Sanger dideoxy methodBoth methods generate DNA fragments that end at each of the four bases (G,

A, T, C) and that are radioactive.

The fragments are subjected to gel electrophoresis in which 4 sample lanes

are featured, one for each base.

Maxim-Gilbert method: used chemicals that break the DNA preferentially at

each of the four nucleotides.

Sanger dideoxy method: sequence is determined by making a copy of the

ssDNA using DNA pol., which used dNTPs as substrates, adding them to a

primer. The dNTPs feature a dideoxy sugar analog that prevent lengthening

of the chain and acts as a specific chain-termination reagent. Fragments of

variable length are obtained. Either the dNTPs or primer are radioactive. This

method can be used to sequence RNA as well.

Sequencing by the Sanger method is now automated and fluorescent labels

have replaced radioactive ones.


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Molecular Cloning= Gene cloningPurpose: isolate large quantities of specific genes or chromosomal fragments

in pure form.

Basic strategy: move the desired gene or region from a large, complex

genome to a small, simple one.

Tools used: restriction enzymes, DNA ligase, synthetic DNA (see #2

below).

Steps:

1. Isolation and fragmentation of the source DNA.2. Joining the DNA fragments to a cloning vector (ex. plasmid or virus) with

DNA ligase. Blunt or sticky ends may be created on the ends of the source

and/or vector DNA - what does this mean and how do you deal with each?

3. Introduction and maintenance in a host organism. What types of organisms

are used as host organisms (what are their characteristics)?


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Molecular Cloning (cont.)What makes plasmids good cloning vectors?

What is plasmid pBR322 a good cloning vector?

How does insertional inactivation work?


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Polymerase Chain Reaction (PCR)PCR requires that the nucleotide sequence of a portion of the desired gene be

known because short oligonucleotide (- what does this mean?) primers

complementary to sequences in the gene or genes of interest must be

available for PCR to work.

Steps:

1. Two oligonucleotide primers flanking the target DNA are made (how?) and

added to excess to heat-denatured target DNA.

2. As the mixture cools, the target strands anneal mostly to a primer, which are

in excess, and not to each other.3. DNA pol. then extends the primers using target strands as template.

4. After an appropriate incubation period, the mixture is heated again to

separate the strands. The mixture is then cooled to allow the primers to

hybridize with complementary regions of newly synthesized DNA, and the

whole process is repeated.


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Polymerase Chain Reaction (PCR)


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PCR (cont.)Taqpol. is stable at 95C and is unaffected by the denaturation step.

However, it has no proofreading activity.

Pfupol. is stable at 100C and has proofreading activity, and is

therefore more accurate.PCR is often conducted in automated thermocycling machines.

It can be used to amplify very small quantities of DNA present in a

sample.

It is not necessary for the organism to be grown in the lab, so it isimportant for environmental studies.

It can also be used for DNA fingerprinting, a powerful forensic tool

permitting ID of individuals (crime scene/suspects) or relationships

between individuals (paternity testing).


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In Vitroand Site-Directed

MutagenesisIn Vitro= in glass, i.e. in the lab external to the organism, as opposed

to in vivo= in the living organism. In other words, you can remove

genes from and organism, manipulate them, engineer in mutations and

put them back into an organism.Site-directed mutagenesis: Mutations can be introduced at precisely

determined sites on genes.

Mutagenesis studies are often done on at the gene level to make amino

acid changes to study protein structure.

Cassette mutagenesis: segments of DNA can be manipulated in which

synthetic fragments of DNA have replaced the wild-type sequence.

These cassettes can be used for insertion inactivation, causing gene

disruption. How is this done?


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The Bacterial ChromosomeThe entire genome of E. coliK-12 has been sequenced

and has been found to be 4,639,221 bp with 4288 open

reading frames, corresponding to 88% of the genome(what are these and what is the rest of the DNA used

for?).

The map distances on this genome are given in minutes

of transfer, in which 0 time = origin of transfer and 100min. = time the whole chromosome takes to be

transferred from an Hfr strain to an F- strain.


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The Bacterial Chromosome

Bacteriology Chapter 10 Part i i

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