Week 3 - Microbiology - Bacterial Genetics

Question 1

Bacterial Chromosome

Answer 1

Circular dsDNA

Question 2

Plasmids

Answer 2

Circular dsDNA (ds double stranded)
Extremely important
Virulence factors located in plasmid

Circular dsDNA
5-100 genes
1-20 copies per cell
Replicates independent of the chromosome
May be transferred from one cell to another.
May contain toxin genes or antibiotic resistance genes, some on transposable elements

Question 3

Bacterial Chromosome

Answer 3

Circular dsDNA
2,000-4,000 genes
1-2 copies per cell
Replicates in concert with cell growth and division
Carries the genes for the all of the functions of the cell

Question 4

Mutation

Answer 4

Arise because of things in physical environment

It effects the chemical nature of nucleic acids and bases

Question 5

Mutagens

Answer 5

Certain man-made molecules can increase the frequency of mutations

Question 6

Transformation

Answer 6

Transfer of naked bacterial chromosomal DNA from dead cell to living cell

Question 7

Conjucation

Answer 7

Transfer of bacterial chromosomal DNA through a pilus from living cell to living cell

Question 8

Transuction

Answer 8

Transfer of bacterial chromosomal DNA in a bacterial virus (phage) from a dead host cell to a live host cell

Question 9

Transposition

Answer 9

Transfer of DNA sequences from one DNA site to another which may be on a plasmid

Question 10

Transformation Steps

Answer 10

When a bacterium dies, its DNA is released into the medium.

Another bacterium may take up that DNA by a process called transformation.

The recipient cell may incorporate that
DNA into its DNA, exchanging its genes for the other bacterium’s genes

ONLY CERTAIN bacteria can go through transformation.. there is a confidence factor..

Question 11

How is tranformation used in recombinant DNA work?

Answer 11

The gene (human gene?) in question is placed in a plasmid and added to a bacterial suspension under conditions that are favorable for DNA uptake.

The recipient bacterium takes up the plasmid DNA bearing the gene in question and expresses the gene. Use the bacteria to produce the gene*

The gene product (for example, IFN- or IFN-) is harvested and employed clinically or for some other purpose. So put gene into bacterial…then infect pts with bacteria..

Question 12

Conjugation - Male vs Female bacteria..

Answer 12

Conjugation occurs between male and female bacteria.

• Male bacteria have an F factor and express F pili on their surface.
• Female bacteria have no F factor and no F pili

Question 13

Conjugation - What is F factor, + vs -

Answer 13

The F factor be found in one of several different states.

• It may be integrated into the host bacterial chromosome: Hfr bacterium. (Hfr = high frequency recombination)
• It may be free in the cytoplasm: F+ bacterium.
• In rare conditions, a previously integrated F factor may excise itself incorrectly from the host bacterial DNA and pick up a piece of host DNA: F’ bacterium

Question 14

Hfr Conjugation

Answer 14

In Hfr bacteria, the F factor is integrated into the host chromosome at one of a very few particular points.

Upon mating with an F- bacterium, the Hfr begins replicating the host bacterial chromosomal DNA, beginning just downstream of the integrated F factor.

The newly replicated DNA is transferred to the F- bacterium.

Thus, the Hfr bacterium transfers first host bacterial chromosomal DNA. Only if conjugation continues for 90 minutes does the F factor get transferred to the F- bacterium, converting it to an Hfr bacterium

Question 15

How does Hfr conjugation occur?

Answer 15

A male bacterium that contains integrated F factor approaches a female bacterium that has no F factor and attaches by an F pilus.

The F factor activatesreplication of the DNA and transfers the DNA either across the F pilus or by means of a cytoplasmic bridge.

The transferred DNA can be incorporatedinto the recipient chromosome, replacing its own DNA

Question 16

F + Conjugation

Answer 16

An F+ bacterium has an F factor that is free in the cytoplasm.

Conjugation occurs as was seen for the Hfr bacterium, except that only the F factor is exchanged, converting the F-bacterium to an F+ bacterium.

If a single F+ bacterium is introduced into a culture of F- bacteria, essentially ALL of the bacteria will become F+

Question 17

F’ Conjugation

Answer 17

Occasionally, an integrated F factor will excise itself from the host bacterial chromosome to become an F+ bacterium.

If the excision is incorrect, a bit of host bacterial DNA may be excised as part of the F factor.

An F factor carrying a piece of host chromosomal DNA is called an F’ factor.

The bacteria with a F’ factor are called F’ bacteria.

Because F factors integrate at only a few specific sites on the host bacterial chromosome, the piece of DNA excised is limited to specific host genes

Question 18

Why do F factors integrate at Specific Sites

Answer 18

Has to do with transposons…

Transposons are sequences of DNA that can recognized complementary sequences in other DNA and hop from one complementary sequence to another (mobile genetic elements).

Bacterial chromosomal DNA (and our DNA) has sequences recognized by transposons.

F factors contain a transposon.

Thus, F factor can use the transposon to integrate into the host DNA, but there are only a limited number of sites in bacterial DNA for the F factor transposon

Question 19

Transposons frequently have the genes for antibiotic resistance on them

Answer 19

Some genes are resistant to multiple antibiotics..

Add pic of this…

As long as they have the correct sequences then the genetic info can jump back and forth between transposon and plasmid..

If have F factor, can transfer the antibiotic resistance to other bacteria..

Question 20

Resistance transfer factors are similar to F factors

Answer 20

Resistance transfer factors (RTFs) are plasmids that contain transposons. Essentially are F factors containing antibiotic resistance genes..

They can be exchanged by conjugation, similar to F factors.

The RTFs get their name because they can contain genes for resistance to antibiotics.

Thus, resistance to antibiotics can be rapidly transferred from one bacterium to another by conjugation

Question 21

How to get different Types of Antibiotic Resistance Genes (3 ways)

Answer 21

By altering the number of porins in their outer cell membranes, certain Gram - bacteria can decrease uptake or increase efflux of an antibiotic.

By altering the target site for the antibiotic, the antibiotic is rendered ineffective (ex. Change in penicillin binding protein reduces ability of penicillin to bind to its target).

By acquiring the ability to destroy or modify an antibiotic, the antibiotic is rendered ineffective (-lactamase destroys penicillin and acetyltransferases alters some antibiotics)

Question 22

A number of genes for resistance occur on what?

Answer 22

The plasmid…

Question 23

Summary of Conjugation

Answer 23

Genes may be transferred from one bacterium to another, without the death of the donor bacterium through the action of a plasmid such as the F factor or the resistance transfer factor (RTF) that confers maleness.

The F factor can exist as a free plasmid or integrated into the bacterial chromosome.
If the F factor is a plasmid (F+ bacterium), the F factor alone may be transmitted.
If the F factor is integrated into the bacterial chromosome (Hfr bacterium), the chromosomal genes alone are transmitted.
If the F factor was integrated and, upon again becoming a plasmid, picked up a few host chromosomal genes (F’ bacterium), the F’ factor carrying those few host chromosomal genes is transmitted.
The RTF is a free plasmid carries genes encoding resistance to certain antibiotics. If it is transferred by conjugation, the recipient bacterium now becomes resistant to those antibiotics

Question 24

What are two types of bacteriophages?

Answer 24

Virulent
Temperate

Diptheria bacter with epsilan phage in it it will sequence and makes toxin and makes disease.. if get phage out of bacteria the bacteria cant cause disease.. .

Question 25

Virulent Phages (bacteriophages)

Answer 25

*These phages infect a bacterium and replicate, killing the bacterium in the process

Each bacterial has unique set of phages

Phages start to replicate in 20 min after cell wall is breached..

replication cycle of the phage uses phage encoded genes to take over control of the host bacterial biosynthetic machinery to make hundreds of progeny

Replication involves separate synthesis of viral genome and viral proteins, with self-assembly creating progeny phages

The phage then lyses the bacterium, releasing hundreds of progeny phages.

In the process of their replication cycle, the phages may degrade bacterial chromosomal DNA to large or small pieces.

These phages may give rise to generalized transduction - if it degrades bacterial chromosomal DNA - and going to assemble phage, you can get a piece of bacterial DNA stuck into the phage DNA - creating defective phage.. so they cant infect a host cell but NOT replicate…

Question 26

Virulent Phage Replication steps

Answer 26

Attachment
Penetration
Transcription
Translation
Nucleic Acid Replicaton
Phage Assembly
Bacterial Lysis
Phage Release

Question 27

Temperate Phages (lysogenic phage)

Answer 27

These phages infect a bacterium and can either replicate, killing the bacterium (lytic cycle) or integrate into the bacterial chromosome to be replicated along with the bacterial genome (lysogenic cycle).

Another name for a temperate phage is a lysogenic phage.

The integrated phage is called a prophage.

The bacterium with an integrated prophage is called a lysogenic bacterium and is said to be lysogenized.

The integrated prophage is repressed by a phage protein and will stay repressed indefinitely, unless the phage protein is degraded by something like UV-light or heat.

If the repressor protein is degraded, the prophage cuts itself out of the host chromosome by a process called induction and begins to replicate in a lytic cycle.

These phages may give rise to specialized transduction. Can only pick of specialized genes from the bacterium.. High probability of specific genes to be transferred

Diptheria is usually a lysogenic phage

Question 28

Diptheria is a __________ phage infected..

Answer 28

Temporate (lysogeny)

Question 29

Specialized Transduction vs Generalized Transduction

Answer 29

Specialized have High probability of specific genes to be transferred

Generalized have EQUAL probability of specific genes to be transferred

Question 30

Temporate Phage Infection Steps

Answer 30

Attatchment
Penetration
Limited transcription
Limited translation
Prophage in lysogenic state
Lytic cycle begins

Question 31

Generalized Transduction

Answer 31

Lytic phage partially degrades host bacterial chromosome during replication cycle.

Pieces of host bacterial chromosome are randomly (and accidentally) encapsidated into phage virions.

These transducing phage virions can enter a second bacterial host and bacterial DNA from the first host can be incorporated into the chromosome of the second host.

Because of the random nature of the encapsidation of host bacterial DNA, any gene can be transduced from one bacterium to another: generalized transduction

Question 32

Specialized Transduction

Answer 32

Lysogenic phages integrate at specific sites in the host bacterial chromosome.

When the prophage is induced to excise itself from the host bacterial chromosome, on rare occasions, the phage inaccurately excises itself from the bacterial chromosome, picking up a piece of the host bacterial chromosome that now becomes a part of the phage genome.

The phage with bacterial DNA replicates and is encapsidated.

A second bacterium is infected by this phage and the phage and the bacterial DNA from the first bacterial host are integrated as a prophage in the second bacterial chromosome.

Because the prophage inserts at a specific point in the host bacterial chromosome, it picks up a specific host gene from the chromosome upon inaccurate excision: specialized transduction

Question 33

Transposition

Answer 33

Are moveable genetic elements incapable of independent replication.
Can involve transposons that contain insertion sequences (ISs),a transposase enzyme gene, and other genes related to antibiotic resistance or virulence.

Can involve integrons that consist of ISs, a transposase enzyme gene, and a series of antibiotic resistant genes controlled by a promoter.

Can involve pathogenicity islands that consist of ISs, a transposase enzyme gene, and groups of virulence-associated genes

Question 34

Pathogenicity Islands*

Answer 34

Are transposons that consist of transposon stuff plus groups of virulence - associated genes

*They are components of GRAM NEG bacteria

Question 35

Genomic (Pathogenicity) Islands

Answer 35

Add pic..

Cassets.. multiple resistance cassets being placed on chromosome or plamid…

Question 36

So resistance genes either exist on..

Answer 36

Phage
or on the
bacterial chromosome in a pathogenicity island*

Question 37

Pathogenicity islands are located…near

Answer 37

tRNA or tRNA like sequences on genes*

Question 38

Pathogenicity Island common factors

Answer 38

Presence of virulence genes

Specific presence in pathogens, absence in benign relatives

Large distinct chromosomal regions (10 to 200 kb)

Characteristic base composition different from core genome

Insertion of PAI adjacent to tRNA genes

Frequent association with mobile genetic elements, i.e., presence of: DR, cryptic or functional integrase or transposase, IS elements, and chromosomally integrated conjugative transposons, plasmids, and phages

Genetic instability (if functional mobility elements are present)

Mosaic structures of several acquisitions

Question 39

Pathogenicity Islands are components of what type of bacteria?

Answer 39

Gram NEG (usually)

Question 40

So there are virulence factors associated with what ?

Answer 40

Pathogenicity Islands..

Question 41

If you can get rid of pathogenicity Island then…

Answer 41

Renders bacteria ineffective

Question 42

What is the only gram POS bacteria that has a pathogenicity island?

Answer 42

Staphylococcus Aureus

Question 43

Gene Cloning..

Answer 43

Plasmids can be used as cloning vectors

There are several plasmids that are currently in use.

We will discuss a gene cloning method that uses a derivative of the pUC plasmid

Question 44

Key features of a Cloning Vector..

Answer 44

It must encode antibiotic resistance genes for use in selection against non-transfected bacteria and against non-transfected eukcaryotic cells.

It must encode replication initiation sites for prokaryotes and eukaryotes.

It must encode a recombination site for adding your gene of interest.

It must encode transcription initiation sites that can be read by prokaryotes and eukaryotes

Question 45

Cloning Prodedure

Answer 45

Obtain gene of interest by reverse transcription

Insert the gene in cloning site of vector

E. coli is transfected with vector and select against non-transfected E.coli using ampicillin

Extract vector

Use sample

Question 46

Using transposons is the basis for

Answer 46

commercial cloning.. no questions on cloning.

Question 47

Bactofection

Answer 47

instead of injecting genetic material from viruses or transponon.. use bacteria etc… jjklsfjaklja