L4: Microbial Genetics and Drug Resistance Flashcards by Ahmed Farag

What is the definition of the genome?

It is the total genetic information in an organism.

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What are the characters of the prokaryotic genome?

 Consists of a single copy (Haploid) circular DNA molecule.

 Range from 580-4600 Kbp

 Many bacteria contain extrachromosomal DNA materials as a part of the genome called plasmids and transposons.

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What is the definition of bacterial extrachromosomal elements?

These are the DNA material present in a cell other than chromosomal DNA
The most famous extrachromosomal DNA are:
A. Plasmid.
B. Transposons.
C. Bacteriophage (virus infecting bacteria)

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What is the definition of plasmids?

Plasmids are pieces of DNA that exist separate from the chromosome, they contain an origin of replication so they replicate independently.

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What are plasmids classified according to?

1- According to the size of the plasmid 
2- According to copy number
3- Shape of plasmid
4- Moving plasmid from cell to cell 
5- Artificial and natural plasmids
6- according to host range
7- according to compatibility

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What are plasmids classified according to size?

Starting from a few hundred base pairs up to 3000 Kbp.

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What are plasmids classified into according to copy number per cell?

1- Stringent plasmids

 1-2 copies / cell
 like F- plasmid and phage – plasmid
hybrid (P1)

2- Low copy number plasmids

 10-15/copies/cell
 such as pSC 101

3- High copy number plasmid
 up to 50 copies/cell
 like ColE, plasmid

4- Extremely high copy number plasmid
 these are specifically engineered to be up to 100-200 copies/cell

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What are plasmids classified into according to the compatibility of plasmids?

Compatible plasmids:
➢ a cell can maintain more than one plasmid in the same cell (if they carry
different origins of replication).

Incompatible plasmids:
➢ The inability of two plasmids to be maintained in the same cell (if they carry the same origin of replication

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What are plasmids classified into according to the shape?

The shape of plasmids can be classified into 3 groups

1- Covalently closed circular (CCC) form:
 The most common form
 Present as ds completely closed circular forms (as in E.coli).

2- Semicircular form:
 Transient form
 Present as one strand is completely closed, the other strand is opened.

3- Linear:
 unstable because it is attacked by exonucleases.

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What are plasmids classified into according to host range?

1- Broad host range plasmids:
➢ Can replicate in a wide range of bacteria.
2- Narrow host range plasmids:
➢ only replicate in one or a few closely related bacteria.

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What are plasmids classified into according to moving from one cell to another?

Conjugative plasmids: which have the tra genes that can mobilize the plasmid from one cell to another by conjugation

Shuttle vectors: plasmids that propagate in 2 diff. Hosts species (yeast and bacteria)

Non–conjugative plasmids: Cannot be mobilized under any known conditions

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What are plasmids classified into according to naturallity or artificiality?

1- natural plasmids:
➢ All the above plasmids are present naturally in bacterial and some yeast cells.

2- Artificial plasmids :
➢ are naturally present plasmid but designed artificially
➢ to be used in genetic cloning as vectors
➢ by adding antibiotic-resistant markers or DNA sequences to be the target of
restriction endonucleases.

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What is the importance of plasmids?

Resistance:
 Antibiotic resistance.
 Heavy metals (metal reductase).
 U/V (DNA repair enzymes).
Conjugation.
Production: Toxins & enzymes and bacteriocin.
Biochemical reactions: Sugar fermentation.
Molecular biology: As a vector.
 Cloning vectors
 Gene therapy: These are plasmids used for the insertion of therapeutic genes to express the protein that is lacking in the cells

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What are transposons?

Extra-chromosomal small pieces of DNA those are capable of moving themselves from one location in DNA to another, (movable elements or jumping genes).

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What are the 3 forms of transposons?

(a) Insertion sequence (IS)
(b) Composite transposons (Tn)
(C) Non – composite transposons

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What are the characters of the insertion sequence?

 The simplest form.

 They encode only proteins needed for their own transposition

 Carry repeated nucleotides at their ends (direct repeats or inverted repeats ~ (15-25).

 Examples: IS 1, 3, and 10.

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What are the characters of composite transposons?

Contain 2 IS at both ends and central piece of DNA which encode for antibiotic resistance, virulence factors.

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What are examples of composite transposons?

Tn5: encodes for kanamycin resistance.

- Tn10: encodes for tetracycline resistance.

What are the characters of non-composite transposons?

 Have no IS at their ends but encode for transposition proteins.
 They carry genes for antibiotic resistance, virulence
factors, and catabolic enzymes.

What are the examples of non-composite transposons?

o Tn 3: carry Ampicillin resistance gene.

o Tn 7: carry streptomycin and trimethoprim resistance

What is the definition of gene transfer?

It is the process of moving apiece of DNA (Either chromosomal or plasmid) from one cell to another using different means.

What are the types of gene transfer?

Vertical Gene Transfer: The Transfer of genetic material from parental organism to progeny

Horizontal Gene Transfer:

known as lateral gene transfer,
organism transfers genetic material to another organism that is not its offspring

What are the types of lateral gene transfer?

a) Conjugation.
b) Transduction.
c) Transformation.

What is the definition of conjugation?

It is a form of gene transfer in which two cells come in direct contact and DNA is transferred from one cell (donor) to the other (recipient).

What are the requirements for conjugation?

 Donor cells should contain F plasmid, which encodes for F pili needed for conjugation.  The donor plasmid should contain tra gene(conjugative type) to mobilize the plasmid.

What are the steps of conjugation?

1- Donor Gram-negative bacilli containing F plasmid come in contact with other F-Gram negative bacilli. 2- The F pili of the donor cell (F +) attach to a specific receptor on the recipient cell (F -). 3- The F pili contract the recipient cell to be in close contact and then canalization occurs through the F pili between the two cells. 4- Once the canal is formed, the F plasmid starts to mobilize one strand of its ds DNA to the recipient cell. 5- New ds DNA is formed from the single strand in both donor and recipient cells so the recipient now contains F plasmid and changed to F+ cell which will act as a donor cell.

What is the method of conjugation in gram-positive organisms?

mediated by signaling molecules called pheromones.

Equation of conjugation

F+ + F- = 2F+

What is the HFr and what are its characteristics?

* If the F plasmid integrates into loci in the chromosome, this integrated F factor creates a high frequency of recombination cell (Hfrcell). * If this integrated plasmid is transferred to another cell by conjugation, it can transfer a segment (locus) of the chromosome during excision and can transfer this locus (gene or genes) from the donor cell (Hfr) to a recipient chromosomal cell

Where does conjugation mostly occur?

Conjugation most frequently occurs in Gram-negative bacilli (only gram-negative have pili) but Gram-positive organisms can make conjugation.

What is the definition of transduction?

It is a form of gene transfer in which bacteriophage (a virus that infects a bacterial cell) mediate the DNA transfer.

What are the types of transduction?

1. Generalized transduction | 2. Specialized transduction

What are the bacteriophage types in transduction?

1- Virulent phage (Lytic cycle) | 2- Tempratephage (lysogenic cycle)

Which type of cycle happens in generalized transduction?

This occurs with the lytic cycle of Bacteriophage (virulent phage).

How does generalized transduction take place?

- The phage infects a bacterial cell and replicates, any piece of bacterial DNA can be packaged inside the phage head. - By cell lysis and release of phages, it can infect another bacterial cell transferring the chromosomal DNA to the recipient cell.

Which type of cycle happens in specialized transduction?

This occurs with the temperate or lysogenic bacteriophage

How does specialized transduction take place? And why is it called "specialized"?

the phage DNA become integrated inside the bacterial DNA at a specific region, When the integrated phage is excised with a piece of bacterial chromosome and infect another bacterial cell transferring this DNA into the recipient cell. (so it is called specialized)

What is the definition of transformation?

It is a method of gene transfer in which direct uptake of DNA by recipient cell either naturally or artificially in the laboratory

What are the methods of transformation?

- Natural transformation - Laboratory induced competence - Transformation by electroporation

Is natural transformation rare or common and give an example for an organism that works with it?

rare occasion e.g., Streptococcus pneumoniae

What are the methods of laboratory-induced competence?

(increasing the permeability of the cell envelopes) by:  Electroporation (exposing a mixture of recipient cells and plasmids to the electrical field to form pores in the cell envelopes)  Adding calcium chloride solution and chilled on ice then heat-shocked

What is the definition of transformation by electroporation?

(by exposing a mixture of recipient cells and plasmids to an electrical field to form pores in the cell envelopes).

Equation of HFr cells

HFr cell + F- = HFr cell + F-