Bacterial Genetics

Question 1

What was the first gnetically modified organism?

Answer 1

1973 - DNA from one antibiotic resistant bacterium, transferred to another.

1981 - rabbit genes spliced into mouse genome.

1990 - Ashani Desilva - gebe therapy for weak immune system.

1994 - Flavr Savr tomato - fruit engineered to more resitant to rottting.

Question 2

What is the bacterial genome?

Answer 2

Chromosome (nucleoid)

• Extrachromosomal genetic elements (if present)
• Plasmids
• Mobile genetic elements
• Transposons
• Insertion sequences

Question 3

What are Bacterial Chromosomes?

Answer 3

• Single, naked, closed circular double stranded chromosome
• Lives freely in the cytoplasm, central region of cell
• Extensively folded and twisted (supercoiled) to fit into the dimensions of the cell

Mycoplasma
500 kb

E. coli
5000 kb

Brucella, Leptospira, Burkholderia, Vibrio spp
Multiple chromosomes

Borrelia, Streptomyces
Linear chromosomes
Linear and circular plasmids

Question 4

What are Plasmids?

Answer 4

Extrachromosomal genetic element, obligate endosymbionts of bacteria with no independent extra-cellular form.

Circular double-stranded DNA molecules, 1-2kb to >1Mb
May also be linear

May exist independently in cytoplasm or integrated into the chromosome

Autonomous – self replicating, replication synchronously with chromosome

Inherited by daughter cells

May be present in multiple copies

Question 5

What are the mobile genetic elements?

Answer 5

Transposons
Insertion sequences

Do not exist as separate entities within the cell, but are incorporated into plasmids and/or chromosome

Ability to move from one location to another
Within the chromosome or plasmid
From plasmid to chromosome and vice versa

Question 6

Functions of gene is bacteria

Answer 6

Morphology, physiology, biochemistry

Replication

Pathogenicity
Ability of a microorganism to maintain viability, adapt, multiply and cause disease

Question 7

Function of Plasmid Genes?

Answer 7

Protection of the Bacterial Cell
- Resistance to antibiotics

Virulence Genes
- Exotoxin production

Genes which code for formation of sex pilus
- Fertility factor, transfer factor

Biochemical properties
- N2 fixation

Question 8

Function of Mobile element genes

Answer 8

Simple genes required for movement among plasmids and chromosomes

Transposons
- Genes that encode other features e.g. drug resistance (TEM2 ß-lactamase)
- Can be recognized phenotypically

Insertion Sequences
- They consist of a unique central sequence flanked by short inverted repeat (IR) sequences or palindromic sequences

Question 9

What are the steps in DNA Replication?

Answer 9

Question 10

Trasncription in bacteria

Answer 10

Question 11

Translation in bacteria

Answer 11

Question 12

What are the 2 genetic changes associated with bacteria?

Answer 12

• Adaptation
  Temporary variability in response to environmental changes
• Mutation
  Genetic change

Question 13

Describe Mutations

Answer 13

Mutation means: “to change” affecting protein end product

• Wild Type: naturally occuring form of gene
• Mutant: progeny with mutation
• Mutagenesis: process of mutation
• Mutagen: agent inducing mutations

Types of Mutations:
-Lethal – kills the organism

-Silent / Neutral – no observable phenotypic change

-Beneficial / Expressed – providing organism with survival advantage

Question 14

What are Point Mutations?

Answer 14

• Addition
• Deletion
• Substitution

There are 2 types:-
- Base pair substitutions – in which a single base pair is altered
- Frame-shift mutations – in which one or few base pairs have been added or removed

Question 15

What are macrolesion mutations?

Answer 15

Macrolesions are alteration of DNA involving large numbers of base pairs

There are 4 types: -
Deletions
Duplications
Inversions
Insertions

Question 16

What is the purpose of Genetic exchange in bactera?

Answer 16

Question 17

How is Genetic Exchange and Genetic Diversity achieved?

Answer 17

Question 18

What is Genetic Recombination?

Answer 18

The RecA gene is a fundamental gene in prokaryotes that encodes the RecA protein, a key player in DNA repair, homologous recombination, and stress responses

Question 19

What are Integrons?

Answer 19

Site specific recombination systems that mediate the movement of small DNA elements called gene cassettes.

Integrons capture open reading frames (genes) from mobile elements called gene cassettes.

They provide a platform to express these captured genes,

Essential components of an integron are:-
- Integrase (int) gene – responsible for integration
- Attachment (att1) gene – used by integrase to capture gene cassettes.
- Sul1 gene – for resistance to sulphonamides

Sulphonamides, also known as sulfa drugs, are a class of synthetic antibiotics that work by inhibiting folic acid synthesis in bacteria.

Question 20

Mechanisms of Gene Acquisition?

Answer 20

Question 21

What is Transformation?

Answer 21

Question 22

How is transformation done?

Answer 22

Natural occurring transformation – seen in S. pneumoniae, B. subtilis and H. influenzae

Artificial competence may be induced in vitro by:
Treating cells (E. coli) with salt solutions (CaCl2) at 00C
Electroporation - subjecting bacterial cells to electrical charge+ to open pores for accepting DNA

Used in genetic engineering to introduce novel genes (even human) into plasmids

Question 23

What is Transduction?

Answer 23

Gene transfer between bacteria can also be mediated by bacteriophages, in which the virus acts as a genetic vector (bringing foreign DNA into the cell).

When the virus infects another bacterial cell, it releases all DNA contents and bacteria becomes the recipient of donor DNA which can be recombined into its genome.

After integration of DNA into chromosome, a phage not only packages its own DNA but also a portion of donor bacterium’s DNA.

Types:
Bacterial DNA may be randomly incorporated into viral DNA (generalized transduction).

Bacterial DNA that was adjacent to viral DNA in the bacterial chromosome is packaged (specialized transduction).

Toxin production in C. diphtheriae and S. pyogenes are examples

Question 24

What is Conjugation?

Answer 24

Conjugation is transfer of DNA from one bacterial cell to another requiring cell to cell contact and mobilization to donor bacterium’s chromosome.

Capacity to donate genetic information depends on possession of the plasmid fertility (F) factor, which allows synthesis of a sex pilus for transfer into a recipient (F-) cell.

Contact is made when thin protein filaments grows out form F+ cell and attaches to surface of F- cell.

Cells are pulled together for direct contact and replicated DNA passes through the pillus.

Question 25

What are the three conjugation patterns in E. Coli

Answer 25

Donor F+ cell makes a copy of its F factor and transmits this to a recipient F- cell, converting it to F+. If the F factor is stably recombined into bacterial chromosome, high frequency recombination (HFr) occurs, in which chromosomal genes are transferred to all recipient cells and high numbers of recombinants are seen. F factors may excise from chromosomal DNA and become a plasmid, taking along pieces of chromosomal DNA on either side and is referred to as a plasmid primer.

Question 26

What is the importance of Genetic Transfer?

Answer 26

Resistance - The emergence of resistance following widespread use of a new antibiotic and its subsequent dissemination among many bacterial species attests to highly efficient gene transfer processes within populations of bacterial cells.

Question 27

What is Genetic Engineering?

Answer 27

Recombinant DNA technology allows: - - Isolation of genes - Manipulation of gene - Re-insertion of genes into bacteria either modified or unmodified

Question 28

What is Restriction Endonuclease?

Answer 28

- Enzymes that cleave dsDNA at specific nucleotide sequences. - Genome is reduced to small linear pieces that can be identified and manipulated. - Recombination then allows bacteria to accept, replicate and express foreign DNA thus cloning the required gene.

Question 29

What are the applications of genetic engineering?

Answer 29

-Transgenic (genetically recombined) microbes, plants and animals -Gene therapy and genetic medicines - Genetic mapping and analysis