Bacterial genetics

Group 15

Objective

By the end of this presentation we expect you to

know:

About bacterial chromosome and plasmids

About translation and transcription

Replication

Genetic variation in bacteria

Uses of bacteria in genetic engineering

Introduction

Genetics is the study of genes including the

structure of genetic materials, what information is

stored in the genes, how the genes are

expressed and how the genetic information is

transferred.

Genetics is the study of heredity and variation.

Two essential function of genetic material is

replication and expression.

The genome of an organism is defined as the

totality of its genetic material.

Genotype vs. phenotype

Introduction

Bacteria are one of the three domains in

taxonomy, they are also prokaryotes;

Bacteria store DNA in two places the

chromosome and the small circular plasmid.

Bacteria’s DNA is about thousand times the size

of the cell.

Has both DNA and RNA

Structure of bacterial genetic materialChromosome

Most bacteria's has a single haploid circular chromosome. But some may have more than one:

For example, Vibrio cholerae and Brucella melitensis,leptospira, have two or more dissimilar chromosomes. There are also exceptions to this rule of circularity because some prokaryotes (eg, Borreliaburgdorferi and Streptomyces coelicolor) have been shown to have a linear chromosome.

Plasmids

are autonomously replicating DNA molecules of varying size located in the cytoplasm. Could contain transposons,

Virulence plasmid

plasmid

Resistance plasmid

Significance of plasmids:

1. Codes for resistance to several antibiotics. Gram-negative

bacteria carry plasmids that give resistance to antibiotics such

as neomycin, kanamycin, streptomycin, chloramphenicol,

tetracycline, penicillins and sulfonamides.

2. Codes for the production of bacteriocines.

3. Codes for the production of toxins

4. Plasmids carry virulence determinant genes. Eg, the plasmid

Col V of Escherichia coli contains genes for iron sequestering

compounds.

5. Codes resistance to uv light (DNA repair enzymes are coded

in the plasmid).

6. Codes for colonization factors that is necessary for their

attachment.

7. Contains genes coding for enzymes that allow bacteria unique

or unusual materials for carbon or energy sources. Some

strains are used for clearing oil spillage.

Replication

Transcription

Transcription enables the DNA to direct the

synthesis of RNA,

Types of RNA:

mRNA rRNA

tRNA snRNA

siRNA miRNA

The process will be explained in the next slide

Initiation

Termination

Translation

Initiation

elongation

From class lecture slide

DNA damageCauses

Endogenous –e.g. Reactive oxygen (metabolic byproducts)

Exogenous - external agents (radiations)

Types of DNA damage:• Deamination: (e.g. C->U )• Depurination : ( purine base A or G lost)• T-T and T-C dimers: (bases become cross-linked)

• Alkaylation : ( an alkyle group e.g. CH3 added to base

• Oxidative damage: (guanine oxidizes to 8 –oxo-guanine• cause SS and DS breaks

• Replication error: wrong nuceotide or modified neuclotideinserted

• Double strand breaks (DSB): induced by ionizing radiations, transposons,, endonucleases, mechanical stress, etc

Repair against damage

Damage caused by UV

Mismatch

Proofreading

Nucleotide Excision repair

SOS response ( there are over set of 17 genes

that are involved tin this)

Base excision repair

mutation

Mutation is random it can happen to any gene

and to any bacteria.

But in bacteria it may seem like the environment

has a role in changing the genotype but it

doesn’t.

Lederberg’s concept

There are different types of mutation

Mutation on a coding stand

Point mutation Nonsense

Missense

Frame shift

Lethal mutation

Conditional

Resistance mutation

Auxotrophic mutation

Reversion

suppression

Mutation on noncoding strand

Polar mutation

Mutation on enhancer, promoter,

Genetic exchange

Bacteria can not adopt simply because of

mutation, because mutation is to rare, they need

other system.

Sharing of genetic material

Donor Vs. recipient, exogenoate Vs.

endogenoate

This along with mutation is the cause of genetic

variation amongst bacteria's.

A type of horizontal transfer

Transformation

Transformation involves the uptake of free or

naked DNA released by donor by a recipient. By

using surface proteins that recognize and snatch

naked DNA.

The competence is determined by genes that

become active under certain conditions. But can

also be artificial.

N.B. not all bacteria’s are capable of carrying out

only competent bacteria’s like ( Bacillus,

Haemophilus, Neisseria, Pneumococcus) can.

The classic example for transformation is the

griffith experiment.

steps The steps involved in transformation are:

1. A donor bacterium dies and is degraded.

2. A fragment of DNA (usually about 20 genes long) from the dead donor bacterium binds to DNA binding proteins

on the cell wall of a competent, living recipient bacterium.

3. Nuclease enzymes then cut the bound DNA into fragments.

4. One strand is destroyed and the other penetrates the recipient bacterium.

3. The Rec A protein promotes genetic exchange (recombination) between a fragment of the donor's DNA and the

recipient's DNA.

Conjugation Bacterial conjugation is the transfer of DNA from

a living donor bacterium to a recipient bacterium.

It requires intimate cell contact

The male donates its plasmid to the female

Conjugate(f) plasmid directs it’s own process, of conjugation

Because it has tra genes.

Transfer replication

Plasmid mobilization

There are various types

Of conjugation

Conjugation Bacterial conjugation is the transfer of DNA from

a living donor bacterium to a recipient bacterium.

It requires intimate cell contact

The male donates its plasmid to the female

Conjugate(f) plasmid directs it’s own process, of conjugation

Because it has tra genes.

Transfer replication

Plasmid mobilization

There are various types

Of conjugation

F+ conjugation

In which plasmid called F plasmid induces f pilli,

formation, and enzymes involved in the process

and the donor bateria gives F plasmid to the

recipient.

the bacterial plasmid to be transferred will be

separated in to two single strands, one to be

exchanged the other will stay with in the cell and

both of them will replicate to be double stranded

DNA,

Exchange occurs across the conjugation tube

Then both bacteria’s will be F+

Hfr conjugation Plasmids may integrate into the bacterial

chromosome by a recombination event depending upon the extent of DNA homology between the two. The plasmid is called episome.

And forms HFR cell because they are able to pass both chromosomal and plasmid genes.

Process is their will be a nick around the area of origin of transfer, and the chromosome will be replicated, and pass through the conjugation tube to the F- cell but most of the time the conjugation bridge collapse.

The integration of the chromosome with the plasmid isn’t stable so the chromosome and the plasmid separate, but through this process the plasmid might take some genes of the chromosome along with it.

Transduction

Transduction is virus-mediated transfer of genetic

information from donor to recipient cell.

This is mediated by bacteriophages.

Bacteriophage’s can be of two types

Lytic

Lysogenic

Depending on the type of bacteriophage used

transduction can be classified as general and

specialized transduction.

General transduction

Lytic bacteriophage infects

the bacteria, the process is

descibed in the image to the

right.

Pseudovirus:- virus that

contains bacterial DNA

Happens 1 in 1000 virus

formed

Specialized transduction

Caused by lysogenic bacteria

The viral DNA attaches to a particular site of the

chromosome, and it may drag along with it the

genes next to it but this is of low probability but as

this integration replicates it becomes of high

probability and then when it infects other bacteria

it would have that gene.

Is valuable for sequencing genes, knowing about

the function and regulation of that gene, cloning

and antibiotic resistance.

transposable DNA elements

Transposable genetic elements are segments of

DNA that have the capacity to move from one

location to another

They synthesize transposase protein that aids

transposition,

The three major kinds of transposable elements

are insertion sequence elements, transposons

and transposable bacteriophage;

Insertion sequences

Transposon are like insertion sequences but they

also cary additional genes between the

transposition gene/ IS segment.

They are important because they might carry

genes for antibiotic resistance

While IS can deactivate a gene by inserting with

in it.

Direct transposition vs. replicative transposition

One such conjugative transposon is Tn916,

found originally in a strain of E. faecalis.

Mechanism of drug resistance Decreased uptake (or increased efflux) of antibiotic: For

example, gram-negative organisms can limit the penetration of certain agents, including β-lactam antibiotics, tetracyclines, and chloramphenicol, as a result of alteration in the number and structure of porins (proteins that form chanels) in the outer membrane.

Alteration of the target site for antibiotic: For example, Staphylococcus pneumoniae resistance to β-lactam antibiotics involves alterations in one or more of the major bacterial penicillin-binding proteins (see p. 75), which results in decreased binding of the antibiotic to its target.

Acquisition of the ability to destroy or modify the antibiotic: Examples of antibiotic inactivating enzymes include: 1) β-lactamases that hydrolytically inactivate the β-lactam ring of penicillins, cephalosporins, and related drugs; 2) acetyltransferases that transfer an acetyl group to the antibiotic, inactivating chloramphenicol or aminoglycosides; 3) esterases that hydrolyze the lactonering of macrolides.

Genetic engineering

Development of vectors or vehicles allowing the

cloning of any DNA sequences

Eucaryotic genes may be expressed in

procaryotic systems

Many genetic diseases are caused by lack of

protein

Production in bacteria of recombinant vaccines

Replacement therapy - bacterial interference

Molecular technologies in diagnosis

Use of nucleic acid (DNA) probes to diagnose

and study diseases

DNA of interest is inserted to bacterium and

amplified to high copy numbers and labeled - in

situ hybridization

PCR - generation of millions copies of specific

pieces of nucleic acid of suspected

microorganism

References

Prescott’s microbiology

Sherri’s microbiology

Lippincott's illustrated review of microbiology

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