Bacterial Genetics

Dr AAROHI PATEL

Objectives:

• Definitions• Structure & functions of genetic material• Protein synthesis• Transformations• Mutation• Types of mutations• Transduction• Conjugation

DEFINITIONS

Genetics: The study of heredity and variation, of the reasons for similarity and differences between parents and their offspring.

Gene :The unit of hereditary is the gene, a segment of DNA that carries in its nucleotide sequence information for a specific biochemical or physiological property.

Genome: The sum total of all the genetic information contained in a cell is its genome.

• DNA: or Deoxyribonucleic acid - the principal molecule responsible for the storage of genetic information.

• Chromosome: The single largest DNA molecule in a cell that contains the information necessary for the cell’s survival.

• Replicon: Any segment of DNA that is capable of encoding its own replication in the cell. Thus, the chromosome is the single largest replicon in the cell.

• Plasmid: Any extra-chromosomal DNA that is capable of encoding its replication. In other words, non chromosomal replicons.

• PHENOTYPE:-Characteristics expressed by a cell in a given environment. Adaptable in response to environment changes.

• GENOTYPE:-Collection of genes encoding the characteristics. Remains Unchanged.

• RECESSIVE :- A gene that does not achieve phenotypic expression in the presence of its homologue is recessive.

• DOMINANT:- A gene that over rides the effect Of its homologue is dominant.

Structure and Function of Genetic Material:

DNA & RNA•DNA=deoxyribonucleic acid•RNA=ribonucleic acid•Basic building blocks:

•Nucleotides•Phosphate group•Pentose sugar•Nitrogenous base

Structure of DNA

• Double stranded (double helix)• Chains of nucleotides• 5’ to 3’ (strands are anti-parallel)• Complimentary base pairing

• A-T• G-C

DNA Structure:

Phosphate-PSugar-blueBases-ATGC

DNA Replication:

• Bacteria have closed, circular DNA• Genome: genetic material in an

organism• E. coli

• 4 million base pairs• 1 mm long (over 1000 times larger

that actual bacterial cell)• DNA takes up around 10% of cell

volume

DNA Replication-occurs at the replication fork :

• 5’ to 3’• DNA helicase-unzips + parental DNA

strand that is used as a template• Leading stand (5’ to 3’-continuous)

- DNA polymerase-joins growing DNA strand after nucleotides are aligned (complimentary)

• Lagging strand (5’ to 3’-not continuous)- RNA polymerase (makes short RNA primer)- DNA polymerase (extends RNA primer then

digests RNA primer and replaces it with DNA)- DNA ligase (seals Okazaki fragments-the newly

formed DNA fragments)

Replication Fork

Protein Synthesis:

• DNA------- mRNA------ protein transcription translation

Central Dogma of Molecular Genetics

Transcription:• One strand of DNA used as a template

to make a complimentary strand of mRNA

• Promoter/RNA polymerase/termination site/5’ to 3’

• Ways in which RNA & DNA differ:• RNA is ss• RNA sugar is ribose• Base pairing-A-U

Transcription:

Types of RNA:

• Three types:• mRNA: messenger RNA

• Contains 3 bases ( codon)

• rRNA: ribosomal RNA• Comprises the 70 S ribosome

• tRNA: transfer RNA• Transfers amino acids to ribosomes for

protein synthesis• Contains the anticodon (3 base sequence

that is complimentary to codon on mRNA)

Genetic Code:

• DNA: triplet code

• mRNA: codon (complimentary to triplet code of DNA)

• tRNA: anticodon (complimentary to codon)

Genetic Code:

• Codons: code for the production of a specific amino acid

• 20 amino acids• 3 base code• Degenerative: more than 1 codon

codes for an amino acid.• Universal: in all living organisms.

Stop codons: -Act as punctuation mark.-Termination the message for the sythesis of the polypeptide. - UAA, UAG, UGA

Translation:

• Three parts:• Initiation-start codon (AUG)• Elongation-ribosome moves along mRNA• Termination: stop codon

reached/polypeptide released and new protein forms

• rRNA=subunits that form the 70 S ribosomes (protein synthesis occurs here)

• tRNA=transfers amino acids to ribosomes for protein synthesis.

Mutation:

• Defination:• It is a heritable change in genotype, that can

occur spontaneously or induced by physical or chemical treatments.

• Organism selected as referance strain is called as wild type.

• Their progeny with mutation is called mutagen.

• Process of mutation is called as Mutagenesis.• The agent inducing mutation is called as

mutagen.

• Spontaneous mutation occurs naturally about one in every million to one in every billion divisions & occurs during the DNA replication.

• Mutation rates of individual genes in bacteria range from 10‾2 to 10‾10 /bactrium/division.

Mechanism of division:

• Substitution of Nucleotides:- Also called as point mutation.

- MC mechanism of the mutation. - It invovles the changing of single base in the

DNA sequence, which is copied during replication, to produce permanent changes.

- If one purine or pyrimidine is replaced by the other , the substitution is called as Transition.

- If a purine is replaced by purine or vice-versa, the substitution is called as Transversion.

• Deletion or addition of nucleotide: - Occurs during the DNA replication. - When a transposon (jumping gene) inserts

itself in to a gene, it leads to distruption of gene , called as Insertional mutation.

Types of the mutation:

• Missense mutation: - Caused by DNA mutatins which leads to changes in to amino acid sequences of the protein products

-Caused by single point mutation or series of mutations.

• Nonsense mutation:- formation of stop codon, which causes termination of protein synthesis leads to incomplete protein products.

• Silent mutation:- Single substitution mutation change in the DNA sequence result in a new codon , still coding for the same amino acid & there is a no change in the product.

• Frameshift mutation:• The addition or deletion of base pair causing a shift in

the “reading frame” of the gene.• Theses causes a reading frame shift & all of the codon

& all amino acids after that mutation are usually wrong.

• Lethal mutation:• Some mutation affect the vital function of the cell, &

bacterial cell become nonviable, & which can kill the cell.

• Suppressor mutation:• It is a reversal of the mutant phenotype by another

mutation at a position on the DNA, differ from that of original mutation.

• True reversion or back mutation results in reversion of mutant to original form, which occurs as a result of mutation occurring at the same spot once again.

• Conditional lethal mutation:• Mutation may occur in such a way that mutation

may survive in certain enviromental condition.

• Inversion mutation:• Segment of the DNA is removed & reinserted in the

reverse direction.

Normal DNA/Missense Mutation

Nonsense Mutation/Frameshift Mutation:

Genetic Transfer in Bacteria:

• Genetic transfer-results in genetic variation

• Genetic variation-needed for evolution• Three ways:

• Transformation: genes transferred from one bacterium to another as “naked” DNA.

• Conjugation: plasmids transferred one bacteria to another via a pilus.

• Transduction: DNA transferred from one bacteria to another by a virus.

Transformation:

•Recipient cell must be competent for uptake of DNA.

•Natural competence versus artificial competence.

•Only certain bacteria are naturally transformable - Streptococcus pneumoniae, Haemophilus influenzae, Neisseria gonorrhoeae, Vibrio.

Transformation:

Transduction by a Bacteriophage:

•Bacteriophage (phage) are viruses of bacteria - can be either lytic or temperate

•1. lytic - always lyse (kill) host bacterial cell temperate - can stably infect and coexist

within bacterial cell (lysogeny) until a lytic phase is induced

• 2. lysogeny•The phage genome during lysogeny is called the

prophage, and the bacterial cell is called a lysogen• If the phage genome encodes an observable

function, the lysogen will be altered in its phenotype - lysogenic conversion (e.g., diphtheria toxin in Corynebacterium diphtheriae)

• 3. Specialized transduction:• Some prophages integrate into the bacterial

genome at a specific location.• When a prophage is induced to lytic phase, it

may drag along a piece of the bacterial genome next to the integration site and move that bacterial sequence into the new recipient host cell, changing the recipient's genome.

not very important medically since only selected genes can be transferred

•4. Generalized transduction:• when a phage lyses the host bacterial cell, it

normally packages phage genome into the capsid.

•sometimes the capsid is accidentally filled with random pieces of bacterial genome, possibly including plasmids

•when the capsid injects the host genes into a new recipient, the new gene can recombine into the recipient genome and cause a change.

•virulence and antibiotic resistance genes can be moved by generalized transduction.

• Difference between lysogeny and generalized transduction1. lysogeny - when a phage lysogenizes a host bacterial cell and brings in a new gene that changes the characteristics of the cell - it is a phage gene that makes the change in lysogenic conversion and a host gene in specialized transduction.

2.Generalized transduction involves the phage particle acting as a vehicle to move bacterial genes from one cell to another, the phage genome is not moved .

Transduction by a Bacteriophage:

conjugation:

Possible states for conjugationi. Conjugative - the plasmid encodes all of the

functions for conjugation and can move itself from the donor cell to the recipient cell.

ii. Mobilizable - the plasmid cannot move itself, but can be moved with help from a conjugative plasmid. Conjugation functions:

i. synthesis of sex pilus.ii. cell to cell contact via pilus.iii. copying plasmid DNA and transfer of copy

into recipient cell.

• Bacteria containing a conjugative plasmid are called donor, male, (F+)

• Bacteria receiving the plasmid are called recipient, female, (F-)

• The F plasmid is a specific E. coli plasmid that has nothing to do with medicine other than its historical and laboratory use (and standardized exams)

i.

• Hfr - a plasmid integrates into the chromosome, conjugation will move part of the chromosome into the recipient (not of medical relevance)• F' - plasmid integrates into the chromosome, excision drags along piece of the chromosome. • F' can move host DNA between cells (not of medical relevance) (similar to specialized transduction)• Phenocopy - a cell with the F plasmid, but lacking pili

•Unlike transduction by phages, most plasmids are more promiscuous in their host range

•Importance of conjugation - moving plasmids encoding multiple antibiotic resistance genes (R plasmids) among bacteria.

Insertion Sequences and Transposons:

1. These sequences are part of other genetic elements - chromosome, plasmid.

-They move from one site in DNA to another WITHIN the same cell (transposition)

2. Insertion sequence (IS) -Gene encoding transposition enzyme

(transposase) flanked by inverted repeats of DNA sequence.

- can interrupt genes if they insert into them.

Conjugation in E. coli:

Conjugation cont…

Conjugation cont…

Referances:

1. Text book of microbiology: anantnarayan. 2. Notes by Shridhar rao