Bacterial Genetics

Review

• Genome: genetic blueprint

• Gene:

• Most organisms-DNA

• Viruses– DNA or RNA

Nucleic acids

• Nucleotides– Sugar– Phosphate – Nitrogenous base

• DNA– Guanine-G; cytosine-C; adenine-A; thymine-T

• RNA– Uracil-U replaces thymine

Review

• DNA –ds helix

• Strands held together by H bonds

• Complimentary base pairing– A pairs with T; in RNA, A pairs with U– C with G

• Linear sequence of bases contains info

• 3 nucleotides code for 1 amino acid

Duplication of DNA

• Replication of chromosome(s)

• Precise duplication of DNA

• DNA polymerase

• Denaturation: separate strands

Terms

• Genotype: set of genes carry instructions

• Phenotype: expression of those genes

• Genome of prokaryotes includes chromosomal DNA and plasmids

Flow of Information

• DNA to RNA to polypeptides

• Transcription-synthesis of complimentary strand of RNA from DNA-mRNA

– Separates strands of DNA– Copies only DNA needed for protein

synthesis-mRNA

Flow of Information

• Translation: protein synthesis– Decodes sequence of nucleotides into

amino acids (20)

– Genetic Code: codons, group of 3 nucleotides-AAU,CGC• AUG-start codon

Degenerate Code

• Each codon specifies a particular amino acid

• Several codons code same aa

Flow of Information

• Translation– Ribosomes-site of protein synthesis– tRNA recognizes the specific codon– tRNA forms complimentary base pairing with

the codon– Has anticodon & carries the required aa

Question

• AUG is first codon on mRNA.

• What is the anticodon on the tRNA?

Regulation of Gene Expression

• Prokaryotes: Where is mRNA transcribed in cell?

– Translation can begin before completion of mRNA molecule

Regulation of Gene Expression

• Eukaryotes: Where?– mRNA contains exons and introns– Exons are expressed, encode for aa– Introns do not– Processing by ribozymes to remove introns &

splice exons

Regulation of Gene Expression

• Conserve energy

• Turn on & off transcription of genes

• Constitutive genes not regulated

• Operon

Operons

• Regulation of metabolic genes

• Uses repressors (regulatory genes)– Block RNA polymerase from attaching

Induction

• Turn on the transcription of gene

• Inducer- induces transcription

• Inducible enzymes

– Synthesized only when substrate is present

• Glycolysis genes constitutive

Lac Operon

• Inducible operon: enzymes to metabolize lactose

• Default is “off”

• Regulatory sites – Promoter- – – Operator-

Lac Operon

• i genes code for repressor-regulatory protein

– Always turned on– Binds to operator

• Structural genes– Lac operon-3 genes

Lactose in Medium

• Binds repressor changing shape• Repressor can’t bind• RNA polymerase can bind• Enzymes for lactose metabolism produced

• To turn on operon:

Repressible Operon

• Tryptophan operon

• Usually occurs in anabolism

• Repressor is inactive so tyrptophan is synthesized

• Default in “on”

Tryptophan Operon

• Amino acid in media– Binds to repressor activating it

• Genes to synthesize amino acid produced

Mutation

• Change (heritable) in base sequence of DNA

– Called mutant– Genotype differs from parent– Phenotype may be altered

Types of Mutations

• Spontaneous mutations– DNA replication errors– Occur at low frequencies

• Induced mutations-mutagens– Alter structure of bases – Errors in base paring

Point Mutations

• Change in 1 base-pair– Single base is replaced with another– Change in genotype– May be change in phenotype

Types of Point Mutations

• Silent mutation- no phenotypic change

• Degenerate code

Missense Mutations

• Change in amino acid

• Can result in significant changes in polypeptide

Examples of Missense

• Sickle cell anemia- hemoglobin– Change from glutamic acid (hydrophilic) to

valine (hydrophobic)

• Change in shape of protein

Nonsense Mutation

• Base-pair substitution

• Create stop codon in middle of mRNA– Premature termination of translation

Frame Shift Mutation

• Bases deleted or inserted

• Shifts translational reading frame

• Large insertions are transposons

Frame Shift

• THE FAT CAT ATE THE BAD RAT

• Remove a C

• THE FAT ATA TET HEB ADR AT

Induced Mutations

• Mutagens

• Increase mutation rate

• Chemical mutagens– Nitrous acid

– Occurs at random sites

Radiation

• UV light -nonionizing

• Covalent bonds between certain bases

• Adjacent thymines(Ts) can cross link– Pyrimidine dimers

• Some enzymes that repair UV damage

Nucleoside Analog

• Structurally similar to normal bases

• Have altered base pairing

• 2 aminopurine replaces A & may pair with C

• 5-bromouracil replaces T but may pair with G

Genetic Recombination

• Physical exchange of genes between 2 homologous DNA molecules

• Contributes to population’s genetic diversity

Horizontal Transfer

• Microbes of same generation

• Involves a donor cell - gives DNA to recipient cell

• Part of DNA incorporated into recipient’s DNA

Transformation

• Free (naked) DNA in solution– Cells after death, release DNA

• Cells may take up DNA– Only in certain stage of cell cycle

Competence

• Cells able to take up DNA & be transformed

• Release competence factor that helps in uptake

Transformation

• Enzymes cut DNA into small pieces

• Recombination between donor & recipient

• Few competent bacteria

First Evidence of Transformation

• Griffith in 1920s

• Streptococcus pneumoniae in mice

Conjugation

• Mediated by one kind of plasmid– F plasmid or F factor

• Genes to control conjugation

• Donor cells must have F plasmid

Differs from Transformation

• Cells must be of opposite mating types– Donor is F plus– Recipient is F minus

• Requires direct cell contact

• Transfers larger quantities of DNA

Conjugation

• Gram negative cells

• Gram positive cells produce sticky surface molecules

– Keeps cells together

Conjugation

• Plasmid is replicated – A copy of plasmid transferred to recipient

• F minus cell becomes F plus

• Receptors on new F plus change

F Plasmid

• Plasmid integrates into the chromosome converts cell to Hfr cell( high frequency of recombination)

• F factor DNA can separate and become plasmid

Conjugation

• Hfr and F- cell• Replication of Hfr begins in middle of

integrated F factor • Small piece leads the chromosome into F-

cell

• Donor DNA can recombine (DNA not integrated is degraded)

Transduction

• Bacterial DNA is transferred via a virus -Bacteriophage

• Virulent phages –lytic cycle

• Generalized transduction– Any gene on donor chromosome transferred

Generalized Transduction

• All genes are equally likely to be packaged inside phage– Virus cannot replicate in new bacteria– Defective virus

• Specialized transduction-only certain bacterial genes transferred

Plasmids

• Extra chromosomal material

• F factor or plasmid is a conjugative plasmid

– Carries genes for sex pili and for transfer

Dissimilation Plasmids

• Enzymes break down unusual sugars and hydrocarbons– Pseudomonas use toluene and petroleum as

carbon and energy sources– Used to clean up oil spills– Allows bug to grow in adverse environments

Virulence Plasmids

• E. coli carries plasmids that code for

toxins –diarrhea

• Bacteriocins- toxic proteins kills other bacteria– E. coli produces colicins

Resistance Factors

• R factors- resistance to antibiotics, heavy metals or cellular toxins

• Wide spread use of antibiotics- led to selection of bacteria with R factors with resistant genes

Transposition

• Segment of DNA moves from one place in chromosome to another– Rare event– Transposable elements– Insert within a gene & inactivate it

Transposons

• Contain information for own transposition

• Insertion sequences (IS) contain only gene for transposase

• Complex transposons carry genes for enterotoxins or antibiotic resistance