Chapter 8:Chapter 8:

Microbial Genetics Microbial Genetics

IntroductionIntroduction GeneticsGenetics is the science of heredity. is the science of heredity.

Study of genes:Study of genes: How they carry informationHow they carry information

How they are replicatedHow they are replicated

How they are passed from one organism to anotherHow they are passed from one organism to another

How they are expressedHow they are expressed

GenesGenes: Segment of DNA (or RNA in some viruses) that : Segment of DNA (or RNA in some viruses) that

codes for functional products.codes for functional products.

ChromosomesChromosomes: : Cellular structures that carry hereditary Cellular structures that carry hereditary

information. They contain all or most of an individual’s information. They contain all or most of an individual’s

genes.genes.

Structure and Function of Genetic Material Structure and Function of Genetic Material

DNA is genetic material in all living DNA is genetic material in all living organisms. RNA is genetic material of some organisms. RNA is genetic material of some viruses.viruses.

DNA is a macromolecule made of repeating DNA is a macromolecule made of repeating units calledunits called nucleotides. nucleotides.

Each DNA nucleotide has a Each DNA nucleotide has a nitrogenous basenitrogenous base (adenine, cytosine, guanine, or thymine), a (adenine, cytosine, guanine, or thymine), a sugarsugar (deoxyribose), and a (deoxyribose), and a phosphatephosphate group. group.

DNA strands are held together by hydrogen DNA strands are held together by hydrogen bonds between nitrogenous bases.bonds between nitrogenous bases. CytosineCytosine pairs with pairs with Guanine (C = G)Guanine (C = G) ThymineThymine pairs with pairs with Adenine (A = T)Adenine (A = T)

DNA strands are DNA strands are complementarycomplementary..

DNA Structure

Gene ExpressionGene Expression DNA codes for proteins and RNA products.DNA codes for proteins and RNA products. The flow of genetic information is:The flow of genetic information is: Transcription TranslationTranscription Translation

DNA ----------> RNA ----------> ProteinDNA ----------> RNA ----------> Protein DNA only has 4 different nucleotides; while DNA only has 4 different nucleotides; while

proteins have 20 different amino acids. proteins have 20 different amino acids. Genetic CodeGenetic Code: Each amino acid is determined : Each amino acid is determined

by a group of 3 nucleotides (triplet or codon). by a group of 3 nucleotides (triplet or codon). There are a total of 64 (4There are a total of 64 (433= 4 x 4 x 4) possible = 4 x 4 x 4) possible codons.codons.

To express genetic information, DNA To express genetic information, DNA structure must be disrupted.structure must be disrupted.

Gene Expression (Continued)Gene Expression (Continued) DNA sequence must beDNA sequence must be replicated replicated (duplicated) (duplicated)

each time a cell divides.each time a cell divides. During DNA replication, two identical During DNA replication, two identical

daughter molecules are made.daughter molecules are made. DNA can change or mutate during replication. DNA can change or mutate during replication.

Some mutations are harmless, others are Some mutations are harmless, others are harmful, and a few may be beneficial.harmful, and a few may be beneficial.

Genotype and PhenotypeGenotype and Phenotype GenotypeGenotype: The genetic makeup of an : The genetic makeup of an

individual. The information that codes for individual. The information that codes for that organism’s genetic characteristics. that organism’s genetic characteristics. Collection of an individual’s genes.Collection of an individual’s genes.

PhenotypePhenotype: Expressed properties of an : Expressed properties of an individual. An individual’s phenotype is a individual. An individual’s phenotype is a function of the genotype (and environment). function of the genotype (and environment).

Collection of an individual’s proteins or gene Collection of an individual’s proteins or gene productsproducts..

DNA and Chromosomes DNA and Chromosomes Bacteria:Bacteria: Typically have a single circular Typically have a single circular

chromosome that is attached to the plasma chromosome that is attached to the plasma membrane.membrane.

E. coliE. coli chromosome is chromosome is 4 million4 million base pairs and base pairs and contains about 2000 genes.contains about 2000 genes.

EucaryotesEucaryotes: Typically have several linear : Typically have several linear chromosomes that are inside the nucleus.chromosomes that are inside the nucleus.

Humans have 46 chromosomes with a total Humans have 46 chromosomes with a total length of length of 3 billion3 billion base pairs, which code for base pairs, which code for up to 100,000 different genes.up to 100,000 different genes.

Circular Chromosome of E. coli

DNA Replication DNA Replication One parent double stranded molecule generates two One parent double stranded molecule generates two

daughter strands.daughter strands. In bacteria, replication begins at an origin of In bacteria, replication begins at an origin of

replication.replication. Replication is Replication is semiconservativesemiconservative. Each strand acts as a . Each strand acts as a

template for the production of a new strand. Each template for the production of a new strand. Each new DNA molecule has one old strand and one new new DNA molecule has one old strand and one new strand.strand.

DNA strands are DNA strands are antiparallelelantiparallelel. . One strand goes from 5’ to 3’ direction.One strand goes from 5’ to 3’ direction. Opposite strand goes from 3’ to 5’ direction.Opposite strand goes from 3’ to 5’ direction.

DNA polymerase only synthesizes in 5’ to 3’ direction.DNA polymerase only synthesizes in 5’ to 3’ direction. LeadingLeading strandstrand is synthesized continuously. is synthesized continuously. LaggingLagging strandstrand is synthesized in small fragments, of about is synthesized in small fragments, of about

1000 nucleotides.1000 nucleotides.

DNA Replication is Semiconservative

Steps in DNA Replication Steps in DNA Replication 1.1. Enzymes Enzymes unwindunwind double stranded DNA molecule. double stranded DNA molecule.

2.2. Proteins stabilize unwound DNA. Proteins stabilize unwound DNA.

3.3. LeadingLeading strandstrand is synthesized is synthesized continuouslycontinuously by by DNA DNA polymerase polymerase in 5’ to 3’ direction.in 5’ to 3’ direction.

4.4. LaggingLagging strandstrand is synthesized is synthesized discontinuouslydiscontinuously..

5.5. RNA primers are made by RNA primers are made by RNA polymeraseRNA polymerase and and extended by extended by DNA polymerase.DNA polymerase.

Okazaki fragments: Okazaki fragments: RNA-DNA fragments.RNA-DNA fragments.

6.6. DNA polymerase digests RNA primers and replaces DNA polymerase digests RNA primers and replaces them with DNA. them with DNA.

7.7. DNA ligaseDNA ligase joins discontinuous fragments of lagging joins discontinuous fragments of lagging strand.strand.

Error RateError Rate: 1 out of 10: 1 out of 101010 bases is changed (mutation). bases is changed (mutation). DNA polymerase has proofreading mechanism.DNA polymerase has proofreading mechanism.

DNA Replication: Leading and Lagging Strands Are Copied Differently

RNA Synthesis (Transcription) RNA Synthesis (Transcription) There are three types of RNA in bacterial cells:There are three types of RNA in bacterial cells:

mRNAmRNA: Messenger RNA. Carries information for : Messenger RNA. Carries information for protein synthesis.protein synthesis.

rRNArRNA: Ribosomal RNA. Forms part of ribosome.: Ribosomal RNA. Forms part of ribosome.

tRNAtRNA: Transfer RNA. Carries amino acids to growing : Transfer RNA. Carries amino acids to growing protein during translation.protein during translation.

Steps of TranscriptionSteps of Transcription1.1. RNA polymeraseRNA polymerase binds to DNA sequence called binds to DNA sequence called

promoterpromoter..

2.2. RNA polymerase makes RNA copy of gene RNA polymerase makes RNA copy of gene ((transcripttranscript).).

3.3. RNA synthesis continues until RNA polymerase RNA synthesis continues until RNA polymerase reaches a reaches a terminatorterminator..

4.4. New RNA molecule and RNA polymerase are released. New RNA molecule and RNA polymerase are released.

Process of Transcription: DNA to RNA

Protein Synthesis (Translation) Protein Synthesis (Translation) mRNA is used to make protein.mRNA is used to make protein. mRNA is read in codons or nucleotide triplets.mRNA is read in codons or nucleotide triplets. Genetic Code was cracked in 1960s.Genetic Code was cracked in 1960s.

There are 64 possible codons, 20 amino acids.There are 64 possible codons, 20 amino acids.

AUGAUG: Start codon (Methionine): Start codon (Methionine)

UAA, UGA, UAGUAA, UGA, UAG: Stop codons: Stop codons Translation occurs on the ribosome, which is made up Translation occurs on the ribosome, which is made up

of two subunits (large and small).of two subunits (large and small). tRNA molecules have an tRNA molecules have an anticodonanticodon, which recognizes , which recognizes

codons. They carry specific amino acids to the codons. They carry specific amino acids to the growing protein chain. growing protein chain.

Universal Genetic Code

Steps of TranslationSteps of Translation1. 1. InitiationInitiation: Ribosomal subunits and mRNA : Ribosomal subunits and mRNA

assemble.assemble.

2. 2. Start codonStart codon ( (AUGAUG) binds to tRNA with ) binds to tRNA with methionine.methionine.

3. 3. ElongationElongation: Subsequent amino acids are : Subsequent amino acids are added by translating one codon at a time.added by translating one codon at a time.

4. 4. RibosomesRibosomes attach each amino acid to growing attach each amino acid to growing protein chain by formation of protein chain by formation of peptide bonds.peptide bonds.

5. 5. TerminationTermination: When a : When a stop codonstop codon is reached, is reached, translation stops, and ribosome-mRNA translation stops, and ribosome-mRNA complex falls apart. complex falls apart.

Translation: Initiation at Start Codon

Translation: During Elongation one Amino Acid is Added at a Time

Elongation: Ribosome Travels Down mRNA, Reading One Codon at a Time

Termination: Once Stop Codon is Reached, Complex Disassembles

Regulation of Bacterial Gene ExpressionRegulation of Bacterial Gene Expression Protein expression requires large amounts of Protein expression requires large amounts of

energy.energy. Cell saves energy by only making necessary Cell saves energy by only making necessary

proteins.proteins. Constitutive genesConstitutive genes: Products made constantly by the : Products made constantly by the

cell, synthesis is not regulated. 60-80% of genes. cell, synthesis is not regulated. 60-80% of genes. Example: Genes for enzymes of major metabolic Example: Genes for enzymes of major metabolic pathways.pathways.

Regulated genesRegulated genes: Products made only when needed by : Products made only when needed by cell. Synthesis is tightly regulated. 20-40% of genes. cell. Synthesis is tightly regulated. 20-40% of genes. Example: Enzymes for lactose digestion (lac operon).Example: Enzymes for lactose digestion (lac operon).

Mechanisms of regulation: Repression and Mechanisms of regulation: Repression and Induction.Induction.

Regulation of Bacterial Gene ExpressionRegulation of Bacterial Gene ExpressionRepressionRepression Inhibits gene expression and decreases enzyme Inhibits gene expression and decreases enzyme

synthesis.synthesis. Response to overabundance of a metabolic Response to overabundance of a metabolic

pathway product.pathway product. Repressors block RNA polymerase from Repressors block RNA polymerase from

transcribing gene(s).transcribing gene(s).

InductionInduction Turns on gene transcription.Turns on gene transcription. Inducers stimulate transcription of gene(s) by Inducers stimulate transcription of gene(s) by

RNA polymerase .RNA polymerase .

Example: Lac operon Example: Lac operon

Operon Model of Gene ExpressionOperon Model of Gene Expression

OperonOperon: Group of metabolically related genes that : Group of metabolically related genes that are transcribed together and a control region that are transcribed together and a control region that regulates their transcription as a unit.regulates their transcription as a unit.

Contains:Contains: Structural genesStructural genes: Code for protein products.: Code for protein products. PromoterPromoter: Site where RNA polymerase initiates : Site where RNA polymerase initiates

transcription.transcription. OperatorOperator: DNA segment that controls passage of : DNA segment that controls passage of

RNA polymerase.RNA polymerase.

Outside of operon:Outside of operon: RepressorRepressor genegene: Codes for repressor protein that : Codes for repressor protein that

blocks operon transcription. blocks operon transcription.

Mutation: Change in Genetic MaterialMutation: Change in Genetic MaterialMutationMutation: Change in the nucleotide sequence of DNA. : Change in the nucleotide sequence of DNA.

These changes may be harmful, beneficial, or have no These changes may be harmful, beneficial, or have no effect (neutral) on the individual or cell.effect (neutral) on the individual or cell.

Silent mutationsSilent mutations: Do not affect activity of gene product. : Do not affect activity of gene product. May or may not change amino acid sequence.May or may not change amino acid sequence.

Spontaneous mutationsSpontaneous mutations: Occur spontaneously during : Occur spontaneously during replication. replication.

Error RateError Rate: Low. In bacteria 1 out of 10: Low. In bacteria 1 out of 101010 bases is bases is mutated during replication. mutated during replication. E. coliE. coli has 4 x 10 has 4 x 106 6 bases, bases, resulting in less than one mutation per replication. resulting in less than one mutation per replication.

DNA polymerase has proofreading mechanism.DNA polymerase has proofreading mechanism. MutagensMutagens: Many chemicals, X rays, ultraviolet light, and : Many chemicals, X rays, ultraviolet light, and

other forms of radiation can cause mutations. Increase other forms of radiation can cause mutations. Increase mutation rate by a factor of 10 to 1000.mutation rate by a factor of 10 to 1000.

Types of MutationsTypes of Mutations Base Substitution (Point Mutation):Base Substitution (Point Mutation): Single nucleotide Single nucleotide

is replaced with a different base. After replication, is replaced with a different base. After replication, base pair changes.base pair changes. Missense mutationMissense mutation: Results in amino acid substitution.: Results in amino acid substitution.

Example: Sickle cell anemia.Example: Sickle cell anemia. Nonsense mutationNonsense mutation: Creates a stop codon which truncates : Creates a stop codon which truncates

protein. Only a fragment is synthesized.protein. Only a fragment is synthesized. Silent mutationSilent mutation: Protein sequence and/or activity is not : Protein sequence and/or activity is not

altered.altered.

Frameshift Mutation:Frameshift Mutation: Several nucleotides are inserted Several nucleotides are inserted or deleted into a gene. These mutations may shift the or deleted into a gene. These mutations may shift the reading frame of translation, resulting in a completely reading frame of translation, resulting in a completely different amino acid sequence after mutation site.different amino acid sequence after mutation site.

Genetic Transfer and RecombinationGenetic Transfer and RecombinationGenetic RecombinationGenetic Recombination: Exchange of genes between two : Exchange of genes between two

DNA molecules to form new combinations of genes on a DNA molecules to form new combinations of genes on a chromosome.chromosome.

Genetic recombination contributes to an organism’s Genetic recombination contributes to an organism’s genetic diversity.genetic diversity.

In eucaryotesIn eucaryotes recombination occurs during meiosis recombination occurs during meiosis through a process called through a process called crossing overcrossing over..

In procaryotesIn procaryotes there are several different mechanisms of there are several different mechanisms of genetic recombination: genetic recombination: TransformationTransformation, , conjugationconjugation, and , and transductiontransduction

In all cases, it involves a DNA donor and a DNA recipient In all cases, it involves a DNA donor and a DNA recipient cell.cell.

Recombination occurs in a small percentage of a bacterial Recombination occurs in a small percentage of a bacterial population.population.

Transformation in Bacteria Transformation in Bacteria Genes are transferred from one bacterial cell to Genes are transferred from one bacterial cell to

another in the form of another in the form of naked DNAnaked DNA.. Initial work done in 1928 by Initial work done in 1928 by Frederick GriffithFrederick Griffith

on two strains of on two strains of Streptococcus pneumoniaeStreptococcus pneumoniae.. Smooth strain: Caused disease due to capsule.Smooth strain: Caused disease due to capsule. Rough strain: Did not cause disease.Rough strain: Did not cause disease.

Experiments with heat killed smooth bacteria Experiments with heat killed smooth bacteria and live rough bacteria, demonstrated the and live rough bacteria, demonstrated the presence of a presence of a transforming factortransforming factor..

In 1944, Avery and others demonstrated that In 1944, Avery and others demonstrated that transforming material was indeed DNA. This transforming material was indeed DNA. This was important in establishing that genetic was important in establishing that genetic material was DNA.material was DNA.

Transformation of Bacteria in Griffith’s Experiment

Transformation: Cells Take up Naked DNA

Transformation in Bacteria (Continued) Transformation in Bacteria (Continued) Only a small percentage of donor DNA is Only a small percentage of donor DNA is

transferred.transferred. Transformation occurs naturally in some bacteria Transformation occurs naturally in some bacteria

((Bacillus, Neisseria, Hemophilus, StreptococcusBacillus, Neisseria, Hemophilus, Streptococcus, , and and StaphylococcusStaphylococcus).).

Other cells can be chemically treated to accept Other cells can be chemically treated to accept foreign DNA (foreign DNA (competent cellscompetent cells). Example: ). Example: E. coliE. coli..

Conjugation in BacteriaConjugation in Bacteria Genetic material is transferred from one Genetic material is transferred from one

bacterial cell to another through bacterial cell to another through direct direct contactcontact..

Gram negative cells form sex pili.Gram negative cells form sex pili. Gram positive cells produce sticky surface Gram positive cells produce sticky surface

molecules.molecules. Requires fairly high cell density. Requires fairly high cell density.

Conjugation Requires Cell to Cell Contact

Transduction in BacteriaTransduction in Bacteria Genetic material is transferred from one Genetic material is transferred from one

bacterial cell to another through a virus bacterial cell to another through a virus ((bacteriophagebacteriophage).).

Transduction may be generalized or Transduction may be generalized or specialized.specialized.

Many genes for toxins are transferred by Many genes for toxins are transferred by specialized transduction:specialized transduction: E. coliE. coli O157:H7: Shiga-like toxin. O157:H7: Shiga-like toxin. Corynebacterium diphtheriaeCorynebacterium diphtheriae: Diphtheria toxin.: Diphtheria toxin. Streptococcus pyogenesStreptococcus pyogenes: Erythrogenic toxin.: Erythrogenic toxin.

Transduction: Bacteriophage Transfers DNA From One Cell to Another