Microbial Genetics

and biotechnology

Define Terms

GeneticsGenome / GenomicsChromosomesGeneGenotypePhenotypeRecombination

DNA StructureDouble strandedNucleotide

Nitrogen BasesSugarPhosphate

Base PairsHydrogen Bonds

A-TC-G

Alpha helix5’ – phosphate group3’ – hydroxyl group

RNA StructureSingle strandNucleotide

Nitrogen baseSugarPhosphate

Base PairsA-UC-G

Three typesmRNArRNAtRNA

Prokaryotic ChromosomesLocation

Nucleoid regionNo membrane

NumberMost have 1Some species have 2, the second linear

AppearanceCircularDsLoops and coils

E. coli genome / chromosome

DNA ReplicationSemiconservative

Replication forkSingle originBidirectional

2 Leading strands2 Lagging strands

EnzymesHelicasesDNA polymerases 5’ to 3’

I for leading strandII digest RNA primerIII for lagging strand

DNA ligaseDNA gyrase

Hydrogen bonds broken and reformedMethylation of adenine bases

Initiation sitesTurn on or Turn off Protect against viral infectionsDNA repair

Polymerase I & II

DNA Replication Overview

Binary Fission

Plasmids

2% of genetic informationDs, circular extra chromosomal DNAIndependent replicationCellular Traits

F-FertilityR-Resistance : inactivate AB, toxins, heavy metals Dissimilation: catabolism of unusual substancesBacteriocinsVirulence : enzymes, toxins, attachment

Rolling Method for DNA replication and F-Plasmid

Rolling MethodOne strand remains in loopSecond strand breaks away and rolls of loopBoth strands serve as templates for daughter strandOccurs during conjugation

TranscriptionDNA RNA

mRNArRNAtRNA

Initiation Sigma factor on RNA polymerase

binds to promoter sequence on DNAWill be release after 10 nucleotides

RNA polymeraseunzips, unwinds DNALacks proof reading ability

Elongation 5’ to 3’, slower

Ribonucleotide sequencesBase pairs :

A-U [instead of Thymine] C-G

TerminationSelf

Terminator sequenceG-C rich area

Protein-dependantTerminator proteinSeparates DNA and RNA polymerase

Sigma Factors for RNA polymerase

Prokaryotic RNATranscription = RNA PolypeptidesRNA

mRNACode for several polypeptides along strandEach code has codons: Start and Stop

tRNAAcceptor stemAnticodonWobble

rRNA70S Ribosomes

50S: 23S + 5S rRNA and 33 proteins30S: 16S rRNA and 21 proteins

Binding Sites on RibosomesA: accepts tRNA with AAP: holds tRNA for base pairing anticodon to mRNA codon for polypeptideE: release [exit] for tRNA

Translation StepsInitiation

30StRNA @ P site50SGTP used

ElongationNew tRNA @ A siteRibozyme in 50S forms peptide bondGTP used

TerminationRelease factor proteinsStop codon on mRNA

Importance of rRNA structures

Regulation of Gene ExpressionConstitutive

Not regulatedAlways “on” at fixed rate

TranscriptionTranslation

60-80%Polypeptides need in large amounts

RegulatedOnly when neededControl synthesis of enzyme : genetic control

InductionRepression

Control enzyme activity: feedbackNoncompetitive inhibitionCompetitive inhibition

Genetic Control of Enzyme Synthesis and formation

Operon ModelOperator (O)Promoter (P)Structural genes

Regulatory genesMakes repressor

Active binds to OInactive unable to bind to O

TypesInducible Operons

Repressor ActiveOperon OffInducer needed

Repressible OperonsRepressor inactiveOperon OnCorepressor needed

Operator and Regulation

Lac Operon: Inducible

Use of Lactose

TRP Operon: Repressible

MutationsDefineTypes

SilentPoint

Mis-senseNon-senseSense [aka silent]

Substitution Transition: purine for purineTransversion: purine for pyrimadine

FrameshiftInsertionsDeletions

CausesSpontaneousInduced

ChemicalPhysical

ConditionalAdaptiveTransposons Thymine dimer

inversion

Transposons

Repair of Mutations

Genetic TransferVertical

Parent to offspring

HorizontalLateral transfer to same generationDonor to recipientDNA transferPlasmid transferTypes

TransformationTransductionConjugation

TransformationOccurance

1%RandomNaturally in certain species

HaemophilusNeisseriaPseudomonasStreptococcusStaphylococcus

Griffith experimentGenetic transfer

Environment Competent cells

Cell wallPlasma membrane

Bacterial lysisDNAPlasmids

Griffith Experiments

Transduction

Transfer of bacterial genes via virusesDonor to recipientVirus: Bacteriophages

Types GeneralizedSpecialized

Replication CycleLyticLysogenic

Generalized Lytic Cycle

Specialized Lysogenic CycleOnly certain bacterial genes are transferredExample: Toxins

CorynebacteriumDiphtheria toxin

Streptococcus pyogenes

Erythrogenic toxinE. coli

Shiga-like toxin

Transduction

Conjugation

DefineBacteria

Gram Neg : F.pilusGram Pos: sticky surface molecules

TypesF+ [plasmid]R [plasmid]Hfr [DNA]

Conjugative Plasmid

Hfr Interrupted Stages

Genetic Recombination

GeneralHomologous chromosomesAny locationDNA breakage and repair

Site SpecificNonhomologousViral genomes in bacterial chromosomes

Replicative

Recombinant DNA

Genetic EngineeringUse

PlasmidsRecombinant DNA

ApplicationsTherapeutic

HormonesEnzymesVaccinesGene therapy

AgriculturalScientific

Southern BlotELISA tests

Biotechnology

Questions?