Chapter 14Chapter 14The Prokaryotic The Prokaryotic

Chromosome: Genetic Chromosome: Genetic Analysis in BacteriaAnalysis in Bacteria

Outline of Chapter 14Outline of Chapter 14 General overview of bacteriaGeneral overview of bacteria

Range of sizesRange of sizes Metabolic activityMetabolic activity How to grow them for studyHow to grow them for study

The bacterial genomeThe bacterial genome StructureStructure OrganizationOrganization TranscriptionTranscription ReplicationReplication Evolution of large, circular chromosomesEvolution of large, circular chromosomes Structure and function of small circular plasmidsStructure and function of small circular plasmids

Gene transfer in bacteriaGene transfer in bacteria TransformationTransformation ConjugationConjugation TransductionTransduction

A comprehensive exampleA comprehensive example Genetic tools to dissect bacterial chemotaxisGenetic tools to dissect bacterial chemotaxis

General overview of bacteriaGeneral overview of bacteria

One of the three major lineages of lifeOne of the three major lineages of life Eukaryotes – organisms whose cells have encased nucleiEukaryotes – organisms whose cells have encased nuclei Prokaryotes – lack a nuclear membraneProkaryotes – lack a nuclear membrane

ArcheaArchea 1996 complete genome of 1996 complete genome of Methanococcus jannaschiiMethanococcus jannaschii sequenced sequenced More than 50% of genes completely different than bacteria and eukaryotesMore than 50% of genes completely different than bacteria and eukaryotes Of those that are similar, genes for replication, transcription, and Of those that are similar, genes for replication, transcription, and

translation are same as eukaryotestranslation are same as eukaryotes Genes for survival in unusual habitats similar to some bacteriaGenes for survival in unusual habitats similar to some bacteria

BacteriaBacteria Similar genome structure, morphology, and mechanisms of gene transfer Similar genome structure, morphology, and mechanisms of gene transfer

to archeato archea

Evolutionary biologist believe earliest single celled organism, Evolutionary biologist believe earliest single celled organism, probably prokaryote existed 3.5 billion years agoprobably prokaryote existed 3.5 billion years ago

A family tree of living organismsA family tree of living organisms

Fig. 14.1

Diversity of bacteriaDiversity of bacteria

Outnumber all other organisms on EarthOutnumber all other organisms on Earth 10,000 species identified10,000 species identified

Smallest – 200 nanometers in diameterSmallest – 200 nanometers in diameter Largest – 500 micrometers in length (10 billion Largest – 500 micrometers in length (10 billion

times larger than the smallest bacteria)times larger than the smallest bacteria) Habitats range from land, aquatic, to parasiticHabitats range from land, aquatic, to parasitic

Remarkable metabolic diversity allows Remarkable metabolic diversity allows them to live almost anywherethem to live almost anywhere

Common features of bacteriaCommon features of bacteria

Lack defined nuclear membraneLack defined nuclear membrane Lack membrane bound organellesLack membrane bound organelles Chromosomes fold to form a nucleoid bodyChromosomes fold to form a nucleoid body Membrane encloses cells with mesosome which Membrane encloses cells with mesosome which

serves as a source of new membranes during cell serves as a source of new membranes during cell divisiondivision

Most have a cell wallMost have a cell wall Mucus like coating called a capsuleMucus like coating called a capsule Many move by flagellaMany move by flagella

Power of bacterial genetics is the Power of bacterial genetics is the potential to study rare eventspotential to study rare events

Bacteria multiply rapidlyBacteria multiply rapidly Liquid media – Liquid media – E. coliE. coli grow to concentration of 10 grow to concentration of 1099 cells per cells per

milliliter within a daymilliliter within a day Agar media – single bacteria will multiply to 10Agar media – single bacteria will multiply to 1077 – 10 – 1088 cells in less cells in less

than a daythan a day Most studies focus on E. coliMost studies focus on E. coli

Inhabitant of intestines in warm blooded animalsInhabitant of intestines in warm blooded animals Grows without oxygenGrows without oxygen Strains in laboratory are not pathogenicStrains in laboratory are not pathogenic Prototrphic – makes all the enzymes it needs for amino acid and Prototrphic – makes all the enzymes it needs for amino acid and

nucleotide synthesisnucleotide synthesis Grows on minimal media containing glucose as the only carbon Grows on minimal media containing glucose as the only carbon

sourcesource Divides about once every hour in minimal media and every 20 Divides about once every hour in minimal media and every 20

minutes in enriched mediaminutes in enriched media Rapid multiplication make it possible to observe very rare genetic Rapid multiplication make it possible to observe very rare genetic

eventsevents

The bacterial genome is composed of The bacterial genome is composed of one circular chromosomeone circular chromosome

4-5 Mb long4-5 Mb long Condenses by supercoiling and looping into a Condenses by supercoiling and looping into a

densely packed nucleoid bodydensely packed nucleoid body Chromosomes replicate inside cell and cell divides Chromosomes replicate inside cell and cell divides

by binary fissionby binary fission

Fig. 14.4 b

E. coliE. coli lysed to release chromosome lysed to release chromosome

Fig. 14.4 a

How to find mutations in bacterial How to find mutations in bacterial genesgenes

Mutations affecting colony morphologyMutations affecting colony morphology Mutations conferring resistance to antibiotics or Mutations conferring resistance to antibiotics or

bacteriophagesbacteriophages Mutations that create auxotrophsMutations that create auxotrophs Mutations affecting the ability of cells to break Mutations affecting the ability of cells to break

down and use complicated chemicals in the down and use complicated chemicals in the environmentenvironment

Mutations in essential genes whose protein Mutations in essential genes whose protein products are required under all conditions of products are required under all conditions of growthgrowth

How to identify mutations by a How to identify mutations by a genetic screengenetic screen

Genetic screens provide a way to observe Genetic screens provide a way to observe mutations that occur very rarely such as mutations that occur very rarely such as spontaneous mutations (1 in 10spontaneous mutations (1 in 1066 to 1 in 10 to 1 in 1088 cells) cells) Replica plating – simultaneous transfer of thousands of Replica plating – simultaneous transfer of thousands of

colonies from one plate to anothercolonies from one plate to another Treatments with mutagens – increase frequency of Treatments with mutagens – increase frequency of

mutationsmutations Enrichment procedures – increase the proportion of Enrichment procedures – increase the proportion of

mutant cells by killing wild-type cellsmutant cells by killing wild-type cells Testing for visible mutants on a petri plateTesting for visible mutants on a petri plate

Bacteria nomenclatureBacteria nomenclature

wild-type – ‘+’ mutant gene – ‘-’ three lower case, italicized letters – a gene

(e.g., leu+ is wild type leucine gene) The phenotype for a bacteria at a specific gene

is written with a capital letter and no italics (e.g., Leu+ is a bacteria with that does not need leucine to grow, and Leu- is a bacteria that does need leucine to grow.)

Structure and organization of Structure and organization of E. coliE. coli chromosomechromosome

4.6 million base pairs open reading frames (ORFs) 90% of genome encodes protein (compare that to humans!) 4288 genes, 40% of which we do not know what they do. almost no repeated DNA 427 genes have a transport function, other classes also

identified bacteriophage sequences found in 8 places (must have been

invaded by viruses at least 8 times during history.

Insertion sequences dot the E. coli chromosome

Transposable elements place DNA sequences at various locations in the genome.

Geneticists use transposable elements to insert DNA at various locations in bacterial genomes.

If you were to insert a piece of DNA into a bacterial genome using a transposable element, can you think of a molecular method that you could use to find out which gene you inserted the DNA into?

Transposable elements in bacteria

Fig. 14.6

Transcription in bacteriaTranscription in bacteria

Transcription machinery moves clockwiseTranscription machinery moves clockwise Different strands code for different genesDifferent strands code for different genes Several genes may be transcribed in one Several genes may be transcribed in one

segmentsegment RNA polymerase may transcribe adjacent RNA polymerase may transcribe adjacent

genes at the same time in a genes at the same time in a counterclockwise directioncounterclockwise direction

Highly transcribed genes generally oriented Highly transcribed genes generally oriented in direction of replication fork movementin direction of replication fork movement

DNA replication in DNA replication in E. coliE. coli

Fig. 14.7

Plasmids: smaller circles of DNA that do not carry essential genes

Plasmids vary in size ranging from 1kb – 3 Mb. Plasmids can carry genes that confer resistance to

antibiotics and toxic substances. Plasmids are not needed for reproduction or normal

growth, but they can be beneficial. Plasmids can carry genes from one bacteria to

another. Bacteria can thus become resistant to a drug, put the resistance gene in the plasmid, and transfer it to other bacteria. This transfer of plasmid DNA can even occur across species.

Some plasmids contain multiple Some plasmids contain multiple antibiotic resistance genesantibiotic resistance genes

Gene Transfer in BacteriaGene Transfer in Bacteria

Fig. 14.9

TransformationTransformation

Fragments of donor DNA enter the Fragments of donor DNA enter the recipient and alter its genotyperecipient and alter its genotype Natural transformation – recipient cell has Natural transformation – recipient cell has

enzymatic machinery for DNA importenzymatic machinery for DNA import Artificial transformation – damage to recipient Artificial transformation – damage to recipient

cell walls allows donor DNA to enter cellscell walls allows donor DNA to enter cells Treat cells by suspending in calcium at cold Treat cells by suspending in calcium at cold

temperaturestemperatures Electroporation – mix donor DNA with recipient Electroporation – mix donor DNA with recipient

bacteria and subject to very brief high-voltage shockbacteria and subject to very brief high-voltage shock

Mechanism of Mechanism of natural natural

transformationtransformation

Fig. 14.10

Conjugation – A type of gene Conjugation – A type of gene transfer requiring cell-to-cell contacttransfer requiring cell-to-cell contact

Fig. 14.11

The F plasmid and conjugationThe F plasmid and conjugation

Fig. 14.12 a

The process of conjugationThe process of conjugation

The F plasmid occasionally integrates into the The F plasmid occasionally integrates into the E. coliE. coli chromosome chromosome

Hfr cells have Hfr cells have integrated part of integrated part of chromosomechromosome

Episomes – plasmids Episomes – plasmids that can integrate into that can integrate into host chromosomehost chromosome

Exconjugate – Exconjugate – recipient cell with recipient cell with integrated DNAintegrated DNA

Integrated plasmid Integrated plasmid can initiate DNA can initiate DNA transfer by transfer by conjugation, but may conjugation, but may take some of bacterial take some of bacterial chromosome as wellchromosome as well

Fig. 14.13

Gene transfer Gene transfer in a mating in a mating between Hfr between Hfr donor and Fdonor and F--

recipientrecipient

Fig. 14.14

Mapping genes in Hfr and FMapping genes in Hfr and F-- crosses crosses by interrupted mating experimentsby interrupted mating experiments

Interrupted mating studies confirm Interrupted mating studies confirm bacterial chromosome is a circlebacterial chromosome is a circle

Cross between Hfr Cross between Hfr and Fand F--

The F plasmid The F plasmid integrates into integrates into different locations different locations in different in different orientations into the orientations into the circular donor circular donor chromosomechromosome

Fig. 14.16 a, b

Partial genetic map of the Partial genetic map of the E. coliE. coli chromosomechromosome

Fig. 14.16 c

Recombination analysis improves Recombination analysis improves accuracy of mapaccuracy of map

Interupted mating experiments accurate to only 2 Interupted mating experiments accurate to only 2 minutesminutes

Frequency of recombination between genes is Frequency of recombination between genes is more accuratemore accurate

Start by considering only exconjugates that have Start by considering only exconjugates that have all of the genes to be mapped (select for the last all of the genes to be mapped (select for the last gene transferred)gene transferred)

Living cells must have even number of crossoversLiving cells must have even number of crossovers Consider as a three-point crossConsider as a three-point cross

Mapping genes using a three-point crossMapping genes using a three-point cross

Fig. 14.17

Different classes of crossovers: quadruple Different classes of crossovers: quadruple crossover is least frequentcrossover is least frequent

Fig. 14.17 c

F’ plasmids can be used for F’ plasmids can be used for complementation studiescomplementation studies

F’ plasmids replicate as discrete circles of DNA F’ plasmids replicate as discrete circles of DNA inside host cells.inside host cells.

Transferred in same manner as F plasmidsTransferred in same manner as F plasmids A few chromosomal genes will always be A few chromosomal genes will always be

transferred as part of the F’ plasmidtransferred as part of the F’ plasmid Can create partial diploidsCan create partial diploids Merozygotes – partial diploids in which two gene Merozygotes – partial diploids in which two gene

copies are identicalcopies are identical Heterogenotes – partial dipoids carrying different Heterogenotes – partial dipoids carrying different

alleles of the same genealleles of the same gene

F’ plasmid F’ plasmid formation and formation and

transfertransfer

Fig. 14.18 a, b

Complementation testing using F’ plasmidsComplementation testing using F’ plasmids

Creation of a Creation of a heterogenoteheterogenote

Phenotype of Phenotype of partial diploid partial diploid establishes establishes whether whether mutations mutations complement each complement each other or notother or not

Fig. 14.18 c

Transduction: Gene transfer via Transduction: Gene transfer via bactgeriophagesbactgeriophages

BacteriophagesBacteriophages Widely distributed in natureWidely distributed in nature Infect, multiply, and kill bacterial host cellsInfect, multiply, and kill bacterial host cells TransductionTransduction - may incorporate some of bacterial - may incorporate some of bacterial

chromosome into its own chromosome and transfer it to chromosome into its own chromosome and transfer it to other cellsother cells

Bacteriophage particles are produced by the lytic Bacteriophage particles are produced by the lytic cyclecycle Phage inject DNA into cellPhage inject DNA into cell Phage DNA expresses its genes in host cell and replicatePhage DNA expresses its genes in host cell and replicate Reassemble into 100-200 new phage particlesReassemble into 100-200 new phage particles Cells lyse and phage infect other cellsCells lyse and phage infect other cells Lysate is population of phage after lytic cyle is completeLysate is population of phage after lytic cyle is complete

Generalized Generalized transductiontransduction

Fig. 14.19

Mapping genes by generalized transductionMapping genes by generalized transduction

Frequency of recombination between genes Frequency of recombination between genes P1 bacteriophage often used for mappingP1 bacteriophage often used for mapping 90kb can be constransduced corresponding 90kb can be constransduced corresponding

to about 2% recombination or 2 minutesto about 2% recombination or 2 minutes First find approximate location of gene by First find approximate location of gene by

mating mutant strain to different Hfr mating mutant strain to different Hfr strainsstrains

P1 transduction then used to map to specific P1 transduction then used to map to specific locationlocation

Fig. 14.20

Temperate phage can integrate into bacterial genome Temperate phage can integrate into bacterial genome through lysogenic cycle creating a prophagethrough lysogenic cycle creating a prophage

Fig. 14.21

Recombination between att sites on the Recombination between att sites on the phage and bacterial chromosomes phage and bacterial chromosomes allows integration of the prophageallows integration of the prophage

Fig. 14.22 b

Errors in prophage excision produce Errors in prophage excision produce specialized transducing phagespecialized transducing phage

Adjacent genes are included in circular Adjacent genes are included in circular phage DNA that forms after excisionphage DNA that forms after excision

Fig. 14.22 c