Chapter 9 Lecture Outline Gene Transfer, Mutations, and Genome Evolution

Covered in this Lecture 9.1 The mosaic nature of genomes 9.2 Gene transfer: Transformation, conjugation, transduction 9.3 Recombination 9.4 Mutations 9.5 DNA repair 9.6 Mobile gene elements Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

The Mosaic Nature of Genomes DNA sequence is not static Over the millennia microbes have undergone extensive gene loss and gain Horizontal gene transfer Recombination events Mutations Single bases Large deletions Large insertions of sequence Transferred from other species New functions useful in particular situations Pathogenicity islands Fitness islands Maintained via interaction with environment Survival determined by having appropriate genes for the specific environment Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Gene Transfer Transformation Uptake of naked DNA Conjugation Uptake of DNA from cell to cell with direct contact Transduction Transfer of DNA via virus Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Transformation: History Griffith: demonstrated the phenomena of transformation in 1928 Avery, MacLeod, and MacCarty: proved that DNA is the transforming element in 1944 Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Gene Transfer: Transformation Uptake of DNA directly from surrounding medium DNA used as nutrient For repair Adjustment to the environment Natural transformation is found in many species Competent cells Gram +: Streptococcus, Bacillus Gram-: Haemophilus, Neisseria Translocasome takes up DNA Located in envelope Competence induced in different species Gram+ cells secrete signal Species specific competence factor Gram- cells do not produce competence factor Always competent Stress induces competence Starvation Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Artificial Transformation In the laboratory Alter membrane to allow DNA to pass Chemical CaCl2 Electroporation Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Gene Transfer: Conjugation Sex pilus connecting 2 E. coli cells. Requires cell to cell contact “Bacterial sex” In gram- bacteria: sex pilus In gram+ bacteria: non-pilus attachment proteins Requires transferable plasmid Contains all the genes for pilus/attachment information and DNA export (ex.: F factor) If plasmid incorporates into host chromosome it may also transfer host genes Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

The Conjugation Process 1. Sex pilus from the F+ donor cell attaches to receptors on recipient cell. Donor (F+) Cell Recipient (F-) Cell 2. Contraction of the pilus draws the two cells together and forms a conjugation bridge. 3. The F factor is nicked at oriT and the 5’ end begins transfer through the bridge. 4. The strand remaining in the donor is replicated by rolling circle with Pol III. 5. Once in the recipient the transferred strand circularizes and replicates. 6. The recipient has been converted to a donor. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. F+ Cell F+ Cell

Gene Transfer: Conjugation Episome Plasmid that can exist in extrachromosomal and integrated form Hfr High frequency recombination F factor integrates on bacterial chromosome Bacterial strain is called Hfr strain More cells are capable to transfer chromosomal DNA Tries to transfer entire chromosome Requires 100 minutes for E. coli (44 kbp/min) Transfers genes in order Can determine order of genes on chromosome by interrupted mating F (F prime) factor Integrated F factor is excised along with some chromosomal DNA F plasmid contains extra genes In addition to genes for pilus, transfer Transfers extra genes to recipient F factor Bacterial chromosome lac genes oriT Flac Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Conjugation across Species Relatively frequently across species Ex.: from E. coli to Salmonella Some bacteria can cross biological domain From Agrobacterium tumefaciens to plants Tumor inducing plasmid Crown gall disease Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Gene Transfer: Transduction Bacteriophage inject DNA into cell After replication and capsid production package DNA into viral capsid Viral DNA Sometimes accidentially package bacterial host DNA Transfer DNA to new host Host DNA Generalized transduction Specialized transduction Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Generalized Transduction During lytic life cycle host DNA is degraded Can be packaged into capsid Can replace entire virus genome Transducing particle is generated Defective: can infect a new host cell but not be replicated Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Specialized Transduction During lysogenic life cycle host DNA is accidentally excised when prophage is reactivated New virus genome contains host genes lying adjacent to the phage insertion site Can be packaged into capsid along with viral DNA Infective virion: can infect a new host cell and will be able to replicate Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Defense against Transferred DNA Bacteria cut entering DNA to pieces Cut at specific restriction sites with restriction enzymes Bacteria add methyl groups to DNA Prevents restriction at those sites Added as cell replicates chromosome Entering DNA is destroyed Unless it comes from a similar species And has methyl groups protecting DNA Prevention of promiscuous DNA transfer EcoRI restriction/ modification site Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Recombination New DNA enters via transformation, conjugation, transduction Fate of DNA Independent existence as plasmid Degradation Integration into host chromosome through recombination Recombination is the process by which DNA sequences can be exchanged between DNA molecules Replaces variable-sized section of DNA DNA repair Enhancement of fitness Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Generalized Recombination Considerable stretch of DNA homology Involves RecA and intermediate ssDNA Cross over sites appear as crosses Holliday junction Deinococcus radiodurans uses RecA homologue for radiation induced DNA repair Has multiple copies of each of its 2 chromosomes Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Site-Specific Recombination Exganche between DNAs with little overall sequence homology Does not require RecA Requires insertion sequences 10 – 20 bp sequence recognizable by restriction enzymes is required Intergrases mediate site-specific recombination Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Click box to launch animation Recombination Animation: Recombination Click box to launch animation Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Mutation Heritable permanent change in DNA sequence Harmful, beneficial, or neutral Change in genotype May result in a change in phenotype Change is base sequence and failure to repair Point mutations Change in one base pair Transition: purine  purine, pyrimidine  pyrimidine Transversion: purine  pyrimidine Larger mutations Insertion addition of 1 or > nucleotides Deletion subtraction of 1 or > nucleotides Inversion flipping a fragment of the DNA Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. (Missense) Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Silent Mutations Normal Most mutations are silent No effect on organism Mutations in regions between genes Mutations that change 3rd base of a codon Mutations that change 1 amino acid into a similar one Protein still retains normal function Mutations that change a protein that is not needed In current environment of organism Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Types of Mutations Silent No effect on organism Missense mutation Change one codon to another Nonsense mutation Change a codon to Stop Frameshift mutation Insert or delete a single base Changes bases read by ribosome Alters all codons downstream of mutation CGA  CGC Arginine  Arginine CGA  CAA Arginine  Glutamine CGA  TGA Arginine  STOP AAA CGA CCC  AAA CTG ACC C Lysine Arginine Proline  Lysine Leucine Threonine Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Mutagens Increase the rate of mutations Number of mutations formed per cell doubling Spontaneous mutation rate is 106 to 108 per generation Increase of mutation frequency Number of mutant cells per population (ratio of mutant cells/ total number of cells) Most mutagens are carcinogens Cancer results from multiple mutations Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Sectored Colonies reflect Mutation Mutant yeasts accumulate red compound Normal Lac- bacteria mutate and revert to Lac+. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Measurement of Mutagens: Ames Test Reversion test Uses bacterial strain auxotrophic for histidine Has mutation in hisG gene Cannot grow unless histidine is supplied Expose bacteria to the mutagen Mutagen causes reversion Changes mutation to normal form Normally rare mutation More colonies = stronger mutagen Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Classical and Modified Ames Test Bacteria are plated on medium w/o his and a disk with mutagen is added Modified Bacteria are incubated with mutagen and liver extract and then plated onto medium w/o his Mutagen on paper disk Revertant colonies Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. DNA Repair Methyl mismatch repair Parent strand is methylated Newer strand is less methylated and more likely to contain errors Nucleotide excision repair Clips out patches of ssDNA Base excision repair Excision of structurally altered bases without cleaving the phosphodiester backbone resulting in an apurinic and apyrimidic site (AP site) AP nucleases follow up Recombinational repair Intact strand is used to replace a homologous damaged strand SOS response Coordinated response to extensive damage Set of genes and repair mechanisms activated Error-Proof Error-Prone Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Mobile Genetic Elements Chromosomes sequence is not fixed Transposable elements insert into chromosome Found in all species Retroviruses (HIV) insert DNA in mammals Can jump from one site to another Nonreplicative transposition Can copy itself to a new site Replicative transposition Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Transposable Elements DNA sequences Are not autonomous Cannot exist outside of a larger DNA molecule Insertion sequences (IS) 500 – 1500 bp Transposase gene Flanked by short inverted repeat sequences Transposons (Tn) More complex than IS Carry additional genes Antibiotic resistance or catabolic genes 5’-AATCGAT……….ATCGATT-3’ Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Basic Transposition and Origin of Target Site Duplication Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Non-Replicative and Replicative Transposition Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Mobile Genetic Elements Animation: Transposition Click box to launch animation Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Concept Quiz Gene transfer by means of a bacteriophage is called: Transformation Conjugation Transduction Answer: C Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Concept Quiz In an Ames Test, a very strong mutagen will cause the appearance of: Many colonies over the plate, with a clear space close to the disk of mutagen. A few colonies throughout the plate up to the edge of the disk. Many colonies covering the whole plate, extending up to the disk. Answer: A Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Concept Quiz Even though transposons disable genes into which they jump, transposons aren’t immediately detrimental for a cell because: Transposons speed evolution through lateral transfer. Transposons are unmethylated, and are destroyed by the recipient cell. Transposons bring antibiotic genes into the cell. Answer: A Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.