Kelly Doran, Ph.D.

• Assistant Professor of Pediatric Infectious Diseases• UCSD, School of Medicine

– kdoran@ucsd.edu

• Lecture 1 – Mutation (239-248)

• Types of mutations• Detection of mutations

– Recombination and Plasmids (285-306)• Recombination• Plasmids• Conjugation• Transposable Elements• Transformation

Microbial Genetics Lectures

• Genetics = branch of biology that deals with heredity, especially the mechanisms of hereditary transmission and the variation of inherited characteristics among similar or related organisms.

• At the most basic level is the study of genes

• Genes are the fundamental unit of heredity– DNA sequence in the

chromosome– Transcribed into mRNA– Translated into proteins

which make cells work

• Genes are copied (DNA replication) almost exactly from parent cell to daughter cell (and from parent to offspring)

• The copying of genes from one generation to the next is crucially important

• Too many mistakes (MUTATIONS) and gene integrity is lost and the system falls apart

• Anyone who has never made a mistake has never tried anything new. - Albert Einstein

• A mistake may turn out to be the one thing necessary to a worthwhile achievement - Henry Ford

Maybe mistakes are not such a bad thing?

In bacterial populations mutations are constantly arising due to errors made during replication.

If there is any selective advantage for a particular mutation (e.g. antibiotic resistance), the mutant will quickly become the major component of the population due to rapid growth rate of bacteria.

Bacteria have mechanisms by which genes can be transferred to other bacteria. Thus, a mutation arising in once cell can be passed on to other cells.

Bacterial Genetics

Mutation = A stable, heritable change in the genomic nucleotide sequence

How do mutations occur?

• Spontaneous mutations - Arise occasionally in all cells; are often the result of errors in DNA replication (random changes)

• Errors in replication which cause point mutations; other errors can lead to frameshifts – Point mutation - mismatch substitution of one

nucleotide base pair for another– Frameshift mutation - arise from accidental

insertion or deletion within coding region of gene, results in the synthesis of nonfunctional protein

Types of Mutations

• Point mutation = affects only 1 bp at a single location

– Silent mutation = a point mutation that has no visible effect because of code degeneracy

– Missense mutation = a single base substitution in the DNA that changes a codon from one amino acid to another

– Nonsense mutation = converts a sense codon to a nonsense or stop codon, results in shortened polypeptide

Base-pair substitution – missense mutation

(Silent mutation)

Missense mutation

Nonsense mutation

Types of Mutations

• Frameshift mutation = arise from accidental insertion or deletion within coding region of gene, results in the synthesis of nonfunctional protein

Frame-shift mutation - Insertion

Frameshift mutation - Deletion

Other Types of Mutations

• Insertion/deletion mutation = Larger stretch of DNA added or deleted from a gene that alters gene expression

• Forward mutation = a mutation that alters phenotype from wild type

• Reverse mutation = a second mutation which may make the mutant appear wt (in same gene)

How do Mutations occur?• Induced mutations-caused by mutagens

• Mutagens – Molecules or chemicals that damage DNA or alter its chemistry and pairing characteristics

– Base analogs are incorporated into DNA during replication, cause mispairing

– Modification of base structure (e.g., alkylating agents)

– Intercalating agents insert into and distort the DNA, induce insertions/deletions that can lead to frameshifts

– DNA damage so that it cannot act as a replication template (e.g., UV radiation, ionizing radiation, some carcinogens)

• Morphological mutations-result in changes in colony or cell morphology

• Lethal mutations-result in death of the organism

• Conditional mutations-are expressed only under certain environmental conditions

• Biochemical mutations-result in changes in the metabolic capabilities of a cell – 1) Auxotrophs-cannot grow on minimal media because they

have lost a biosynthetic capability; require supplements – 2) Prototrophs-wild type growth characteristics

• Resistance mutations-result in acquired resistance to some pathogen, chemical, or antibiotic

Mutations affect bacterial cell phenotype

Mutant Detection• In order to study microbial mutants, one must be able

to detect them and isolate them from the wild-type organisms

• Visual observation of changes in colony characteristics

• Mutant selection-achieved by finding the environmental condition in which the mutant will grow but the wild type will not (useful for isolating rare mutations)

• Screen for auxotrophic mutants: A lysine auxotroph will only grow on media that is supplemented with lysine

Mutant Detection

Mutants are generated by treating a culture of E. coli with a mutagen such as nitrosoguanidine

The culture will contain a mixture of wild-type and auxotrophic bacteria

Out of this population we want to select for a Lysine auxotrophic mutant

minus lysinecomplete

Lysine auxotrophsdo not grow

All strains grow

Isolation of a Lysine Auxotroph

Microbial Recombination and Plasmids(p285-306)

•Recombination•Plasmids•Conjugation•Transposable Elements•Transformation

Bacterial RecombinationA process by which one or more nucleic acid molecules are

rearranged or combined to produce a new nucleotide sequence

• Types of recombination – General recombination involves exchange between

homologous DNA sequences

– Site-specific recombination is the nonhomologous insertion of DNA into a chromosome; often occurs during viral genome or transposon integration into the host, a process catalyzed by enzymes specific for the host sequence

– Replicative recombination accompanies replication and is used by some genetic elements that move about the genome

DNA Recombination:

Horizontal gene transfer-transfer of genes from one independent organism to another

Vertical gene transfer-transmission of genes from parents to offspring)

Mechanisms of horizontal gene transfer

• Conjugation is direct transfer from donor bacterium to recipient while the two are temporarily in physical contact

• Transformation is transfer of a naked DNA molecule

• Transduction is transfer mediated by a bacteriophage (viruses that infect bacteria)

Plasmids

• Plasmids are small ds DNA molecules, usually circular that can exist independently of the host chromosome. They have their own replication origin so can replicate automonously (episomes) and have relatively few genes (<30) that may or may not be essential to the host.

Bacterial cell

Plasmid DNAChromosomal

DNA

Types of Plasmids

• Conjugative plasmids have genes for pili and can transfer copies of themselves to other bacteria during conjugation

• Fertility factor or F factor - These plasmids can also intergrate into the host chromosome or be maintained as an episome (independent of chromosome)

• R factor - Also conjugative plasmids which have genes that code for antibiotic resistence for the bacteria harboring them. These do not integrate into the host chromosome.

• Col Plasmids - harbor Bacteriocins which are proteins that destroy other bacteria (eg cloacins kill Enterobacter species)

• Virulent plasmids - have genes which make bacteria more pathogenic because the bacteria is better able to resist host defenses or produce toxins/invasins

Bacterial Conjugation

• The transfer of genetic information via direct cell-cell contact

• This process is mediated by fertility factors (F factor) on F plasmids

• Video Clip…

QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.

Lederberg

Tatum

Evidence for Bacterial Conjugation1946

Demonstrated genetic recombination

Met- Thr+ Leu+ Thi+ Met+ Thr- Leu- Thi-

The U-Tube Experiment

Genetic recombination by conjugation requires direct physical contact between bacteria

Basic Bacterial Conjugation

• F+ / F- mating• An F plasmid moves from the donor (F+) to a recipient (F-)• The F plasmid is copied and transferred via a sex pilus, the

recipient becomes F+ and the donor remains F+

• The F factor codes for pilus formation which joins the donor and recipient and for genes which direct the replication and transfer of a copy of the F factor to the recipient

• The F factor can remain as a plasmid or it can integrate into the bacterial chromosome via IS sequences. This type of donor is called and Hfr strain (High frequency recombination)

• F’- When the F factor in an Hfr strain leaves the chromosome, sometimes is makes an error in excision and picks up some bacterial genes

Bacterialchromosome

F plasmid

Homologousrecombination

F Factor

Bacterialchromosome

HFR StrainF+ Strain

High Frequency Recombination

Transposable Elements • Transposons - DNA segments that carry genes that allow them to move about the

chromosome (transposition)– Unlike plasmids or phages, they are unable to reproduce or exist apart from the host

chromosome

Tn

plasmid

Transposable Elements• Insertion sequences - IS elements- short sequence of DNA

containing only genes required for transposition Flanked by inverted repeats (IR) - identical or similar sequences 15-25 bp in reversed orientation– Transposase - enzyme that recognizes the IR and promotes

transposition

Transposase (402 amino acids)

Invertedrepeat (IR) IR

IS101329 bp

Bacterial chromosome

Bacterial chromosome

IS10L IS10R

Tn109,300 bp Tetracycline

resistancegene

Composite transposon (Tn)- contains other genes in addition to transposase like antibiotic resistance genes or toxins

Transposable Elements

Transposition Mechanism

IR IR

ACAGTTCAGTGTCAAGTC

CTGAACTGTGACTTGACA

Transposase

TCGATAGCTA

ChromosomalDNA

Cut

Cut

Insertion of Tninto chromosomal

DNA target sequencecatalysed by transposase

IR IR

ACAGTTCAGTGTCAAGTC

CTGAACTGTGACTTGACA

TransposaseTCGATAGCTA

IR IR

ACAGTTCAGTGTCAAGTC

CTGAACTGTGACTTGACA

Transposase TCGATAGCTA

TCGATAGCTA

Gap filled byDNA polymeraseand DNA ligase

Transposition Mechanism

Transposable Elements

Importance

• Can insert within a gene to cause a mutation or stimulate DNA rearrangement leading to deletions of genetic material

• Can have termination sequences to block translation or transcription

• Can have promoters which activate genes near pt of insertion

• Can move antibiotic resistance genes around

• Can be on plasmids to aid in insertion of F plasmids into host chromosome

• Some bear transfer genes (Tn916) and can move between bacteria through conjugation (conjugative transposon)

DNA Transformation

• Transformation-a naked DNA molecule from the environment is taken up by the cell and incorporated in some heritable form. This process is random and any portion of the genome may be transferred

• A competent cell is one that is capable of taking up DNA

• Competent bacteria must be in a certain stage of growth (usually exponential) and secrete a small protein (competency factor) that stimulates production of new protein required for DNA uptake

• Gene transfer by this process occurs in soils and marine environments so it is an important route of genetic exchange in nature

• Artificial transformation - carried out in laboratory to transfer plasmid DNA, a common method for introducing recombinant DNA into bacterial cells. eg CaCl2 or electroporation

QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.

Oswald T. Avery

Competent cell

S strain

R strain S strain

Treatment

Plasmid

Competent cell

Hfr X F– Mating

• Similar to the F+ X F– cross

Sex pilus

F - cell

conjugalbridge

HfrF - cell

Ames Test for MutagenicityAuxotroph (tryp- mutant)

Looking for reversion mutants

Selective media to look for mutants