Microbial Genetics

Lecture 1

Introduction

Genetics

Human genetics Plant genetics Animal Genetics Microbial genetics

Cytogenetics Quantitative Genetics Immunogenetics Biochemical Genetics


)(haploid )(transformation-conjugation-transduction


Esherichia coli Sallmonella typhimurium Streptococcus pneumonia Bacillus subtilis

Genetic material

DNA = genetic material

Griffith (1920)

Hershey-Chase experiment

Microbial Genetics

Lecture 2

DNA Replication

 DNA

 -1

 -2 A

 -3 DNA RNA -4 DNA -5 RNA

 -6

Primosome -7

Microbial Genetics

Lecture 3

Gene Expression

DNA methylation

Gene expression

Transcription
DNA is transcribed to make RNA (mRNA, tRNA, and rRNA) Transcription begins when RNA polymerase binds to the promoter sequence Transcription proceeds in the 5' 3' direction Transcription stops when it reaches the terminator sequence

Transcription

Figure 8.7

The Process of Transcription

The Process of Transcription

Figure 8.7

The Process of Transcription

Figure 8.7

The Genetic Code

Figure 8.8

Initiation

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

The Process of Translation

Figure 8.9

Simultaneous Transcription & Translation

The Regulation of Gene Expression

Constitutive genes are expressed at a fixed rate Other genes are expressed only as needed
Metabolism Response to environmental stress Cell division

Operon

Lactose present, no glucose

Lactose + glucose present

Microbial Genetics

Lecture 4

Mutation

Mutants

MUTATION
Mutation : Is any heritable change in the genetic material Mutations may be neutral, beneficial, or harmful Mutant : organism or strain whose genome carries a mutation Mutagen: Agent that causes or accelerates rate of mutations Wild type: the usual (native) form of the organism Phenotype : all the observed properties Genotype : The entire set of genes in an organism.

Other phenotype in bacterial genetics

Auxotrophy
An auxotroph needs some nutrient that the wild type strain (prototroph) can make for itself. For example,
trp- auxotroph cant make its own tryptophan (an amino acid). To grow trp- bacteria, you need to add tryptophan to the growth medium. Prototrophs are trp+; they dont need any tryptophan supplied since they make their own.

Conditional Lethal Mutations

Temperature Sensitive Mutations Mesophilic bacteria (20-42 C) Thermophilic bacterica (42-60 C)
45 C

Incubation Temperature 30 C


confer resistance antibiotics Example: AmpR causes bacteria to be resistant to ampicillin, a common antibiotic related to penicillin

Microbial Genetics

Lecture 5

Mutation

Mutations
Spontaneous Induced

Occur in the absence of a mutagen

Occur in the presence of a mutagen

 Penicillin Selection Method Kills Growing Cells

FREQUENCY OF MUTATION
Spontaneous mutation rate = 1 in 109 replicated base pairs
1 in 106 replicated genes

Mutagens increase to 105 or 103 per replicated gene

TYPES OF MUTATION
Base substitution (point mutation)
Change in one base

Frame shift mutation

Insertion or deletion of one or more nucleotide pairs

Base substitution

Frame Shift Mutation

Mutation
Frameshift mutation Insertion or deletion of one or more nucleotide pairs

Other Mutations
Deletions Lose of a Piece of DNA Insertions Additions of a Piece of DNA Translocation Movement of a Piece of DNA to a New Location in the Genome

Inversion
Orientation of a Piece of DNA is Reversed in the Genome

Mutation Back mutation Suppressor mutation

Prototroph

Auxotroph

Back Mutation

Mutation

Back Mutation

Normal Protein

Ames Test for Chemical Carcinogens

Ames Test for Chemical Carcinogens

Mutagens
Chemicals Base Analogs Chemically Resemble Base

Intercalating Agent Inserts Between Base Pairs

Physical mutagens
Ionizing radiation (X rays and gamma rays) causes the formation of ions that can react with nucleotides and the deoxyribosephosphate backbone
Nucleotide excision repairs mutations

Non ionizing radiation

UV radiation causes thymine dimers Light-repair separates thymine dimers

Radiation
Ionizing radiation (X rays and gamma rays) causes the formation of ions that can react with nucleotides and the deoxyribosephosphate backbone

 Radiation UV Light Ionizing Radiation

TRANSPOSONS
Biological mutagen Segments of DNA that can move from one region of DNA to another Contain insertion sequences for cutting and resealing DNA (transposase) Complex transposons carry other genes

Transposon mutagenesis
Insertion elements (IS)

ORF

IR
ORF encodes the transposase

IR

Inverted repeats are identical and of variable length Different IS exist (IS1, IS2, IS50.) Function unknown?

Segments of DNA that can move from one region of DNA to another Contain insertion sequences for cutting and resealing DNA (transposase) Complex transposons carry other genes

Transposons

Figure 8.30a, b

Transposon: a piece of short DNA that replicates by inserting into other pieces of DNA (plasmids, chromosomes, etc) Useful for studying gene function because when the transposon moves into different location in the DNA it may cause a disruption in a gene or a set of genes. Transposons also have many useful properties for mutagenesis: Cause clean mutations Can be random or specific mutations Typically encode for antibiotic resistance or some other advantageous gene. Can use a transposon that inserts at a high frequency When used in bacteria it causes selectable phenotypes

Mini-transposons
km tnp

Only resistance gene and ends transpose

Reduced size No possibility of secondary transposition No excision (stable insertions)

Transposon mutagenesis

Transposon mutagenesis

No protein

Transposon mutagenesis
Donor (E. coli) Recipient (Pseudomonas)

Tn5
Suicide vector Conjugation Selection Chromosome

Vector lost

Tn5 inserted

Chromosome

Selection of transposon insertion clones

Conjugation mix

Plating on rich medium plus Km and Sm

Replica-plating

Auxotroph Rich medium Minimal medium

Strategy

PCR Sequencing

Advantages of transposon mutagenesis

Transposon
Safe All clones are mutants Single mutation Identification of insertion

Chemical
Point mutations

Chemical Mutagens

Chemical Mutagens

Radiation

Microbial Genetics

Lecture 6

Gene transfer in Bacteria

Genetic Recombination
Vertical gene transfer: Occurs during reproduction between generations of cells. Horizontal gene transfer: The transfer of genes between cells of the same generation.

Horizontal gene transfer

Transformation
Transfer of isolated donor DNA (either chromosomal DNA fragments or plasmid DNA) to a recipient cell. Successful transformation depends on the presence of double-stranded donor DNA molecules that are large enough, as well as cells that are competent for transformation

Conjugation

Figure 8.26

Conjugation
A process of gene transfer from a living donor cell to a living recipient cell Typically, the donor cell will possess conjugative structures on its surface that attach the donor cell to the recipient cell. The conjugative structures will also mediate the transfer of DNA from the donor to the recipient. The ability to conjugate is often encoded on a plasmid. For example: In Escherichia coli, conjugation is mediated by the F pili that are encoded for by genes on the F plasmid.

Conjugation in E. coli

Figure 8.27a

A strain of E. coli having F plasmids and pili is called an F+ strain; a strain lacking F plasmids or pili is F. When an F+ cell (the donor) is mated with an F cell (the recipient), a copy the F plasmid is transferred to the F cell, so that after the process is complete, both cells will be F+.

Conjugation in E. coli

In some strains of E. coli, an F plasmid DNA sequence has become inserted into the chromosome through genetic recombination. These are called Hfr strains. Different Hfr strains have the F sequence inserted at different locations on the chromosome.
Figure 8.27b

Conjugation in E. coli

The cells of Hfr strains have F pili, and are capable of conjugating with F cells. In an Hfr x F mating, the F sequence is transferred first, followed by the chromosomal DNA. Genes from the Hfr (donor) chromosome can replace genes in the F chromosome by genetic recombination.

Figure 8.27c

The order of genes near the F insertion site on the chromosome can be determined in an interrupted mating cross between Hfr x F strains.
Select an Hfr strain and an F strain that differ in specific phenotypes. For example, an Hfr with the phenotypes gal+, trp+, lac+, tsx+ could be mated to an F that is gal, trp, lac, tsx. Mix together broth cultures of the Hfr & F cells. The two strains will begin the conjugation process. This is time 0 of the interrupted mating experiment. At time intervals, remove a sample from the culture & gently shake it to break up the conjugating pair (interrupted mating)

Plate the sample onto selective media to determine the number of Hfr phenotypes found among the exconjugants. The order that the genes are transferred from the Hfr to F strains reflects their order on the chromosome; in the example given the order would be lac, tsx, gal, trp

Transduction
Transfer of genes from a donor cell to a recipient cell through a bacteriophage intermediate. Bacteriophage: A bacterial virus

Transduction by a Bacteriophage

Fig. 14.3 Daviss U-tube experiment