Chair of Microbiology, Virology, and Immunology GENETICS OF BACTERIA AND VIRUSES BASES OF BIOTECHNOLOGY AND GENE ENGENEERING.

Chair of Microbiology, Virology, and Immunology

GENETICS OF BACTERIA AND VIRUSES BASES OF BIOTECHNOLOGY

AND GENE ENGENEERING

Lectures schedule

1. Structure of bacterial genome.

2. Extrachromosomal elements.

3. Mutations.

4. Recombinations.

5. Gene engineering.

F. Crick i J. Watson – described DNA

structure

DNA structure

E. coli DNA

The chromosome of E. coli has a contour length of approximately 1.35 mm, several hundred times longer than the bacterial cell, but the DNA is supercoiled and tightly packaged in the bacterial nucleoid. The time required for replication of the entire chromosome is about 40 minutes

E. coli DNA

Definition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon)Episome - a plasmid that can integrate into the chromosome

They are usually much smaller than the bacterial chromosome, varying from less than 5 to more than several hundred kbp.

Most plasmids are supercoiled, circular, double-stranded DNA molecules, but linear plasmids have also been demonstrated in Borrelia and Streptomyces.

Plasmid

Classification of Plasmids• Transfer properties

– Conjugative (This plasmids code for functions that promote transfer of the plasmid from the donor bacterium to other recipient bacteria)

Nonconjugative (do not)

Phenotypic effects– Fertility– Bacteriocinogenic plasmid– Resistance plasmid (R factors)

Phenotypic effects

Structure of R factors

• RTF– Conjugative

plasmid– Transfer genes

Tn 9

Tn

21

Tn 10

Tn 8

RTF

R determinant

• R determinant– Resistance

genes– Transposons

The average number of molecules of a given plasmid per bacterial chromosome is called its copy number. Large plasmids (40 kilobase pairs) are often conjugative, have small copy numbers (1 to several per chromosome).

Plasmids smaller than 7.5 kilobase pairs usually are nonconjugative, have high copy numbers (typically 10-20 per chromosome), rely on their bacterial host to provide some functions required for replication, and are distributed randomly between daughter cells at division.

Some plasmids are cryptic and have no recognizable effects on the bacterial cells that harbor them

Transposable Genetic Elements

• Definition: Segments of DNA that are able to move from one location to another

• Properties– “Random” movement

– Not capable of self replication

– Transposition mediated by site-specific recombination

• Transposase

– Transposition may be accompanied by duplication

Types of Transposable Genetic Elements

• Insertion sequences (IS)– Definition: Elements that carry no other genes

except those involved in transposition

– Nomenclature - IS1

– Structure

– Importance

• Mutation

•Plasmid insertion

•Phase variation

TransposaseABCDEFG GFEDCBA

The known insertion sequences vary in length from approximately 780 to 1500 nucleotide pairs, have short (15-25 base pair) inverted repeats at their ends, and are not closely related to each other.

Phase Variation in Salmonella H Antigens

ISH1 gene H2 gene

H1 flagella

H2 flagella

Types of Transposable Genetic Elements

• Transposons (Tn)– Definition: Elements that carry other genes

except those involved in transposition– Nomenclature - Tn10– Transposons can move from one site in a DNA

molecule to other target sites in the same or a different DNA molecule.

– Structure

IS ISResistance Gene(s)

IS ISResistance Gene(s)

Transposons are not self-replicating genetic elements, however, and they must integrate into other replicons to be maintained stably in bacterial genomes

Complex transposons vary in length from about 2,000 to more than 40,000 nucleotide pairs and contain insertion sequences (or closely related sequences) at each end, usually as inverted repeats. The entire complex element can transpose as a unit.

Importance

they cause mutations, mediate genomic rearrangements, function as portable regions of genetic homology, and acquire new genes, contribute to their dissemination within bacterial populations. insertion of a transposon often interrupts the linear sequence of a gene and inactivates it, transposons have a major role in causing deletions, duplications, and inversions of DNA segments as well as fusions between replicons.

In medically important bacteria, genes that determine production of adherence antigens, toxins, or other virulence factors, or specify resistance to one or more antibiotics, are often located in complex transposons.

Well-known examples of complex transposons are Tn5 and Tn10, which determine resistance to kanamycin and tetracycline, respectively.

Mutation is 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)

• Frequency of naturally occurring (spontaneous) mutation varies from 10-6 to 10-9 (avg = 10-8)

• This means that if a bacterial population increases from 108 to 2 x 108, on the average, one mutant will be produced for the gene in question.

Induced mutations - Arise under an influence of some factors

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

Types of MutationsMissense mutation: a single base

substitution in the DNA that changes a codon from one amino acid to another

Types of MutationsNonsense mutation: converts a sense

codon to a nonsense or stop codon, results in shortened polypeptide

Types of Mutations• Frameshift mutation: arise from accidental insertion or deletion

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

Insertion

Frameshift mutation - Deletion

Other Types of Mutations Forward mutation: a mutation that

alters phenotype from wild type Reverse mutation: a second

mutation which may reverse wild phenotype and genotype (in same gene)

Suppressor mutation: a mutation that alters forward mutation, reverse wild phenotype (in same gene - intragenic, in another gene - extragenic)

• 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

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)

N. meningitidis genes with high mutation rates include those

involved in:

capsule biosynthesis

LPS biosynthesis

attaching to host cells

taking up iron

Examples of mutagensCHEMICAL

AGENTACTION

HNO2

Nitrogen mustard NTG

React chemically with one or more bases so that they pair improperly

Intercalating agents (acridine dyes)

Insert into DNA and cause frame-shift mutations by inducing an addition or the subtraction of a base

Base analogs:

5-bromouracil

2-amino purine

Incorporate into DNA and cause mispairing

Analog of T which can pair with C

Analog of A which can pair with C

Examples of mutagensPHYSICAL

AGENTACTION

UV irradiation Causes formation of adjacent T-T dimers that distorts the DNA backbone, altering the binding properties of bases near the dimer

X-ray Alters bases chemically, causes deletions and induces breaks in DNA chain

Examples of mutagensBIOLOGICAL

AGENTACTION

Insertion sequences (IS)

Pieces of DNA about a thousand nucleotide bases in length which can insert into a genetic sequence

Transposons genetic elements goverened by IS which can insert into the chromosome within a gene

Viruses Some bacteriophage (e.g. phage µ) can integrate their DNA into random positions in

the bacterial chromosome

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

Reparation

Light-requiring

Dark

SOS- reactivation

Light-requiring Reparation

Dark Reparation

Exchange of Genetic Information

Recombination

Transformation

TransformationDefinition: Gene transfer resulting from the uptake of

DNA from a donor.

• Factors affecting transformation– DNA size and state (DNA molecules must be at least 500

nucleotides in length)

• Sensitive to nucleases (deoxyribonuclease)

– Competence of the recipient (Bacillus, Haemophilus, Neisseria, Streptococcus)• Competence factor• Induced competence

Transformation

– Recombination• Legitimate,

homologous or general

• recA, recB and recC genes

• Significance– Phase variation in Neiseseria– Recombinant DNA technology

• Steps

– Uptake of DNA

• Gram +

• Gram -

Competent cell

S strain

R strain S strain

Transduction

• Definition: Gene transfer from a donor to a recipient by way of a bacteriophage

Phage Composition and Structure

• Composition– Nucleic acid

• Genome size

• Modified bases

– Protein• Protection• Infection

• Structure (T4)

– Size– Head or capsid– Tail

Tail

Tail Fibers

Base Plate

Head/Capsid

Contractile Sheath

Transduction

Types of transduction– Generalized - Transduction in which

potentially any donor bacterial gene can be transferred

Generalized Transduction

• Release of phage

• Phage replication and degradation of host DNA

• Assembly of phages particles

• Infection of recipient• Legitimate recombination

• Infection of Donor

Transduction

Types of transduction

–Specialized - Transduction in which only certain donor genes can be transferred

Specialized TransductionLysogenic Phage

• Excision of the prophage

gal

bio

gal bio

gal bio

gal

bio

bio

gal

• Replication and release of phage

• Infection of the recipient

• Lysogenization of the recipient– Legitimate

recombination also possible

TransductionTypes of transduction

Abortive transduction refers to the transient expression of one or more donor genes without formation of recombinant progeny, whereas complete transduction is characterized by production of stable recombinants that inherit donor genes and retain the ability to express them.

• In abortive transduction the donor DNA fragment does not replicate, and among the progeny of the original transductant only one bacterium contains the donor DNA fragment. In all other progeny the donor gene products become progressively diluted after each generation of bacterial growth until the donor phenotype can no longer be expressed.

Transduction

• Significance– Common in Gram+ bacteria– Lysogenic (phage) conversion

Bacterial ConjugationDefinition: The transfer of genetic

information via direct cell-cell contact

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

In conjugation, direct contact between the donor and recipient bacteria leads to establishment of a cytoplasmic bridge between them and transfer of part or all of the donor genome to the recipient. Donor ability is determined by specific conjugative plasmids called fertility plasmids or sex plasmids.

The F plasmid (also called F factor) of E coli is the prototype for fertility plasmids in Gram-negative bacteria. Strains of E coli with an extrachromosomal F plasmid are called F+ and function as donors, whereas strains that lack the F plasmid are F- and behave as recipients.

Conjugation• Gene transfer from a donor to

a recipient by direct physical contact between cells

• Mating types in bacteria– Donor

• F factor (Fertility factor)– F (sex) pilus

Donor

Recipient

– Recipient• Lacks an F factor

Physiological States of F Factor

• Autonomous (F+)– Characteristics of F+ x F-

crosses• F- becomes F+ while F+ remains

F+

• Low transfer of donor chromosomal genes F+

Physiological States of F Factor

•Integrated (Hfr)–Characteristics of Hfr x F- crosses•F- rarely becomes Hfr while Hfr remains Hfr•High transfer of certain donor chromosomal genes

F+ Hfr

Physiological States of F Factor

•Autonomous with donor genes (F′)–Characteristics of F’ x F- crosses•F- becomes F’ while F’ remains F’•High transfer of donor genes on F’ and low transfer of other donor chromosomal genes

Hfr F’

Mechanism of F+ x F- Crosses

• DNA transfer– Origin of

transfer– Rolling

circle replication

• Pair formation

– Conjugation bridge

F+ F- F+ F-

F+ F+F+ F+

Mechanism of Hfr x F- Crosses

• DNA transfer– Origin of

transfer

– Rolling circle replication

• Homologous recombination

• Pair formation

– Conjugation bridge

Hfr F- Hfr F-

Hfr F-Hfr F-

Mechanism of F′ x F- Crosses

• DNA transfer– Origin of

transfer

– Rolling circle replication

• Pair formation

– Conjugation bridge

F’ F’F’ F’

F’ F- F’ F-

Conjugation

• Significance– Gram - bacteria

• Antibiotic resistance

• Rapid spread

– Gram + bacteria• Production of adhesive material by donor cells

Map of chromosome

Gene cloning is the process of incorporating foreign genes into hybrid DNA replicons.

Cloned genes can be expressed in appropriate host cells, and the phenotypes that they determine can be analyzed. Some key concepts underlying representative methods are summarized here.

Recombination DNA and Gene Cloning

Bacterial plasmids in gene cloning

Steps for eukaryotic gene cloning• Isolation of cloning vector

(bacterial plasmid) & gene-source DNA (gene of interest)

• Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase

• Introduction of cloning vector into cells (transformation by bacterial cells)

• Cloning of cells (and foreign genes)

• Identification of cell clones carrying the gene of interest

DNA Cloning• Restriction enzymes (endonucleases):

in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific

• Restriction site:recognition sequence for a particular

restriction enzyme

• Restriction fragments:segments of DNA cut by

restriction enzymes in a reproducable way

• Sticky end:short extensions of

restriction fragments

• DNA ligase: enzyme that can join the

sticky ends of DNA fragments

• Cloning vector: DNA molecule that can carry foreign

DNA into a cell and replicate there (usually bacterial plasmids)

Restriction endonucleases

Practical DNA Technology Uses

• Diagnosis of disease

• Human gene therapy

• Pharmaceutical products (vaccines)

• Forensics

• Animal husbandry (transgenic organisms)

• Genetic engineering in plants

• Ethical concerns?

GENES THERAPY

Biotechnology practical use