Lectures of Dr.Mohammad Alfaham. The Bacterial Genetics

Transcription

1 Lectures of Dr.Mohammad Alfaham The Bacterial Genetics

2 is the total collection of genes carried by a bacterium both on its chromosome and on its extrachromosomal genetic elements (plasmids)

3 A Gene A gene (unit of heredity) is a nucleotide sequence of DNA which determines the synthesis of polypeptide, or replication of suitable molecule of RNA Genes essential for bacterial growth are carried on a single chromosome - nucleoid All the bacterial cells are therefore haploid

4 DNA Structure a) A schematic, nonhelical model b) A schematic drawing of the Watson-Crick structure of DNA, showing helical sugar-phosphate backbones of the two strands held together by hydrogen bonding between the basis c) Space-filling model of DNA

5 DNA Forms a) The DNA double helix of most procaryotes has the shape of a closed circle b) The circular DNA strands, already coiled in a double helix, are twisted a second time to produce supercoils

6 DNA Forms The total length of E.coli chromosome is about 1 mm The Bacterium itself is only several micrometers in length The DNA is about 1000 times longer than bacterium and must be condensed (supercoiling)

7 DNA Replication Semiconservative Replication of DNA The replication fork of DNA showing the synthesis of two progeny strands. Each copy contain one new and one old strand. Bacterial chromosome is called Replicon Replicon a part of the genome that contains an origin site and is replicated as a whole unit

8 DNA Replication An autoradiograph of a replicating E.coli chromosome; about one-third of the chromosome has been replicated

9 Replication of circular DNA Vegetative

10 Replication of circular DNA Conjugative Mechanism used by some bacterial viruses, plasmids and in chromosomal transfer

11 Transposable elements IS Insertion sequences Tn - Transposones IS are small DNA pieces, about 1-2 kilobases long with 2 distinctive traits: - CORE area containing genes for transposition - Specific INVERTED REPEATS at their ends Tn is a genetic element that containing genes for transposition and additional extragenes, which provide resistance against antibiotics or synthesis enzymes of specific metabolic pathways These elements can transfer from chromosomal location to another, and from a chromosome to a plasmid or back

12 Transposable elements

13 Transposable elements Main roles: Cause deletion and inversions of DNA sequences (internal or biological mutagenic agents) Insert into genes and inactivate those genes Spread of antibiotic resistance genes Mobile genetic elements are responsible for the major part of genetic variability in natural bacterial populations Tn-s may enter other genera of bacteria during transfer of plasmids or via transducing phage

14 Plasmids Plasmids is a small genetic elements capable of independent replication in bacteria. Most plasmids are circular doublestranded DNA molecule, but some are linear. F-Plasmid Plasmids made their presence by conferring phenotypes of cell harboring them. F - fertility factor R - antibiotic resistance Col - colicin production Virulence plasmids: Ent - enterotoxin production Hly - hemolysin production CFA-I; CFA-II - adhesins production

15 Bacteriophages Litic cycle Lysogenic cycle

16 Bacteriophages Virulent bacteriophage a bacteriophage which always causes the lytic cycle, resulting in a death of a cell and production of new phage particles Temperate (or lysogenic) bacteriophage a bacteriophage whose DNA integrates into bacterial chromosome, but doesn t cause the lysis of the cell and production new phage particles. The integrative phage s DNA is called prophage A bacterial cell with prophage is called lysogenic cell

17 Bacteriophages Lysogenic conversion is a state when bacterial cell exhibit new properties, that are coded by the prophage genes Example: - Toxigenic (tox+) and nontoxigenic strains of Corynebacterium diphtheriae - Production of erythrogenic toxin by Streptococcus pyogenes - Production botulinal toxin of Clostridium botulinum

18 Genetic Variation Mutations are stable hereditary changes in the coding sequence of DNA Occur spontaneously or are induced by different mutagens Various kind of mutations

19 Genetic Variation Genetic recombination at the molecular level is the process by which DNA from a donor cell and DNA from a recipient cell combine to yield a new genome containing information from both sources Molecular Mechanisms of Recombination Homologous (legitimate) Nonhomologous (illegitimate)

20 Genetic Variation Homologues recombination occurs between closely related DNA sequences and generally substitutes one sequence to another. The process requires a set of enzymes produced by the group rec genes, and homology in n.p. Nonhomologues recombination occurs between dissimilar DNA sequences and generally produces insertions or deletions or both. This process usually requires specialized (site-specific) recombination enzymes, such as those produced by many transposons and lysogenic bacteriophages

21 Gene Transfer The transfer of genetic material between procaryotes is called horizontal (or lateral) gene transfer. It takes place in one of 3 ways: 1. Transformation 2. Conjugation 3. Transduction

22 Gene Transfer Transformation Was discovered by Griffits in 1928 during the experiments with Streptococcus pneumoniae. Later Avery, MacLeod and McCarty identified the DNA as the transforming agent and as the genetic material Transformation is the process by which bacteria take up fragment of naked DNA and incorporate this molecule into recipient chromosome in a heritable form

23 Gene Transfer Transformation The requirement for the DNA fragment from the donor bacterium Molecular weight (M) D Double-stranded fragment of DNA The state of recipient cell is called competence Competency is a complex phenomenon which is dependent on several conditions: - Certain stage of bacterial culture s growth (exponential phase) - Secretion of a small protein, called competence factor, that stimulates the production of 8 to 10 new proteins is required for transformation (DNA-biding protein, endonucleases, autolysins)

24 Gene Transfer Conjugation Was discovered in 1946 by Lederberg and Tatum. In 1952 Hayes demonstrated that the gene transfer was polar. Polarity is mediated by plasmid, known as F-factor. Donor (F +, or fertile) and recipient (F -, or nonfertile) were defined. F plasmid contains tra-operon. Genes of tra-operon encode proteins for building the sex pili and proteins needed to construct the type four secretion system that will transfer DNA from donor to the recipient

25 Gene Transfer Conjugation IS3 γδ tra F F-plasmid 100 kb IS3 IS2 orit oriv

26 Gene Transfer Conjugation Donor Types: 1. F + cells with free F - plasmid F + x F - F + 2. Hfr cells with integrated plasmid Hfr x F - F - (F + 100) 3. F free F - plasmid with a portion of chromosomal genes F x F - F

27 Gene Transfer Cell-to-cell transfer of a conjugative plasmid Integration of conjugative plasmid into the bacterial chromosome an Hfr The order of genes on the bacterial chromosome can be determined by the time of entry of the genes into a recipient cell

28 Gene Transfer Conjugation is very useful for genetic mapping of bacteria A circular genetic map of E.coli K12 with the location of selected genes. The map is divided into 100 minutes

29 Gene Transfer Transduction Was discovered by Zinder and Lederberg in Transduction is a process of transfer of the bacterial DNA with bacteriophages. Transduction can be classified as: - Generalized any gene of the host bacterium can be transferred and doesn t require lysogeny (Salmonella enterica P-22) - Specialized only specific genes near the attachment sites of a lysogenic phage in the host chromosome can be transferred (E.coli- λ) - Abortive the transferred DNA is not integrated but often is able to temporary survive and express. The fragment inherits linearly and lost in progeny

30 Transduction

31 Genetic Engineering Genetic Engineering a combination of methods which allows to conduct artificial recombination of DNA and produce chimerical molecules, non-typical for nature

32 Genetic Engineering Main aims of Genetic Engineering: 1. The production of medically useful proteins (somatostotine, insulin, human growth harmone, interferons, interleukin-2, etc.) 2. Recombinant vaccines (hepatitis B vaccine) 3. Gene therapy (involves the insertion of a normal gene into cell to correct a defective gene

33 Genetic Engineering The basic tools of Genetic Engineering: 1. Cloning vectors which can be used to deliver the DNA sequences into receptive bacteria 2. Restriction enzymes which are used to cleave DNA at defined sequences 3. DNA ligase the enzyme that links the fragment to the cloning vector

34 Genetic Engineering Types of vector: 1. Plasmid (puc, pbr322, pgem) are used for DNA fragments up to 20Kb 2. Bacteriophages (λ, T7) are used for larger fragments up to 25Kb 3. Cosmid (combination of plasmid and phage genes, pjc720) for fragments up to 45Kb 4. Viruses (poxvaccine)

35 Genetic Engineering The specific properties of plasmid cloning vectors: 1. Small size to be easy inserted into bacteria 2. High number of copies to be easily purified in sufficient quantities 3. Ability to replicate within a host cell 4. Selectable traits (resistance to an antibiotic) 5. One or few sites for restriction endonucleases which cut DNA and allow the insertion of foreign DNA

36 Genetic Engineering Steps in Cloning a Gene: Isolate a DNA to be cloned Use restriction enzyme to generate fragments of DNA Generate a recombinant molecule by inserting DNA fragments into a cloning vector Introduce recombinant molecule into new host Select bacterial clones carrying specific genes

37 Thank you for your attention! Questions?