BACTERIAL GENETICS

1. BACTERIAL GENETICS Lectures of Dr. Orubakhalid

2. Key Words • Genetics • Bacterial genetics • Mutation & its types • Bacteriophage • Mechanisms of gene transfer • Transformation • Transduction • Lysogenic conversion • Conjugation

3. Bacterial Genetics • Genetics is the study of heredity and variation. The unit of heredity is gene, which is a segment of DNA specifying for a particular polypeptide. Introns - non coding sequences on a gene. Exons - coding sequences on a gene translated into gene products. • Bacterial genetics is used as a model to understand DNA replication, genetic characters, their changes & transfer to next generations.

4. Nucleic Acids • DNA ( deoxy ribonucleic acid ) : stores information for protein synthesis. • RNA ( ribonucleic acid ) : transcription & translation of information for protein synthesis. • Central Dogma : DNA RNA Protein

5. Structure Of DNA • Proposed by Watson & Crick. • Double helix model. • Composed of 2 chains of polypeptides, each chain has a backbone of deoxyribose sugar and phosphate residues arranged alternately. • 4 nitrogenous bases: Adenine (A) Purine Guanine (G) Thymine(T) Pyrimidine Cytosine (C)

6. Double helical structure of DNA by Watson & Crick

7. DNA • DNA is copied by DNA polymerase • In the 5 3 direction • Initiated by an RNA primer • Leading strand synthesized continuously • Lagging strand synthesized discontinuously • Okazaki fragments • RNA primers are removed and Okazaki fragments joined by a DNA polymerase and DNA ligase

8. DNA Figure 8.6

9. Replication of this DNA molecule always starts at a certain point (the origin of replication) and it is “semi-conservative” meaning that one strand in each of the two resulting double strands is conserved .

10. DNA Replication: • -The identical duplication process of DNA is termed semi-conservative because the double strand of DNA is opened up during replication and each strand serves as the matrix for synthesis of a complementary strand. Thus each of the two new double strands “conserves” one old strand. • -The doubling of each DNA molecule begins at a given starting point called origin of replication. This process continues throughout the entire cycle.

12. DNA • DNA replication is semiconservative Figure 8.7

13. Translation • mRNA is translated in codons (3 nucleotides) • Translation of mRNA begins at the start codon: AUG • Translation ends at a STOP codon: UAA, UAG, UGA Figure 8.2

14. The Genetic Code Figure 8.9

15. Structure Of RNA • Structurally similar to DNA, except for 2 major differences: • ribose sugar • uracil in place of thymine. • 3 types of RNA • m RNA (messenger RNA) • t RNA ( transfer RNA ) • r RNA ( ribosomal RNA )

16. Genetic Information In Bacteria Chromosome Carries properties like virulence, pathogenicity & resistance Plasmid Extrachromosomal genetic material in the cytoplasm Replicate independently Bacteriophage Virus infecting bacteria

17. PLASMIDS • Circular DNA molecules • Important vectors in genetic engineering • EPISOME • Plasmid DNA integrated with chromosomal DNA. • Types of plasmids • R plasmid (drug resistance): RTF + r determinant • F plasmid (maleness )

18. Types of plasmids • Sex factor plasmids: the cell that possess this plasmid is called F+, male or the donor cell, while the one that do not possess it is called; F-, or the recipient cell. • R-plasmids (resistance plasmids) responsible for resistance to drug • Col- plasmids: responsible for production of bacteriocins. • Heavy metal ion resistant plasmids: responsible for resistance to heavy metal ions that the bacteria may get exposed in the envirnment • Plasmids of catabolic activity: responsible for degredation of highly complex compounds, such as: hydrocarbons • Virulence plasmids: reponsible for production of certain virulence factors such as: toxin , hemolysin, adhesive factors,… etc Genetic Engineering

19. Mechanisms Of Genetic Variations • Mutation • Transfer or exchange of genetic material • Transformation • Transduction • Conjugation • Lysogenic conversion • Transposition

20. Genotype: is the gatalogue of gene arranged on the DNA molecule. • Phenotype: the collection of characters as result of the expression of these genes. • Mutation: hereitiditary changes occur in the genotype which may or may not lead to phenotypic change.

21. The physical or the chemical agent that leads to mutation is called mutagen, and the bacteria produced with such genotypic change are called mutants. • Mutation occur either spontaneously or by induction. • Induced mutation occur as a result of the effect of one of the followings:

22. A-Physical: such as U.V. light, X-ray…etc. • B-chemical:such as 5-bromouracil, nitrous acid, hydroxylamine, nitrogen mustard, • acridines, nitrosoguanidine… etc. • C-Biological: such as transposons.

23. Types of mutations: • Deletion mutation: this result in the loss of a piece of DNA. • A B C D E F G______________A B C D F G • 2. Inversion mutation:this result in recombining the cut piece in revese order. • A B C D E F G_____________A B C E D F G

24. 3. Insertion mutation: a totally new base sequence is synthesized and inserted in the DNA molecule. • A B C D E F G_____________A B C K D E F G • 4.Substitution mutation: a newly different base sequence is synthesized instead of the lost one • A B C D E F G______________A B C N E F G • 5. Duplication mutation: an insertion of a base sequence similar to a sequence already existed. • A B C D E F G______________A B C D B E F G

25. Bacterial genetics • Experiments byNature of the genetic material • Griffith (1928); working on S- forms and R-forms of the strep. Pneumonia • Avery (1944); mixing DNA extract of the S-forms with and without Dnase, then mixing it with R-forms • Hershyand Chase (1952), used radioactive isotops on bacteriophages (S25-for head protein ) and (P32- for nucleic acids)

26. Gene transfer: • Recombination ; is the reassortment of nucleotide sequences within the DNA molecule • Recombination may occur between

27. Sometimes rearrangement occurs within the DNA molecule itself without an external DNA • Any recombination or spontaneous rearrangement leads to what is called genotypic change, this may or may not leads to phenotypic change • The fate of the transferred DNA depends on: • Its capability to be taken by the host cells • Its stability within the host chromosome

28. There are three mechanisms for gene transfer in bacteria Transformation Conjugation Transduction

29. Transformation (Griffith, 1928) Transfer of genetic information by free DNA. i.e. by direct uptake of donor DNA by the recipient DNA. Live noncapsulated (R) pneumococci + heat killed capsulated (S) pneumococci Injected into mice Death of mice • Live capsulated pneumococcus isolated from the blood of mice.

31. 3. Transduction • It is the transfer of DNA from a donor to a receptor with • the help of transport bacteriophages. • Bacteriophages • Infection of another bacterium • Transfer of host bacterial DNA to the new bacterium • Acquisition of new characteristics coded by the donor DNA.

32. Bacteriophage replication occur in 5 stages: • Adsorption: where the virus is adsorped on specific receptor on the bacterial cell wall. • Penetration: the virus make a hole in the bacterial cell wall using its core and its DNA is passed through this hollow core to inside the bacteria • Replication : the viral nucleic acid controls the bacterial cell activity and mechanisms for its benefit and synthesize several numbers of the viral parts. • Maturation : during this stage, each head of a virus will be surround one part of the viral DNA, then the other parts will be joined to each other forming several numbers of viruses. • Release : finally the virus release a lysozyme that leads to cell lysis and the release of the viruses, then each virus will infect other bacteria, and so on until the lysis of the whole colony.

34. Bacteriophages Definition:- Bacteriophages are viruses that infect bacteria . They are therefore obligate cell parasites. They possess only one type of nucleic acid, either DNA or RNA, have no enzymatic systems for energy supply and are unable to synthesize proteins on their own. Morphology:- Similar to the viruses that infect animals and vary widely in appearance.

35. Transduction • Transfer of bacterial genes via viruses • Donor to recipient • Virus: Bacteriophages • Types • Generalized • Specialized • Replication Cycle • Lytic • Lysogenic

36. Composition: Phages are made up of protein and nucleic acid. The proteins form the head, tail, and other morphological elements, the function of which is to protect the phage genome. The nucleic acid in most phages is DNA, which occurs as a double stranded DNA .

37. Sometimes, the recombination of the viral DNA with the chromosomal DNA may lead to a drastic change in the character of the bacteria, such as becoming a powerful toxin producer (e.g. Clostridium botulinum,, Corynebacterium diphtheriae… etc). • The bacteria are called Lysogenic cell, and the process is called Lysogenic conversion

39. 2. Conjugation It is the transfer of DNA from a donor to a receptor in a conjugation process involving cell-to-cell contact. Conjugation is made possible by two genetic elements: the conjugative plasmids and the conjugative pilli . Conjugation is seen frequently in Gram-negative rods (Enterobacteriaceae), in which the phenomenon has been most thoroughly researched, and enterococci

41. Plasmid Chromosome Transposon Transposon (Jumping Genes, Barbara McClintock) DNA segment that can move between chromosome & plasmids Insertion of transposon into a functional gene would destroy the function of the gene (internal mutagenic agents) Transposons are not self replicative, they depend on chromosomal or plasmid DNA for replication

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

43. Steps in • DNA from any desired source is cleaved into fragments by restriction endonuclease. • The fragments are spliced into vector such as plasmid or viral genome. • By transformation, the vector is introduced into a host cell in which it can replicate. • - After replication of the vector & amplification of the original DNA fragment, vector is isolated. • The inserted fragment is cleaved back out & purified. • see fig.

44. Recombinant DNA

45. Application of of genetic engineering (molecular cloning):- • Gene structure \mapping \function e.g. globin. • Biosynthesis e.g. insulin, GH (growth hormone), interferon. • Control of genetic disease. • Using PCR in detection & identification of specific organism e.g. detection of HIV by PCR. • Gene therapy. • NOTE: the field of genetic engineering (molecular cloning) involves the introduction of new genes into the cells.

46. References: • 1- Jawetz, Melnick, & Adelberg’s.( 2013). Medical Microbiology (Twenty-Sixth Edition). • 2- Kenneth Todar. (2008).Todar’s Online Textbook of Bacteriology ,University of Wisconsin.