1. GENETICS OF MICROORGANISMS

2. QUESTIONS OF LECTURE 1. Introduction 2. Genetics of microorganisms 3. Genotypic and phenotypic variations 4. Transmission of genetic material

3. GENETICS Genetics is the study of heredity and variation, seeking to understand the causes of the resemblances and differences between parents and their progeny

4. Genetics of microorganisms The «central dogma» of molecular biology is that DNA carries genetic information, which is transcribed on to RNA and then translated as the particular polypeptide (DNA - RNA - polypeptide) An exception exists in the case of some viruses genetic material is RNA instead of DNA

5. Genetics of microorganisms The DNA molecule is composed of two chains of nucleotides bound together in the form of a «double helix» Each chain has a backbone of desoxyribose and phosphate residues arranged alternately Attached to each desoxyribose is one of four nitrogenous bases, the purines: adenine (A) and guanine (G), and the pyrimidines: thymine (T) and cytosine (C) Adenine is always linked to thymine, and guanine to cytosine: A=T, G=C

6. Genetics of microorganisms Genetic information is stored in the DNA as a code, the unit of the code (codon) consisting of a sequence of three bases (the code is triplet) Each codon specifies for a single aminoacid, but the code is «degenerate» so that more than one codon may exist for the same aminoacid A segment of DNA carrying codons specifying for a particular polypeptide is called a «cistron» or gene The bacterial chromosome carries about 1000- 4000 cistrons.

7. Genetics of microorganisms Structure of operon Lac Lac P Lac O Lac Z - Y -A Regulator Promotor Operator Structural genes For transcription of RNA for the enzyme synthesis, the RNA polymerase has to attach to the promotor region and travel along the structural genes in sequence. The transfer of genetic information from DNA to RNA is called transcription and from RNA to protein is called translation.

8. Genetics of microorganisms RNA is structurally similar to DNA except for three major differences: It contains the sugar ribose (desoxyribose in DNA) One of pyrimidine base is uracile instead thymine in DNA There are 3 distinct types of RNA on the basis of structure and function: 1.Messenger RNA (m RNA) 2.Ribosomal RNA (r RNA) 3.Transfer RNA (t RNA) DNA acts as the template for the synthesis of mRNA.

9. Genotypic and phenotypic variations The sum total of genes the make up the genetic apparatus of a cell establishes its genotype. The phenotype («phaeno»: display) is the physical expression of the genotype in a given environment.

10. Genotypic and phenotypic variations Phenotypic variations are influenced by the environment, limited in range by the genotype, temporary and not heritable. Variations are genotypic when they are due to alterations in the genome. Genotypic variations are stable, heritable and not influenced by the environment. They may occur by mutation, or by one of the mechanisms of genetic transfer or exchange, such as transformation, transduction, lysogenic conversion and conjugation.

11. Mutation Mutation is a heritable variation caused by an alteration in the nucleotide sequence at some point of the DNA The sequence of nucleotides in DNA can change in either of 2 ways: (a) by substitution of one base pair for another as the result of a replication error – transition or transvertion. by breakage of the sugar phosphate back bone with subsequent deletion or insertion of a DNA segment.

12. MUTATIONS IN BACTERIA –Spontaneous (replication error ) –Induced (mutations are induced by a variety of physical, chemical and biological agents) Physical agents are radiations, heats etc. Chemical agents are nitroso compounds, alkylating agents, base analogs, and others.

13. TRANSFORMATION Definition: It is the transfer of genetic information through the agency of free DNA. Pieces of DNA involved in transformation may carry 10 to 50 genes. Factors affecting transformation –DNA size and state –Competence of the recipient (Bacillus, Haemophilus, Neisseria, Streptococcus)

14. TRANSFORMATION –Recombination Significance – Phase variation – Recombinant DNA technology Steps –Uptake of DNA Gram + Gram -

15. TRANSDUCTION Definition: Gene transfer from a donor to a recipient by way of a bacteriophage Bacteriophage (phage): A virus that infects bacteria

16. Phage Composition and Structure Composition –Nucleic acid Genome size Modified bases –Protein Protection Infection Structure (T 4 ) –Size (80 X 100 nm) –Head or capsid –Tail Tail Tail Fibers Base Plate Head/Capsid Contractile Sheath

17. Infection of Host Cells by Phages Irreversible attachment –Base plate Adsorption –Tail fibers – Receptor is LPS for T4 Nucleic acid injection Sheath Contraction DNA uptake

18. Microbe Library, American Society for Microbiology www.microbelibrary.org

19. TRANSDUCTION Types of transduction –Generalized - Transduction in which potentially any dornor bacterial gene can be transferred. – Specialized (Restricted) - Transduction in which only certain donor genes can be transferred

20. Generalized Transduction Release of phage Phage replication and degradation of host DNA Assembly of phages particles Infection of recipient Homologous recombination Infection of Donor Potentially any donor gene can be transferred

21. BACTERIOPHAGES EXHIBIT TWO TYPES OF LIFE CYCLE: In the virulent or lytic cycle, large numbers of progeny phages are built up inside the host bacterium, which ruptures to release them. In the temperate or nonlytic cycle, the phage DNA becomes integrated with the bacterial chromosome as the prophage and is transferred to the daughter cells. This process is called lysogenic or phage convertion.

23. CONJUGATION Definition: Gene transfer from a donor to a recipient by direct physical contact between cells Mating types in bacteria –Donor F factor (Fertility factor) is a transfer factor that contains the genetic information necessary for the synthesis of the sex pilus and for self-transfer. Cells carrying the F factor are called F+ cells. F factor may exist in the «integrated state» or inserted into the host chromosome. Such cells are known as Hfr cells. Donor Recipient –Recipient Lacks an F factor

24. Mechanism of F + x F - Crosses DNA transfer –Origin of transfer –Rolling circle replication Pair formation – Conjugation bridge

25. Microbe Library, American Society for Microbiology www.microbelibrary.org

26. 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 (not a replicon) –Transposition mediated by site-specific recombination Transposase –Transposition may be accompanied by duplication

27. 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 Transposase ABCDEFG GFEDCBA

28. Types of Transposable Genetic Elements Transposons (Tn) –Definition: Elements that carry other genes except those involved in transposition –Nomenclature - Tn10 –Structure Composite Tns – Importance Antibiotic resistance IS Resistance Gene(s) IS Resistance Gene(s)

29. Most of the transposons in the genome are stably silenced and many of them have been “tamed” and do important cellular jobs

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

31. Classification of Plasmids Transfer properties –Conjugative are transferred from bacterium to bacterium (usually members of the same species or of very closely related species) through conjugation –Nonconjugative Phenotypic effects –Fertility (F-plasmids) –Bacteriocinogenic plasmid (Col-plasmid) –Resistance plasmid (R factors) (R-plasmid) –Enterotoxin plasmid (Ent-plasmid) –Haemolysin plasmid (Hly-plasmid)

32. Structure of R Factors RTF –Conjugative plasmid –Transfer genes Tn 9 Tn 21 Tn 10 Tn 8 RTF R determinant –Resistance genes –Transposons

33. Genetic Engineering It is now possible to isolate the genes coding for any desired protein from microorganisms and introduce them into suitable microorganisms, in which the genes would be functional directing the production of the specific protein. This is known as the Recombinant-DNA technology or Genetic engineering. Plasmid DNA vaccination Multivalent DNA vaccine for malaria. Against tuberculosis Against hepatitis B virus.