Lecture 6 Bacterial-Genetics-Lecture-Slides.pptx

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1. BACTERIAL GENETICS Write a subtitle of this presentation here LECTURE SLIDES

2. Understanding Genetics We resemble and differ because of Genetic configurations Parents - Son - Daughter , how they resemble each other. They breed true from Generation to Generation But vary in small proportions in progeny. Bacteria too obey the laws of Genetics

3. Watson - Crick Discovery of DNA

4. DNA A Complex Structure Makes Life

5. Prokaryotes Vs Eukaryotes Genetics Prokaryotes Eukaryotes Prokaryotes are haploid eukaryotes are often diploid contain a single circular chromosome . eukaryotes have linear chromosomes, usually more than 1 Prokaryotes often contain “plasmids” . Doesnot contain plasmids In prokaryotes, translation is coupled to transcription: translation of the new RNA molecule starts before transcription is finished In eukaryotes, transcription of genes in RNA occurs in the nucleus, and translation of that RNA into protein occurs in the cytoplasm . The two processes are separated from each other.

6. DNA ( Deoxyribonucleic Acid ) DNA is composed of Many Units of Adenine – Thymine A - T Guanine – Cytosine G - C A+ T G+C proportion differ for each species DNA replicates first unwinding at one end to form a fork Each strand of fork acting as template for the synthesis of complementary strand

7. DNA A DNA molecule is composed of two chains of Nucleotides wound together in the form of a Double Helix Each chain has back bone of Deoxyribose and Phosphates residues arranged alternatively

8. Structure of DNA Attached to each Deoxyribose and phosphate residues arranged alternatively Attached to each Deoxyribose are of four nitrogen bases Purines - Adenine, Guanine Pyrimidines Thymidine and Cytosine

9. How RNA differs from DNA RNA contains - Sugar Ribose instead of Deoxyribose Uracil is present instead of Thymine Types of RNA Messenger RNA mRNA Ribosomal RNA rRNA Transfer RNA tRNA

10. What is a Code in Genetics? Code is a unit consists of sequence of three Bases Code is triplet A-T- C A code can make single Amino acid More than one code present for making similar sequence of Amino acid AGA make Arginine AGC, CGU, CGG, also code for similar Amino acid Some Codons UAA don't code for any Amino acid called as Nonsense codon

11. What is a Gene? Gene is a sequence of DNA carrying codons specifying for particular polypeptide. DNA contains many Genes( combinations of hundreds and thousands of Nucleotides )

12. Bacterial Chromosome Contains a Double stranded molecules of DNA arranged in circular form. Length 1,000 microns . Bacterial DNA contains about 4,000kilobases 1 kb = 1000 base pairs (A-T) (G-C)

13. Extra chromosomal Genetic Elements Bacteria posses Extra chromosomal genetic elements Not Essential for survival of Bacteria But makes the Bacteria Resistant to antibiotics , and makes them survive & also able to produce toxins

14. Plasmids Plasmids are circular DNA molecules present in the cytoplasm of the Bacteria Their size varies from 1 kbp to over 400 kilobase pairs (kbp). Capable of Autonomous replication Can transfer genes from one cell to other Act as vectors in Genetic engineering.

15. Plasmids Plasmid seem to be ubiquitous in bacteria, May encode genetic information for properties 1 Resitance to Antibiotics 2 Bacteriocins production 3 Enterotoxin production 4 Enhanced pathogenicity 5 Reduced Sensitivity to mutagens 6 Degrade complex organic molecules

16. Plasmids are classified 1. by their ability to be transferred to other bacteria Conjugative The sexual transfer of plasmids to another bacterium through a pilus. Non-conjugative Non-conjugative plasmids don’t initiate conjugaison. They can only be transferred with the help of conjugative plasmids.

17. Mobilisable An intermediate class of plasmids are mobilisable, and carry only a subset of the genes These plasmids can 'parasitise' another plasmid, transferring at high frequency presence of a conjugative plasmid Incompatibility groups : Several types of plasmids could coexist in a single cell. On the other hand, related plasmids are often 'incompatible', resulting in the loss of one of them from the cell line.

18. 2. Function 1. Fertility-(F) plasmids, They are capable of conjugation (they contains the genes for the pili). Resistance-(R) plasmids , Contain gene (s) that can build resistance against one or several antibiotics or poisons . Col-plasmids , Contain genes coding for colicines, proteins that can kill other bacteria . .

19. Degradative plasmids , able to digest unusual substances, e.g., toluene or salicylic acid 5. Virulence plasmids turn a bacterium into a pathogen. Addiction system. These plasmids produce both a long-lived poison and a short-lived antidote. Daughter cells that retain a copy of the plasmid survive while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell .

20. Plasmids Can be integrated with Chromosomal DNA Episomes -Integrated form of plasmid with DNA

21. Potentials of Plasmids Plasmids can be self transmissible and Non transmissible Transfers the Sex and Drug resistance with the help of restriction end nucleases

22. Genotypic & Phenotypic variation Genome – Sum total of Gene that make up the genetic apparatus of cell established as Genotype. Hereditary constitution of cell this transmitted to its progeny Phenotype – is the physical expression of genotype in a environment & can change according to environment.

23. Phenotypic variation Exhibit – different phenotypes Appearance differs in different situations. Eg: Typhoid bacilli flagellated normally But grown in Phenol agar don't grow flagella so flagella are lost physical variation Lactose fermentation in E.coli dependent on Beta Galactosidase When lactose present - test is positive When lactose is absent - test turns negative

24. Genotypic variations Stable, heritable and not influenced by environment May occur by Mutations Genotypic by transfer of genes Transformation Transduction(Lysogenic conversion) Conjugation

25. Mutations Mutation is a Random, Undirected, Heritable variation Caused by alteration in the Nucleotide sequence at some point of DNA which can occur due to Addition Deletion Substitution of one or more bases

26. Mutation Rate Different types of mutations can occur at different frequencies . For a typical bacterium, mutation rates of 10 –7 to 10 –11 per base pair are generally seen Although RNA and DNA polymerases make errors at about the same rate, RNA genomes typically accumulate mutations at much higher frequencies than DNA genomes .

27. Mutations can occur in any sequence, inveitable , useful for Survival

28. Multiple Mutations Causes extensive chromosomal rearrangement Missense mutation Triplet code is altered so as to specify an aminoacid different from that normally located at particular position in the protein. Nonsense mutation Deletion of nucleotide within a gene may cause premature polypeptide chain termination by nonsense codon Transversion is Substitution of purine for pyramidine or vice versa in the base pairing

29. Possible effects of bp substitution

30. Replica Plating Method A common way to find bacterial mutants is replica plating , which means making two identical copies of the colonies on a petri plate under different conditions. For instance, if you were looking for trp- auxotrophs , one plate would contain added tryptophan and the other plate would not have any tryptophan in it. Bacteria are first spread on the permissive plate , the plate that allows both mutants and wild type to grow, the plate containing tryptophan in this case..

31. They are allowed to grow for a while, then a copy of the plate is made by pressing a piece of velvet onto the surface of the plate then moving it to a fresh plate with the restrictive condition (no tryptophan). The velvet transfers some cells from each colony to an identical position on the restrictive plate.

32. Colonies that grow on the permissive plate but not the restrictive plate are (probably) trp - auxotrophs , because they can only grow if tryptophan is supplied

33. Replica Plating

34. Transmission of Genetic material ( Gene Transfer ) Different Mechanisms Transformation Transduction Conjugation

35. Transformation Transformation is defined as transfer of Genetic information through the activity of free DNA first experiment that showed DNA was the genetic information Griffith experiment Mice injected with Live non capsulated ( R ) Pneumococci & with heat killed capsulated (S) Pneumococci Lead to death of Mice with isolation of Live capsulated Pneumococci It means that some factor from dead pneumococci transferred to live non pathogenic Pneumococci

36. Griffith Phenomenon

37. Certain prokaryotes exhibit competence, a state in which cells are able to take up free DNA released by other bacteria. Incorporation of donor DNA into a recipient cell requires the activity of single-stranded binding protein, RecA protein, and several other enzymes. Only competent cells are transformable

38. Conjugation A process by which a Donor cell or male cell makes contact with another cell, the recipient or Female cell. DNA is directly transferable Plasmid Carry genetic information necessary for conjugation to occur. Only cell that contain such plasmids can act as donor . the cell lacking a corresponding plasmid act as recipient. Requires direct contact between donor and recipient

39. Conjugation - Transferring genes with plasmids Plasmids mediating conjugation carry genes coding for properties, of 1-2 microns long protein appendage termed Pilus on the Donor cell

40. Conjugation

41. Pilus helps Conjugation Different types of Pilus are specified by different types of plasmids and can help in aid of plasmid classification. Only one strand of circular DNA of the plasmid nicked upon at a specific site and passed into a recipient. Spread to all other cells.

42. F factor Transfer factor that contains the genetic information necessary for synthesis of Sex Pilus and for self transfer without any other identifiable genetic materials such as drug resistance

43. F + called as Donor bacteria can transform F - into F + cell Can be Episomes able to exist in some cells in the integrated state in the donor cell chromosome Can transform chromosomal genes to recruitment with high frequency are known as Hfr cells Conversion of F + cells into Hfr state is reversible. F factor incorporates some chromosomal genes and is called as F’ Sexduction : process of transfer of host genes through F’ factor

44. Transduction Transduction is defined as transfer of portion of DNA from one bacteria to another by Bacteriophages, is known as Transduction

45. 48 Historical Lederberg & Zinder Transduction was first discovered in 1952 by Joshua Lederberg and Norton Zinder Joshua Lederberg Norton Zinder

46. How transduction was discovered 49 Studied in  Salmonella typhimurium Plated two auxotrophic strains (LA-2 and LA-22) individually on minimal medium, no cells grew. Plated a mixture of the two auxotrophic strains on minimal medium, cells grew into colonies. Thus, genetic exchange was taking place between the two cell types.

47. U-tube Experiment www.2dix.com 50

48. TYPES OF TRANSDUCTION GENERALIZED TRANSDUCTION SPECIALIZED TRANSDUCTION

49. GENERALIZED TRANSDUCTION process by which any bacterial gene may be transferred to another bacterium via a bacteriophage. typically carries only bacterial DNA and no viral DNA

50. Generalised Transduction 53 1. A lytic bacteriophage adsorbs to a susceptible bacterium. 2. The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes

51. 54 3. Occasionally, a bacteriophage head or capsid assembles around a fragment of donor bacterium's nucleoid instead of a phage genome by mistake. steps in Generalised Transduction (cont’d) 4. The bacteriophages are released.

52. steps in Generalised Transduction (cont’d) 55 5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium 6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium .

53. 56 steps in Generalised Transduction (contd) 7. The donor bacterium's DNA is exchanged for some of the recipient's DNA.

54. SPECIALIZED TRANSDUCTION Discovered by Morse et al Process by which a restricted set of bacterial genes are transferred to another bacterium Partially coded prophage DNA is called a "heterogenote".

55. Specialised Transduction 58 1. A temperate bacteriophage adsorbs to a susceptible bacterium and injects its genome . 2. The bacteriophage inserts its genome into the bacterium's nucleoid to become a prophage.

56. steps in Specialised Transduction (cont’d) 59 3. Occasionally during spontaneous induction, a small piece of the donor bacterium's DNA is picked up as part of the phage's genome in place of some of the phage DNA which remains in the bacterium's nucleoid. 4. As the bacteriophage replicates, the segment of bacterial DNA replicates as part of the phage's genome. Every phage now carries that segment of bacterial DNA.

57. steps in Specialised Transduction (cont’d) 60 5. The bacteriophage adsorbs to a recipient bacterium and injects its genome. 6. The bacteriophage genome carrying the donor bacterial DNA inserts into the recipient bacterium's nucleoid.

58. Genetic Mechanisms of Drug Resistance Bacteria acquire drug resistance through several Mechanisms Mutations Genetic transfer Transformation, Transduction Conjugation Several Biochemical Mechanisms Decreasing permeability of drugs, Attaining alternative pathways Produce enzymes and inactivate drugs

59. Genetic Mechanisms in Bacteria helps to spread the Infectious diseases

60. Transposons and Insertion Sequences Transposons and insertion sequences are genetic elements that can move from one location on a chromosome to another by a process called transposition. a type of site-specific recombination.

61. Transposition is linked to the presence of special genetic elements called transposable elements . Transposition can be either replicative or conservative

62. Summary A DNA molecule is composed of two chains of Nucleotides wound together in the form of a Double Helix Gene is a sequence of DNA carrying codons specifying for particular polypeptide Plasmids are circular DNA molecules present in the cytoplasm of the Bacteria Phenotype variation – is the physical expression of genotype in a environment & can change according to environment Genotype variation – Stable, heritable and not influenced by environment

63. Mutation – alteration in the Nucleotide sequence at some point of DNA which can occur due to Addition, Deletion, Substitution of one or more bases Replica plating Transformation – is defined as transfer of Genetic information through the activity of free DNA Conjugation – process by which a Donor cell or male cell makes contact with another cell, the recipient or Female cell.

64. Transduction – is defined as transfer of portion of DNA from one bacteria to another by Bacteriophages Generalised transduction – process by which any bacterial gene may be transferred to another bacterium via a bacteriophage Specialised transduction – Process by which a restricted set of bacterial genes are transferred to another bacterium

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