BACTERIAL GENETICS

1. BACTERIAL GENETICS Bakteriologi dan Mikologi Drh. Maxs U. E. Sanam, M.Sc.

2. Genetics Bacterial genetics Gene structure & control Mutation & its types Point mutation Frameshift mutation Lethal mutation Suppressor mutation Missense & nonsense mutation Bacteriophage Lysogenic cycle Mechanisms of gene transfer Transformation Transduction Lysogenic conversion Conjugation Transposition (Jumping Genes) Key Words

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. Structure of procaryotic gene

5. Structure of eucaryotic gene

7. 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

8. 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)

9. Double helical structure of DNA by Watson & Crick

10. 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 )

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

12. 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 )

13. Genotypic & Phenotypic Variations • Genotype – genetic constitution of a cell that is transmitted to its progeny • Phenotype – physical expression of the genotype in a given environment • Variations • Phenotypic variations – • influenced by the environment • temporary & not heritable • Genotypic variations – • Not influenced by the environment • Stable & heritable

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

15. Mutation • Random, undirected heritable variation • Caused by a change in the nucleotide base sequence of the DNA • Types of mutation: • Point mutation • Frame shift mutation • Lethal mutation • Suppressor mutation • Mutagens - Agents which can induce mutation e.g. UV rays, 5 bromouracil, alkylating agents, etc.

16. 1. Point Mutation • Cause - due to addition, deletion or substitution of one or more bases. • Types - • Transition : a purine base is replaced by a purine base or a pyrimidine base is replaced by another pyrimidine base. Most common type. • Transversion : substitution of a purine base by a pyrimidine base & vice versa

17. 1. Point Mutation • Results of mutation - • Missense mutation – triplet code is altered so that a different amino acid is present at a particular position in the protein. • Nonsense mutation – converts a codon that specifies an amino acid into a termination codon. 

18. 2. Frame Shift Mutation • Cause - Deletion or insertion of a base - changes all of the codons downstream from the change

19. 3. Lethal Mutation • Mutation which resulting involve vital functions in the death of the organism – nonviable mutation. • A conditional lethal mutant may be able to live under certain conditions – permissive conditions. • Commonest type of conditional mutant is the temperature sensitive (ts) mutant which is able to live at the permissive temperature of 35C but not at the restrictive temp (39C).

20. 4. Suppressor Mutation • Reversal of a mutant phenotype by another mutation at a position on the DNA, distinct from that of the original mutation.

21. 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.

24. Transduction • Transfer of a portion of the DNA from one bacterium to another by a bacteriophage. Packaging error within the infected bacteria during the assembly of progeny phages – presence of a segment of host DNA along with the phage nucleic acid in the core of phage Infection of another bacterium Transfer of host bacterial DNA to the new bacterium Acquisition of new characteristics coded by the donor DNA.

27. Transduction • Types of Transduction – • Generalized - When it involves any segment of the DNA • Restricted - When a specific bacteriophage transduces only a particular genetic trait. • Episomes & plasmids can also be transduced. • Most widely used mechanism of gene transfer among prokaryotes • Any group of bacteria for which bacteriophages exist, can be subjected to transduction.

28. Lysogenic Conversion Phage DNA itself is the new genetic element. Bacteriophages – 2 Types of life cycle • Lytic or virulent cycle– progeny viruses build up inside host bacterium, which rupture to release them. • Temperate or nonlytic or lysogenic cycle– host bacterium is unharmed.

30. This process is called Lysogeny & the bacteria harbouring these prophages are called Lysogenicbacteria. Prophage behaves as an additional segment of bacterial chromosome, coding for new characteristics. This process by which prophage confers genetic information to a bacterium is called Lysogenic conversion . • By lysogenic conversion diphtheria bacilli acquire toxigenicity & therefore virulence. Elimination of phage renders a toxigenic strain non- toxigenic.

31. Conjugation • First described by Lederburg & Tatum in 1946 in a strain of E.coli called K12. • A donor or male bacterium passes DNA directly to a recipient or female bacterium by a conjugation tube (sex pili). The female bacterium attains donor status & in turn can conjugate with other female cells. • Maleness is determined by the presence of a plasmid which codes for sex pili. • The plasmid is called the sex factor or fertility factor (F factor) • R (resistance) factor can also be transferred by conjugation

32. Process of Conjugation

33. 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

34. Gene Control in Prokaryotes • In bacteria, genes are clustered into operons: gene clusters that encode the proteins necessary to perform coordinated function, such as biosynthesis of a given amino acid. • RNA that is transcribed from prokaryotic operons is polycistronic a term implying that multiple proteins are encoded in a single transcript.

35. In bacteria, control of the rate of transcriptional initiation is the predominant site for control of gene expression. • As with the majority of prokaryotic genes, initiation is controlled by two DNA sequence elements that are approximately 35 bases and 10 bases, respectively, upstream of the site of transcriptional initiation and as such are identified as the -35 and -10 positions.

36. These 2 sequence elements are termed promoter sequences, because they promote recognition of transcriptional start sites by RNA polymerase. • The consensus sequence for the -35 position is TTGACA, and for the -10 position, TATAAT. (The -10 position is also known as the Pribnow-box.) • These promoter sequences are recognized and contacted by RNA polymerase.

37. The activity of RNA polymerase at a given promoter is in turn regulated by interaction with accessory proteins, which affect its ability to recognize start sites. • These regulatory proteins can act both positively (activators) and negatively (repressors). • The accessibility of promoter regions of prokaryotic DNA is in many cases regulated by the interaction of proteins with sequences termed operators.

38. The operator region is adjacent to the promoter elements in most operons and in most cases the sequences of the operator bind a repressor protein. • However, there are several operons in E. coli that contain overlapping sequence elements, one that binds a repressor and one that binds an activator.

39. lac operon • The lac operon (see diagram below) consists of one regulatory gene (the i gene) and three structural genes (z, y, and a). • The i gene codes for the repressor of the lac operon. • The z gene codes for β-galactosidase (β-gal), which is primarily responsible for the hydrolysis of the disaccharide, lactose into its monomeric units, galactose and glucose. • The y gene codes for permease, which increases permeability of the cell to β-galactosides.