C hair of M icrobiology, V irology, and I mmunology

1. Chair of Microbiology, Virology, and Immunology GENETICS OF BACTERIA AND VIRUSES BASES OF BIOTECHNOLOGY AND GENE ENGENEERING

2. Lectures schedule 1. Structure of bacterial genome. 2. Extrachromosomal elements. 3. Mutations. 4. Recombinations. 5. Gene engineering.

3. F. Crick i J. Watson – described DNA structure

4. DNA structure

5. E. coli DNA The chromosome of E. coli has a contour length of approximately 1.35 mm, several hundred times longer than the bacterial cell, but the DNA is supercoiled and tightly packaged in the bacterial nucleoid. The time required for replication of the entire chromosome is about 40 minutes

6. E. coli DNA

7. Plasmid Definition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon) Episome - a plasmid that can integrate into the chromosome They are usually much smaller than the bacterial chromosome, varying from less than 5 to more than several hundred kbp. Most plasmids are supercoiled, circular, double-stranded DNA molecules, but linear plasmids have also been demonstrated in Borrelia and Streptomyces.

8. Classification of Plasmids • Transfer properties • Conjugative (This plasmids code for functions that promote transfer of the plasmid from the donor bacterium to other recipient bacteria) Nonconjugative (do not) Phenotypic effects • Fertility • Bacteriocinogenic plasmid • Resistance plasmid (R factors)

9. Phenotypic effects

10. RTF Tn 10 Tn 8 Tn 9 Tn 21 R determinant Structure of R factors • RTF • Conjugative plasmid • Transfer genes • R determinant • Resistance genes • Transposons

11. The average number of molecules of a given plasmid per bacterial chromosome is called its copy number. Large plasmids (40 kilobase pairs) are often conjugative, have small copynumbers (1 to several per chromosome). Plasmids smaller than 7.5 kilobase pairs usually are nonconjugative, have high copy numbers (typically 10-20 per chromosome), rely on their bacterial host to provide some functions required for replication, and are distributed randomly between daughter cells at division. Some plasmids are cryptic and have no recognizable effects on the bacterial cells that harbor them

13. 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 • Transposition mediated by site-specific recombination • Transposase • Transposition may be accompanied by duplication

14. GFEDCBA ABCDEFG Transposase 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 The known insertion sequences vary in length from approximately 780 to 1500 nucleotide pairs, have short (15-25 base pair) inverted repeats at their ends, and are not closely related to each other.

15. H2 gene H1 gene H1 flagella H2 flagella Phase Variation in Salmonella H Antigens IS

16. IS Resistance Gene(s) IS IS Resistance Gene(s) IS Types of Transposable Genetic Elements • Transposons (Tn) • Definition: Elements that carry other genes except those involved in transposition • Nomenclature - Tn10 • Transposons can move from one site in a DNA molecule to other target sites in the same or a different DNA molecule. • Structure Transposons are not self-replicating genetic elements, however, and they must integrate into other replicons to be maintained stably in bacterial genomes

17. Complex transposons vary in length from about 2,000 to more than 40,000 nucleotide pairs and contain insertion sequences (or closely related sequences) at each end, usually as inverted repeats. The entire complex element can transpose as a unit.

18. Importance • they cause mutations, • mediate genomic rearrangements, • function as portable regions of genetic homology, and acquire new genes, • contribute to their dissemination within bacterial populations. • insertion of a transposon often interrupts the linear sequence of a gene and inactivates it, • transposons have a major role in causing deletions, duplications, and inversions of DNA segments as well as fusions between replicons.

19. In medically important bacteria, genes that determine production of adherence antigens, toxins, or other virulence factors, or specify resistance to one or more antibiotics, are often located in complex transposons. Well-known examples of complex transposons are Tn5 and Tn10, which determine resistance to kanamycin and tetracycline, respectively.

20. Mutation is a stable, heritable change in the genomic nucleotide sequence

21. How do mutations occur? • Spontaneous mutations - Arise occasionally in all cells; are often the result of errors in DNA replication (random changes) • Frequency of naturally occurring (spontaneous) mutation varies from 10-6 to 10-9 (avg = 10-8) • This means that if a bacterial population increases from 108 to 2 x 108, on the average, one mutant will be produced for the gene in question. Induced mutations - Arise under an influence of some factors Errors in replication which cause point mutations; • other errors can lead to frameshifts • Point mutation - mismatch substitution of one nucleotide base pair for another • Frameshift mutation - arise from accidental insertion or deletion within coding region of gene, results in the synthesis of nonfunctional protein

22. Types of Mutations • Point mutation: affects only 1 bp at a single location • Silent mutation: a point mutation that has no visible effect because of code degeneracy

23. Types of Mutations Missense mutation: a single base substitution in the DNA that changes a codon from one amino acid to another

24. Types of Mutations Nonsense mutation: converts a sense codon to a nonsense or stop codon, results in shortened polypeptide

25. Types of Mutations • Frameshift mutation: arise from accidental insertion or deletion within coding region of gene, results in the synthesis of nonfunctional protein Insertion

26. Frameshift mutation - Deletion

27. Other Types of Mutations • Forward mutation:a mutation that alters phenotype from wild type • Reverse mutation: a second mutation which may reverse wild phenotype and genotype (in same gene) • Suppressor mutation: a mutation that alters forward mutation, reverse wild phenotype (in same gene - intragenic, in another gene - extragenic)

28. Mutations affect bacterial cell phenotype • Morphological mutations-result in changes in colony or cell morphology • Lethal mutations - result in death of the organism • Conditional mutations - are expressed only under certain environmental conditions • Biochemical mutations - result in changes in the metabolic capabilities of a cell • 1) Auxotrophs - cannot grow on minimal media because they have lost a biosynthetic capability; require supplements • 2) Prototrophs - wild type growth characteristics • Resistance mutations-result in acquired resistance to some pathogen, chemical, or antibiotic

29. Induced mutations-caused by mutagens • Mutagens – Molecules or chemicals that damage DNA or alter its chemistry and pairing characteristics • Base analogs are incorporated into DNA during replication, cause mispairing • Modification of base structure (e.g., alkylating agents) • Intercalating agents insert into and distort the DNA, induce insertions/deletions that can lead to frameshifts • DNA damage so that it cannot act as a replication template (e.g., UV radiation, ionizing radiation, some carcinogens)

30. N. meningitidis genes with high mutation rates include those involved in: capsule biosynthesis LPS biosynthesis attaching to host cells taking up iron

34. Mutant Detection • In order to study microbial mutants, one must be able to detect them and isolate them from the wild-type organisms • Visual observation of changes in colony characteristics • Mutant selection - achieved by finding the environmental condition in which the mutant will grow but the wild type will not (useful for isolating rare mutations) • Screen for auxotrophic mutants: A lysine auxotroph will only grow on media that is supplemented with lysine

35. Mutant Detection Mutants are generated by treating a culture of E. coli with a mutagen such as nitrosoguanidine The culture will contain a mixture of wild-type and auxotrophic bacteria Out of this population we want to select for a Lysine auxotrophic mutant

36. minus lysine complete All strains grow Lysine auxotrophs do not grow Isolation of a Lysine Auxotroph

37. Reparation Light-requiring Dark SOS- reactivation

38. Light-requiring Reparation

39. Dark Reparation

40. Exchange of Genetic Information Recombination

41. Transformation

42. Transformation Definition: Gene transfer resulting from the uptake of DNA from a donor. • Factors affecting transformation • DNA size and state (DNA molecules must be at least 500 nucleotides in length) • Sensitive to nucleases (deoxyribonuclease) • Competence of the recipient (Bacillus, Haemophilus, Neisseria, Streptococcus) • Competence factor • Induced competence

43. Transformation • Steps • Uptake of DNA • Gram + • Gram - • Recombination • Legitimate, homologous or general • recA, recB and recC genes • Significance • Phase variation in Neiseseria • Recombinant DNA technology

44. S strain R strain Competent cell S strain

45. Transduction • Definition: Gene transfer from a donor to a recipient by way of a bacteriophage

46. Head/Capsid Contractile Sheath Tail Tail Fibers Base Plate Phage Composition and Structure • Composition • Nucleic acid • Genome size • Modified bases • Protein • Protection • Infection • Structure (T4) • Size • Head or capsid • Tail

47. Transduction • Types of transduction • Generalized - Transduction in which potentially any donor bacterial gene can be transferred

48. Generalized Transduction • Infection of Donor • Phage replication and degradation of host DNA • Release of phage • Assembly of phages particles • Infection of recipient • Legitimate recombination

49. Transduction • Types of transduction • Specialized - Transduction in which only certain donor genes can be transferred

50. gal gal bio bio gal bio gal bio bio gal Specialized TransductionLysogenic Phage • Excision of the prophage • Replication and release of phage • Infection of the recipient • Lysogenization of the recipient • Legitimate recombination also possible