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. Structure of R factors RTF R determinant Conjugative plasmid
Transfer genes
Tn 9
Tn 21
Tn 10
Tn 8
RTF
R determinant
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 copy numbers (1 to several per chromosome).
Plasmids smaller than 7.5 kilobase pairs usually are nonconjugative, have high copy numbers (typically 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. Types of Transposable Genetic Elements
Insertion sequences (IS)
Definition: Elements that carry no other genes except those involved in transposition
Nomenclature - IS1
Structure
Transposase
ABCDEFG
GFEDCBA
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. Phase Variation in Salmonella H Antigens
H1 gene
H2 gene IS
H1 flagella
H2 flagella

16. 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 IS
Resistance Gene(s)
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. attaching to host cells
N. meningitidis genes with high mutation rates include those involved in:
capsule biosynthesis
LPS biosynthesis
attaching to host cells
taking up iron

31. Examples of mutagens CHEMICAL AGENT ACTION HNO2 Nitrogen mustard NTG
React chemically with one or more bases so that they pair improperly
Intercalating agents (acridine dyes)
Insert into DNA and cause frame-shift mutations by inducing an addition or the subtraction of a base
Base analogs:
5-bromouracil
2-amino purine
Incorporate into DNA and cause mispairing
Analog of T which can pair with C
Analog of A which can pair with C

32. Examples of mutagens PHYSICAL AGENT ACTION UV irradiation
Causes formation of adjacent T-T dimers that distorts the DNA backbone, altering the binding properties of bases near the dimer
X-ray
Alters bases chemically, causes deletions and induces breaks in DNA chain

33. Examples of mutagens BIOLOGICAL AGENT ACTION Insertion sequences (IS)
Pieces of DNA about a thousand nucleotide bases in length which can insert into a genetic sequence
Transposons
genetic elements goverened by IS which can insert into the chromosome within a gene
Viruses
Some bacteriophage (e.g. phage µ) can integrate their DNA into random positions in the bacterial chromosome

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

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 Recombination Steps Uptake of DNA Gram + Gram -
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. Phage Composition and Structure
Nucleic acid
Genome size
Modified bases
Protein
Protection
Infection
Head/Capsid
Contractile Sheath
Tail
Structure (T4)
Size
Head or capsid
Tail
Tail Fibers
Base Plate

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
Assembly of phages particles
Release of phage
Infection of recipient
Legitimate recombination

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

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

51. Transduction Types of transduction
Abortive transduction refers to the transient expression of one or more donor genes without formation of recombinant progeny, whereas complete transduction is characterized by production of stable recombinants that inherit donor genes and retain the ability to express them.
In abortive transduction the donor DNA fragment does not replicate, and among the progeny of the original transductant only one bacterium contains the donor DNA fragment. In all other progeny the donor gene products become progressively diluted after each generation of bacterial growth until the donor phenotype can no longer be expressed.

52. Transduction Significance Common in Gram+ bacteria
Lysogenic (phage) conversion

53. Bacterial Conjugation
Definition: The transfer of genetic information via direct cell-cell contact
This process is mediated by fertility factors (F factor) on F plasmids

54. In conjugation, direct contact between the donor and recipient bacteria leads to establishment of a cytoplasmic bridge between them and transfer of part or all of the donor genome to the recipient. Donor ability is determined by specific conjugative plasmids called fertility plasmids or sex plasmids.

55. The F plasmid (also called F factor) of E coli is the prototype for fertility plasmids in Gram-negative bacteria. Strains of E coli with an extrachromosomal F plasmid are called F+ and function as donors, whereas strains that lack the F plasmid are F- and behave as recipients.

56. Conjugation
Gene transfer from a donor to a recipient by direct physical contact between cells
Mating types in bacteria
Donor
F factor (Fertility factor)
F (sex) pilus
Donor
Recipient
Recipient
Lacks an F factor

57. Physiological States of F Factor
Autonomous (F+)
Characteristics of F+ x F- crosses
F- becomes F+ while F+ remains F+
Low transfer of donor chromosomal genes F+

58. Physiological States of F Factor
Integrated (Hfr)
Characteristics of Hfr x F- crosses
F- rarely becomes Hfr while Hfr remains Hfr
High transfer of certain donor chromosomal genes F+
Hfr

59. Physiological States of F Factor
Autonomous with donor genes (F′)
Characteristics of F’ x F- crosses
F- becomes F’ while F’ remains F’
High transfer of donor genes on F’ and low transfer of other donor chromosomal genes
Hfr F’

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

61. Mechanism of Hfr x F- Crosses
Pair formation
Conjugation bridge
Hfr F-
DNA transfer
Origin of transfer
Rolling circle replication
Homologous recombination

62. Mechanism of F′ x F- Crosses
Pair formation
Conjugation bridge F’ F-
DNA transfer
Origin of transfer
Rolling circle replication

63. Conjugation Significance Gram - bacteria Gram + bacteria
Antibiotic resistance
Rapid spread
Gram + bacteria
Production of adhesive material by donor cells

64. Map of chromosome

65. Recombination DNA and Gene Cloning
Gene cloning is the process of incorporating foreign genes into hybrid DNA replicons.
Cloned genes can be expressed in appropriate host cells, and the phenotypes that they determine can be analyzed. Some key concepts underlying representative methods are summarized here.

66. Bacterial plasmids in gene cloning

67. Steps for eukaryotic gene cloning
Isolation of cloning vector (bacterial plasmid) & gene-source DNA (gene of interest)
Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase
Introduction of cloning vector into cells (transformation by bacterial cells)
Cloning of cells (and foreign genes)
Identification of cell clones carrying the gene of interest

68. DNA Cloning
Restriction enzymes (endonucleases): in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific
Restriction site: recognition sequence for a particular restriction enzyme
Restriction fragments: segments of DNA cut by restriction enzymes in a reproducable way
Sticky end: short extensions of restriction fragments
DNA ligase: enzyme that can join the sticky ends of DNA fragments
Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

69. Restriction endonucleases

70. Practical DNA Technology Uses
Diagnosis of disease
Human gene therapy
Pharmaceutical products (vaccines)
Forensics
Animal husbandry (transgenic organisms)
Genetic engineering in plants
Ethical concerns?

71. GENES THERAPY

72. Biotechnology practical use