1. Chair of Medical Biology, Microbiology, Virology, and Immunology
GENETICS OF BACTERIA AND VIRUSES. BASES OF BIOTECHNOLOGY AND GENE ENGENEERING
Lecturer Prof. S.I. Klymnyuk

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. The genetic material of bacteria and plasmids is DNA.
The two essential functions of genetic material are replication and expression.
Expression of specific genetic material under a particular set of growth conditions determines the observable characteristics (phenotype) of the organism.

5. Nucleic Acid Structure
Nucleic acids are large polymers consisting of repeating nucleotide units.
Each nucleotide contains one phosphate group, one pentose or deoxypentose sugar, and one purine or pyrimidine base.
In DNA the sugar is D-2-deoxyribose; in RNA the sugar is D-ribose.
In DNA the purine bases are adenine (A) and guanine (G), and the pyrimidine bases are thymine (T) and cytosine (C).
In RNA, uracil (U) replaces thymine.

6. The double helix is stabilized by hydrogen bonds between purine and pyrimidine bases on the opposite strands.
The two strands of double-helical DNA are complementary. Because of complementarity, double-stranded DNA contains equimolar amounts of purines (A + G) and pyrimidines (T + C), with A equal to T and G equal to C, but the mole fraction of G + C in DNA varies widely among different bacteria.
Information in nucleic acids is encoded by the ordered sequence of nucleotides along the polynucleotide chain, and in double-stranded DNA the sequence of each strand determines what the sequence of the complementary strand must be. The extent of sequence homology between DNAs from different microorganisms is the most stringent criterion for determining how closely they are related.

7. DNA structure

8. E. coli DNA

9. E. coli DNA

10. DNA Replication
During replication of the bacterial genome, each strand in double-helical DNA serves as a template for synthesis of a new complementary strand. Each daughter double-stranded DNA molecule thus contains one old polynucleotide strand and one newly synthesized strand. This type of DNA replication is called semiconservative. Replication of chromosomal DNA in bacteria starts at a specific chromosomal site called the origin and proceeds bidirectionally until the process is completed.

11. Gene Expression
Genetic information encoded in DNA is expressed by synthesis of specific RNAs and proteins, and information flows from DNA to RNA to protein. The DNA-directed synthesis of RNA is called transcription. Because the strands of double-helical DNA are antiparallel and complementary, only one of the two DNA strands can serve as template for synthesis of a specific mRNA molecule.
Messenger RNAs (mRNAs) transmit information from DNA, and each mRNA in bacteria functions as the template for synthesis of one or more specific proteins.
The process by which the nucleotide sequence of an mRNA molecule determines the primary amino acid sequence of a protein is called translation.

12. Ribosomes, complexes of ribosomal RNAs (rRNAs) and several ribosomal proteins, translate each mRNA into the corresponding polypeptide sequence with the aid of transfer RNAs (tRNAs), amino-acyl tRNA synthesases, initiation factors and elongation factors.
All of these components of the apparatus for protein synthesis function in the production of many different proteins.

13. The genetic code determines how the nucleotides in mRNA specify the aminoacids in a polypeptide.
Minimum of three nucleotides is required to provide at least one unique sequence corresponding to each of the 20 amino acids. The "universal" genetic code employed by most organisms is a triplet code in which 61 of the 64 possible trinucleotides (codons) encode specific amino acids, and any of the three remaining codons (UAG, UAA or UGA) results in termination of translation.
The chain-terminating codons are also called nonsense codons because they do not specify any amino acids.
The genetic code is described as degenerate, because several codons may be used for a single amino acid, and as nonoverlapping, because adjacent codons do not share any common nucleotides.

14. Exceptions to the "universal" code include the use of UGA as a tryptophan codon in some species of Mycoplasma and in mitochondrial DNA, and a few additional codon differences in mitochondrial DNAs from yeasts, Drosophila, and mammals.
Translation of mRNA is usually initiated at an AUG codon for methionine, and adjacent codons are translated sequentially as the mRNA is read in the 5' to 3' direction. The corresponding polypeptide chain is assembled beginning at its amino terminus and proceeding toward its carboxy terminus. The sequence of amino acids in the polypeptide is, therefore, colinear with the sequence of nucleotides in the mRNA and the corresponding gene.

15. Genome Organization
DNA molecules that replicate as discrete genetic units in bacteria are called replicons. In some Escherichia coli strains, the chromosome is the only replicon present in the cell. Other bacterial strains have additional replicons, such as plasmids and bacteriophages

16. Chromosomal DNA
Bacterial genomes vary in size from about 0.4 x 109 to 8.6 x 109 daltons (Da), some of the smallest being obligate parasites (Mycoplasma) and the largest belonging to bacteria capable of complex differentiation such as Myxococcus.
The amount of DNA in the genome determines the maximum amount of information that it can encode. Most bacteria have a haploid genome, a single chromosome consisting of a circular, double stranded DNA molecule. However linear chromosomes have been found in Gram-positive Borrelia and Streptomyces spp., and one linear and one circular chromosome is present in the Gram-negative bacterium Agrobacterium tumefaciens.
The single chromosome of the common intestinal bacterium E coli is 3 x 109 Da (4,500 kilobase pairs [kbp]) in size, accounting for about 2 to 3 percent of the dry weight of the cell. The E coli genome is only about 0.1 % as large as the human genome, but it is sufficient to code for several thousand polypeptides of average size (40 kDa or 360 amino acids).

17. The chromosome of E coli has a contour length of approximately 1
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,

19. Plasmids
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.

20. 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)

21. Phenotypic effects

22. 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

23. 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.

25. Some plasmids are cryptic and have no recognizable effects on the bacterial cells that harbor them.
Comparing plasmid profiles is a useful method for assessing possible relatedness of individual clinical isolates of a particular bacterial species for epidemiological studies.

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
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
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.

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

29. 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

30. 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.

31. 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.

32. 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.

33. Transposone

34. Tn917 encodes tetracycline resistance
Most transposons in bacteria can be separated into four major classes.
Insertion sequences and related composite transposons comprise the first class.
The second class of transposons consists of the highly homologous TnA family (ampicillin resistance transposon Tn3 and Tn1000 (the gamma-delta transposon) found in the F plasmid.
The third class of transposons consists of bacteriophage Mu and related temperate phages)
A fourth class of transposons, discovered in Gram-positive bacteria and represented by Tn917, consists of conjugative transposons (Gram-positive bacteria the host strain carrying the transposon can act as a conjugal donor).
Tn917 encodes tetracycline resistance

35. Mutation and Selection
Variant forms of a specific genetic determinant are called alleles.
Genotypic symbols are lower case, italicized abbreviations that specify individual genes, with a (+) superscript indicating the wild type allele.
Phenotypic symbols are capitalized and not italicized, to distinguish them from genotypic symbols.
For example, the genotypic symbol for the ability to produce β-galactosidase, required to ferment lactose, is lacZ+, and mutants that cannot produce β-galactosidase are lacZ. The lactose-fermenting phenotype is designated Lac+, and inability to ferment lactose is Lac-.

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

37. 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

38. 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

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

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

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

42. Frameshift mutation - Deletion

43. 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)

44. Other Types of Mutations
Suppressor mutation: a mutation that alters forward mutation, reverse wild phenotype (in same gene, in another gene)

45. Suppressor mutations can be intragenic or extragenic.
Intragenic suppressors are located in the same gene as the forward mutations that they suppress. The possible locations and nature of intragenic suppressors are determined by the original forward mutation and by the relationships between the primary structure of the gene product and its biologic activity.
Extragenic suppressors are located in different genes from mutations whose effects they suppress. The ability of extragenic suppressors to suppress a variety of independent mutations can be tested. Some extragenic suppressors are specific for particular genes, some are specific for particular codons, and some have other specificity patterns. Extragenic suppressors that reverse the phenotypic effects of chain-terminating codons have been well characterized and found to alter the structure of specific tRNAs..

46. 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

47. 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)

48. 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

49. 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

50. 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

51. 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

52. 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

53. 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

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

55. Isolation of a motility mutant by direct selection

56. Reparation
Light-requiring
Dark
SOS- reactivation

59. Exchange of Genetic Information
Recombination

60. Transformation

61. 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

62. 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

63. S strain
R strain
Competent cell
S strain

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

65. 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

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

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

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

69. 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

70. 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.

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

73. 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

74. 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.

75. 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.

77. Basic Bacterial Conjugation
F+ / F- mating
An F plasmid moves from the donor (F+) to a recipient (F-)
The F plasmid is copied and transferred via a sex pilus, the recipient becomes F+ and the donor remains F+
The F factor codes for pilus formation which joins the donor and recipient and for genes which direct the replication and transfer of a copy of the F factor to the recipient
The F factor can remain as a plasmid or it can integrate into the bacterial chromosome via IS sequences. This type of donor is called and Hfr strain (High frequency recombination)
F′- When the F factor in an Hfr strain leaves the chromosome, sometimes is makes an error in excision and picks up some bacterial genes

78. 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

79. 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+

80. 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

81. 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’

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

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

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

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

86. Map of chromosome

88. Recombination DNA and Gene Cloning
Many methods are available to make hybrid DNA molecules in vitro (recombinant DNA) and to characterize them. Such methods include isolating specific genes in hybrid replicons, determining their nucleotide sequences, and creating mutations at designated locations (site-directed mutagenesis). A clone is a population of organisms or molecules derived by asexual reproduction from a single ancestor. 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.

89. Bacterial plasmids in gene cloning

90. 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

91. 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)

92. Restriction endonucleases

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

94. GENES THERAPY

95. Biotechnology practical use