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

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

F. Crick i J. Watson – described DNA structure

DNA structure

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

E. coli DNA

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.

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)

Phenotypic effects

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

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 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

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

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.

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

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

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.

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.

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.

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

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

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

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

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

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

Frameshift mutation - Deletion

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)

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

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)

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

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

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

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

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

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

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

Reparation Light-requiring Dark SOS- reactivation

Light-requiring Reparation

Dark Reparation

Exchange of Genetic Information Recombination

Transformation

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

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

S strain R strain Competent cell S strain

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

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

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

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

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

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

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.

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

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

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.

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.

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

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+

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

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’

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

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

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

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

Map of chromosome

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.

Bacterial plasmids in gene cloning

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

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)

Restriction endonucleases

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

GENES THERAPY

Biotechnology practical use