Chair of Medical Biology, Microbiology, Virology, and Immunology

GENETICS OF BACTERIA AND VIRUSES. BASES OF BIOTECHNOLOGY AND GENE ENGENEERING

Lecturer Prof. S.I. Klymnyuk

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

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.

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.

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.

DNA structure

E. coli DNA

E. coli DNA

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.

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.

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.

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.

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.

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

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).

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,

PlasmidsDefinition: 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– 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.

Comparing plasmid profiles is a useful method for assessing possible relatedness of individual clinical isolates of a particular bacterial species for epidemiological studies.

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

– Importance

• Mutation

•Plasmid insertion

•Phase variation

TransposaseABCDEFG GFEDCBA

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

ISH1 gene H2 gene

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 ISResistance Gene(s)

IS ISResistance Gene(s)

Transposons are not self-replicating genetic elements, however, and they must integrate into other replicons to be maintained stably in bacterial genomes

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.

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.

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.

Transposone

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

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-.

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 MutationsMissense 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)

Other Types of Mutations

• Suppressor mutation: a mutation that alters forward mutation, reverse wild phenotype (in same gene, in another gene)

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..

• 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

Mutations affect bacterial cell phenotype

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)

N. meningitidis genes with high mutation rates include those

involved in:

capsule biosynthesis

LPS biosynthesis

attaching to host cells

taking up iron

Examples of mutagensCHEMICAL

AGENTACTION

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 mutagensPHYSICAL

AGENTACTION

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 mutagensBIOLOGICAL

AGENTACTION

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

minus lysinecomplete

Lysine auxotrophsdo not grow

All strains grow

Isolation of a Lysine Auxotroph

Isolation of a motility mutant by direct selection

ReparationLight-requiring

Dark

SOS- reactivation

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• Legitimate,

homologous or general

• recA, recB and recC genes

• Significance– Phase variation in Neiseseria– Recombinant DNA technology

• Steps

– Uptake of DNA

• Gram +

• Gram -

Competent cell

S strain

R strain S strain

Transduction

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

Phage Composition and Structure

• Composition– Nucleic acid

• Genome size

• Modified bases

– Protein• Protection• Infection

• Structure (T4)

– Size– Head or capsid– Tail

Tail

Tail Fibers

Base Plate

Head/Capsid

Contractile Sheath

Transduction

Types of transduction– Generalized - Transduction in which

potentially any donor bacterial gene can be transferred

Generalized Transduction

• Release of phage

• Phage replication and degradation of host DNA

• Assembly of phages particles

• Infection of recipient• Legitimate recombination

• Infection of Donor

Transduction

Types of transduction

–Specialized - Transduction in which only certain donor genes can be transferred

Specialized TransductionLysogenic Phage

• Excision of the prophage

gal

bio

gal bio

gal bio

gal

bio

bio

gal

• Replication and release of phage

• Infection of the recipient

• Lysogenization of the recipient– Legitimate

recombination also possible

TransductionTypes 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.

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

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

• DNA transfer– Origin of

transfer– Rolling

circle replication

• Pair formation

– Conjugation bridge

F+ F- F+ F-

F+ F+F+ F+

Mechanism of Hfr x F- Crosses

• DNA transfer– Origin of

transfer

– Rolling circle replication

• Homologous recombination

• Pair formation

– Conjugation bridge

Hfr F- Hfr F-

Hfr F-Hfr F-

Mechanism of F′ x F- Crosses

• DNA transfer– Origin of

transfer

– Rolling circle replication

• Pair formation

– Conjugation bridge

F’ F’F’ F’

F’ F- F’ F-

Conjugation

• Significance– Gram - bacteria

• Antibiotic resistance

• Rapid spread

– Gram + bacteria• Production of adhesive material by donor cells

Map of chromosome

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.

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