Download Genetics of bacteria

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, doublestranded 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 doublestranded 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 Gramnegative 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,
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.
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
RTF
– Conjugative
plasmid
– Transfer genes
• R determinant
– Resistance
genes
– Transposons
R determinant
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
GFEDCBA
ABCDEFG
Transposase
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.
– Importance
• Mutation
•Plasmid insertion
•Phase variation
Phase Variation in Salmonella H Antigens
H1 gene
H1
flagella
IS
H2 gene
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)
IS
IS
Resistance Gene(s)
IS
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 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)
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 chainterminating codons have been well characterized and found to alter the
structure of specific tRNAs..
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)
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:
Incorporate into DNA and cause mispairing
5-bromouracil
2-amino purine
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
All strains grow
Lysine auxotrophs
do not grow
Isolation of a motility mutant by direct selection
Reparation
Light-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
• Steps
– Uptake of DNA
• Gram +
• Gram -
– Recombination
• Legitimate,
homologous or
general
• recA, recB and
recC genes
• Significance
– Phase variation in
Neiseseria
– Recombinant DNA
technology
R strain
Competent cell
S 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
Head/Capsid
– Protein
• Protection
• Infection
• Structure (T4)
– Size
– Head or capsid
– Tail
Contractile
Sheath
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
• Replication and
release of phage
• Infection of the
recipient
• Lysogenization of
the recipient
bio
gal
gal
gal
bio
bio
– Legitimate
recombination
also possible
gal
bio
bio
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.
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
Donor
• F factor (Fertility factor)
– F (sex) pilus
– Recipient
• Lacks an F factor
Recipient
Physiological States of F Factor
• Autonomous (F+)
– Characteristics of F+ x Fcrosses
• 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 Fcrosses
• 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
• DNA
transfer
– Origin of
transfer
– Rolling
circle
replication
F+
F-
F+
F-
F+
F+
F+
F+
Mechanism of Hfr x F- Crosses
• Pair formation
– Conjugation
bridge
• DNA transfer
Hfr
F-
Hfr
F-
– Origin of
transfer
– Rolling circle
replication
• Homologous
recombination
Hfr
F-
Hfr
F-
Mechanism of F′ x F- Crosses
• Pair formation
– Conjugation
bridge
• DNA transfer
– Origin of
transfer
– Rolling circle
replication
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 (sitedirected 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) & genesource 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