Download bacterial genetics

BACTERIAL GENETICS
Structure of DNA
•
•
•
•
Double stranded (double helix)
Chains of nucleotides
5’ to 3’ (strands are anti-parallel)
Complimentary base pairing
– A-T
– G-C
DNA Structure
Phosphate-P
Sugar-blue
Bases-ATGC
DNA Replication
• Bacteria have closed, circular DNA
• Genome: genetic material in an organism
• E. coli
– 4 million base pairs
– 1 mm long (over 1000 times larger that actual
bacterial cell)
– DNA takes up around 10% of cell volume
DNA Replication-occurs at the
replication fork
• 5’ to 3 ‘
• DNA helicase-unzips + parental DNA strand that is
used as a template
– Leading stand (5’ to 3’-continuous)
*DNA polymerase-joins growing DNA strand after
nucleotides are aligned (complimentary)
– Lagging strand (5’ to 3’-not continuous)
*RNA polymerase (makes short RNA primer)
*DNA polymerase (extends RNA primer then digests RNA primer and
replaces it with DNA)
*DNA ligase (seals Okazaki fragments-the newly formed DNA
fragments)
Replication Fork
Protein Synthesis
• DNA------- mRNA------ protein
transcription
translation
Central Dogma
of Molecular Genetics
Transcription
• One strand of DNA used as a template to make a
complimentary strand of mRNA
• Promoter/RNA polymerase/termination site/5’ to 3’
• Ways in which RNA & DNA differ:
– RNA is ss
– RNA sugar is ribose
– Base pairing-A-U
Transcription
Translation
• Three parts:
– Initiation-start codon (AUG)
– Elongation-ribosome moves along mRNA
– Termination: stop codon reached/polypeptide released
and new protein forms
• rRNA=subunits that form the 70 S ribosomes (protein
synthesis occurs here)
• tRNA=transfers amino acids to ribosomes for protein
synthesis)
PHENOTYPIC VARIATION
• Change in the colony characters , capsule or
flagella
GENOTYPIC VARIATION
• Change in the genes resulting in change in
protein synthesis
Mutations – changes in the DNA
• Point mutation – addition, deletion or
substitution of a few bases
• Missense mutation – causes change in a single
amino acid
• Nonsense mutation – changes a normal codon
into a stop codon
• Silent mutation – alters a base but does not
change the amino acid
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Mutations – changes in the DNA
• Point mutation – addition, deletion or
substitution of a few bases
• Missense mutation – causes change in a single
amino acid
• Nonsense mutation – changes a normal codon
into a stop codon
• Silent mutation – alters a base but does not
change the amino acid
18
Mutations
• Changes in base sequence of DNA/lethal and
inheritable
• Can be:
– Harmful
– Lethal
– Helpful
– Silent
Normal DNA/Missense Mutation
Nonsense Mutation/Frameshift
Mutation
Ames Test
22
General Features of
Gene Transfer in Bacteria
• Unidirectional
– Donor to recipient
• Donor does not give an entire chromosome
– Merozygotes
• Gene transfer can occur between species
Transformation
• Definition: Gene transfer resulting from the
uptake of DNA from a donor.
• Factors affecting transformation
– DNA size and state
• Sensitive to nucleases
– 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
–
Transduction
• Definition: Gene transfer from a donor to a
recipient by way of a bacteriophage
Phage Composition and Structure
• Composition
– Nucleic acid
Head/Capsid
• Genome size
• Modified bases
– Protein
• Protection
• Infection
• Structure (T4)
– Size
– Head or capsid
– Tail
Contractile
Sheath
Tail
Tail Fibers
Base Plate
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
– Generalized - Transduction in which potentially
any dornor bacterial gene can be transferred.
– 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
• Definition
• Types of transduction
• Significance
– Common in Gram+ bacteria
– Lysogenic (phage) conversion
Conjugation
• Definition: 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 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
• 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
F’
F-
F’
F-
F’
F’
F’
F’
– Origin of transfer
– Rolling circle
replication
Conjugation
• Significance
– Gram - bacteria
• Antibiotic resistance
• Rapid spread
– Gram + bacteria
• Production of adhesive material by donor cells
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
– 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
– Structure
• Composite Tns
– Importance
• Antibiotic resistance
IS
Resistance Gene(s)
IS
IS
Resistance Gene(s)
IS
Plasmids
• Definition: Extrachromosomal genetic
elements that are capable of autonomous
replication (replicon)
• Episome - a plasmid that can integrate into
the chromosome
Classification of Plasmids
• Transfer properties
– Conjugative
– Nonconjugative
• Phenotypic effects
– Fertility
– Bacteriocinogenic plasmid
– Resistance plasmid (R factors)
Structure of R Factors
• RTF
RTF
– Conjugative
plasmid
– Transfer genes
• R determinant
– Resistance genes
– Transposons
R determinant
Genetic basis of drug resistance
• Mutational drug resistance - Chromosomal
• Transferable drug resistance – Plasmid
mediated
Genetic engineering
• Genetic engineering is the manipulation of
genetic material to alter the characteristics of an
organism.
• Genetic fusion: allows transposition from one
location on a chromosome to another, sometimes
deleting a portion, thereby causing the joining of
genes from two different operons.
• Protoplast Fusion: combines protoplasts (
organisms without cell walls) and allows mixing
of genetic information.
• Gene Amplification: involves the addition of plasmids
to microorganisms to increase yield of useful
substances .
• Recombinant DNA Technology: is DNA produced when
genes from one kind of organism are introduced into
the genome of a different kind of organism. The
resulting organism is transgenic, or recombinant
organism.
• Recombinant DNA has proven especially useful in
medicine, industry and agriculture.
• Hybridisms: are genetic recombinations involving cells
of higher organisms.
• DNA PROBES: Labeled (Radioactive, Biotin etc.)
copies of single-stranded DNA fragments
containing unique nucleotide sequences, which
are used to detect homologous DNA by
hybridization. Highly specific detects even lninute
amounts of target DNA.
• PCR - POLYMERASE CHAIN REACTION: Rapid
automated method for amplification of specific
DNA sequences. A single PCR cycle consists of;
• Denaturation of sample DNA to obtain a single
strand
• Annealing of sequence specific oligonucleotide
primers. . Extension of primers by DNA Polymerase
enzyme to form a new double stranded DNA.
• This cycle is repeated for 20 - 50 cycles in a
thermocycler to obtain several thousands of copies
of Sample DNA that can be identified later by DNA
probes.
• Diagnosis of Infectious diseases, genetic disorders,
cancer, forensic investigations and biotechnology.
• Restriction endonucleases: Enzymes which can
cleave DSDNA at specific oligonuleotide seqences.
• e.g: Taq I, Hind III.
• RECOMBINANT DNA TECHNOLOGY:
• Isolation of genes coding for any desired protein
from cells or microorganisms –
• Synthesis- small sequences; Enzymes - restriction
endonucleases.
• mRNA - DNA (SS) synthesis by reverse transcriptase
&
• polymerization by DNA polymerase to yield DS DNA.
• Vectors - Plasmids, Bacteriophages;E.coli K12 &
Yeast's.