Subject: Microbiology
BATCH 2019
Topic: Microbial Genetics
MICROBIAL GENETICS
GENE
Basic unit of hereditary
A segment of DNA that encodes in its nucleotide
sequence information for a specific physiologic
property.
PHENOTYPE
The collective structural & physiologic properties of an
organism.
E.g., Eye color in human or Resistance to antibiotic in
a bacterium
GENOTYPE
Chemical basis for variation in phenotype
Alteration in the DNA sequence, w/in a gene or w/in
the organization of genes
DNA
Fundamental element of hereditary (suggested by
Frederick Griffith)
Transformed the live, nonvirulent strain to the virulent
phenotype
RESTRICTION ENZYMES
Proteins that cleave DNA at speicifc sites, giving rise to
DNA RESTRICTION FRAGMENTS
PLASMIDS
Small genetic elements carrying genes and capable of
independent repication in bacteria & yeast
Are most commonly transferred by Conjugation
Introduction of DNA restriction fragment into a plasmid =
AMPLIFICATION OF DNA FRAGMENTS many times
Polymerase Chain Reaction (PCR)
Is a bacterial enzyme that cause amplification of
specific region of DNA
PROMOTERS
Allow encoded proteins to be expressed at increased
levels
ORGANIZATION OF GENES
Genetic information in bacteria is stored as a sequence
of DNA BASES
Genetic info in bacteriophages & viruses, stored as a
sequence of RNA
Most DNA molecules are double stranded, with
COMPLEMENTARY BASES
(A-T; G-C)
ANTIPARALLEL is the orientation of 2 DNA strands
o CHEMICALLY ORIENTED: 5 3
o COMPLIMENTARY STRAND: 35
TEMPLATE STRAND provide information for copying or
expression of information in the CODING STRAND
Reference: Murray 7th Ed & Jawetz 26th Ed
Transcribed by: Acosta
Each turn of helix produces:
o 1 MAJOR groove where BASES are more
exposed
o 1 MINOR groove
Each of the 4 bases is bonded to phospho-2-deoxyribose
to form NUCLEOTIDE
The negatively charged phosphodiester backbone of DNA
faces the SOLVENT
The length of DNA molecule is usually expressed in:
o THOUSANDS of base pairs or
o KILO base pairs (kbp)
Small virus contain single DNA molecule of <0.5kbp
Escherichia Coli is >4000 kbp
Because the bacterial cell are roughly 1000 fold smaller
tham this length, it is evident that SUPERCOILING,
contributes to the physical structure of the molecule in
vivo
Ribonucleic Acid (RNA)
Single stranded
Complementary bases: A-U ; C-G
RNA molecules ranges in size:
o Small tRNA w/c contain <100 bases
o mRNAs w/c carry genetic messages
extending to several thousand bases
The MOST GENERAL function of RNA is communication of
DNA gene sequences in the form of messenger RNA
(mRNA) to ribosomes.
Messenger RNA (mRNA)
aka +ssRNA
transcribed as the RNA complement to the
coding DNA strand
it is then translated by ribosomes
Ribosomes
Contains:
o Proteins
o Ribosomal RNA (rRNA)
Translate this message into the primary
structure of proteins via aminoacyl-transfer
RNAs (tRNA)
Bacterial ribosomes contains 3 kinds of rRNA,
w/ respective sizes:
o 120 (bases & # of proteins)
o 1540 (bases & # of proteins)
o 2900 (bases & # of proteins)
Ribozymes when RNA molecule function as an enzyme
TRANSCRIPTION
The information carried in the genetic memory of the
DNA is transcribed into a useful mRNA for a subsequent
translation into protein
RNA synthesis is performed by a DNA-dependent RNA
polymerase
Subject: Microbiology
BATCH 2019
The process begins when sigma factor
recognizes the promoter (a particular
sequence of nucleotides in the DNA) and binds
tightly to this site
Promoter sequences occur just before the start of the
DNA that actually encodes a protein
Sigma factors bind to these promoters to provide a
docking site for the RNA polymerase
Topic: Microbial Genetics
Once the polymerase has bound to the appropriate site on
the DNA, RNA synthesis proceeds w/ ribonucleotides
complementary to the sequence of the DNA
Once an entire gene or operon (group of genes) has been
transcribed, the RNA polymerase dissociates from the DNA
o This bacterial DNA-dependent RNA polymerase
- is inhibited by RIFAMPIN (an antibiotic, tx
for TB)
The need for expression of individual gene changes in
response to: physiologic demand, and requirements of
flexible gene expression are reflected in the rapid
metabolic turnover of most mRNAs.
tRNA & rRNA
tRNA - associated w/ the universally required
function of protein synthesis
Stable & together accounts for more than 95%
of the total RNA in a bacterial cell.
PROMOTERS & OPERATORS
Control the expression of a gene
By Influencung w/c sequence will be transcribed to
mRNA
OPERONS
Groups of one or more structural genes expressed
from a particular promoter
Ending at a: transciptional terminator
Operons w/ many structural genes are:
POLYCISTRONIC
Has 2 types:
o INDUCIBLE
Introduction of a substrate (inducer)
into the growth medium may induce
an operon to the expression of
enzymes necessary for metab
o REPRESSIBLE
An abundance of the end products (corepressor) may signal that a pathway
should be shut down/repressed by
the synthesis of its enzyme
In E. coli when glucose in the cell, cAMP to
promote usage of sugars for metabolism
Binding of cAMP to a protein (Catabolite geneactivator protein: CAP) allows it to bind to a specific
DNA sequence present in the promoter
The CAP-cAMP complex enhances binding of the
RNA polymerase to the promoter allowing in
frequency of transcription initiation
lac operon gene associated w/ fermanetation of
lactose in E. coli
This operon has 3 structural genes:
Reference: Murray 7th Ed & Jawetz 26th Ed
Transcribed by: Acosta
lacY mediates the transport of lactose into
the cell
o lacZ encodes Beta-Galactosidease ( enzyme
that hydrolyzes lactose to galatose & glucose)
o lacA produces a transacetylase
ALLOLACTOSE inducer of lac operon; because Lactos
itself cant influence transcriptional regulations
o
TRYPTOPHAN OPERON (trp operon)
Contains structural genes necessary for tryptophan
biosynthesis
Under dual transcriptional control mechanisms
Although tryptophan is essential for protein synthesis,
TOO MUCH TRYPTOPHAN in the cell can be TOXIC
TRYPTOPHAN TYPES/CLASS:
o DNA LEVEL repressor protein is activated by
intracellular proteins of tryptophan to
PREVENT TRANSCRIPTION
o PROTEIN SYNTHESIS LEVEL rapid
translation of test peptide at the beginning
of mRNA in the presence of tryptophan
allows the formation of a double stranded loop
in the RNA w/c TERMINATES TRANSCRIPTION
o mRNA LEVEL the term attenuation:
regulates tryprophan synthesis at this level,
wherein mRNA synthesis is prematurely
terminated
INITIATION OF TRANSCRIPTION may be under:
POSITIVE control
o Genes that are NOT transcribed unless an
active regulator protein APOINDUCER is
present
NEGATIVE control
o Genes that are expressed unless they are
switched off by repressor protein
TRANSLATION
Is the process by w/c the language of genetic code (in
the form of mRNA) is converted (translated) into a
sequence of Amino Acids (AA; the protein product)
Each AA is written as sets of 3 nucleotides known as
CODONS
DEGENERACY OF THE GENETIC CODE
It occurs, when some of the AA are encoded by more
than 1 triplet codon
May function in protecting the cell from the effects of
minor mutations in the DNA or mRNA
Each tRNA molecule contains 3-nucleotide sequence
complementary to one of the codon sequence this tRNA
sequence is known as ANTICODON; it allows the base
pairing & binds to the codon sequence on the mRNA.
Attached to the opposite end of tRNA is an AA that
corresponds to the particular codon-anticodon pair
Subject: Microbiology
BATCH 2019
Topic: Microbial Genetics
PROCESS OF PROTEIN SYNTHESIS
AUG start codon
Starts w/ binding of 30S ribosomal subunit & special
initiator of tRNA for fMET at the methionine codon
(AUG) to form INITIATION COMPLEX
The 50S ribosomal subunit binds to the complex to
initiate mRNA synthesis
Ribosome contains 2 tRNA binding sites:
- w/c allows base pairing between tRNA and mRNA
o A (aminoacyl) site
o P (peptidyl) site
TRANSPEPTIDATION
A reaction of the amino group of the AA attached to
the A site, forms a peptide bond w/ the carboxyl group
of the AA in the P site
And the empty tRNA in the P site (uncharged tRNA) is
released from the ribosome
Translation continues until the new codon in the A site is
one of the 3 termination codons: (STOP CODON)
in RNA
in DNA
UAG
TAG
UAA
TAA
UGA
TGA
At that point: NEW protein is released to the cytoplasm &
translaction complex may be disassembled or ribosome
shuffles to the next start codon (AUG) and initiates a new
protein
Ribosomal shuffling is a characteristic of 70S bacterial
but not the 80S eukaryotic ribosome
small interfering RNA (siRNA)
New class of RNA
Described in plants
Double stranded RNA
20-25 nucleotides in length
Functions as regulators
o Binding near the 5 end of an mRNA
Preventing ribosomes from
translating
o Base pairing directly with a strand of
DNA near the promoter
Preventing transcrption
EUKARYOTIC GENOME
Almost all of the eukaryotic genome is carried on 2 or
more linear chromosomes separated from the cytoplasm
w/in the membrane of the nucleus
GENOME
Totality of genetic information in an organism
DIPLOID
Eukaryotic cells that contain TWO HOMOLOGUES
(divergent evolutionary copies) of each chromosome
MUTATIONS
Genetic Changes ( change in base sequence)
Cannot be detected on diploid cells
Reference: Murray 7th Ed & Jawetz 26th Ed
Transcribed by: Acosta
Because the contribution of one gene copy
compensates for changes in function of its
homologue
RECESSIVE
A gene that does not achieve phenotypic expression in
the presence of its homologue
DOMINANT
A gene that overrides the effect of its homologue
HAPLOID CELLS
Discern the effects of mutations
Carry single copy of most gene
E.g. YEAST CELLS (eukaryotic)
EUKARYOTIC CELLS
Contains:
o Mitochondria
o Chloroplasts (for plants)
EUKARYOTIC CHROMOSOMES
Where most genes associated w/ organelle function
are carried on
PLASMIDS / EPISOMES
Frequently associated w/ prokaryotes
Because of its small size, renders them amenable to
genetic manipulation
Commonly used in genetic engineering
Repetitive DNA
Occurs in large quantities in eukaryotic cells
short-sequence repeats (SSRs) or
short tandemly repeated sequences (STRs)
Presence of prokaryotic SSRs & STRs shows
extensive-length polymorphism
Caused by slipped-strand mispairing
IMPORTANT prerequisite for bacterial phase variation &
adaptation
INTRONS
It interrupts many eukaryotic genes
Observed in archaebacterial genes
o Methanococcus jannaschil
o Archaeglobus fulgidus
NOT FOUND in eubacteria
PROKARYOTIC GENOME
Mostly consist of single circular DNA molecule
580 kbp to 5220 kbp of DNA
A few bacteria have genomes cosisting of 2 circular
DNA molecules
o Brucella melitensis
o Burkjolderia pseudomallel
o Vibrio cholerae
REPLICONS
Subject: Microbiology
BATCH 2019
A covalently closed DNA circles (plasmids & bacterial
chromosomes), w/c contains genetic information
necessary for their own replication
Cleavage of circles produces linear DNA that is
packaged inside protein coats to form daughter
phages
Prokaryotes- do not contain a nucleus
Eukaryotes a membrane doesnt separate bacterial
genes from cytoplasm
Bacteria might produce more than 6 different sigma
factors to provide global regulation in response to:
STRESS, SHOCK, STARVATION, or to coordinate production
of complicated structures such as FLAGELLA
cAMP indicates glucose levels need to utilize
alternative metabolic pathways
QUOROM SENSING
When a sufficient # of bacteria are present &
producing a specific small molecule, virulence & other
genes are turned on
PSEUDOMONAS spp.
The trigger for biofilm production: is the critical
concentration of N-acyl homoserine lactone (AHL)
It is produced when sufficient number of bacteria
(quorom) are present
STAPHYLOCOCCUS AUREUS
Activates toxin production & more virulent behavior w/
an INCREASE in concentration of a cyclic peptide
the genes for some virulence mechanisms are organized
into a pathogenicity island
PATHOGENICITY ISLANDS
Some bacterial species causes disease because they
possess specific genes for pathogenic determinants;
these genes are often clustered together in DNA and
referred to as pathogenicity islands.
Large (up to 200kbp) & encode a collection of
virulence genes
1. Have different G+C content
2. Closely linked on the chromosome to the tRNA
genes
3. Flanked by direct repeats
4. Contain diverse genes important for pathogenesis:
o Antibiotic resistance
o Adhesins
o Invasins
o Exotoxin
HOUSEKEEPING GENES
Genes essential for bacterial growth
Carried on the chromosome
Plasmid - Carry genes associated w/ specialized
function
- Also encodes genetic sequences (e.g.,
involved w/ sex pili)
Reference: Murray 7th Ed & Jawetz 26th Ed
Transcribed by: Acosta
Topic: Microbial Genetics
EXAMPLES OF METABOLIC ACTIVITIES BY PLASMIDS:
ORGANISM
ACTIVITY
Pseudomonas spp.
Degradation of Camphor,
Toluene, Octane, Salicylic Acid
Bacillus
- Amylase
stearothermophilus
Alcaligenes eutrophus Utilization of H2 as oxidizable
energy source
Escherichia coli
Sucrose uptake & metabolism,
Citrate uptake
Klebsiella spp.
Nitrogen fixation
Streptococcus (group
Lactose utilization, galactose
N)
phosphotransferase system,
Citrate metabolism
Rhodospirillum
Synthesis of photosynthetic
rubrum
pigment
Flavobacterium spp.
Nylon degradation
TRANSPOSONS
Jumping genes
Genetic elements that contain several genes, including
those necessary for their migration from one genetic
locus to another. In doing so, they create insertion
mutations
Complex transposons carry genes for specialized
functions like antibiotic resistance; flanked by Insertion
Sequence (IS)
DO NOT CARRY the genetic information required to
couple their own replication to cell division
Their propagation depends on their physical
integration w/ a bacterial replicon
Due to LOW specificity of sequence at the site of
insertion: Transposons insert in a random pattern, but
they tend to favor regions encoding tRNAs.
INSERTION ELEMENTS
Aka Insertion Sequence [IS] elements
Involvement of relatively short transposons (0.75-2.0
kbp long)
PRODUCE MAJORITY of the insertion mutations
Carry only the genes for enzymes needed to promote
their own transposition to another genetic locus but
cannot replicate on their own
Almost all bacteria carry IS elements
Related IS elements (found in some bacteria)
indicates that at some point in evolution they have
crossed species barriers
PLASMIDS also carry IS elements important in the
formation of high-frequency recombinant (Hfr)
strains
VIRAL GENOME
In the absence of cell host: viruses - capable of
survival, NOT GROWTH
This form of genetic parasitism, results in debilitation
or death of host cells
Virus CANNOT multiply through the division of cells
because they are ACELLULAR
They seek host cell in w/c they replicate & assemble
themselves
Subject: Microbiology
BATCH 2019
REPLICATION of viral genome depends on:
Metabolic energy
Macromolecular synthetic machinery of the host
SUCCESSFUL PROPAGATION of the virus requires:
A stable form allows virus to survive w/o a host
Mechanism of invasion of a host cell
Genetic information required for replication w/in the
cell
Additional information required for packaging the viral
components & liberating the resulting virus from the
host cell
BACTERIOPHAGE or PHAGE
Virus associated w/ prokaryotes
Group of viruses that infects bacteria
Constitute the largest of all viral groups
The nucleic acid molecule of bacteriophages is
surrounded by a protein coat
Hydroxymethylcytosine sometimes found in the
phage nucleic acid
Contain specialized syringe-like structures (tails) that bind to receptors on the cell surface, & inject the
phage nucleic acid into a host cell
Type of bacteriophages:
o M13
o T phage
o Lambda phage BEST CHARACTERIZED
temperate phage ([Link])
o MS2
o G4
o Phi174
PASSIVE REPLICATION of bacteriophage occurs when
daughter bacterial cells are produced
TEMPERATENESS OR TEMPERATE PHAGE
One of the characteristics of bacteriophage
It refers to the ability of some bacteriophage
(particularly LAMBDA PHAGE), to choose between two
cycles:
o LYSOGENIC
o LYTIC
Temperate phages reel towards the lysogenic cycle
especially when phage absorption in the infected
bacteria is apparent
They are integrated into the genome of the bacteria, &
produce prophages (w/c are created w/o disrupting
the bacterial cell)
UV light causes depression of prophage
When temperate phage undergoes LYTIC CYCLE, it
becomes a VIRULENT PHAGE
LYTIC PHAGES
Produce many copies of themselves in a single burst of
growth, as they KILL their host cell
Most studied lytic phages: the T-even (e.g., T2, T4) of
E. coli
o Demonstrate the need for precisely timed
expression of viral genes
Reference: Murray 7th Ed & Jawetz 26th Ed
Transcribed by: Acosta
Topic: Microbial Genetics
dsDNA of many lytic phages is linear
FIRST STAGE: formation of circular DNA This process
depends on:
o COHESIVE ENDS complementary ssDNA
(tails) that hybridize
o LIGATION formation of phosphodiester bonds
between 5-3
LYOSGENIC CYCLE
Term for bacteria carrying prophages
Genomes of temperate phages are NOT expressed
LYSOGENS
Is the prophage-containing bacteria cells
It can exist as a dormant DNA w/in its host cell
Have the ability to stay in the lysogenic cycle for a
VERY LONG TIME
But w/ INDUCTION directed to LYTIC CYCLE
FILAMENTOUS PHAGES (Ff phage)
Filament or Rod like shape
SINGLE-stranded DNA
Infect gram (-) bacteria
Types of Ff phage:
o M13 infects E. coli
o F1
o Fd
These phages undergo a non-lytic life cycle, where
infected bacteria are not killed but continuously
release viral particles
Only ONE END binds to the F pilus & can inject the
DNA to the host cell
ssDNA of Ff phage is converted to a circular doublestranded replicative form
REPLICATION
DOUBLE-stranded DNA is synthesized by
semiconservative replication
Parental duplex unwinds each strand serves as a
template for DNA replication.
New strands are synthesized w/ their bases
complementary to the pre-existing strands
When synthesis is COMPLETE:
o Each DAUGHTER molecule contains
1 PARENTAL strand
1 NEWLY synthesized strand
Chromosome replication is initiated at the membrane
Bacterial membrane, peptidoglycan synthesis, & cell
division LINKED together such that peptidoglycan
synthesis will also inhibit cell division
ALARMONES
A chemical that is triggered by depletion of
metabolites (starvation) or a buildup of toxic
byporducts (e.g., ethanol)
Causes synthesis to stop, but degradative processes
continues
POPULATION DYNAMICS
Subject: Microbiology
BATCH 2019
Topic: Microbial Genetics
LAG PHASE of growth
o When bacteria are added to a new medium,
they require time to adapt to the new
environment before they begin to divide
Logarthmic (LOG) or exponential phase
o Bacteria will grow & divide with a doubling
time a characteristic of the strain &
determined by the conditions
o The number of bacteria will to 2n
n - is the # of generations (doublings)
Stationary phase
o Bacteria stops growing due to:
Culture eventually runs out of
metabolites
Toxic substance builds up in the
medium
BACTERIAL DNA
Bidirectional replication
Semi conservative replication
o 2 OLD strands of DNA are separated & used as
templates to synthesize: 1 parental & 1 new
strand
Dispersive replication
o DNA only copied itself for a short chunks at a
time, & synthesize: Alternated parent &
daughter dna
Conservative replication
o DNA DID NOT split open at all, but somehow
kept the parent strands intact while creating
an entirely new & separate copy
ORI locus where DNA begins to replicate (w/ several
proteins)
TER where chromosomal replication of [Link]
terminates
o Iron levels can activate expression of
hemolysin in [Link] or diptheria toxin from
Corynebacterium Diptheria
e.g., TAG (refer to page 3 table)
Cause the ribosome to fall off the mRNA & end
the protein prematurely
CONDITIONAL MUTATIONS
o May result from a conservative mutation w/c
changes the structure or function of an
important protein at elevated temperatures
o e.g., Temperature-sensitive mutation
FRAMESHIFT MUTATION
o Introduction/Removal of single base pairs from
DNA
Caused by: slight slippage of DNA
strands
o Is produced if a small deletion or insertion- is
not in multiples of three
o This results in change in reading frame
leading to a useless peptide & premature
truncation of the protein
NULL MUTATION
o Completely destroy gene function
o Arise when there is an extensive insertion,
deletion, or gross rearrangement of
chromosome structure
o
o
MUTAGEN
Frequency of mutation is greatly enhance by exposure of
cells to mutagen
o PHYSICAL MUTAGEN
Ultraviolet light (UV)
Damages DNA by linking neighboring
Thymine bases to form dimers
o CHEMICAL MUTAGEN
Act by altering either the
chemical/physical structure of DNA
This type of mutation occurs because more than one
codon may encode an AA:
Chemical mutagens can be grouped into three classes:
NUCLEOTIDE-BASE ANALOGUES
o Lead to mispairing & frequent DNA replication
mistakes
o e.g., incorporation of 5-bromouracil in the DNA
produces a change from T-A base pair to G-C
base pair
FRAMESHIFT MUTAGENS
o Such as polycyclic flat molecules
Ethidium Bromide
Acridine Derivatives
o Insert or intercalate between the bases as
they stack w/ each other in the double helix
DNA-REACTIVE CHEMICALS
o Act directly on the DNA to change the
chemical structure of the base
MUTATIONS OF DNA & THEIR CONSEQUENCES:
TRANSITION a result of single base change; example:
o
1 purine is replaced by another purine
o 1 pyrimidine is replaced by another pyrimidine
TRANSVERSION Purine is replaced by pyrimidine (or
vice versa)
SILENT MUTATION - change that does not result in any
change of AA in the encoded protein at the DNA level
MISSENSE MUTATIONS
o Results in a different AA being inserted in the
protein
CONSERVATIVE MUTATION
o New AA has similar properties
o e.g., VALINE replacing ALANINE
NONSENSE MUTATION
o Changes a codon, encoding an AA to stop a
codon
Reference: Murray 7th Ed & Jawetz 26th Ed
Transcribed by: Acosta
TOPOISOMERASE
Enzymes that alter the supercoiling of ds-DNA
(e.g., GYRASE)
Acts by cutting 1 or both strands to relax coil &
extend DNA
Targets of antibiotics (e.g., QUINOLONES)
REVERSION & SUPPRESSION
PHENOTYPIC REVERSION: regaining an activity lost as
a consequence of mutation
Subject: Microbiology
BATCH 2019
Topic: Microbial Genetics
GENOTYPIC REVERSION: Restoration of original DNA
sequence
SUPRESSOR MUTATION: Mutation at 2nd Locus;
Restores lost activity
INTRAGENIC MUTATION: at 1o mutation lost activity; 2nd
at different site RESTORES
EXTRAGENIC SUPPRESION: caused by 2nd mutation
lying outside the affected gene
REPLICATION OF DNA
Initiated at a specific sequence in the chromosome
called oriC
Replication process requires many enxymes:
o HELICASE unwind the DNA
o PRIMASE to synthesize primers to start the
process
o DNA-dependent DNA polymerases
synthesize a copy of the DNA (5 to 3 direction
ONLY)
REPLICATION FORK
It refers to the structure where 2 strands are
separated & the new synthesis is occuring
Created by HELICASES w/c breaks HYDROGEN bonds
holding the 2 strands together
LEADING STRAND
Strand of DNA w/c is being synthesized in the same
direction as the growing replication fork
A POLYMERASE reads the leading strand template &
adds complementary nucleotides
DNA POLYMERASE III is the polymerase involved in
the leading strand synthesis
Pol / Pol is involved in the leading strand synthesis
of EUKARYOTES
LAGGING STRAND
Strand of DNA whose direction of synthesis is opposite
the direction of the growing replication fork
Synthesized in SHORT, SEPARATED segments
A PRIMASE reads the template DNA & initiates
synthesis of short complementary RNA primer.
PHAGE
ssRNA phages smallest extracellular particles,
containing information that allows for their own
replication
MS2 RNA of phage
Contains 4 proteins:
GENE
GENE PRODUCT
MAT
Maturation A(MS2g1)
protein
CP
Upstream gene
Coat protein
(MS2g2)
LYS
Gene encoding
Lysis protein
(MS2g3)
(Overlaps 3 end
of cp & 5 end of
rep)
1st known
overlapping
genes
Reference: Murray 7th Ed & Jawetz 26th Ed
Transcribed by: Acosta
REP
(MS2g4)
Downstream
gene
RNA replicase,
Beta subunit
TRANSFER OF DNA
HAPLOID NATURE of bacterial genome LIMIT the
genomic plasticity of a bacterium
Replication can be achieved by:
o Integration of the donor DNA to the recipients
chromosome
o Establishment of donor DNA as an
independent replicon
RESTRICTION ENZYMES
aka Restriction endonucleases
Provides the bacteria w/ a mechanism that can
distinguish its own DNA from other biologic
DNA
Restriction-modification system has 2 broad
classes:
o Type 1 restriction & modification
activities are combined in a single
multisubunit protein
o Type 2 consist of separate
endonucleases & methylases
Direct biologic CONSEQUENCE: cleavage of
donor DNA
PLASMIDS
Small genetic elements that replocate independently
of the bacterial chromosome
Circular, dsDNA
1500 400k base pairs
Exhibit a narrow host range & are able to replicate
only in closely related set of bacteria
2 or more plasmids can: stably coexist or will interfere
one another
o When these plasmids are introduced at the
same cell, this phenomenon is called: PLASMID
INCOMPATIBILITY
Incompatibility (Inc) group
Different (Inc) group
2 plasmids CANNOT stably
coexist
2 plasmids CAN stably
coexist
LINEAR PLASMID
These 2 are unique eubacteria (because of linear
plasmid)
o Borrelia burgdorferi Causative agent of Lyme
Disease
o Borrelia hermsii
DRUG-RESISTANCE PLASMIDS
Replicate in a broad range of bacterial genera
LARGE PLASMID
20 to 120 kb
Can often mediate their own transfer from one cell to
another by: CONJUGATION
o Such as fertility factor F found in E. coli
o Resistance transfer factor (80 kbp)
Subject: Microbiology
BATCH 2019
Topic: Microbial Genetics
MECHANISM OF RECOMBINATION
Mediated by rec gene products is reciprocal
HOMOLOGOUS Recombination
LEGITIMATE
Involves exchange between genes that share common
ancestry
Consequence of close similarity in the sequences of
donor & recipient DNA
NONHOMOLOGOUS Recombination
ILLEGITIMATE
The result of enzyme-catalyzed recombination
between two dissimilar DNA sequences
Depends on enzyme encoded by integrated DNA &
clearly exemplified by insertion of DNA into a recipient
to form a copy of transposon
TRANSFORMATION
o First mechanism of genetic transfer to be
discovered in bacteria
o Direct uptake of naked donor DNA by the
recipient cell
Facilitate uptake by: electroporation
o Gram + & Gram bacteria: can take up &
stably maintain EXOGENOUS DNA (H.
influenza, Strep. Pneuomniae, Bacillus spp,
Neisseria spp)
o This process is common in bacteria, less so in
eukaryotes
o may be NATURAL or FORCED
o Forced transformation is induced in the
laboratory
o The capacity of force bacteria to incorporate
extracellular plasmids by transformation is
fundamental to genetic engineering
MECHANISM OF GENE TRANSFER
DNA composition of microorganism is FLUID
HORIZONTAL GENE TRANSFER
aka Lateral gene transfer
Movement of genetic material between unicellular
and/or multicellular organism other than vertical
transmission (e.g., parent to offspring)
3 broad mechanism mediate efficient movement of
DNA:
o CONJUGATION
o TRANSDUCTION
o TRANSFORMATION
CONJUGATION
o Requires donor cell-to-recipient cell contact
o Transfer only 1 strand of DNA
o One way transfer of DNA from DONOR (male)
to RECIPIENT (female): thorugh the SEX
PILUS
o Recipient completes the structure, results in
dsDNA
o Mode of transfer of plasmids
o Encoded by tra gene
o F PLASMID of E. coli
it carries all the genes necessary for its
own transfer, ncluding the ability to make
sex pili & to initiate DNA synthesis at the
transfer origin (oriT) of the plasmid
TRANSDUCTION
o Donor DNA is carried in a phage coat
o Mediated by bacterial viruses
(BACTERIOPHAGES)
o w/c pick up fragments of DNA & package them
into bacteriophage particles
o Transferred into the recipient
o CLASSIFICATION
SPECIALIZED
If the phages in question,
transfer particular genes
GENERALIZED
Reference: Murray 7th Ed & Jawetz 26th Ed
Transcribed by: Acosta
If incorporation of DNA
sequences is random (because
of accidental packaging of host
DNA into phage capsid)
Generalized transducing
particles VALUABLE IN
GENETIC MAPPING
GENETIC ENGINEERING
aka recombinant DNA technology
Uses the techniques & tools to
o Purify
o Amplify
o Modify
o Express specific gene sequences
BASIC COMPONENTS OF GENETIC ENGINEERING:
1. CLONING & EXPRESSION VECTORS
-Used to deliver DNA sequences into
receptive bacteria & amplify the desired
sequence
- Must allow foreign DNA to be inserted
into them, but still must be able to
replicate normally in a bacterial/eukaryotic
host
MANY TYPES OF VECTORE ARE CURRENTLY
USED:
PLASMID VECTORS:
Used for DNA fragments up to 20 kb
pUC
pBR322
pGEM
Used for larger fragments up to 25
kb
Bacteriophage: LAMBDA
Used for fragments up to 45 kb
COSMID VECTORS (combines
plasmids & phages)
CLONING VECTORS
o Have been engineered to have a SITE OF
INSERTION of foreign DNA
EXPRESSION VECTORS
Subject: Microbiology
BATCH 2019
Have DNA sequences to facilitate their
replication in bacteria & eukaryotic cells & also
the transcription of the gene into mRNA
2. DNA SEQUENCE
o Are to be amplified & expressed
o Polymerase Chain Reaction (PCR) is a
technique where the DNA to be cloned
can be obtained by purification of
chromosomal DNA from cells, viruses,
or other plasmids or by the selective
amplification of DNA sequences
3. RESTRICTION ENZYMES
o Used to cleave DNA preproducibly at
defined sequences
o Cleaves Vector & foreign DNA
o Cleaves Multiple Cloning Site
o Recognize a specific palindromic
sequence & make a staggered cut, w/c
generates sticky ends, or blunt cut, w/c
generated blunted ends
4. DNA LIGASE
o Enzyme that links the fragment to the
cloning vector
o Ligation of the vector with the DNA
fragments generates a molecule called
recombinant DNA, w/c is capable of
replicating the inserted sequence
GENOMIC LIBRARY
It is the total # of recombinant vectors obtained when
cloning all the fragments that result from cleavage of
chromosomal DNA
REVERSE TRANSCRIPTASE
RNA-dependent DNA polymerase
A RETROVIRUS ENZYME that is used as an alternative
approach to cloning the gene for a protein to
convert the mRNA into DNA to produce a
complementary DNA (cDNA)
cDNA LIBRARY
Represents the genes that are expressed as mRNA in a
particular cell
The vaccine against HEPA B virus represents the 1st
successful use of recombinant DNA technology. The HEPA
B surface antigen is produced by the yeast:
SACCHAROMYCES CEREVISIAE
Topic: Microbial Genetics
(VANCOMYCIN RESISTANT S. AUREUS)
VANCOMYCIN
LAST RESORT DRUG FOR S. AUREUS strains resistant
to B-lactam
S. Aureus acquired vancomycin resistance gene,
during mixed infection w/ Enterococcus Faecalis
The gene for vancomycin resistance was contained
w/in a transposon (TN1546) on a multi-resistance
conjugative plasmid
This plasmid transferred by CONJUGATION (between
E.F. & S.A)
After lysis of E. Faecalis S. Aureus acquired the DNA
by TRANSDUCTION and became TRANSFORMED by the
new DNA
Transposon jumped from E. Faecalis plasmid,
RECOMBINED, & INTEGRATED into the S. Aureus multiresistance plasmid E. Faecalis DNA was degraded
The new S. Aureus can be transferred to other S.
Aureus strains by CONJUGATION. And now resistant
to:
o B-lactams
o Vancomycin
o Trimethoprim
o Gentamycin/ Kanamycin/ Tobramycin
o Quarternary ammonium disinfectants
REPAIR MECHANISMS OF DNA
1. DIRECT DNA REPAIR
ENZYMATIC REMOVAL OF DAMAGE
Such as : PYRIMIDINE DIMERS & ALKYLATED
BASES
2. EXCISION REPAIR
Removal of a DNA segment containing the
damage
Followed by, synthesis of new DNA strand
2 TYPES: Generalized & Specialized
3. RECOMBINATION / POSTREPLICATION REPAIR
Retrieval of missing information when both
DNA strands are damaged
4. SOS RESPONSE
Induction of many genes after DNA damage or
interruption of DNA replication
Post replication DNA repair system that allows
DNA replication to bypass lesions or error in
the DNA
5. ERROR-PRONE REPAIR
Last resort of a bacterial cell before it dies
Used to fill in gaps w/ a random sequence
when a DNA template is not available for
direct accurate repair
MULTIPLE GENETIC MANIPULATIONS
Reference: Murray 7th Ed & Jawetz 26th Ed
Transcribed by: Acosta
