ntroduction to Microbial

Genetics
Microbial Genetics
concerned with the transmission of hereditary
characters in microorganisms
deals with the genotype and phenotype of
microbial species. t studies how genes are
organized and regulated in microbes.
t also involves the study of genetic processes
taking place in micro organisms
#ecombination
icroorganisms have the ability to acquire
genes and thereby undergo the process of
recombination.
n recombination, a new chromosome with a
genotype different from that of the parent
results from the combination of genetic
material from two organisms.
This new arrangement of genes is usually
accompanied by new chemical or physical
properties.
General #ecombination
&sually involves a reciprocal exchange
of DNA between a pair of DNA
sequences.
t occurs anywhere on the microbial
chromosome and is typified by the
exchanges occurring in bacteriaI
transformation, bacteriaI
recombination, and bacteriaI
transduction.
General #ecombination
$ite-specific #ecombination
involves the integration of a viral genome
into the bacterial chromosome.
also known as conservative site-specific
recombination
$ite-specific #ecombination
#eplicative #ecombination
which is due to the movement of genetic
elements as they switch position from
one place on the chromosome to
another.
%e Bacterial Cromosome and
Plasmid
!rokaryotes such as bacteria possess a
single chromosome composed of
double-stranded DNA in a loop.
The DNA is located in the nucleoid of the
cell and is not associated with protein.
Bacterial Cell
Plasmids
any bacteria (and some yeasts or other
fungi) also possess looped bits of DNA
known as pIasmids, which exist and
replicate independently of the chromosome.
!lasmids behave like a mini-chromosomes
!lasmids have relatively few genes (fewer
than 30).
The genetic information of the plasmid is
usually not essential to survival of the host
bacteria.
Plasmid
are circular DNA molecules present in
the cytoplasm of the Bacteria
Capable of Autonomous replication
Can transfer genes from one cell to other
!lasmid Carry genetic information
necessary for conjugation to occur.
Only cell that contain such plasmid can
act as donor. the cell lacking a
corresponding plasmid act as recipient.
Plasmid
!lasmids contain genes that impart antibiotic
resistance. &p to eight genes for resisting eight
different antibiotics may be found on a single
plasmid. Genes that encode a series of bacteriocins
are also found on plasmids. Bacteriocins are
bacterial proteins capable of destroying other
bacteria. Still other plasmids increase the
pathogenicity of their host bacteria because the
plasmid contains genes for toxin synthesis.
Plasmid
Curing
!lasmids can be removed from the host cell
in the process of curing.
Curing may occur spontaneously or may be
induced by treatments such as ultraviolet
light.
Chemical agents used for plasmid
curing were, Acridine orange,
Acriflavine, Ethidium bromide
(Intercalating dyes) and Rifampin
inds of Plasmid
Certain plasmids, called episomes, may
be integrated into the bacterial
chromosome.
onjugative pIasmids contain genes
for certain types of pili and are able to
transfer copies of themselves to other
bacteria.
inds of Plasmid
#esistance PIasmid (R factor)
Carry genes that confer resistance to
certain antibiotics
&suaIIy has two types of genes:
1. #-determinant: resistance genes that code
for enzymes that inactivate certain drugs
2. #% (#esistance %ransfer actor): genes
for plasmid replication and conjugation.
ertility { actor
A special plasmid that plays an important role in
conjugation.
contains genes that encourage cellular attachment
during conjugation and accelerate plasmid transfer
between conjugating bacterial cells.
Those cells contributing DNA are called (donor)
ceIIs or maIe, while those receiving DNA are the -
(recipient) ceIIs or femaIe.
The F factor can exist outside the bacterial
chromosome or may be integrated into the
chromosome.
fr Cell
hen the pIasmid is integrated
within the bacteriaI chromosome, the
ceII is caIIed an Hfr ceII (high
frequency of recombination cell).
%ransposable elements
also known as transposons or "jumping
genes", are segments of DNA that move
about within the chromosome and establish
new genetic sequences.
They exhibit the so called "cut and paste
mechanism
First discovered by Barbara cClintock in the
1940s, transposons behave somewhat like
lysogenic viruses except that they cannot
exist apart from the chromosome or
reproduce themselves.
Not self replicating and depend on !lasmid
or Chromosome for replication.
they assisted in the transfer of bacterial
resistance to antibiotics. Furthermore, it soon
became evident that they caused most of the
spontaneous mutations occurring in
laboratory populations of more sophisticated
organisms
%ransposable elements
%ransposons
Bacterial #ecombinations
Three types of bacterial
recombination result in
a change in the DNA of
recipient organisms.
The proteins expressed
by the new genes lead
to new physiological
characteristics in the
bacteria.
Bacterial conjugation
as first postulated in the 1940s by Joshua
Lederberg and Edward Tatum.
The essential feature of the process is that two
bacterial cells come together and mate such that a
gene transfer occurs between them.
One cell, the donor cell (F+), gives up DNA; and
another cell, the recipient cell (F~), receives the
DNA.
%he DNA is directIy transferabIe.
The transfer is nonreciprocal, and a special
pilus called the sex piIus joins the donor and
recipient during the transfer.
The DNA most often transferred is a copy of
the F factor plasmid.
The factor moves to the recipient, and when
it enters the recipient, it is copied to produce
a double-stranded DNA for integration.
The channel for transfer is usually a special
conjugation tube formed during contact
between the two cells
Bacterial conjugation
Naked DNA molecule from the
environment is taken up by the cell and
incorporated into its chromosome in
some heritable form
Defined as transfer of Genetic
information through the activity of DNA
Bacterial transformation
During transformation, a competent cell
takes up DNA and destroys one strand
of the double helix.
A single-stranded fragment then
replaces a similar but not identical
fragment in the recipient organism, and
the transformation is complete.
Bacterial transformation
Bacterial transformation
as discovered by Frederick Griffith in 1928.
riffith Experiment
Griffith worked with the pneumococci that
cause bacterial pneumonia.
e discovered that if he mixed fragments of
dead pathogenic pneumococci with
specimens of live harmless pneumococci, the
harmless bacteria took on genes of the
bacterial fragments and became pathogenic.
Griffit Experiment
Bacterial transduction
n transduction, bacteriaI viruses (also
known as bacteriophages) transfer
DNA fragments from one bacterium (the
donor) to another bacterium (the
recipient).
The viruses involved contain a strand of
DNA enclosed in an outer coat of
protein.
After a bacteriophage (or phage, in brief)
enters a bacterium, it may encourage the
bacterium to make copies of the phage.
At the conclusion of the process, the
host bacterium undergoes lysis and
releases new phages.
This cycle is called the Iytic cycIe.
Bacterial transduction
ytic Cycle
States that the virulent phages are those
phages that can multiply only on
bacterial cells. At the end of their life
cycle, they cause cell lysis that kills the
host bacteria.
ytic Cycle $teps
1. Attachment - Attachment sites on the phage adsorb
to receptor sites on the host bacterium
2. Penetration - phage injects its genome into the
bacteriaI cytopIasm
3. #epIication - %he phage repIicates its genome and
uses the bacterium's metaboIic machinery to synthesize
phage enzymes and phage structuraI components
4. AssembIy - %he phage repIicates its genome and
uses the bacterium's metaboIic machinery to synthesize
phage enzymes and phage structuraI components
5. #eIease - %he ceII breaks open and each repIicated
virus can now infect other ceIIs.
ysogenic cycle
&nder other circumstances, the virus may
attach to the bacterial chromosome and
integrate its DNA into the bacterial DNA.
t may remain here for a period of time before
detaching and continuing its replicative
process.
The virus is called a temperate phage, also
known as a prophage.
ysogenic Cycle $teps
1.) Viral genome enters cell
2) Viral genome integrates into host cell
genome
3) ost cell DNA polymerase copies viral
chromosomes
4) Cell divides, and virus chromosomes are
transmitted to cell's daughter cells
5) At any moment when the virus is
"triggered", the viral genome detaches from
the host cell's DNA and enters stage 2 of the
lytic cycle.
any part of the bacterial genome can be
transferred; occurs during the lytic cycle
of virulent and temperate bacteriophages
because the host's chromosome is
broken down into fragments.
Generalized transduction
Generalized transduction
1. A phage attaches to ceII waII of bacterium and
injects DNA.
2. %he bacteriaI chromosome is broken down and
biosynthesis of phage DNA and protein occurs.
3. %he ceII Iyses, reIeasing viruses.
4. %he phage carrying bacteriaI DNA infects
another ceII.
5. rossing over between donor and recipient
DNA can occur producing a recombinant ceII.
$pecialized %ransduction
Transfer of only specific portions of the
bacterial genome; carried out only by
temperate phages that have integrated
their DNA into the host chromosome at a
specific site in the chromosome
$pecialized %ransduction
1. #emember that in the Iysogenic cycIe, phage
DNA can exist as a prophage integrated in the
bacteriaI chromosome)
2. OccasionaIIy when the prophage exits it can
take adjacent bacteriaI genes with it.
3. %he phage DNA directs synthesis of new
phages(%he phage particIes carry phage DNA
and bacteriaI DNA.)
4. %he ceII Iyses, reIeasing the phages.
5. A phage carrying bacteriaI DNA infects another
ceII.
Mutation
A permanent alteration in the sequence of
nitrogenous bases of a DNA molecule.
The result of a mutation is generally a
change in the end-product specified by that
gene.
n some cases, a mutation can be beneficial
if a new metabolic activity arises in a
microorganism, or it can be detrimental if a
metabolic activity is lost.
Mutation
Caused by alteration in the Nucleotide
sequence at some point of DNA which
can occur due to
Addition
Deletion
Substitution
of one or more bases
%ypes of mutations
Missense Mutation
the new nucleotide alters the codon so as to
produce an altered amino acid in the protein
product. (ex. sickle cell anemia)
the new nucleotide changes a codon that specified an
amino acid to one of the $%OP codons (%AA, %A,
or %A)
onsense Mutation
is Substitution of purine for pyramidine or
vice versa in the base pairing
%ransversion
ramesift Mutation
pairs of nucleotides are either added to or
deleted from the DNA molecule, with the
result that the "reading frame is shifted.
Cemical mutagens
include nitrous acid, substance converts
adenine to hypoxanthine, a molecule that will
not pair with thymine, and thus interrupts the
genetic code.
base anaIog is a chemical mutagen that
resembles a nitrogenous base and is
incorporated by error into a DNA molecule.
Such a DNA molecule cannot function in
protein synthesis.
Certain dyes and fungal toxins (for example,
aflatoxin) are known to be mutagens.
Pysical mutagens
rays and gamma rays break the covalent bonds
in DNA molecules, thereby producing fragments.
&ItravioIet Iight binds together adjacent thymine
bases, forming dimers. These dimers cannot function
in protein synthesis, and the genetic code is thereby
interrupted.
#adiation damage can be repaired by certain
bacterial enzymes, a process known as photo
reactivation.
'#&$ '#D$ and P#$
'#&$
Viruses contain DNA or RNA
And a protein coat
Some are enclosed by an envelope
Some viruses have spikes
Cause infection and disease
Obligate intracellular parasites
Require hosts to multiply
$izes of 'irus
elical viruses
consist of nucIeic acid surrounded by a hoIIow
protein cyIinder or capsid and possessing a heIicaI
structure
Polyedral viruses
consist of nucIeic acid surrounded by a poIyhedraI
(many-sided) sheII or capsid, usuaIIy in the form of an
icosahedron
Complex 'iruses
have neither
helical nor
polyhedral forms,
are pleomorphic
(irregular
shaped), or have
complex
structures
Enveloped 'iruses
consist of
nucleic acid
surrounded by
either a helical
or polyhedral
core and
covered by an
envelope
'iral $tructure
Nucleic acid
DNA or
RNA
Single or double stranded
Linear or circular
Several separate segments
nfluenza
Few thousand to 250,000 nucleotides
Bacteria have 4 million
'iral $tructure
Capsid
!rotein coat
surrounding nucleic
acid
ost of mass of virus
Composed of
capsomeres
Arrangement
characteristic for a
particular virus
Single protein type
Several protein types
'iral $tructure
Envelope
Covers capsid in
some viruses
Combination of
Lipids
!roteins
Carbohydrates
Can be derived from
host cells plasma
membrane
Components can
determined by viral
nucleic acid
'iral $tructure
Spikes
Carbohydrate
protein complexes
!roject from envelope
Attachment
mechanism
eans of identification
emagglutination
Clumping of RBC's
'iral %axonomy
Family names end in -viridae
Genus names end in -virus
Viral species: A group of viruses sharing
the same genetic information and
ecological niche (host). Common names
are used for species
Subspecies are designated by a number
erpesviridae
erpesvirus
uman
herpes virus
1, V 2,
V 3
W Retroviridae
W Lentivirus
W uman
mmunodeficie
ncy Virus 1,
V 2
'iral %axonomy
Growing Bacteriopages
Bacteriophages
Grow in
Liquid
suspensions of
bacteria
Solid media
bacterial
cultures
!laque method
Bacteriophage
mixed with bacteria
ixed in melted
agar
!oured in !etri plate
over layer of
hardened agar
ix solidifies into
one cell thick layer
Growing Bacteriopages
Growing Animal 'iruses
Three methods used
Living animals
Embryonated eggs
Cell cultures
Growing 'iruses in iving Animals
Some viruses only grow in living animals
(ice, rats, rabbits, and guinea pigs)
Best to study immune responses
&sed for diagnostic purposes
ice inoculation for rabies
Some diseases lack adequate animal models
V-1
ice have been genetically engineered to act
as animal model
Growing 'iruses in Embryonated
Eggs
Embryonated eggs
Embryo present
ole drilled in shell
Viral suspension
injected into fluid in
egg
Viral growth
Embryo death
Embryo cell
damage
Typical lesions on
membrane
Growing 'iruses in Embryonated
Eggs
Embryonated eggs
Four types on
inoculation
Yolk sac
Allantoic
Amniotic
Chorioallantoic
Growing viruses in Cell Cultures
Consist of homogenous cells grown in culture media
ore convenient than living animals or embryonated
eggs
Cell cultures can be propagated like bacterial
cultures
Growing 'iruses in Cell Cultures
Virus infects cells
Cells in monolayer deteriorate as virus multiplies
C!E cytopathic effect or cytopathogenic effect
Can be detected and counted similar to
plaques
'irus dentification
ethods of identification
Serological
estern blot method
ost commonly used
s detected and identified by reactions to
antibodies
Cytological changes
olecular methods
Restriction fragment length polymorphisms
(RFL!s)
!olymerase chain reactions (!CR)
Multiplication of Bacteriopages
{ytic Cycle
Two types of multiplication
Lytic cycle
Ends with death and lysis of cell
T-even bacteriophage
Lysogenic cycle
ost cell remains alive
Bacteriophage /
ytic Cycle
States that the virulent phages are those
phages that can multiply only on bacterial
cells. At the end of their life cycle, they cause
cell lysis that kills the host bacteria.
ytic Cycle $teps
1. Attachment - Attachment sites on the phage adsorb to
receptor sites on the host bacterium
2. Penetration - phage injects its genome into the bacteriaI
cytopIasm
3. #epIication - %he phage repIicates its genome and uses
the bacterium's metaboIic machinery to synthesize phage
enzymes and phage structuraI components
4. AssembIy - %he phage repIicates its genome and uses the
bacterium's metaboIic machinery to synthesize phage enzymes
and phage structuraI components
5. #eIease - %he ceII breaks open and each repIicated virus
can now infect other ceIIs.
ysogenic cycle
&nder other circumstances, the virus may
attach to the bacterial chromosome and integrate
its DNA into the bacterial DNA.
t may remain here for a period of time before
detaching and continuing its replicative process.
The virus is called a temperate phage, also
known as a prophage.
ysogenic Cycle $teps
1.) Viral genome enters cell
2) Viral genome integrates into host cell genome
3) ost cell DNA polymerase copies viral
chromosomes
4) Cell divides, and virus chromosomes are
transmitted to cell's daughter cells
5) At any moment when the virus is "triggered", the
viral genome detaches from the host cell's DNA
and enters stage 2 of the lytic cycle.
ne - $tep Growt Experiment
Burst time
Time elapse from attachment to release
20-40 minutes
Burst size
Number synthesized particle released
50-200
Multiplication of Animal viruses
Attachment Viruses attaches to cell membrane
Penetration By endocytosis or fusion
&ncoating By viral or host enzymes
iosynthesis !roduction of nucleic acid and
proteins
aturation Nucleic acid and capsid proteins
assemble
#eIease By budding (enveloped viruses) or
rupture
Prions
!rions are "infectious proteins
They are normal body proteins that get converted into
an alternate configuration by contact with other prion
proteins
They have no DNA or RNA
The main protein involved in human and mammalian
prion diseases is called "!r!
Prion Diseases
scrapie a fatal disease of sheep and
goats
ad cow disease- incurable, fatal brain disease that
affects cattle and possibly some other animals, such
as goats and sheep. The medical name for mad cow
disease is bovine spongiform encephalopathy or
BSE for short. t's called mad cow disease because it
affects a cow's nervous system, causing a cow to act
strangely and lose control of its ability to do normal
things, such as walk.
Prion Diseases
reutzfeIdt-Jacob disease is a rare, degenerative
brain disorder. Symptoms usually start around age
60. emory problems, behavior changes, vision
problems and poor muscle coordination progress
quickly to dementia, coma and death. ost patients
die within a year.
Prion Diseases
uru is among the fatal neurodegenerative prion
protein (!r!) diseases in humans t is now widely
accepted that Kuru was transmitted among members
of the Fore tribe of !apua New Guinea via
cannibalism.
he term "kuru" derives from the Fore word
"kuria/guria", 'to shake', a reference to the body
tremors that are a classic symptom of the disease; it
is also known among the Fore as the laughing
sickness due to the pathologic bursts of laughter
people would display when afflicted with the disease
Prion Diseases
'iroids
SmoII, circuIor PMA
moIecuIes wifhouf o
profein coof
Infecf pIonfs
Pofofo fomine in
IreIond