Bacteria

Structure:
Internal structure:
A. Chromosome:
- Double stranded
- Circular DNA molecule
- Associated with proteins (not histones)
- DNA + proteins = loop domains supercoiling (high
condensed to fit only part of cell)
- Within region: nucleoid
- May have plasmids
B. Nucleoid:
- Region where chromosomal DNA confined to
- No membrane, visibly distinct
C. Ribosomes:
- 70S
- Give cytoplasm granular look
D. Storage granules:
- Nutrients and chemical reserves (glycogen, lipids, ions)
stored in form of granules
E. Plasmids:
- Small circular autonomously replicating extra chromosomal
DNA molecule
- Confer non-essential genes not required for cell
metabolism, genes that may confer advantages on bacteria
living in stressful environments e.g. antibiotic resistance
genes
- Fewer genes
- Multiple copies in a cell
Surface structure:
A. Cell membrane:
- Phospholipid bilayer
- Normal role +
- Electron transport chains and enzyme ATP synthase
embedded produce ATP during photosynthesis and
respiration
B. Cell Wall:
- Peptidoglycan (not cellulose): long chains of sugars, crosslinked by short peptide chains
- Protects cell from osmotic lysis
- May be classified as gram-positive or gram-negative
C. Capsule:
- Layer of polysaccharides glycocalyx (sugar coat) just
beyond the cell wall
o Organized: capsule, diffused mass: slime layer

May contain proteins

Capsule protects bacteria from being taken in via
phagocytosis by white blood cells which are unable to
recognize bacteria due to capsule
Enables bacteria to adhere to particular surface e.g.
mucous membrane
Contains water to prevent desiccation

Appendages:
A. Fimbriae:
- Short bristle like fibres, evenly distributed over entire cell
surface
- For attachment to surfaces or other bacteria
B. Pili:
- Longer and few in numbers
- Involved in motility and DNA transfer
o Motility: pilus makes contact with surface and
retracts to pull bacteria forward in jerky motion
o DNA transfer: sex pilus allows two bacterial cells to
be drawn close to each other such that mating bridge
can be forme
C. Flagella:
- Long appendages for motility
- Hollow cylindrical protein thread that propels bacterium by
rotation (propulsion, swimming propulsion)
- 1, more than 1, all over cell, one pole, opposite poles

Binary Fission: Replication

1. Begins at Ori, made up for specific seq of nucleotide bases
2. Double helix separates to form replication bubble made up of
two single DNA strands. Replication takes place outward from
the origin in both directions with 2 replication forks. Each fork:
leading strand and lagging strand being synthesized
3. As chromosome replicates, 2 newly formed ori move to
opposite sides of cell and attach to plasma membrane
4. Cell elongates to prepare for division
5. As DNA is circular with no free ends, topoisomerase is needed
to cut, separate and reseal the interlocking structure made up
of the 2 daughter DNA molecules
6. When daughter DNA molecules are separated, bacterium will
have reached twice it initial size.
7. Invagination of the plasma membrane and deposition of new
cell wall (division septum) eventually divide the parent cell
into two daughter cells with each inheriting a genetically
identical genome
Asexual means of producing genetically identical offspring

 Selective advantage in stable favourable environment, allows

successful genotypes to rapidly colonise + (fewer resources
needed)
Difference from mitosis:
- No spindle fibres, no specific position of chromosomes
- Formation of entangled rings
- Attachment of chromosome to plasma membrane

Methods by which New DNA is introduced into bacteria

- Rapidly changing environment, genetic variation through
combination of new alleles is crucial for enhancing
reproductive success ( individuals selected for and survive
to reproduce)

A. Transformation

- Uptake of naked foreign DNA from surround environment

o May be from dead lysed neighbouring cells in
-

medium
Competent cells: possess cell-surface proteins that can
bind to and transport DNA into cells
Those that lack these proteins: can be made artificially
competent immerse in culture medium with high
concentrations of CaCl2 + heat shock (used in genetic
engineering) (Cl: permeable and causes influx of water,
priming it for heat shock= transient pores. Ca: cation binds
to negatively charged DNA to neutralize it and prevent
repulsion by negatively charged membrane)
Foreign DNA can be incorporated into chromosome through
crossing over at 2 homologous regions = homologous
recombination
If foreign DNA contains different allele that is now
expressed in cell, it has transformed and is a recombinant
cell( DNA double stranded and as either strand is
repaired, it could remove the original strand or the new
allele, need for plating)
change in phenotype as new allele expressed

B. Transduction
- phages randomly carry bacterial genes from one host cell
to another

B.1 Generalized Transduction

(any part of DNA lytic cycle)

- when phage undergoes lytic cycle, phage enzymes
hydrolyze bacterial chromosome

- low fidelity: during assembly of phage genome in capsid,

-

small piece of host cells degraded DNA is randomly

packaged instead of phages own genetic material
following lysis, defective phage released and can infect
another bacterium and inject piece of bacterial DNA from
first cell
(viral genes replaced by bacterial genes, no new phages
can be synthesized in bacterial cell)
The foreign bacterial DNA can subsequently replace the
homologous region of the recipient cells chromosome if
crossing over and homologous recombination take place
becomes recombinant if new alleles integrated and
expressed
(can also be 1. Absorbed and used for spare parts 2.
Plasmid, recircularize)

B.2 Specialized Transduction

(parts adjacent to prophage lysogenic cycle)
- Carried out by temperate phages
- Phage attaches to host cell and injects viral genome into
-

host
Phage DNA integrates into host chromosome forming
prophage and phage enters lysogenic pathway. As cell
divides, a large population of bacteria carry the virus in
prophage form
Upon induction, the viral DNA may be improperly excised
such that bacterial DNA adjacent to the prophage is
excised as well.
Phage-host hybrid DNA is replicated which become new
viral progeny. All viral progeny become transducing
particles (capable of infecting new bacteria)
New alleles can be incorporated into bacterial genome by
crossing over at homologous regions or integration of the
bacterial-phage hybrid as defective phage enters lysogenic
cycle (results in double copy of bacterial genes)

C. Conjugation:
- Direct transfer of genetic material from one bacterial cell to
another through temporary link between 2 cells

- F+ bacteria which possess a F plasmid produces sex pili

which make contact with the F- cell and retract, drawing

cells together allowing the formation of a cytoplasmic a
mating bridge
One strand of F plasmid of F+ cell nicked by nuclease and
transferred from F+ cell to the F- cell through mating
bridge (via rolling circle mechanism) as the other DNA
strand used as a template for elongation
o Rolling circle mechanism:
3 end of nick extended by DNA polymerase for
synthesis of new complementary strand using
intact strand as template
Newly synthesized strand displaces the nicked
strand which is transferred concurrently via the
5 end across mating bridge into recipient cell
(like lagging strand, in fragments)
Upon completion of a unit length of plasmid
DNA another nick occurs to release the original
strand and end the replication
The single strand F plasmid re-circularizes and serves as a
template to synthesize a complementary strand for a
double stranded F plasmid DNA resulting in F+ cell.
Can be between same of different species, success rate
decreases with decreasing relatedness

Gene Regulation:
- All somatic cells carry identical gene but
o cells in multicellular organism show wide variation in
structure and function
o Rate at which certain protein molecules synthesized
different according to circumstances and demand:
constitutively expressed and regulated

Lac operon in E. coli:

-

In intestines
Not normally exposed to milk sugar lactose, except from
mothers milk
Regulate expression of genes for lactose-metabolizing
enzymes to prevent wastage of energy and materials
Benefits of gene regulation:
- Economical use of energy and resources
- Responsiveness to environment

Operon: cluster of genes with related functions that are turned on

and off together
Promoter
Operator: site on DNA at which repressor proteins binds to
prevent transcription from initiating by preventing RNA
polymerase from binding at the adjacent/overlapping
promoter
One or more structural genes
Regulatory genes (not part of operon): nucleotide sequence
that codes for a protein involved in control of expression of
structural genes e.g. repressor proteins, CAP (activator
protein)
Produces single polycistronic mRNA: base sequence coding or
amino acids sequence of several proteins
Usually in prokaryotes and simple eukaryotes
Structural genes: codes for a protein that forms part of a structure
or has enzymatic function
Lac operon:
- Structural genes
o lacZ: B-galactosidase: hydrolyses lactose to glucose
and galactose (for food) + converts lactose to
allolactose
o lacY: permease: membrane transport protein that
enables cells to take up lactose efficiently
o lacA: transacetylase (metabolises certain
disaccharides)
- Promoter upstream: for RNA polymerase to bind and
initiate transcription
- Operator: between promoter and structural genes
(overlaps), controls transcription of structural genes.
Regulatory gene lacI (not part of operon):
- Short distance upstream from operon
- Has own promoter and terminator sequences
- Produces repressor protein
- (constitutively expressed)

How it operates:
Default mode:
- Regulatory gene lacI constitutively transcribed, continued
-

production of small amounts of lac repressor protein

Repressor protein produced in active form and binds
specifically to the lac operator via its DNA-binding site

In absence of lactose, repressor nearly always occupies

operator site, denying RNA polymerase access to the
promoter
Lac operon switched off transcription blocked
However, binding of repressor to operator mediated by
weak interactions: repressor sometimes dissociates from
operator basal level of operon products: B-galactosidase,
permease and transacetylase

Lactose present:
Inducer/effector molecule = lactose/allolactose
- A few molecules of lactose will enter the cell with help of
-

basal level of permease and converted to allolactose by Bgalactosidase

Binding of allolactose to allosteric site on repressor protein
alters its tertiary structure such that its DNA-binding site no
longer recognizes and cannot bind to the operator
When all the repressor molecules have allolactose bound to
them they are inactivated and RNA polymerase is free to
bind to the promoter
Structural genes are transcribed as single polycistronic
mRNA
All 3 enzymes translated from single mRNA molecule
(translated separately but expressed in unison)

Lactose and glucose present:

-

As there is considerable energy expenditure required to

synthesize additional lactose-metabolizing enzymes such
as B-galactosidase, it makes sens for E coli cells to utilize
all supplies of glucose first before metabolizing lactose

Lac operon promoter has low affinity for RNA polymerase,

even when protein inactivated by allolactose (unable to
fully activate lac operon)
Second regulatory mechanism sensitive to presence of
glucose involved
(low cAMP levels, does not activate activator protein)

Lactose but absence of glucose:

-

Involves activator protein catabolite activator protein CAP:

has allosteric and DNA-binding site like repressor proteins
Binds to activator site within promoter
Allosteric site specific for cyclic AMP (cAMP)
When cells become depleted of glucose, cAMP levels
increase (glucose , cAMP)

cAMP binds to allosteric site of CAP and resulting active

complex then binds to CAP-binding site within promoter
binding of CAP strengthens affinity of promoter region for
RNA polymerase the rate of transcriptional initiation
accelerates (frequency of transcription not elongation rate)

dual control: for lactose metabolizing enzymes to be produced

in appreciable quantity : lactose present + glucose short
supply
o negative regulation by lac repressor: whether
transcribed or not
o positive regulation by CAP : rate of transcription
(ensures other alternative carbon sources used first)

trp operon::
effector molecule/co-repressor(works with repressor
protein): tryptophan
-

tryptophan: repressible operon

o normally on by default, turned off by effector
molecule
o involved in synthesis of amino acid tryptophan which
is essential for protein synthesis ,usually on unless
mammal consumes protein rich diet, turned off in
presence of tryptophan (effector molecule)
repression: transcription level, inhibition enzymatic level
tryptophan repressor synthesized in inactive form with little
affinity for trp operator
as tryptophan accumulates, it binds to the trp repressor at
its allosteric site
the repressor protein changes to its active form that binds
to the trp operator at its DNA-binding site
RNA polymerase cannot bind to the promoter
Operon turned off: transcription cannot proceed

Inducible Operon
Usually off but can be
induced/stimulated
and turned on
Catabolic pathways: break
down of molecules:
induced when molecule
present

Repressible Operon
Usually on but can
be repressed and
turned off
Anabolic pathways:
synthesis of amino acids,
nucleotides etc. from
simpler materials
(construct molecules) :
repressed when enough of

Negative Regulation:
Regulatory repressor
protein normally in active
form bound to operator
inducer molecule present,
binds to repressor and
changes conformation
(inactivate), unable to bind
to operator = expression of
operon
Allolactose repressor
protein
Positive Regulation:
Activator proteins normally
in inactive form, unable to
bind to DNA inducer
present, binds to activator
protein and changes
conformation (activate),
binds to ( region within
promoter) = activate
transcription
Glucose = cAMP CAP
LAC OPERON (dual control)

the end product available

(can be close to
constitutively expressed
e.g. RNA polymerase)
Negative Regulation:
Regulatory repressor
proteins normally in
inactive form, unable to
bind to DNA corepressors (molecule)
binds to repressor
proteins and changes
conformation (activate),
binds to operator =
prevent transcription
Tryptophan repressor
proteins
Positive Regulation:
Activator proteins
normally bound to
pertinent DNA segment
inhibitor (molecule) binds
to activator protein and
changes conformation
(inactivate), unable to
bind to DNA = prevents
transcription
TRP OPERON

Transcripti
on or not
on/off

Rate of
transcripti
on