OCPG Flashcards
Gram-positive vs Gram-negative bacteria
- distinguish using gram stain
fyi
(i) Gram-positive bacteria
Have cell walls with a relatively large amount of peptidoglycan
(ii) Gram negative bacteria
Have less peptidoglycan in their cell walls
Have an outer membrane that contains lipopolysaccaharide.
movement of bacterial cells
Some prokaryotes are motile, able to move toward nutrients and away from toxic substances.
Some prokaryotes swim by means of flagella; other twitch or glide by the use of thread-like
pili, or adjust their buoyancy in water by means of intracellular vesicles.
Organisation of prokaryotic genome
Most bacterial species contain a large, circular chromosome.
1. The chromosome inside a bacterial cell is highly compacted and found within the nucleoid
and is not bounded by a membrane
2. A typical chromosome is a double-stranded DNA (a few million base pairs in length) that
is associated with DNA binding proteins (non-histone scaffolding proteins).
3. Several thousand different genes are interspersed throughout the chromosome.
Structural genes (i.e. sequences that encode proteins) account for majority of bacterial
genome.
- Each chromosome possesses only one origin of replication. The origin of replication is a
sequence that functions as an initiation site for the assembly of several proteins that
are required for DNA replication - In addition to the chromosome, bacteria also have several plasmids, which are small,
circular pieces of DNA that exist independently of the bacterial chromosome.
Plasmids are self-replicating, i.e. their replication is independent of the bacterial
chromosome, as it contains its own origin of replication.
- Plasmids are not necessary for survival of bacteria. However, in many cases,
certain genes within a plasmid confer advantages to bacteria’s survival in stressful
environments
- Fertility plasmids (F plasmids), also known as F factors, allow bacteria to mate with each other. Through the mating process, F plasmid facilitates genetic recombination, which may be advantageous in a changing environment that no
longer favours existing strains in a bacterial population.
- Resistance plasmids, also known as R factors, contain genes that confer
resistance against antibiotics and other types of toxins.chromosome.
Structure of bacterial genome
To fit within the bacterial cell, the chromosomal DNA must be compacted several folds. Part of
this compaction process involves the formation of loop domains. A loop domain is a
segment of chromosomal DNA that is folded into a structure that resembles a loop. DNA
binding proteins anchor the base of these loops.
Supercoiling leads to further compaction of the looped bacterial chromosome.
The chromosome in living bacteria is negatively supercoiled. The force of negative
supercoiling may promote DNA strand separation in small regions, enhancing genetic
activities such as replication and transcription.
DNA gyrase and topoisomerase I control the degree of supercoiling in bacterial
chromosome.
Binary fission
Bacterial cells divide by binary fission. Binary fission is a form of asexual reproduction. Repeated cellular
divisions of the bacterial cell may form a clone of genetically identical cells called a bacterial
colony.
Before the bacterial cell divides, semi-conservative replication of parental DNA begins at
the origin of replication to give rise to two origins.
As the chromosome continues to replicate, each origin moves rapidly toward the
opposite end of the cell and adhere to the cell surface membrane.
Both DNA strands are the template to synthesize the complementary strand
While the chromosome is replicating, the cell elongates. Elongation of the cell also
separates the two identical copies of the chromosomes.
When replication is complete and the cell has reached about twice its initial size, its cell
surface membrane invaginates, and deposits new cell wall materials. Two daughter
cells are formed which are genetically identical to the parent cell. Each cell inherits a
parental strand of DNA.
Genetic variation in prokaryotes
strain - lineage that has genetic differences compared to another strain
First, spontaneous mutations can occur that alter the bacterial genome and affect the traits of bacterial cells.
Second, diversity can arise by genetic transfer / horizontal gene transfer, in which genetic material is transferred from one bacterial cell to another. Genetic transfer can occur in three very different ways: transformation, transduction and conjugation. The transfer of genetic material gives rise to genetic variation within a bacteria population.
Transformation
- Transformation is the uptake of naked, foreign DNA from the surrounding environment.
- Many bacteria possess cell surface proteins which recognises and transport DNA from closely related species into the cell.
- This foreign DNA can be incorporated into the genome, either by integration or homologous recombination. the cell is now a recombinant
Transduction
- occurs when a phage infects a bacterial cell (donor) and then transfers some of the bacterial DNA to another bacterial cell (recipient)
- If some of this DNA is then incorporated into the recipient cell’s chromosome by homologous recombination, a recombinant cell is formed.
generalised and specialised transduction
generalised transduction
- Generalized transduction results from an error in a phage lytic cycle.
- Bacterial genes are randomly transferred from one bacterial cell to another.
- During the synthesis of phage DNA and proteins, the bacterial chromosome is degraded into small pieces.
- During generalized transduction, a random small piece of the host cell’s degraded DNA could be accidentally packaged within a phage capsid in place of the phage genome during assembly.
- Some of this DNA can subsequently replace the homologous region of the recipient cell’s chromosome, if crossing-over takes place, resulting in genetic recombination.
Specialised transduction
- Specialized transduction results from an error in a phage lysogenic cycle.
- Only bacterial genes adjacent to the prophage site are efficiently transferred to another bacterium. Prophage refers to the phage DNA that is inserted as part of the bacterial genome.
- -During induction, the prophage is excised and phage enters lytic cycle.
- During specialized transduction, the prophage is incorrectly excised from bacterial chromosome and the phage DNA incorporated some bacterial genes adjacent to the prophage.
- As the phages are released from its host cell (via the lytic cycle), this mistake creates a phage carrying bacterial chromosomal DNA.
- After its release from the lysed host, the phage can attach to another bacterium (the recipient) and inject the piece of phage DNA carrying bacterial genes from the first cell (the donor) into the recipient bacterium
- Some of this DNA can replace the homologous region of the recipient cell’s DNA by homologous recombination / the phage DNA and bacterial genes are integrated into the recipient cell’s DNA.
comparison between generalised and specialised transduction
g vs s
- involves virulent bacteriophages (T4 phage) vs involves temperate bacteriophages (lambda phage)
- host cell’s DNA is destroyed vs host cell’s DNA not hydrolysed during lysogenic cycle but Host cell’s DNA is eventually hydrolysed during lytic cycle
- viral DNA is not integrated into the bacterial chromosome vs viral DNA is integrated into the bacterial chromosome to form a prophage
- occurs during the lytic cycle vs occurs during the lysogenic cycle for integration of viral DNA and when under environmental stress, switches to lytic cycle
- bacterial DNA is randomly packaged within a capsid vs prophage with adjacent bacterial genes are packaged into a capsid
conjugation
Conjugation involves a direct physical interaction between two bacterial cells and the transfer of genetic material from a donor bacterium to a recipient bacterium.
The DNA transfer is one-way, i.e. one cell donates DNA, and the other cell receives the DNA.
The donor uses appendages called sex pili (singular: pilus) to attach to the recipient.
After contacting a recipient cell, a sex pilus retracts, drawing the donor and
recipient cells closer together.
A temporary cytoplasmic mating bridge then forms between the two cells, providing
an avenue for DNA transfer.
In most cases, the ability to form sex pili and donate DNA during conjugation is due to
the presence of an F factor (F = fertility).
F factor can exist either as a segment of DNA within the bacterial chromosome
(refer to extra information on Hfr cell on page 12) or as a plasmid.
The F plasmid consists of several genes that are required for the production of
sex pili and may carry genes that confer a growth advantage for the bacterium.
Bacterial cells containing the F plasmid are F+ cells and function as DNA donors during conjugation.
Bacterial cells lacking the F factor are designated F- cells. These cells function as DNA recipients during conjugation as the F+ condition is transferable (i.e. F+ cell converts an F- cell to F+ during conjugation).
Successful contact between a donor and a recipient cell stimulates the donor cell to begin the transfer process.
Genes within the F factor encode proteins that promote the transfer of one strand of F factor DNA.
This DNA strand is cut at the origin of transfer, and then the strand travels through the cytoplasmic mating bridge into the recipient cell.
The other strand remains in the donor cell and is replicated, restoring the F factor DNA to its original double-stranded condition.
In the recipient cell, the two ends of the newly acquired F factor DNA strand are joined to form a circular molecule, which is then replicated to become double- stranded.
Each parental strand acts as a template for synthesis of the second strand in its respective cell.
The end result of conjugation is that the recipient cell has acquired an F factor, converting it from an F- to an F+ cell. The genetic composition of the donor strain has not changed.
control of prokaryotic gene expression
- transcriptional control (mainly this)
- translational control
Defn. Transcriptional control
In prokaryotes, the cluster of structural genes that encode enzymes of the same metabolic pathway under the transcriptional control of a single promoter and operator, in a region on the chromosome, is called an operon.
operon
operon:
- single promoter
- operator
- structural genes (lac Z, lac Y, lac A)
regulatory gene not part of operon
Operons are categorised as either inducible or repressible:
The lac operon is an inducible operon.
its transcription is usually off.
transcription can be turned on (induced) by the presence of a small effector molecule, i.e. lactose.
The structural genes in this operon code for inducible enzymes.
The trp operon is considered to be a repressible operon.
its transcription is usually on.
transcription is turned off (repressed) by the presence of a small effector molecule i.e. tryptophan.
The structural genes in this operon code for repressible enzymes.
Advantages of operons
In operons, genes that function together or have similar functions are regulated together. As prokaryotes are simple unicellular organisms, organizing their genome into operons can allow them to respond to changes in the environment (eg. composition of growth medium) more quickly.
Operons ensure that the cell does not waste energy synthesizing unneeded enzymes or other proteins. Eg. The lac operon is transcribed only when the substance to be broken down (i.e. lactose) is present.
Eg. The trp operon is transcribed only when the substance required by the cell (i.e. tryptophan) is absent.
lac operon
Lactose metabolism begins with its hydrolysis into monosaccharides (glucose and galactose), a reaction catalysed by β-galactosidase. By producing the appropriate enzymes only when the nutrient is available, the cell avoids wasting energy and resources making proteins that are not needed.
The regulatory sequences found in the lac operon are:
Promoter
is found upstream of structural genes.
is the site where RNA polymerase binds to DNA prior to transcription of the structural genes.
Terminator
is found downstream of structural genes.
is the site which signals the end of transcription.
CAP site
is a DNA sequence recognised by an activator protein.
Operator,
is situated between the promoter and structural genes.
is a binding site for the repressor.
The structural genes in an operon code for enzymes and lie adjacent to one another. When the RNA polymerase moves from one structural gene to the next, the genes are transcribed into a single long mRNA.
The mRNA that is transcribed is described as a polycistronic mRNA as it contains the coding sequences of two or more structural genes. Introns are absent in the mRNA. This extended mRNA is then translated into the various enzymes of a particular metabolic pathway.
This is possible because the mRNA is punctuated with start and stop codons that signal where the coding sequence for each polypeptide begins and ends.
The lac operon contains three structural genes:
lacZ gene
codes for β-galactosidase
β-galactosidase hydrolyses lactose into glucose and galactose.
A side reaction of this enzyme is to convert a small percentage of lactose into allolactose, a structurally similar lactose analogue;
lacY gene,
codes for lactose permease,
lactose permease is a membrane protein required for transport of lactose into the cell;
lacA gene,
codes for galactoside transacetylase
galactoside transacetylase’s physiological role is unclear, although it has been suggested that it prevents the toxic build up of non-metabolizable lactose analogues in the cytoplasm.
regulatory gene (lacI gene)
Not part of the lac operon, a regulatory gene codes for a specific protein product that regulates the expression of the structural genes.
A regulatory gene, lacI gene, lies adjacent to the lac operon.
codes for the lac repressor.
lac repressor is important for the regulation of the lac operon.
lacI gene is constitutively expressed at fairly low levels.
lacI gene has its own promoter called the i promoter. It is considered to be a regulatory gene because the sole function of the repressor protein is to regulate the expression of structural genes.
The lacI gene is not considered a part of the lac operon.
The lac repressor is synthesised in an active form and binds to the operator.
When the lac repressor binds to the operator, the promoter is blocked from the RNA polymerase, and transcription of the structural genes is prevented.
However, the operon is not permanently switched off as the binding of the repressors to operators is reversible.
The ability of the repressor to bind the operator and inhibit transcription depends on the protein’s conformation, which is allosterically regulated by an inducer. Thus, the concentration the inducer determines the activity of the operon.
lac operon control thing
The lac operon is an inducible operon. Its transcription is usually switched off but can be turned on when a specific small effector molecule (lactose/allolactose) binds allosterically and inactivates the repressor.
In the absence of lactose or when lactose concentration is low:
No allolactose binds to the lac repressor.
lac repressor binds to the operator site and blocks RNA polymerase from transcribing the structural genes, thus inhibits transcription.
In the presence of lactose:
A small amount of lactose is transported into the cytoplasm via lactose permease.
β-galactosidase will convert the lactose to allolactose.
The cytoplasmic level of allolactose will gradually rise until allolactose binds to the repressor, which has four identical subunits, each one recognising a single allolactose molecule.
This results in a conformational change of the repressor, preventing it from binding to the operator site.
vRNA polymerase is then able to transcribe the structural genes, synthesising enzymes that catabolise lactose molecules.
The lac operon is under dual control:
Negative control by lac repressor and
Positive control by catabolite activator protein (CAP), also known as the cAMP
receptor protein (CRP).
positive, negative control
Catabolite repression is a form of transcriptional regulation influenced by the presence of glucose (which is a catabolite).
The ability of glucose to repress the lac operon depends on a small effector molecule, cAMP, that is converted from ATP via adenylate cyclase.
cAMP accumulates when the intracellular concentration of glucose is low. When cAMP accumulates, it binds to CAP.
This activates CAP and causes it to bind to the CAP site.
Because CAP is an activator, it enhances the rate of transcription of the structural genes in the operon and more enzymes are synthesized for lactose metabolism.
Thus, two factors regulate the synthesis of enzymes of this pathway:
The state of the lac repressor determines whether transcription of the
structural genes occurs or not.
The state of CAP determines the rate of transcription of the structural
genes, only when the operator is not bound by a repressor.
trp operon
The trp operon is a repressible operon because its transcription is usually turned on but can be inhibited (repressed) when a specific small molecule (tryptophan) binds allosterically to a regulatory protein.
A regulatory gene, trpR gene, located some distance away from the operon.
codes for the trp repressor.
trp repressor is important for the regulation of the trp operon.
trpR gene has its own promoter. It is considered to be a regulatory gene because the sole function of the repressor protein is to regulate the expression of structural genes.
The trp repressor is synthesised in an inactive form with little affinity for the trp operator.
Only if tryptophan binds to the trp repressor does the repressor protein change to the active form that can bind to the operator, inhibiting transcription of the structural genes.
Tryptophan functions as a co-repressor (as opposed to the inducer in lac operon) that cooperates with a repressor protein to switch an operon off. As more tryptophan accumulates, more tryptophan molecules can then bind to the trp repressor, which can then bind to the trp operator and inhibit the synthesis enzymes involved in the tryptophan biosynthetic pathway.
translational control
(i) Translational repressors
A translational repressor recognises sequences within the mRNA, acting to inhibit translation. These proteins bind to the mRNA to inhibit translation by:
Binding near the ribosome-binding site and / or start codon and strategically block the ribosome from initiating translation.
Binding to the secondary mRNA structures, thereby stabilising these secondary structures, thus preventing initiation of translation by ribosomes.
(ii) Synthesis of antisense RNA
Double-stranded RNA can form if a second strand of RNA whose sequence of bases is complementary to the first strand is available. An antisense RNA is an RNA strand that is complementary to a strand of mRNA. It can be synthesised from the non- template (antisense) strand of the double-stranded DNA. When mRNA forms a
duplex with a complementary antisense RNA sequence, translation is blocked.
