Prokaryote genes/growth

What are the regions along DNA strand that can regulate genes

Promoter region - where RNA polymerase binds, so other binding can control gene expression

Activator region - can activate the gene

What groups have operons and what are they

Bacteria and archaea

Several genes clumped together controlled by one promoter region. They have separate RBS (ribosome binding sites) so are translated separately

What are regulators

Proteins that interact w/ nuclei acids (DNA, RNA etc.)

Usually are dimers (made of 2 protein monomers) that bind to DNA, binding to inverted repeat sequences on diff. strands so structure is attached to both.

Each monomer usually has a recognition helix (which interacts with the DNA) and a stabilising helix

One type are transcription factors

Types of transcription factors

Activator protein - turn transcription on

Repressor protein - turn transcription off

What are transcription factors (activators and repressors) controlled by and how

Controlled by effectors - can bind and cause conformational change, rendering them able/unable to bind to DNA

How is negative regulation carried out in prokaryotes, difference between anabolic vs. catabolic genes

Repressor transcription factor binds to the operator region (downstream of promoter) and blocks RNA polymerase binding

For anabolic genes - the end-product acts as a co-repressor and activates the repressor to block transcription

For catabolic genes - the starting substrate inactivates the repressor protein, so transcription begins and genes expressed

Example of negative regulation in bacteria

Lac operon - repressor protein ‘LaccI repressor’ usually binds to operator and prevents transcription. When lactose is present, forms allolactose which acts as an inducer, binding and changing shape of repressor, preventing it binding to the operator, allowing RNA polymerase to bind and transcription to occur.

How is positive regulation carried out in prokaryotes

Activator protein binds to activator site to allow recruitment of RNA polymerase

Activator binding is itself caused by the binding of an inducer

Activator site is upstream of the promoter region but can be close/distant as the DNA strand is able to bend

Example of positive regulation in bacteria

Mal operon - maltose acts as the inducer. Binds to maltose activator protein which then binds to the activator site and recruits RNA polymerase.

What is a regulon

Multiple dispersed genes controlled by the same regulator protein (activator/repressor) - can contain multiple operons

Interactions between lac operon and cAMP - regulation on both a specific and global level

1. Cells growing on glucose have exponential growth until glucose used up

2. Glucose inhibits adenylate cyclase and therefore cAMP production, so when glucose is used up lots of cAMP is made

3. cAMP activates lacZYA operator

4. If lactose → allolactose is also present and acts as an inducer to cause repressor to fall off, then the lac operon is expressed

5. Lactose can be used for second period of cell growth

How do archaeal repressor and activator proteins work

Repressor proteins block binding of TBP (TAT binding protein) and TFB (transcription factor B) which RNA polymerase needs to bind, or block RNA polymerase directly

Activator proteins recruit the proteins like TBP and TFB

Example of negative gene regulation in archaea

Nitrogen metabolism genes - NrpR is the repressor and blocks binding of TFB and TBP to two sites: BRE and TATA box. Alpha-ketoglutarate acts as the inducer, and binds to NrpR to release it from DNA and allow TFB and TBP to bind, letting genes for nitrogen metabolism be transcribed.

How can nitrogen status be determined by cells

Using the ratio of alpha ketoglutarate to glutamine

When nitrogen levels are low (and ammonia), there are lots of keto acids and alpha-ketoglutarate, and less glutamine → which causes genes for nitrogen metabolism to be transcribed to increase levels.

alpha ketoglutarate + ammonia → glutamine

Example of regulator having dual control in archaea

TrmBL1 - simultaneously acts as a repressor for sugar uptake and an activator for gluconeogenesis genes.

Maltose is the inducer - binds to TrmBL1 and causes it to fall off DNA.

On one level this allows RNA polymerase to bind to promotor region for sugar uptake genes, and they are expressed.

On the other side, TrmBL1 is no longer able to recruit TFB and TBP for transcription of gluconeogenesis genes, and they are no longer expressed.

Components of the bacterial two-component regulatory system

Specific sensor kinase protein in membrane

Response regulator protein in cytoplasm

How does the two-component regulatory system work

Sensor is an auto-kinase which phosphorylates itself in response to signal.
The phosphoryl group is transferred to the response regulator, its phosphorylated form binds to DNA - either acting as activator or repressor.

What is the consensus/canonical sequence

The sequence that is the ‘standard’

The closer a gene’s promoter sequence is to the consensus, the stronger the promotor, and the more the gene will be expressed

How can gene expression be controlled in relation to the consensus

Changing the promotor sequence to be more/less similar to the consensus, therefore changing how much it is expressed

What is a sigma factor

Subunit of bacterial RNA polymerase

Recognises and binds to promoter, and recruits the ‘holo-enzyme’

What is sigma 70

Sigma factor for housekeeping genes (controls the main genes needed for functionality)

What are the prokaryotic mechanisms of gene exchange

Transformation, transduction, conjugation

What happens to the DNA that enters the recipient cell

Either degraded, self-replicated, or recombines with recipient cell’s chromosomes

What is recombination

Physical exchange of DNA between genetic elements

What is the major recombination molecule for single strand invasion

RecA protein - essential for nearly all homologous pathways

Process of recombination

1. endonuclease nicks donor DNA

2. one strand separated using helicase-like enzymes

3. the single strand binds to SSB (single strand binding) protein, and then RecA

4. this complex promotes complementary base pairing with one strand of recipient DNA, displacing the other strand —> strand invasion

5. cross-strand exchange develops

6. molecules separated by enzymes that cut and rejoin the DNA strand

Different outcomes of recombination

Can lead to patches and splices, depending on resolution at different sites

What is an auxotroph

Organism that cannot synthesise specific compounds

What is a prototroph

Organism that can synthesise all compounds

What is transformation

Method of prokaryote horizontal gene transfer where bacteria take up genetic material from their environment

How can transformation occur

Can occur naturally in gram +ve bacteria → extracellular DNA is taken up as a single stranded molecule and recombined

Can be induced in gram -ve bacteria using Ca2+ treatment at a low termperature

Two types of transduction

Generalised and specialised

What is transduction

Method of prokaryotic gene transfer (HGT) where genetic material is spread via a bacteriophage

What is generalised transduction

There is a phage in its lytic cycle in a bacterium. A mistake in the phage DNA causes bacterial DNA to be packaged, producing a transducing particle. This particle attaches to another cell, injects its DNA (carrying the previous bacterium’s DNA) and it undergoes homologous recombination and integrates into new cell’s DNA

What is specialised transduction

Phage in lysogenic cycle has its DNA integrated into host DNA forming a prophage. Mistake in the phage DNA excising and takes some neighbouring host DNA along with it. The phages produced are specialised transducing phages, often cannot infect other cells and instead need to piggyback off wild types.

Example of specialised transduction

Lambda phage which infects E. coli. Integrates into the host bacterium genome at specific sites - binding of attP in the phage and attB in bacteria chromosome, integrated using recombinase.

The lambda phage DNA has cohesive end sites that are complementary, therefore bind together and the DNA circularises. Replicates using rolling circle replication.

What is phage conversion

Benign bacterial strain converted to harmful strain by infection of a phage

What are gene transfer agents

Defective viruses hijacked by bacteria to transfer DNA

What is conjugation

Form of HGT through cell-to-cell contact. A conjugative plasmid can transfer genes from a donor to recipient cell.

What is the common mechanism for bacterial conjugation, and how does it work

The F plasmid —> transferred from F+ donor to F- recipient. Encodes genes involved in conjugation in its tra region (for producing the pilus and type IV secretion system)

Pilus joins cells together to form mating junction. Type IV system secretes DNA into neighbouring cell. TraL protein nicks the DNA and unwinds it (helicase properties). Plasmid DNA begins replicating by rolling circle replication as other strand feeds into recipient cell, and complementary strand is synthesised on the other side. Then cells separate.

What are high frequency recombination strains

Have the F plasmid integrated into their chromosomal DNA - the F plasmid is transferred in high frequencies to recipient cells, along with some of the host DNA.

The F plasmid being in different locations in different bacterial strains can lead to different genes being transferred.

What is an episome

Plasmid that can integrate into the chromosome

Archaeal gene transfer methods

Some use transformation, few shown to use transduction and conjugation

Example of archaeal conjugation - difference with bacterial

Sulfolobus olfataricus has unidirectional conjugation

Uses nanotubes instead of a pilus

What are transposons, their problem, and two types

Sequences that randomly hop to different positions in DNA

Can be transferred between bacteria and could quickly spread antibiotic resistance

Conservative vs. replicative - former disrupts the host DNA by a section leaving while latter makes a copy to leave in original location and leaves host DNA intact

How are transposons used in research

Add transposons to wildtype bacteria, wait for growth, and select those mutants with the transposons. Can sequence the mutant DNA and see where the transposons have ended up.

Where do restriction enzymes come from

Bacteria produce restriction enzymes to protect their own DNA from foreign DNA. They recognise foreign unmethylated DNA and digest it by cutting with restriction enzymes.

What system do bacteria and archaea use to recognise specific foreign DNA sequences, and how does it work

Crispr-Cas system - during viral infection part of the viral DNA integrates into host genome and is copied unto RNA. The RNA attaches to a Cas protein and acts as a surveillance system. If the complex encounters the same sequence - i.e. the invading virus - it destroys the DNA.

Structure of peptidoglycan

Sugar backbone of two amino sugars (N-acetylglucasamine, NAG, and N-acetylmuramic acid, NAM) joined by 1-4 beta glycosidic link.

NAM has a tetrapeptide (4 peptides) side chain, and the different side chains are joined together by peptide cross bridges.

Covalent bonds between the different polymers.

How does peptidoglycan structure differ between +ve and -ve

Differs in the cross-bridges between side chains.

In +ve → Diaminopimelic acid joins d-glutamic acid to form peptide crosslink. Also the peptidoglycan form cables with cross-links.

In -ve → l-lysine connects to inter-bridge connected to d-alanine

Difference between bacterial and eukaryotic cytoplasmic membrane

Bacterial membrane strengthened by hopanoids, not sterols like in eukaryotes

Cytoplasmic membrane function

Permeability barrier, protein anchor, energy conservation - can cause generation and dissipation of proton motive force

Structure of the G -ve outer membrane

Components known as lipopolysaccharides (LPS)

Very long carbohydrate chains extending away from the membrane

3 sections: highly variable O-specific polysaccharide, core polysaccharide, and lipid A (disaccharide of glucosamine)

What are lipopolysaccharides

Components of outer membrane of G-ve bacteria

Are endotoxins, cause sickness. LPS units switched regularly to evade immune system

What is the space between the outer and inner membrane of G-ve bacteria

Periplasm - site of enzymes and binding sites

What do many bacteria have as well as cell wall and membrane

Extracellular capsule - a polysaccharide that is secreted to the outside of the cell

Structure of archaeal membranes

Made up of lipids formed of a glycerophosphate head and phytanyl/biphytanyl tail

Different structures of archaeal cell walls

Most common are S-layers - protein coat made from repeating protein subunits of pseudomurein. Some have pseudopeptidoglycan.

What are the different types of flagella

Peritrichous - flagella all over

Lophotricous - lots of flagella at one pole

Polar - one flagellum at one pole

How do bacterial flagella move

Rotate in one direction to move. If one flagellum (in a group) rotates the other direction it causes a tumble and the bacteria stops.

Polar flagellum can reverse the direction of their rotation and therefore reverse direction of movement. Otherwise the bacteria has to reorient itself to change direction.

Structure of flagella

Base is formed of lots of rings situated in the cell wall and membrane: L ring in the LPS (outer membrane), P ring in peptidoglycan, MS ring in cytoplasmic membrane, C ring in cytoplasm

Filament stretches out of the basal body

How do flagella move

Energy from the flow of protons through the stator (Mot proteins) causes the rotational movement

Chemotaxis of prokaryotes

Have a biased, 3-D, random walk.

With no attractant/repellant present, movement is random.

If attractant is present, frequency of tumbles increase until bacteria is oriented towards it and then the frequency of tumbles decrease. Opposite occurs for repellants.

What are prokaryotic pili used for

Used to make bridges between other prokaryotes and themselves

Methods of bacterial cell division

Binary fission, simple budding, budding from hyphae, cell division from stalked organism, polar growth

Features of binary fission

Most common method of bacterial cell division, produces equal products.

Production of the new cell wall is intercalatory as the new components and material are inserted into the existing peptidoglycan all around the cell

Features of simple budding and budding from hyphae

Budding cell wall formation is polar, with new material and components added at one end only. Asymmetrical growth.

How to calculate bacterial growth rate

Mean generation time (g) = time / number of generations (n)

Mean generation time refers to the time taken to double numbers of cells.

Trick for calculating growth rate - on log10 and log2 graph

log10 plot: g = 0.301 / slope

log2 plot: g = 1 / slope

Stages of bacterial growth

Lag (need to adapt and regulate genes), log / exponential, stationary, death

Ways to measure bacterial growth

Turbidity and viable counts

What is turbidity and what is one problem

Measure of the optical density using a spectrometer, how much light passes through the solution. Bacteria scatter light so the denser/more populated the solution is, the higher the optical density/absorbance.

Problem: dead bacteria still scatter light and affect optical density, so turbidity is less accurate for the death phase

Classifications of bacteria by optimum temperatures

Psychrophiles → mesophiles → thermophiles → hyperthermophiles

Classifications of bacteria by optimum pH

acidophiles → neutrophiles → alkaliphiles

Classifications of bacteria by optimum osmolarity of salts

halotolerant (optimum at low/no salt conc.) → halophile → extreme halophile (need high salt conc.)

Classifications of bacteria by optimum osmolarity of sugars

Osmophiles (grow in high sugar conc.) → xerophiles (grow in dry conditions / low sugar conc.)

How do bacteria deal with different osmolarities and osmotic pressure

Bacteria accumulate compatible solutes or synthesise them to change their inner osmolarity and osmotic pressure.

Instead of accumulating sodium which would damage/mess with the cell’s biochemistry.

Classifications of bacteria by optimum oxygen conc.

Aerobes (use oxygen): obligate (need O2) → facultative (better w/ O2) → microaerophilic (optimum at low O2 levels, high levels are poisonous)

How can bacteria detect cell density

Quorum sensing - enables them to affect their gene regulation based on population.

G -ve bacteria make AHLs (N-acyl homoserine lactones) which activate gene expression at high cell density.

G +ve bacteria make peptides instead of AHLs

Example of AHLs used in quorum sensing

LuxI produces AHL and it diffuses out of the cell.

When population size gets large, there are v. high levels of AHL in the environment and some begins to diffuse back into the bacteria cells.

AHL acts as an inducer, binding to LuxR (activator) and causes transcription of lux operon, which includes LuxI. Therefore more AHL is produced → positive feedback.

Luc also expressed which produces light - enables bioluminescence controlled by AHL.

What is activated if growth of bacteria is halted

Stringent response activated: translation slowed down so tRNA and rRNA synthesis reduced, cell division halted, stress response increases (e.g. amino acid biosynthesis)

What is the heat shock response - specific example

Heat shock proteins can refold proteins that have denatured due to high temperatures.

In stress conditions heat shock proteins HSP70 or DnaK refold proteins.

They usually degrade RpoH sigma factor, but in stress conditions are used for refolding and therefore RpoH is available to replace sigma 70, and bind to promotors.

Replication time of chromosomes

100 minutes

The variety of genes found on plasmids

Lots of genes for resistance (including antibiotic resistance) - because bacteria produce dangerous toxins/chemicals and need resistance mechanisms

Pathogenic, metabolic, symbiotic genes

Bacteriocins → kill related (other strains) bacteria, biggest competition

Tra genes → make plasmid self-transmissable, code for genes for pilus mating bridge and type IV secretion system

What are incompatibility groups

Two plasmids that use the same replication mechanism, therefore cannot be in the same cell

Steps of the bacterial cell cycle

Replication (initiated by DnaA binding to oriC)

Regions blocked by SeqA and cell elongates

Chromosome segregates

Z-ring forms

Cell divides

How does chromosome replication occur

Occurs under very specific circumstances - both SeqA and HdaA activity prevent replication

If DNA is fully methylated, then DnaA-ATP can bind to DnaA box on oriC (and replication can begin)

SeqA prevention: But if DNA is hemi-methylated (just after chromosomal replication) seqA binds to oriC and displaces DnaA

HdaA prevention: hydrolyses DnaA-ATP to DnaA-ADP to prevent replication

How is separation time of cells 20 minutes but replication is often much longer - example of length in E. coli

Multiple replication forks form

In E. coli replication takes 40 minutes

How are chromosomes partitioned without a spindle - one example

Caulobacter - ‘old pole’ of cell has PopZ which binds to ParB. ParB binds to DNA at the parS sequence. ParA activity at the opposite pole draws ParB at the new parS sequence (during replication) to new pole.

Plasmid replication and separation

Replicate independently of chromosomes, separate to the two poles

Cell division process

FtsZ (tubulin like) assembles at the centre of the cell as a ring.

ZipA attaches the FtsZ to the cell membrane. FtsA (actin like) recruits the FtsZ and other proteins (like FtsI which is needed for peptidoglycan biosynthesis) to form the structural divisome.

The FtsZ ring contracts down, pulling the two sides of the cell down to form two separate cell walls, eventually two different cells.

How does the FtsZ ring find the centre

MinE disperses MinCD at one pole, and the MinCD proteins oscillate from one end of the cell to the other.

MinCD prevents FtsZ polymerisation and MinCD levels end up being lowest at the middle of the cell, so FtsZ ring forms in the middle.

How is bacterial cell shape dictated

Often by MreB - forms perpendicular bands to the cell wall (in rod-shaped bacteria), and it connects the cell membrane and directs peptidoglycan synthesis

Caulobacter has ‘crescentin’ protein that localises at concave surface, causing the bacterium’s curve

Effect of MreB on type of bacterial cell division (examples of bacteria)

MreB directs peptidoglycan synthesis - if growth and adding of new components/material (lateral synthesis) occurs at multiple points along cell wall → binary fission (e.g. E. coli)

Alpha-proteobacteria lack MreB → tend to be polar / budding

Streptococcus lack MreB → grow at band from middle

Steps of petidoglycan biosynthesis

Peptidoglycan precursor NAG-NAM pentapeptide linked to C55 alcohol bactopronel

Bactopronel carries peptidoglycan unit across the cytoplasmic membrane

Autolysins hydrolyse the glycosidic backbone of peptidoglycan

Transglycosylases add in new unit, transpeptidases link DAP to D-ala to form cross links

Why was penicillin so effective (related to peptidoglycan biosynthesis)

Inhibits the transpeptidation step of peptidoglycan biosynthesis

Different antibiotics and what they target

Actinomycin → blocks production of RNA polymerase (DNA replication)

Puromycin → binds to termination factors (inhibiting translation)

Streptomycin → binds to ribosome (inhibiting translation)

Others target cell wall synthesis, DNA gyrase, membrane