Microbial Genetics (Ian Bloomfield) Flashcards
Are bacterial prokaryotes or eukaryotes?
Prokaryotes
What are some characteristics of prokaryotes?
- Cytoplasm does not contain membrane bound organelles
- In some bacteria the plasma membrane can form extensive folded structure that extends into cytoplasm
- Lack nucleus
- Transcription and translation occur simultaneously in the same place
How many bacterial cells are there on earth? and how many different types are there?
~ 5 x 10^30
- >10^7 different types
How much of the worlds carbon biomass is made up by bacteria?
Around half
also, majority of nitrogen and sulfur in living material
Approx how many bacteria have been cultured in the lab?
~9,300
What are the 3 domains of life?
Common ancestor: - Bacteria - Archaea - Eukarya > Algae and plants > Fungi and animals > Protists
What are prokaryotes and eukaryotes?
Pro:
- Archaea and Bacteria
Eu:
Eukarya
Describe the changes in earth atmosphere composition and organism changes from origin of earth to present
1) Origin of Earth (4.6bya)
2) Bacteria (~4bya)
3) Anoxygenic phototrophic bacteria (~3.5bya)
In these steps the earth is anoxic (without oxygen) (N2, CO2, CH4 atmosphere)
4) Origin of cyanobacteria (~3bya)
Earth becomes slowly oxygenated
5) Origin of eukaryotes (~2bya)
6) Algae diversity (~1.5bya)
7) Within last 0.5by, in order:
Shelly invertebrates
Vascular plants
Mammels
Humans
What are autotrophic cyanobacteria?
Conduct photosynthesis by splitting water and releasing oxygen
What is endosymbiosis and what is the endosymbiotic theory?
Endosymbiosis is when one partner population grows within the body of another organism
Endosymbioti theory is a theory of how eukaryotes evolved from prokaryotes
- Eukaryotes underwent one (animals) or 2 (plants) rounds to endosymbiosis
What do archaea include?
Many extremophiles
- None are pathogenic (as far as they know)
What 2 groups of bacteria do most pathogenic bacteria belong to?
Proteobacteria and Gram positive bacteria
What are the typical sizes of pro vs eu cells?
Pro: 1-10µm
Eu: 10-100µm
How long is a typical E.coli?
2µm
How many more bacterial cells do humans have than their own cells?
~10 fold
10 bacteria to 1 cell
Give some examples of the ecological importance of bacteria
1) Chemolithotrophs
- able to oxidise inorganic ions (e.g Fe2+) as a source of electrons to generate a membrane potential for ATP biosynthesis and other functions
2) Phototrophs (carry out photosynthesis)
- Autophototrophs (use CO2 like plants)
- Heterophototroph (need an organic carbon source)
3) Chemoprganotrophs
- oxidise organic molecules in orfer to produce NADH needed for PMF required for ATP biosynthesis
4) Some bacteria produce cellulase - needed for utilisation of cellulose
5) Involved in nitrogen and sulfur cycles
- Fixing (generate ammonia from atmospheric NH2)
- Nitrifying (oxidise ammonia)
- Denitrifying (convert nitrate to NH2)
Give some examples of the medical importance of bacteria
Some bacteria pathogenic: important to study to prevent disease
- Robert Koch pioneered scientific study of pathogenesis
1) Mycobacterium tuberculosis causes tuberculosis (discovered by Koch) - around 25% of worlds population infected
2) Enterobacteriaceae major cause of intestinal infections - E.coli
- Salmonella
3) MRSA world threat to human health
What is Escherichia coli and who discovered it?
- Gram negative enteric bacterium
- Discovered by Theodor Escherich
- Important model organism
What macromolecules are bacterial cells composed of?
Larger macromolecules: - Murein or peptidoglycan - Cell membrane - Flagella - LPS Smaller macromolecules: Proteins, DNA, RNA, lipids etc
How are these components produced?
- By polymerisation reactions in which building blocks are joined by enzymatic reactions
What are the building blocks?
Amino acids Fatty acids Amino sugars Sugars Nucleotides
Where do the building blocks come from?
Either
1) Made de nova
2) Taken up from the environment
What are the bacterial building blocks synthesised from?
Made in biosynthetic pathways from one or more of 12 precurosr metabolites
e.g acetyl-CoA, Succinyl-CoA, pyruvate, G6P
Where do the 12 precursor metabolites come from?
Central metabolism: TCA cycle Pentose phosphate pathway Glycolysis e.g pyruvate from glycolysis, succinyl CoA from TCA cycle
What are the main elements missing from the precursor metabolites that are present in one or more of the building blocks?
Nitrogen and Sulfur
How do bacteria assimilate nitrogen? (incorporate nitrogen into usable organic molecules)
Using the nitrogen cycle
How does the nitrogen cycle work?
1) Nitrogen fixing bacteria
- Fix dinitrogen gas by enzyme nitrogenase to ammonium (NH4+)
2) Nitrifying bacteria
- Oxidise ammonium first to nitrites (NO2-) then to nitrates (NO3-)
3) Denitrifying bacteria
Denitrification of NO3- to N2
What is the only source of nitrogen that can be assimilated into bacterial cell constituents?
Ammonia (NH3)
What are the 3 key enzymatic systems used in assimilation of nitrogen in E.coli?
1) L-glutamate dehydrogenase (GDH) catalyses the combination of ammonia with alpha-ketoglutarate to produce glutamate
2) Glutamate reacts with second molecule of ammonia to produce glutamine (catalysed by glutamine synthase)
3) To complete cycle, glutamine then reacts with alpha-ketoglutarate to generate 2 molecules of glutamate (catalysed by glutamate synthase)
- 1 can be used in bacteria, 1 used to bind with ammonia
(Diagram good if confusing)
What else is required for the biosynthesis pathways to operate?
ATP
NADPH
1 carbon units
How, and in what form, are the 1 carbon units generated?
1 carbon unit generation requires folic acid in the form of tetrahydrofolate
What drugs inhibit the biosynthesis of folic acid?
The sulfa drugs (sulphanilamide)
Inhibit the biosynthesis of folic acid
Therefore sulfa drugs are toxic to bacteria
How does the antibiotic trimethoprim work?
Inhibits the enzyme dihydrofolate reductase required for the generation of tetrahydrofolate
Selectively toxic to bacteria
What is the doubling time of E.coli in a simple ‘minimal medium’ that contains an organic carbon source (glucose preferred) and a variety of inorganic salts e.g ammonia?
around 1 hour
What is the doubling time of E.coli in a rich medium that contains many building blocks?
20 minutes (much faster)
What does this tell you about the relative energy and resource efficiency of utilising building blocks present in the environment as opposed to making them by biosynthesis?
Utilising building blocks in environment has better relative energy and resource efficiency
How are wild type E.coli able to grow without the presence of any building blocks in the environment?
It is a prototroph
What is an auxotroph?
Auxotrophic bacteria require the presence of building blocks in environment in order to grow (opposite of prototroph)
What is an essential nutrient?
Specific building blocks that cannot be synthesised in the organism
- All bacteria require each building block
- Difference of requirements depending on which they can or cannot synthesis
What are fuelling reactions and what do they produce?
Variable reactions that provides the diversity of bacterial metabolic activity
Produce:
- Precursor metabolites in central metabolism
- NADH2 required as a hydrogen donor in energy transfer reactions
- NADPH2 - required for the biosynthetic pathway
- Energy transfer - including proton gradients across membranes
- Sulfide and ammonia for assimilation
How do fuelling reactions contribute to diversity of bacterial metabolic activity?
Ability to use different carbon sources
Use different terminal electron acceptors
(Humans can only use oxygen as a terminal electron acceptor)
See summary diagram for lecture 2
please its helpful
What are the outer structurea of a gram positive bacteria?
- Surrounded by a single (cytoplasmic) membrane
- Multi-layer cell wall lies on top of membrane
- Membrane and cell wall associated with protein and (lipo)teichoic acid
What are the outer structures of a gram negative bacteria?
- Has both a cytoplasmic and an outer membrane
- Outer membrane and inner peptidoglycan later make up cell wall
- Peptidoglycan layer within the periplasmic space
- Periplasmic space filled with matrix called periplasm
- Outer membrane contains phopspholipids and lipopolysaccharides (LPS)
Describe the inner structures of bacteria
No nucleus or other internal membrane bound organelles
- Chromosomes attach to cell membrane and become looped coils known as the nucleoid
- Transcription and translation occur simultaneously - known as coupled
What is attenuation?
Regulation of gene expression in bacteria
- Can occur due to coupled transcription and translation
- A control mechanism in which the efficiency of translation determines if transcription terminates or not
What are the features of the bacterial genome?
- Composed of DNA and is contained in nucleoprotein structures called the nucleoid
- Genome is haploid and is typically a single circular chromosome
Describe the DNA replication and cell replication in E.coli
- Replication of the 4.6 x 10^6 bp genome proceeds bi-directionally around the circular chromosome and takes around 40 minutes
- Can undergo binary fission under favorable conditions every 20 minutes
What is the main constituent of bacterial cell walls?
A peptidoglycan called Murein (unique to bacteria)
- consists of parallel polymers of disaccharides called glycan chains cross linked with peptides of 4 AAs
What does murein form in gram positive bacteria?
Multilayer, highly cross linked layer that lies on top of cytoplasmic membrane
What does murein form in gram negative bacteria?
Thin peptidoglycan layer that is less cross linked
- Found between inner and outer membrane in periplasmic space
What is the structure of the peptidoglycans
Backbone of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)
- Peptide links between NAMs of different chains
- Nature of peptides differs between 2 groups
Why is the cell wall of bacteria necessary?
- Cytoplasm is under high turgor pressure
- Cell wall prevents bacteria from lysing
Although it is used for structure, why is it important that the cell wall is dynamic?
Allow for cell division (elongation and septation)
Describe the properties of the bacterial cytoplasmic membrane
- Lipid bilayer with proteins imbedded
- Selectively permeable barrier
- allows only passage of uncharged molecules up to the size of ~ 100 Da by passive diffusion
- Transport of nutrients into cell needed for growth and division
What main things can be found on and in the cytoplasmic membrane?
1) Transporters
- for active transport of nutrients (e.g sugars and AAs) and ions (e.g K+) across the membrane
2) Components of the electron transport chain (acts as a proton pump to generate membrane potential) and the ATP synthetase
3) Proteins involved in export
4) Signal transduction proteins
5) Basal body of flagella, type IV pillus and type II secretion system
What are the main 3 difference between gram positive and gram negative cell membrane/wall structures?
Pos: Single membrane
Neg: Outer plus cytoplasmic membran seperated by periplasmic space
Pos: Thick cell wall, fully cross linked
Neg: Thin cell wall, less cross links
Pos: (Lipo)teichoic acid on cell wall
Neg: LPS on cell wall
Describe the properties of the outer membrane of gram negative bacteria
- Allows diffusion of molecules up to the size of disaccharides through it
- ## This is because the membrane contains relatively non-selective channels called porins
How do LPS’s influence transport?
Negatively charged and prevent hydrophobic substances (such as detergents and some antibiotics) from coming into contact with membrane
- Cations (Mg2+) associate with LPS to diminish overall negative charge
- Gram negative bacteria insensitive to many hydrophillic substances
What is Murein and what is its structure?
- A paptidoglycan that is an essential component of the eubacteria
- Composed of long, stricly alternating polymers of aminosugars N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) in a beta(1–>4) linkage
- MurNAc is initially substituted with a penta-peptide at C3 position
What happens to peptidoglycans during cell division?
Peptidoglycan fragments are released and new fragments added as cell grows
- During exponential growth, around 60% of the cell wall of E.coli is released in this way every generation
- Many of released fragments are transported back into the cytoplasm as NAG-NAM-peptide units by the AmpG transporter
- Components are partially degraded and recycled to build new peptidoglycans
What are the 2 main steps in peptidoglycan synthesis?
1) Synthesis of the basic unit in the cytoplasm followed by attachment to a lipid carrier in the inner surface of the cytoplasmic membrane and its subsequent transport through the cytoplasm membrane
2) Polymerisation of the monomer unit into pre-existing peptidoglycan outside of the cytoplasmic membrane
What is the basic unit in peptidoglycan synthesis and what is the lipid carrier?
- GlcNAc-MurNAc pentapeptide
- undecaprenol phosphate
How is the basic unit formed?
Stepwise pathway:
1) Starts with UDP-GlcNAc
2) Synthesis of UDP-MurNAc
3) Addition of amino acids (e.g L-Alanine) and D-glucose to form UDP-MurNAc-tripeptide
(UDP-MurNAc-tripeptide can be synthesised by de nova synthesis or peptidoglycan recycling)
4) Addition of further amino acids produced the GlcNAc-MurNAc petapeptide bound to the lipid undecaprenol phosphate
What 2 reactions occur outside of the cytoplasmic membrane?
1) Monomer subunit is joined to glycan chain in transglycosylation reactions catalysed by a family of enzymes called Penicillin Binding Proteins (PBPs)
2) Cross linking between adjacent peptidoglycan chains involves transpeptidation reactions
(Membrane carrier is recycled post transglycosylation)
How does transpeptidation occur in E.coli?
- Involves cleavage of the terminal D-alanine on one chain
- Formation of an enzyme-substrate covalent complex
- Cross-link formation by reaction with an amino group of diaminopimelic acid (DAP) of another chain
- Transpeptidation is also catalysed by the PBPs
How do beta-lactam antibiotics work?
Structural homologs of D-alanine
- They form an irreversible complex with the PBPs to inhibit transpeptidation
- Destabilises bacterial cell wall
What does Lytic transglycosylase catalyse?
Cuts the glycosidic linkages between MurNAc and GlcNAc groups
- Generates the products 1,6-anhydroMurNAc and GlcNAc
- Works in combination with PBPs to allow biosynthesis of new cell walls
Where is lipopolysaccharise (or endotoxin) found?
Essential part of the outer portion of the outer membrane in gram negative bacteria
What is the structure of LPS?
- Very large, amphipathic molecule
- Acyl groups form a hydrophobic anchor that lies within the membrane
- Sugar groups form the hydrophilic outer component
- Sugar componens block access of hydrophobic components like detergents and antibiotics
How can LPS cause toxicity?
- Is an endotoxin
- Interaction with mammalian pattern recognition receptors (PRRs) stimulates a vigorous cytokine response that can lead to septic shock and ultimately multiple organ failure and death
How do LPS molecules compare in different species and between different strains?
Differs in size and composition
What are the 3 components of LPS (all covalently linked)
1) Lipid A (endotoxin)
2) Core polysaccharide
3) O antigen repeats
Which parts of the LPS are essential for variability?
Lipid A plus the first 2 sugar groups
What is lipid A composed of?
2 beta1–>6 linked glucosamine residues that are further acylated, phosphorylated and glycosylated
- Attached fatty acids are linked to GlcN by an ester-amine bond
What is the core composed of?
- Branched, non-repeating oligosaccharide of 6-10 sugars in length, partly substituted at various positions
What do substitutions of phosphate groups on lipid A with L-4 amino arabinose cause?
Increased resistance to cationic antimicrobial peptides such as polymyxins
What do mutations that disrupt the core structure cause?
Prevents the correct insertion of outer membrane proteins into the outer membrane - leading to induction of the sigmaE stress response pathway
What is the structure of the O antigen repeat?
Composed of between 1 and 40 oligosaccharide repeat units of around 4-5 sugars
- O antigen is extremely variable between different strains (O157 of E.coli O157:H7 refers to O antigen)
- O antigen group is called the serogroup
What is the name for bacteria that can resist killing by complement action?
Bacteria are called serum resistance
How is complement activated in bacteria?
Binding of protein C3 to LPS (lipoteichoic acid in gram positive)
What are the 2 main alterations to LPS that can lead to serum resistance?
1) O antigen composed of sialic acid blocks complement activation
2) Large number of O antigen repeats block MAC formation
How is LPS assembled?
1) Lipid A, with core attached, syntesised in the cytoplasm via a series of steps - lipid A-core flips through membrane, with acyl groups remaining within the hydrophobic environment of the membrane
2) Each O antigen repeat is also synthesised in cytoplasm attached to undecaprenol phosphate (i.e in analogous matter to peptidoglycan) before transport through the cytoplasmic membrane
- repeats are added onto growing chain one at a time
- O unit repeats added onto core via transferase reaction within the periplasm
Describe the biosynthesis of Lipid A
Starts with GlcNAc
Followed by (in sequence):
- addition of acyl groups
- formation of the disaccharide
- addition of the first 2 sugar groups of core
- further acylation of the core-linked glucosamine and so on
Describe the transfer of lipid A
Transfer of lipid A with core attached (Ra-LPS) to outer layer of cytoplasmic membrane requires the ABC transporter MsbA and involves the hydrolysis of ATP
- Release of LPS from cytoplasmic membrane requires a second ABC transporter composed of LptBCF and G (also involves ATP hydrolysis)
- Transport of amphipathic LPs across the periplasm requires LptA while final insertion into outer membrane requires LptD and E
What is the purpose of bacterial capsules?
1) Thought to prevent desiccation (extreme dryness)
2) Blocking the activation of complement (K1 capsule in E.coli is composed of sialic acid)
3) Blocking access of antibodies to antigen on cell surface
Where is the capsule produced?
Produced in cytoplasm and transported to outside the cell
What are the 3 regions of genes encoding for capsule sysnthesis and what does each do?
II - encodes the genes for specific biosynthetic pathway
I and III - involved in transport of materials
(I and III highly conserved, II much more variable)
What is needed for transport?
Requires a dedicated ABC transporter (KpsMT for the E.coli capsule)
- Also requires other capsule specific products as well as a non-capsule specific outer membrane porin
(Protein export)
How are lipoproteins anchored in bacteria?
Anchored to the cytoplasmic and outer membrane by diacylglycerol
- The diacylglycerol is attached to an N-terminal cysteine
- The presence of aspartate at second position = lipoprotein remians attached to inner membrane
What are the common destinations of exported proteins from the cytoplasm?
- Cytoplasmic membrane
- Periplasm (GN) or cell surface (GP)
- Outer membrane (GN)
- Released from cell
What is an example of something transported by the cytoplasmic membrane transporter?
Periplasmic binding proteins
- Part of ABC (ATP binding cascade) transport system
- System contains periplasmic binding protein (binds substrate in the periplasm), membrane spanning transporter (through cytoplasmic membrane) and a cytoplasmic ATP-hydrolysing protein to provide energy
- Moves substrate from periplasm into cytoplasm
What other proteins are transported out of the cytoplasm to form membrane transporters?
Porin proteins
- associate in the outer membrane of GN bacteria
- allow permeability to small molecules
Sec and Tat proteins
- Involved in forming protein export pathways
What enzymes are involved in protein export?
- Enzymes required for cell wall biosynthesis etc
- Folding catalysts (e.g disulphide bond formation)
- Exoenzymes (e.g pectinase)
What proteins are needed to be transported that are important for energy transduction and PMF?
Proteins that act as proton and electron carriers in the cytoplasmic membrane
- Proteins that make up ATP synthetase
What proteins are needed to be transported in order for bacterial attraction?
Chemotaxis proteins
Give some examples of structural and functional proteins that need transporting
- Lipoproteins
- Flagella proteins
- Adhesins
- Toxins e.g cholera toxin
In what 2 states can proteins be when secreted?
Unfolded or folded state
What are the main 2 pathways in which most proteins are exported from the cytoplasm?
1) General secretory pathway (Sec; transports proteins in their unfolded state)
2) Tat pathway (twin arginine translocase; proteins in their folded state)
What are the 2 main types of more specialised pathways?
Type I and IV secretory pahways
What do unfolded proteins need in order to be exported using the general secretary pathway?
Requires an N-terminal signal sequence, that is removed during export
- Signal sequence has a N-terminal positive charge, a hydrophobic (H) domain and a polar (C) domain
What are the main destination of proteins exported using the GSP?
Either inserted into cytoplasmic membrane or pass though membrane
- Proteins inserted into the cytoplasmic membrane contain 1 or more hydrophobic regions that become embedded into the membrane
Describe the path of proteins excreted by the GSP
- Cytoplasmic SecA ATPase binds proteins destined for export
- Delivers them in their unfolded for, to a membrane channel composed of SecYEG
- Some proteins are translocated post translationally with the assistance of chaperon SecB (usually pass through membrane)
- Others bind signal recognition particle (SRP) during translation and complec is delivered to translocase complex
- Energy for export is provided both by SecA-dependant ATP hydrolysis and the PMF
What are the characteristics of the TAT (twin-arginine targeting translocate) pathway?
- Transports fully folded proteins
- Mant TAT substrates contain cofactors that must be inserted before export
- Involves the TatABC proteins
- Signal sequence similar to Sec pathway except all tat exported proteins contain a characteristic twin arginine motif within signal sequence
What are the 2 systems in which most proteins are secreted from the periplasm?
Type 2 secretion system (aka the main terminal branch of the general secretion pathway)
ABC transporter
Give examples of a protein that is secreted by the type 2 system?
Type 2 SS: Cholera toxin
How do some pathogenic bacteria inject proteins into host cells?
Using a molecular syringle (type III secretion system)
- Proteins exported directly from cytoplasm across both bacterial membranes and the host cytoplasmic membrane
Examples in many GN bacteria (E.coli, Salmonellae, Shigella)
What is the structure of Fimbriae (pili)?
Hollow, hair-like protein structures that include an adhesin (often lectin or sugar binding protein) that recognise specific receptors (e.g mannose-containing glycoproteins on eukaryotic cell surfaces)
What are the 2 impotant classes of fimbriae?
Class I fimbriae and type IV pili
What are the 2 main types of Class I fimbriae? What are their structures?
- Pap (pylenonephritis associated pilus) and fim (type 1 fimbriae)
- Anchored in the outer membrane of GN bacteria
- Subunits exported to periplasm by the Sec pathway
- Contains an adhesin at the tip (PapG or FimH)
- Adapter proteins form a link between the major subunit (PapG or FimH) and the adhesin
- Chaperon proteins (PapD/FimC) bind the subunits in periplasm and transport them to outer membrane usher channel (PapC/FimD)
- Not dynamic structures
What are the characteristics of the type IV pili?
Example: toxin co-regulated pilus of vibrio cholerae
- Do not require general secretory pathway for their synthesis
- Span both inner and outer membranes of GN bacteria
- Dynamic structures
- often involved in twitching motility
- Like class I fimbriae Pap and Fim, type IV pili are often virulence factors
(Flagella and chemotaxis)
What is the basic structure of flagella?
- Span both membranes in GN bacteria
- Structure composed of a filament (the bulk of the visible structure), a hook and basal body
- Long (10µM), thin (20nM) filaments
- Filaments are helical structures, composed of protein subunits
How are flagella synthesised?
Using a pathway that is homologous to the type III secretion systems
Production starts at base
How do flagella cause movement?
They rotate to produce a propeller-like action
What is the structure of the basal body?
Spans the 2 membranes and includes both a rotor (C ring) and membrane anchors in both the cytoplasmic and outer membranes
What were the 3 findings after tethering cells to a microscope by anti-flagella antibody?
1) Flagella rotate (tethered bacteria = bacteria rotate)
2) Flagella of bacteria such as E.coli and Salmonella can rotate either clockwise or counter clockwise
3) Counter clockwise rotation is stimulated by chemoattractants whereas chemorepellents stimulate clockwise rotation
How do E.coli and Salmonella flagella rotate?
- Requires the transport of H+ ions across the cytoplasmic membrane
- Transport of H+ by Mot proteins leads to conformational change that causes the C ring rotor to spin
How do bacteria respond to chemoattractants/repellents?
1) Sense the presence of chemoattractants or chemorepellents in environment
2) Sense a concentration gradient of chemoattractants/repellents
3) Transduce the information about substance concentration into signal transduction pathway that brings about the suitable change in motility
How do bacteria sense the concentration gradient and react to it?
- Bacteria are too small to sense a concentration gradient directly (don’t have front or back end)
- Therefore they use temporal information ([at t1] versus [at t2])
- Chemotaxis is achieved via a ‘biased random walk’
> In this mode of locomotion, bacteria swim smoothly (movement in a straight ish line) or tumble on the spot (leading to locomotion in a new direction at random)
How are the concentration of chemoattractant/repellent and smooth swimming related?
- Increase in conc of cheoattractant leads to an increase in the period of smooth swimming
- Chemotaxis is achieved by altering the ratio of smooth swimming to random tumbling to produce net locomotion towards or away from stimuli
What are the 2 classes of mutations that cause loss of chemotatic behaviour?
1) Loss of substrate specific chemotaxis (e.g tsr-serine, tsr-aspartate etc)
2) Loss of all chemotactic responses (in E.coli; cheA, cheB etc)
How is the concentration of substances detected in chemotaxis?
- Detected by the interaction of specific chemicals with protein receptors that span the inner membrane
- These include the methyl-accepting chemotaxis proteins (MCP), phosphotransferase transport system (PTS) and the electron transport chain
- Cytoplasmic domain of MCP highly conversed, whereas periplasmic domain shows no similarity between different receptors
What occurs in the MCPs upon interaction with a ligand in the periplasm?
Conformational changes occur that changes the ternary complex of MCP-CheW-CheA
- CheA is able to catalyse its own autophosphorylation and then the transfer of the phosphate group from itself to CheY
- Interaction of attractants with MCPs leads to inhibition of phosphotransferase activity from CheA
What does phosphorylation of CheY cause?
Affects the interaction of this protein with flagella motor components FliM to bring about a change in the direction of the flagella rotation
- CheY-P causes motor to rotate in a clockwise direction leading to tumbling
- CheZ dephosphorylates CheY to acts as a homeostatic regulator
What does methylation of MCPs cause?
Inhibits signal transduction from the sensor domain of the protein CheA-P, and is itself modulated to provide negative feedback control and adaption
What is the organisation of DNA in bacteria?
- Bacteria are haploid
- Double stranded DNA typically a single covalently closed circle
- Circle negatively supercoiled by the enzyme DNA gyrase (a topoisomerase II)
- Genome supercoiled into supercoiled domains with associated proteins to form the nucleoid
- Nucleoid-associated proteins such as IHF (integration host factor) and H-NS are required for this
How does E.coli K-12 replicate?
- E.coli is model for study
- Genome of E.coli K-12 is 4.6 million bp
- Replicated bi-directionally from a single origin of replication called oriC
- Origin of replication forms a complex nucleoprotein structure that directs initiation
- DnaA plays key role in initiation
- Replication initation requires the synthesis of an RNA primer
- Leading and laggind strand
It takes E.coli around 40 to replicate its genome but bacteria in a rich medium can divide every 20 minutes. How can this be explained?
Initiation of replication must occur more than once per division
- A new round of replication begins at the “future origin” before the current round of replication is completed
- Each daughter must inherit more than 1 copy of genome before septation occurs
Most highly expressed genes are transcribed in the same direction as replication occurs. Why is this?
Prevents collision of the replication machinery with RNA polymerase that would inhibit transcription
What does the E.coli K-12 genome encode?
Estimated to encode 4288 polypeptides
- 88% encodes known or predicted orfs
- 0.5% non-coding repetitive DNA
- 1% encodes stable RNA (tRNA and rRNA)
- 10% of DNA contains regulatory regions such as promoters etc
Some genes are organised into operons and encode polycistronic mRNA (~6% of gene)
How many genes in E.coli K-12 are thought to be essential? How many of these essential genes are functionally unknown (FUN)?
303
37
What is the name of the computer analysis of functional genomics and how does it work/.
Bioinformatics
- genes often encode proteins that belong to families with similar function that have arised by gene duplication
- Function of genes thus often predicted by their homology to other genes that encode proteins of known function
What are bacterial plasmids?
- DNA that can replicate as extra-chromosomal elements
- Small circular DNA, some of which are conjugative, that encodes genes for auxiliary functions (ability to utilize carbon source, pathogenesis, antibiotic resistance etc)
- Must contain 1 or more replication origins
What are broad range plasmids?
Plasmids that contain a number of origins that allow replication in different organisms
Can a bacteria contain many types of plasmid?
Yes, but plasmids belonging to the same incompatibility group will not coexist in same cell
What are plasmid cloning vectors used for?
Recombinant DNA technology
Cloning relies on what things?
- Ability to readily isolate purified plasmid DNA from bacterial cells
- Cut DNA at specific sequences with restriction endonucleases
- Ligate DNA fragments into the cut vector with DNA ligase
- Transform plasmid DNA into bacterial cells, selecting for a plasmid-encoded antibiotic resistance
How many introns do bacteria have compared to eukaryotes?
Do not normally have introns, or very rare
- However, bacterial genome may contain other genetic elements
What other genetic elements do bacteria contain?
May contain genomes of lysogenic bacteriophage (prophage or defective prophage) and/or integrated plasmids (episomes)
What types of transposable elements may be in bacterial genomes?
1) Insertion sequences (IS elements)
- Short genetic elements that encode functions needed for their transposition only
- When multiple copies are present in the cell, homologous recombination between elements can cause variety of recombinations e.g inversions, deletions
2) Transposons
- More complex elements that encode additional functions such as antibiotic resistance
What post replicative modifications of DNA are important in regulation?
Covalent mods, especially methylation of specific bases, can play regulatory roles and act as immunity mechanisms against foreign DNA
