molecular microbiology L7-9

identifying membrane constituents process?

1. lysozyme breaks cell walls to form a spheroblast 2. use chromatographic methods to identify what they are or french press can be used to produce inside out vesicles then use chromatographic methods to see which proteins are present on the inside leaflet of the membrane

How does the Sec system work?

1. proteins bind sec protein which stops them from folding and translocates them to the inner membrane. 2. they're brought to another sec membrane protein which recognises the signal sequence and uses ATP to push the protein through the membrane through a channel

has 1 target has multiple targets

local regulator global regulator

phosphorelays

phosphate can be passed from one 2 component system to another until the final one does the regulatory job

all the genes that fall under the control of a specific regulator

regulon

how were riboswitches discovered?

used in industry so they were looked for in nature and the were found

two component signalling system -example -what are they used for and in which organism -what do they use?

• Phosphorelays • bacteria to respond to stimulus outside of the cell • use kinases • sensor kinases have sensor domains and transmitter domains. response regulator only binds DNA after signalling for it to do so has occurred. - activated by phosphorylation of the response regulator • sensor protein autophosphorylates then transfers phosphate to asp of regulator activating it

example in nature larger picture

• V.fischeri are bacteria that glow in the dark when in high density together • LuxI : produces acylate signalling molecule • cytoplasmic transcription factor LuxR binds cognate signalling molecule directly and activates gene transcription

quorum sensing -example of quorum sensing -example in gram +ve bacteria -example in gram -ve bacteria

• accumulation of a number of molecules reaches a threshold and a response is activated due to the accumulation of their signalling • biofilms are formed • in gram +ve bacteria autoinducer peptides can control the uptake of DNA • in gram -ve bacteria use acylated HSLs

second messengers -examples

• cAMP - effector is CAP (CRP) • cyclic-di-GMP - riboswitch regulation and c-di-GMP binding protein regulation

regulation of expression after transcription initiation

• elongation of transcription: attenuation (translation, riboswitch, RNA binding regulatory protein) • translation initiation (sRNA, riboswitch, RNA binding regulatory protein • mRNA stability (sRNA) • controlling protein activity (protein folding, interaction with ligand, modification)

designer bacteria - experiment did what?

• experiment reprogrammed E.coli to move to a compound of choice • a synthetic theophylline aptamer was used which controlled expression of the cheZ gene that regulates directed swimming on a plate of different compounds. E.coli moved towards the theophylline sample as opposed to similar molecules

outer membrane proteins - what is the function of the outer membrane - how does the cell know to translocate the outer membrane proteins to the outer membrane -why can't the protein fold in the periplasm?

• function of outer membrane is to allow movement of small molecules into the periplasm this requires porins. (large proteins with hole in the middle) • porins tend to have a beta barrel structure, lending it to being a channel therefore the cell recognises it is to go to the outer not inner membrane • high coordinated process requires lots of proteins to help formation

tangible signals - which carbon source is used first?

• glucose has a higher yield of ATP than nearly all molecules so tends to be primary carbon source

cell membranes - how are they formed

• hydrophobic tails of lipids coalesce to exclude water as a bilayer • alot of lateral diffusion go phospholipids in the same leaflet of a membrane. changing from one leaflet to the other isn't common

glucose metabolism - global effects in the body

• level of cAMP in a cell is inversely correlated to levels of glucose in the cell. this effects CRP which is a global regulator

Riboswitch what domains does the RNA of a riboswitch contain where are they found?

• metabolite directly interacts with RNA, inducing structural changes that result in the regulation of gene expression - effect transcription termination, translation initiation or RNA processing • aptamer (metabolite + sensing domain) and expression platform • found in 5' area of untranslated RNA regions

gram -ve bacterial cell structure

• outer membrane • inner membrane • cell wall • periplasm • high proportion of cellular proteins associated with envelope • macromolecular crowding enables: nucleoid to be condensed, macromolecular diffusion to be limited, protein folding to be stabilised

catabolite repression - what is it called when there are two stage of growth defined by two carbon sources? - in E.coli what are these sources generally? -why is there a lag while the carbon source changes?

• preferential utilisation of best carbon + energy source - Diauxic growth - glucose and lactose • transcription and translation account for the lag in growth between carbon sources

protein assembly in periplasm

• proteins destined for the periplasm are kept in an unfolded state and use a signal sequence at N terminal for proteins destined for aqueous compartment outside the cytoplasmic membrane

alternative system for periplasm translocation

• proteins folded in cytoplasm and transfered via the tat system to the membrane where they move through a hole in it. • this can cause leakage into and from the cell however

protein assembly in the membrane

• proteins have a run of hydrophobic residues which run across the plasma membrane 1. SRP binds partially translated protein to stop translation + transports whole complex to correct location on the membrane 2. translation then continues • hydropathy plots used to identify membrane proteins as there are usually alternating hydrophobic and -philic runs of amino acids

riboswitch example 1 -which molecules participate? - how is it found?

• purine biosynthesis genes - guanine and adenine aptamers • using atomic res. structure of guanine riboswitch + views of ligand-binding pocket of G + A riboswitches it can be observed that - 20,000 x greater affinity for G with G aptamer compared to A

connecting peptidoglycan

• single molecule polymer NAG/NAM inserted into pre-existing wall structure • breaking bonds: autolysin • making bonds: transpeptidases, transglycosylases

what conditions do microorganisms have to respond to?

• temp • pH • antimicrobials • oxygen conc • nutrients

What else can be used to control RNA structure change? -give an example

• temperature can be used • human pathogenic bacteria the gene PrfA controls virulence protein production. RNA can only correctly fold at 37 degrees this means it saves energy by only making the virulence proteins when inside the body

PhoP/PhoQ regulon -example of what? -what is it activated by?

• two component regulatory system • activated by Mg2+ and some anti-microbial peptides • PhoP phosphorylated by PhoQ after autophosphorylation which then goes on to activate the gene for production of the magnesium transporter protein

riboswitch example 2 - evidence of conservation of the sequence in elements

• two different riboswitches worked with the same metabolite (SAM) • SAM riboswitch I - transcript elongation premature attenuation is activated by the binding of the SAM molecule • SAM riboswitch II - acts -vely by inhibiting translation initiation - loop formed so ribosome can't form • conservation of the sequences in elements of structural importance in the SAM binding motif that are found in two bacterial species

secretion systems - example?

• type 3 secretion system (T3SS) -uses a molecular syringe that is able to cross two bacterial membranes + the membrane of a host cell to transfer proteins

GFP - what is it used for?

GFP is used as a fusion reporter protein as shine dalgarno sequence is made available to ribosome by breaking of H bonds in watson-crick pairing

how does glucose - lactose diauxic growth work?

INACTIVE LAC 1. Glucose binds to cell surface receptor 2. IIA(glc) isn't phosphorylated and binds LacY transporter protein 3. [cAMP] low and LacI repressor is bound to promoter ACTIVE LAC 1. phosphorylated IIA(glac) activated AC 2. cAMP increases 3. lac repressor not bound and lacY is made and translocates to the cell surface membrane to increase flux of lactose into the cell

experimental approaches to study a riboswitch or confirm its regulatory role

• examine gene expression in vivo +/- ligand • determine structure of RNA +/- ligand • bioinformatics: predict riboswitch based on predicted structures of mRNA from genome sequences