Lecture 8 - Sensing and responding to environmental signals Flashcards
What carbon source is the best for getting ATP and why is this important?
glucose
- links to physiology, growth, energy yeild
- most ATP from a single molecule
What is catabolite repression?
-preferential utilisation of the best carbon and energy source
How can you test if a carbon source is preferntially used by a bacteria?
Growth curve experiment
- plot the bacterial growth curve, when given two (or multiple) carbon source concentrations e.g. glucose and lactose
- if show diauxic growth then the bacteria are performing catabolite repression
- concentration of the carbon source to run out first is the one preferentially used
What is diauxic growth?
when the growth goes in two phases with an adaptive phase between the two exponential bacterial growth phases
-e.g. adpative phase - allows e.coli to utilise lactose expression
What is the molecular mechanism of preferential glucose use when both glucose and lactose are present in E.coli’s environment?
- when glucose enters the cell through EII[Glc] transporter, it interacts with IIA[Glc]
- this begins a chain reaction where Pi gets passed from IIA[Glc] to glucose (Glu-6-P), phosphorylating glucose and dephosphorylating IIA[Glc]
- phosphorylation state of IIA[Glc] transmits information about glucose transport (lots of dephosphorylated IIA[Glc] = lots of glucose)
- In the dephosphorylated state, IIA[Glc] means that adenyl cyclase is inactive, and therefore cAMP is low
- non phosphorylated IIA[Glc] blocks LacY (lactose permease) so lactose cannot enter the cell and Lacl is activated, repressor binds to the LacZYA operator
- the absence of CRP activation (requires cAMP) which would normally assist RNA pol in binding to the Lac operator means that even if lactose were in the cell, the LacZYA genes could not be transcribed
- therefore lacOperon transcription is low
What is the molecular mechanism of lactose use wen only lactose is present in E.coli’s environment?
- IIA[Glc] remains phosphorylated (no final electron acceptor present)
- in the phosophorylated form IIA[Glc] activates AC which converts ATP to cAMP, incresaes cAMP levels
- in the phosphorylated form IIA[Glc] cannot block lacY
- lactose enters the cell and the LacI repressor comes off DNA through interaction with lactose
- cAMP interacts with CRP to bind the regulatory region of the lac operon and activate transcription
- more lacY is produced and LacZYA transcription is activated
What are the features of the Lac operon (lacZ,Y,A)?
one regulatory region controls the expression of more than one gene
- facilitates a coordinated response, advantageous as the gene products are functionally linked
- has a local regulator (LacI at lac operon) and a Global regulator (cAMP - CRP at lac operon (also regulates as many as 359 other genes)
What is the difference between a local regulator and a global regulator?
Local
-has one target
-e.g. LacI at the lac operon
Global
-has many targeted sites across the genome for synchronised regulation
-e.g. cAMP-CRP at lac openon (also regulates as many as 359 other genes)
What is the regulon of a global regulatory protein?
all the genes that fall under the control of a specific global regulatory protein
- e.g. the cAMP-CRP regulon consists of more than 200 genes
- facilitates coordinated expression
Give two examples of second messengers
cAMP
Cyclic-di-GMP
-cAMP and c-di-GMP regulons are integrated
What are the features of cAMP as a second messenger?
- nucleotide based second messenger
- The effector is CAP-cAMP activator protein (CRP-cAMP receptor protein)
- cAMP needs crp to be attached to be activated
What are the features of Cyclic-di-GMP as a second messenger?
- nucleotide based 2nd messenger
- levels of c-di-GMP vary and excert regulation associated with vir
- c-di-GMP acts as a regulator through: c-di-GMP binding protein mediated regulation and riboswitch mediated regulation
- controlled by synthesis and breakdown mediated by sensor proteins
- large range of signals sensed
- e.g. involved in biofilm formation for virulence
What is two component signalling?
Requires: -Sensor kinase (sensor domain(outside)TM domain, transmitter domain(inside)) -Response regulator (sits within the cell, has DNA binding domains, reciever domain)
- can be no signal/stimulus or signal/stimulus states
- Singal kinase autophosphorylates itself and passes Pi onto reciever domain of the RR to allow it to bind to DNA and activate transcription
- RR must be activated/repressed e.g. by phosphorylation
- gram negative bacteria
- large range of gene/operons
- good for sensing items that cannot move across the membrane
What occurs in two-component signalling pathway of histidine?
What activation mechanisms can occur after phosphorylation?
- sensor protein (through histidine kinase activity) autophosphorylates histidine (using ATP->ADP)
- then transfers the phosphoryl group to the aspartate of the aspartate residues of the response regulator, rendering it active
Activation mechanisms:
- inhibition of effector domains by unphosphorylated regulatory domains
- allosteric activation of effector domains by phosphorylated regulatory domains
- dimerisation or higher order oligomerisation of phosphorylated RRs
- interaction of RRs with heterologous target proteins
What is the features of the PhoP/PhoQ regulon?
-PhoQ = sensor
-in E.coli and salmonella
-two component regulatory system
-PhoQ can be activated by Mg2+ and by some antimicrobial peptides
-it passes a phosphate to PhoP
Phosphorylated PhoP:
-activates the expression of 18 genes in E.coli
-represses 5
-regulated genes associated with magnesium transport, outer membrane modification, acid resistance, and pathogenesis
-PhoPQ mutant of Salmonella is avirulent
