Featuring four authors from our Open Access journal #microLife, join us as we dive into the microscopic realm with our webinar on bacterial nucleotide second messengers!
In this session, we unveil the mysteries behind Streptomyces‘ response to osmotic stress, shedding light on alternative sigma factors, two-component systems, and the role of c-di-AMP in osmoregulation. Explore the intricate world of c-di-GMP signaling networks, dissecting the criteria for local signaling in Escherichia coli and Pseudomonas. Unravel the unique role of c-di-AMP in Bacillus subtilis, acting as both a survival essential and a potential toxin, impacting potassium homeostasis and amino acid regulation. Lastly, journey into the diverse functions of (p)ppGpp alarmones in bacterial stress responses, unveiling their overlap with the transcriptional regulator Spx in Bacillus subtilis.
Please also read the accompanying Thematic Issue from microLife on Nucleotide Second Messenger Signaling in Bacteria: https://academic.oup.com/microlife/pages/second-messenger-signaling-in-bacteria
Originally broadcast on Thursday 29 February 2024, this webinar features:
Chair: Regine Hengge, Editor of microLife Thematic Issue on Nucleotide Second Messenger Signaling in Prokaryotes
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Speaker 1: Natalia Tschowri, Leibniz-Universität Hannover | Talk title: c-di-GMP and c-di-AMP signaling in Streptomyces development and osmotic response | Author of: Osmotic stress responses and the biology of the second messenger c-di-AMP in Streptomyces: https://academic.oup.com/microlife/article/doi/10.1093/femsml/uqad020/7115319
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Speaker 2: Eike Junkermeier, Humboldt-Universität zu Berlin | Talk title: Local signaling in c-di-GMP signaling networks | Author of: Local signaling enhances output specificity of bacterial c-di-GMP signaling networks: https://academic.oup.com/microlife/article/doi/10.1093/femsml/uqad026/7160452
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Speaker 3: Jörg Stülke, Georg-August-Universität Göttingen | Talk title: c-di-AMP signaling in the physiology of Bacillus subtilis | Author of: The many roles of cyclic di-AMP to control the physiology of Bacillus subtilis: https://academic.oup.com/microlife/article/doi/10.1093/femsml/uqad043/7326004
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Speaker 4: Kürsad Turgay, Max Planck Unit for the Science of Pathogens, Berlin | Talk title: (p)ppGpp and stress response in Bacillus subtilis | Author of: (p)ppGpp – an important player during heat shock response: https://academic.oup.com/microlife/article/doi/10.1093/femsml/uqad017/7110398
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This event is part of a series of webinars by FEMS with OUP
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Hello and welcome to this webinar organized by microlife my name is Regina h and I’m a professor of microbiology at HW World University and I’m going to share this um session so our topic today are second messenger nucleotide second messengers in bacteria and uh there is actually a thematic issue of microlife
Um that will tell you uh the state-of-the-art of This research uh in in the current issues there’s a link on on the journal and you can get all these articles as a package basically and uh this thematic issue on second messengers in in bacteria actually goes back to a
To a um seven year old or long research program that has been funded by the German Research Foundation the FDA and uh where more than 20 people or more than 20 groups in Germany have have done research on this topic so this thematic issue is basically our final report and
As I said it gives you the state-of-the-art of of this issue now what is a second messenger in principle a second messenger is an intercellular molecule in most cases it’s a nucleotide sometimes a single dinucleotide sometimes they are also cyclic but what is really uh important is that second messengers are
Informational molecules that means they stand for something else so let’s say a condition outside the cell or something that changes inside the cell and so they are representative of this change and they inform the cell to actually mount a certain response that is to react appropriately and that means so that the
Cells can adapt so basically second meth Mees are right in the middle of signal transaction Pathways between sensing and and generating a certain appropriate response so they’re really essential and uh their production and their degradation is actually highly Dynamic so there are enzymes at least one enzyme that makes a certain messenger in
Response to a certain incoming signal and then there are also enzymes that can degrade it and it’s the balance between these enzymes basically that determines the level of of the second messenger and then of course the second messenger is sensed by some other component in the
Cell that we call an infector and that actually triggers an output response mounted by a certain Target component and to actually um um produce an adap Adaptive response so basically there are two principles which are important the first is representation and that’s what I’ve already been talking about the
Second is specificity so um because a certain signal has to generate a certain output reaction and in the middle is this second mesenger which is a diffusible molecule but this is actually guaranteed by molecular interaction and recognition and um as you’re going to see uh in many cases it’s not that
Simple but it’s even possible that bacteria have parallel acting responses that react to different signals and generate different outputs but still use the same second messenger even though this is a diffusible molecule and this is kind of a paradox uh but actually cells manag to generate this kind of
Specificity and you’re going to hear in one of our talks now historically the first second messenger that was discovered was cyclic and it was uh in bacteria it was first discovered in eoli which was very fortunate because this is in a nutshell the most simple system you can think of
It’s just one enzyme that makes it one enzyme that breaks it and one protein that can sense it and that’s all basically so that was very good for the beginning beginning but actually in parallel another molecule ppgpp was also discovered and which over the years turned out to be much more complex and
Also cyclic signaling in other bacteria is more complex but uh as a general rule cyclic is usually involved in sensing and reacting and determining the status of carbon metabolism whereas ppgpp has a much more General role basically it links the nutrient availability to appropriate responses with go from
Growth if nutrient uh signaling or nutral availability is great to a non-growth and survival so it’s a general stress signal molecule basically and then actually another molecule was discovered which is CYCC D GMP and this is the one that really made it clear that second messenger signaling bacteria
Is much more complex because about 20 years ago it was discovered that most bacteria that use cyclic DMP and that’s almost all of them that they have multiple enzymes that that can produce it and degrade it so why and how and what does it mean for specificity was an
Important question a few years later then cyc D was discovered and it was over the years it was found that it it’s mainly involved in osmo balancing and um and linking osmo balance to Associated Central metabolic reactions and during recent years actually a number of additional second messenger mole were
Discovered some of them very specific some of them actually playing an important role in Fage uh resistance systems which we’re not going to cover in in this uh session now here but if you’re interested in that um I can refer you to the review that we have written
Uh in that thematic issue so it’s h and and several other authors including the ones that uh who are talking here and there this topic is also covered so let’s get to the speaker so so our first Speaker today uh will be Natalia Chri Natalia here she is she is a professor
Of microbiology at Hanover University in Germany and she is a leading expert on strepto misis molecular biology and strepto misis is a very interesting bacteria because it has a a very complex and interesting life cycle and as you’re going to see especially CYCC die GMP plays a key role in controlling this
Life cyle CLE but in addition strepto misis uses also CYCC DM for osmo balancing and and other P purposes and and this is actually connected so so Natalia give you will give you a very broad picture of the physiology and how this is linked to to molecular regulation and then we’re going to
Proceed to Ike Yuna who is also here and Ike is a a grate student in my group and he works on the specificity problem of parallel signaling bicyclic DMP uh which he does in eoli so this is the topic of of his talk and the third talk will be
Given by yor yor is a professor at hello uh at Goan University and he is an aado of pilosis so Grand positive soil bacteria and and yur is one of the leaders in the field of Cy DM and discovered many of the important functions of cycl DM in basila sais and
The final talk will be given by K toai who is a professor both at Hanover University and also at the max Blanc unit uh for the science of pathogens in bin and in recent years kushad has has been working a lot on ppgpp and its role in the general stress response and in
Particular in in termal tolerance in basilla satellit so these are our first our four speakers and without further using up your time I would like to give the stage to Natalia please so hello everybody um thank you Rina for this very nice introduction into this important topic and the event today and
Thanks everyone for joining so we are many that’s very nice um and yeah so now it’s my pleasure um to present you some of our key discoveries um about cyclic die GMP and cyclic D signaling inisis so um streptomyces are gr positive actino bacteria and these bacteria are
Very important members of the soil microbiome um and soil is actually um quite harsh environment to live in um so they experience here many challenges and develop many different strategies to adapt um and to cope with these challenges so soil is for example a very um nutrient poor environment um but
Sertom misis can survive prolonged periods of starvation by simply switching into the existence um as sport um it is a very competitive environment um and this is one of the reasons at least this is what we believe uh why these bacteria makes so many different antibiotics um so to to kill the
Competitors and of course these molecules are very valuable for the medicine and for the biotechnology and uh soil is actually also an environment that uh where changes happen quite often and rapidly especially in osmolarity because soil is exposed to weather changes so to rainfall and rad and surises um evolv
Many strategies to um adapt to these changes in osmolarity and in all these um key processes um or in all these processes these two molecules that I will be talking about today cyclic diag mp play a key role in controlling cellular response and survival so in the first few slides um I
Will start with cyclic diet and give you an overview about what we know um about the role of this mole Strom Isis and I will then switch to cyclic DMP so when we started um to work on cylic D M um the only certainty we had was that actino bacteria
Um including styes have exactly one so-called di adelite Cycles domain containing protein which is called this a and at that time um we knew from work in basilis that this a is capable of C MP production so it converts ATP to CM but we had no idea how this bacteria
Actually terminate um side mediated signaling because um at that time time um there was no actually known phosphor so the two enzymes that were known from other bacteria they not present in styes and we also had no idea what are the targets of this molecule and which functions uh cyclic di actually controls
In styes so by simply um screening proteins that were predicted to have some sort of phosph activity we identified an enzyme that we renamed atic um and this enzyme actually is a very specific phosph Ras for caamp and hydrolyzes the cyclic dinoti to and once we had the two enzymes we
Started to look um into phenotypes um and we learned that um similar to what we know from firmicutes and this is uh this will be explained in more detail by y inisis side is also um involved in osmo stress regulation because um that this a mutant which has low levels of
Cyclic DMP has troubles um in in growing and surviving in presence of high salt on nutrient AA on the other hand um when we look at the phenotypes caused by high side so um we looked at the atic mutant so this Trin has high levels of this molecule we
Realized that high side MP is somehow connected to defect in development so normally when styes complete the life cycle they produce such beautiful sport chains but as you can see here the atic mutant um produces non-differentiated and very flat and sick appearing highi and this train also fails to produce the
Typical sport pigment that strpis is normally produce so at the moment we don’t have a detailed mechanistic understanding how C MP actually contributes to Salt resistance and in and how it controls development in these bacteria but we um have identified um one side M Target protein so this is a
An rckc domain containing protein that we call CPA so CPA vind side the m and upon binding of the liant this protein interacts with a transporter that is predicted to be a Kon proton antiport and studies from the lab from RE Elliot have shown that side MP also binds to a
So-called yo like ribos switch which is located in the five Prime utr of a gene um which encodes a cell wall hydrolase um and we also know that um most of the stromy strains contain other or additional wi like Ral switches uh which also likely um and likely controls genes that that
Encode several hydrolases so this is basically the context that we know about now that c m controls iron transport and cell wall modulating enzymes um and at the moment we are trying to find additional targets and um try to understand the mechanism behind the the
Function of side NP and this is uh the summary where we are now with our knowledge in this area and now I would like to switch to some GMP and the set of the enzymes that control s GMP is far more complex than the one that we know from side
Metabolism so most of um stomy train in in average have 10 of these enzymes so which have either ggdf or an e domain and our lab model which is trisis Venezuela um has 10 of these enzymes and we know that four of these proteins are um important for developmental
Regulation because deletion of rmda and rmdb which are active phosphorized Rises or cdgb and cdgc which are active d c lases um the this affects or has has a consequence on developmental regulation and um at the moment we know that high cyclic ding blocks differentiation and this is nicely
Visualized here in this image so this is how a cisis wild type so Venezuela wild type looks at the end um of the so when when the spores mature and are formed on this um aerial H and the colony becomes green um however in comparison a strain
In which I overexpressed a Dand Cy leading to high side P this train is heavily blocked in spiration so here we just see vegetative High which are covered in some sort of Matrix material and this train cannot go further in the development on the other hand um a
Strain in which I have expressed a phosphor race leading to low cyclic DP this train um sporulates like crazy so this train even skips one developmental stage so so the spores are not formed on on this aerial highy but rather so they are formed as a mass on on the AG plate
Yeah um so um which I also want to to mention here is that cylic digp not only controls development but also antibiotic biosynthesis in these bacteria so our model for example produces Clon fenical and when we look at the Clon fenical production in a strain with high cagp
You can see that that this string produces much more clam fenicol than the walp so it’s like 25 fold more clam fenicol after seven days of incubation which mean that this signaling molecule can also be used as a um yeah as a switch on device for antibiotic biosynthesis but in the following slides
I would like to elaborate on the mechanism um so so our picture from today what we know about how cyclic DP actually inhibits the transition from multicellular filaments um to unicellular spores in this bacteria and using the um cylic dig capture compound uh I have identified um a developmental Master regulator a
Protein called Bal as a cyclic D effector so we see here the structure of this protein in complex with DNA and cyp and this is a very unique example in biology because Bal binds actually a tetramic form of CP so four molecules of of CP and this is the C terminal domain
Of the protein and this is the end terminal domain the D binding domain and actually when Bal binds ATP it dimerizes and when it is a dimer it becomes active and Baldi is a repressor of um developmental genes so um at high Cogen P BDI dimerizes and represses the
Expression of genes needed for arum formation and spiration so that styes um are AR rested in this vegetative phase so in the second um transition phase so in the aerium formation phase um high side AGP also blocks uh differentiation so the transition from aerial H to spores but by a completely different
Mechanism so here a zigma factor um a zigma factor called YG is inhibited um and Kelly Giga Post in the lab of mar bner has discovered that a diic form of Cogen p stabilizes a complex between an anti- zigma Factor called rsig and the zigma factor Factor YG and because of
This the zigma factor becomes um inaccessible to the arip polymerase and therefore in under such conditions uh late spiration genes cannot be transcribed and the cells remain caught in this stage okay so knowing that um High Cogen P um activates b d and leads to sequestration of YG we would assume that
At a certain time point the Bal dmer dissociates and the YG the zigma factor becomes free so that developmental genes can be can be transcribed normally and the developmental um program can proceed and if we think about this model and also consider cycli d p levels in the
Cell we would um expect that when the bacteria decide to sporulate the levels of the molecule has to drop so we um Quantified Cogen p in the cell and this is indeed uh so this is what we see and this is kind of in line with our model
So in the vegetative phase the levels are elevated to repress poration but then there’s a drop of the signaling molecule to allow the the expiration program um to be initiated uh but then we were surprised to see that in the late developmental stage cylic d p levs rise again and initially we believed
Maybe this is just resetting um program let’s say because the cycle starts with the Spore and ends with the Spore so the beginning at the end actually has have to be the same um but we also considered that maybe there’s an additional effector protein in this late developmental stage
Which senses the second increase of cyclic DMP um so uh again using the cyclic D capture compound um we identified um a glycogen debranching enzyme which is called glyx as a cyclic DP effector which is um particularly important in the late developmental stage because this protein accumulates
During spiration and here we see the structure of glyx dier so this is one monomer and this is the other one and it is an anti- parallel head to tail dimer and you see that two molecules of Cogen are bound here at the end or here at at
These areas of the DI and um the the mo actually stabilizes the dimer and activates the enzyme so and here um is just thin layer chromatography results so where we used purified glyx in presence and absence of Cogen p and glycogen and a sub as a substrate and
You can see that Cogen P activates this enzyme to degrade um glycogen and liix is also a very important enzyme in um the glycogen metabolism in Vivo um so this is quantif ification of glycogen um for example in the mutant and you see that glycogen in um accumulates or we
Can detect increased levels in the mutant uh however in a strain where we overexpress GX um glycogen can be hardly detected and it looks like glycogen is somehow um connected to normal development because a strain with uncontrolled degradation of glycogen due to overexpression of cix this train has
A developmental defect so it does not Spate as fast or as normal or in the same speed as the wild type and this train also produces less viable spores okay so I’m already at the end and would like to give you a little summary um about the sagp part um so
What we know is that streptomyces use three different side GP Factor proteins which control um sporulation and differentiation Associated functions in a specific Aura and this AA is actually determined by the cyclic DP levels in the cell and by the affinities of these proteins for CMP so in the early stage
So during the vegetative growth um high side GMP um um activates both these so the molecule binds to b d the b d dimerizes and becomes active as a repressor and also this cylic dig in this stage uh favors sequestration of YG so this Mo so YG is inactive and
Inaccessible for the an poies but then the SLI dig P levels drop and b d dissociates becomes inactive and YG um can be released and and um become part of the AR polymerous Hol complex and uh initiate Gene transcription and this Aura is uh supported by the fact that
Baldi has actually a lower affinity for S GMP than the RS gyg complex and in the later stages um the Glick ex protein accumulates and this second increase of cogp activates this enzyme um to degrade glycogen in order to give access to the to to to the bacteria to distort glucose
In this polymer okay so at the end I would like to thank my great team at the life University in hanova uh and also all the wonderful collaborators um and YC and d g for funding and of course all you for your attention and interest to join us
Here thank you very much Natalya for this beautiful talk which really gave a summary of I know so many years of hard work in in a very nicely understandable way thank you so much uh and before we switch to the next talk um I I would like to men mention because I forgot
That in the beginning if for any questions you have please type them in the question and answer session so we’re going to collect these uh these questions and uh after the four talks each of them will be 15 minutes we’re going to have a 30 minute minute uh joint discussion session where we’re
Going to u to respond to your questions thank you very much so our next speaker will be AE yuner and he is now uh focusing on CYCC DMP and he talks about um local signaling and that it enhances output specificity of uh of cyc digmp signaling so the floor is yours
I yeah thank you hello everybody I’m going to talk about the topic of the review which we contributed about local signaling in psychic DP signaling networks so a very simple uh signaling model the intercellular concentration of the second messenger is controlled via the antagonistic activity of a single cyclace and a single
Phosphodiesterase and upon reaching a certain concentration within the cell cylic d p binds to specific eector components and thereby activates or sometimes deactivates specific um receptors and creates an output reaction however there are a couple of observations which do not align well with this standard model of of cylic DP
Signaling the most striking one is that one can find a multitude of the enzymes involved in making and breaking cyclic DP encoded in the chromosomes of most bacterial species just to just to to give you some numbers we’ve heard this already from Natalia on average one can
Find uh 10 diagonal cyes encoded in the chromosome of a single species but the numbers can go up to 50 and the same goes for phosph erases in um many or in some model organisms we know that these Co um PGC and pde encoding genes are not only um
Expressed but they are present and active at the same time within the cell also quite frequently um there have been in the literature reports of specific phenotypes of knockout mutations of U um particular Cycles or foder rases which often involved direct protein protein interactions also in eoli we have a
Really interesting finding which is that eoli maintains basically at all time points along its growth really really low intracellular cying P concentrations um of around or which never really exceed 150 nanom within the cell there is however one exception which is a really specific layer within the biofilm
But in general one can say that eoli does not not really increase its intracellular cyc digp concentration to activate the eector or Target systems present in the cell although one can clearly see an output from these eector components also quite strikingly in many cases there are multiple of these
Effector components present at the same time within the cell so the key question is here how can multiple eector or Target systems be controlled in a specific manner if all of them use the same diffusable second messenger in the cytool one uh explanation which has been proposed already many years ago is the
Um formation of so-called local signaling modules and the idea here is quite straightforward um which is that specific Cycles or phosphodiesterases do directly team up with really specific eector or Target systems and a multiprotein complex to form a so-called signaling module within this signaling module now psychic DP gets produced
Right next to its receptor binding site and um based on um the findings which I’ve uh shown on the previous slide um one can or weefine three criteria which have to be met to uh conclusively demonstrate such a case of local signaling within the lab so uh within such a locally controlled um
Module one can find really specific phenotypes for single or specific Cycles or phosphodiesterases which do phenocopy the phenotypes of the respective eector Target systems which are controlled within this module also as I’ve already mentioned there are direct protein protein interactions in in involved so the specificity between the cyclace and
The eector target system is facilitated by direct protein protein interactions and also under conditions where one can clearly see see an output reaction meaning a a phenotype um the intracellular cyclic DP levels can be significantly lower than the eector binding or The Binding affinities of the of the ectors or the intracellular
Concentrations they do remain unchanged in active side mutants of cyclases or phosph rases which however do show a clear phenotype in the case of eoli these strikingly or its strikingly low intracellular concentrations are um are produced by the activity of one uh strongly active so-called Master
Phosphor Ras called PD and PD is in the case of eoli constantly acting within the cytool to or by degrading cyclic DMP and maintaining these really low levels and these really low levels in the case of eoli are also a preit for these local signaling modules to act on a local
Level now the idea for for the talk now is that I would like to guide you through some examples uh of such locally controlled systems and we will see if we can apply these criteria to these um systems most um prominent one or the best studied one um might be the anob
Bacterial cello secretion system and as most of you know the secretion and also um modification of cellulose is allosterically activated by cyclic d in eoli and also many other bacteria celos in eoli is an exopolysaccharide which is um secreted and is part of the biofilm Matrix of eoli and together with
Am curly fibers it forms the The bofilm Matrix here you can see uh such a model which one can use in the lab to study equal biofilms this is a strain which is producing both components curly and cose and you can see here that um this this strain was incubated for 5
Days on lb agop plates which um contains a certain red dye which stains curly and cellulose and as you can see here these two um these two components do form a composite material which in or in in which these cells are embedded and um it has some really interesting patterns of
A buckling or folding up and wrinkling colony and um it also has some interesting uh tissue or almost tissue like properties now um in a variant which is not able to produce cellulose which is or which can be for example achieved by knocking out bcsa the glyos
Transfer race or the um catalytic core basically of the cellulose secretion Machinery one ends up with a microc colony which only consists of the Amo curly fibers and they look clearly different from the strain producing both curly and cellulose you can only see a pattern of concentric Rings now the
Really interesting uh uh finding was that between the 12 diagonal cyes of Eola it is specifically just The Knockout of one specific cycl dgcc which phenocopies this cellulose free macrocolony morphology also I don’t know if you can see it on the screens a knockout of one specific cyclace pdk has
Been found to slightly uh increase the uh cellulose pro pro pro production uh maybe if you don’t see here you just have to to trust me and these knockout mutations in DCC and pdk they do correspond also to active side mutations in the cyclace OR phosphodiesterase
Domains of dgcc and PD now to make this long story a little bit shorter DCC and PD have indeed been found to directly interact with the cello secretion Machinery by interacting uh with bcsb which is a component within this uh or of the C secretion machinery here I’ve only shown the uh key
Components um basically important for this talk the cello secretion Machinery is of course way more complex there are also other BCS components involved but I’m not showing them here and there are also multiple copies of bcsb however dgcc and pdk by directly interacting with bcsb they have been
Found to um or to to function as a local source and sync for cyclic DMP in the activation of the uh cus secretion machinery and this is all this um has also been shown in a um in silico or in a mathematical modeling approach which um also to make this long story short
Showed that the hitting probability of uh cyc dig p for its targets uh the P domain of pcsa and pcse drastically increases if the components The Source sync and target of Cy DP are in a or up on a distance lower than 10 nanometers so this on the one hand this this
Mathematical modeling approach showed that in yes it’s this this signaling princip in in principle works but it also showed that uh the signaling here Works without the formation of a closed micro compartment within the cell so what this means is that CYCC Dent P which is produced locally here um has um
Just in principle two options it can either bind to the uh P domain of bcsa or BCS or it can diffuse away into the cytoplasm where however it gets degraded um via pdh um another example of such a um locally controlled um exop polysaccharide secretion system is the um production of another
Exopolysaccharide by the NFR system which is Al also stimulated in a Cy DP dependent manner so um here this this NFR system it produces a mon based exopolysaccharide however um also to make this long story short we found that this exopolysaccharide is not involved in the biofilm formation which is why we
Are not looking at eoli mic colonies here um we only found out that it’s uh yeah in principle not involved in the in the biof information but interestingly we found that this exop poly zaride is used by a certain phage as an initial receptor for absorption so instead of
Looking at macro colonies we are looking at the ability of the fages to infect eoli which indirectly tells us about the presence of this of the Gin interestingly also here again we can see the same principles so a knockout of one specific cycl dcj in in this case leads
To a plating defect uh while none of the other 12 enzymes or ddcs in equal in in EA showed an effect and here again this plating effect could only be restored VI the expression of the W of dgcg D dcj but not a catalytically inactive variant
Here we also found that D dcj indeed directly interacts with nfrb and functions as a local source of cyclic digp in the activation of the system and here in principle the same um uh the same principles apply which is that this close V vicinity between the source and
The target of cyclic digent P um enable a a specific activation of the system and this also could could explain why nrb although it has a high affinity for cyclic DP or the main of nrb uh why this KD is still 10 times higher than the intracellular concentration within the
Cell so at the D time points where we clearly see an output of this um of this um of the system one thing I would also like to point out here is that the protein protein interaction between um the source of cyclic d p and its target has no further regulatory role between
No further regulatory role than just Pro providing the specificity and the vicinity between the source and the sink there are however examples where such protein protein interaction uh does have a regulatory function and such an example is the control of the expression of csgd csgd is a transcriptional regulator in eoli
Which drives the expression of the curly subunits and dgcc and thereby indirectly controls the activity of the cell secretion Machinery so csgd is a key player in the formation of of equoli biofilms now the expression of csgd is controlled WI a module consisting of a phosphodiesterase PDR a cyclace tgcm and
Ml which is a transcription Factor it is not a cyc DP effector component so ml is not binding cyc d p but we’ll see in a second how this how this regulation works now here um as I’ve mentioned um PR ddcm and ml they do form a or all of
These components they do interact within this module and as long as PD PDR is not active as a phosph erase it um ues or it it blocks the DCM ml or a driven um um activation of expression of cs3d however if PDR get active as a so-called trigger enzyme it releases its
Inhibitory role on the on DCM and ML and thereby enables the um expression of of csgd so here PDR has on the one hand a regulatory role and it functions as a trigger enzyme because or it functions as a trigger enzyme and thereby by a cyclic DP effector component which does
Not bind cyclic DP but it degrades cyclic DP here again interestingly PDR degrades CYCC DP which is specifically produced by another cyl called dgc um with this um done and I would like to sum up what I’ve shown you today so uh we or I talked about the three
Criteria which one can Define for local cyp signaling which are specific knockout phenotypes of specific Cycles or phosph races direct protein protein interactions between cycles and eector or Target systems and binding affinities which are higher than the intracellular Global concentration of cyclic digp and I’ve um told you today that there are
Two types of local signaling on the one hand where the protein protein interaction uh just Associates a local acting Source or s think of cyclic DP to a specific effector Target system and also um another type in which this protein protein interaction has a regulatory function exampled by the
Trigger PD PDR there are however other examples um as you’ve maybe realized in the um in this talk today I was only giving examples from eoli if you’re interested now in in this uh kind of signaling in local signaling you can find many more examples also from other
Bacter in the review so if you’ve not checked it out or if you’ve not read it yet maybe you are now interested in for example um another regulatory interaction between a cyclace in the case of ponas where the LA system also is controlled on a local level one thing
I would like to highlight in the end is that um if there is a case of local signaling within the cell this does not mean that these cells do only uh uh uh sit in this local signaling mode so there definitely is a switching from a
Local to a global from a local to a global signaling mode um for example also in eoli and um something which I’ve not told you in due to the interest of time is that specificity can also be achieved by fine-tuning the global inell levels if you’re interested in that also
I can highly recommend to check out the review um and with this I’m done I would like to thank of course all of the members of our group foremost of course Regina for being my pi and R for being an awesome labmate and all of the
Members of reg’s lab from the last uh or the the many years in regin’s lab which um did all the work and uh which I used to or which I um summarized here so and also I would like to thank you for your attention thank you very very much a for
This beautiful summary of a very difficult topic um we’re actually accumulating questions this is very nice and we’re going to answer them at the at the end of uh the talking session okay so let’s switch to the to the next talk um that will be given by yor
Stul and Y are you ready yes okay okay so this is a talk about cyc die and why this is actually an essential uh nucleotide second messenger and what it actually does in basilla cist so please go ahead y okay thank you very much Regina for the introduction and thank you for the
Organizers who put together this nice webinar well we are studying pyic DMP signaling in theosis a gr positive model bacterium this molecule has been discovered in 2008 by gor V whom you can see here in unic and the enzyme is synthesized the the molecu is synthesized by enzymes that are called deaden
Cyclases cyclic DM is present in most gr positive bacteria in many gram negatives including cyano IIA also many proteobacteria but not in enterobacteria like eal and it’s even found in some AA cylic DM is essential for filus and upon accumulation in the cells it can become toxic so we DT it in essential
Poison cyclic DM can also be secreted from the cell and you know many C positive bacteria are pathogens like stus orus or leria monocytogenes so we do not our body does not produce cyclic DM and if cyclic DM is released by the bacteria then our immune system
Recognizes the molecule and knows now we have to deal and to fight in invader okay in theosis there are actually three enzymes that can produce cyclic DM this a which has already been briefly introduced by Natalia which is also present in stepto misis cdaa and the third enzyme CD is only
Active during spiration in the C and there are two enzymes to phosphodiesterases which can degrade cyclic DM and they are shown here and you see several of these proteins are located in the membrane now the key question and what really triggered my interest in cm is what makes this molecule essential and
Also what makes it toxic and what we saw is that cyclic demp might be essential because it stimulates the AC ity of an essential enzyme like for example enzymes of Salo biosynthesis and alternatively it could also counteract something that is toxic and this we know for rna3 in eoli this
Is essential because it degrades a toxic RNA molecule and of course it could also be a combination of both now but now the problem is that none of the known targets is essential and also none of the known targets is toxic so what might be the reason so one
Might be that the essential target has has has escaped detection the second that essentiality and toxicity of cyclic D are the two sides of the same coin and that cyclic DM might control a complex process that is essential but must be limited at the same time and I will show you that it’s
Exactly uh the last two points that are correct and what we found and also others is that the control of potassium homeostasis um is the essential function of cyclic D in bosis and for other gr positives we can also say it’s the control of osmo homeostasis and potassium is one of
Them so if you look at Target molecules of cyclic DMP then you see there are quite a lot of different proteins and there’s even an RNA molecule a ribos switch so cyclic DP binds both proteins and RN and what you also can see there are two potassium up systems the
Potassium export system and the ribos switch that controls the expression of potassium Transporters and then we have osoi Transporters and two signal construction PS so what one thing that is already interesting is that cyclic DMP is the only second messenger that controls the biological process and this is potassium
Upcap by binding a enzyme or transporter in this case and the ribos switch that controls its expression so two completely different molecules both have evolved to be controlled by cyclic IP binding now we thought we should have a closer look to potassium and cyclic DM and it
Turned out that in indeed at low concentration of potassium in the medium the cyclic DP concentrations are rather low and at high concentrations there’s also more cyclic I and here we have strains where only one theate Cycles is present and you see the same result so we can conclude that potassium
Availability is the primary signal for which Cy DM serves as the second messenger now we also have observed that in fact it’s potassium that can be toxic for cells that lack cyclic DM so we have is two strains a wild type strain and a strain Laing cyclic DM we cultivate them
At low potassium concentration everything is fine over time now if we have a high potassium concentration you see after 5 hours the cells of the strain lacking cyclic DM they are all ghost cells so they have died so potassium is toxic in the absence of cyclic Di and this brings me
To a small model so if there’s a lot of cyclic d a lot of potassium in the medium there’s a lot of cyclic D which in fact activates potassium export systems and inhibits potassium uptake systems and all their expression so that the amount of potassium in the cell remains limited
Now in contrast if the cyclic D if the potassium concentration is low then there’s also a low concentration of of Cy DM in the cell and then the molecule cannot bind its interacting proteins and we have a effective uptake of pottassium so that the cell get enough potassium in
The cell so we can say that cyclic D EMP inhibits toxic potentially toxic potassium export uh uptake and it’s required for the export potassium P now let me turn to something different there are two signal transduction proteins D A and D B that don’t have any specific activity like the potassium
Transporter so these proteins only serve for signal transduction in the case of P2 of d a it’s a so-called P2 like protein the largest class of signal transduction proteins that are particularly important in nitrogen regulation and are B is a protein composed of so socalled CBS domains that are known to bind Adine
Nucleotides and we have identified interaction partners of Da and we found two pot candidates the Rel is the PPP PP gpp synthesizing and degrading enzyme and big a is the Pyro carboxy which replenishes the TCA cycle and we did an alternative experiment a just a control experiment
Where we um did a pull down of proteins of pilosis using a so-called streptactin colum to which biotinated proteins can bind and in billos there are two biotinated proteins so they both bound of course to our column but there was one additional protein and this was theb
And of course now if you see this here it’s obvious that this is most likely mediated via the interaction with the Pyro carboxilate so we can conclude that the Rel and pig a proteins are attractive candidates for further study and we did this and here we have an interaction study between strap Tech
Re and d poim and what you can see here the interaction takes place in the absence of cyclic DM we can detect dby but if cyc DM is added there is no daby so the interaction corresponds to the availability of cyclic DM and specifically cyclic DP inhibits The
Binding of w tol a and this has also been shown by ITC experiments now I told you that cyclic DM is the second messenger for the availability of potassium so we had a look at the link between potassium concentration and the interaction between the two proteins and again it
Turned out that the interaction takes place in the absence of potassium or very low potassium concentration and the interaction is very weak at a high potassium concentration so this now tells us that da a binds to Rel a in the absence of cyclic DM under conditions of potassium
Starvation and this brings me already to the model so at low potassium concentrations the apod dby without cyclic DM binds to Rel and we have also shown that this triggers Cy uh ppgpp synthesis and inhibits the hydras domain so that the ppgpp concentration in the cell increases so we can conclude note that
Potassium starvation triggers a stringent response in a c the dependent Manner and this makes a lot of sense because potassium is required for many cellular processes like translation in the ribosome and if there’s not enough potassium then proteins cannot be synthesized so it makes a lot of sense
That then uh this is perceived like amino acid starvation just in another molecular way and I told you we also saw an interaction between theb and the Pyro carboxylase and what we have shown before is that potassium starvation requires the synthesis of positively charged amino acids because
Potassium is the counter ion for the negative charges of DNA and if there’s no poten iium then the cells needs other positively charged molecules to buffer the negative charge of DNA and these These are positively charged amino acids and they are made from glutamate which
Is derived from the TCA cycle so it that means in the absence of potassium we have a high demand for glutamate and this means we have a high demand to replenish to refill the TCA cycle and now you see a essay an entic essay of pyate carox
ASE uh in the absence and presence of the B and we can conclude that D B again the cyclic DMP free form the ared in activates Pig a under conditions of potassium starvation and now what is really interesting in is that a link between cyclic DM and pyate carox has
Already been described in leria monocytogenes but there cyclic DM at high potassium concentration directly binds the pyrovate carboxylase and inhibits the enzyme and the outcome is that at high potassium concentration we have a reduced p a activity in billos the the mechanism is different here it’s potassium starvation which needs to
Enhance expression of pyate carboxy and enhanced activity of the enzyme but the general logic behind this regulation is very similar we have a link between the extracellular potassium concentration and the intracellular activity of the bovate carox so this shows that even in very closely related organisms the specific
Mechanism can bit can differ a bit but the logic often Remains the Same so now to sum up I can say that um cyclic di or that the control of potassium homeostasis is the essential function of cyclic DMP in pilosis at low potassium concentrations that the interaction between abodi and Rel Triggs the
Stringent response and arodi also activates the Pyro carboxy to refill the PCA Suter and so we can conclude that cyclic DM really controls several aspects of cellular homeostasis and these are the people involved in this work Larissa Kruger Jan G where PhD students Dennis Vick still is and castina as a technical assistant
Who was involved in this project over the years and we have excellent lab members and collaboration Partners in gingan in hanova in chal Frankfurt and also in the United States without whom this work would not be possible and of course I’m also really grateful to Regina for putting together this
Fantastic um SP Pro program which was funded by the DFG and with this I’m at the end and would like to thank you for your interest thank you very much y for this beautiful summary and talk um I I would like to say this talk also nicely
Illustrates where the whole field of of second messenger signaling is actually moving to so we’re no longer looking at single processes only but now we look at the connection between the processes so so basically the field moves towards the systemic view of of regulation in in the
Cell so this is really interesting and uh you you also gave the keyword for the next talk by mentioning the relationship between CYCC DM and ppgpp so the last talk will be by K toai and this is about ppgpp again in basillo CIS and now this
Is about the heat choke resp resp and I should say that ppgpp has been known for a long time it has mainly been studied in eoli and it was it was known to bind to AR polymerase but in recent years and especially in basil so many new functions and above all mechanisms have
Been discovered in basilla St so this is an example here and so kot you can start okay so um I will go on after the nice introduction from yor and the general theme of stress response and ppgpp was already highlighted by Y and we are more
Interested in or my lab Works more on the heat shock response in basilo satus where we also look as Regino already said in processes like thot toolerance and thmo resistance and heat shock response is all about at least what we look on is proteins and proteins
Uh have to get folded to be functional and upon protein folding stress like heat proteins can unfold and misfold and then they can form protein aggregates which in general is bad for the cell and uh it helps them or induces that they cannot function very well anymore and here I
Want to show you an experiments where we took the subtler cells R them at 37 degree where they can grow perfectly and then we uh edit a heat stress or Expos them to a heat stress which is uh very high 53° in this case and then you can
See that the batler cells are dying so they cannot respond strong enough against that heat stress but if we put up a little pre-shock at 48 degree which is heat but um can be stand by the cells very easily then what we see is that almost all the cells survives the
Otherwise toxic heat stress at 53 degree so by putting a little heat shock at the beginning um we kind of prime the cells they induce a kind of response to the heat shock which then allows the cells to survive in otherwise toxic heat chock and here you can see an experiment where
We just looked at the proteins and prepared the protein aggates the pellets here and you can see uh if we have the 53 degree stress then the cells produce a lot of agregate protein aggregates and when we have the 48 degree pre-shock before the 53 degree then we don’t see a
Lot of uh agates and we can also detect these agregates by FL sense microscopy with a marker like this small heat chock marker or here in DH gfp and we can also see this dark face uh in um cells and a normal microscopy okay so what is what are the
Processes which um go against this and this is called the protein quality control system or the heat shock response and one important heat shock response is regulated by a repressor called hrca and hrca is a repressor of an operon or of different operons which en quote chaperons like gr l or DNA k
And it’s a repressor and when heat is going up hrca um is aggregating and uh the chaperon are induced interestingly one of the chaperons glory L can then activate hrca uh again and turn off the system but once there’s a lot of stress the CH L hydrates away to the aggregated
Proteins and thereby the system is always on and running unless enough Sheron are made and then um the whole system is turned off again another important system is besides the chaperon which can repair and refold misfolded proteins and also inhibiting aggregation is a system where there’s proteas which
Can recognize help by adapter proteins and kinases mcsb and mcsa which um then can Target the uh the transcription factor which can also be inactivated just by heat and thereby again these proteas complexes are made they can Target unfolded misfolded protein or protein aggregates which can get
Unfolded and then also degraded and this is all controlled by this transcription factor or again a repressor ctsr which um is then targeted also to CP or degradation and thereby we can also control in a similar mechanism of a titration model the activity of this proteas which can then remove protein
Nega Gates and can also um uh repair misfed proteins okay there’s another system called Sig B which is like the general stress Sigma factor and billos which en quods also a lot of many genes and this is all controlled by a sigma B is a sigma Factor that’s controlled by anti
Sigma factors and anti anti Sigma factors which can also sense environmental stress or energy stress which can also prepare the cells during heat stress heat shock response against um stresses which come from the environment and from the outside and the last but not least system is also a heat chock response
Actually also an oxidative press response which is um mediated also by a transcription Factor an unusual transcription Factor SX SX is usually Target by an adapter protein to another prote system clip XP and is getting degraded there but when there is heat or oxidative stress this adapter protein
Goes to an Aggregates and cannot Target SPX anymore and then SPX is active uh it interacts with the alpha CDD of an R a poas and it can turn on the SP SPX regulons which are Sharons and Cyro oines but it can also inhibit the transcription of proteins which are connected to
Translation and here you can also see in an experiment that the Delta sdx knockout has a terot toolerance phenotype and you can also see that in a Delta SX the priming doesn’t happen we get a lot of aggregated proteins which is not good for the cell also toxic and here’s an
Experiment which I like a lot that we put SPX under the induction of an ipdg inducible promoter and then we follow ribosomal RNA and when we add the RPG SP SPX is seduced and the ribosomal RNA transcription is is um Turned down and at the same time the transcription and
Translation of Redux chaperon like C redox on B is turned up so SBX is really a kind of dual role that it can induce heat shock and general stress response and the oxidative stress with tricks A and B but at the same time it can downregulate translation on the
Transcriptional level of our RNA operons and roral proteins a transcription of the genes of them it also has a quite uh biotrophic influence on other processes in billos okay and when we look at the transcriptor changes during thermotolerance in billus then um we can clearly see that SP SPX regulated genes
Are regulated very highly under these conditions they are both down regulated as I just mentioned in Upper regulated genes and um the the other interesting part what we saw here is that many string and response gen were affected under these conditions in addition to many translation regulated genes which are also downregulated upon
Um the um thermotolerance conditions is heat shock um with a pre-shock okay so what is String’s response you already heard from from York and others that we have here ppgpp which is uh can be active as pgpp ppgpp or PP pgpp and it’s a an alamon which is synthesized in billos satus by
Synthetases ppgp synthetase there’s first of all re which yor already mentioned which has a regulatory domain a hydas and a synthetase domain and we also have small synthetases ra p and ra Q which only has the synthetase domain which can also synthesize ppgp and um here’s a little scheme where you
Can see the model on um what we think how that system works is that um when we have amino acid station we get a lot of uh many Dil trnas and these Dil trnas can interact with well and then real can go to the ribosome and sits on the
Ribosome and there its caste domain is turned on and it can make ppgpp while when it is off the ribosome most of the time the hydrolac is on and ppgpp actually get get uh degraded so small synthetases can also synthesize ppgpp and we also know of the hydrol lasers
Which can uh change a ppgpp and a pgpp for example we um know that in our system these are not so important and what um y already just mentioned is this well protein which in his model can interact with star a with the star a protein can form this dier and then of
Course it cannot also not go to the citas to the ribosome and CTI um ppgpp and so it it’s a sensing system so this ra protein can apparently also sense other protein which by direct interaction with well then can also control or interfere with the or use the ppgpp
System okay and normally here’s just a little growth curve and you can see when the bad cells get into stationary phase ppgpp goes up and uh the reason for that is that nutrients are getting scarce once nutrients are getting scarce one way to react to it especially when you
Go into stationary phas is to uh shut down translation that not because the cells stop to grow stop to uh differentiate and that is an important way that it’s a kind of break which slows down for example translation but also other processes in in these cells under other conditions when there
Are not enough amino acids also amino acid czes can be turned on and uh here you can see um little scheme so when string responsive billos is turned on it can first inhibit transcript replication it can inhibit translation at many steps because ppgpp can bind to GTP Asis and
Inhibited the GTP Asis with this additional dual phosphate there and um it also interferes with the synthesis of GTP in the cells at the GTP level can also interfere with the transcriptional regulation and they also other proteins called K and uh so therefore inhibition of purin and we now
Also know pyramidan bio citizens can appear where pbtp the transcription can be reprogrammed to Copus translation and of course if there is an amino acid if there not Amino of amino acids there then the amino acid czis can also be turned on in these cells okay so what happens in terot tolerance
When we get rid of ppgpp and we could observe that it didn’t have a big influence on terot tolerance or Tero resistance but we could observe that there were more higher translation and you saw stronger formation of agregates um and normal uh when you switch to 53 degree under strong stress which could
Be alleviated why versus pre-stress but most interestingly when we made a mutant we could make different ways and just one example where we deleted well which is see only hydol and therefore we get a lot of ppgpp in these cells and what you can see is that all these cells with
High ppgpp level can basically survive this strong heat shock and only the wild type cell cells are dying under the 53 um each shock stress and the other interesting thing is we almost saw no eates in these cells so they have higher PPT PP list which increased results in increased
Thermotolerance less aggates and they seem to appear always strant okay then um we looked at the translation we could see that in Y type cells if you just grow them we can measure translation and when they go actually to 50° they grow even faster you see a higher translation
And when we look at this in ppgp Z we even saw another additional increase of translation um especially in the beginning when the uh 50° started the growth and under the real mutant we saw lower trans ation in in general so ppgpp when it goes up can shut down translation and
Um uh we also observed that in the real mutant we saw here we look at the ribosome profiles in billos in in cells and it’s sross gradient and then you can see the 70s the 50s the 30s and here the chomes which are translating and when we
Have a real mutant we see much less of a translation and when we go to the 4850 C to the terance conditions we also see the formation of so-called diomes or 100s ribosomes which get dimerized and this dimerization is also depending on ppgpp and this is a kind of ribosome
Which is parked because the translation is sh is shut down but the ribosomes which are not translating can also be put in in this um yeah form where they can be reactivated when you want to come back later to translation okay so as ppgpp really
Turned up in an an up on heat chock and here we can see that at 50 degree we see a induction of ppgpp which then goes down again so the it goes down after 10 minutes and this is an an induction of ppgpp adding normal novaline or here is a data ra
Mutant and the other interesting thing we observed was that under heat shock conditions we only have this brief short peak of ppgpp and we could see in all our results we did all kind of experiments to test that that these processes were not uh um affected by the short pgbp
Cause but the translation was really strongly affected and uh one reason could be I just showed you that ppgpp is cized on the ribosome by real sitting on the ribosome so could represent also um a kind of syn or local synthes local induction of ppgpp because only a
Percentage a low percentage of the ribosomes actually can interact with ra and they synthesize this ppgpp but around them are many other romes which then can also sense ppgpp so in a way it’s a little bit like the this local synthesis and local syn model we heard
From AA and uh from Regina which uh can Happ for second pgmp and here in a similar way we have the activity of pbgb kind of under heat stress conditions only on the ribosomes and we also know that only red is important for the PBG synthesis up on heat stress in the small
C days don’t produce that much more ppgpp because of this okay and then we also were interested because we know that sdx is also downregulating translation whether there’s an interaction between them and in if you look at ppgpp Z the growth is not really affected it’s almost like wild type at
50° Delta SP SPX has a a gross effect but the the combination of ppgpp Z and Delta s have even a stronger um gross defect which kind of argues that these two system might interact they they appear to be independent but both uh uh can also be involved in the down regulation of
Translation so our simple model is in a way that when we have normal grown cells that um translation is up rabal prot are made heat shock proteins are down then the heat shock comes up it’s sensed by the different systems I just introduced as thex HCA ctsr Sigma B the protein
Quality control capacity is induced because the number of sherons and rux shroads are made but at the same time ppgpp is induced and SPX inhibits a transcription of translation related proteins and PPG gpp on a um direct level uh on the protein level post translation you can in take with
These gtpases in the translation and can thereby inhibit translation and the inhibition of translation under stress conditions of course very important because if we wouldn’t inhibit the translation under these stress conditions we would have many more proteins made but then under the heat conditions they would just agregate and
They wouldn’t become functional so so if and also for the Shon systems you just reduce a load of proteins which could misfold or Aggregate and thereby also allows the Shon system to work work better and more efficiently on the already misfolded and aggregated proteins without getting um new proteins
Synthesized at the same time which would increase the load and uh would not help the protein quality control system the protein hom stes under these stress conditions okay this this I’m finished I would just want to acknowledge Shan rund who worked a lot on the SPX system
Hinrich worked on SPX at ppgpp and now we have Christina and Fabian which also work on heat and ppgpp systemsis and maybe in another seminar later I can talk about their work and Ingo and Drina also work together with Engish and U help a lot okay with this I’m ready to take
Questions okay thank you kirad very much for your talk so you can see how complicated it can get with second messengers it started very simple but now we are really at at a very complicated picture so thanks to all the uh the speakers we have a number of
Interesting questions in the talk uh in in the question and and answer um session now um we are a bit over time but we have time for a few questions so some of these questions are very specific others are a little more General so I I’d like to start with a um
A bit more general question that is maybe something for um for Natalia and or and or IE to answer so what is uh generally considered to be high or low concentration of cyclic die GMP or cyclic die so because we always speak in these relative terms what is high and
What is low and what does it depend upon uh that we call a concentration high or low okay great question that I actually answered by typing H yes unfortunately inisis we don’t know the exact concentration of these molecules because these bacteria change the morphology the volume of the cell and it’s very
Difficult to calculate but in cisis we we estimated it to be in the low microl range so let’s say up to 5 microl which is already high if we compare it to 150 nanomolar in a cooli so I would say this is the range we are about talking about high and
Low yeah maybe I can add something to it because I have also worked on that for a long time I mean um um what is really what really matters is actually the KD of the effector systems so if you have a concentration which stays way below the
KD you would need in order to the concentration you would need to activate based on the KD of an effector then we talk of a low concentration and a high concentration is basically a concentration that is beyond the KD for certain effectors so so this might be very different in different bacteria so
So like in Eola it’s very low um and um and what we can consider high in eoli would still be low in strepto misis for instance because it depends on the KDs of of the of the effect Vector Systems then I have here another interesting question um this is one something for Ike does N4 phage Target the nfra directly or other nfra Associated proteins because you have talked about um that its primary target is um the the polysaccharide yeah so um the Fage does
Um so this is like a like a general mechanism which can often be observed for for pages to initially basically reversibly bind to a glycon on the cell surface and then find in a second step a most of the cases protein receptor and this is most likely also the case for
The N forage so it binds in the first step most likely the exopolysaccharide secreted by the system and then in a in a second step the n a protein and um this has been shown many years ago that it indeed binds um to the um to the N
Protein so this has been shown in VTO and um we based on on our results most likely um consider that it binds also the exite okay thank you I there was actually another question for natala but it seems that natala has answered the question so they have disappeared but I
Think that might be interesting for a general audience as well and there was a question about clickx so whether this is general that clickx is targeted by cycl DMP or can you recognize that at the molecule um in other strepto mises Etc yes yes that was a question from khet
Who is here so um yes so we actually purified GX from Eola which also has it right but it does not have the binding signature and it also does not bind side GP so this is what we tested uh but some stripto mises have a second copy of big
X which does not have the motive um but some of them are not even expressed so there are these two types of gigs with the motive and reaction or binding of CP and without and all the stomi trains that we checked have one version of the protein with the side side
Binding signature and also a few other actino bacteria so this is more something seems to be specific for actino bacteria okay thank you um then there’s a a question for York about cyclic DM and its interaction with ppgpp so if cyclic DM regulates the levels of ppgpp how do you justify the activation
Of the TCA cycle secondly does this a have has any role in sporulation in basilla species as potassium concentration also impacts biofilm and sporulation machinery okay yes uh this a was reported to have an impact on the onset of of sporulation in response to DNA damage but this is a field which
Certainly needs more research um the first question was how we justify the role of cyclic D and the control of the TCA cycle right yeah okay yes was it what was the first question the first question was is cyc D if cyc DM regulates the levels of ppgpp
How do you justify the activation of the TCA cycle okay um one thing is that genes of the TCA cycle are also activated by the stringent response for example this very Gene of the Pyro carboxy is subject to a positive stringent response so if ppgpp accumulates also the pyrovate carboxilate gene expression is
Stimulated and um we know if pottassium gets low then the cell really is only viable if if it gets excess to other positively charged molecules and the way to to get access to such molecules is the production of positively charged amino acids and this works via lutar
Made in the first part at least of the TCA cycle okay thank you y um then I have a question for K this is about SPS uh and its role in redo homeostasis and and the question is have you looked into the levels of glutathione and other essential sulfur metabolites that maintain Redux
Homeostasis in response to speaks we can’t hear you Kad switch On um we did not look at gluta directly but we looked at thyoxin and they are upregulated and they’re really important then also for the repair because there’s an important thing that cins which get oxidized can also stabilize agregates and we also have I think a nice experiment where we grew the cells
Anerobic and heat chock them and when you heat CH anerobic uh you have much less Aggregates so the oxidation is another important part of the stabilization and formation of of Aggregates and therefore the whole SPX pathway which is also Cy oxine and also diesi metabolism is quite important for that
Okay thank you and uh another question for Natalia in the context of osmotic stress what specific changes in gene expression or cellular processes are modulated by CYCC DM signaling in strepto misis so this about the Targets effector target system and output and osmotic stress conditions yeah okay in the context of osmotic
Stress um well so we we did some aresc experiments u i mean harvested cells from these plates and there are many many different genes that are um differentially expressed including for example ectoin biosynthesis genes which is an osmoprotectant for example but I think uh it is also relevant how
Proteins are affected on protein level on activity level and stpes have many different Iron Transporters so really a big set and we are trying to understand how all this fits together within this cyclic diet signaling network uh but definitely um osmoprotectant systems are also affected and we will look into this
In more detail in future I think it was a good question about signals that activate the enzymes Regina oh yeah that was a very general question actually for all you can answer so this is difficult to answer uh so basically all these or most of these enzymes that make a certain a certain
Make or degrade a certain second messenger have sensory input domains so they sense different things and and you you name it I mean it can be almost anything yeah that can be sense different molecules it can be light it can be membrane tension it can be another protein a specific state of
Another protein so so it’s a whole zoo of possible inputs yeah so so it’s hard to give that question to someone yeah I think actually y had a nice example with the star a d b the interaction of the two Pathways and that say it’s protein protein detection then in the end
Between well and this new protein which can sense the other metabolite so yeah but that’s more of the output for the output the question was more about the sensory input yeah that’s more difficult yeah yeah and that’s actually the uh in in many cases it’s the it’s
The main thing that we don’t know about these enzymes what what they actually sense what they respond to that’s where a lot of of research is actually going on and then there’s also another question that I found here in the chat and and this is um actually so yor
Mentioned that cyc die under specific condition in specific cases can be extracellular seen from the perspective of the bacterium and now there’s a question where somebody asked can you ever find cyclic D GMP also outside the cell Natalia do you know any example because I don’t know of any I well if I
Remember correctly there was this dict to steum uh case right which has this transporter that can um I mean the the very low UK carot reartic system that produces side and can ex export it but otherwise I think the side Transporters I mean this is we don’t
Know and the mo also cannot diffuse through the membrane right due to the um because it’s negatively changed structure and size and so it would not go spontaneously through the membranes much less across two mens in in a gram negative so maybe y can comment on the
Cy D case well there’s just one little thing that I would like to add so in fact cyclic DM can be inced and it is inced by pathogens and I as I mentioned it’s recognized by a protein of our immune system system this poin is called sting
And what I really find extremely amazing is that we got this stin proteins from bacteria so originally this was kind of designed as a protein that bacteria use to fight against Fage infection and we adopted this proteim The Sting Pim who sends bacterial infections to sense cyclic DM and in
Fact it can even sense cyclic D GMP yes and use it to stimulate our immune system so this is really something I find absolutely fascinating and unfortunately I didn’t have the time to mention it on the talk so I’m happy I have the chance here yes so so this is
Really a nice example that a part of our own human immune system is a descendant of a bacterial immune system that bacteria used to immunize themselves against pages so it’s really really amazing yeah so I think we are we are already a bit overtime so maybe I have
To close the session now uh I would uh like to thank all four speakers for their wonderful presentations and everybody who joined in for uh for staying for the whole session and for those of you who could not make it for the whole time there there will be a
Record of it and it will be available via the website of microl live so thank you very much to everybody who was involved and also for the people who did uh all the technical work in the background whom you could not see thank you very much I’m happy that everything
Worked so nicely and I hope to see you soon again at least the speakers okay byebye thank you byebye take care