Advanced School on Quantitative Principles in Microbial Physiology: from Single Cells to Cell Communities | (smr 3879)
Speaker: Marco MAURI (Jena University, Germany)
of the day with marari and so so thank you okay welcome back after lunch um I’m Marco M Po in Ros lab in yena before we were in Edinburgh so we mve recently to yenna and today I’m going to present something regarding alga bacteria interaction so dynamics of alga bacteria interaction in artificial planton communities uh first of all something regard indana because uh last week many people asked where is yena so yen is a small City in the middle of Germany in the East part and is actually surrounded by now we will try to move a mouse it works yes surrounded by Nature so very nice and in there is this balance of a microverse is an excellent cluster recently established and the idea is to gather together different different expertise uh to study principle and interaction of different microbial communities so uh we study for example microbial commities from the gut in water soil plants and how human interact with them and how they interact with each other so vetan is our embedded environment and uh our group actually focus in different uh topics with different scales so these are just some topics in which I’m involved to we study individual microbes for example we are interested in uh antimicrobial resistance specific uh so we do modeling at the level of micros scales we study also microbial communities here you see for example meba that are interacting with eoli cells and then we scale up also to uh population of microbes for example biofilms and inide this cluster we had the possibility to interact with other people from other groups actually the connection and the exchanges are really uh really nice established and this group from G porer works with algae they are expert in understanding the chemical signal signals that shape the interaction between algae and bacteria especially in aquatic environment so uh they take care of studying this kind of stuff so algae and the chemical release in the in the water and this time I’m talking about this small algae that are algae that were already mentioned this morning and they are diatoms so basically they have an EXO exoskeleton uh that I mean uh constrain the the algae to be unicellular and uh yeah they can do photosynthesis and create biomass from from that so why are they important so also this morning was already mentioned something uh they are in a a soup of um microorganism called phop planton so these Fanon are made by unicellular algae and not only because there are also bacteria with them so they actually interact a lot they are important because they fix up to 30% of the global CO2 into nutrients this morning they said 20% actually I mean it’s just an estimate and this fixation after is able to fuel the um marine food web so it’s the basis of the um food web in marine environment algae have uh a peculiar characteristics so because they change the cycle over the different seasons and these cycling is made by a blooming part after they kind of die they go up a little bit during the summer and uh they do the same in Winter uh they die out and they start again the cycle the next year so vison depends obviously from uh some abiotic condition like light and other nutrients that are around and also diet to have a life cycle and it’s quite interesting and visible because that also change color of over the life cycle so they start with a light green color and if you measure the chlorophile content that is represented here over time you see that they have an exponential phase exactly like bacteria do and then they decay in a death phase or late exponential phase depending on how they they go up or down depending on the condition and when they age they change the color to Brown so this is because the chlorophyll changes the functionality inside the cell and we are basing our research at the moment so what we will see in the next uh upon measuring this chlorophile content because this one is more or less aoxy of the biomass of Al okay so chlorophile content uh is related to the amount of Al that you have in the in your system and uh some motivation regarding the this kind of talk so as I told you this group study uh samples from Marine water uh taken in Germany especially from a Consortium called aquad Diva very similar to T ocean the same stuff and they sample these uh water and they found that there are OB algae and bacteria interacting living together and we are interested in the composition of this uh uh environment that is actually complex because there are many players together and the interaction that defines this composition which are the interaction interaction can be interaction within the same species or between bacteria and algae so for example uh they compete for the same nutrient uh in a bacteria compete for the same nutrient then the same happens also in among a and they have exchanges exchanges that can be positive or negative for example ARG are giving carbon sources to nutrients to grow the same happens also in for bacteria that provides other micronutrients to the U algae most of them metabolized and in some cases there are also negative repression in which bacteria would like to get rid of algae so they promote something to uh down regulate the growth of the AG and that is to understand this kind of interaction obviously ah yeah a question sorry um so they don’t um compete for the same Nutri sources it’s always like carbon Source are always flowing from the phop plon to the bacteria no that’s not completely true okay most of the time uh they have a common source of carbon uh but really depends on the species that you look at uh well bacteria prefer obviously glucose pieces or all this stuff that contain a lot of carbon and while algae are able also to rely on light obviously so that might be a but when the neutron are actually not enough or light is not enough uh they can compete for the same yeah actually fop planton use a lot of other stuff so complex nutrients that are more difficult to be uh internalized by bacteria so can be a competitive uh process also uh okay so we are interested in this kind of interaction and the idea was let’s get simple so sample something from the water extract what we need and reproduce it in the lab uh it’s not so easy because this kind of stuff grow in very complex media and they are not able to grow in a the well established media that we have the lab so usually you do an extraction of uh Marine water you purify it and you use it independently of what inside uh to make B grow and uh to study the the kind of V simple system to understand what are the kind of influences on each other we have couple of question to understand to that you like to answer and uh obviously is a very regarding interaction secondly the Dynamics that is something also not so easy in this case because communities are quite complex and um the first question that was moving this kind of research is a common belief that was there in the in the field that of algae especially diatoms have a universal role what does it mean universal means that if you take one specific diom Bon will have always a positive or A negative effect on this specific bacteria and the bacteria does the same on an Aly obviously if you think a little bit uh it’s very difficult that they might have the same Universal behavior and uh to test whether was real or not we went on with our experiment well I can already tell you that there is not it’s not Universal so I mean it’s not so difficult to guess it so it’s a work in progress because we started last year we just published the first result at the end of last year and now uh we are transiting from uh the more experimental methods to uh the theoretical and the modeling part so I will give you just a glimpse of initial part not everything uh what is the research method that we are using uh if I’m late just tell me stop okay couple of minutes before thank you and so research method are the following first of all as a to you screen by sampling in Marine water for Relevant bacterial interaction why they have to screen because not all of them are able to work to to grow in the lab uh they request a lot of specific abiotic condition temperature uh and shaking condition of salt and so on that are not not very known and you have to screen also because uh you don’t know what you pick up from the water so there are many bacteria that you have no idea what they are they’re difficult to identify and the same also happens for algae although algae are actually much easier because um they’re big so you can see them in the under the microscope they go from 10 to 100 microns so actually you don’t really need to uh do specific processes to identify them while for bacteria is much more complex and then we did a a choice of four bacteria and one datom and we did growth corves first of all of isolated culture so one bacteria alone one one bacterial species alone one diom species alone with the same medium obviously and then we did the Parise culture okay so you might already guess where we are going to so kind of lotter idea and um after the idea is actually develop a mathematical model to predict the community Dynamics and from this one you would like to understand what happen if you put together all these stuff so the four different bacteria and the DM uh once we understand how they grow in isolation and pair wise we should be able to predict what’s going on uh for the whole community so we are creating artificial Community inferring some Properties by uh the single growth C and per wise growth CS how we did this stuff so it’s actually a huge work so something like six six years of work on on these things because uh growing in uh in Le this these bugs are complex and uh Vision was done by uh yundang postdoc in Geor corer lab and what he did was to uh first of all uh check the growth of the of a datom how you do it I already told you basically you measure the should I move this Stu yeah yeah the chlorophile content in the algae H over the time so Bon could be a kind of core that you obtain uh and you do the same in the presence of bacteria so the black one is in the absence of bacteria in the presence of bacteria since there is an interaction uh can have a different change basically the chlorophile content gives you a proxy for the number of cells that you have number of atoms that you have okay and what you would like to do is to compare um the case we with out the bacteria into different time Point why that because there are different obviously um phase one growth phase here and one death phase here and so you measure just basically the difference between this point and this point with and without bacteria and you say there is a positive or negative interaction so it’s a yes no answer at the moment once you do it you obain something like that so they did it for you did it for many different algae different diatoms that you see here in uh comparison with other bacteria that usually lives in the same environment for the growth phase uh that is actually Vis one and late stationary or death phase that is actually Vis one and if you see uh yellow means that the interaction is positive so the bacteria is helping in growing the algae negative means that the presence of bacteria represses the algae and as you already seen from this kind of uh it’s not random but I mean looks like random metrics uh this one is different from this one uh so you already see that probably there is no universality for two reason the first one is that a single bacteria or a single algae is not uh giving the same effect uh on the other partner every time so you see yeah sorry yeah I was wondering about this this measure right because the way it’s drawn it suggests that you always take it at particular times but you could imagine that when they’re together the time Dynamics just is shifted or trans scaled in some way and and then it’s sort of hard for me to say what can happen in your M that’s totally true even even if they go up and down the same height that’s totally true so they choose for different couples always the same time basically the one in which uh we saw that most of them were in the exponential phase so make it more comparable but you are to in the other one a fixed time independently whether we are dying or not so um but yeah you you you’re right so it’s difficult to to infer the same uh uh a general condition uh they just took a single time point to be to have a for screen understanding which uh bacteria and which Al should decide to take in the experiment later so was a force screening but yeah I mean it’s just a proxy to have an idea I agree that is not the best option but they have Dynamics complex Dynamics but this one is shifted after in the idea of studing yeah so I was saying that you see already that a single Al or single bacteria are not having a complete yellow or a complete blue row or column uh so Vis are means already that there is no Universal effect on the species but the uh effect depends on the given bacteria or the given alal partner and then you see also that can I switch oh here exactly so if you compare some of them for example here to here you see that from the early growth phase to the late growth phase you go from Blue to negative to Yellow positive means that the behavior changes most of the time also during the two phases and so depends uh the Dynamics and the the effect on the growth phase uh that you’re looking at so after Vision they choose uh the most representative um actually they’re not the most representative but they are the one that are easy to collect in the and make it grow in the lab uh that are the algae cin discus radiatus that I will call datom because I’m not able to uh yeah there’s a question uh is sorry this that it becomes more positive generally the interaction do you think that’s just because there’s more algae dying and there’s more bursts or am I completely missing something we we don’t know what is actually we don’t know whether it is dying or decreasing for other reason especially algae have a kind of exos exoskeleton so uh you don’t know whether I mean even if a die stays very algae because it’s kind of empty you just have to measure the chlorophile to see whether it’s dead or not we don’t know why happens we don’t know only that in the culture if you sample and you measure it you see less chlorophile we don’t know what’s going on but if you do a life de stain of the algae or a count I don’t know we counted in the microscope that’s how we did it so B basically you see d whether is dead or alive from the color and you can already inir how many cell are there and uh yeah that’s what is done so what the question was whether they are dying so removing or maybe they are uh sedimenting or maybe they are keeping fix the number of cells so we we we don’t know because this stuff is happening in a flask and after we sample out at two time point we are not monitoring everything over time completely but only these two time points and so it’s difficult to infer what is going on in between and I think also vison is the same question that he had before can kind of connected to yeah another are micr allow to change or is the media buffered uh okay the media is very complex actually I don’t know uh the answer precisely but um I don’t think it’s buffered no it’s just an extraction of marine water whatever it is inside because whenever you modify it they tend not to grow uh and regarding what’s going on with bacteria and algae is what we are doing now so we are actually taking also extra of media and uh looking at the metabolites the properties to understand what’s going on so I cannot give you an answer for for B another moment another r no leardo so sorry I don’t think I understood so you’re using like filter sea water or synthetic medium for these experiments uh I think it’s filter yes okay okay no no I mean well synthetic you would know what’s going on inside and you know all the components here we are not sure about everything uh yeah yeah so here are the name that we CH sorry what’s going on here Vis uh so the datum is cisus radiatus just to give you a name and the four bacteria are called for short CS4 Ros A1 cross A1 and cs1 okay so I won’t give you the name for long because I don’t remember them yeah so and is also quite exciting to do in y because the first guy I mean one of the first guy that systematically inspect and describe the shape of the D was cyle and he lived in y operated in Yen studied there so it’s quite connected also to environment inside DN and so how Yun did this kind of experiment basically already told you more or less what’s going on so first of all It Grow separately uh the algae and the bacteria in the same medium uh here you see on the x-axis the days uh of population inide the medium so basically we grow in the medium we sample out we count under Microsoft number of algae alive and also dead looking at the color of the of the algae by ey and um here you can see the number of cells so the concentration and this curve in Orange that is the same curve in all these four plots is the case when Al are growing alone okay so we just to reproduce four plots with the same curve just to give a guide for the eye and then you do the same by adding bacteria and Counting again the number of cell over time of atoms and for example when you add CS4 you see that this is the count of algae in green this one in yellow is again the count of algae this one in red and this one in Violet I think uh with a presence of for bacteria You Can Count also bacteria obviously so uh this is done by qpcr so it’s quite complex uh was difficult to make a standard so that’s why you see up and down in the curves and here you measure the amount of bacteria actually the concentration and over time in Gray again the bacteria loone so CS4 cross A1 Ros A1 and cs1 alone in Gray and with the presence of the algae you can measure again bacteria and you see that you obtain this green yellow red and violet counting what you can notice already from the this kind of plot is quite clear so uh here when you add bacteria to the algae uh algae are grow are growing better so they are rescue in the death phase here instead when you ad algae this guy is basically killing cross a one is killing the partially the growth of the of the algae vison again is increasing vison is doing something very crazy but we are not really able to understand of the moment we have some ins is Shifting the starting point of the growth and also for bacteria this one happens it’s a little bit more difficult to understand what’s going on but Vis one is very clear so basically this bacterium is not able to grow without algae but in the presence of algae is able to grow so probably algae are supplying something to uh necessary to the growth of bacteria uh so they have different growth Dynamics obviously and Depends also on the alal Aging because you see that for example this rescue doesn’t happen here but only on this late uh phase and this only something that we already knew from the first uh uh very first inspection now going to the model very simple lot voltera so we discussed a lot about Lota last week I don’t go in detail why it’s good or not was the only option that we had obviously because we don’t know anything regarding the physiology OB stuff okay so we know something regarding the algae we don’t know much about this kind of bacteria uh about the specific physiology about the metabolite that are uh released and so we use a lot voltera model just to give you an idea again there are the before different bacteria that interact with the datom x is the density of the cell and then you have a growth rate the interaction term that are divided between self interaction and power was interaction and uh uh you can uh I mean you know already that if a term is positive means that there is a positive interaction so one enhances the other one negative there is a repression and zero means that there is no interaction so from F of axenic culture so culture alone glowing alone you obtain the growth rate and the self interaction term nothing special from F of the peras culture you obtain the interaction terms and here there is also kind of a trick because we have to include a carry capacity for Val apparently uh they have uh they are they are using a lot of nutrient so we had to to fix the amount of caring capacity that we reached and once you do that you obtain this kind of interaction so apparently CS4 and DM are repressing each other Ros one is helping datom to grow diom helps cross A1 and cs1 interact negatively with the datom so these are the main interaction and as I told you there are two set of parameters because we have a condition for the early growth phase and the late stationary phase that are different H especially in the late stationary phase is quite boring nothing happens apart from Ros one that is repressing by DM and now the question is what happen if you put actually all together this stuff uh and the datom is the model representing well the the data so this one was just the um fitting of the model and the prediction of the model but we have not tested yet so you can test it and here I represent it again once more uh The Matrix of interaction here times after inoculation of the algae alone or bacteria and algae in this case it’s just algae alone so just to guide the ey for you and then you have V the number of a okay so this one again is the fit of the datom in absence of algae sorry in absence of bacteria and when you put instead all the bacteria together with the algae the model gives this prediction in black and the measurements gave these dotted line so actually we can see that uh they’re quite in good agreement so lot here uh works pretty nicely and to be sure we also did um uh okay here is another stuff yeah um first of all we have also to say that um there are no interaction between bacteria bacteria so no no higher harder interaction and since the model already fit very nicely the data we think that probably per per interaction are negligible although the kind of error that you have are not really able to distinguish between the two cases probably even if the the bacteria very weak interaction you Cann not see them then we check that neutral case so no interaction gave a completely different ASW in the green one here and finally uh you can actually divide the the system in uh excluding all the interaction but two and are this cs1 and this cross a one so this error and this error are already able to give this magenta line that is very night very nicely uh next to the uh black line with all the interaction so only two are leading the Dynamics and actually are the faster Growers so you can do the same for bacteria we can give a prediction for the growth of bacteria because you can also measure them I don’t know what’s happening here H these are the prediction of a modeling solid line and the measurement with qpcr are the dotted line so we are more over Less in the in the same hierarchy since we are out of time I will give you also the summary so here we studied the algen bacteria interaction uh they don’t present any universal uh effect the community Dynamic depends on the growth phase of the algae and the Outlook I can also give you an in about the Outlook we would like to investigate the communities that are more similar to the Natural one but to do that one we have to understand better the physiology of algae the physiology of bacteria and what they uh how they interact so what they exchange and that’s why uh the group of pet is actually the best place to be because they are able to measure all the metabolite inside the the system and just to give you an idea uh that’s the last slide uh the the model Works more or less in this way I mean we’re already uh on the mathematical part but I won’t present the result about Vis one so basically alga are using some nutrients nitrogen silicate especially and light and they release some subst subate that is used by bacteria so bacteria is able to grow in this substrate but over time algae are dying so they age and they produce less and less nutrient they start dying so bacteria at this point are not receiving any substrate they are happy they start dying too and but bacteria are not stupid they release some metabolites that stimulate algae to produce some vesicle that shuttle out some um actually our redox uh product ofo stuff in in the algae so basically you get rid of of what make you old and they get back to be younger at this point we are able to produce more substrate and the bacteria R again and they stop in producing the metabolites so this one is kind of a uh loop obviously depend on the AL bacteria that you have we are investigating the specific substrate and metabolite that are released uh so as I told you is a work in progress with Vis I would like to acknowledge the people that work with me so in yena in Max blank for chemical ecology still in yena our funding agency and I would like to advertise something so we have a uh Twitter theoretical biophysics uh we have also a a podcast so if people would like to listen to the podcast and participate and help us in enlarging this community would be very nice and we have also a population Dynamics virtual seminar every uh uh three weeks online open to everyone uh I know that already some people here already had subscribe and other were speaker by us so this stuff is actually published in a thispaper and we Vis would like also to say say that next week not in two weeks 9 anyway uh we have this uh small one day seminar in one day Symposium in N New Frontiers in model ecology and evolution of microb microbiomes you can uh come to Yen or it’s for free or you can follow it online and for example yakobo will be one of the invited speaker uh so with Reon I would like to thank you for the [Applause] attention there are any more questions yeah it’s very short um I know nothing about algae but I was wondering in your natural samples do you see your microorganisms in like agglomerates in clusters or do they swim freely I don’t know that I I’ve not done the experiment I’ve not seen it I’ve seen just what happen after uh I I know that actually in the the theory is that in Marine water since nutrients are so diffuse and so lighting concentration you must have agglomeration of alga and bacteria because in the other case nutrients would be too much uh disperse and not usable so for sure there are glomeration and this the basic idea of this fop Plankton is that one uh I don’t know if you sample it and you put it in my microscope immediately you see it they don’t create cluster in sense of biofilms or snowflakes or stuff so so the followup question is that you just like you did not observe this forming in your artificial actually everything is very well shaked there so there is a reason and the reason is to keep it simple at the moment yeah thank you uh just a couple of questions one is uh regarding the modeling so you do put bacteria bacteria interactions and they turn out to be negligible uh okay no I’ve have not put it I tried to put it okay it was not fitting uh in any sense because uh was overfitting okay uh so I cannot see any given veror that we have I cannot distinguish them so it’s already done and fine in that way we said no maybe it’s not useful to put it okay the other thing is since in these experiments I know you haven’t done the experiments but since in these experiments you’re using filter sea Waters there’s a possibility that this filter sea water contains lots of fages that might affect the bacteria they purified before in that case yeah they they get rid of the yes they get rid of it yes one more question I don’t know how can no I don’t know I have no idea I have no idea I can I can ask and tell you but I don’t know oh okay I I I know that there is a part of microfiltration several times several layer of microfiltration but they don’t know after how they get get rid of all leaving and interacting stuff I can ask right thanks the speaker again