Hey, how you doing? So, we are going to finish up transport today. And today, the thing that we're really going to focus closely on in terms of transport is this very specialized transport system found in gram negative bacteria called the toneb transport apparatus. Okay? And we're going to walk through that transport system molecule by molecule so that I can better explain how this system works to transport a very uh defined set of substrates in bacteria. Some reminders, you have a homework assignment on ELC. That homework assignment is due tomorrow by 11 p.m. You should be able to finish the homework assignment with the material that you received today. There's at least one question on there that you probably can't answer effectively without the material from today. And then you have a paper online. You will need to read that paper prior to Wednesday. You'll also need to read it to actually get a good idea of what I'm asking in those questions on your homework assignment. If I can be of any assistance as you try to dissect what's happening in that paper, please do let me know. Any questions about anything, especially with your first uh homework assignment too. Okay. Oh, question over here. Yes, please send it to me by email. You can print it out if you want and hand it to me. You can do whatever you want, but I usually grade them electronically and then I will send them back to you. Last thing, starting next week, our departmental seminar series will begin under general classroom information. I have uploaded a uh PDF file that has a list of all of the seminar dates. They will be held in the bio the biological sciences building, which is now Cedar Street building B. and they are usually from 11:10 to noon on Thursdays. I will upload a one page or a a sample um summary online. Essentially, it's a one page, one and a half page max summary of the seminar series. Now, if you can't make those seminars on Thursdays, you can attend a different seminar in one of the other biolog uh division of biology departments. So, uh biochemistry, cellular biology, genetics, sometimes entomology. If you send me uh a question by email saying, "Hey, can I attend this seminar to make up for the one that I can't see for uh the microbiology program, please send me an email so that I can confirm that that seminar would be worth you going to for an extra credit experience." Okay. Does anybody have any questions about the extra credit? Right on. Okay. So, we're going to finish up talking about active transport today. And remember, these are the transport systems that are going to require the use of energy. In one case, we're going to use the power of ATP hydraysis in the case of these ABC family transporters. And in the ion solute transporters, we're going to harness the energy of an ion ingredient to do the transport work for us. Okay. So ABC family transporters like I explained to you on Friday have two really important domains plus another protein that we're going to talk about. So the important domains of an ABC family transporter are the pore itself. Okay. So in this cartoon the uh image or the the green blob that is the channel that's going through the lipid billayer. This is an aram negative bacterium. So this is the paraplasm at the top and then the inner membrane would be at the very top of the slide and then the cytoplasm is on the inside. The other important domain for these particular proteins is the ATP hydraysis domain shown in yellow. This domain of the protein is going to respond to the presence of solute. That is the thing that this transporter is attuned to transport. Once it feels that solute binding to this channel, we're going to begin to hydrayze ATP in this yellow domain. And that's going to cause a confirmational shift in our channel, which will open the pore allowing the transport of that solute into the cell. Now I said something about an sort of an accessory protein or another protein that's used and that protein is this protein in blue. This is called a solute binding protein. It turns out that this is the protein that's going to pick up the solute. thing that you want to transport out of the paraplasm of a gram negative bacterium. This solutebinding protein once bound to a solute for example if this is iron or something like that or an ironbound compound it will then be picked up by the solute binding protein which then directs it to the actual channel that's sitting in the membrane. Upon successful binding of the solute binding protein with bound solute, we initiate ATP hydraysis on the inside of the cell and we use that energy to physically open up that pore or the channel to allow the solute to disengage from the solute binding protein and then enter the cell. These ABC family transporters have been found to work in biology and therefore they are conserved. You can find members of this family of AT uh ABC family transporters essentially throughout biology proarotes up to ukareotes. This is in contrast to transporting some molecule with ions. Okay, this is also sometimes known as secondary active transport where ATP uh ABC family uh transporters are primary active transport. They can exist in a couple of different flavors. One is a so-called simporter. The other is the so-called antiporter. Simporters move ions and solutes in the same direction. Anti-porters move them in opposite directions regardless of Shiloh. What is the benefit of what? I'm sorry. Ah, so what is the benefit of having an anti-porter over a simporter? And really the key is going to be in what direction we want these the solute to move as well as what does our environment look like in terms of ionic concentration. So if this bacterium finds itself in maybe a high sodium environment, it's going to want to move sodium from outside the cell to inside the cell. So that's going to define the way the ion's going to move. Is your solute in high concentration on the outside? If it's high concentration on the outside as well, maybe it'll be a simporter. If it's at low concentration, maybe it'll be an anti-porter. There is no necessarily benefit or disadvantage to using one over the other, but the type that you use is almost certainly going to depend on your environmental ion concentration. So for example, this sim porter, we're going to move substrate, which is the blue square or solute along with an ion. As you can see where this cell finds itself, it has a lot of ions on the outside. So pretend that's something maybe like sodium in red. at relatively low concentration on the inside of the cell. Now, these blue squares or blue cubes on the inside, you can see they're actually at higher concentration in the inside than they are on the outside. And because of this, we need to use active transport because we can't follow the concentration gradient here. So, the channels remain closed to the outside until we want to transport a solute. And then what happens is this solute and the ion bind in the active site of this transporter at the same time. Once bound inside the transporter in that aquous tunnel through the middle of the the transporter that causes a confirmational shift in the protein structure which then opens up the bottom allowing both the ion as well as the solute to move inside the cell. Antiporters work in a very similar manner. However, in this case, the substrate is red. So, we're moving from low concentration to higher concentration out here. We're going to move a um this in concert with another ion. In this case, red moves in as blue moves out. Okay, so both thin porter and antiporters essentially work in the same way. However, it all depends on where your solute concentration lies as well as your ion pool. But they both can serve the same function. The substrates or the energy sources for these transporters tend to be protons, sodium or potassium. Okay. So based on our knowledge of these active transporter families, we're going to take a look at this conserved transport pathway in gram negative bacteria called toneb dependent transporters. This particular system actually utilizes kind of both types of active transport. We're going to see the use of an ABC family transporter, but we're also going to see the use of an ion gradient to do some transport events. Okay, so these toneb dependent transporters in gram negative bacteria are used for transporting a variety of different substrates, sugars, co-actors, other metal containing compounds. And we're going to see that while the cell has multiple different transporters for these these uh different substrates on the outside of the cell, it turns out that the machinery that allows for the the transport event to occur is actually shared between each of these different transport events. Okay. So I'm going to walk you through a transport event caused by a tone B dependent transporter. Then we'll talk a little bit about what's happening here. So while we're all we're going to use ATP binding as one step in here. The initial transport event for any tone B dependent substrate is powered by the proton motive force. That's the initial transport event. Remember, this is a gram negative bacterium. So, we've got an outer membrane that we've got to transport across and an inner membrane that we have to transport across. So, we have to have two distinct transport events to get something from the outside of the cell all the way inside. Okay. So here is an example of a toneb dependent transport event and we're going to focus in on the specific uptake of this interobactin iron 3 complex. Interabactin is a bacterial cederaphor and these small molecules very tightly bind iron in the environment. This is a molecule that lots of bacteria use to try to acquire iron from the environment. Okay, as we work through this sort of molecular system here, I don't want you to get caught up too much on the names of these proteins other than tone B, EXBB, and EXBD. And the reason for that is that the protein names I have up here are different for the different types of substrates that we're going to transport. But the only complex that stays the same across all of those different transport events is tone B, EXBB, and EXBD. Okay, so we're out in the environment. We're lacking some iron. Our bacterium is making interabactin. It's shooting it out into the environment. And now we have this pool of interabactin that's bound to iron 3. And the bacteria really want to eat this stuff. So initially what happens is there is an outer membrane transporter or an outer membrane receptor for the interabactin iron complex. This is the protein that's going to get this interabactin iron 3 complex across the outer membrane. We're also going to use the power of a proton motive force. Higher concentrations of protons on the outside of the cell and we're going to bring them into the cell which is now going to allow us to do some fun biology. And what happens is with the FEP A protein, which is this outer membrane transporter for interobactin iron 3, it turns out we're going to take protons, move them through the cell from outer membrane to the inner membrane, and they're actually going to go through this EXBB exp. And that's going to cause the tone B protein now to slide up and actually touch that FAP A transporter. And as you could see from that small animation, as tone B gets organized and touches that FEP a transporter, now all of a sudden that interabactin iron molecule can cross the outer membrane. Once the interabactin iron is brought across the membrane, it is then picked up by this fat B protein which is the solute binding protein for an ABC family transporter. So from our previous slide that showed when a solutebinding protein binds its solute or its target substrate in the paraplasm and then directs it to an inner membrane transporter. This inner membrane transporter at least for interobactin iron 3 is a protein called feep C. FEP C is normally closed to the paraplasm. But when the fat B protein binds with its interabactin iron complex that causes the ATP binding domain of FEPS to begin hydraizing ATP and we're going to use that energy to change the shape of FEPS which is now going to allow that interobactin iron 3 to come inside the cell. So the first part of this transport event is getting our solute across the outer membrane. To do that, we're going to use a toneb dependent outer membrane transporter or outer membrane receptor. That outer membrane receptor when when it's bound by our substrate will then signal protons to be paged through the outer and inner membrane. We're going to use those protons to change the way that EXBB and EXBD interact with tone B. Tone B will interact with the outer membrane transporter which then opens up that transporter to allow the solute through. And then we're going to use the power of an ATP binding cassette transporter and ABC family transporter to finish the transport event. Remember how I said that there are multiple different transport pathways for toneb dependent substrates. So we can have a different family of outer membrane receptors as well as inner membrane ABC family transporters that recognize a different substrate. Yet due to the fact that they're tone B dependent, they will share the activity of tone B, EXBB and EXBD. So this one protein complex tone B, EXBB, EXBD is responsible for opening lots of different transporters and not just FEP A. Okay. So how did this how does this work? Well, to better understand how this works, we actually are going to look at the structure of a tone B dependent transporter. So shown on your screen is the protein feck A from E.coli. This protein unlike the previous one that was transporting interabactin iron 3, this one is transporting feric citrate. So it is specific for binding feric citrate. You can see we have these lovely beta sheets wrapping around this the the central, you know, channel. This is what's making up that outer membrane hole in the membrane. So here's outside, here's inside. This is a pretty typical we call a beta barrel protein. But there is something a little bit different when we look at this structure. When we look at this structure, we see these sort of beta sheets in blue sitting in the middle of that transporter. This is the aquous channel where we're expecting solutes to come moving through. And yet there is this portion of the protein that's sitting right in the center. And this is acting like a plug. This is the part of the protein that's keeping that channel closed. At the very end of this plug domain, we see this little region in red. That region in red is known as the tone B box. This is a sequence of amino acids that is recognized by tone B. So when tone B binds this transporter to initiate a transport event, it actually grabs onto that little piece of of protein there in red and pulls it and it actually removes the plug from the center of the transporter allowing transport to occur. And of course, if you are a different tone B dependent transporter, for example, that FEP A protein on the previous slide, not only will your structure look the same overall, you will absolutely have a tone B box sequence because you are going to be opened by tone B. Okay. So, tone B, EXBB, EXBD activity actually kind of works like a cycle. So, here in position number one, we've got our outer membrane receptor. So, this is like that FEP A protein. And in the inner membrane, we have tone B being held with EXBB and EXBD. When substrate comes around, we can move protons through that EXBB exper. And that causes EXBB and EXBD to actually release tone B. So now tone B can float up and interact with that outer membrane receptor, which allows the transport of these delicious black triangles. After the transport event has finished, EXBB and EXBD will recapture tone B. And by pulling tone B away from that transporter, tone B will now release that tone B box and that outer membrane receptor or outer membrane channel will snap closed. And then the cycle can begin again once we have more substrate in the environment. Do I have any questions about the activity of tone B dependent transporters? All right. Okay. So, TOEMBB expB and EXBD talks with several different receptors. So, here's just an example for the types of proteins that uh tone B will interact with in a typical gram negative. The system depends both on the proton mode of force as well as ATP hydraysis to carry out transport and we only see it in gram negative bacteria due to the presence of an outer membrane and an inner membrane and tone B expBD is a system that has evolved to allow the transporters on both sides that is the outer membrane and inner membrane to talk to each other and to regulate their activity basically at the same time. Okay. The last type of active transport I want to talk about is something called group transport. Now, this type of transport is not going to directly use either an ion gradient or ATP hydraysis to regulate some transport event. But what we are going to do is we're going to use the phosphate from a high energergy phosphate intermediate to regulate a transport event. And because we're using this high energy phosphate intermediate, we could have used that to generate ATP. but instead we're going to bypass the creation of ATP and use that phosphate to help regulate this particular transport event. So what we're going to do is we're going to use phosphorilation to modify incoming sugars when the cell is trying to transport them. So it turns out that the actual transport event doesn't require any energy. In fact, we're going to be following concentration gradients here. But to keep that compound inside the cell and begin metabolism on it, we must phosphorolate it. So, we're going to use this particular system to regulate sugar transport. Now, this system is not found in all bacteria. Archa do not have it nor do strict aerobic bacteria. However, this system is a wellescribed system that drives this process of group transport. So, we're going to use a phosphorilation event to modify incoming sugars. This system is also modular. So, we're going to talk about a couple of different modules that are used in this group transport system. And some of these modules are shared with other group transport systems. The best example of this group transport process is the so-called phosphoeal pyuvate sugar phosphot transansferase system or the PTS system. Okay. And what this system is going to allow us to do, so here's the punchline. This system is going to allow us to transport and accumulate sugars in a ranked order manner. That is, I'm going to acquire my favorite sugar first. Even if I have three or four or five different sugars out in the environment that I can catabolize, if glucose is floating around, I'm going to eat the out of that glucose first and then wait for the glucose to go away before I start on everybody else. And it's going to be that transfer of that phosphate that's going to dictate which sugar is transported and metabolized. Okay, so here is a general representation of how the PTS system works to transport and accumulate sugars. So there are a group of sugars that we know as PTS sugars like manos and manitol and glucose and so on and so forth. And many of these sugars are transported by these so-called PTS systems. There is an outer membrane, excuse me, in this case it's an inner membrane transporter. This transporter is a permease. And remember with permeases we use facilitated diffusion. These transporters are made up of families of proteins that we call enzyme 2B and enzyme 2 C. Once the sugar is transported, a phosphate that was existing on the transporter will get transferred to that sugar and that will help the cell accumulate that sugar and force it into metabolic pathways. But how does that phosphate get there in the first place? Well, it turns out that the cell has a system to pluck that high energy phosphate off of phosphoenal pyuvate. Remember, we could make ATP from phosphoenal pyuvate. Instead though, we're going to take this high energy phosphate and pass it along the series of other proteins. And we'll also talk about this again after I work through this slide. The first protein that picks up the phosphate from phosphoenal pyuvate is a protein called enzyme 1 or E1. When E1 gets phosphorolated, it can then pass its phosphate onto this next protein called HPR. HPR stands for histadine protein because it has a lot of histadines in it. After HPR becomes phosphorolated and then passes the phosphate onto enzyme 2A which then passes the phosphate onto enzyme 2B. And only when that sugar comes inside the cell through this enzyme 2 C2B channel, then the phosphate gets transferred from enzyme 2B to the sugar. I told you that pieces of this system are modular and they're going to be shared with some of these other components. And the modular proteins are enzyme one and HPR. So if you draw a line between HPR and enzyme 2A, everything on the top of that line, enzyme 2 A, 2 B, 2 C, those proteins are sugarpecific. They are looking for a specific sugar substrate and the enzyme 2A component is looking for specific enzyme 2B2C components. So if you have three different PTS sugars on outside of the cell, you also have three different enzyme 2 A, 2B, 2C components. So these are kind of like those tone B outer membrane receptors we just talked about. Now the shared piece of this is the enzyme one and HPR proteins. They can interact with all of the different enzyme 2 components. But it turns out we're going to see that the phosphate flow can only go one way at a time. And this is going to be the molecular reason why we can't transport multiple PTS sugars at the same time because that phosphate flow is only going to go to one set of these enzyme 2 components at a time and that depends on how much of that substrate is out in the environment. Okay, so here is a little bit simpler model in terms of there's not so much stuff all over the place for this cartoon. We have this system that's going to transport PTS sugars. So we've got an enzyme 2B2C component here for glucose and an enzyme 2B2C system for manitol. So we have distinct enzyme 2B 2C systems for each of those different sugars. For both of these transport events, the phosphate donor again is phospholenal pyuvate. What's missing in the middle here in all these arrows is what was on the previous slide. enzyme 1 HPR and then the enzyme 2A component that will be specific for either the glucose 2B2C or it'll be specific to the manitol 2B 2C. So in this case we've got these substrates out there glucose and manitol. As glucose is transported, phosphate will flow from PEP to enzyme one to HPR to enzyme 2A glucose, make it to enzyme 2B2C, which will then phosphorolate glucose becoming glucose 6 phosphate, which can then be metabolized. If phosphate is flowing in this direction on the top because glucose is present in the environment that phosphate will not be going this way to manitol cuz remember those enzyme 1 and HPR proteins are shared and if they're dedicating their activity to sending phosphate down the enzyme 2 glucose components it's not going to be interacting with the enzyme 2 manitol. components. So if both glucose and manitol are present in the environment, the cell in this case will transport glucose first. And they're going to transport glucose first because these cells prefer glucose. and to they prefer glucose because they have engineered their enzyme 2 components to have a much higher affinity for both glucose compared to the other sugars as well as having the en the HPR enzyme would prefer to interact with the enzyme 2A for glucose. So all of these affinities have been encoded to make the phosphate flow happen one way preferentially as long as that preferred sugar is in place and then if it's not then the enzyme uh one and HPR system can look for the next best enzyme 2 components that will eventually allow manitol to come in and be phosphorolated. Okay, so the enzyme 2 components, enzyme 2 A, B, and C, they're specific for their substrates, glucose or manitol or a different PTS sugar. Enzyme 1 and HPR are shared and this is going to regulate the uptake of sugar through a phosphot transfer event. We're going to shuttle this phosphate down to at least four different proteins before it makes it onto the incoming sugar. It turns out that enzyme 2A for glucose could actually have downstream regulatory roles. This has helped us drive catabolite repression in bacteria. We'll talk about that a little bit later on. So it turns out strict arobes strict aerobic bacteria don't have phospholeneal fy pyuvate phosphot transferase systems. Um they so only organisms that can perform anorobic glycolysis can actually have these uh pts family proteins. Archa don't have them either and ukarotes certainly don't have them. Okay. So this kind of wraps up our transport summary. We covered three different types of transport, facilitated diffusion, and active transport. Simple and facilitated diffusion must follow the concentration gradient. The cells can have some impact on that gradient by changing the water concentration, moving it in and out, or by running metabolism to take away something that's on the inside of the cell. The active transport series can use energy either the proton mode of force ATP hydraysis or a high energy intermediate. All transport events regardless of the type used has some specificity built into it. Even simple diffusion or passive diffusion has specificity built in because most molecules can't cross that lipid billayer. You have to have a very specific composition to be able to move across that lipid billayer. Question. So how does the cell move water out of itself? Well, water moves by simple diffusion. But in lots of bacteria and other cell types, we have these proteins in the membrane called aquaporins. And those aquaporins allow water to leave to basically cross the the billayer more easily. And the cell can actually change the pore size in the membrane of those of those aquaporins to change how quickly water might move out of the cell or move into the cell. Again, based on the osmotic uh strength of the surrounding environment. Does that make sense? Okay. All right. So now we're going to switch from transport events into starting to talk about bacterial growth and the cell cycle with a focus on the mechanisms that drive binary fision or cell division. Okay. So proarotes are hloid right they function with one copy of the total genome. There are some billayers. Most of the time we think about bacterial division as being binary fision where one cell splits down the middle into two. There are outliers of course there are some that do sort of fungal like growth where the cells bud. Some do sporulation but when it comes down to it when cells want to divide basically they've got to make at least two copies of everything. make sure those things get separated, move to opposite ends of the cell, and then we got to figure out how to divide that one cell into two. All of these processes that drive bacterial cell division are highly regulated and highly coordinated because defective division events for example closing the cell too early or pinching off the cell not at the middle more towards the end. Those are can be lethal events. So, we really have to make sure that all of our machinery that's used to divide the cell is at the right place and at the right time. Okay. So, during binary fision, we're going to figure out how to replicate and separate genomes. Some of these cells are going to carry other genetic material like plasmids. We're going to have to figure out whether or not we also need to move those plasmids into daughter cells. So, while we're making extra copies of everything on the inside, we also our cells are growing. They're getting bigger. And we got to take this one cell and turn it into two cells. So, we're also making all of these cell wall components. We're making membranes, lipids, pepidoglycan. We got to figure out how do we open up the pepidoglycan layer and add more pepidoglycan subunits because remember that's a rigid mesh and we're going to have to manipulate that rigid mesh in order to divide the cell. And then finally the most major protein involved in cell division is the FTSZ protein. And this protein is going to make up the so-called constricting ring which is directly involved in splitting one cell into two. Okay. And so I am going to move on to the next one if I can find it. One, two, three. Oh my god. Just like my house computer. Too many icons. underneath there. Okay. So, in this lecture, we're going to talk about a few events that are going to happen during cell division. We're certainly not going to talk about all of them. The first thing we're going to talk about is the so-called plasma segregation pathway. We're going to use it as a model for how cells can actively partition their genetic material into two different sides of the cell. So that when the cells split down the middle, they can be pretty sure that the daughter cell is going to have the same stuff in it that the mother cell had. so that both of these uh cells will have the exact same stuff. Now remember that an FTSZ constricting ring, this is the protein polymer that's really going to close down the cell and pinch one cell into two. So many cells have a number of systems to ensure that the FTSZ protein only sets up and begins to polymerize at the midpoint of the cell. One of those systems that we'll talk about is the so-called min system. And then finally, we'll talk about how all these proteins work together to bring in cell wall production and and modification proteins to the site of division to make sure that the pepidoglycan layer is modified appropriately during division. Okay, so here is a simple cartoon of what happens during cell division. We've got some mother cell out there that decides, hey, I think I've got enough energy. I've got a enough nutrients around me. I want to make a baby. So, here is our genome. We have an origin of replication. This is where DNA replication is going to occur. We'll talk a lot about this in a couple of weeks. So in this case, we begin to replicate our DNA to forming two copies of that genome. And they're kind of pushed off into it sort of opposite sides. So that if we were to split the cell down the middle, we would then have one cell with one genome and the other cell with another genome. And we're going to repeat this process over and over and over again. Okay, so these first couple steps that are involved in genomic replication and eventually genomic partitioning, the initiation of bacterial genomic replication depends upon the concentration, the intracellular concentration of a protein called DNA. We're going to talk a lot more about DNAa later. But as the concentration of that protein builds up and sufficient ATP exists in the cell, we will initiate genomic replication. Then it's this next step, these are the processes which eventually end in cytochinesis or the division of those cells. So in this set of of cartoons here, the cells are building this FTSZ ring. We're going to see that the FTSZ ring is not uh a ring that works on its own. There are many protein players involved in building this Z-ring at the right place and at the right time. In the background, we're also working on making more of the cell envelope, lipids, pepidoglycan, and starting to get them into the right place at the right time so that when we divide our cells, we don't have breaks in our cell wall. And then finally, we have to have replication resolve so that we have now two loose genomes, one for each cell. We'll talk about how we get here on Wednesday. Remember to read that paper. We'll have a paper discussion for the second half of class on Wednesday. Get out of here. We'll see you around.