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Molecular Genetics and Microbiology
William Navarre

Lecture 11- Transcript MGY377 NOTE: The number prior to the information describes a certain point in the lecture slides the lecturer addresses. Bacterial Cell cycle and Growth Slide 2 1. What is bacterial growth? 3. Why is this important? Food production I.e. pasteurization, as bacteria can grow rapidly in food. Biotechnology: we want to harness microbes to grow certain factors such as hormones I.e. insulin. Fighting disease: understanding how microbes grow in us to learn how to fight infection better I.e. antibiotics Slide 3 2. Chromosomes once duplicated will separate. This separation is dependent on membranes 3. Time varies between species to species. Good example is with E. Coli. It replicates very rapidly in lab conditions but much more slowly in the human gut (lots of competition) Slide 4 1. Many bacterial cells divide by binary fission. This is where the cell divides into two cells by first elongating to double its size and forming a partition in the middle called the septum. This is due to an inward growth of the cytoplasm, membrane and cell wall. Occurs simultaneously. Eventually this septation fuses with itself to give you two daughter cells Slide 5 1.Movie showing dividing bacteria on agar septating into more and more bacteria (bacterial replication). Under ideal conditions can take over the planet in two days- damn. Slide 6 -That’s the surface of an agar plate- what about the inside of our cells? -Salmonella are intracellular pathogens. They breach the barrier and get into our cells to cause persistent infection. Salmonella can replicate rapidly in a specific region of our cells called the cytosol -Looking at the diagram: Salmonella in green (GFP) replicate very fast, whereas salmonella in yellow (found in the vacuole) replicate very slowly. Red shows where the vacuoles are. Rapid replication during early infection drives this inflammatory response by our immune system and hence the intense wave of symptoms. Slide 7 -Also showing binary fission -Cell will increase the number of macromolecules -cell elongates, DNA will be replicated. Both copies of the chromosome will be anchored to the cell membrane so that when the septum forms, it will split the two copies of the DNA to each of the two daughter cells. Eventually, you have septation and the formation of the two daughter cells. The time it takes to create two daughter cells is called one generation. Slide 8 1. Many bacterial factors in cell division. Many of these proteins were isolated via mutagenic screens 2. Idenitifed group of proteins called fts proteins. The mutation of these genes cause cell division defects. These proteins act together to form the complex called the divisome. Divisome drives cell division. 3. FtsZ is important for starting off the complex for division. 4. FtsZ is highly conserved in prokaryotes. It is structurally similar to tubulin present in eukaryotic cells (tubulin is important for microtubules, essential for cell division) Tubulin is important for splitting the chromosomes apart during mitosis (cell division) Slide 9 -FtsZ in bacteria is a GTPase. Forms a membrane associated ring structure in the mid cell called the FtsZ ring. -Here we have a schematic of a bacterial cell. The FtsZ ring is associated with the cell membrane. These triangular shaped divisome proteins associate with the FtsZ ring, scaffold, and drive the cell division process. -Here we can see fixed images of the bacterial cell cycle (showing different stages). -FtsZ is pretty diffuse in the cytosol. -In the early stages, you can see it being pulled apart. After division, the FtsZ ring breaks apart. FtsZ ring goes to the midpoint of the two cells to make daughter cells. Slide 10 -Schematic of the sequence of events occurring -First you have the FtsZ ring form in the midpoint of the cell. Next, you have the ring serving as a scaffold to recruit these other FtsZ proteins. These through various activites will cause cell division. Slide 11 -Some of the protein that interacts with the FtsZ ring. Slide 12 -When we’re talking about binary fission of bacterial cells, we often assume that the daughter cells are equal. They are not, as described in the paper. Important concept to keep in mind especially when you’re looking at population of bacteria. When you are doing infections, you see populations of bacteria with different fates inside of our cells. Part of the heterogeneity that you see is attributed to the bacterial culture (heterogeneous group of bacteria) -Authors explore the concept if the two daughter cells are equal. How did they do this? Tracking the growth characteristics of individual cells by microscopy. Slide 13 -Here you have a microbe that’s dividing. -in order to generate a septum you need to generate new cell wall material in order to divide a cell wall into two you end up with two daughter cells that have a new pole with new material and the old pole as it divides again, you have an old pole cell and a new pole cell they showed that the cell that contains the old pole develops a little bit slower than the cell that contains the new one (the older material is therefore having an impact on the growth of the microbes) Slide 14 -This is what they did -Old pole cell progeny tended to elongate 2.2% slower than the new pole old pole cells divided less and less frequently than the new pole cells and therefore gave less biomass concept: aging (every time you divide, there is some sort of carryover from the old growth in the life span) Slide 15 -FtsZ is a tubulin homolog that makes up the microtubules in our cells. Important in separation of chromosomes in cell division -Another homolog of eukaryotic cytoskeleton protein is MreB. This is an actin homolog (actin has an important role in controlling cell shape in our cells as well as in vesicular transport) -MreB helps bacteria maintain a rod shape (rod-shaped bacteria). If you knockout MreB, the bacteria are not rod shaped any more but cocci. (Use the protein to attain the rod shape) -Another important protein is crescentin expressed by some bacteria. This is an intermediate filament homolog (intermediate filaments are important in our cells for certain scaffold functions) In these bacteria (Caulobacter crescentus), use this protein to help bend the bacteria Slide 16 1. Recall that we said that the FtsZ ring always falls in the middle. How does this occur? 2. Use of Min family of proteins to exclude FtsZ ring from the formation of certain regions of cells, so therefore by exclusion- only forms in the middle. 4. Min D is a membrane anchor 5. Min C/D inhibits FtsZ and Min E inhibits these two. What is it mean by oscillating? C/D will oscillate in the microbe Slide 17 -The concept of oscillation is shown here. GFP tagged min-C and at time zero it’s at one pole of the bacteria. 24 seconds later its on another pole and so forth. If you look at the cell, the complex is usually at one pole of the cell. It is rarely at the midpoint. By excluding FtsZ ring at the poles, it only forms in the middle. Slide 18 -Movie of localization of MinD Slide 19 -How does this all work? Here you see FtsZ trying to form a ring. At one pole you have the MinC/D complex. MinC is inhibiting FtsZ and knocks it off. MinE forms a ring and moves inward, inactivating the MinC/D complex. MinC/D go back and forth preventing FtsZ rings from forming. The only place the FtsZ can form a ring, unmolested, is in the midpoint Slide 20 1. We need to keep synthesizing the cell wall for continuous cell divisions 2. Lysozyme is a molecule that can punch holes in the cell wall of microbes as is a key element of our innate immune defense (found in saliva, tears, sweat etc.) 3. Scar formation occurs at the junction of the new and old cell wall Slide 21 -here you have a microbe dividing. -new cell wall material is added in the green region, or the growth zones. The scar or the wall bands are shown- this is the interface between the new cell wall and the old cell wall material Slide 22 2. Bactroprenol binds to peptidoglycan precursors in the cytoplasm, carries them across the cytoplasmic membrane, so they can be incorporated into the peptidoglycan meshwork. *Refer to diagram on slide 23 (to see what the structure it looks like) Slide 23 -Looking at the schematic: first Bactroprenol binds to peptidoglycan precursors and carries them along the cytoplasmic membrane. Then it interacts with precursors that insert these precursors in the growing cell wall *Really good drug target for a
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