MCB 502A-2014. Lecture #11. The Cell Cycle. The prokaryotic cell cycleThe prokaryotic versus eukaryotic cell cycleChromosomal replication is the central event of the cell cycle, which is the sequence of events that initiates with the birth of a new cell and culminates with its division to give rise to two daughter cells. The familiar outline of the eukaryotic cell cycle is represented by the circular diagram —>G1—>S—>G2—>M—>. If we follow the rate of DNA synthesis through the cycle in synchronized cells, we will detect it only during the S-phase. Another way to present the cell cycle is via the genome content of the cell: it is stable in Gap-1, duplicates throughout S, stable again in Gap-2 and is halved by cell division. We know that the eukaryotic cell cycle is driven by the cycline-dependent kinases (CDKs) and is subdivided into four major blocks of activities. When CDK activates a particular block of activities, it then waits for the information about completion of these activities. Once all the activities of the block have been successfully completed, CDK activates the next block of the cell cycle, and so on. The critical feature of the eukaryotic cell cycle is that any particular step cannot begin before the previous step has been reported as accomplished. Can we study the bacterial cell cycle the same way we study the eukaryotic one? In particular, can we detect the period of DNA synthesis by following incorporation of a radiolabeled DNA precursors into DNA? If we just add label to a growing culture and measure the rate of label incorporation into DNA, we will end up with an exponential curve, which wouldbe superimposable with the growth curve of the culture. This result is uninterpretable (and wouldbe similarly uninterpretable in eukaryotic systems), because the culture is asynchronous: some cells have just started chromosomal replication, some are finishing it while still others are engaged in chromosomal segregation and cell division. Synchronization is a prerequisite for studying the cell cycle in any system. As was discussed at the beginning of the lecture, we can synchronize the culture if we prevent replication initiation by blocking protein synthesis due to withdrawal of a required aminoacid, waiting until all cells complete the ongoing rounds of DNA replication and then releasing the protein synthesis block. In slow-growing synchronized cells of E. coli, we see the familiar features of the eukaryotic cell cycle. After the cell division, there is a period of no DNA synthesis (G1), followed by a period during which DNA is synthesized (S), followed by a short lull in activities (G2), followed by cell division (D). Essentially the same curve can be obtained for HeLa (~human) cultured cells. What is the mechanism driving this bacterial cell cycle? Since it looks superficially similar to the eukaryotic one, we may start with a simple idea that the bacterial cell cycle is driven by an analog of cycline-dependent kinase, with a similar format of orders to execute specific stages and decisions made on the basis of reports of successful completion of prior stages. This system assumes the existence of a group of dedicated signal-generating and 1information-processing proteins (kinases) that do nothing else besides driving the cell cycle, and whose inactivation is lethal (like CDKs in eukaryotic cells). No such system has been revealed sofar in bacteria; in particular, there is no group of conditionally-lethal mutants in suspected protein kinases. The majority of lethal mutations in E. coli directly inactivate important metabolic functions, like DNA ligase or the initiation protein DnaA, that execute, rather than order, a particular stage of the cell cycle. Perhaps we can get a glimpse into the bacterial cell cycle if we study bacteria growing at different division rates? We know that in the lab, in the most favorable growth conditions, entericbacteria like E. coli can divide as fast as every 24 minutes. In natural habitats, enteric bacteria grow much slower. E. coli is distributed 50:50 between the two natural habitats: the guts of vertebrates, where bacterium is thought to live a stable though very slow life, and natural waters of lakes and ponds, where bacterium is starved and dying. When in the water, the bacterium doesnot multiply and simply tries to stay alive, waiting to be swallowed by an animal with an appropriate gut. Once in an appropriate gut, with plenty of food around, a lucky bacterium will try to colonize it. However, there is a lot of competition in established gut communities from the resident species. As a result, bacteria in the gut are thought to divide only once a day, — judging by the total mass of bacteria there and by the portion of this mass that gets "evacuated" on the regular basis. Thus, under the most optimal conditions in the lab, E. coli divides once every 24 minutes, whereas during the stable growth in the gut it divides once every 24 hours, with a 60 times longer cell cycle. We may further presume that the chromosomal replication probably takesless than 20 minutes when cells are dividing every 24 minutes, or it could easily take 20 hours when cells are dividing every 24 hours. The way to test this notion would be to measure the timing and length of chromosomal replication in synchronized cultures, grown at different cell division rates. In certain eukaryotes, like budding yeast, synchronization is easy because there is the cellcycle machinery: one just has to send a specific signal to this machinery that the previous stage isincomplete (even though it could be in fact complete), and the cell cycle freezes at a specific point without much disturbance of the overallcellular metabolism. Not so in bacteria, lackingthe cell cycle engine. The procedures forsynchronization in bacteria involve such drasticmeasures as blocking protein synthesis byremoval of required aminoacid. These grossperturbations of the cellular metabolism shouldundoubtedly perturb the cell cycle in bacteria.Here we ran into a paradox: in order to study thecell cycle, we have to synchronize the cells, butsince synchronization procedures in bacteria2Concept-in-the-box. Synchronization without interference with growth of the culture is achieved by fractionation. In general, the newly-born cells are two times smaller than the cells that are about to divide, and therefore can be separated from the latter by gradient centrifugation as, correspondingly, the slowest- and
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