LectNotes&Reading_Motility

LectNotes&Reading_Motility - Structures and Functions...

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Unformatted text preview: Structures and Functions of Microbial Motility Overview The driving force for the evolution of motility appears to be acquisition of food. Thus, motility has also evolved with a mechanism to sense the environment and control motility in response. Almost all microorganisms have mechanisms for motility, both prokaryotic and eukaryotic. Despite the widespread nature of motility, the mechanisms for motility have independently evolved several times. Some forms of motility are very well understood (e.g. the bacterial flagella), but scientists continue to identify microorganisms that exhibit motility through unknown mechanisms. The study of motility, specifically the bacterial flagella, has made fundamental breakthroughs in how motors, environmental sensing, and protein secretion function. Many microbial pathogens are motile, and this motility plays a major role in their ability to cause disease. Reading. 11.5 Structure and Function of Cell Appendages (pg. 307-314) E.coli and Experiments that Lead to Our Understanding of Bacterial Flagellar Motility Here are presented some of the key experimental methods and observations that lead to our understanding of chemotaxis in E. coli. The point here is not to memorize the particular experiments. These are presented to elucidate the scientific method. Quantification of Chemotaxis Chemotaxis=movement in response to a chemical. This chemical could be either an attractant or repellent. The experiment described in the textbook demonstrated that bacteria exhibited chemotaxis. With an assay to study chemotaxis, scientists could then make perturbations to the system and then quantify what effect those perturbations had on the chemotactic response. Key points: 1. Understand how to measure chemotaxis. 2. Know that the concentration of bacteria in the capillary tube relative to the exterior of the tube will change whether there is an attractant or repellent present in the tube or whether the solution in the tube is the same as the solution in the beaker. 3. Be able to explain why these different results would be obtained. Microscopic Analysis of Chemotaxis Models for how detecting the presence of an attractant or repellent affected the motility of E. coli was developed through the use of time-lapsed photography of microscopic observations. This analysis revealed that E. coli moved in a series of runs and tumbles (see movie of the course web site). This movement did not change as long as E. coli was in a homogeneous solution, whether or not an attractant or repellent was present. But remember, in the tube experiment (above), a gradient of attractant or repellent would have been set up as the 1 chemical diffused from the tube. Thus, when a gradient is established under the microscope a change in the motility of E. coli was observed. This behavior is described in the textbook. Key points: 1. Be able to explain the difference between a spatial and temporal gradient. Know that bacteria respond to a temporal gradient. For an example of response to a spatial gradient, see the movie on the course website of the eukaryotic microbe, Dictyostelium. 2. Be able to explain how bacteria can, using only random movement, effectively swim towards an attractant. Isolation of Chemotaxis or Motility Defective Mutants Isolation and characterization of mutants defective for a process of interest is a powerful method to reveal the molecular basis of a process. Thus, to reveal the molecular mechanism of bacterial motility, scientist isolated mutants of E. coli that exhibited a defect in motility or chemotaxis, called Mot- and Che-, respectively. The key step in the isolation of mutants is the development of conditions that allow the screening of thousand of mutant cells for the desired, altered phenotype. To isolate Mot- and Che- E. coli mutants, an agar plate-based assay was developed. The use of agar plates allows thousands of cells to be analyzed simultaneously. The agar plate used to isolate the Mot- and Che- mutants contained soft agar (i.e. semi-solid). E. coli can swim in this soft agar. To generate a gradient for chemotaxis, the plates also contained a very small amount of a carbon source. E.coli cells are inoculated in a spot on the plate. These bacteria use the carbon compound present near the spot and grow and divide. As they exhaust the carbon compound at the site of inoculation, they create a gradient, with the highest concentration always being outside the zone of the bacteria. Thus, E. coli will chemotax outward, to the higher concentration of carbon compound, use that compound to grow and divide, starting the cycle again. To generate mutants, a culture of wild-type bacteria is mutagenized (do not worry about how). This creates a culture of cells that each have different mutations. The culture of mutagenized cells can then be spread on the plates to separate the cells, so that each cell will give rise to a colony. Mot- mutants are unable to move from the spot of inoculation. These mutants form pinpoint colonies on the plates as compared to the wild-type cells that spread outward to form large colonies. Che- mutants are able to move, but are not able to sense the gradient of the carbon compound. These Che- mutants have undirected, random motility. By this random movement, the Che2 mutants move away from point of inoculation, but not to the extent that wild-type cells move. Thus, Che- mutants form colonies that are larger than the Mot- mutants, but not as large as those formed by the wild-type cells (see above figure). Characterization of Mot- Mutants To learn what mutant bacteria reveal about the molecular mechanism of a process, scientists have to characterize them further. In what gene is the mutation? We will not go into how to do this, just suffice it to know that this is an essential part of characterizing mutants. If the mutation is in a gene of known function, then this information will provide obvious models to test further. But, if the mutation is in a gene that belongs to a family of genes that has never been characterized, then scientists need to design experiments to gain a more finedetailed knowledge of the phenotype of the mutant strain. This additional information should help to generate testable models regarding the role of the mutated in gene in the process under study. An example of this is how Mot- mutants were characterized. The Mot- mutants were analyzed for changes in structure by electron microscopy. Figure 4.54 on page 92 shows examples of flagellated bacterial cells observed by electron microscopy. Many of the Mot- E. coli cells lacked observable flagella. This lead to the hypothesis that the gene that were mutated in the Mot- mutants encoded proteins that were either part of the flagellum or involved in the assembly of the flagellum. Through these types of experiments we have gain a detailed knowledge of the bacterial flagella. Motility Structures Bacterial Extracellular Flagella “The mucus layer associated with epithelium forms inevitable physical and chemical obstacles for pathogens. Similarly, dynamic processes, such as the upward flow of mucus from the bronchial epithelia or peristalsis in the intestine, have to be counterbalanced by pathogens if colonization is to be achieved. In the host, motility combined with chemotaxis enables fine-tuned access of pathogens to the target mucosal tissues. Motility functions of Helicobacter pylori (cause of stomach ulcers) and Pseudomonas aeruginosa (cause of mortality of cystic fibrosis patients) are crucial for infection of the stomach and lungs, respectively. Colonization of intestinal mucosa by Vibrio cholerae (cause of cholera) strictly requires motility. Because motility increases the occurrence of host– pathogen interactions, this feature contributes to the main role of the flagellum in pathogenesis.” Adapted from Ramos HC, Rumbo M, and Sirard JC (2004) Trends Microbiol. 12(11):509-17. Key points: 1. Understand that the flagellum is a rigid helix. 2. A motor in the cytoplasmic membrane turns this rigid helix to propel the bacterium forward. 3. The energy for the motor is derived from pumping protons into the cell. 3 4. Know that there are many different described arrangements for flagella. Periplasmic Flagella Spirochaetes have periplasmic flagella, and these flagella are believed to contribute to the corkscrew shape of these organisms (for a picture, see page 408). These periplasmic flagella also contribute to motility. Spirochaete species include those that cause Lyme disease and syphilis. During the course of these diseases, these bacteria move from the bloodstream into tissue. Their particular type of motility may contribute to their ability to make this transfer. Type IV Pili Twitching motility is a form of surface motility mediated by type IV pili. Pili work through an extrusion, adhesion, and then retraction mechanism. For movies demonstrating the use of type IV pili in movement, see the course web site. Many questions remain regarding the mechanism of twitching motility. How does sensing the presence of an attractant or repellent control pili movement? What is the energy source for this motility? Slime Nozzles Slime nozzles are used for surface movement. Hoe they work is unclear, but it has been proposed that as the extruded slime hydrates, the bacterium is pushed in the opposite direction. Actin Tails Intracellular pathogenic bacteria use this form of motility. Many of these bacteria do not appear motile outside of a host cell. In the host cell, a tail of host-derived actin forms, which appears to constantly assemble and disassemble as the bacterium move forward. The purpose of this motility appears to be to move from one cell to another. See some fun movies on the course web site to gain a better appreciation of this form of motility. Miscellaneous There are many bacteria that are motile, but do not appear to use any of the above described structures. Some example that are being researched are the Ratchet structure used by members of the Cytophaga-Flavobacterium group of bacteria, the contractile cytoskeleton of Sprioplasma, and the cili-like structures of Synechococcus. Archea Archea have structures on their surface that resemble flagella and that appear to mediate swimming motility. Despite this similarity, the genes that encode the flagella components are similar to those for Type IV pili. What modifications to these genes has occurred to create swimming flagella in the Archea and to create surface-moving pili in the Bacteria? 4 Eukaryotes Movies of eukaryotic microbial motility are available on the course website. Three types of motility are known for eukaryotic microbes: flagella and cili for swimming and amoeboid motion for surface translocation. These forms of motility are not related to the prokaryotic forms at the molecular level. Mechanisms for motility seem to have independently evolved many times. 5 ...
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This note was uploaded on 03/06/2012 for the course MIMG 100 taught by Professor Lazazzera during the Summer '10 term at UCLA.

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