Experiment 3: Bacterial Behavior- Motility and Chemotaxis One of the distinguishing characteristics of animals is their ability to move in response to stimuli that originate from within their own bodies or from the outside world. Many of us are attracted to the smell of fresh-baked chocolate chip cookies and repelled by the aroma of a recently antagonized skunk. An animal moving rapidly toward an object it recognizes as food or fleeing a harmful chemical are examples from the repertoire of responses known as animal behavior. Even the simplest and smallest animals exhibit behaviors within their own sensory capabilities. But what about microbes? Microbes don t have what we typically associate with behavior, such as eyes, ears, noses, arms, legs, let alone a nervous system to interpret sensory signals. We spend a lot of time in the lab looking at colonies on hard agar surfaces. The bacteria are seemingly stationary and remain within the boundaries of the colony. You rarely see bacteria climbing over the side of the petri dish and onto the bench (although at least one species of bacteria, Proteus mirabilis, can do just that). Other than Proteus, most bacteria appear to be sedentary. However, this picture is misleading for many bacterial species. Not only can bacteria move under their own power, but they can also decide what to move towards and what to avoid. The Proteus that crawled over the side of their petri dish were seeking light. Bacteria have several means of self-propelled locomotion: swimming, swarming, gliding, and twitching. The most common form of bacterial motility is powered by whip-like appendages called flagella that extend from the surface of the bacterium. Bacterial flagella are made completely of proteins. They are anchored in the cytoplasmic membrane by a protein assembly that works much like a rotary motor. In E. coli, the motor powers rotation of the long flagellum; counterclockwise rotation propels the bacterium forward, while clockwise rotation results in tumbling, non-directional behavior. Therefore, swimming E. coli move in a run and tumble fashion, moving in one direction, then tumbling and moving in a different direction. For all motile microbes studied to date, the ability to move is coupled with the ability to move toward attractants and away from repellents. Bacteria use specialized membranebound sensory proteins to detect small changes in the concentration of chemicals in the environment. When the surrounding environment is homogenous, the bacteria move in random directions. However, when a localized attractant or repellent is present, the sensory receptors bind to the attractant or repellent initiating a signal transduction pathway that ultimately controls flagellar rotation. Attractants cause the flagella to rotate in the counterclockwise direction, prolonging the run phase of run-and-tumble. As a result, bacteria move toward an attractant in a biased random walk. Repellents cause the flagella to rotate in the clockwise direction, stopping motion and favoring tumbling, until the direction of the run moves the bacterium away from the repellent. Bacteria respond to a diverse range of stimuli including nutrients, toxins, light, temperature, osmolarity, and even electrochemical potential. Directed motility, or taxis, is used not only to find that perfect environment- enough food, not too hot, not too cold - 1
but also to allow bacteria to find niches in a host. Helicobacter pylori, the bacteria responsible for stomach ulcers, use chemotaxis to find the mucus lining of the stomach. Rhizobial species use chemotaxis to find roots of legumes with which they form a symbiotic relationship, getting nutrients from the host plant and providing nitrogen to the plant. How do you know if a bacterium is motile? Bacterial flagella are too narrow to be seen in light microscopes like those we have for the lab class. However, flagella can be seen with electron microscopy or with special stains that coat the flagella making them visible. You can also determine if a bacterium is motile by observing its behavior. Does it zip around on your microscope slide, instead of flowing gently in the suspension? Motile bacteria appear to be moving with a purpose. You can also observe bacterial motility on semisolid agar in which the bacteria swim through holes in the agar. Because many bacteria move at once, areas that they occupy become cloudy. Non-motile bacteria remain in one place and don t spread across a plate of semisolid agar. If you have a motile bacterium, what chemicals, signals, and conditions attract or repel it? These are fundamental questions that we will explore in this lab module. Resources You can find basic information about flagella and chemotaxis in any microbiology textbook. Online resource with an overview of bacterial motility, flagella, and taxis: http://textbookofbacteriology.net/structure_2.html Key concept Bacteria behave in response to stimuli. Challenge We will use two different assays to determine if your pets are motile and chemotactic. We will also provide you with a plate of swimming E. coli and an assay to determine if a substance is an attractant or a repellent. Develop a hypothesis about E. coli s responses to different compounds- we will provide you with some sugars and amino acids. You are also welcome to bring in other substances that you would like to test. 2
Key Questions What advantages does chemotaxis provide for a bacterium? High concentrations of repellents increase the tumbling time of E. coli. How does this help the bacteria move away from the repellent? Different bacteria chemotax towards different chemicals. Why might this be? Some bacteria are not motile. What factors in their environment might allow them to flourish without self-propelled motility? Penicillium notatum is not motile. However, it spreads over the surface of a petri dish and can even spread from one piece of bread to another. How might either of these behaviors occur in the absence of self-propelled motility? References: Adler, J. 2011 My life with nature. Ann Rev Biochem 80: 42-70. Wadhams, G.H., Armitage, J.P. (2004) Making sense of it all: bacterial chemotaxis. Nat Rev Mol Cell Biol 5:1024-37. DeLoney-Marino, C.R., Wolfe, A.J., Visick, K.L. (2003) Chemoattraction of Vibrio Fischeri to Serine, Nucleosides, and N-Acetylneuraminic Acid, a Component of Squid Light-Organ Mucus. Appl Environ Microbiol. 69:7527-30. 3
Motility Assay 1: Sectioned plates GYE agar (for 1 liter) 10.0 g of tryptone 10.0 g of yeast extract 5.0 g of glucose 5.0 g of K2HPO4 15.0 g of agar (1.5% agar) Water agar (for 1 liter) 15.0 g of agar (1.5% agar) Method 1. Pass your forceps through the flame. 2. Center 2 sterile filter strips bridging the ridge of a petri dish - see diagram below. 3. Gently press the paper strips into place on the agar. 4. Using sterile technique and an inoculating loop, place a small amount of bacteria from the slant onto opposite ends of the paper strips, so that one strip is inoculated on each side of the plate. 5. On the underside of the plate, mark the ends of the strips that were inoculated. 6. Incubate 30 C for 48 hours. Example: GYE H2O agar bacteria inoculated here 4
Motility Assay 2: Chemotaxis in semi-solid agar When inoculated in the center of a semi-solid tryptone agar plate, E. coli will begin to consume nutrients in the tryptone, forming a pattern of concentric rings (figure on left). They first consume L-serine in the area around the point of inoculation. Some bacteria move outward in search of L-serine, forming a ring tracking L-serine. Bacteria that are left behind, where there is no L-serine, consume L-aspartate and form a second ring searching for L-aspartate. The next nutrient to be tracked is L-threonine, and bacteria searching for this amino acid form the third innermost ring. If a disk with an attractant or repellent is placed near one of the migrating rings, the ring shape will deform near the disk as the bacteria move towards or away from the compound (figure on right). Chemotactic rings of E. coli Deformation of the chemotaxis pattern by a disk of acetate, a repellent, placed outside of the L-serine ring (left) and by a disk of Lserine, an attractant (right). Materials semisolid tryptone agar (1% tryptone, 0.5% NaCl, 0.25% agar) inoculated with E. coli sterile filter disks solutions of potential and known attractants and repellents (acetate, sucrose, glucose, maltose, L-serine, L-aspartate, L-glutamate, L-histidine). forceps If you bring in a compound to test and it is not already liquid, mix it with a small amount of sterile water. Methods ** Be very careful with the plates. Do not turn them upside-down. The agar is semi-solid, which means semi-liquid too. It can slop around and disturb the chemotactic rings.*** 5
1. Place a drop of compound onto a filter disk. 2. Repeat on separate disks with each compound that you will test. 3. Flame your forceps. 4. Carefully place one of the filter disks near but outside one of the chemotactic rings formed by the E. coli in the plate. 5. Label the underside of the plate so you know what disk has each compound. 6. Place the plates at 35 C and check every 15-20 min. for evidence of chemotaxis. *** Be very careful with the plates. Do not turn them upside-down. The agar is semisolid, which means semi-liquid too. It can slop around and disturb the chemotactic rings.*** Test your pet to determine if it is ch6emotactic to components of tryptone or is motile, but does not respond to the tryptone growth medium. 1. Pick a colony of your pet microbe with a sterile inoculating loop. 2. Stab the loop into the agar at the center of the semi-solid agar plate. 3. Incubate at room temperature 48 h. Growth on a semisolid tryptone plate. Chemotactic E. coli spread forming rings. Motile, nonchemotactic E. coli move a little from the point of inoculation. Non-motile, nonchemotactic E. coli do not move from the point of inoculation. 6