Exercise 17 Rhizobium -- implementation of Koch's Postulate

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Learning Objectives for Lab Exercise #17: At the end of Exercise #17, students will be able to: 1. Recall Koch’s Postulate. 2. Describe costs and benefits of nitrogen reduction to nitrogen-fixing bacteria. 3. Explain the value of the rhizobia/legume relationship in Iowa agriculture. 4. Recognize the macroscopic and microscopic properties of a legume root nodule. 5. Compare bacteroids to bacteria in the group known as rhizobia. 6. Explain the answers to the pre-lab and Conclusions/Applications questions. 7. Draw appropriate conclusions from a class experiment to test host specificity for rhizobia isolated from pea and clover nodules. Exercise #17: Rhizobium : Implementation of Koch’s Postulate To prepare for Lab Exercise 17 , visit the blog about Koch’s Postulates ( http://fungalscience.blogspot.com/2010/11/kochs-postulates.html ). Background Information: Why Do Rhizobia and Legumes Form a Symbiosis? Among the many, many essential services that prokaryotes provide for the continuation of life on Planet Earth, one of the most important is nitrogen fixation : the reduction of biologically inert nitrogen gas (N 2 ) to biologically valuable ammonium (NH 4 + ). While N 2 is everywhere (78% of Earth’s atmosphere), it is useless for amino acid synthesis. In contrast, NH 4 + is rare and precious — ready for assimilation into amino acids. Once fixed, a nitrogen atom will be recycled many, many times as cells consume one another and digest their proteins. In other words, virtually all protein on Earth—in any creature, big or small — includes nitrogen atoms which first entered the biological realm via prokaryotic nitrogen-fixing enzymes. at neutral pH, [NH 4 + ] >> [NH 3 ] ≥16 ATP Nitrogen fixation: in living cells, the ATP cost is 20-30 ATP per molecule of N 2 reduced: nitrogenase useless precious https://www.nature.com/scitable/knowledge/library/ biological-nitrogen-fixation- 23570419 The importance of N 2 -fixation. Blue lines on the phylogenetic tree (left) indicate 8 Bacterial phyla and 1 Archael phylum in which homologous genes for nitrogen- fixation have been found. (LUCA, Last Universal Common Ancestor). Of the four plants (photo at right), only one has been allowed to establish a symbiotic relationship with nitrogen-fixing bacteria. The three smaller, pale-looking plants either lack the bacteria and/or carry mutations that prevent a relationship with the bacteria. https://www.frontiersin.org/articles/ 10.3389/fmicb.2013.00201/ful l

Exercise 17 (Koch’s Postulate) 2 General Microbiology Lab, MICR:2158 Nitrogen fixation demands extensive genetic resources and enormous amounts of energy per NH 4 + molecule, but the guaranteed access to such a precious nutrient is worth the cost, as evidenced by diversity of prokaryotes on the evolutionary tree that can fix N 2 (blue lineages on diagram at left on page 1). Many eons after the evolution of nitrogen-fixing enzymes by ancient prokaryotes, plants evolved. Then, relationships developed between some nitrogen-fixing bacteria and some plants — both share the critical need for fixed nitrogen (amino acids, nucleotides, etc.) and have pooled their resources to acquire it. A variety of plant species have evolved to become hosts, while bacteria from diverse gram-positive and gram-negative lineages have evolved to partner with specific plants. The partnership between rhizobia and plants in the pea/bean family ( legumes ) is today’s most-studied nitrogen-fixing symbiosis. In this bacterium-plant relationship, both members work cooperatively to construct unique nodules on the legume’s roots where nitrogen fixation will occur. How might their complex partnership have arisen? Recent molecular insights suggest that ancient rhizobia originally had a disease-causing relationship with their plant hosts. As time passed, the pathogenic mechanisms used by the bacteria to invade and cause damage in the plant tissues mellowed, adapting towards mutualistic nitrogen fixation within special compartments in the plant’s roots instead. Economically speaking, the most important nitrogen-fixing symbioses take place between legumes (peas, beans, clover, alfalfa, peanuts, etc.) and several Proteobacterial genera which have “r hizobium ” in their names (informally known as rhizobia). For example, much of Iowa’s agricultural wealth depends upon the activities of two genera, Sinorhizobium and Bradyrhizobium , in soybean roots; livestock are fed soybeans as a protein source while humans eat tofu and edamame. Additionally, through the practice of crop rotation , farmers save on the expense and environmental damage of commercial nitrogenous fertilizers by alternating crops year after year: a field planted in legumes one year is planted with a non-legume, such as corn, the next: the corn is able to build its own proteins and nucleic acids from nitrogen remaining in the soil courtesy of the previous summer’s soybean rhizobia. Also, livestock can naturally obtain the amino acids they need in their diet by grazing in Each “bump” is a root nodule on a pea plant root. Every nodule contains thousands of specially adapted plant cells, within which are rhizobia that have become dedicated to “fertilizing” this plant through nitrogen fixation. Evidence of crop rotation in a soybean field near North Liberty, Iowa. Each tall plant germinated from a corn kernel left behind from the previous autumn harvest.

Exercise 17 (Koch’s Postulate) 3 General Microbiology Lab, MICR:2158 legume-rich pastures thanks to Sinorhizobium and Rhizobium activities in alfalfa and clover roots, respectively. Certain free-living rhizobia in soil (black rods) become attracted to chemicals secreted from a legume’s root hair. Drawn by the plant’s chemical invitation, rhizobia swim over, attach, and secrete Nod factors, which trigger a curl in the root hair. The curled root hair tip invaginates to form a cave-like opening (red) into the root hair itself. Rhizobia enter and grow. The plant responds by extending a hollow passageway for them (in red) now called an “infection thread”. Continued growth of rhizobia in the infection thread “pushes” the plant to extend a network of threads more deeply into the root root soil Branching infection threads provide access for rhizobia into many plant cells (more than the 7 cells shown here). The partnership continues to develop as infected root cells and intracellular rhizobia adapt symbiotically. Once fully-developed, a nodule will include a core of modified plant cells, each packed with bacteroids in symbiosomes. Also, the root is modified to maintain flows of molecules between the nodule and the rest of the plant. soil root soil 1 2 3 6 5 4 zoom out: A TEM view inside one plant cell in a soybean root nodule. The plant cell cytoplasm is packed with bacteroids (b), each enclosed in a symbiosome . A portion of the plant’s cell wall (CW) and endoplasmic reticulum (ER) are visible in the TEM. Each bacteroid is ~ 1 micrometer. TEM from http:// en.academic.ru/pictures/enwiki/82/Root- nodule01.jpg ER b b b

Exercise 17 (Koch’s Postulate) 4 General Microbiology Lab, MICR:2158 Background Information, continued: How Do Rhizobia and Legumes Form their Symbiosis? Once bacteria enter into the rhizobia-legume partnership, they differentiate from normal, free-living rods into bent, swollen, N 2 -fixing factories that will remain embedded within the root nodules for the rest of the plant's life. These misshapen, plant-dependent prokaryotes have lost their cell walls and are now referred to as bacteroids . Bacteroids live in root cells within symbiosomes , membrane-bound chambers that are optimized for nitrogen fixation. In the symbiosome, rhizobial genes are transcribed to make the enzymes for reducing N 2 to NH 4 + . Also, a protein called “leghemoglobin” binds to O 2 in order to protect one key enzyme, nitrogenase, which would otherwise become inactivated by oxygen. Ironically, N 2 -fixation in a root nodule is a process that requires oxygen-free conditions even though it’s carried out by a partnership of two O 2 -loving (aerobic) organisms! Thanks to photosynthesis, the plant has plenty of ATP to support its side of the partnership: light energy and CO 2 are captured by the legume in its leaves, converted into small organic molecules which pass down the stem and across symbiosome membranes to feed bacteroids in the roots. Within bacteroids, the Kreb’s Tricarboxylic Acid Cycle metabolizes these organic compounds to meet all the microbial carbon and energy requirements for survival and N 2 fixation. Ample NH 4 + from bacteroids provides an excellent return on the plant’s investment of resources. When the plant dies and plant material decays, the net result is nitrogen- enriched soil and a robust food web in the soil. Substantial genetic resources are devoted to this symbiosis by both partners. The rhizobia must use numerous genes to encode the enzymes of nitrogen fixation. In addition, both organisms must be able to signal each other with the molecular equivalent of a series of “secret handshakes” — after all, the plant cannot afford to mistakenly admit a potentially dangerous microbe into such an intimate relationship within its root cells. Meanwhile, the rhizobia respond only in the case of a plant that can support its needs. The specific recognition between the partners is so finely tuned that each species of legume will admit only certain strains of nitrogen-fixing bacteria (see step 2 on the previous page). The rhizobia carry genes that allow them to work together with the host plant to build the root nodule. These genes are often organized into “ fix ” and “ nif” operons (for nitrogen fixation) and “nod” operons (for nodulation). Some rhizobia encode these functions on plasmids that are so large they’re called megaplasmids. In one completely sequenced example of a megaplasmid (pSym, found in Rhizobium etli ), the length is over 371,000 base pairs and includes many ORFs of unknown function in addition to fix , nif , and nod operons. Experimental Overview and Goals Our goals for Exercise 17 are to observe the natural partnership between legumes and rhizobia and also see if we can develop this highly specific symbiosis under laboratory culture conditions. In Part A, students will dissect root nodules from living legumes. Microorganisms from within the nodule will be examined using microscopic approaches and plated onto Rhizobium Isolation Agar. Students will also inoculate pea plants with rhizobia cultures in a controlled study to test the specificity of the root nodule symbiosis.

Exercise 17 (Koch’s Postulate) 5 General Microbiology Lab, MICR:2158 in Part B, students will evaluate the results of their streak plates: colonies of rhizobia should be easily recognizable because rhizobia convert the mannitol from Rhizobium Isolation Agar into a glistening, white polymer: the resultant colonies are round, shiny and mucoid. In Part C — ongoing until the end of the semester — students will monitor the growing pea plants for formation of root nodules. In the week 15, numbers of root nodules will be tallied for analysis by the class. Interactions between legumes and rhizobia constitute a unique example of infection . Although the usual outcome of this infection appears to be mutually beneficial, the legume/ rhizobium interaction resembles the type of specific interactions that occur when a pathogenic bacterium infects its host to elicit damage and disease. Thus, students should understand that with Exercise 17, we are pursuing the classical microbiological test of cause and effect known as Koch’s Postulates . Part A MATERIALS per student: 1. Rhizobium isolation agar plate (recipe on p. 4) 2. a razor blade (for dissecting) 3. sterile water (~1 mL in Eppendorf tube) 4. methylene blue and staining tray 5. ethanol jar (for sterilizing razor blade) 6. a pea seedling on a large slant of plant growth agar 7. a sterile swab on benchtop (share with neighbors): 8. Eppendorf tubes of rhizobia cultures (might be from clover or peas originally; numbered 1-8) available per section: 9. nodulated pea plants (6- to 8-weeks old; grown indoors in garden soil to which rhizobia (previously isolated from pea nodules) had been added at the time of planting) and/or clover plants, freshly dug 10. tin labeled for incubation at 30°C 1 2 3 4 6 7 8 # 9 5

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