1. About Frankia Literature Review Suzanna Kruger Biology 523 Microbial Ecology Dr. Anna-Louise Reysenbach 3 June 2003

2. Overview Frankia taxonomy Host species Role in nitrogen cycle Methods of isolation Anatomy of nodule formation Anatomy and Metabolism Ecological relationships

3. Frankia Taxonomy Filamentous, High GC, Gram-Positive Bacteria Group III Actinomycete –Nitrogen-fixing symbiont of plants (Madigan, 2003) http://www.msu.edu/~batzli/whats_frankia.html http://helios.bto.ed.ac.uk/bto/microbes/microbes.htm#Microorganisms% 20and%20the%20"Tree%20of%20Life"

4. Host Species Hosts are non- leguminous, woody angiosperms 8 Families, 23 genera including: –Alnus (Betulacaceae) –Ceanothus (Rhamnaceae) –Rubus (Rosaceae) http://www.msu.edu/~batzli/graphics/alder_forest.jpg Red Alder Forest, Washington

5. Role in Nitrogen Cycle (Madigan, 2003) Nitrogen fixation: N 2 + 8H+ + 8e - + 16 ATP = 2NH 3 + H 2 + 16ADP + 16 Pi Catalyzed by nitrogenase Reasons for interest in Frankia are related to the Nitrogen Cycle: Forestry management Land reclamation Soil improvment

6. Methods of Isolation and Culture of Frankia ISOLATION of: First isolated 1978 (Cervantes and Rodríguez-Barrueco, 1992) Sterilization, mechanical disruption, or lytic enzymes (Cervantes and Rodríguez- Barrueco, 1992) CULTURE of: Simple culture media which includes glucose and propionate as a carbon source, casein hydrolysate as a nitrogen source, and vitamins, salts and trace elements. Lipids may also be helpful. (Cervantes and Rodríguez-Barrueco, 1992) WHY: It’s not an obligate relationship, so having these tools allows scientists and those interested in forestry management or soil amelioration to inoculate stands of alder or other sp.

7. Identification of Frankia strains 11th International Conference on Frankia and Actinorhizal Plants (1998) 1. 16s rRNA (Ritchie et al., 1999) 2. PCR + DNA hybridization (Nalin et al., 1999) 3. PCR + RFLPs (Lumini and Bosco, 1999) 4. Antibiotic resistance patterns (Tisa et al., 1999)

8. Anatomy of Nodule Formation Berg, 1999: Nodule development is under the plant’s control…great variety of nodule morphology across species Three steps to invasion of host by Frankia : – Infection threads with “interfacial matrix”, formation of cytoplasmic bridges (filamentous) – Vegetative hyphae (filamentous) – Symbiotic vesicles - where N 2 - fixation occurs (multicellular and spherical) http://helios.bto.ed.ac.uk/bto/microbes/nitrogen.htm

9. Anatomy and Metabolism: O 2 and Nitrogenase Silvester et al., 1999 Frankia grows best at atmospheric pO 2. In most species, symbiotic vesicles develop an envelope to protect nitrogenase. –Example: In Alnus, envelope thickness adjusts to ambient thickness. Coriaria and Dastica do not have a multilaminate envelope - how do they protect nitrogenase? Methods: Miscroscopy High concentrations of mitochondria around the base of the symbiotic vesicles may protect nitrogenase. Tjepkema et al., 1999 Dastica has a similar morphology to legumes, which have a variable diffusion barrier to O 2. Does Dastica ? Methods: Nitrogenase activity was measured via C 2 H 2 reduction and the evolution of CO 2. Results: Nitrogenase activity was not restored to predecline values by increasing external pO 2. Furthermore, calculations of O 2 uptake by mitochondria are not sufficient to reduce the diffusion of O 2 past the mitochondria… H A : Nitrogenase activity is compartmenalized in multiple vesicles so that if activity is decreased in one, it is increased in another.

10. Ecological Relationships Clawson et al., 1999 What are the interactions and relationships that address dominance, competition and distribution among Frankia strains growing in a variety of conditions? Methods: Obtained strains and developed clades based on 16s rRNA. Results: Diversity in a single alder stand is low; instead it is dominated by a single strain or set of similar strains. Further research: Are there different ecotypes associated with different soil conditions? Markham and Chanway, 1999 Does past contact reduce the degree of mutualism in the Alnus rubra - Frankia symbiosis? Methods: Three low-elevation and three high-elevation populations of Red Alder were inoculated with Frankia, either a familiar or an unfamiliar strain. Results: –Low elevation: High nitrogen in soils. Plants inoculated with the unfamiliar strain grew larger than those with the familiar strain. –High elevation: Low nitrogen in soils. Plants inoculated with the familiar strain grew larger. Conclusion: The degree of mutualism depends on environmental conditions and may decrease with time.

11. Relationship to Mycorrhizal co-Symbionts Cervantes, E. and C. Rodríguez-Barrueco. 1992. “Relationships between the Mycorrhizal and Actinorhizal Symbioses in Non-legumes,” in Methods in Microbiology, Volume 24: Techniques for the Study of Mycorrhiza, Norris J.R., D.J. Read and A.K. Varma, Editors. Academic Press: New York. 417-432. Alnus sp. forms symbiotic relationships with fungi also, including: –Cortinariaceae –Hygrophoraceae –Russulaceae –Boletaceae –Fungi Imperfecti Presence of Frankia and N-fixing activity may put increased pressure on the plant for the production of ATP. Ectomycorrhizal association as been shown to enhance phosphate uptake for Alnus viridis.

12. References Berg, R. Howard. 1999. “Frankia forms infection threads.” Canadian Journal of Botany. 77:1327-1333. Berg, R. Howard. 1999. “Cytoplasmic bridge formation in the nodule apex of actinorhizal root nodules.” Canadian Journal of Botany. 77:1351-1357 Cervantes, E. and C. Rodríguez-Barrueco. 1992. “Relationships between the Mycorrhizal and Actinorhizal Symbioses in Non- legumes,” in Methods in Microbiology, Volume 24: Techniques for the Study of Mycorrhiza, Norris J.R., D.J. Read and A.K. Varma, Editors. Academic Press: New York. 417-432. Clawson, Michael L., Jeffrey Gawronski and David R. Benson. 1999. “Dominance of Frankia strains in stands of Alnus incana subsp. Rugosa and Myrica pensylvanica.” Canadian Journal of Botany. 77:1203-1207. Lumini, Erica and Marco Bosco. 1999. “Polymerase chain reaction - restriction fragment length polymorphisms for assessing and increasing biodiversity of Frankia culture collections.” Canadian Journal of Botany. 77:1261-1269. Madigan, M.T., J.M. Martinko and J. Parker. Brock Biology of Microorganisms, 10th Edition. Prentice-Hall: Upper Saddle River, NJ, 2003. Markham, John H. and Chris P. Chanway. 1999. “Does past contact reduce the degree of mutualism in the Alnus rubra - Frankia symbiosis?” Canadian Journal of Botany. 77:434-441. Nalin, R., P Normand, P. Simonet and A.M. Domenach. 1999. “Polymerase chain reaction and hybridazation on DNA extracted from soil as a tool for Frankia spp. Population distribution studies in soil.” Canadian Journal of Botany. 77:1239-1247. Ritchie, Nancy J. and David D. Myrold. 1999. “Phylogenetic placement of uncultured Ceanothus microsymbionts using 16s rRNA gene sequences.” Canadian Journal of Botany. 77:1208-1213. Silverster, Warwick B., Birgit Langenstein, R. Howard Berg. 1999. “Do mitochondria provide the oxygen diffusion barrier in root nodules of Coriaria and Dastica?” Canadian Journal of Botany. 77:1358-1366 Tisa, Louis S., Matthew Chval, Glenn D. Krumholz and Joel Richards. 1999. “Antibiotic resistance patterns of Frankia stains.” Canadian Journal of Botany. 77:1257-1260. Tjepkema, John D., Gangyi Du and Christa R. Schwintzer. 1999. “Response of respiration and nitrogenase activity in Dastica glomerata (Presl.) Baill. to changes in pO 2.” Canadian Journal of Botany. 77:1367-1372.