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Applied and Environmental Microbiology, November 2009, p. 6721-6728, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.01559-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Diminished Exoproteome of Frankia spp. in Culture and Symbiosis {triangledown} ,{dagger}

J. E. Mastronunzio, Y. Huang, and D. R. Benson*

Department of Molecular and Cell Biology, University of Connecticut, 91 North Eagleville Rd., Unit 3125, Storrs, Connecticut 06269

Received 2 July 2009/ Accepted 7 September 2009

Frankia species are the most geographically widespread gram-positive plant symbionts, carrying out N2 fixation in root nodules of trees and woody shrubs called actinorhizal plants. Taking advantage of the sequencing of three Frankia genomes, proteomics techniques were used to investigate the population of extracellular proteins (the exoproteome) from Frankia, some of which potentially mediate host-microbe interactions. Initial two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatants indicated that cytoplasmic proteins appeared in supernatants as cells aged, likely because older hyphae lyse in this slow-growing filamentous actinomycete. Using liquid chromatography coupled to tandem mass spectrometry to identify peptides, 38 proteins were identified in the culture supernatant of Frankia sp. strain CcI3, but only three had predicted export signal peptides. In symbiotic cells, 42 signal peptide-containing proteins were detected from strain CcI3 in Casuarina cunninghamiana and Casuarina glauca root nodules, while 73 and 53 putative secreted proteins containing signal peptides were identified from Frankia strains in field-collected root nodules of Alnus incana and Elaeagnus angustifolia, respectively. Solute-binding proteins were the most commonly identified secreted proteins in symbiosis, particularly those predicted to bind branched-chain amino acids and peptides. These direct proteomics results complement a previous bioinformatics study that predicted few secreted hydrolytic enzymes in the Frankia proteome and provide direct evidence that the symbiosis succeeds partly, if not largely, because of a benign relationship.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, University of Connecticut, 91 North Eagleville Rd., Unit 3125, Storrs, CT 06269. Phone: (860) 486-4258. Fax: (860) 486-4331. E-mail: david.benson{at}uconn.edu

{triangledown} Published ahead of print on 11 September 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, November 2009, p. 6721-6728, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.01559-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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