Diminished Exoproteome of Frankia spp. in Culture and Symbiosis

Abstract

Frankia species are the most geographically widespread gram-positive plant symbionts, carrying out N 2 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 peptidecontaining 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.

Significant Natural Product Biosynthetic Potential of Actinorhizal Symbionts of the Genus Frankia, as Revealed by Comparative Genomic and Proteomic Analyses

“…CcI3, of which three (FC03, FC12, and FC19) contained two or more protein hits with two peptide identifications per protein. Furthermore, other transcriptome and proteome studies show expression of some of these biosynthetic clusters (1,3,5,33,34,44). These data together indicate that Frankia has both the genetic capacity and the biosynthetic capacity to produce secondary metabolites.…”

“…43 Mb for the narrow-host-range Frankia strain CcI3 to 7.50 Mb for the medium-host-range Frankia strain ACN14a (ACN) to 8.98 Mb for the broad-host-range Frankia strain EAN1pec (EAN). Since the elucidation of these Frankia genomes, bioinformatic approaches have illuminated codon usage patterns (47), predicted secretosome profiles (35), and led to genomeguided studies on the Frankia transcriptome (3,44) and proteome (1,5,33,34). Genome mining also provides an opportunity to identify important physiology and metabolic functions, including secondary metabolism.…”

Wild nodules can be broken: proteomics of Frankia in field-collected root nodules

“…Two putative secreted lipases were found in A. incana nodules. A bioinformatics analysis predicted relatively few secreted proteins in Frankia, compared to soil organisms such as Bacillus and Streptomyces that secrete numerous degradative enzymes (Mastronunzio et al 2008(Mastronunzio et al , 2009). …”

The Determinants of the Actinorhizal Symbiosis

“…Symbiotic cells can also be studied with this approach, which permitted to identify 42 signal peptide containing proteins in Frankia 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, in particular putative branched-chain amino acids and peptides transporters (62). Few of the proteins recovered under these symbiotic condition were present in the three genomes.…”