Comparative secretome analysis suggests low plant cell wall degrading capacity in Frankia symbionts

Abstract

BackgroundFrankia sp. strains, the nitrogen-fixing facultative endosymbionts of actinorhizal plants, have long been proposed to secrete hydrolytic enzymes such as cellulases, pectinases, and proteases that may contribute to plant root penetration and formation of symbiotic root nodules. These or other secreted proteins might logically be involved in the as yet unknown molecular interactions between Frankia and their host plants. We compared the genome-based secretomes of three Frankia strains representing diverse host specificities. Signal peptide detection algorithms were used to predict the individual secretomes of each strain, and the set of secreted proteins shared among the strains, termed the core Frankia secretome. Proteins in the core secretome may be involved in the actinorhizal symbiosis.ResultsThe Frankia genomes have conserved Sec (general secretory) and Tat (twin arginine translocase) secretion systems. The potential secretome of each Frankia strain comprised 4–5% of the total proteome, a lower percentage than that found in the genomes of other actinobacteria, legume endosymbionts, and plant pathogens. Hydrolytic enzymes made up only a small fraction of the total number of predicted secreted proteins in each strain. Surprisingly, polysaccharide-degrading enzymes were few in number, especially in strain CcI3, with more esterolytic, lipolytic and proteolytic enzymes having signal peptides. A total of 161 orthologous proteins belong to the core Frankia secretome. Of these, 52 also lack homologs in closely related actinobacteria, and are termed "Frankia-specific." The genes encoding these conserved secreted proteins are often clustered near secretion machinery genes.ConclusionThe predicted secretomes of Frankia sp. are relatively small and include few hydrolases, which could reflect adaptation to a symbiotic lifestyle. There are no well-conserved secreted polysaccharide-degrading enzymes present in all three Frankia genomes, suggesting that plant cell wall polysaccharide degradation may not be crucial to root infection, or that this degradation varies among strains. We hypothesize that the relative lack of secreted polysaccharide-degrading enzymes in Frankia reflects a strategy used by these bacteria to avoid eliciting host defense responses. The esterases, lipases, and proteases found in the core Frankia secretome might facilitate hyphal penetration through the cell wall, release carbon sources, or modify chemical signals. The core secretome also includes extracellular solute-binding proteins and Frankia-specific hypothetical proteins that may enable the actinorhizal symbiosis.

Diminished Exoproteome of Frankia spp. in Culture and Symbiosis

“…Conversely, in Streptomyces species grown in minimal medium, the lack of complex nutrients stimulates cells to secrete proteins in order to scavenge resources (10). Bioinformatics analysis of the three Frankia genomes predicted few degradative enzymes, so it is unlikely that the medium alone accounts for the minimal secretome in strain CcI3 (22).…”

“…Unlike related soil actinobacteria, such as Streptomyces spp., that secrete an array of degradative enzymes, Frankia strains have been predicted to secrete comparatively few proteins, according to an extensive bioinformatics study that predicted frankial secreted proteomes based on consensus signal peptides and transmembrane domains (12,22). While several Streptomyces species are predicted to secrete more than 100 hydrolases, the Frankia secreted proteomes have only 10 to 20, and these are primarily lipases, esterases, and proteases, rather than putative polysaccharide-degrading enzymes (22).…”

“…While several Streptomyces species are predicted to secrete more than 100 hydrolases, the Frankia secreted proteomes have only 10 to 20, and these are primarily lipases, esterases, and proteases, rather than putative polysaccharide-degrading enzymes (22). This observation raises the hypothesis that frankiae are successful symbionts at least partly because they present an innocuous visage to the plant host.…”

“…To provide a more complete overview of secreted proteins in light of our previous bioinformatics study (22), we investigated both free-living and symbiotic cells of another Frankia strain, the Casuarina symbiont CcI3, using 2D SDS-polyacrylamide gel electrophoresis (PAGE) and liquid chromatography cou-pled to tandem mass spectrometry (LC MS/MS) of trypsindigested protein samples.…”

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

“…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.…”