Progress 08/01/03 to 01/31/06
Outputs
The goal of this research was to elucidate the molecular biology of the protein glomalin, which is produced by arbuscular mycorrhizal fungi (AMF). Glomalin-related protein in soils is highly positively correlated with soil aggregate water stability, a major ecosystem function of AMF. We have succeeded in this project, and have identified that this protein has high sequence identity with heat shock protein 60. The strategy adopted to identify the gene(s) responsible for Glomalin synthesis entailed (a) performing random sequencing of the glomalin protein produced under in vitro culture, (b) designing PCR primers of various degeneracy based on this sequence, and (c) obtaining the gene from a cDNA library prepared from AM fungi. First we significantly improved the culture system for AMF (Gadkar et al. 2006) to provide starting material sufficient for this study. Another major impediment to research in this area is obtaining sufficient DNA from AMF spores. We hence
developed a method to greatly improve DNA amount produced from AMF spores using a polymerase with strand-displacement activity (Gadkar and Rillig 2005); this development has the potential to substantially improve molecular biology work with AMF in general. During our efforts to extract glomalin from in vitro cultures of AMF, we were able to demonstrate that, contrary to our working model, glomalin resides predominantly (80%) in the mycelium of AMF, not the culture supernatant (Driver et al. 2005). We finally modified the protocol of protein extraction from fungal cultures to produce distinct protein bands on an SDS-PAGE gel. Immunoblot analysis using the glomalin-specific and glomalin-defining monoclonal antibody MAb32B11 revealed a single protein band of ~65 kDa. Based on these developments and observations, we employed two amino acid sequencing strategies on the excised band, of which only nano LC MS was successful, while N-terminal sequencing proved to be a dead end. Using a nested
primer design, we were eventually able to isolate and sequence the full length gene for glomalin. We also succeed in in vitro expression of the protein. We showed that glomalin has high amino acid sequence identity with, and based on initial phylogenetic analysis likely homology to, fungal heat shock protein 60 (hsp 60) (Gadkar and Rillig 2006). Comparing with pieces of information that had been obtained in work with proteinaceous material from soil, we were able to confirm several hypothesized features of the actual protein, while not finding evidence for others: for example, we found putative N-glycosylation sites, but we found no sequence similarity with hydrophobins (Rillig 2005) or Fe-binding domains. This result puts research on glomalin and glomalin-related soil protein (Rillig 2004; Rillig and Mummey 2006) on a much firmer footing.
Impacts
There are several impacts of our work. (1) Defining the molecular biology of glomalin will have a large impact on both our understanding of soil aggregation and the biology of the important symbiotic fungi that produce it. Arbuscular mycorrhizal fungi (AMF) have long been known to be important in soil aggregation, yet the exact mechanisms involved are poorly understood. Glomalin-related soil protein is very highly correlated with soil aggregation, which is why we try to learn more about it. With gene for glomalin known, novel applications may be possible, such as biotechnological production of glomalin in bioreactors for field application; or just better management of the fungi to improve their production of glomalin. (2) Our work on improving DNA yield from AMF, conducted as part of this project, will have wide-ranging impact on research with these fungi. Molecular biology of these fungi is extremely limited by the availability of DNA; by removing this bottleneck we
hope to have greatly stimulated further understanding of these key symbionts. (3) As part of this project we have taken on the task of clearly distinguishing between glomalin-related protein (GRSP) from soil, and the actual protein, as part of a review on this subject. This new nomenclature is now being widely adopted in the literature, and serves to eliminate widespread ambiguity. (4) In the course of the research on this project we have also developed a new protocol for growing AMF in vitro. This will likely have far-reaching consequences for the study of the biochemistry of fungal products.
Publications
- Rillig MC. 2004. Arbuscular mycorrhizae, glomalin and soil quality. Canadian Journal of Soil Science 84: 355-363.
- Driver JD, Holben WE, Rillig MC. 2005. Characterization of glomalin as a hyphal wall component of arbuscular mycorrhizal fungi. Soil Biology & Biochemistry 37: 101-106.
- Gadkar V, Rillig MC. 2005. Application of Phi29 DNA polymerase mediated whole genome amplification on single spores of arbuscular mycorrhizal fungi. FEMS Microbiology Letters 242: 65-71.
- Rillig MC. 2005. Polymers and microorganisms. In: Encyclopedia of Soils in the Environment (Ed. Hillel D). Elsevier Ltd., Oxford, UK, pp.287-294.
- Rillig MC. 2005. A connection between fungal hydrophobins and soil water repellency. Pedobiologia 49: 395-399.
- Rillig MC, Rosier CL, Piotrowski JS, Gadkar V, Driver JD. 2005. Glomalin: a novel proteinaceous substance produced by arbuscular mycorrhizal fungi. In: Varma A, Podila GK (Eds), Biotechnological applications of microbes. IK International Pvt. Ltd., New Delhi, pp. 67-85.
- Rillig MC, Mummey DL. 2006. Tansley review. Mycorrhizas and soil structure. New Phytologist (in press)
- Gadkar V, Driver JD, Rillig MC. 2006. A novel in vitro cultivation system to produce and isolate soluble factors released from hyphae of arbuscular mycorrhizal fungi. Biotechnology Letters (in press)
- Gadkar V, Rillig MC. 2006.The arbuscular mycorrhizal fungal protein glomalin is a putative homolog of heat shock protein 60. FEMS Microbiology Letters (in revision)
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Progress 10/01/03 to 09/30/04
Outputs
The goal of this research is to elucidate the molecular biology of the protein glomalin, which is produced by arbuscular mycorrhizal fungi (AMF). Glomalin-related protein in soils is highly positively correlated with soil aggregate water stability, a major ecosystem function of AMF. The strategy adopted to fish out the gene(s) responsible for Glomalin synthesis entailed (a) by performing random sequencing of the Glomalin protein,(b) designing PCR primers based on this sequence, and (c) fishing out the gene from a cDNA library prepared from AM fungi. Sequencing of putative Glomalin protein: Using improved in vitro culture systems of AMF, we have been able to extract and partially purify this protein. In order to sequence the protein, we adopted two sequencing strategies: N-terminal and tandem MS. The N-terminal sequence gave us a 20 amino acid and the MS sequencing gave us a set of six peptides with length from 7 to 18 amino acids. Based on the N-terminal sequence, we
designed a total of four sets of degenerate PCR primers with a degeneracy factor ranging from 64 to a maximum of 256. These degenerate primers were used for PCR based screening of cDNA library synthesized from the AM fungus Glomus intraradices. Two different libraries were used, one prepared from extraradical mycelium and one from germinating spores. A nested PCR strategy was used where one end was the putative glomalin specific degenerate primer and the other end was the vector primer. Sequencing of all the amplified fragments was done, out of which two sequences have been selected for further characterization. These sequences are being used for designing primers and obtaining the complete sequence of the fragment from the cDNA library. A major impediment to research in this area, we have developed a method to greatly improve DNA amount produced from AMF spores (Gadkar and Rillig 2005); this development has the potential to substantially improve molecular biology work with AMF in
general. During efforts to extract glomalin from cultures of AMF, we were able to demonstrate that, contrary to our working model, glomalin resides predominantly in the mycelium of AMF, not the culture supernatant (Driver et al. 2005), providing important clues concerning production of glomalin for analysis purposes.
Impacts
Defining the molecular biology of glomalin is expected to have a large impact on both our understanding of soil aggregation and the biology of the important symbiotic fungi that produce it. Arbuscular mycorrhizal fungi (AMF) have long been known to be important in soil aggregation, yet the exact mechanisms involved are poorly understood. Glomalin-related soil protein is very highly correlated with soil aggregation, which is why we try to learn more about it. If the gene for glomalin were known, novel applications may be possible, such as biotechnological production of glomalin in bioreactors for field application; or just better management of the fungi to improve their production of glomalin. Our work on improving DNA yield from AMF, conducted as part of this project, will have wide-ranging impact on research with these fungi. Molecular biology of these fungi is extremely limited by the availability of DNA; by removing this bottleneck we hope to have greatly
stimulated further understanding of these key symbionts.
Publications
- Rillig MC. 2004. Arbuscular mycorrhizae, glomalin and soil quality. Canadian Journal of Soil Science (in press)
- Rillig MC. 2005. Polymers and microorganisms. In: Encyclopedia of Soils in the Environment (Ed. Hillel D). Elsevier Ltd., Oxford, UK, pp.287-294.
- Driver JD, Holben WE, Rillig MC. 2005. Characterization of glomalin as a hyphal wall component of arbuscular mycorrhizal fungi. Soil Biology & Biochemistry 37: 101-106.
- Gadkar V, Rillig MC. 2005. Application of Phi29 DNA polymerase mediated whole genome amplification on single spores of arbuscular mycorrhizal fungi. FEMS Microbiology Letters 242: 65-71.
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