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ABSTRACT: The above- and below-ground parts of rice plants create specific habitats for various microorganisms. In this study, we characterized the phyllosphere and rhizosphere microbiota of rice cultivars using a metaproteogenomic approach to get insight into the physiology of the bacteria and archaea that live in association with rice. The metaproteomic datasets gave rise to a total of about 4600 identified proteins and indicated the presence of one-carbon conversion processes in the rhizosphere as well as in the phyllosphere. Proteins involved in methanogenesis and methanotrophy were found in the rhizosphere, whereas methanol-based methylotrophy linked to the genus Methylobacterium dominated within the protein repertoire of the phyllosphere microbiota. Further, physiological traits of differential importance in phyllosphere versus rhizosphere bacteria included transport processes and stress responses, which were more conspicuous in the phyllosphere samples. In contrast, dinitrogenase reductase was exclusively identified in the rhizosphere, despite the presence of nifH genes also in diverse phyllosphere bacteria.
The ISME Journal 12/2011; 6(7):1378-90. · 7.38 Impact Factor
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ABSTRACT: Acetyl-CoA assimilation was extensively studied in organisms harboring the glyoxylate cycle. In this study, we analyzed the metabolism of the facultative methylotroph Methylobacterium extorquens AM1, which lacks isocitrate lyase, the key enzyme in the glyoxylate cycle, during growth on acetate. MS/MS-based proteomic analysis revealed that the protein repertoire of M. extorquens AM1 grown on acetate is similar to that of cells grown on methanol and includes enzymes of the ethylmalonyl-CoA (EMC) pathway that were recently shown to operate during growth on methanol. Dynamic 13C labeling experiments indicate the presence of distinct entry points for acetate: the EMC pathway and the TCA cycle. 13C steady-state metabolic flux analysis showed that oxidation of acetyl-CoA occurs predominantly via the TCA cycle and that assimilation occurs via the EMC pathway. Furthermore, acetyl-CoA condenses with the EMC pathway product glyoxylate, resulting in malate formation. The latter, also formed by the TCA cycle, is converted to phosphoglycerate by a reaction sequence that is reversed with respect to the serine cycle. Thus, the results obtained in this study reveal the utilization of common pathways during the growth of M. extorquens AM1 on C1 and C2 compounds, but with a major redirection of flux within the central metabolism. Furthermore, our results indicate that the metabolic flux distribution is highly complex in this model methylotroph during growth on acetate and is fundamentally different from organisms using the glyoxylate cycle.
Journal of Biological Chemistry 11/2011; 287(1):757-66. · 4.77 Impact Factor
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ABSTRACT: Diverse bacterial taxa that live in association with plants affect plant health and development. This is most evident for those bacteria that undergo a symbiotic association with plants or infect the plants as pathogens. Proteome analyses have contributed significantly toward a deeper understanding of the molecular mechanisms underlying the development of these associations. They were applied to obtain a general overview of the protein composition of these bacteria, but more so to study effects of plant signaling molecules on the cytosolic proteome composition or metabolic adaptations upon plant colonization. Proteomic analyses are particularly useful for the identification of secreted proteins, which are indispensable to manipulate a host plant. Recent advances in the field of proteome analyses have initiated a new research area, the analysis of more complex microbial communities. Such studies are just at their beginning but hold great potential for the future to elucidate not only the interactions between bacteria and their host plants, but also of bacteria-bacteria interactions between different bacterial taxa when living in association with plants. These include not only the symbiotic and pathogenic bacteria, but also the commensal bacteria that are consistently found in association with plants and whose functions remain currently largely uncovered.
Proteomics 03/2011; 11(15):3086-105. · 4.43 Impact Factor
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ABSTRACT: Rhizobia and legume plants establish symbiotic associations resulting in the formation of organs specialized in nitrogen fixation. In such organs, termed nodules, bacteria differentiate into bacteroids which convert atmospheric nitrogen and supply the plant with organic nitrogen. As a counterpart, bacteroids receive carbon substrates from the plant. This rather simple model of metabolite exchange underlies symbiosis but does not describe the complexity of bacteroids' central metabolism. A previous study using the tropical symbiotic model Aeschynomene indica/photosynthetic Bradyrhizobium sp. ORS278 suggested a role of the bacterial Calvin cycle during the symbiotic process. Herein we investigated the role of two RuBisCO gene clusters of Bradyrhizobium sp. ORS278 during symbiosis. Using gene reporter fusion strains, we showed that cbbL1 but not the paralogous cbbL2 is expressed during symbiosis. Congruently, CbbL1 was detected in bacteroids by proteome analysis. The importance of CbbL1 for symbiotic nitrogen fixation was proven by a reverse genetic approach. Interestingly, despite its symbiotic nitrogen fixation defect, the cbbL1 mutant was not affected in nitrogen fixation activity under free living state. This study demonstrates a critical role for bacterial RuBisCO during a rhizobia/legume symbiotic interaction.
PLoS ONE 01/2011; 6(7):e21900. · 4.09 Impact Factor
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ABSTRACT: In this article, we introduce a method using nanoscale ion-pair reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC) hyphenated to nanoelectrospray ionization high-resolution mass spectrometry (nano-ESI-HRMS) to separate and identify metabolites in cell extracts. Separation of metabolites was performed on a 100 μm i.d. C18 column with tributylamine (TBA) as the ion-pairing reagent and methanol as the eluent. Basic pH (9.4) of the mobile phase was critical to achieve sufficient retention and sharp metabolite elution at a low concentration of TBA (1.7 mM). Limits of detection were determined for 54 standards with an LTQ-Orbitrap mass spectrometer to be in the upper attomole to low femtomole range for key metabolites such as nucleotides, phosphorylated sugars, organic acids, and coenzyme A thioesters in solvent as well as in a complex matrix. To further evaluate the method, metabolome analysis was performed injecting different amounts of biomass of the methylotroph model organism Methylobacterium extorquens AM1. A (12)C/(13)C labeling strategy was implemented to improve metabolite identification. Analysis of three biological replicates performed with 1.5 ng of cell dry weight biomass equivalents resulted in the identification of 20 ± 4 metabolites, and analysis of 150 ng allowed identifying 157 ± 5 metabolites from a large spectrum of metabolite classes.
Analytical Chemistry 12/2010; 83(3):850-5. · 5.86 Impact Factor
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ABSTRACT: Rhizobia are able to infect legume roots, elicit root nodules, and live therein as endosymbiotic, nitrogen-fixing bacteroids. Host recognition and specificity are the results of early programming events in bacteria and plants, in which important signal molecules play key roles. Here, we introduce a new aspect of this symbiosis: the adaptive response to hosts. This refers to late events in bacteroids in which specific genes are transcribed and translated that help the endosymbionts to meet the disparate environmental requirements imposed by the hosts in which they live. The host-adaptation concept was elaborated with Bradyrhizobium japonicum and three different legumes (soybean, cowpea, and siratro). Transcriptomes and proteomes in root-nodule bacteroids were analyzed and compared, and genes and proteins were identified which are specifically induced in only one of the three hosts. We focused on those determinants that were congruent in the two data sets of host-specific transcripts and proteins: seven for soybean, five for siratro, and two for cowpea. One gene cluster for a predicted ABC-type transporter, differentially expressed in siratro, was deleted in B. japonicum. The respective mutant had a symbiotic defect on siratro rather than on soybean or cowpea. This result demonstrates the value of the applied approach and corroborates the host-specific adaptation concept.
Molecular Plant-Microbe Interactions 06/2010; 23(6):784-90. · 4.43 Impact Factor
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ABSTRACT: Bradyrhizobium japonicum, a gram-negative soil bacterium that establishes an N(2)-fixing symbiosis with its legume host soybean (Glycine max), has been used as a symbiosis model system. Using a sensitive geLC-MS/MS proteomics approach, we report the identification of 2315 B. japonicum strain USDA110 proteins (27.8% of the theoretical proteome) that are expressed 21 days post infection in symbiosis with soybean cultivated in growth chambers, substantially expanding the previously known symbiosis proteome. Integration of transcriptomics data generated under the same conditions (2780 expressed genes) allowed us to compile a comprehensive expression profile of B. japonicum during soybean symbiosis, which comprises 3587 genes/proteins (43% of the predicted B. japonicum genes/proteins). Analysis of this data set revealed both the biases and the complementarity of these global profiling technologies. A functional classification and pathway analysis showed that most of the proteins involved in carbon and nitrogen metabolism are expressed, including a complete set of tricarboxylic acid cycle enzymes, several gluconeogenesis and pentose phosphate pathway enzymes, as well as several proteins that were previously not considered to be present during symbiosis. Congruent results were obtained for B. japonicum bacteroids harvested from soybeans grown under field conditions.
Proteomics 04/2010; 10(7):1391-400. · 4.43 Impact Factor
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ABSTRACT: Aerial plant surfaces represent the largest biological interface on Earth and provide essential services as sites of carbon dioxide fixation, molecular oxygen release, and primary biomass production. Rather than existing as axenic organisms, plants are colonized by microorganisms that affect both their health and growth. To gain insight into the physiology of phyllosphere bacteria under in situ conditions, we performed a culture-independent analysis of the microbiota associated with leaves of soybean, clover, and Arabidopsis thaliana plants using a metaproteogenomic approach. We found a high consistency of the communities on the 3 different plant species, both with respect to the predominant community members (including the alphaproteobacterial genera Sphingomonas and Methylo bacterium) and with respect to their proteomes. Observed known proteins of Methylobacterium were to a large extent related to the ability of these bacteria to use methanol as a source of carbon and energy. A remarkably high expression of various TonB-dependent receptors was observed for Sphingomonas. Because these outer membrane proteins are involved in transport processes of various carbohydrates, a particularly large substrate utilization pattern for Sphingomonads can be assumed to occur in the phyllosphere. These adaptations at the genus level can be expected to contribute to the success and coexistence of these 2 taxa on plant leaves. We anticipate that our results will form the basis for the identification of unique traits of phyllosphere bacteria, and for uncovering previously unrecorded mechanisms of bacteria-plant and bacteria-bacteria relationships.
Proceedings of the National Academy of Sciences 09/2009; 106(38):16428-33. · 9.68 Impact Factor
