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 Methylobacterium) 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.metaproteomics ͉ methylotrophy ͉ plant phyllosphere ͉ Pseudomonas ͉ Sphingomonas F or terrestrial plants, the phyllosphere represents the interface between the above-ground parts of plants and the air. Conservative estimates indicate that the roughly 1 billion square kilometers of worldwide leaf surfaces host more than 10 26 bacteria, which are the most abundant colonizers of this habitat (1, 2). The overall microbiota in this ecosystem is thus sufficiently large to have an impact on the global carbon and nitrogen cycles. Additionally, the phyllosphere inhabitants influence their hosts at the level of the individual plants. To a large extent, interest in phyllosphere microbiology has been driven by investigations on plant pathogens. Their spread, colonization, survival, and pathogenicity mechanisms have been the subject of numerous studies (2). Much less understood are nonpathogenic microorganisms that inhabit the phyllosphere. The composition of the phyllosphere microbiota has been analyzed in only a few studies by cultivation-independent methods (e.g., refs. 3-5); however, such methods are essential in light of the yet uncultivated majority of bacteria existing in nature (6), or more specifically on plant leaves (7). Not only their identity, but in particular the physiological properties of phyllosphere bacteria, their adaptations to the habitat, and their potential role (e.g., with res...

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.

A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange x ion-pair reversed-phase (SCX x IP-RP-HPLC) approach, the RP x IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification. About 13% more peptides and 7% more proteins could be identified with the alternative approach in 30% less analysis time, enabling the analysis of the proteome of Corynebacterium glutamicum with a coverage of 24.9% (745 proteins). Combining the identification results both of the SCX- x IP-RP-HPLC-ESI-MS/MS and RP- x IP-RP-HPLC-ESI-MS/MS methods, a total of 871 proteins were identified in a cytosolic protein preparation, which represented 29.1% of all proteins annotated in the genome of C. glutamicum.

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.

Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.infectious diseases | host-pathogen interactions