Highlights of Research Progress
The Shewanella Federation
The Shewanella Federation, a multi-institutional consortium assembled by DOE, is applying high-throughput approaches for measuring gene and proteome expression of Shewanella oneidensis MR-1. The federation seeks to achieve a systems-level understanding of how this respiration-versatile microorganism regulates energy and material flow and uses its electron-transport system to reduce metals and nitrate. Leveraging substantial DOE investments in capability development and scientific knowledge, the Shewanella Federation employs an approach to systems that capitalizes on the relative strengths, capabilities, and expertise of each federation group. The federation conducts integrated and coordinated investigations that incorporate many facets of biological research and technologies across a number of disciplines and, hence, serve as a model for systems biology studies within the Genomics:GTL program. Federation members share information and resources and collaborate on projects consisting of a few investigators focused on a defined topic and on larger experiments combining their capabilities to address complex scientific questions. Several recent accomplishments are provided as examples below.
Combining Computational and Experimental Approaches to Enhance Shewanella Genome Annotation
Genomics, the study of all the genetic sequences in living organisms, has leaned heavily on the blueprint metaphor. A large part of the blueprint unfortunately has been unintelligible, requiring a way to link genomic features to what’s happening in the cell. The Shewanella Federation has taken a significant step toward improving the interpretation of the blueprint for S. oneidensis MR-1. Federation members have applied a powerful new approach that integrates MR-1. Federation members have applied a powerful new approach that integrates experimental and computational analyses to ascribe cellular function to genes that had been termed “hypothetical”—sequences that appear in the genome but whose biological expression and purpose previously were unknown. This approach currently offers the most-comprehensive “functional annotation,”a way of assigning biological function to the mystery sequences and ranking them based on their similarity to genes known to encode proteins. Before this study, 1988 (nearly 40%) of the predicted 4931 genes in S. oneidensis were considered hypothetical.
To gain insight into whether the sequences in fact produced proteins and the importance and function of any expressed hypothetical genes, a rigorous experimental approach was used. This approach involved growing the cells under a range of conditions to elicit expression of as many genes as possible, followed by comprehensive comparative analyses using a wide assortment of databases. High-throughput proteome and transcriptome analyses of MR-1 cells grown under a variety of conditions revealed that 538 of the hypothetical genes were expressed (proteins and mRNA) under at least one condition. The analyses confirmed that these are true genes used for one or more cellular processes.
Searches were undertaken to determine if existing databases could provide high-confidence insights into putative functions for these expressed genes (initially hypothetical). Of the 538 genes, 97% were identified as having homologs in other genomes, and general functional assignments were possible for 256 of them. Given the current amount and quality of experimental data in public genome databases, however, assigning exact biochemical function was possible for only 16 genes. These results and other arguments (Roberts 2004; Roberts et al. 2004) point to the need for new methods for understanding gene, protein, and, ultimately, organism function.
The ability to rank hypothetical sequences according to their likelihood to encode proteins will be vital for any further experimentation and, eventually, for predicting biological function. The method not only portends a way to fill in the blanks in any organism’s genome but also to compare the genomes of different organisms and their evolutionary relationships. In many cases, it is not known if a computationally annotated gene expresses a protein. With growing confidence that many hypothetical genes are expressing proteins, follow-on analyses now can be used to establish the role these proteins play.
Reference
E. Kolker et al., “Global Profiling of Shewanella oneidensis MR-1: Expression of Hypothetical Genes and Improved Functional Annotations,”Proc. Natl. Acad. Sci. USA 102, 2099-2104 (2005).
Physiologic, Genetic, and Proteome Response of Shewanella oneidensis to Electron Acceptors
As a facultative anaerobe and dissimilatory metal-reducing bacterium, S. oneidensis MR-1 can shift its metabolism MR-1 can shift its metabolism and flexible electron-transport system to allow it to thrive in environments with steep redox gradients. It can accommodate O2 as a terminal electron acceptor, or it can generate energy from anaerobic respiration using a variety of soluble (e.g., nitrate, thiosulfate) and insoluble electron acceptors such as Fe(III) and Mn(IV). This major shift in lifestyle probably requires rewiring of electron transport and metabolism by sensing changes in the environment and making the necessary changes in cellular proteins or the proteome. To begin to understand how MR-1 cells respond at the whole-cell or “system”level to this transition to anaerobicity, the federation initiated a series of experiments in which MR-1 was grown under changing conditions in continuous culture. These experiments revealed that MR-1 cells growing at high oxygen concentrations formed cell aggregates—the precursor to biofilms. They also exhibited elevated expression levels of genes involved in attachment and autoaggregation including fimbrae (curli, pili, flagella), extracellular polysaccharides, lectins, and surface antigens. These studies indicated that aggregation in S. oneidensis MR-1 may serve as a mechanism to facilitate reduced O2 tensions to cells within the aggregate interior, avoiding the oxidative stress associated with production of reactive oxygen species during metabolism.
To gain insight into the complex structure of the energy-generating networks in MR-1, global mRNA patterns were examined in cells exposed to a wide range of metal and nonmetal electron acceptors. Gene-expression patterns were similar regardless of which metal ion was used as electron acceptor, with 60% of the differentially expressed genes showing similar induction or repression relative to fumarate-respiring conditions. Several groups of genes exhibited elevated expression levels in the presence of metals, including those encoding putative multidrug efflux transporters, detoxification proteins, extracytoplasmic sigma factors, and PASdomain regulators. Only one of the 42 predicted c-type cytochromes in MR-1, SO3300, displayed significantly elevated transcript levels across all metal-reducing conditions. Genes encoding decaheme cytochromes MtrC and MtrA, which were linked previously to reduction of different forms of Fe(III) and Mn(IV), exhibited only slight decreases in relative mRNA abundances under metal-reducing conditions. In contrast, specific transcriptome responses were displayed to individual nonmetal electron acceptors, resulting in identification of unique groups of nitrate-, thiosulfate- and TMAO-induced genes including previously uncharacterized multicytochrome gene clusters. Collectively, gene-expression results reflect the fundamental differences between metal and nonmetal respiratory pathways of S. oneidensis MR-1, in which the coordinate induction of detoxification and stress-response genes play a key role in adaptation of this organism under metal-reducing conditions. [Shewanella Federation]
Reference
A. S. Beliaev et al., “Global Transcriptome Analysis of Shewanella oneidensis MR-1 Exposed to Different Terminal Electron Acceptors,”J. Bacteriol., accepted for publication.
