Shewanella knowledgebase: integration of the experimental data and computational predictions suggests a biological role for transcription of intergenic regions.

Shewanellae are facultative gamma-proteobacteria whose remarkable respiratory versatility has resulted in interest in their utility for bioremediation of heavy metals and radionuclides and for energy generation in microbial fuel cells. Extensive experimental efforts over the last several years and the availability of 21 sequenced Shewanella genomes made it possible to collect and integrate a wealth of information on the genus into one public resource providing new avenues for making biological discoveries and for developing a system level understanding of the cellular processes. The Shewanella knowledgebase was established in 2005 to provide a framework for integrated genome-based studies on Shewanella ecophysiology. The present version of the knowledgebase provides access to a diverse set of experimental and genomic data along with tools for curation of genome annotations and visualization and integration of genomic data with experimental data. As a demonstration of the utility of this resource, we examined a single microarray data set from Shewanella oneidensis MR-1 for new insights into regulatory processes. The integrated analysis of the data predicted a new type of bacterial transcriptional regulation involving co-transcription of the intergenic region with the downstream gene and suggested a biological role for co-transcription that likely prevents the binding of a regulator of the upstream gene to the regulator binding site located in the intergenic region. Database URL: http://shewanella-knowledgebase.org:8080/Shewanella/ or http://spruce.ornl.gov:8080/Shewanella/

Conserved synteny at the protein family level reveals genes underlying Shewanella species' cold tolerance and predicts their novel phenotypes.

Bacteria of the genus Shewanella can thrive in different environments and demonstrate significant variability in their metabolic and ecophysiological capabilities including cold and salt tolerance. Genomic characteristics underlying this variability across species are largely unknown. In this study, we address the problem by a comparison of the physiological, metabolic, and genomic characteristics of 19 sequenced Shewanella species. We have employed two novel approaches based on association of a phenotypic trait with the number of the trait-specific protein families (Pfam domains) and on the conservation of synteny (order in the genome) of the trait-related genes. Our first approach is top-down and involves experimental evaluation and quantification of the species' cold tolerance followed by identification of the correlated Pfam domains and genes with a conserved synteny. The second, a bottom-up approach, predicts novel phenotypes of the species by calculating profiles of each Pfam domain among their genomes and following pair-wise correlation of the profiles and their network clustering. Using the first approach, we find a link between cold and salt tolerance of the species and the presence in the genome of a Na(+)/H(+) antiporter gene cluster. Other cold-tolerance-related genes include peptidases, chemotaxis sensory transducer proteins, a cysteine exporter, and helicases. Using the bottom-up approach, we found several novel phenotypes in the newly sequenced Shewanella species, including degradation of aromatic compounds by an aerobic hybrid pathway in Shewanella woodyi, degradation of ethanolamine by Shewanella benthica, and propanediol degradation by Shewanella putrefaciens CN32 and Shewanella sp. W3-18-1.

A general system for studying protein-protein interactions in Gram-negative bacteria.

One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged "bait" proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.

Statistically inferring protein-protein associations with affinity isolation LC-MS/MS assays.

Affinity isolation of protein complexes followed by protein identification by LC-MS/MS is an increasingly popular approach for mapping protein interactions. However, systematic and random assay errors from multiple sources must be considered to confidently infer authentic protein-protein interactions. To address this issue, we developed a general, robust statistical method for inferring authentic interactions from protein prey-by-bait frequency tables using a binomial-based likelihood ratio test (LRT) coupled with Bayes' Odds estimation. We then applied our LRT-Bayes' algorithm experimentally using data from protein complexes isolated from Rhodopseudomonas palustris. Our algorithm, in conjunction with the experimental protocol, inferred with high confidence authentic interacting proteins from abundant, stable complexes, but few or no authentic interactions for lower-abundance complexes. The algorithm can discriminate against a background of prey proteins that are detected in association with a large number of baits as an artifact of the measurement. We conclude that the experimental protocol including the LRT-Bayes' algorithm produces results with high confidence but moderate sensitivity. We also found that Monte Carlo simulation is a feasible tool for checking modeling assumptions, estimating parameters, and evaluating the significance of results in protein association studies.