EMSL: Mary S Lipton's Publications

Konstantinidis K, MH Serres, MF Romine, JLM Rodrigues, JM Auchutung, LA McCue, MS Lipton, A Obraztsova, CS Giometti, KH Nealson, JK Fredrickson, and JM Tiedje. 2009. "Comparative systems biology across an evolutionary gradient within the Shewanella genus ." Proceedings of the National Academy of Sciences of the United States of America 106(37):15909-15914. doi:10.1073/pnas.0902000106 Abstract To what extent genotypic differences translate to phenotypic variation remains a poorly understood issue of paramount importance for several cornerstone concepts of microbiology such as the species definition. Here, we take advantage of the completed genomic sequences, expressed proteomic profiles, and physiological studies of ten closely related Shewanella organisms to provide quantitative insights into this issue. Our analyses revealed that, despite the extensive horizontal gene transfer characterizing these genomes, the genotypic and phenotypic similarities among the organisms were generally predictable from their evolutionary relatedness. The power of the predictions depended, however, on the degree of ecological specialization of the organisms evaluated. Using the unprecedented genetic gradient formed by these genomes, we were able to isolate the effect of ecology from the effect of evolutionary divergence and rank the different cellular functions in terms of their rates of evolution. Our ranking also revealed that whole-cell protein expression differences among these organisms when grown under identical conditions were relatively larger than differences at the genome level, suggesting that similarity in gene regulation and expression should constitute another important parameter for (new) species description. Collectively, our results provide important new information towards beginning a system level understanding of bacterial species and genera.

Sowell SM, L Wilhelm, AD Norbeck, MS Lipton, CD Nicora, DF Barofsky, C carlson, RD Smith, and SJ Giovannoni. 2009. " Transport Functions Dominate the SAR11 Metaproteome at Low-Nutrient Extremes in the Sargasso Sea." The ISME Journal 3(1):93-105. Abstract The northwestern Sargasso Sea is part of the North Atlantic subtropical oceanic gyre that is characterized as seasonally oligotrophic with pronounced stratification in the summer and autumn. Essentially a marine desert, the biological productivity of this region is reduced during stratified periods as a result of low concentrations of phosphorous and nitrogen in the euphotic zone. To better understand the mechanisms of microbial survival in this oligotrophic environment, we used capillary LC-tandem mass spectrometry to study the composition of microbial proteomes in surface samples collected in September 2005. A total of 2279 peptides that mapped to 236 SAR11 proteins, and 3208 peptides that mapped to 404 Synechococcus proteins, were detected. Mass spectra from SAR11 periplasmic binding proteins accounted for a disproportionately large fraction of the peptides detected, consistent with observations that these extremely small cells devote a large proportion of their volume to periplasm. Abundances were highest for periplasmic substrate-binding proteins for phosphate, amino acids, phosphonate, sugars, and spermidine. Although the data showed that a large fraction of microbial protein synthesis in the Sargasso Sea is devoted to inorganic and organic nutrient acquisition, the proteomes of both SAR11 and Synechococcus also indicated that these populations were actively growing. Our findings support the view that competition for multiple nutrients in oligotrophic systems is extreme but sufficient to sustain microbial community activity.

Ansong C, SO Purvine, JN Adkins, MS Lipton, and RD Smith. 2008. "Proteogenomics: the needs and roles to be filled by proteomics in genome annotation." Briefings in Functional Genomics and Proteomics 7(1):50-62. Abstract While genome sequencing efforts reveal the basic building blocks of life, a genome sequence alone is insufficient for elucidating biological function. Genome annotation – the process of identifying genes and assigning function to each gene in a genome sequence – provides the means to elucidate biological function from sequence. Current state-of-the-art high throughput genome annotation uses a combination of comparative (sequence similarity data) and non-comparative (ab initio gene prediction algorithms) methods to identify open reading frames in genome sequences. Because approaches used to validate the presence of these open reading frames are typically based on the information derived from the annotated genomes, they cannot independently and unequivocally determine whether a predicted open reading frame is translated into a protein. With the ability to directly measure peptides arising from expressed proteins, high throughput liquid chromatography-tandem mass spectrometry-based proteomics, approaches can be used to verify coding regions of a genomic sequence. Here, we highlight several ways in which high throughput tandem mass spectrometry-based proteomics can improve the quality of genome annotations and suggest that it could be efficiently applied during the initial gene calling process so that the improvements are propagated through the subsequent functional annotation process.

Callister SJ, LA McCue, JE Turse, ME Monroe, KJ Auberry, RD Smith, JN Adkins, and MS Lipton. 2008. "Comparative Bacterial Proteomics: Analysis of the Core Genome Concept." PLoS One 3(2):e1542. Abstract Comparative bacterial genomic studies commonly predict a set of genes indicative of common ancestry. Experimental validation of the existence of this core genome requires extensive measurement and is not typically undertaken. Enabled by an extensive proteome database development over a six year period, we experimentally verified the expression of proteins predicted from genomic ortholog comparisons among 17 environmental and pathogenic bacteria. More exclusive relationships were observed among the expressed protein content of phenotypically related bacteria, which is indicative of the specific lifestyles associated with these organisms. While genomic studies establish relative orthologous relationships among a set of bacteria and propose a set of ancestral genes, our proteomics study establishes expressed lifestyle differences among conserved genes and proposes a set of expressed ancestral traits.

Ding YHR, KK Hixson, M Aklujkar, MS Lipton, RD Smith, DR Lovley, and T Mester. 2008. "Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor." Biochimica et Biophysica Acta--Proteins and Proteomics 1784(12):1935-1941. doi:10.1016/j.bbapap.2008.06.011 Abstract e(III) oxides are the most abundant source of reducible Fe(III) by microorganisms in most soils and sediments, yet few studies on the physiology of Fe(III)-reducing microorganisms during growth on Fe(III) oxide have been conducted because of the technical difficulties in working with cell growth and harvest in the presence of Fe(III) oxides. Geobacter sulfurreducens is a representative of the Geobacter species that predominate in a variety of subsurface environments in which Fe(III) oxide is important. In order to better understand the physiology of Geobacter species during growth on Fe(III) oxide, the proteome of G. sulfurreducens grown on Fe(III) oxide was compared with the proteome of cells grown with soluble Fe(III) citrate. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) revealed 19 proteins that were more abundant during growth on Fe(III) oxide than on soluble Fe(III). These included proteins related to protein synthesis, electron transfer and energy production, oxidative stress, protein folding, outer membrane proteins, nitrogen metabolism and hypothetical proteins. Further analysis of the proteome with the accurate mass and time (AMT) tag method revealed additional proteins associated with growth on Fe(III) oxide. These included the outer-membrane c-type cytochrome, OmcS and OmcG, which genetic studies have suggested are required for Fe(III) oxide reduction. Furthermore, several other cytochromes, as yet unstudied, were detected to be significantly up regulated during growth on Fe(III) oxide and other proteins of unknown function were more abundant during growth on Fe(III) oxide than on soluble Fe(III). PilA, the structural protein for pili, which is required for Fe(III) oxide reduction, and other pilin-associated proteins were also more abundant during growth on Fe(III) oxide. Confirmation of the differential expression of proteins known to be important in Fe(III) oxide reduction was observed, and an additional number of previously unidentified proteins were found with significant abundance in the cells grown under conditions of Fe(III) oxide reduction.

Du X, SJ Callister, NP Manes, JN Adkins, RA Alexandridis, X Zeng, JH Roh, WE Smith, TJ Donohue, S Kaplan, RD Smith, and MS Lipton. 2008. "A Computational Strategy to Analyze Label-Free Temporal Bottom-up Proteomics Data." Journal of Proteome Research 7(7):2595-2604. doi:10.1021/pr0704837 Abstract Motivation: Biological systems are in a continual state of flux, which necessitates an understanding of the dynamic nature of protein abundances. The study of protein abundance dynamics has become feasible with recent improvements in mass spectrometry-based quantitative proteomics. However, a number of challenges still re-main related to how best to extract biological information from dy-namic proteomics data; for example, challenges related to extrane-ous variability, missing abundance values, and the identification of significant temporal patterns. Results: This article describes a strategy that addresses the afore-mentioned issues for the analysis of temporal bottom-up proteomics data. The core strategy for the data analysis algorithms and subse-quent data interpretation was formulated to take advantage of the temporal properties of the data. The analysis procedure presented herein was applied to data from a Rhodobacter sphaeroides 2.4.1 time-course study. The results were in close agreement with existing knowledge about R. sphaeroides, therefore demonstrating the utility of this analytical strategy.

Elias DA, SL Tollaksen, DW Kennedy, HM Mottaz, CS Giometti, JS Mclean, EA Hill, GE Pinchuk, MS Lipton, JK Fredrickson, and YA Gorby. 2008. "The Influence of Cultivation Methods on Shewanella oneidensis Physiology and Proteome Expression ." Archives of Microbiology 189(4):313-324. doi:10.1007/s00203-007-0321-y Abstract High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O2 deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell metabolic variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.

Eraso JM, JH Roh, X Zeng, SJ Callister, MS Lipton, and S Kaplan. 2008. "Role of the global transcriptional regulator PrrA in Rhodobacter sphaeroides 2.4.1: combined transcriptome and proteome analysis." Journal of Bacteriology 190(14):4831-4848. doi:10.1128/JB.00301-08 Abstract The PrrBA two-component regulatory system is a major global regulator in Rhodobacter sphaeroides 2.4.1. In this study we have compared the transcriptome and proteome profiles of the wild type (WT) and mutant PrrA2 cells grown anaerobically, in the dark, with DMSO as electron acceptor. Approximately 25% of the genes present in the genome are PrrA-regulated, at the transcriptional level, either directly or indirectly, by ≥ 2-fold relative to wild type. The genes affected are widespread throughout all COG functional categories, with previously unsuspected “metabolic” genes affected when in the PrrA mutant background. PrrA was found to act both as an activator and a repressor of transcription, with more genes being repressed in the presence of PrrA (9:5 ratio). An analysis of the genes encoding the 1,536 peptides detected through our chromatographic study, which corresponds to 36% coverage of the genome, revealed that approximately 20% of the genes encoding these proteins were positively regulated, whereas approximately 32% were negatively regulated by PrrA, which is in excellent agreement with the percentages obtained for the whole genomic transcriptome profile. In addition, comparison of the transcriptome and proteome mean parameter values chosen between WT and PrrA2 showed good qualitative agreement, indicating that transcript regulation paralleled the corresponding protein abundance, although not one for one. The microarray analysis was validated by direct mRNA measurement of randomly selected, both positively and negatively regulated genes. lacZ transcriptional and kan translational fusions enabled us to map putative PrrA binding sites, as well as revealing potential gene targets for indirect regulation by PrrA.

Gupta N, J Benhamida, V Bhargava, D Goodman, E Kain , I Kerman, N Nguyen , N Ollikainen, J Rodriguez, J Wang, MS Lipton, MF Romine, V Bafna, RD Smith, and PA Pevzner. 2008. "Comparative proteogenomics: combining mass spectrometry and comparative genomics to analyze multiple genomes." Genome Research 18(7):1133-1142. Abstract While bacterial genome annotations have significantly improved in recent years, techniques for bacterial proteome annotation (including post-translational chemical modifications, signal peptides, proteolytic events, etc.) are still in their infancy. At the same time, the number of sequenced bacterial genomes is rising sharply, far outpacing our ability to validate the predicted genes, let alone annotate bacterial proteomes. In this study, we use tandem mass spectrometry (MS/MS) to annotate the proteome of Shewanella oneidensis MR-1, an important microbe for bioremediation. In particular, we provide the first comprehensive map of post-translational modifications in a bacterial genome, including a large number of chemical modifications, signal peptide cleavages and cleavage of N-terminal methionine residues. We also detect multiple genes that were missed or assigned incorrect start positions by gene prediction programs and suggest corrections to improve the gene annotation. This study demonstrates that complementing every genome sequencing project by an MS/MS project would significantly improve both genome and proteome annotations for a reasonable cost.

Romine MF, T Carlson, AD Norbeck, LA McCue, and MS Lipton. 2008. "Identification of Mobile Elements and Pseudogenes in the Shewanella oneidensis MR-1 Genome." Applied and Environmental Microbiology 74(10):3257-3265. Abstract Shewanella oneidensis MR-1 is the first of 22 different Shewanella spp. whose genomes have been or are being sequenced and thus serves as the model organism for studying the functional repertoire of the Shewanella genus. The original MR-1 genome annotation revealed a large number of transposase genes and pseudogenes, indicating that many of the genome’s functions may be decaying. Comparative analyses of the sequenced Shewanella strains suggest that 209 genes in MR-1 have in-frame stop codons, frameshifts, or interruptions and/or are truncated and that 65 of the original pseudogene predictions were erroneous. Among the decaying functions are that of one of three chemotaxis clusters, type I pilus production, starch utilization, and nitrite respiration. Many of the mutations could be attributed to members of 41 different types of insertion sequence (IS) elements and three types of miniature inverted-repeat transposable elements identified here for the first time. The high copy numbers of individual mobile elements (up to 71) are expected to promote large-scale genome recombination events, as evidenced by the displacement of the algA promoter. The ability of MR-1 to acquire foreign genes via reactions catalyzed by both the integron integrase and the ISSod25-encoded integrases is suggested by the presence of attC sites and genes whose sequences are characteristic of other species downstream of each site. This large number of mobile elements and multiple potential sites for integrasemediated acquisition of foreign DNA indicate that the MR-1 genome is exceptionally dynamic, with many functions and regulatory control points in the process of decay or reinvention.