Independent genome reduction and phylogenetic reclassification of the oceanic SAR11 clade

The SAR11 clade, here represented by Candidatus Pelagibacter ubique, is the most successful group of bacteria in the upper surface waters of the oceans. In contrast to previous studies that have associated the 1.3 Mb genome of Ca. Pelagibacter ubique with the less than 1.5 Mb genomes of the Rickettsiales, our phylogenetic analysis suggests that Ca. Pelagibacter ubique is most closely related to soil and aquatic Alphaproteobacteria with large genomes. This implies that the SAR11 clade and the Rickettsiales have undergone genome reduction independently. A gene flux analysis of 46 representative alphaproteobacterial genomes indicates the loss of more than 800 genes in each of Ca. Pelagibacter ubique and the Rickettsiales. Consistent with their different phylogenetic affiliations, the pattern of gene loss differs with a higher loss of genes for repair and recombination processes in Ca. Pelagibacter ubique as compared with a more extensive loss of genes for biosynthetic functions in the Rickettsiales. Some of the lost genes in Ca. Pelagibacter ubique, such as mutLS, recFN, and ruvABC, are conserved in all other alphaproteobacterial genomes including the small genomes of the Rickettsiales. The mismatch repair genes mutLS are absent from all currently sequenced SAR11 genomes and also underrepresented in the global ocean metagenome data set. We hypothesize that the unique loss of genes involved in repair and recombination processes in Ca. Pelagibacter ubique has been driven by selection and that this helps explain many of the characteristics of the SAR11 population, such as the streamlined genomes, the long branch lengths, the high recombination frequencies, and the extensive sequence divergence within the population.     

A proteomic study of the HUPO Plasma Proteome Project's pilot samples using an accurate mass and time tag strategy

Characterization of the human blood plasma proteome is critical to the discovery of routinely useful clinical biomarkers. We used an accurate mass and time (AMT) tag strategy with high-resolution mass accuracy cLC-FT-ICR MS to perform a global proteomic analysis of pilot study samples as part of the HUPO Plasma Proteome Project. HUPO reference serum and citrated plasma samples from African Americans, Asian Americans, and Caucasian Americans were analyzed, in addition to a Pacific Northwest National Laboratory reference serum and plasma. The AMT tag strategy allowed us to leverage two previously published "shotgun" proteomics experiments to perform global analyses on these samples in triplicate in less than 4 days total analysis time. A total of 722 (22% with multiple peptide identifications) International Protein Index redundant proteins, or 377 protein families by ProteinProphet, were identified over the six individual HUPO serum and plasma samples. The samples yielded a similar number of identified redundant proteins in the plasma samples (average 446 +/- 23) as found in the serum samples (average 440 +/- 20). These proteins were identified by an average of 956 +/- 35 unique peptides in plasma and 930 +/- 11 unique peptides in serum. In addition to this high-throughput analysis, the AMT tag approach was used with a Z-score normalization to compare relative protein abundances. This analysis highlighted both known differences in serum and citrated plasma such as fibrinogens, and reproducible differences in peptide abundances from proteins such as soluble activin receptor-like kinase 7b and glycoprotein m6b. The AMT tag strategy not only improved our sample throughput but also provided a basis for estimated quantitation.     

Accurate mass tag retention time database for urine proteome analysis by chromatography-mass spectrometry

Information about peptides and proteins in urine can be used to search for biomarkers of early stages of various diseases. The main technology currently used for identification of peptides and proteins is tandem mass spectrometry, in which peptides are identified by mass spectra of their fragmentation products. However, the presence of the fragmentation stage decreases sensitivity of analysis and increases its duration. We have developed a method for identification of human urinary proteins and peptides. This method based on the accurate mass and time tag (AMT) method does not use tandem mass spectrometry. The database of AMT tags containing more than 1381 AMT tags of peptides has been constructed. The software for database filling with AMT tags, normalizing the chromatograms, database application for identification of proteins and peptides, and their quantitative estimation has been developed. The new procedures for peptide identification by tandem mass spectra and the AMT tag database are proposed. The paper also lists novel proteins that have been identified in human urine for the first time.     

Energy starved Candidatus Pelagibacter ubique substitutes light-mediated ATP production for endogenous carbon respiration

Previous studies have demonstrated that Candidatus Pelagibacter ubique, a member of the SAR11 clade, constitutively expresses proteorhodopsin (PR) proteins that can function as light-dependent proton pumps. However, exposure to light did not significantly improve the growth rate or final cell densities of SAR11 isolates in a wide range of conditions. Thus, the ecophysiological role of PR in SAR11 remained unresolved. We investigated a range of cellular properties and here show that light causes dramatic changes in physiology and gene expression in Cand. P. ubique cells that are starved for carbon, but provides little or no advantage during active growth on organic carbon substrates. During logarithmic growth there was no difference in oxygen consumption by cells in light versus dark. Energy starved cells respired endogenous carbon in the dark, becoming spheres that approached the minimum predicted size for cells, and produced abundant pili. In the light, energy starved cells maintained size, ATP content, and higher substrate transport rates, and differentially expressed nearly 10% of their genome. These findings show that PR is a vital adaptation that supports Cand. P. ubique metabolism during carbon starvation, a condition that is likely to occur in the extreme conditions of ocean environments.     

The proteomics of quiescent and nonquiescent cell differentiation in yeast stationary-phase cultures

As yeast cultures enter stationary phase in rich, glucose-based medium, differentiation of two major subpopulations of cells, termed quiescent and nonquiescent, is observed. Differences in mRNA abundance between exponentially growing and stationary-phase cultures and quiescent and nonquiescent cells are known, but little was known about protein abundance in these cells. To measure protein abundance in exponential and stationary-phase cultures, the yeast GFP-fusion library (4159 strains) was examined during exponential and stationary phases, using high-throughput flow cytometry (HyperCyt). Approximately 5% of proteins in the library showed twofold or greater changes in median fluorescence intensity (abundance) between the two conditions. We examined 38 strains exhibiting two distinct fluorescence-intensity peaks in stationary phase and determined that the two fluorescence peaks distinguished quiescent and nonquiescent cells, the two major subpopulations of cells in stationary-phase cultures. GFP-fusion proteins in this group were more abundant in quiescent cells, and half were involved in mitochondrial function, consistent with the sixfold increase in respiration observed in quiescent cells and the relative absence of Cit1p:GFP in nonquiescent cells. Finally, examination of quiescent cell-specific GFP-fusion proteins revealed symmetry in protein accumulation in dividing quiescent and nonquiescent cells after glucose exhaustion, leading to a new model for the differentiation of these cells.     

SAR11 bacteria have a high affinity and multifunctional glycine betaine transporter

Marine bacterioplankton face stiff competition for limited nutrient resources. SAR11, a ubiquitous clade of very small and highly abundant Alphaproteobacteria, are known to devote much of their energy to synthesizing ATP-binding cassette periplasmic proteins that bind substrates. We hypothesized that their small size and relatively large periplasmic space might enable them to outcompete other bacterioplankton for nutrients. Using uptake experiments with ¹⁴C-glycine betaine, we discovered that two strains of SAR11, Candidatus Pelagibacter sp. HTCC7211 and Cand. P. ubique HTCC1062, have extraordinarily high affinity for glycine betaine (GBT), with half-saturation (K _s) values around 1 nM and specific affinity values between 8 and 14 L mg cell⁻¹ h⁻¹. Competitive inhibition studies indicated that the GBT transporters in these strains are multifunctional, transporting multiple substrates in addition to GBT. Both strains could use most of the transported compounds for metabolism and ATP production. Our findings indicate that Pelagibacter cells are primarily responsible for the high affinity and multifunctional GBT uptake systems observed in seawater. Maximization of whole-cell affinities may enable these organisms to compete effectively for nutrients during periods when the gross transport capacity of the heterotrophic plankton community exceeds the supply, depressing ambient concentrations.     

Nutrient requirements for growth of the extreme oligotroph ‘Candidatus Pelagibacter ubique' HTCC1062 on a defined medium

Chemoheterotrophic marine bacteria of the SAR11 clade are Earth''s most abundant organisms. Following the first cultivation of a SAR11 bacterium, ‘Candidatus Pelagibacter ubique'' strain HTCC1062 (Ca. P. ubique) in 2002, unusual nutritional requirements were identified for reduced sulfur compounds and glycine or serine. These requirements were linked to genome streamlining resulting from selection for efficient resource utilization in nutrient-limited ocean habitats. Here we report the first successful cultivation of Ca. P. ubique on a defined artificial seawater medium (AMS1), and an additional requirement for pyruvate or pyruvate precursors. Optimal growth was observed with the collective addition of inorganic macro- and micronutrients, vitamins, methionine, glycine and pyruvate. Methionine served as the sole sulfur source but methionine and glycine were not sufficient to support growth. Optimal cell yields were obtained when the stoichiometry between glycine and pyruvate was 1:4, and incomplete cell division was observed in cultures starved for pyruvate. Glucose and oxaloacetate could fully replace pyruvate, but not acetate, taurine or a variety of tricarboxylic acid cycle intermediates. Moreover, both glycine betaine and serine could substitute for glycine. Interestingly, glycolate partially restored growth in the absence of glycine. We propose that this is the result of the use of glycolate, a product of phytoplankton metabolism, as both a carbon source for respiration and as a precursor to glycine. These findings are important because they provide support for the hypothesis that some micro-organisms are challenging to cultivate because of unusual nutrient requirements caused by streamlining selection and gene loss. Our findings also illustrate unusual metabolic rearrangements that adapt these cells to extreme oligotrophy, and underscore the challenge of reconstructing metabolism from genome sequences in organisms that have non-canonical metabolic pathways.     

Quantitative proteome analysis of breast cancer cell lines using 18O-labeling and an accurate mass and time tag strategy

Proteome comparison of cell lines derived from cancer and normal breast epithelium provide opportunities to identify differentially expressed proteins and pathways associated with specific phenotypes. We employed 16O/18O peptide labeling, FT-ICR MS, and an accurate mass and time (AMT) tag strategy to simultaneously compare the relative abundance of hundreds of proteins in non-cancer and cancer cell lines derived from breast tissue. A cell line reference panel allowed relative protein abundance comparisons among multiple cell lines and across multiple experiments. A peptide database generated from multidimensional LC separations and MS/MS analysis was used for subsequent AMT tag-based peptide identifications. This peptide database represented a total of 2299 proteins, including 514 that were quantified in five cell lines using the AMT tag and 16O/18O strategies. Eighty-six proteins showed at least a threefold protein abundance change between cancer and non-cancer cell lines. Hierarchical clustering of protein abundance ratios revealed that several groups of proteins were differentially expressed between the cancer cell lines.     

Proteomic analyses using an accurate mass and time tag strategy

An accurate mass and time (AMT) tag approach for proteomic analyses has been developed over the past several years to facilitate comprehensive high-throughput proteomic measurements. An AMT tag database for an organism, tissue, or cell line is established by initially performing standard shotgun proteomic analysis and, most importantly, by validating peptide identifications using the mass measurement accuracy of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) and liquid chromatography (LC) elution time constraint. Creation of an AMT tag database largely obviates the need for subsequent MS/MS analyses, and thus facilitates high-throughput analyses. The strength of this technology resides in the ability to achieve highly efficient and reproducible one-dimensional reversed-phased LC separations in conjunction with highly accurate mass measurements using FTICR MS. Recent improvements allow for the analysis of as little as picrogram amounts of proteome samples by minimizing sample handling and maximizing peptide recovery. The nanoproteomics platform has also demonstrated the ability to detect >10(6) differences in protein abundances and identify more abundant proteins from subpicogram amounts of samples. The AMT tag approach is poised to become a new standard technique for the in-depth and high-throughput analysis of complex organisms and clinical samples, with the potential to extend the analysis to a single mammalian cell.     

Quantitative proteome analysis of human plasma following in vivo lipopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach

Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes postdigestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional LC-FTICR mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC elution time AMT tag data base was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag data base contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion of high abundance proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses, and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.     

Global detection and characterization of hypothetical proteins in Shewanella oneidensis MR-1 using LC-MS based proteomics

The availability of whole genome sequences has enabled the application of powerful tools for assaying global expression patterns in environmentally relevant bacteria such as Shewanella oneidensis MR-1. A large number of genes in prokaryote genomes, including MR-1, have been annotated as hypothetical, indicating that no similar protein has yet been identified in other organisms. Using high-sensitivity MS coupled with accurate mass and time (AMT) tag methodology, 1078 tryptic peptides were collectively detected in MR-1 cultures, 671 of which were unique to their parent protein. Using only these unique tryptic peptides and a minimum of two peptides per protein, we identified, with high confidence, the expression of 258 hypothetical proteins. These proteins ranged from 3.5 to 139 kDa, with 47 being 100 amino acid residues or less. Using a combination of information including detection in cells grown under specific culture conditions, presence within a specific cell fraction, and predictive algorithms such as PSORT and PSORT-B, possible/plausible functions are proposed for some hypothetical proteins. Further, by applying this approach a number of proteins were found not only to be expressed, but only expressed under certain culturing conditions, thereby suggesting function while at the same time isolating several proteins to distinct locales of the cell. These results demonstrate the utility of the AMT tag methodology for comprehensive profiling of the microbial proteome while confirming the expression of a large number of hypothetical genes.     

The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions

The proteome of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high-pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under six different growth conditions in order to enhance the potential that a wide range of genes would be expressed. The AMT tag approach was able to identify a much greater number of proteins than could be detected with the 2-D PAGE approach. With the AMT approach over 3,000 gene products were identified, representing about 90% of the total predicted gene products in the genome. A high proportion of predicted proteins in most protein role categories were detected; the highest number of proteins was identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT tag approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.     

The minimal genome: a metabolic and environmental comparison

The field of Synthetic Biology seeks to apply engineering principles to biology in order to produce novel biological systems. One approach to accomplish this goal is the genome-driven cell engineering approach, which searches for functioning minimal genomes in naturally occurring microorganisms, which can then be used as a template for future systems. Currently a prototypical minimal genome has not been discovered. This review analyzes the organisms Mycoplasma pneumoniae, Pelagibacter ubique, Vesicomyosocius okutanii and Prochlorococcus marinus as models of heterotrophic symbiont, heterotrophic free-living, autotrophic symbiont and autotrophic free-living organisms respectively and compares them to the current minimal cell model in order to determine which most closely resembles a true minimal genome. M. pneumoniae possesses a genome of 816 394 base pairs (bp) with 688 open reading frames (ORF) and a severely limited metabolism. Pelagibacter ubique possesses a 1 308 000 bp genome with 1354 ORF and has a fully functional metabolism but requires a reduced form of sulphur. Vesicomyosocius okutanii possesses a 1 020 000 bp genome with 975 ORF and is deficient in the production of threonine, isoleucine and ubiquinone. Prochlorococcus marinus possesses a 1 751 080 bp genome with 1884 ORF and has a complete metabolism with no deficiencies. The current minimal cell model requires a genome to be of limited size, culturalble and having minimal media requirements as such it is the conclusion of this review that P. marinus best fits this model. Further, future research should concentrate on genome reduction experiments using P. marinus and the search for additional minimal genomes should concentrate on autotrophic free-living organisms.     

Application of the accurate mass and time tag approach to the proteome analysis of sub-cellular fractions obtained from Rhodobacter sphaeroides 2.4.1. Aerobic and photosynthetic cell cultures

The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-nagtive bacterium Rhodobacter sphaeroides 2.4.1. In addition, we analyzed the proteins within purified chromatophore fractions that house the photosynthetic apparatus from photosynthetically grown cells. In total, 8,300 peptides were identified with high confidence from at least one subcellular fraction from either cell culture. These peptides were derived from 1,514 genes or 35% percent of proteins predicted to be encoded by the genome. A significant number of these proteins were detected within a single subcellular fraction and their localization was compared to in silico predictions. However, the majority of proteins were observed in multiple subcellular fractions, and the most likely subcellular localization for these proteins was investigated using a Z-score analysis of estimated protein abundance along with clustering techniques. Good (81%) agreement was observed between the experimental results and in silico predictions. The AMT tag approach provides localization evidence for those proteins that have no predicted localization information, those annotated as putative proteins, and/or for those proteins annotated as hypothetical and conserved hypothetical.     

Application of the accurate mass and time tag approach in studies of the human blood lipidome

We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC-MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput LC-MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance.     

A combinatorial synthesis of tyrphostins via the "directed sorting" method

Using solid-phase organic synthesis, we have prepared a 432-member (18 x 8 x 3) sample library based on the AG 490 "tyrphostin" template. By utilizing 432 reactors each equipped with a unique radiofrequency memory ID tag, the 432 products could be obtained as discrete entities (i.e., not as mixtures) via 18 + 8 + 3, or 29 reactions. Reading each ID tag after each reaction step permitted the "directed sorting" of reactors into appropriate reaction vessels containing multiple reactors. After synthesis, all products were cleaved from the solid-phase support and lyophilized to afford powders. Characterization of 5% of the library members by NMR and mass spectrometry provided verification of structure. In addition, TLC analysis of every library member provided evidence that most (or all) are composed of a single major organic compound. Some 88% of these samples were obtained in amounts of between 5 and 19 mg. Using this reaction sequence and the "directed sorting" approach, the synthesis of much larger AG 490-based libraries can be envisioned.     

Dynamics alignment: comparison of protein dynamics in the SCOP database

A novel methodology for comparison of protein dynamics is presented. Protein dynamics is calculated using the Gaussian network model and the modes of motion are globally aligned using the dynamic programming algorithm of Needleman and Wunsch, commonly used for sequence alignment. The alignment is fast and can be used to analyze large sets of proteins. The methodology is applied to the four major classes of the SCOP database: "all alpha proteins," "all beta proteins," "alpha and beta proteins," and "alpha/beta proteins". We show that different domains may have similar global dynamics. In addition, we report that the dynamics of "all alpha proteins" domains are less specific to structural variations within a given fold or superfamily compared with the other classes. We report that domain pairs with the most similar and the least similar global dynamics tend to be of similar length. The significance of the methodology is that it suggests a new and efficient way of mapping between the global structural features of protein families/subfamilies and their encoded dynamics.     

NanoLC-FT-ICR MS improves proteome coverage attainable for approximately 3000 laser-microdissected breast carcinoma cells

Proteomics assays hold great promise for unraveling molecular events that underlie human diseases. Effective analysis of clinical samples is essential, but this task is considerably complicated by tissue heterogeneity. Laser capture microdissection (LCM) can be used to selectively isolate target cells from their native tissue environment. However, the small number of cells that is typically procured by LCM severely limits proteome coverage and biomarker discovery potential achievable by conventional proteomics platforms. Herein, we describe the use of nanoLC-FT-ICR MS for analyzing protein digests of 3000 LCM-derived tumor cells from breast carcinoma tissue, corresponding to 300 ng of total protein. A total of 2282 peptides were identified by matching LC-MS data to accurate mass and time (AMT) tag databases that were previously established for human breast (cancer) cell lines. One thousand and three unique proteins were confidently identified with two or more peptides. Based on gene ontology categorization, identified proteins appear to cover a wide variety of biological functions and cellular compartments. This work demonstrates that a substantial number of proteins can be detected and identified from limited number of cells using the AMT tag approach, and opens doors for high-throughput in-depth proteomics analysis of clinical samples.     

The SAR11 group of alpha-proteobacteria is not related to the origin of mitochondria

Although free living, members of the successful SAR11 group of marine alpha-proteobacteria contain a very small and A+T rich genome, two features that are typical of mitochondria and related obligate intracellular parasites such as the Rickettsiales. Previous phylogenetic analyses have suggested that Candidatus Pelagibacter ubique, the first cultured member of this group, is related to the Rickettsiales+mitochondria clade whereas others disagree with this conclusion. In order to determine the evolutionary position of the SAR11 group and its relationship to the origin of mitochondria, we have performed phylogenetic analyses on the concatenation of 24 proteins from 5 mitochondria and 71 proteobacteria. Our results support that SAR11 group is not the sistergroup of the Rickettsiales+mitochondria clade and confirm that the position of this group in the alpha-proteobacterial tree is strongly affected by tree reconstruction artefacts due to compositional bias. As a consequence, genome reduction and bias toward a high A+T content may have evolved independently in the SAR11 species, which points to a different direction in the quest for the closest relatives to mitochondria and Rickettsiales. In addition, our analyses raise doubts about the monophyly of the newly proposed Pelagibacteraceae family.