Cell Isolation and Systems Analysis

The function of proteins, cells and cell communities can be investigated using super resolution, quantitative fluorescence microscopy with single molecule sensitivity, cell sorting and transcriptomic analysis, together with proteomics, metabolomics and electron microscopy.

Resources and Techniques

  • High resolution fluorescence, electron and ion microscopy can image living systems for unprecedented molecular- and atomic-level resolution.
  • Transcriptomics generate massive parallel sequencing to support whole-transcriptome analysis, gene expression profiling, small RNA analysis and novel transcript identification.
  • Cell isolation and fractionation detect and isolate distinct cells and organelles for analyses from complex cultures, communities or tissues.
  • High-throughput data interpretation can be employed for intensive data analysis.

Capability Details
• CISA offers a microscopy system that integrates nonlinear two-photon excitation, laser scanning confocal microscopy, and FLIM, enabling minimally invasive and deeply penetrating laser excitation for three-dimensional imaging and detection of molecular interactions in living tissues and cells.
• The stochastic optical reconstruction microscope (STORM) is a unique instrument for imaging intact cells at nanoscale resolution using single-molecule fluorescence techniques to construct super-resolution images with software that acquires and analyzes data in real time.
• Two Applied Biosystems SOLiD systems offer a non-biased, highly quantitative and accurate method to sequence hundreds of distinct DNA samples in parallel within a single run, generating up to 700 million, 50-base pair reads with extremely high accuracy; multiplexing capabilities under varied conditions; and epigenetic modifications.
• The influx flow cytometer/cell sorter provides powerful detection capabilities and diverse sorting modes using advanced multiple-parameter sorting technologies based on the presence and content of distinct genes, proteins and other molecules or intracellular structures. Influx technology supports detection and sorting of nanoscale particles, making it highly suited for sorting and analyzing organelles and microorganisms.

Two massively parallel next-generation sequencing platforms (SOLiD4) are currently incorporated in users' research for transcriptomics analysis. The...
Custodian(s): Galya Orr
The atomic force microscope (AFM) compound microscope, is designed primarily for fluorescence imaging in the study of nanoscale chemical processes,...
This FEI Tecnai T-12 cryo-transmission electron microscope (TEM) complements EMSL's broader microscopy suite and JEOL 2010 analytical high-...
Custodian(s): Alice Dohnalkova
High-sensitivity total internal reflection fluorescence (FLIM) microscopes support time-lapse single-molecule fluorescence imaging of individual...
Custodian(s): Galya Orr
The single-molecule optical microscope is designed to study complex reaction dynamics such as enzymatic reactions, protein-protein interactions, and...
Nitric oxide chemistry and photochemistry on the Cr-terminated surface of α-Cr2O3(0001) was examined using temperature programmed...
Olivines, a significant constituent of basaltic rocks, have the potential to immobilize permanently CO2 after it is injected in the deep subsurface,...
Magnetic and density separation methods have been applied to composite sediment sample from the Hanford formation from sediment recovered during...
Based on combined FTIR and XRD studies, we report here that H2O induces a morphological change of KNO3 species formed on model K2O/Al2O3 NOx storage-...
NOx uptake experiments were performed on a series of alkaline earth oxide (AEO) (MgO, CaO, SrO, BaO) on γ-alumina materials. Temperature...
Posted: June 17, 2014
The Science Many bacterial species have genes called mraZ and mraW, which are located in a cluster of genes that regulate cell division and cell...
Posted: May 13, 2014
EMSL and Pacific Northwest National Laboratory scientists isolated two bacterial consortia from a microbial mat in Hot Lake, in north-central...
Posted: September 16, 2013
A new transcriptomics-based model accurately predicts how much isoprene the bacterium Bacillus subtilis will produce when stressed or nourished....
Posted: March 19, 2013
One of the most noteworthy concerns for the U.S. Department of Energy is controlling atmospheric carbon dioxide to mitigate its effects on global...
Posted: May 02, 2012
Researchers at Pacific Northwest National Laboratory revealed new knowledge about the effects ionizing radiation has on human skin by using mass...

The function of proteins, cells and cell communities can be investigated using super resolution, quantitative fluorescence microscopy with single molecule sensitivity, cell sorting and transcriptomic analysis, together with proteomics, metabolomics and electron microscopy.

Resources and Techniques

  • High resolution fluorescence, electron and ion microscopy can image living systems for unprecedented molecular- and atomic-level resolution.
  • Transcriptomics generate massive parallel sequencing to support whole-transcriptome analysis, gene expression profiling, small RNA analysis and novel transcript identification.
  • Cell isolation and fractionation detect and isolate distinct cells and organelles for analyses from complex cultures, communities or tissues.
  • High-throughput data interpretation can be employed for intensive data analysis.

Proteogenomic Analysis of a Thermophilic Bacterial Consortium Adapted to Deconstruct Switchgrass.

Abstract: 

Thermophilic bacteria are a potential source of enzymes for the deconstruction of lignocellulosic biomass. However, the complement of proteins used to deconstruct biomass and the specific roles of different microbial groups in thermophilic biomass deconstruction are not well-explored. Here we report on the metagenomic and proteogenomic analyses of a compost-derived bacterial consortium adapted to switchgrass at elevated temperature with high levels of glycoside hydrolase activities. Near-complete genomes were reconstructed for the most abundant populations, which included composite genomes for populations closely related to sequenced strains of Thermus thermophilus and Rhodothermus marinus, and for novel populations that are related to thermophilic Paenibacilli and an uncultivated subdivision of the littlestudied Gemmatimonadetes phylum. Partial genomes were also reconstructed for a number of lower abundance thermophilic Chloroflexi populations. Identification of genes for lignocellulose processing and metabolic reconstructions suggested Rhodothermus, Paenibacillus and Gemmatimonadetes as key groups for deconstructing biomass, and Thermus as a group that may primarily metabolize low molecular weight compounds. Mass spectrometry-based proteomic analysis of the consortium was used to identify .3000 proteins in fractionated samples from the cultures, and confirmed the importance of Paenibacillus and Gemmatimonadetes to biomass deconstruction. These studies also indicate that there are unexplored proteins with important roles in bacterial lignocellulose deconstruction.

Citation: 
D'haeseleer P, JM Gladden, M Allgaier, P Chain, SG Tringe, S Malfatti, JT Aldrich, CD Nicora, EW Robinson, L Pasa-Tolic, P Hugenholtz, BA Simmons, and SW Singer.2013."Proteogenomic Analysis of a Thermophilic Bacterial Consortium Adapted to Deconstruct Switchgrass."PLoS One 8(7):e68465. doi:10.1371/journal.pone.0068465
Authors: 
P D'haeseleer
JM Gladden
M Allgaier
P Chain
SG Tringe
S Malfatti
JT Aldrich
CD Nicora
EW Robinson
L Pasa-Tolic
P Hugenholtz
BA Simmons
SW Singer
Facility: 
Publication year: 
2013

Evidence supporting dissimilatory and assimilatory lignin degradation in Enterobacter lignolyticus SCF1.

Abstract: 

The anaerobic isolate Enterobacter lignolyticus SCF1 was initially cultivated based on anaerobic growth on lignin as sole carbon source. The source of the isolated bacteria was from tropical forest soils that decompose litter rapidly with low and fluctuating redox potentials, making it likely that bacteria using oxygen-independent enzymes play an important role in decomposition. We have examined differential expression of the anaerobic isolate Enterobacter lignolyticus SCF1 during growth on lignin. After 48 hours of growth, we used transcriptomics and proteomics to define the enzymes and other regulatory machinery that these organisms use to degrade lignin, as well as metabolomics to measure lignin degradation and monitor the use of lignin and iron as terminal electron acceptors that facilitate more efficient use of carbon. Proteomics revealed accelerated xylose uptake and metabolism under lignin-amended growth, and lignin degradation via the 4-hydroxyphenylacetate degradation pathway, catalase/peroxidase enzymes, and the glutathione biosynthesis and glutathione S-transferase proteins. We also observed increased production of NADH-quinone oxidoreductase, other electron transport chain proteins, and ATP synthase and ATP-binding cassette (ABC) transporters. Our data shows the advantages of a multi-omics approach, where incomplete pathways identified by genomics were completed, and new observations made on coping with poor carbon availability. The fast growth, high efficiency and specificity of enzymes employed in bacterial anaerobic litter deconstruction makes these soils useful templates for improving biofuel production.

Citation: 
DeAngelis KM, D Sharma, R Varney, BA Simmons, NG Isern, LM Markillie, CD Nicora, AD Norbeck, RC Taylor, JT Aldrich, and EW Robinson.2013."Evidence supporting dissimilatory and assimilatory lignin degradation in Enterobacter lignolyticus SCF1."Frontiers in Microbiology 4:Article No. 280.
Authors: 
KM DeAngelis
D Sharma
R Varney
BA Simmons
NG Isern
LM Markillie
CD Nicora
AD Norbeck
RC Taylor
JT Aldrich
EW Robinson
Facility: 
Publication year: 
2013

The Toxoplasma gondii cyst wall protein CST1 is critical for cyst wall integrity and promotes bradyzoite persistence.

Abstract: 

Toxoplasma gondii infects up to one third of the world’s population. A key to the success of T.gondii is its ability to persist for the life of its host as bradyzoites within tissue cysts. The glycosylated cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii cysts. Deletion of CST1 results in a fragile brain cyst phenotype revealed by a thinning and disruption of the underlying region of the cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal cyst wall structure, the ability of the cyst to resist mechanical stress and binding of DBA to the cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that reinforces the cyst wall structure and confers essential sturdiness to the T. gondii tissue cyst.

Citation: 
Tomita T, DJ Bzik, YF Ma, BA Fox, LM Markillie, RC Taylor, K Kim, and LM Weiss.2013."The Toxoplasma gondii cyst wall protein CST1 is critical for cyst wall integrity and promotes bradyzoite persistence."PLoS Pathogens 9(12):e1003823. doi:10.1371/journal.ppat.1003823
Authors: 
T Tomita
DJ Bzik
YF Ma
BA Fox
LM Markillie
RC Taylor
K Kim
LM Weiss
Publication year: 
2013

Changes in Translational Efficiency is a Dominant Regulatory Mechanism in the Environmental Response of Bacteria.

Abstract: 

To understand how cell physiological state affects mRNA translation, we used Shewanella oneidensis MR-1 grown under steady state conditions at either aerobic or suboxic conditions. Using a combination of quantitative proteomics and RNA-Seq, we generated high-confidence data on >1000 mRNA and protein pairs. By using a steady state model, we found that differences in protein-mRNA ratios were primarily caused by differences in the translational efficiency of specific genes. When oxygen levels were lowered, 28% of the proteins showed at least a 2-fold change in expression. Altered transcription levels appeared responsible for 26% of the protein changes, altered translational efficiency appeared responsible for 46% and a combination of both were responsible for the remaining 28%. Changes in translational efficiency were significantly correlated with the codon usage pattern of the genes and measurable tRNA pools changed in response to altered O2 levels. Our results suggest that changes in the translational efficiency of proteins, in part caused by altered tRNA pools, is a major determinant of regulated protein expression in bacteria.

Citation: 
Taylor RC, BJM Webb-Robertson, LM Markillie, MH Serres, BE Linggi, JT Aldrich, EA Hill, MF Romine, MS Lipton, and HS Wiley.2013."Changes in Translational Efficiency is a Dominant Regulatory Mechanism in the Environmental Response of Bacteria."Integrative Biology 5(11):1393-1406. doi:10.1039/C3IB40120K
Authors: 
RC Taylor
BJM Webb-Robertson
LM Markillie
MH Serres
BE Linggi
JT Aldrich
EA Hill
MF Romine
MS Lipton
HS Wiley
Facility: 
Volume: 
5
Issue: 
11
Pages: 
1393-1406
Publication year: 
2013

Field evidence of selenium bioreduction in a uranium-contaminated aquifer.

Abstract: 

Removal of selenium from groundwater was documented during injection of acetate into a uraniumcontaminated aquifer near Rifle, Colorado (USA). Bioreduction of aqueous selenium to its elemental form (Se0) concentrated it within mineralized biofilms affixed to tubing used to circulate acetate-amended groundwater. Scanning and transmission electron microscopy revealed close association between Se0 precipitates and cell surfaces, with Se0 aggregates having a diameter of 50-60 nm. Accumulation of Se0 within biofilms occurred over a three-week interval at a rate of c. 9 mg Se0m-2 tubing day-1. Removal was inferred to result from the activity of a mixed microbial community within the biofilms capable of coupling acetate oxidation to the reduction of oxygen, nitrate and selenate. Phylogenetic analysis of the biofilm revealed a community dominated by strains of Dechloromonas sp. and Thauera sp., with isolates exhibiting genetic similarity to the latter known to reduce selenate to Se0. Enrichment cultures of selenate-respiring microorganisms were readily established using Rifle site groundwater and acetate, with cultures dominated by strains closely related to D. aromatica (96-99% similarity). Predominance of Dechloromonas sp. in recovered biofilms and enrichments suggests this microorganism may play a role in the removal of selenium oxyanions present in Se-impacted groundwaters and sediments.

Citation: 
Williams KH, MJ Wilkins, AL N'Guessan, BW Arey, EN Dodova, A Dohnalkova, D Holmes, DR Lovley, and PE Long.2013."Field evidence of selenium bioreduction in a uranium-contaminated aquifer."Environmental Microbiology Reports 5(3):444-452. doi:10.1111/1758-2229.12032
Authors: 
Williams
MJ Wilkins
AL N'Guessan
BW Arey
EN Dodova
A Dohnalkova
D Holmes
DR Lovley
PE Long
Facility: 
Volume: 
5
Issue: 
3
Pages: 
444-452
Publication year: 
2013

Discovery of a Splicing Regulator Required for Cell Cycle Progression.

Abstract: 

In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

Citation: 
Suvorova ES, M Croken, S Kratzer, LM Ting, M Conde de Felipe, B Balu, LM Markillie, LM Weiss, K Kim, and MW White.2013."Discovery of a Splicing Regulator Required for Cell Cycle Progression."PLoS Genetics 9(2):Article No. e1003305. doi:10.1371/journal.pgen.1003305
Authors: 
ES Suvorova
M Croken
S Kratzer
LM Ting
M Conde de Felipe
B Balu
LM Markillie
LM Weiss
K Kim
MW White
Publication year: 
2013

Coregulation of terpenoid pathway genes and prediction of isoprene production in Bacillus subtilis using transcriptomics.

Abstract: 

The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding the system level regulation and control of the pathway. To address this limitation, we examined Bacillus subtilis grown under multiple conditions and then determined the relationship between altered isoprene production and the pattern of gene expression. We found that terpenoid genes appeared to fall into two distinct subsets with opposing correlations with respect to the amount of isoprene produced. The group whose expression levels positively correlated with isoprene production included dxs, the gene responsible for the commitment step in the pathway, as well as ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. This analysis showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model which accurately predicts production of this secondary metabolite across many simulated environmental conditions.

Citation: 
Hess BM, J Xue, LM Markillie, RC Taylor, HS Wiley, BK Ahring, and BE Linggi.2013."Coregulation of terpenoid pathway genes and prediction of isoprene production in Bacillus subtilis using transcriptomics."PLoS One 8(6):e66104. doi:10.1371/journal.pone.0066104
Authors: 
BM Hess
J Xue
LM Markillie
RC Taylor
HS Wiley
BK Ahring
BE Linggi
Facility: 
Publication year: 
2013

Amino acid treatment enhances protein recovery from sediment and soils for metaproteomic studies .

Abstract: 

Characterization of geomicrobial protein expression provides information necessary to better understand the unique biological pathways that occur within soil microbial communities and the role they play in regulating atmospheric CO2 levels and the Earth’s climate. A significant challenge in studying soil microbial proteins is their initial dissociation from the complex mixture of particles found in natural soil. Due to bias of the most robust cells, the removal of intact bacterial cells limits the characterization of the complete representation of a microbial community. However, in-situ lysis of bacterial cells leads to the expulsion of proteins to the soil surface, which can lead to potentially high levels of adsorption due to the physicochemical properties of both the protein and the soil. We investigated various compounds for their ability to block protein adsorption soil sites prior to in-situ lysis of bacterial cells, as well as their compatibility with both tryptic digestion and mass spectrometric analysis. The treatments were tested by adding lysed Escherichia coli proteins to representative treated and untreated soil samples. The results show that it is possible to significantly increase protein identifications through blockage of binding sites on a variety of soil textures; use of an optimized desorption buffer further increases the number of identifications.

Citation: 
Nicora CD, BJ Anderson, SJ Callister, AD Norbeck, SO Purvine, JK Jansson, OU Mason, M David, DD Jurelevicius, RD Smith, and MS Lipton.2013."Amino acid treatment enhances protein recovery from sediment and soils for metaproteomic studies ."Proteomics 13(18-19):2776-2785. doi:10.1002/pmic.201300003
Authors: 
CD Nicora
BJ Anderson
SJ Callister
AD Norbeck
SO Purvine
JK Jansson
OU Mason
M David
DD Jurelevicius
RD Smith
MS Lipton
Facility: 
Instruments: 
Volume: 
13
Pages: 
2776-2785
Publication year: 
2013

Enhanced top-down characterization of histone post-translational modifications .

Abstract: 

Background: Multiple post-translational modifications (PTMs) on core histones often work synergistically to fine tune chromatin structure and functions, generating a “histone code” that can be interpreted by a variety of chromatin interacting proteins. Although previous bottom-up and middle-down proteomic approaches have been developed for limited characterization of PTMs on histone N-terminal tails, high-throughput methods for comprehensive identification of PTMs distributed along the entire primary amino acid sequence are yet to be implemented. Results: Here we report a novel online two-dimensional liquid chromatography - tandem mass spectrometry (2D LC–MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The metal-free LC system with reverse phase separation followed by weak cation exchange – hydrophilic interaction chromatography (WCX-HILIC) and online Orbitrap Velos tandem mass spectrometry allowed for unambiguous identification of over 700 histone isoforms from a single 2D LC–MS/MS analysis of 7.5 µg of purified core histones. In comparison with previous offline top-down analysis of H4, this online study identified 100 additional isoforms from 100-fold less sample. This platform enabled comprehensive characterization of histone modifications, including those beyond tail regions, with dramatically improved throughput and sensitivity compared to more traditional platforms. Isoforms identified included those with combinatorial PTMs extending well beyond the N-terminal tail regions as well as a large number of phosphorylated isoforms.

Citation: 
Tian Z, N Tolic, R Zhao, RJ Moore, SM Hengel, EW Robinson, DL Stenoien, S Wu, RD Smith, and L Pasa-Tolic.2012."Enhanced top-down characterization of histone post-translational modifications ."Genome Biology 13(10):R86. doi:10.1186/gb-2012-13-10-r86
Authors: 
Z Tian
N Tolic
R Zhao
RJ Moore
SM Hengel
EW Robinson
DL Stenoien
S Wu
RD Smith
L Pasa-Tolic
Publication year: 
2012

Proteome Analyses of Strains ATCC 51142 and PCC 7822 of the Diazotrophic Cyanobacterium Cyanothece sp under Culture Conditions

Abstract: 

Cultures of the cyanobacterial genus Cyanothece have been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N2-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes and we performed quantitative proteome analysis of Cyanothece ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N2-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochrome c oxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose-phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited in Cyanothece ATCC 51142 in the presence of glycerol under H2 producing conditions, suggesting a competition between these sources of carbon. However, in Cyanothece PCC 7822, the Calvin cycle still played a role in cofactor recycling during H2 production. Our data comprise the first comprehensive profiling of proteome changes in Cyanothece PCC 7822, and allows an in-depth comparative analysis of major physiological and biochemical processes that influence H2-production in both the strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large scale H2 production.

Citation: 
Aryal UK, SJ Callister, S Mishra, X Zhang, JI Shutthanandan, TE Angel, AK Shukla, ME Monroe, RJ Moore, DW Koppenaal, RD Smith, and L Sherman.2013."Proteome Analyses of Strains ATCC 51142 and PCC 7822 of the Diazotrophic Cyanobacterium Cyanothece sp under Culture Conditions Resulting in Enhanced H-2 Production."Applied and Environmental Microbiology 79(4):1070-1077. doi:10.1128/AEM.02864-12
Authors: 
UK Aryal
SJ Callister
S Mishra
X Zhang
JI Shutthanan
TE Angel
AK Shukla
ME Monroe
RJ Moore
DW Koppenaal
RD Smith
L Sherman
Facility: 
Instruments: 
Volume: 
79
Issue: 
4
Pages: 
1070-1077
Publication year: 
2013

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