Introduction

1. High Throughput Methods To Identify Underlying Molecular Signature Pathways In Desulfovibrio vulgaris Masood Hadi1,3, Yooli Kim-Light1,3, Sara Gaucher1,3 ,Julie Kaiser1,3, Julia Dibble1,3, Pamela Lane,, Gabriela Chirica1,3, Dominique Joyner2,3, Mary Singer2,3,Terry Hazen 2,3, Adam Arkin2,3 and Anup Singh1,3 1Biological and Microfluidic Sciences, Sandia National Laboratories, 7011 East Avenue, Livermore, CA, USA, 2Lawrence Berkeley National Laboratory, Berkeley, CA, USA, 3Virtual Institute for Microbial Stress and Survival 1 2 3 4 5 6 7 8 9 10 11 12 A B C D Introduction Interactomix by exogenously tagged proteins Putative Target Stress ORFs From Computational Core (E. Alm) Design, Synthesize Primers and PCR ORFs Exogenous Tagged-Protein Pull Down Clone Into Ligation Independent Cloning Vector (Gateway) Representative Image of First PCR to amplify the target ORFs from D. vibrio genome. Confirm ORF and Transform Into Expression Cell Lines Grown in Liquid Culture to Determine Induction/expression Conditions Determine Solubility Conditions and purify protein Couple Bait Protein to Magentic Affinity Beads Capture Interacting Proteins Representative Image of M13-PCR analysis used to confirm the presence of ORFs in bacterial colonies. Representative image of dot-blot western analysis to determine solubility conditions Mass Spec Identification Results and Discussion Targeted proteomics is the systematic study of protein-protein interactions through the isolation of protein complexes. Several approaches have been developed over the years for purification and identification of protein complexes such as yeast two hybrid methods, co-immunoprecipitaions and endogenous expression of affinity tagged bait proteins (tandem affinity tags –TAPTAG). The TAPTAG approach has been successfully used to build a protein interaction map of the yeast genome. All of these approaches have one thing in common – they are cumbersome, time consuming, require considerable manual handling and are not amenable to automation. We have been developing novel generic high throughput technologies that will enable systematic identification, characterization and eventual understanding of molecular machines that function during stress responses in Desulfovibrio vulgaris [Strain, Hildenborough] from a targeted proteomics perspective. This sulfate reducing bacteria has been shown to reduce toxic metals (such as chromium and uranium) to insoluble species making them a good model system for understanding molecular machines involved in bioremediation of contaminated soils and ground water. Computationally identified open reading frames (ORFs) that are involved in stress response (including oxygen, heat, pH and salt) based on homology to known stress related genes from other prokaryotic species and have used these ORFs as bait proteins to isolate molecular machines. The Exogenous Tagged-Protein method for identification of interacting protein is the most amenable to high throughput and automation. We are now focusing on a set of bait proteins that are unique to Desulfovibrio vulgaris and other sulfate reducer, i.e. "signature" genes which are expected to yield novel complexes related to sulfate/metal reduction. We discuss our results and the application to these technologies towards proteome wide interaction mapping. ClpX is the ATP-dependent specificity component of clpP serine protease, ClpX and ClpA subunit bind clpP serine protease. Functional communication between ClpXP rings depends on the ATPase activity of ClpX and seems to be transmitted through structural changes. Proteins identified in the complex are: t ATP-dependent Clp protease, ATP-binding subunit ClpX (clpX), ransketolase (tkt), carboxyl-terminal protease, ACT domain protein, GAF domain protein, HlyD family secretion protein, putative, glutamyl-tRNA synthetase (gltX), translation elongation factor Ts (tsf), tyrosyl-tRNA synthetase (tyrS), malonyl CoA-acyl carrier protein transacylase (fabD), ketol-acid reductoisomerase (ilvC), 2-oxoglutarate dehydrogenase, E3 component,, lipoamide dehydrogenase (lpdA), serine protease, HtrA/DegQ/DegS family (htrA), decarboxylase family protein, ribose-phosphate pyrophosphokinase (prsA), acetoin utilization protein AcuB, putative, 6-phosphofructokinase (pfkA), acetyl-coenzyme A carboxylase, biotin carboxylase (accC), aminotransferase, class I, hydrogenase, putative, heterodisulfide reductase, A subunit (hdrA), pyruvate kinase (pyk), DNA gyrase, B subunit (gyrB), universal stress protein family family, NAD-dependent epimerase/dehydratase family protein, aromatic aminotransferase, chorismate mutase/prephenate dehydratase, ADP-L-glycero-D, mannoheptose-6-epimerase (rfaD) Iron is essential to virtually all organisms, but poses problems of toxicity and poor solubility. Bacteria have evolved various mechanisms to counter the problems imposed by their iron dependence, allowing them to achieve effective iron homeostasis under a range of iron regimes. Highly efficient iron acquisition systems are used to scavenge iron from the environment under iron-restricted conditions. Ferritin(ftn) is an iron-binding protein that prevents toxic levels of ionized iron (Fe2+) from building up in cells. Bacterioferritin (bfr) gene encodes an iron storage protein. Roles of the bacterial ferritin-like compounds are not well understood. A few studies conducted with mutants indicated that ferritin-like compounds can protect bacterial cells from iron-overload, serve as an iron source when iron is limited, protect the bacterial cells against oxidative stress and/or protect DNA against enzymatic or oxidative attack. There is very little information available concerning the roles that ferritin-like compounds might play in the survival of bacteria in food, water, soil or eukaryotic host environments Using parallel liquid handelling and 96 well plates we have streamlined key steps of the cloning process, including PCR reactions, recombination reactions and gel electrophoresis. Researchers still manually perform transformations and colony picking which can be automated in 96-well microtiter plates using a recently acquired colony picker. Initial clones are sequenced and data analyzed by database search. We have engineered our process to be cost-effective and HT by replacing costly and time-consuming analytical steps, such as replacing traditional gel electrophoresis with E-96 gels. From loading the gel to obtaining results, the process takes only 30 minutes. Using the automated system described, our clone-generating throughput has been enhanced while tedious, labor-intensive manipulations have diminished. All processes are tracked using LIMS. QmoABC complex encodes a novel membrane-bound respiratory complex. The ubiquitous presence of the qmoABC genes in sulfate-reducing prokaryotes and their presence in some sulfite-reducing and phototrophic sulfur-oxidizing bacteria emphasize their relevance for general sulfur-based energy metabolism. • The emerging field of functional proteomics is driven by a need to understand the biological relevance of newly discovered genes and the encoded proteins. Gaining this understanding requires development of methods to produce thousands of proteins per genome in parallel for high throughput analysis —a major challenge. Protein function can be described based on its role in the behavior of an organism (phenotype) or a cell type (cellular function), as well as in terms of its interactions with other molecules (molecular function). We have been trying addresses several key challenges to isolating protein complexes: • Many stable complexes in the proteome are as yet unknown. • The available data about complexes are error prone. • Many stable complexes in the proteome involve membrane proteins. • There is no proven technology that can achieve isolation of even 60% of globular cytosolic proteins. • We have been using the Exogenous Tagged-Protein (interactomix) method for identification of interacting proteins in a protein complex. Interactomix does not need mutants or established genetic systems, is applicable to un-culturable and high-risk organisms and is most amenable to high throughput and automation. The goal is to establish a fully functional and integrated pipeline for high throughput protein-protein interaction mapping in order to establish a cost-effective production center for high-throughput interactomix that will enable systematic identification, characterization and eventual understanding of molecular machines that function in Desulfovibrio from a targeted proteomics perspective. • Target open reading frames (ORFs) that are putatively involved in stress response or unique to D. vibrio or other sulfate reducers are computationally identified. The ORFs are amplified, cloned into modular vectors containing affinity tags, and then transferred into surrogate expression systems (E coli or cell-free). The tagged proteins are expressed and purified. These purified proteins are used as baits by attaching them to magnetic beads and incubating them with Desulfovibrio lysates grown under anaerobic conditions. Interacting proteins assemble on the bait and are captured, washed to remove background and non-specifically interacting (sticky) proteins. Interacting proteins are eluted using denaturing conditions followed by 1-D gel analysis and Mass. Spec. identification. • . Representative Image of Second PCR preformed with adapter primers to add the recombination signal sequence onto the target ORFs. Using autoinduction methods (F.W. Studier, Brookhaven National Lab, personal communication), we have developed a screen for soluble expression in E coli. Briefly, for in vivo expression screening, clones are transformed in E. coli BL21(DE3) (Novagen) and arrayed in a 96-well deep-well plate with 1 ml of Studier auto induction media, which allows growth to high densities in a shaker incubator. We typically assay expression yields by Dot blot westerns and SDS page electrophoresis. Approximately 70% of clones yield detectable full-length expression. We regrow transformed cells that show expression in 1 ml cell culture 96-well plates and express target proteins. Following expression, the cells are aliquoted into five 96-well plates for screening against five lysis buffers. Each sample is then pelleted, resuspended in lysis buffer, and cells disrupted to release proteins. After lysis, we assess the soluble fraction for protein yield by western blot analysis. Greater than 90% of clones that yield detectable expression also have detectable protein in the soluble fraction for at least one of the lysis buffers. In two days, one to two people can screen 96 clones for soluble expression and lysis conditions. Targets that show detectible yield of soluble expression are then prioritized for scaled-up expression. Data from individual pull-down experiment was used to build an interactomix network. Two separate clusters were observed (comprising of about five hundred proteins). We are currently in the process of cloning and tagging the connecting partners in order to confirm our initial results. During these experiments we have identified a few issues that need further attention: Reasonable yield of bait proteins are difficult to obtain in conventional multi-well format. Methods do not exist to (predict/confirm) interacting partners, although for some model organisms a combination of literature, microarray and bioinformatics analysis can be helpfull. Experimental repeats may simply confirm "sticky" proteins. It is difficult to design proper controls for interactomix. Data handling and sample tracking can become the major bottleneck. 49kb 12kb