# Presentation: 525187

## Overview

**The goal of the FGC is to develop the experimental methods to elucidate the regulatory networks in the stress responses of *****Desulfovibrio vulgaris*****, *****Shewanella oneidensis*****, and *****Geobacter metallireducens*****. In order to accomplish these goals we will: **
**Use existing DNA arrays for *****S. oneidensis*****, and to develop DNA arrays for *****D. vulgaris***** and *****G. metallireducens***** to measure the transcript profile (transcriptome) during the response to various environmental stresses**
**Use HPLC-MS-MS to measure the protein profile (proteome) of *****D. vulgaris*****, *****G. metallireducens*****, and *****S. oneidensis***** during the response to various environmental stresses**
**Measure the metabolite profile (metabolome) of *****D. vulgaris*****, *****G. metallireducens*****, and *****S. oneidensis***** during the response to various environmental stresses**
**Determine protein-protein interactions in the signaling cascade of *****D. vulgaris*****, *****G. metallireducens*****, and *****S. oneidensis***** during the response to various environmental stresses**
**Generate mutants in the various genes whose gene products are found to be responsible for a particular stress response, and to compare transcript, protein, and metabolite profiles in the mutant and isogenic wild-type strains and**
**Biopan the environment for stress response pathways.**
**The stresses to be examined initially are oxygen, heavy metal exposure (U, Co, Hg, Ni, and Cr), reductant limitation, phosphate restriction, and pH****.**

**Profiling of Low-pH Stress**
**Tolerance in S. oneidensis MR-1**

_**Genes Induced at pH 4**_**:**
**~3,300 genes (~ 60% of total predicted genes) displayed changes in expression in response to 30-minute exposure to pH 4.**
**outer membrane proteins/lipoproteins**
**ABC transporter proteins (e.g., phosphate transport system)**
**metabolic proteins, including those related to amino acid synthesis**
**hypothetical proteins**

**Review of the Protein Identification**
**from a LC****n****-MS/MS Run**

**1% SDS**

**Sonication**

**Ultracentrifugation**

**Supernatant**

**Membrane pellet**

**Soluble Proteins**

**4M urea**

**Ultracentrifugation**

**Supernatant**

**Membrane pellet**

**Urea Soluble Proteins**

**SDS Soluble Proteins**

**3D LC-MS/MS**

**Protease Digestions**

**Select a pathway involved in stress response**
**Identify protein(s) involved in the pathway to be used as a bait**

**Attach “tags” to genes of interest**

**Transform and select positive clones**

**Tandem affinity purification**

**Cultivate, apply stress**

**Information on proteins and protein interaction**

**Graph Database System**

**Grown aerobically to mid exponential phase **

**pH 4 (MES)**

**Trizol reagent and RNA isolation**

**Microarray hybridization **

**Label with Cy3 or Cy5 **

**pH 7 (HEPES)**

**pH 10 (CAPS)**

- Harvest and suspend cells in buffer

- Incubate 30 min.

**Matthew Fields****1****,***** *****Jay Keasling****2****, Martin Keller****3****, Vincent Martin****2****, Anup Singh****4****, Dorothea Thompson****1****, Judy Wall****5****, Jizhong Zhou****1**
**1****Oak Ridge National Laboratory, ****2****Lawrence Berkeley National Laboratory & University of California, Berkeley, ****3****Diversa, Inc., ****4****Sandia National Laboratory, ****5****University of Missouri, Columbia**

**Outline of physiological monitoring**_** **_

**Overview**

**Transcription Profiling**

**Protein Expression Profiling**

**Metabolite Profiling**

**Protein Complex Identification**

**Stable**
**Intermediates**

**Cells**

**Supernatant**

**Secreted**
**Metabolites**

**Cytoplasm**

**Insoluble **
**Fraction**

**Metabolite Amounts**
**And Labeling Patterns**

**Amino Acid**
**Labeling Pattern**

**Total Macromolecule**
**Content (DNA, RNA, Lipid,**
**Storage Polymers**

**Centrifugation**

**HPLC-MS**

**Lysis**

**HPLC-MS**
**CE-MS**

**Derivitization**
**GC-MS**

**GC-MS**
**HPLC-MS**

**Quench Metabolism**

- Functional Genomic Core

**The Virtual Institute of Microbial Stress and Survival **
- Rapid Deduction of Stress Response Pathways in Metal/Radionuclide Reducing Bacteria