1. Concerns and Possible Solutions From Harms 2007

2.  If metabolites are cycled by a single organism, or within a community of organisms, how can we reproducibly measure a change in transcriptome, proteome, or metabolome that represents a level of stress we should act upon by monitoring or remediation?  How are we defining “ecological stress” for this discussion.  Life at the edge takes advantage of chemical and redox gradients that typify conditions of ‘ecological stress’ ◦ Evidence of microbial stress response may not indicate a substantial change in “ecological health”  What level of enzyme activity is an indication of “stress” in this context?  Growth dependent? ◦ Other stresses (nutrition) would need control ◦ Scale dependence of stress concept  Toxicity

3.  Multiple signals can trigger any given response ◦ Operons (packages of genes) concurrently regulate multiple genes ◦ Universal stress regulator ◦ Selenate reductase example  How to ID optimal analytes to target? ◦ Metal Homeostasis (Hobman, et al, 2007) ◦ Importer/Exporter Proteins ◦ Intracellular vs. Extracellular proteins  Extraction, isolation, quantification ◦ Metabolites that are unique to the microbial stress response of interest. Metal reporter genes (Harms, 2007)

4.  Time consuming and expensive  Potential method biases – extraction, amplification  Reproducibility, precision vary with individual analyst and method  Data management ◦ Require enormous amount of data to resolve key proteins, metabolites that provide a unique environmental signal. ◦ Each monitoring effort would generate a large amount of data requiring significant analysis  How to distinguish background in natural environment?

5.  Select Indicator Organisms  Identify key transcriptome and proteome pathways that will offer unique evidence of significant (actionable) stress  Use only for ◦ strictly xenobiotic compounds without natural analogs ◦ Detoxification pathways only (e.g., not respiration) ◦ Not metals  Stick with measuring metabolites and toxicology