Mechanisms of Cellular Response to Environmental Changes

     Research in the laboratory is centred around mechanisms that underpin cellular response(s) to changes in the environment.  The research has two quite distinct components: the one uses Saccharomyces cerevisiae as a model eukaryotic system to evaluate the cellular response to heavy metal exposure, such as Copper, Chromium (VI) and Cadmium.  The other investigates the role of transcriptional regulatory circuits within Pseudomonas putida, which coordinate both the cell's metabolic and chemotactic response(s) to the presence of aromatic hydrocarbons. 

Oxidative Targeting of Enzymes in S. cerevisiae: While both cellular response mechanisms differ dramatically in their mechanisms of action, they each share a significant metabolic component.  The first level response of S. cerevisae to exposure to sub-lethal concentrations of heavy metals involves targeted oxidation of enzymes predominantly within the glycolytic pathway (see Figure 1).  

    Such targeting is believed to result in an immediate -but transient- decrease in glycolytic enzyme activity, possibly resulting in a catabolic shift of carbon flow away from glycolysis and into the pentose-phosphate pathway -to generate NADPH2 that can be utilized to combat the oxidative insult.  Work in the lab is presently focused upon this and other cellular responses to heavy metals that affect protein expression (Figure 2); namely transcriptional levels of key enyzmes and regulatory proteins (Figure 2: underlay).

 

Figure 1: The Glycolytic and Pentose Phosphate pathways in S. cerevisiae, showing the key enzymes and reactions (in red) that appear to be specifically targeted for oxidation in response to the presence of heavy metals, such as Cu, Cd and Cr.  
              

Figure 2: A 2-dimensional display of cellular protein expression from S. cerevisiae cells, following exposure to sub-lethal levels of Chromium.

Underlay: A graphical representation of RTPCR analyses, showing the temporal expression of key glycolytic enzymes, pentose phosphate pathway enzymes and transcriptional / translational regulatory genes in response to the presence of heavy metals.

 
 

Transcriptional Regulation of Aromatic Metabolism in P. putida:   Microbial degradation of aromatic hydrocarbons, such as benzoate and its substituted derivative -para hydroxybenzoate, occurs in Pseudomonas putida primarily through the ß-ketoadipate pathway (Figure 3).  Consequently, it is not too surprising that this pathway responds towa wealth of regulatory controls, finely tuned to handle the diverse metabolism of Pseudomonas. With this in mind the laboratory's research interests in this area centre around various aspects of gene regulation within and consequential to this "central" pathway, with particular emphasis on the characterization of two of the regulatory genes involved in the induction of both branches of the pathway, CatR and PcaR.

 

 

 

 

     Figure 4.  A swarm plate assay of a number of P. putida wild-type and pathway mutant strains; showing the importance of the pca genes to chemotaxis of the cells toward POB.

     PcaR, in particular, appears to be of significant interest. We have shown it to be a unique, prokaryotic transcriptional activating protein, which simultaneously binds the -35 and -10 promoter elements, as well as having direct, specific contacts with the initiating RNA polymerase, whose activity it is enhancing. The potential, therefore, for genetic manipulation of this regulatory system to shed some light on the precise mechanisms involved in RNA polymerase/promoter DNA interactions at the very threshold of transcriptional initiation is quite considerable.

     

Figure 3. depicts the ß-ketoadipate pathway from Pseudomonas putida: showing how the catechol degradation (cat) branch of the pathway merges with the protocatechuate degradation (pca) pathway (green) to form Krebs' cycle intermediates.

Also shown: how POB and the major inducer of the pathway (ß-ketoadipate itself) enter the cell.

                      
           

 

MOST RECENT PUBLICATIONS:

Nargund, A. M, S. V. Avery, S. A. and J. E. Houghton (2008)
Cadmium induces a heterogeneous and caspase dependent apoptotic response in Saccharomyces cerevisiae
Apoptosis 13: 811-821

Sumner, E. R., A. Shanmuganathan, T. C. Sideri, S. A. Willetts, J. E. Houghton and S. V. Avery (2005)
Oxidative Protein Damage Causes Chromium Toxicity in Yeast
Microbiology. 151: 1939-1948

Shanmuganathan, A., S. V. Avery, S. A. Willetts and J. E. Houghton (2004)
Copper-induced Oxidative Stress in Saccharomyces cerevisiae Targets Enzymes of the Glycolytic Pathway
FEBSletters 556: 2253-2259

Sumner, E. R., A. M. Avery, J. E. Houghton, R. A Robbins, and S. V. Avery (2003)
Cell Cycle and Age-Dependent Activation of Sod1p Drives the Formation of Stress Resistant Cell Subpopulations within Clonal Cultures.
Molecular Microbiology. 32: 253-264

Herman, H. H., Lin, W, Petrecca, P., Herman, T. M., Bates, C., Simmons, R. and Houghton, J. E. (2001)
Centrifugal Bioreactors and their Application in Wastewater Remediation
In Remediation 11: 15-33