Studies on Copper Homeostasis in Methylococcus Capsulatus based on the complete genomic sequence

Copper is an essential element required by all living systems. Copper can also cause serious cell damage through radical formation. Therefore, careful regulation of the intracellular copper concentration is required. Essentially no free copper is availabl e in the cytoplasm; the copper ions are bound with high specificity to proteins. We have sequenced and annotated the complete genomic sequence of Methylococcus capsulatus. Based on this novel information, we will study copper ion regulation in M. capsula tus at a global level, i.e. how the cells accumulate copper from the environment, copper transportation over the outer and inner membranes, how copper ions are delivered to copper-requiring enzymes/proteins, and how the bacterium regulates the expression of the proteins involved. Using specific M. capsulatus microarrays and proteomics, we intend to investigate copper-regulated gene expression and to identify proteins involved in copper homeostasis and to study the molecular and structural properties of th ese proteins, and their specific function in copper homeostasis. The genome will be analysed for putative proteins with homology to proteins known to be involved in uptake, distribution, and sequestering of copper ions. Different protein abundance between cells grown in high- and low-copper media will be analysed by 2-D gel electrophoresis and mass spectrometry and by microarray and RT-PCR mRNA expression analyses. Structural and functional studies will be continued on three of the proteins, MopE, SapE, a nd McHem, that we already know from our genomic investigations are relevant for copper homeostasis. These will be cloned and purified for functional, biophysical and structural analysis. Molecular functions will be analysed by genetic tools, e.g. by knock out mutants, protein-protein interaction and metal-binding studies. Bioinformatics tools will be used and new such tools will be developed where the project requires.