K-095. Analysis of Phosphoproteins from Dissimilatory Metal Reducers

C. Giometti, J. Krieger, J. Smotrys, B. Lindberg, G. Babnigg;
Argonne Natl. Lab., Argonne, IL.

The role of serine, threonine, and tyrosine phosphorylation in bacterial systems is as yet undefined. Unlike the phosphorylation events mediated by histidine kinases, which are transient and involved in bacterial signal transduction processes, pSer, pThr, and pTyr events are believed to be relatively stable and related to the regulation of protein function in a manner similar to that described for eukaryotic phosphoproteins. Recent reports of phosphoproteins in Corynebacterium glutamicum and Bacillus subtilis suggest that such phosphorylations are involved in the control of protein synthesis and in response to cold, heat, and osmotic shock. We have been investigating the possibility that phosphorylation of serine residues in metal-reducing bacteria such as Shewanella and Geobacter serves as a regulatory mechanism in the metabolic processes related to energy metabolism. Using a combinatorial proteomics approach that includes the use of affinity chromatography for enrichment of phosphoproteins, one- and two-dimensional gel electrophoresis for protein separation, immunoassay and specific staining for detection of total phosphoproteins and phosphoserine proteins, and, finally, tandem mass spectrometry to identify proteins, we have identified a set of putative phosphoproteins in Shewanella oneidensis MR-1. In addition, we have demonstrated that the level of protein phosphorylation in both S. oneidensis and G. sulfurreducens is dramatically lower than that found in eukaryotic cells. This latter finding necessitates the optimization of existing methods for the detection of phosphoserine proteins in order to monitor differential expression of these bacterial phosphoproteins. Comparison of the phosphoserine profiles from S. oneidensis grown with or without oxygen as the terminal electron acceptor show distinct differences in the level of specific protein phosphorylation. These results confirm that phosphorylation of serine residues does occur in S. oneidensis and G. sulfurreducens and that differences in phosphorylation on serine correlate with differences in electron acceptor availability in S. oneidensis.