Abstract:
The photosynthetic purple bacterium Rubrivivax (R.) gelatinosus possesses an original copper resistance system thanks to an operon encoding 3 periplasmic proteins: CopI, CopJ and CopH. These proteins have homologs in other environmental as well as pathogenic bacteria, and some of these bacteria lack systems such as Cu oxidase or the Cus export complex. In order to gain a better understanding of this mechanism, and to complement the in vitro studies carried out at BIP, it is important to carry out in vivo studies as well. In this context, proteomic studies are ideally suited to determine which part of the R. gelatinosus proteome is affected by copper or other metal stress. However, difficulties can be encountered in detecting certain expressed proteins. This is particularly true of the CopH protein, whose sequence has very few trypsin cleavage sites (K (0), R (3), on 97 AA) and the only R residues are located at the end of the sequence, generating only 1 trypsin peptide that can be analyzed under conventional conditions.
In order to improve this, we have established new protocols using trypsin/chymotrypsin to digest CopH in peptides identifiable by LC- MS/MS. This biochemical method combined with targeted proteomics experiments allowed a significant improvement in the detection of CopH. We applied those methods to study the effect of a Cu stress in vivo on the whole proteome of R. gelatinosus under photosynthetic or microaerobic conditions. Overexpression of several proteins is detected under Cu stress including all those of the Cop operon except CopH. The results obtained led us to hypothezise that either CopH is used as a primary barrier against Cu and is then degraded or that it is exported outside of the cell. Further analyses of the global proteomic experiments in vivo are underway to help deciphering the Cu resistance mechanism of R. gelatinosus.
Co-supervisors :
- Régine LEBRUN (IMM)
- Pierre DORLET (BIP)