Examinations of Cell Surface Layer Proteins in the Thermophilic Thermotogales Bacteria to Determine Their Roles in Polysaccharide Hydrolysis
EMSL Project ID
39965
Abstract
We propose to characterize the protein constituents of the outer surface layer of cells of the hyperthermophilic bacterium Thermotoga maritima and its relatives and examine their roles in the hydrolysis of complex polysaccharides. Thermophilic bacteria belonging to the order Thermotogales are among the best sugar-fermenting thermophiles for biohydrogenesis because their catabolism produces hydrogen at the thermodynamic limit, i.e. four moles of hydrogen per mole of hexose consumed. The Thermotogales are distinguished by their outer cell envelope or toga. A few sugar hydrolyzing enzymes have been isolated from the toga fraction and these likely allow those cells to depolymerize insoluble polysaccharides. However, there has been no comprehensive examination of the toga-associated sugar hydrolases or other depolymerizing enzymes or how their arrangement there enhances polysaccharide hydrolysis. This study, in part, seeks to address this gap in our knowledge. Only two hydrolytic enzymes have been isolated from the toga of the best-studied species, Thermotoga maritima, a beta-amylase and a xylanase. Only two other proteins have been conclusively found to be within the toga fraction. OmpB is a 40 kDa porin and OmpA is a 43 kDa rod-shaped spacer protein and anchor to the periplasmic membrane. There is no published evidence for lipid in the toga fraction. The aim of the proposed research is to characterize the constituents of the togas of cells grown under a variety of conditions and to determine the toga's role in the hydrolysis of insoluble growth substrates. We intend to identify toga-associated proteins to better define its role when cells grow on complex polysaccharides. We would like to understand how hydrolases are organized on the surface of the toga to efficiently hydrolyze complex substrates. If proteins with binding domains for complex polysaccharides are involved in this process, we would like to identify them. These data will provide essential information for future efforts to capitalize on biohydrogenesis by Thermotogales species. Working in collaboration with EMSL scientists Drs. Ljiljana Pasa-Tolic and Mary Lipton, we propose to use EMSL instrumentation to examine the questions posed in the proposal. Potential instrumentation applicable to these projects includes FT-ICR or Orbitrap MS for identification of extracted toga proteins and Cryo-TEM to visualize binding of cells to insoluble polymers. Not only will the information gathered enhance our understanding of how insoluble polysaccharides are hydrolyzed by these cells, but the data will also allow correct gene annotations to be applied to genes encoded in the several Thermotogales genome sequences currently available.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2010-10-06
End Date
2013-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Petrus AK, KS Swithers, CR Ranjit, S Wu, HM Brewer, JP Gogarten, L Pasa-Tolic, and KM Noll. 2012. "Genes for the Major Structural Components of Thermotogales Species’ Togas Revealed by Proteomic and Evolutionary Analyses of OmpA and OmpB Homologs." PLoS One 7(6):e40236. doi:10.1371/journal.pone.0040236