|Droleskey, Robert - Bob|
Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 11/8/2002
Publication Date: N/A
Technical Abstract: The stcABCD genes form a putative fimbrial operon which was identified in Salmonella enterica serovar Typhimurium by hybridization and later by whole genome sequence analysis. Fimbriae expression is regulated by phase variation, that results in expression of fimbriae only in a fraction of bacterial cells present in a culture (phase on), while a fraction of bacteria is afimbriated (phase off). We studied in vitro expression of stc operon in S. Typhimurium by flow cytometry. These data suggested low fraction of "phase on" cells detected under different growth conditions would complicate further characterization of the encoded surface structure. To overcome this problem, the stc operon was cloned and expressed in a non-fimbriated E. coli strain (ORN172, Delta fim E. coli) from the heterologous T7 promotor. Expression of StcA, the putative major subunit of the operon, was detected by flow cytometry, transmission electron microscopy and immunogold labeling. ORN172 and ORN172 expressing the pef operon were used as negative and positive controls, respectively. Expression of StcA was detected by flow cytometry in above 60% of E. coli ORN172, after expression of the stc operon was induced by activation of the T7 polymerase. The extracellular localization of StcA was confirmed by immunoglold labeling. Thin and short fimbrillae were visualized by transmission electron microscopy in only a small fraction of cells, suggesting that StcA may assemble structures that are difficult to visualize by electron microscopy.