A microbial model for visualization of surface proteins using cryoimmobilization and high-resolution cryo-SEM

Abstract

Numerous studies on high resolution visualization of surface membrane proteins have utilized colloidal gold markers in conjunction with SEM, TEM, and by freeze-fracture cytochemistry using conventional or fracture flip methods, but in each case only information on the distribution of the marker complex, and not the actual structure of individual surface protein(s) was obtained.To investigate the detection of individual surface proteins, we have used a microbial model to examine surface proteins involved in conjugation or the transfer of DNA inEnterococcus faecalis. Two surface proteins encoded by the pCF10 plasmid, aggregation substance (Asc10; globular shape) and surface exclusion protein (Sec10; helical shape), mediate the aggregation of donor and recipient cells and prevent aggregation of like donor cells, respectively. Isogenic strains of E. faecalis that constitutively express neither Asc10 or Sec10 (pWM401) or both (pINY1801) were processed by 1) conventional chemical fixation, dehydration, critical point drying with CO2, ion beam sputtering with a discontinuous platinum film (<1 nm), and examination in a Hitachi S-900 FESEM operated at low voltage (<3 keV), or 2) cryoimmobilization of cells by high pressure freezing (Balzers HPM 010), mounting in a Gatan cryoholder followed by etching for 60 minutes at −100°C, cryocoating by planar magnetron sputtering (Balzers MED 010), and examination at −100°C in the S-900 FESEM.