Revealing the ultrastructure of the membrane pores of intact Serratia marcescens cells by atomic force microscopy

Abstract

This study aimed to characterize the surface ultrastructure of intact Serratia marcescens cells under physiological conditions. Topographic information of membrane pores of the cells was obtained by atomic force microscope (AFM). Three types of membrane pores (CH-1-Pore A, CH-1-Pore B and CH-1-Pore C) were observed and the spatial arrangements of membrane-spanning subunits in membranes were defined. High-resolution images revealed that the doughnut-shaped structures of CH-1-Pore A and CH-1-Pore B were composed of six-to-eight and four transmembrane subunits. The inverted teepee-shaped structure of CH-1-Pore C was segmented into two transmembrane subunits straddling a single funnel-like pore. This study, to the best of authors' knowledge, represents the first direct characterization of the surface ultrastructure of the membrane pores of Serratia marcescens CH-1 cells at the nanometer scale and offers new prospects of mapping membrane pores on intact prokaryotic cells.

Keywords: Atomic force microscopy; Biomechanics; Biomedical engineering; Cell biology; Medical imaging; Microbiology; Serratia marcescens; Surface membrane pore; Transmembrane subunit channel.

Fig. 1

(A) An SEM image of the S. marcescens CH-1 cells. (B–E) Three-dimensional AFM topographic images of the S. marcescens CH-1 cells adsorbed on poly-L-lysine-coated mica. The AFM scanning areas are 10 × 10 (B), 3 × 3 (C), 1.5 × 1.5 (D), and 0.5 × 0.5 (E) μm² for the low- and higher-resolution images, respectively. Images acquired by zooming into the boxed areas of Fig. 1B, C and D are displayed in Fig. 1C, D and E, respectively. The higher-resolution image (E) shows that many protrusions like mountain ranges and hills exist on the surface and (F) the corresponding cursor profile reveals the presence of an individual membrane pore.

Fig. 2

Exemplary imaging of the first type of membrane pore CH-1-Pore A observed with AFM. (A) and (B) Two- and three-dimensional top view of an individual pore in the outer membrane of S. Maccensis from the extracellular side. (C) A contour view of Fig. 2B. (D) View from within the membrane with the extracellular region above and the intracellular region below. (E–H) (depicted from red lines in Fig. 2C). Profiles of the internal canal indicating the measurable inner diameters. The doughnut-like structure displayed in Fig. 2B is segmented into six to eight subunits. The blue dot line indicates the approximately surface height (R_mean = 9.95 nM) of the outer membrane.

Fig. 3

Exemplary imaging of the second type of membrane pore CH-1-Pore B. (A) Three-dimensional top view of an individual pore in the outer membrane of S. Maccensis from the extracellular side. (B) A contour view of B. (C–E) (red lines in B) Profiles of the internal canal indicating the measurable inner diameters. The doughnut-like structure is apparently segmented into four subunits. The blue dot line indicates the approximately surface height (R_mean = 4.72 nM) of the outer membrane.

Fig. 4

Exemplary AFM imaging of the third type of membrane pore CH-1-Pore C. (A) Three-dimensional top view of an individual pore in the outer membrane of S. Maccensis from the extracellular side. (B) A contour view of B. (C–D) (see red lines in B) Profiles of the internal canal indicating the measurable inner diameters. The inverted teepee-shaped structure is apparently segmented into two subunits. The blue dot line indicates the approximately surface height (R_mean = 6.23 nM) of the outer membrane.