Ultrastructural analysis of the rugose cell envelope of a member of the Pasteurellaceae family

Abstract

Bacterial membranes serve as selective environmental barriers and contain determinants required for bacterial colonization and survival. Cell envelopes of Gram-negative bacteria consist of an outer and an inner membrane separated by a periplasmic space. Most Gram-negative bacteria display a smooth outer surface (e.g., Enterobacteriaceae), whereas members of the Pasteurellaceae and Moraxellaceae families show convoluted surfaces. Aggregatibacter actinomycetemcomitans, an oral pathogen representative of the Pasteurellaceae family, displays a convoluted membrane morphology. This phenotype is associated with the presence of morphogenesis protein C (MorC). Inactivation of the morC gene results in a smooth membrane appearance when visualized by two-dimensional (2D) electron microscopy. In this study, 3D electron microscopy and atomic force microscopy of whole-mount bacterial preparations as well as 3D electron microscopy of ultrathin sections of high-pressure frozen and freeze-substituted specimens were used to characterize the membranes of both wild-type and morC mutant strains of A. actinomycetemcomitans. Our results show that the mutant strain contains fewer convolutions than the wild-type bacterium, which exhibits a higher curvature of the outer membrane and a periplasmic space with 2-fold larger volume/area ratio than the mutant bacterium. The inner membrane of both strains has a smooth appearance and shows connections with the outer membrane, as revealed by visualization and segmentation of 3D tomograms. The present studies and the availability of genetically modified organisms with altered outer membrane morphology make A. actinomycetemcomitans a model organism for examining membrane remodeling and its implications in antibiotic resistance and virulence in the Pasteurellaceae and Moraxellaceae bacterial families.

Fig 1

Transmission electron micrographs of whole-mount A. actinomycetemcomitans stained with Nano-W at low (top row) and high (bottom row) magnifications. (A and D) Wild-type strain; (B and E) morC mutant strain; (C and F) complemented strain. Black arrows indicate the location of EmaA adhesins easily recognizable by their characteristic shape (see, for example, reference 22) and black arrowheads show convolutions on the surface of the bacterium. White arrows point to the regions of the low-magnification images (top row) that have been imaged at a higher magnification (bottom row). Bar, 100 nm.

Fig 2

Transmission electron micrographs of whole-mount A. actinomycetemcomitans stained with Nano-W. (A) Wild-type strain; (B) morC mutant strain; (C) complemented strain. Arrows indicate the location of outer membrane vesicles. Bar, 100 nm.

Fig 3

3D tomograms of A. actinomycetemcomitans strains acquired from whole-mount preparations of the bacteria viewed in perspective along an axis 20° off the imaging axis. (A) Wild-type strain; (B) morC mutant strain. Arrows indicate grooves on the bacterial surface. Bar, 100 nm.

Fig 4

Analysis of the cell surface of A. actinomycetemcomitans strains using AFM. (A to C) The left panels show the wild-type strain, and the right panels show the morC mutant strain. (A) Topography images; (B) 3D images; (C) section profiles indicating the depth of the grooves on the bacterial surface corresponding to the black lines in the topography images. (D) Comparison of the groove depth between wild-type and morC mutant strains measured by section profiles. The data are means ± SD of groove depth. ***, P < 0.001 versus the wild type.

Fig 5

Transmission electron micrographs of ultrathin sections from A. actinomycetemcomitans strains prepared by high-pressure freezing and freeze substitution. (A) Wild-type strain; (B) morC mutant. Arrows indicate the inner and outer membranes separated by the periplasmic space. White arrows in the middle column point to the regions of the images that have been magnified four times and are shown in the last column of each row. IM, inner membrane; OM, outer membrane. Bar, 100 nm.

Fig 6

Electron tomography and segmentation of the membrane of A. actinomycetemcomitans strains in ultrathin sections after high-pressure freezing and freeze substitution. Central slices of a tomogram from the wild-type (A) and morC mutant (C) strains and segmentation of a small area of the inner and outer membranes from the tomograms of the wild-type (B) and morC mutant (D) strains are shown. The membranes clearly separated by periplasmic space are indicated by arrows. IM, inner membrane; OM, outer membrane. Bar, 100 nm.

Fig 7

Z-slices extracted from the tomogram of the ultrathin sections after high-pressure freezing and freeze substitution of the wild-type A. actinomycetemcomitans strain showing the connection between the inner and outer membranes. White arrows indicate the connection site. Bar, 10 nm.