RESOLVING THE HIGH-RESOLUTION ARCHITECTURE, ASSEMBLY AND FUNCTIONAL REPERTOIRE OF BACTERIAL SYSTEMS BY IN VITRO ATOMIC FORCE MICROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 86

sheets) and leave other (here, perpendicular) residues or structures intact.
Identiication of the gene(s) encoding the rodlet structure and the enzymes
responsible for rodlet degradation are important areas for future research.

4.2.2 Emergence of Vegetave Cells

Etch pits were the initiation sites for early germination-induced spore coat
issure formation. During intermediate stages of germination, small spore
coat apertures developed that were up to 70 nm in depth ( Fig. 4.10a,b ) .
During late stages of germination, these apertures dilated ( Fig. 4.10c-e ) ,
allowing vegetative cell emergence (data not shown).
In vitro AFM visualization of germling emergence allowed high-resolution

visualization of nascent vegetative cell surface structure ( Fig. 4.10e-g ).
Vegetative cell wall structure could be recognized through the apertures
approximately 30-60 minutes prior to germ cell emergence. During the
release of vegetative cells from the spore integument, the entire cell surface
consisted of a porous ibrous network ( Fig. 4.10g ) .
To compare the cell wall structure of germling and mature vegetative
cells, we carried out separate experiments in which cultured vegetative B.

atrophaeus and imaged with

AFM in water. As seen in Fig. 4.10h , the cell wall of mature vegetative cells
contained a porous, ibrous structure similar to the structure observed on the
surface of germling cells ( Fig. 4.10g ) .
The bacterial cell wall consists of long chains of peptidoglycan that are
cross-linked via lexible peptide bridges. 55 While the composition and chemical
structure of the peptidoglycan layer vary among bacteria, its conserved
function is to allow bacteria to withstand high internal osmotic pressure. 55
The length of peptidoglycan strands varies from 3-10 disaccharide units in
Staphylococcus aureus

cells were adhered to a gelatin-coated surface

4

to ~100 disaccharide units in B. subtilis , with each

unit having a length and diameter of ~1 nm. The ibrous network observed

on the germ cell surface with 5-100 nm pores ( Fig. 4.10e,g ) and the ibrous
network observed on mature vegetative cells with 5-50 nm pores ( Fig. 4.10h )
appear to represent the nascent peptidoglycan architecture of newly formed
and mature cell wall, respectively, and is composed of either individual or
several intertwined peptidoglycan strands. The cell wall density of mature
cells appears to be higher with, on average, smaller pores and more ibrous
material, as compared with the germ cells. These results are consistent with
murein growth models whereby new peptidoglycan is inserted as single
strands and subsequently cross-linked with preexisting murein. 57 The AFM-
resolved pore structure of the nascent

56

germ and vegetative cell
surfaces is similar to the honeycomb structure of peptidoglycan oligomers
determined by NMR.

B. atrophaeus

55