A Structural Framework for a Near-Minimal Form of Life: Mass and Compositional Analysis of the Helical Mollicute Spiroplasma melliferum BC3

Abstract

Spiroplasma melliferum is a wall-less bacterium with dynamic helical geometry. This organism is geometrically well defined and internally well ordered, and has an exceedingly small genome. Individual cells are chemotactic, polar, and swim actively. Their dynamic helicity can be traced at the molecular level to a highly ordered linear motor (composed essentially of the proteins fib and MreB) that is positioned on a defined helical line along the internal face of the cell’s membrane. Using an array of complementary, informationally overlapping approaches, we have taken advantage of this uniquely simple, near-minimal life-form and its helical geometry to analyze the copy numbers of Spiroplasma’s essential parts, as well as to elucidate how these components are spatially organized to subserve the whole living cell. Scanning transmission electron microscopy (STEM) was used to measure the mass-per-length and mass-per-area of whole cells, membrane fractions, intact cytoskeletons and cytoskeletal components. These local data were fit into whole-cell geometric parameters determined by a variety of light microscopy modalities. Hydrodynamic data obtained by analytical ultracentrifugation allowed computation of the hydration state of whole living cells, for which the relative amounts of protein, lipid, carbohydrate, DNA, and RNA were also estimated analytically. Finally, ribosome and RNA content, genome size and gene expression were also estimated (using stereology, spectroscopy and 2D-gel analysis, respectively). Taken together, the results provide a general framework for a minimal inventory and arrangement of the major cellular components needed to support life.

Variable Field Analytical Ultracentrifugation: II. Gravitational Sweep Sedimentation Velocity

“…Within the existing initial delay for the onset of scanning (caused by delay calibrations and/or requirement for optical adjustments) on the order of a few minutes, this limits observations of particles to those sedimenting more slowly than a few hundred S. With the lowest limit for detectable sedimentation coefficients being~0.1 S, the standard experiment therefore spans~3 decades of s values. On the other extreme, if a very low rotor speed of 3000 rpm is used in a conventional constant-field experiment (cyan), very large s values of~10,000 S and higher can be detected (30), but the lower limit is~100 S.…”

Dynamics of a Protein Chain Motor Driving Helical Bacteria under Stress

“…The mean diameter of Spiroplasma's helical center line d hd t ¼ 2R h z 370 nm, the diameter of the helix d h ¼ 570 nm, the cell tube diameter d t z 190 nm, and the helical pitch p ¼ k h /2p z 900 nm (see Fig. 1 A) were estimated by others though analysis of dark field images (30). The initials in c gs (x) stand for the ground state (gs) of the mechanical energy.…”

“…They are obtained by a single scan along x, as presented at the top of Fig. 1 A, showing a measured mean diameter of the cell's centerline (30). To analyze the deformations of the cell caused by its linear chain motor, we plot the temporal course of the shape signals c y (x L (t)) and c z (x L (t)) in kymographs.…”

“…However, the average initial repetition time of 1/R cyc z (4 Hz) À1 ¼ 0.25 s cannot be explained by the protein switching times ð1=k 0 Þexpð1=2bG A Þ z 0.09 s predicted by our model without considering the fluctuationlimited or diffusion-limited binding of the ligand to the first switching protein. Using a diffusion coefficient D ¼ 150 mm 2 /s for a ligand such as ATP in the cytoplasm (49) and an average ATP concentration of c lig z 700 ATP/mm 3 deduced from data found in the literature (30,46,50), the mean free passage time t Diff ðc lig Þ ¼ c À2=3 lig ,1=2D would be less than 1 ms. Remarkably, this would lead to an ATP concentration in Spiroplasma that is approximately two magnitudes lower than that in E. coli bacteria (51).…”

Sedimentation coefficient distributions of large particles

“…In the present work we recapitulate the theoretical relationships underpinning the different differential and integral approaches for determining sedimentation coefficient distributions of particles with negligible diffusion from experimental sedimentation data. These are of interest for the characterization by AUC of large particles including protein complexes, 3,4 fibrils, 5,6 viral particles, 7,8 entire organisms, 9 , nanoparticles such as carbon nanotubes, 10–12 nanocrystals, 13 gold nanoparticles and their conjugates, 14 quantum dots, 15 and other colloids, 16 both in research and in regulatory environment. 17,18 Furthermore, sedimentation coefficient distributions have been the basis for determining molar mass distributions of such particles, 19,20 and apparent sedimentation coefficient distributions of diffusing species have been used for estimating molar mass 21–23 and assess protein conformational changes.…”