Kinetic studies of temperature-induced helix hand inversion in Bacillus subtilis macrofibers.

The inversion of Bacillus subtilis macrofibers from right to left handedness induced by a temperature upshift was compared with inversion from left to right handedness induced by a temperature downshift. Following an upshift the new steady-state growth rate was achieved prior to inversion of helix orientation. There was no discernible perturbation of growth rate at the time of inversion. The time required after a temperature shift up or down for fiber rotation in the original sense to cease was dependent on the temperature to which the fibers were transferred and was always shortest when this temperature was highest. The results suggest a basic asymmetry in the two inversion processes. Cessation of rotation in the right-to-left inversion appeared to reflect contributions of the old and new wall materials that depended on their twist values, whereas the left-to-right inversion appeared to require that a specific amount of newly made wall material be inserted into the cell surface. The degree of twist of the newly inserted right-handed material appeared not to influence the timing of inversion.     

Effect of cortisol on the synthesis of lamellar body glycerophospholipids in fetal rabbit lung tissue in vitro

The effect of cortisol on the rate of choline incorporation into tissue phosphatidylcholine was investigated in lung explants from fetal rabbits of 19-28 days gestational age. The explants were incubated in medium with or without fetal calf serum for up to 7 days. When lung tissues were incubated in serum-free medium, a stimulatory effect of cortisol on tissue phosphatidylcholine synthesis was found in explants from 21-, 24-, 26- and 28-day fetal rabbits; a stimulatory effect of cortisol was observed in 19-day fetal lung explants only if fetal calf serum was present in the culture medium. To assess directly the effect of cortisol on the synthesis of lamellar body phosphatidylcholine, choline incorporation into phosphatidylcholine associated with a purified lamellar body fraction isolated from lung explants of 21- and 28-day fetal rabbits was also investigated. Cortisol caused a marked stimulation of synthesis and accumulation of lamellar body phosphatidylcholine in lung explants from both 21- and 28-day fetal rabbits. The magnitude of the stimulatory effect of cortisol on the rate of synthesis of lamellar body phosphatidylcholine was always greater than the effect of cortisol on the rate of choline incorporation into lipids of tissue homogenates. The relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol were also significantly altered in lung explants from 21- and 28-day fetal rabbits by cortisol treatment. Lamellar bodies that were formed initially in the fetal lung explants were enriched in phosphatidylcholine and phosphatidylinositol and had a relatively low phosphatidylglycerol content. With cortisol treatment there was a decrease in the relative rate of synthesis of lamellar body phosphatidylinositol and an increase in the relative rate of synthesis of phosphatidylglycerol. The stimulatory effect of cortisol on the synthesis of lamellar body phosphatidylcholine was observed at an earlier time-point of incubation than was the effect of cortisol on the relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol. The temporal sequence of the cortisol-induced changes in the synthesis of lamellar body glycerophospholipids, therefore, reflects that which occurs with maturation in vivo.     

The shapes of the motor domains of two oppositely directed microtubule motors, ncd and kinesin: a neutron scattering study.

The shapes of the motor domains of kinesin and ncd, which move in opposite directions along microtubules, have been investigated. Using proteins expressed in Escherichia coli, it was found that at high salt (> 200 mM) Drosophila ncd motor domain (R335-K700) and human kinesin motor domain (M1-E349) were both sufficiently monomeric to allow an accurate determination of their radii of gyration (Rg) and their molecular weights. The measured Rg values of the ncd and kinesin motor domains in D2O were 2.06 +/- 0.06 and 2.05 +/- 0.04 nm, respectively, and the molecular weights were consistent with those computed from the amino acid compositions. Fitting of the scattering curves to approximately 3.5 nm resolution showed that the ncd and kinesin motor domains can be described adequately by triaxial ellipsoids having half-axes of 1.42 +/- 0.38, 2.24 +/- 0.44, and 3.65 +/- 0.22 nm, and half-axes of 1.52 +/- 0.23, 2.00 +/- 0.25, and 3.73 +/- 0.10 nm, respectively. Both motor domains are described adequately as somewhat flattened prolate ellipsoids with a maximum dimension of approximately 7.5 nm. Thus, it appears that the overall shapes of these motor domains are not the major determinants of the directionality of their movement along microtubules.     

Deoxyribonucleic Acid Distribution in Bacillus subtilis Independent of Cell Elongation

A temperature-sensitive DNA(-) mutant of Bacillus subtilis has been studied during the resumption of deoxyribonucleic acid (DNA) synthesis following a 45 to 30 C temperature shift. For several hours after return to 30 C, DNA synthesis proceeds although the cells fail to elongate appreciably. Autoradiographs of cell populations synthesizing DNA during the recovery period demonstrate that DNA can become distributed to previously unoccupied regions along the cell length. By varying the labeling regime, newly synthesized DNA as well as DNA present at the time of transfer from 45 to 30 C were followed independently. Measurements of the percent of cell length covered by grains ((3)H-thymine in DNA) demonstrate the progressive refilling of DNA-vacant cell regions by both newly synthesized and original DNA. These data indicate that cell surface growth is not an absolute requirement for segregation of bacterial DNA.     

Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid

Bacillus subtilis 168ts-200B is a temperature-sensitive mutant of B. subtilis 168 which grows as rods at 30 C but as irregular spheres at 45 C. Growth at the nonpermissive temperature resulted in a deficiency of teichoic acid in the cell wall. A decrease in teichoic acid synthesis coupled with the rapid turnover of this polymer led to a progressive loss until less than 20% of the level found in wild-type rods remained in spheres. Extracts of cells grown at 45 C contained amounts of the enzymes involved in the biosynthesis and glucosylation of teichoic acids that were equal to or greater than those found in normal rods. Cell walls of the spheres were deficient also in the endogenous autolytic enzyme (N-acyl muramyl-l-alanine amidase). Genetic analysis of the mutant by PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation demonstrated that the lesion responsible for these effects (tag-1) is tightly linked to the genes which regulate the glucosylation of teichoic acid in the mid-portion of the chromosome of B. subtilis.