Mechanics of bacterial macrofiber initiation.

The twisting and writhing during growth of single-cell filaments of Bacillus subtilis which lead to macrofiber formation was studied in both left- and right-handed forms of strains FJ7 and RHX. Filament bending, touching, and loop formation (folding), followed by winding up into a double-strand fiber, were documented. Subsequent folds that produced multistrandedness were also examined. The rate of loop rotation during winding up was measured for 26 loops from 16 clones. In most cases, the first loop formed turned at a lower rate than those produced by the following cycles of folding. The sequence of folding topologies differed in FJ7 and RHX strains and in left- versus right-handed structures. Right-handed FJ7 routinely gave rise to four-stranded helices at the second fold, whereas left-handed FJ7 and both left-handed and right-handed forms of RHX made structures with predominantly two double-stranded helical regions. Left-handed RHX structures frequently produced second folds within the initial loop itself, resulting in T- or Y-shaped fibers. Sixteen cases in which the initial touch of a filament to itself produced a loop that snapped open before it could wind up into a double-strand fiber were found. The snap motions were used to obtain estimates of the forces generated by helical growth of single filaments and to investigate theoretical models involving the material properties of cell filaments. In general, the mechanical behavior of growing single-cell filaments and fibers consisting of two-, three-, or four-strand helices was similar to that described for larger, mature, multifilament macrofibers. The behavior of multicellular macrofibers can be understood, therefore, in terms of individual cell growth.

Mechanical properties of Bacillus subtilis cell walls: effects of removing residual culture medium.

Experiments are described in which the tensile strength, the initial (Youngs') modulus, and other mechanical properties of the bacterial cell wall were obtained as functions of relative humidity (RH) in the range of 20 to 95%. These properties were deduced from tensile tests on bacterial thread, a fiber consisting of many highly aligned cells of Bacillus subtilis, from which residual culture medium had been removed by immersion in water. Reasons are given to support the idea that the mechanical properties of bacterial thread relate directly to those of the cylinder wall and that they are not influenced by septa, cytoplasm, or the thread assembly. The data show that the cell wall, like many other heteropolymers, is visco-elastic. When dry, it behaves like a glassy polymer with a tensile strength of about 300 MPa and a modulus of about 13 GPa. When wet, its behavior is more like a rubbery polymer with a tensile strength of about 13 MPa and a modulus of about 30 MPa. Thus, the cell wall is stronger than previously reported. Walls of this strength would be able to bear a turgor pressure of 2.6 MPa (about 26 atm). The dynamic behavior suggests a wide range of relaxation times. The way in which mechanical behavior depends strongly on humidity is discussed in terms of possible hydrogen bond density and the ordering of water molecules. Cell walls in threads containing residual culture medium TB are, except at low RH, 10 times more flexible and about 4 times less strong. All of their mechanical properties appear to vary with change in RH in a manner similar to those of walls from which the culture medium has been washed, but with a downshift of about 18% RH.

Mechanical properties of Bacillus subtilis cell walls: effects of ions and lysozyme.

Bacterial threads of Bacillus subtilis have been immersed in, and redrawn from, water of various pH values, in solutions of (NH4)2SO4 and NaCl of various concentrations, and in lysozyme solutions. The changes in the tensile strength, elastic modulus, and other mechanical properties of the bacterial cell wall due to these treatments were obtained. The data show that change in pH has little effect but that as the salt concentration is increased, the cell walls become more ductile. A high salt concentration (1 M NaCl) can reduce the modulus by a factor of 26 to 13.5 MPa at 81% relative humidity and the strength by a factor of only 2.5. Despite attacking the septal-wall region of the cellular filaments, lysozyme has no effect on the mechanical properties. There is no significant change in the stress relaxation behavior due to any of the treatments. The dependence of mechanical properties on the salt concentration is discussed in terms of the polyelectrolyte nature of cell walls. The evidence presented in this and the accompanying paper (J. J. Thwaites and U.C. Surana, J. Bacteriol., 173:197-203, 1991) supports the idea that the peptidoglycan in bacterial cell wall is an entanglement network with a large degree of molecular flexibility, with some order but no regular structure.

Mechanical properties of peptidoglycan as determined from bacterial thread.

Experiments are described in which the tensile strength, the extensibility and the initial Young's modulus of bacterial cell wall have been determined as functions of relative humidity in the range 11-98%. Data on stress relaxation and recovery are also given. Standard fibre-measuring technique has been used on 'bacterial thread', made from a cell-separation-suppressed mutant of Bacillus subtilis. The data show that peptidoglycan, the load bearing polymer in the cell wall, behaves very much like other viscoelastic polymers. Its mechanical behaviour when dry is that of a glassy polymer with tensile strength about 300 MPa and modulus about 20 GPa. When wet, it is weaker and much less stiff with tensile strength about 3 M Pa and modulus 10 M Pa. The relaxation data indicate a wide spectrum of relaxation times. The results are discussed in terms of the structure of peptidoglycan and its orientation in the bacterial cell wall. The way in which mechanical behaviour depends strongly on humidity is compared with that of other biopolymers in terms of possible hydrogen-bond density and the ordering of water molecules. The possibility of a well-defined glass transition is briefly examined.

Cell wall mechanical properties as measured with bacterial thread made from Bacillus subtilis.

Engineering approaches used in the study of textile fibers have been applied to the measurement of mechanical properties of bacterial cell walls by using the Bacillus subtilis bacterial thread system. Improved methods have been developed for the production of thread and for measuring its mechanical properties. The best specimens of thread produced from cultures of strain FJ7 grown in TB medium at 20 degrees C varied in diameter by a factor of 1.09 over a 30-mm thread length. The stress-strain behavior of cell walls was determined over the range of relative humidities between 11 and 98%. Measurements of over 125 specimens indicated that cell wall behaved like other viscoelastic polymers, both natural and man-made, exhibiting relaxation under constant elongation and recovery upon load removal. This kinetic behavior and also the cell wall strength depended greatly on humidity. The recovery from extension observed after loading even up to a substantial fraction of the breaking load indicated that the properties measured were those of cell wall material rather than of behavior of the thread assemblage. Control experiments showed that neither drying of thread nor the length of time it remained dry before testing influenced the mechanical properties of the cell walls. Specimens drawn from TB medium and then washed in water and redrawn were found to be stiffer and stronger than controls not washed. However, tensile properties were not changed by exposure of cells to lysozyme before thread production. This suggests that glycan backbones are not arranged along the length of the cell cylinder. The strength of the cell wall in vivo was estimated by extrapolation to 100% relative humidity to be about 3 N/mm2. Walls of this strength would be able to bear a turgor pressure of 6 atm (ca. 607.8 kPa), but if the increase in strength of water-washed threads was appropriate, the figure could be 24 atm (ca. 2,431.2 kPa).

Relaxation motions induced in Bacillus subtilis macrofibres by cleavage of peptidoglycan.

Bacillus subtilis macrofibres exposed to lysozyme underwent characteristic rotations, termed relaxation motions, in which their twist changed. Intact macrofibres and macrofibre fragments devoid of loop ends responded in the same way. Macrofibre strains for which the helix hand is temperature-dependent and also those of fixed-hand (both left and right) underwent initial relaxation motions towards the right-hand end of the twist spectrum, the only exception being those in which the initial twist state was at or near the right-hand maximum. Often when the initial relaxation motions were completed immediately before structure breakdown the macrofibres underwent one or a few rotations in the opposite direction (towards the left-hand end of the twist spectrum). Crude autolysin extract obtained from wild-type B. subtilis also caused macrofibre relaxation motions at pH 5.6 but at pH 8.0 macrofibre breakdown occurred as a result of septal cleavage. This resulted in the release of helically shaped individual cellular filaments. These findings suggest that strain in the cell wall associated with helical shape was dependent on the integrity of the glycan backbone rather than peptide cross-bridges. In contrast, cleavage of peptide cross-bridges apparently was instrumental in the cell separation process. Left- and right-hand macrofibres, when exposed to lysozyme, exhibited different rates of relaxation, breakdown of fibre structure and protoplast formation. Similarly, the rate of macrofibre breakdown during the lag between temperature shift and inversion reflected the replacement of septal wall material by that of a new conformation corresponding to the new helix hand.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Biomechanics of bacterial walls: studies of bacterial thread made from Bacillus subtilis.

Bacterial threads of up to 1 m in length have been produced from filaments of separation-suppressed mutants of Bacillus subtilis. Individual threads may contain 20,000 cellular filaments in parallel alignment. The tensile properties of bacterial threads have been examined by using conventional textile engineering techniques. The kinetics of elongation at constant load are indicative of a viscoelastic material. Both Young's modulus and breaking stress are highly dependent upon relative humidity. By extrapolation to 100% relative humidity, it appears that cell walls may be able to bear only internal osmotic pressures of about 2 atmospheres (2.03 X 105(5) Pa) in living cells. Similarly, the strength of wall material limits the amount of cell-surface charge permissible to only a small fraction of that known to be carried by the negatively charged wall polymers.

Factors contributing to helical shape determination and maintenance in Bacillus subtilis macrofibres.

Bacillus subtilis, normally a rod-shaped organism, can grow in the form of a helix with pitch ranging over a spectrum from tight right-handed to tight left-handed depending upon the growth environment and genetic composition of the strain. Five factors have been identified which contribute either to the helical shape deformation or its maintenance: 1) a biomechanical component involving blocked rotation during growth; 2) cell wall polymer conformation; 3) a protein(s) concerned with the left-hand form produced at high temperature; 4) electrostatic aspects of the cell wall; and 5) water, as it affects the mechanical properties of cell walls and the structure of cell wall polymers. The findings are compatible with a model in which the cell wall polymers are inserted in helical orientation along the cylindrical portion of the cell during growth.

Twisted states of Bacillus subtilis macrofibers reflect structural states of the cell wall.

Static and dynamic studies of helical Bacillus subtilis macrofibers reveal that a spectrum of twisted states exists ranging from tight left-handed structures with twist equal to approximately equal to 40 left turns per mm to tight right-handed structures with twist equal to 57 right turns per mm. In the lytic-deficient strain FJ7 , twist varies as a function of growth temperature above or below 39 degrees C, where there is zero twist. The relationship between the temperature (below 39 degrees C) at which right-hand structures are produced to the time it takes for them to begin the inversion process in which they become left-handed following transfer to 48 degrees C reveals that structures with less twist are more rapidly converted to left-handedness than are those with higher values of twist. The initial response of live macrofibers to digestion by lysozyme consists of "relaxation" motions in which the twist of both left- and right-handed structures changes towards the right-hand end of the spectrum. The rate of relaxation is approximately equal to 5-fold higher at the left-hand end than at the right-hand end. These findings suggest that cell wall polymers can assume a range of structural states during helical growth and that these determine the quantitative aspects of macrofiber shape as well as the sensitivity of walls to attack by lysozyme.