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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.The bacterial cell wall has a substantial mechanical role in addition to other functions. It must be strong enough to protect the cytoplasmic membrane from forces originating outside of the cell and to stabilize it against turgor. The wall must be stiff enough to maintain cell shape but ductile enough to allow growth. It must also be elastic enough to recover from environmentally induced changes. In vivo the wall is under stress and is also stretched (7,17). Evidence for this has been obtained in several ways, including subjecting cells to electrochemical changes and osmotic shocks (1, 3, 5, 9, 18), but until recently estimates of cell wall mechanical properties have been largely qualitative. To understand how bacteria maintain their characteristic shape during growth and even during division, for example, to understand why rod shape is so stable, an investigation of the states of stress and deformation in the cell wall is necessary. For this, quantitative estimates of mechanical properties are required, but measurement, even indirectly, is difficult and often impossible in normal cultures. Bacterial thread is a fibrillar fiber consisting of many cellular filaments which lie parallel to the fiber axis and which adhere together very strongly. A thread contains hundreds of millions of highly aligned cells but closely resembles a textile fiber (whereas a macrofiber resem...

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 ='40 left turns per mm to tight right-handed structures with twist equal to 57 right turns per mm. In the lyticdeficient strain FJ7, twist varies as a function of growth temperature above or below 39°C, where there is zero twist. The relationship between the temperature (below 390C) at which right-hand structures are produced to the time it takes for them to begin the inversion process in which they become lefthanded following transfer to 480C 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 =-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.The shape deformation responsible for the production of helical macrofibers of Bacillus subtilis has been interpreted as a ramification of the organization of the cell wall and the interplay of forces in the cell wall associated with growth (1, 2). Key features of macrofiber behavior are failure of cell separation leading to formation of cellular filaments, twisting about the cylindrical axis of the cells during growth, whiplike motions of filaments and subsequently fibers, which eventually cause a given fiber to touch itself, and folding of the structure by the twisting together of the two arms so created. The folding process is repeated numerous times resulting in the construction of a macrofiber in which all of the cellular filaments are packed into an organized structure of the same helix hand (3).Macrofibers can exist as either right-or left-handed structures (2). In this communication we report that a spectrum of macrofiber states exists in which the twist ranges from rightthrough neutral to left-handed. For any given strain, the position in the spectrum depends on the growth medium, the concentration of divalent cations, and temperature, but for any set of conditions the structures grow with constant twist. All cells within a given structure must therefore assemble their walls following the same rules of geometry.In some strains macrofibers can be made to invert their helix hand (4). We report here the kinetics of temperatureinduced inversion in one of themn. The twist of macrofibers can also be changed as the result of attack by enzymes. The cell wall of B. subtilis consists primarily of two polymers: peptidoglycan and teichoic acid (5). In this paper we show that cleavage by lysozyme of the peptidoglycan backbone in live macr...

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 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.A fundamental understanding of the relationship between growth and form in bacterial cells requires, in addition to details concerning genetic and physiological regulation of the cell cycle, knowledge of the mechanical properties of cells. The interplay of forces within the cell wall is of particular importance; consequently, the material properties of the wall must be known. Such information is difficult to obtain from individual cells because of their minute dimensions. Nevertheless, it has been shown that bacterial cells can be stretched appreciably (1) and that the strength-bearing wall polymer, peptidoglycan, is elastic (2, 3). The role of stress in the cell surface also has been recognized as a factor in cell-shape determination (4, 5).Direct measurement of the mechanical properties of bacterial cells has hitherto proved impossible. To circumvent this difficulty, we have developed a means to produce thread-like structures of Bacillus subtilis that are suitable for direct measurement by techniques used in the study of textile threads. This communication describes the construction, structure, and properties of bacterial thread, and the results of initial studies of their mechanics.Basically, the cell walls behave as viscoelastic materials. Both the initial elastic modulus and the breaking stress are highly dependent upon the degree of hydration of the bacterial thread. The strength of cell walls determined in this manner places limitations on the degree of internal osmotic pressure that can be withstood by growing cells. Permissible values are appreciably lower than those currently accepted on the basis of indirect measurements. Similarly, the electrostatic structure of cell walls must be compatible with the strength of wall material as measured in bacterial thread. The results indicate that nearly all of the cell-wall teichoic acid phosphates must be neutralized in order to maintain cell wall integrity. Therefore, bacterial thread appears to provide a unique way in which to study the mechanics of bacteria. MATERIALS AND METHODSBacterial threads have so far been produced only from cultures of cell-separation-suppressed (lyt) mutants of the 168 strain ...

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