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...

The steady-state twist of Bacillus subtilis macrofibers produced by growth in complex medium was found to vary as a function of the magnesium and ammonium concentrations. Four categories of macrofiber-producing strains that differed in their response to temperature regulation of twist were studied. Macrofibers were cultured in the complex medium TB used in previous experiments and in two derivative media, T (consisting of Bacto Tryptose), in which most strains produced left-handed structures, and Be (consisting of Bacto Beef Extract), in which right-handed macrofibers arose. In nearly all cases, increasing concentrations of magnesium led to the production of macrofibers with greater right-handed twist. Some strains unable to form right-handed structures as a function of temperature could be made to do so by the addition of magnesium. Inversion from right-to left-handedness in strain FJ7 induced by temperature shift-up was blocked by the addition of magnesium. The presence of magnesium during a high-temperature pulse did not block the establishment of "memory," although it delayed the initiation of the transient inversion following return to low temperature. The twist state of macrofibers grown without a magnesium supplement was not instantaneously affected by the addition of magnesium. Such fibers were, however, protected from lysozyme attack and associated relaxation motions. Lysozyme degradation of purified cell walls (both intact and lacking teichoic acid) was also blocked by the addition of magnesium. Ammonium ions influenced macrofiber twist development towards the left-hand end of the twist spectrum. Macrofiber twist produced in mixtures of magnesium and ammonium was strain and medium dependent. FJ7 was much more responsive to ammonium than magnesium ions, producing left-handed structures over most of the interaction matrix. In contrast, strain 734 was more responsive to magnesium in medium T, producing right-handed structures over most of the matrix, but in medium Be responded preferentially to ammonium ions and produced left-handed structures over most of the matrix. The results suggest that charge interactions at the time of cell wall assembly influence the conformational states of cell wall polymers and consequently affect the geometry of growth as well as cell shape determination.

~~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. The difference in the rates of protoplast formation indicates asymmetry in the overall rate of cleavage by lysozyme which may reflect the activity of left-twist protein(s). I N T R O D U C T I O NThe spectrum of possible twists in macrofibres of Bacillus subtilis strain FJ7, from tight righthanded through neutral to tight left-handed, indicates a range of states of differentiation, all of which can be achieved by the same starting cell. We have begun to explore the major factors responsible for the determination and maintenance of the macrofibre twist state and have identified five : (i) a biomechanical factor, (ii) the peptidoglycan state, (iii) left-twist protein(s), (iv) electrostatic interactions in the cell wall and (v) water effects in terms of hydrophobic/hydrophilic interactions of cell wall polymers . We believe that understanding the factors that govern helical growth in the macrofibre system will also provide insight into those governing normal cell growth, shape determination and maintenance.In previous work (Mendelson et al., 1984) we presented evidence that macrofibre geometry reflects individual cell wall organization in an amplified form, and we have argued that because of this relationship it is possible to use the macrofibre system to explore properties of the cell wall. We have suggested that the cell wall conformation can vary over a spectr...

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