Michael L. Higgins

Procaryotic Cell Division with Respect to Wall and Membranes

(1971). Procaryotic Cell Division with Respect to Wall and Membranes. CRC Critical Reviews in Microbiology: Vol. 1, No. 1, pp. 29-72.

Surface tension-like forces determine bacterial shapes: Streptococcus faecium.

The same tendency that causes soap bubbles to achieve a minimum surface area for the volume enclosed seems to account for many of the features of growth and division of bacteria, including both bacilli and cocci. It is only necessary to assume that growth takes place in zones and that only in these zones does the tension caused by hydrostatic pressure create the strain that forces the cell to increase the wall area. The stress developed by osmotic pressure creates strains that significantly lower the free energy of bond splitting by hydrolysis or transfer. We believe this is sufficient to make growing wall have some of the properties ordinarily associated with surface tension. The feature common to all bacterial cell wall growth is that peptidoglycan is inserted under strain-free conditions. Only after the covalent links have been formed are the intervening stressed peptide bonds cleaved so that the new unit supports the stress due to hydrostatic pressure. The present paper analyses the growth of Streptococcus faecium in these terms. This is a particularly simple case and detailed data concerning morphology are available. The best fit to the data is achieved by assuming that growth takes place in a narrow region near the splitting septum and that the septal material is already under tension as it is externalized and is twice as thick as the external wall throughout the development of the nascent poles. Constancy of the ratio of hydrostatic pressure to the effective surface tension, P/T, is also consistent with electron microscopic observations.

The role of surface stress in the morphology of microbes.

The shapes of many prokaryotes can be understood by the assumption that the cell wall expands in response to tension created by the osmotically derived hydrostatic pressure. Different organisms have different shapes because wall growth takes place in different regions. A previous paper (Koch et al., 1981 a) considered the simplest case of prokaryotic growth, i.e. that of Streptococcus faecium. In the present paper, an elaboration of this theory is applied to two further cases - the more perfectly spherical cocci and the rod-shaped bacteria. These cases are more complex mathematically, because growth over a considerable fraction of the surface must be considered. Such diffuse growth cannot be treated analytically, but can be simulated on a computer or handled by geometric arguments. The spherical form of the cocci may result from either diffuse growth over their entire external surface, or from zonal growth in which the addition of new material only occurs in the immediate vicinity of the splitting septum. In the zonal model, it must be assumed that the least amount of previously laid down septal peptidoglycan consistent with wall growth is reworked in the formation of the new external wall. For Gram-positive rods, where the body of the rod is truly cylindrical, three kinds of growth zones are required: (1) the inward edge of the ingrowing septum, (2) the junction of septum and nascent pole, and (3) the cylindrical walls. Two modes for cylindrical elongation ara possible: (a) new wall is added in one or a few narrow annular zones, or (b) new wall material is added continuously all over the innermost surface and the outer layer is degraded. It is shown that the latter case applies to Bacillus subtilis. Also summarized in this paper are results, developed in more detail elsewhere, concerning the morphology of fusiform bacteria, Gram-negative rods and the hyphal tips of fungi.

PROBLEMS OF CELL WALL AND MEMBRANE GROWTH, ENLARGEMENT, AND DIVISION*

Not many years ago, growth and division of bacteria was thought of as a rather casual process. Cells merely increased in size and, after a more or less appropriate increase, divided into two smaller units. In fact, the observations of Henrici (published in 1928) were not fully appreciated until relatively recently. In cultures of Escherichia coli, Henrici observed that cells from the lag phase were both larger on the average and less uniform in size than cells from the exponential or stationary phases of growth. More recent observations, of balanced exponentially growing cultures, showed that both cellular composition and the average size of bacterial cells are dependent on growth rate.?, Such observations served as a basis for defining some of the factors involved in the regulation of the synthesis of informational macromo1ecules.3 Later, it became apparent that the processes that lead to cell division also must be coupled to, and interrelated with, the synthesis of informational macromolec u l e ~ . ~ ~ In addition to the cell division process itself, the enlargement of the bacterial cell surface (cell wall and membrane) also must be somehow related to the synthesis of other cellular constituents. I t is necessary for the cell surface not only to expand in area to enclose an increasing volume of protoplasm but also to undergo a series of changes in shape (morphogenesis), while maintaining the osmotic integrity of the cell. As cells increase in mass and volume, all of the cellular dimensions do not increase proportionately. During balanced growth of rod-shaped species, such as E. coli, at any specific growth rate the diameter of the cylinder remains fixed,9 so that increases in cell volume can be attributed to an increase in cell length. In the process of cylindrical elongation, there is a continuous decrease in the surface area to volume ratio.lo Such potential changes in surface area to volume ratio are illustrated in FIGURE 1. The formation of new poles and cell division result in the surface area to volume ratio reverting to that at the start of the cycle. Immediate separation of newly formed cross walls, to become one of the poles of each of the daughter cells, is not, however, essential to the process. When newly divided cells remain in chains, the potential surface area of their poles remains hidden, so that the ratio of cellular surface area to volume is considerably less than that of fully separated cells. Therefore, consideration must be given not only to exposed surface area but also to total

Inactivation of mecA prevents recovery from the competent state and interferes with cell division and the partitioning of nucleoids in Bacillus subtilis.

The development of genetic competence in Bacillus subtilis requires the synthesis of ComK, a transcription factor, which is normally produced as a culture enters the stationary phase. This synthesis is known to be regulated in part by the protein MecA. Loss‐of‐function mutations in mecA result in overexpression of ComK and its appearance early during exponential growth. We show here that mecA inactivation also causes a loss of colony‐forming ability, especially during stationary phase. This loss is accompanied by the appearance of cells in which normal nucleoid separation has failed to occur. Renografin gradient fractionation of mecA cultures grown to competence reveals that nearly 100% of the cells band at the low buoyant density characteristic of competent cells, and that this low density is competence‐related. The loss of viability, the low buoyant density and the nucleoid separation defect, are all comK‐dependent. The loss of viability can be reversed by even the transient introduction of mecA+. It is proposed that these effects of ComK overexpression are related to the DNA replication arrest normally exhibited by the competent cell fraction and that MecA is needed to reverse this arrest and to permit escape from the competent state. The shift of nearly 100% of the cells to light buoyant density in a mecA mutant culture strongly suggests that the MecA protein is a regulator of the cell‐type‐specific expression of competence.

The division during bacterial sporulation is symmetrically located in Sporosarcina ureae

Immunofluorescence microscopy was used to visualize the FtsZ band that marks the site of septation in Sporosarcina ureae. Image analysis indicated that the vegetative division was symmetrically located with respect to the ends of the cells. Fusions of lacZ to the sporulation loci, spoIIA and cotE, of Bacillus subtilis were introduced into S. ureae by mobilization of plasmids containing the fusions from Escherichia coli. The fusions showed similar patterns of sporulation‐associated expression in S. ureae to those observed in B. subtilis. Formation of β‐galactosidase encoded by the spoIIA–lacZ fusion made it possible to identify early sporulating cells by immunofluorescence microscopy. Analysis of the position of FtsZ bands in cells expressing spoIIA–lacZ indicated that the location of sporulation division was symmetrical with respect to the ends of the cells, in sharp contrast to the asymmetrical location of septation in sporulating Bacilli. It is inferred that asymmetry of location of the sporulation division is not essential for the compartmentalization of gene expression that follows the division.

The putative DNA translocase SpoIIIE is required for sporulation of the symmetrically dividing coccal species Sporosarcina ureae

The spoIIIE gene of Sporosarcina ureae encodes a 780‐residue protein, showing 58% identity to the SpoIIIE protein of Bacillus subtilis, which is thought to be a DNA translocase. Expression of the S. ureae spoIIIE gene is able to restore sporulation in a B. subtilis spoIIIE mutant. Inactivation of the S. ureae spoIIIE gene blocks sporulation of S. ureae at stage III. Within the limits of detection, the sporulation division in S. ureae shows the same symmetry, or near symmetry, as the vegetative division (in contrast to the highly asymmetric location of the sporulation division for B. subtilis), and so it is inferred that SpoIIIE facilitates chromosome partitioning during sporulation, even when the division is not grossly asymmetric. It is suggested that chromosome partitioning lags behind division during sporulation but not during vegetative growth.

Inability of purified Pseudomonas aeruginosa exopolysaccharide to bind selected antibiotics.

It has been proposed that the exopolysaccharide (alginate) of mucoid Pseudomonas aeruginosa strains which infect cystic fibrosis patients might bind and hence protect this pathogen from antibiotics. To test this hypothesis, we employed equilibrium dialysis to measure the binding between several antibiotics and purified Pseudomonas alginate. Binding was calculated from the residual concentrations of antibiotics in free solution by a biological assay. The detectable binding of antibiotics to alginate was consistent with expectations; the positively charged antibiotics steptomycin and tobramycin, bound to the polyanion (0.047 and 0.024 mumol/mg of alginate, respectively), whereas the neutral species, clindamycin and penicillin, bound negligibly or not at all (0.0011 and 0 mumol/mg of alginate, respectively). When these experiments were performed in the presence of physiological concentrations of saline, none of the antibiotics bound to the polysaccharide. Since the binding observed was abrogated by salt concentrations typical of the tracheobronchial secretions of cystic fibrosis patients, the data suggest that tight binding of antibiotics to the exopolysaccharide of a mucoid P. aeruginosa strain does not provide increased antibiotic resistance.

Septal membrane fusion - a pivotal event in bacterial spore formation ?

Formation of the asymmetrically located septum divides sporulating bacilli into two distinct cells: the mother cell and the prespore. The rigidifying wall material in the septum is subsequently removed by autolysis. Examination of published electron micrographs indicates that the two septal membranes then fuse to form a single membrane. Membrane fusion would be expected to have profound consequences for subsequent development. For example, it is suggested that fusion activates processing of pro‐σE to σE in the cytoplasm by exposing it to a membrane‐bound processing enzyme. Asymmetry of the fused membrane could restrict processing to one face of the membrane and hence explain why σEthe tanscription in the mother cell but not in the prespore. Asymmetry of the fused membrane might also provide a mechanism for restricting the activity of another factor, σF to the prespore. Attachment of the flexible fused septal membrane to the condensing prespore nucleoid could help drive the engulfment of the prespore by the mother cell.

Evidence that Streptococcus mutans Constructs Its Membrane with Excess Fluidity for Survival at Suboptimal Temperatures

When cells from cultures of Streptococcus mutans strain FA-1 grown at 37 degrees C were exposed to incubation temperatures of 26 degrees C or less for 5 min or more, an extensive aggregation of particles was observed on the convex fracture faces of their freeze-cleaved membranes. Aggregation of particles was accompanied by a parallel increase in the activation energy for growth. By shifting the growth temperature from 37 to 24 degrees C for one doubling of culture mass, the transition temperature for membrane particle aggregation could be lowered from about 26 to 0 degrees C. Although membrane lipids became enriched with unsaturated fatty acids during this period of growth at 24 degrees C, this enrichment was not accompanied by an increased growth rate of the culture. However, the period of growth at 24 degrees C did result in bacteria that could grow more rapidly at 10 degrees C than could bacteria directly transferred from cultures grown at 37 degrees C. These observations suggest that the increase in membrane fluidity that occurs when bacteria are grown at 24 degrees C doses not allow bacteria to grow faster at 24 degrees C, but rather allows them to adapt more readily to further decreases in growth temperature.