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Biomedical subjects

A L Koch

Publications and source records attributed to A L Koch.

At least 91 records · Page 5Linked to original sources
PubMed1983

The protein burden of lac operon products.

A new approach to measuring the slowing of growth due to the manufacture of proteins not needed by a bacterium is presented. An entire single colony of Escherichia coli was used to start a chemostat culture that was then given a selective pressure by the addition of phenylgalactoside (phi-gal). This enriched the population for constitutive mutants that produced beta-galactosidase without induction and could split phi-gal, consume the galactose, and grow faster. When the phi-gal was removed, the constitutives grew slower than the parental strain and were gradually lost. This procedure allows competition experiments to be carried out with minimum effects due to genetic drift. Experiments with both strains having wild-type and mutant permease genes were conducted. With the former the selective disadvantage was initially much greater than expected from the simplest hypothesis that extra unused proteins would slow growth in proportion to their fraction of the total protein synthesis. This phase was followed by a second phase where the selective disadvantage was smaller than predicted by this simple hypothesis. With a very slowly reverting permease negative strain the selective disadvantage, and therefore the protein burden, was found to be much smaller and not statistically different from zero. Thus, while one would expect under carbon and energy limitation in the chemostat the protein burden to be larger than under unlimited conditions, it is so small that even the refined technique used here could not measure it accurately. It is certainly less than the fraction of 'waste' protein synthesis; but it could be between zero and the fraction of the cells' energy and carbon budget spent on manufacture of the proteins of the lac operon.

Bacterial Proteins↗
PubMed1983

The surface stress theory of microbial morphogenesis.

From the physics of the situation, one might conclude that the osmotic pressure within most prokaryotes creates a sufficiently high tension in the wall that organisms are at risk of ripping themselves apart. The Surface Stress Theory holds that they avoid this, and are able to carry out certain morphogenetic processes by linking the cleavages of appropriate bonds to enzymes that are sensitive to the stress in the bonds under attack. This tends to maintain the internal pressure and couples wall growth to cytoplasmic growth. Mechanisms with widely different geometry function for different organisms, but they have in common the requirement that new murein be covalently linked, and usually in an unextended conformation. Organisms differ in the site of wall addition and site of cleavage. In the Gram-positive Streptococcus, septum formation, and septal splitting occurs with little stretching of the unsplit septum. In Gram-positive bacilli, the cylinder grows by the inside-to-outside mechanism, and the poles appear to be formed by a split-and-stretch mechanism. Gram-negative rods, with their much thinner wall, resist a spherical shape and are capable of cell division by altering the biochemical mechanism so that initially one-third to one-fifth of the pressure-volume work required to increase the area of the side wall is needed to increase that in a developing pole. The growth of hyphae is a separate case; it requires that much less work is needed to force growth of the apex relative to the side wall. Some other bacterial shapes also can be explained by the theory. But at present, it is only a theory, although it is gradually becoming capable of accounting for current observations in detail. Its importance is that it prescribes many experiments that now need to be done.

Bacillus↗
PubMed1983

Cell wall-DNA association in Bacillus subtilis.

Autolysis of cell walls of Bacillus subtilis 168 resulted in solubilization of wall-associated DNA. Most of the DNA was solubilized only in the later stages of autolysis. Solubilization of up to 70% of the wall by autolysins resulted in only 25 to 30% solubilization of wall-associated DNA. When the wall fragments remaining after 70% autolysis were analyzed by electron microscopy, it was observed that the preparations were highly enriched for completed septa, or poles. Partial autolysis at pH 5.2 or pH 8.6, both of which reflect hydrogen ion levels that permit either N-acetylglucosaminidase or N-acetylmuramyl-L-alanine amidase, but not both, to act, gave rise to enrichment of cell poles. When walls were incubated with subtilisin, DNase, or RNase, release of DNA (or DNA fragments) was accelerated. Density gradient centrifugation patterns of lysates of cells pulse-labeled with N-[3H]acetylglucosamine and then chased revealed that a small, but significant, proportion of the radioactivity sedimented to a density position equivalent to that of DNA-membrane complexes. Because the pulse-chase sequence enriched for radioactivity in cell poles, the results suggest that at least some molecules from polar cell walls have an affinity for DNA-membrane complexes. We suggest that DNA binds strongly, possibly via a DNA-membrane complex, to cell poles of B. subtilis. The results provide support for a view offered previously (Koch et al., FEMS Microbiol. Lett. 12:201-208, 1981) that some special structure in or very near the poles of gram-positive bacilli is involved in the segregation of DNA during cell division.

Bacillus subtilis↗
PubMed1982

How close to the theoretical diffusion limit do bacterial uptake systems function?

Using a 10 cm flow-through cuvette in a high precision spectrophotometer linked to a mini-computer, the growth rate dependence of Escherichia coli on glucose concentration has been studied. The specific growth rate vs bacterial mass of single cultures consuming small amounts of glucose was followed. The data were analyzed with the computer programs described previously. For neither batch nor chemostat-cultured organisms did growth follow the Monod growth law. Rather, the growth rate vs residual glucose concentration has an almost abrupt change in slope, indicative of a passive diffusion barrier prior to an uptake system possessing hyperbolic dependency. Calculations showed that the diffusion through the outer membrane via the porin channels could quantitatively account for the deviations from hyperbolic dependency. Long term chemostat culture alters the bacteria so that the maximum specific growth rate is reduced, but the initial dependence on glucose concentration is increased approaching more closely the theoretical limit. Therefore there was both a change inthe outer membrane channels and the uptake activity of the cytoplasmic membrane.

Biological Transport↗
PubMed1982

Spatial resolution of autoradiograms of rod-shaped organisms.

Isotope-containing rod-shaped bacteria approximate a line-segment source for purposes of autoradiography. This is because the width is small and bacteria are usually cylindrically symmetric. For this reason published work has only classified the silver grains longitudinally, independent of the transverse position. While the theoretical integral distribution from a variety of geometric shapes has been calculated, the line-segment has been overlooked because long narrow objects well separated from other sources occur infrequently in eukaryotic biology. This oversight is corrected here. In addition, a simple program for line-segments and ways to combine distributions from separate line-segment sources is presented. Some typical distributions are depicted. The program is used elsewhere in the analysis of wall growth patterns of bacteria.

Autoradiography↗
PubMed1982

Cell cycle dynamics inferred from the static properties of cells in balanced growth.

The duration of a morphological phase of the cell cycle is reflected in the steady state distribution of the sizes of cells in that phase. Relationships presented here provide a method for estimating the timing and variability of any cell cycle phase. It is shown that the mean size of cells initiating and finishing any phase can be estimated from (1) the frequency of cells exhibiting the distinguishing morphological or autoradiographic features of the phase; (2) the mean size of cells in the phase; and (3) their coefficient of variation. The calculations are based on a submodel of the Koch-Schaechter Growth Controlled Model which assumes that (i) the distribution of division sizes is Gaussian; (ii) there is no correlation in division sizes between successive generations; and (iii) every cell division gives rise to two daughter cells of equal size. The calculations should be useful for a wider range of models, however, because the extrapolation factors are not sensitive to the chosen model. Criteria are proposed to allow the user to check the method's applicability for any experimental case. The method also provides a more efficient test of the dependence of growth on cell size than does the Collins-Richmond method. This is because the method uses the mean and coefficient of variation of the size of the total population, in conjunction with those of the cells in a final phase of the cell cycle, to test potential growth laws. For Escherichia coli populations studied by electron microscopy, an exponential growth model provided much better agreement than did a linear growth model. The computer simulations were used to generate rules for three types of cell phases: those that end at cell division, those that start at cell division, and those totally contained within a single cell cycle. For the last type, additional criteria are proposed to establish if the phase is well enough contained for the formulae and graphs to be used. The most useful rule emerging from these computer studies is that the fraction of the cell cycle time occupied by a phase is the product of the frequency of the phase and the ratio of the mean size of cells in that phase to the mean size of all cells in the population. A further advantage of the techniques presented here is that they use the 'extant' distributions that were actually measured, and not hypothesized distributions nor the special distributions needed for Collins-Richmond method that can only be calculated from the observed distributions of dividing or newborn cells on the basis of an assumed growth law.

Bacteria↗
PubMed1982

Incorporation of diaminopimelic acid into the old poles of Escherichia coli.

The surface stress theory of the ontogeny of the bacterial rod depends critically on whether the old poles continue to incorporate new material into the stress-bearing murein. If insertion of peptidoglycan continues, then seemingly the shape must become gradually rounder due to the surface stress resulting from the internal hydrostatic pressure. We have reanalysed our earlier experimental data by classifying grains with respect to distance from the nearest pole, and not from the cell centre as was done previously, and conclude that old poles do incorporate new diaminopimelic acid residues. This eliminates the model we have proposed for Gram-positive rods, which assumed diffuse growth on the cylindrical sides and that poles once formed would be rigid. The new results are consistent with another model (presented elsewhere) in which insertion of new murein occurs all over the surface, although not equally. This new model leads to elongation and division if the energetics of wall expansion is altered by the cell in a control region at a particular point of the cycle by the cell.

Amino Acids, Diamino↗
PubMed1982

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.

Bacillus subtilis↗
PubMed1982

Estimation of the most probable number with a programable pocket calculator.

The most-probable-number method has many potential applications, particularly if many tubes per dilution and many dilution levels are used. Increasing the number of cultures is possible with modern automatic and semiautomatic equipment. However, available tables are not sufficiently detailed to handle data from a large number of culture tubes used in an assay. This paper provides a computer program capable of handling the necessary arithmetic and written for a hand-held, advanced programable calculator.

Bacteria↗
PubMed1981

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.

Cell Division↗
PubMed1980

The inefficiency of ribosomes functioning in Escherichia coli growing at moderate rates.

It is generally agreed that ribosomes function with reduced efficiency (i.e. a smaller proportion is actually engaged in protein synthesis) in bacteria growing at low growth rates (doubling times greater than 2 h). This paper examines whether the efficiency is constant in bacteria growing at various rates corresponding to doubling times of less than 2 h. Because isotopic methods cannot be used in very rich media, turbidimetric methods have been extended to follow the kinetics of growth immediately following the shift-up of cultures of Escherichia coli ML308 growing in glucose minimal medium or succinate minimal medium into a very rich medium supporting a balanced doubling time of 17.4 min. It is concluded that the efficiency of ribosome participation in protein synthesis is higher in the very rich medium than in the two minimal media, which support doubling times of 43 and 65 min, respectively, at 37 degrees C.

Bacterial Proteins↗