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A L Koch

Publications and source records attributed to A L Koch.

At least 37 records · Page 2Linked to original sources
PubMed1996

What size should a bacterium be? A question of scale.

There are living prokaryotes (Bacteria and Archaea) that have cell sizes that range from 0.02-400 microns3. Over this tremendous range, various abilities to cope with the environment are needed. This review attempts to formulate some of the problems and some of the solutions. The smallest size for a free-living organism is suggested to be largely set by the catalytic efficiency of enzymes and protein synthetic machinery. Because of fluctuations in the environment, cells must maintain machinery to cope with various catastrophes; these mechanisms increase the minimum size of the cell. On the other hand, the largest cell is reasonably assumed to be limited by the ability of diffusion to bring nutrients to the appropriate part of the cell and to dispose of waste products. To explore the limitation imposed by diffusion, analysis is developed of diffusion processes through stirred and unstirred media, diffusion through media that contains obstacles, and the effect of size and shape.

Adaptation, Biological↗
PubMed1995

Buoyant density of Escherichia coli is determined solely by the osmolarity of the culture medium.

In previous studies, we had shown that the buoyant density of Escherichia coli is determined by the osmolarity of the growth medium by varying the osmolarity of the medium with NaCl or sucrose. However, the buoyant density of the cells always exceeded that of the growth medium. Here we determined the effect of medium with a buoyant density greater than the expected buoyant density of cells by adding Nycodenz to Luria broth. Percoll gradients of cells were analyzed by laser light scattering. The buoyant density for 125- and 375-mOsM-grown cells was 0.002 g/ml and 0.003 g/ml more, respectively, for cells grown in the presence of Nycodenz than those grown without Nycodenz, while the buoyant density of 250-mOsM-grown cells was 0.005 g/ml less for cells grown in the presence of Nycodenz than those grown without Nycodenz. Cells grown in 500-mOsM medium with or without Nycodenz had the same buoyant density. The buoyant density of cultures grown in defined medium was the same as those grown in rich medium, with only the medium osmolarity correlating to buoyant density. We conclude from these experiments that neither buoyant density nor chemical make-up of the medium determines the buoyant density of cells grown in that medium. Only the medium osmolarity determines cell buoyant density, suggesting that E. coli has no mechanisms to sense buoyant density.

Centrifugation, Density Gradient↗
PubMed1995

Phase and electron microscopic observations of osmotically induced wrinkling and the role of endocytotic vesicles in the plasmolysis of the Gram-negative cell wall.

When a Gram-negative bacterium is challenged with a sufficient concentration of a non-penetrating solute such as sucrose, water is sucked out of the cell. Plasmolysis spaces may form if the cell's cytoplasmic membrane (CM) separates from the murein wall (M) and the outer membrane (OM). However, we suggest that first wrinkling of the wall envelope, forced by dehydration of the cytoplasm, occurs. The cryofixation, freeze-substitution electron microscope studies used here are much too slow to study the kinetics of shrinkage, wrinkling and plasmolysis. However, they are consistent with faster phase microscope studies and previous stopflow experiments. For the electron microscopy studies reported here, only sucrose was used as the osmotic agent and under conditions that do not cause extreme plasmolysis. Plasmolysis spaces were associated with the formation of small membrane-bound vesicles in the nearby cytoplasm. Such vesicles formed by osmotic challenge are called 'endocytotic' in plant cell systems. They had been recorded in earlier plasmolysis studies in bacteria, but not interpreted as a concomitant part of plasmolysis space formation in certain locations of the cell. We suggest that the endocytotic vesicles form because the phospholipid membranes are capable of very little contraction so extra membrane must be disposed of when plasmolysis spaces form. In the case of plasmolysis spaces forming at poles and constriction sites, for geometric reasons the surface area of the CM may be conserved without disposition of excess membrane. We suggest that it is this biophysical property of lipid membranes that leads to the frequent formation of plasmolysis spaces at a pole and at the site of future division. We also observed a novel structure, this is seen only under mild osmotic up-shock, and consists of very thin, straight, uniform and long plasmolysis spaces which were called 'lamellar spaces'; these commonly formed inside the sidewalls and were usually associated with the formation of endocytotic vesicles. Since lipoprotein links the M to the OM layers and thus could affect plasmolysis, we examined both wild-type and deficient strains. Some effects were observed, but they were minimal. The volume of the periplasmic space of growing unshocked cells was determined to be about 7%.

Cell Wall↗
PubMed1995

Growth and buoyant density of Escherichia coli at very low osmolarities.

The growth and buoyant densities of two closely related strains of Escherichia coli in M9-glucose medium that was diluted to produce osmolarities that varied from as low as 5 to 500 mosM were monitored. At 15 mosM, the lowest osmolarity at which buoyant density could be measured reproducibly in Percoll gradients, both ML3 and ML308 had a buoyant density of about 1.079 g/ml. As the osmolarity of the medium was increased, the buoyant density also increased linearly up to about 125 mosM, at which the buoyant density was 1.089 g/ml. From 150 up to 500 mosM, the buoyant density again increased linearly but with a different slope from that seen at the lower osmolarities. The buoyant density at 150 mosM was about 1.091 g/ml, and at 500 mosM it was 1.101 g/ml. Both strains of E. coli could be grown in M9 medium diluted 1:1 with water, with an osmolarity of 120 mosM, but neither strain grew in 1:2-diluted M9 if the cells were pregrown in undiluted M9. (Note: undiluted M9 as prepared here has an osmolarity of about 250 mosM.) However, if the cells were pregrown in 30% M9, about 75 mosM, they would then grow in M9 at 45 mosM and above but not below 40 mosM. To determine which constituent of M9 medium was being diluted to such a low level that it inhibited growth, diluted M9 was prepared with each constituent added back singly. From this study, it was determined that both Ca2+ and Mg2+ could stimulate growth below 40 mosM. With Ca2+ - and Mg2+ -supplemented diluted M9 and cells pregrown in 75 mosM M9, it was possible to grow ML308 in 15 mosM M9. Strain ML3 would only haltingly grow at 15 mosM. Four attempts were made to grow both ML3 and ML308 at 5 mosM. In three of the experiments, ML308 grew, while strain ML3 grew in one experiment. While our experiments were designed to effect variations in medium osmolarity by using NaCl as an osmotic agent, osmolarity and salinity were changed concurrently. Therefore, from this study, we believe that E. coli might be defined as an euryhalinic and/or euryosmotic bacterium because of its ability to grow in a wide range of salinities and osmolarities.

Adaptation, Physiological↗
PubMed1994

Development and diversification of the Last Universal Ancestor.

The majority of evolutionary steps in the development of basic cellular processes took place in the time interval after the "First Cell" arose until the time of the "Last Universal Ancestor". During this period, life evolved in a monophyletic way in which no stable diversity arose; i.e. although side branches developed, only one survived because of simple "survival of the fittest". The myriad of enzymes and processes developed in this time interval can be grouped in eight qualitatively different categories. In many cases, the evolution of a particular gene was dependent on the concomitant improvement of the cellular machinery generally, including developments in other categories. Eventually several individuals arose (the immediate descendants of the Last Universal Ancestor) that made stable diversity possible because they developed alternative non-competing strategies. These diverse strategies subsequently led to eubacteria, archaebacteria, and eukaryotes (and viruses, plasmids, etc). This paper considers the evolutionary developments in this monophyletic epoch. It depends on three assertions: first, that there is no trivial spontaneous mechanism for the introduction of polynucleotides into a living cell and thus transfer of genes from organism to organism did not occur; second, that the number of accessible habitats and niches was very limited; and third, that the major factor that led to stable diversity was three nearly simultaneous developments. Two were radically different and independent solutions of the problem of overcoming cellular osmotic stress and the third was the development of methanogenesis. Sufficient osmotic pressure could create a high turgor pressure and destructive tension in the wall. However, osmotic stress only became a problem with development of improved metabolism, which resulted in greater success in the accumulation of cellular macromolecules and soluble constituents. One solution preventing osmotic rupture of the cell was the development of mechano-proteins and associated elements of the cytoskeleton by the predecessors of future eukaryotes. The second solution was the development of the murein sacculus (i.e. a covalently closed, cross-linked fabric made of peptidoglycan) by the predecessors of future eubacteria. The former allowed larger cells with flexible cell membranes to evolve and the latter to the development of a strong elastic "exoskeleton" providing ability to survive in extreme environments. Each of these diverse strategies allow both cell types to resist turgor pressure and led to independent non-competing organisms. Interwoven with these developments was the concomitant development of methanogenesis (the third change needed to generate three Kingdoms), which provided the first truly large-scale generation of metabolic energy.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

A change in a single gene of Salmonella typhimurium can dramatically change its buoyant density.

The growth rates and buoyant densities of a Salmonella typhimurium mutant, TL126 (proB74A+), with enhanced osmotolerance caused by proline overproduction were measured and compared with the growth rates and buoyant densities of an isogenic (wild-type) strain, TL128 (proB+ A+), with normal control of proline production. Growth rates were determined in a rich medium (Luria broth) with added NaCl to produce various osmotic strengths ranging from 300 to 2,000 mosM. At low concentrations of NaCl, there was little variation in doubling times between the two strains. However, as the osmotic strength of the medium approached and exceeded 1,300 mosM, the doubling times of TL126 (osmotolerant) were 1.5 to 2 times faster than those of TL128 (wild type), confirming the osmotolerance of TL126. Buoyant densities were determined by equilibrium sedimentation in a Percoll gradient of osmotic strength equal to that of the growth medium. The osmolarity of the Percoll gradient was adjusted by the addition of NaCl. At low osmolarities (300 to 500 mosM), the buoyant density of TL126 (osmotolerant) was slightly but consistently lower than that of TL128 (wild type). As the osmotic strength was increased, the buoyant density of TL126 (osmotolerant) increased in proportion to the osmotic strength. In contrast, the buoyant density of strain TL128 (wild type) did not increase as much. At high osmolarities (1,600 to 2,000 mosM), the buoyant density of TL126 (osmotolerant) was consistently higher than that of TL128 (wild type). These results suggest that the intracellular accumulation of proline by TL126, the osmotolerant strain, increases both the growth rates and buoyant densities at osmolarities of 1,300 mosM and above.

Centrifugation, Isopycnic↗
PubMed1993

Anomalies in cell wall turnover associated with the growth temperature of Bacillus subtilis.

Cell wall turnover appeared to be anomalously fast in Bacillus subtilis when the cells were grown at temperatures below 29 degrees C. Turnover rates k(generation-1), of exponential cultures at 25 degrees were approximately double those of cells grown at 37 degrees C. When autolysin levels were assayed in cell walls, it was found that the enzyme activities were constant between 25 degrees C and 40 degrees C, suggesting that there was no greater synthesis of autolysin at the lower temperature. Analyses of walls for individual components, extent of aminosugar substitution and extent of crosslinking, did not reveal significant differences between samples obtained from 25 degrees C or 37 degrees C cultures. The N-acetylmuramoyl-L-alanine amidase was stable over the temperature range studied. Lysis of cells, induced by carbonylcyanide-m-chlorophenylhydrazone, occurred at a faster rate for cells obtained at 25 degrees C than for cells obtained at 37 degrees C. In addition, the lysis of cells by hen egg white lysozyme was slightly faster when the cells were obtained from 25 degrees C cultures than from 37 degrees C cultures. It is possible the autolysin(s) responsible for cell wall turnover are cold-activated.

Bacillus subtilis↗
PubMed1993

Genetic response of microbes to extreme challenges.

Bacteria are capable of surviving in a wide range of seemingly impossible situations. In the main part this is because very large numbers of individual cells are involved, so that even very rare mutations will occur often enough to solve most acute problems and permit growth. But in addition, bacteria have evolved in an environment that has fluctuated so often in the past that they may have evolved a group of "last ditch" mechanisms to meet these challenges. These include developing a metabolically inactive state; activating previously evolved, but silent genes; increasing rates of mutation under dire condition; and favoring movement of exogenous and endogenous genetic elements. Some of these processes can function to make it appear that directed "Lamarckian" mutations have occurred. Together these survival mechanisms constitute the "catastrophe insurance" of the cells.

Bacteria↗
PubMed1993

The growth kinetics of B. subtilis.

There has been considerable discussion by Kubitschek and Cooper concerning the growth rate of cells of E. coli throughout the cell cycle. Consequently, it is relevant to test Kubitschek's linear model against the exponential model espoused by Cooper (and many others) with another organism and another technique. Burdett et al. measured, by electron microscopy and computer analysis of the microphotographs, the distribution of lengths of a population of cells of Bacillus subtilis grown in 0.4% succinate in a minimal medium. The data were fitted to the extended Collins-Richmond method of Kirkwood & Burdett which subdivided the cell cycle into several phases. I have taken their results and compared them with the linear and exponential growth models for the entire cell cycle after applying correction to the data for the shape of completed and forming poles; i.e., to put the data on a cell-volume basis instead of a cell-length basis. Most of the correction involves no arbitrary assumptions. The conclusion is that global volume growth rate is nearly proportional to cell volume; i.e. growth of Bacillus subtilis is nearly exponential for almost every cell in the growing culture.

Bacillus subtilis↗
PubMed1993

Proton motive force may regulate cell wall-associated enzymes of Bacillus subtilis.

Bacterial metabolism excretes protons during normal metabolic processes. The protons may be recycled by chemiosmosis, diffuse through the wall into the medium, or bind to cell surface constituents. Calculations by Koch (J. Theor. Biol. 120:73-84, 1986) have suggested that the cell wall of gram-positive bacteria may serve as a reservoir of protons during growth and metabolism, causing the wall to have a relatively low pH. That the cell wall may possess a pH lower than the surrounding medium has now been tested in Bacillus subtilis by several independent experiments. When cultures of B. subtilis were treated with the proton conductors azide and carbonylcyanide m-chlorophenylhydrazone, the cells bound larger amounts of positively charged probes, including the chromium (Cr3+) and uranyl (UO2(2+) ions and were readily agglutinated by cationized ferritin. In contrast, the same proton conductors caused a decrease in the binding of the negatively charged probe chromate (CrO4(2-)). Finally, when levansucrase was induced in cultures by the addition of sucrose, the enzyme was inactive as it traversed the wall during the first 0.7 to 1.0 generation of growth. The composite interpretation of the foregoing observations suggests that the wall is positively charged during metabolism, thereby decreasing its ability to complex with cations while increasing its ability to bind with anions. This may be one reason why some enzymes, such as autolysins, are unable to hydrolyze their substrata until they reach the wall periphery or are in the medium.

Bacillus subtilis↗
PubMed1993

Biomass growth rate during the prokaryote cell cycle.

The rate of biomass growth throughout the cell cycle of prokaryotes is important in the study of global regulation. Two limiting cases have generally been considered: the exponential model and the linear model. The exponential model is a logical expectation because protein, the main component of biomass of a bacterial cell, increases continuously during the cell cycle and therefore the means for synthesis of other cell components and metabolites also increases. In addition, during the cell cycle, ribosomes, the means of production of proteins, increase monotonically. As a consequence, the increase of all should be autocatalytic and the content of cell substance should be an exponential function of time. Two cellular components would not be expected to increase exponentially: the DNA and the cell envelope. The former because of the intermittent synthesis of the chromosome, and the latter because of changes in the surface-to-volume ratio with growth and division. In contrast to the exponential model, the linear model of Kubitschek postulates that the cell only increases its membrane transport capability over a brief period during the cell cycle, and, thus limited by transport, all cell components can increase only at a constant linear rate during most of the cell cycle. Other proposed models are intermediate and assume that the growth rate of the cell depends on some cell cycle event, such as the initiation of chromosome replication. The models have relevance to prokaryotes undergoing balanced growth; they may not be relevant to eukaryotic microbes or to eukaryotic cells in tissue culture that have endogenous rhythms or are controlled by protein growth factors. Logically, the models could possibly apply to a free-living cell that does not respond to environmental cues. Even under rigidly constant conditions, however, cells may try to respond to a stimulus that was periodic or regulatory under natural conditions, but is present at a constant level under the experimental culture condition. There are four classes of experiments that have been used to measure the accumulation of dry biomass or its components during the cell cycle of a bacterium, as typified by Escherichia coli. For the first class of experiments, the dimensions of living cells are measured under the microscope. So far, the experiments have been limited by the resolving power of the phase microscope, but adequate resolution should be possible with the confocal scanning light microscope or various video computer systems. Such experiments are called integral because augmentation of cell constituents is followed. The second class involves pulse-chase labeling of cells and then their separation into different phases of the cycle or age groups and measurement of the radioactivity per cell in the fractions. Such experiments are called differential in that the rate is measured directly instead of being deduced by comparing the total size at different times.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Cycle↗
PubMed1993

Marketing prevention to elderly Medicare beneficiaries enrolled in an HMO. The San Diego Medicare Preventive Health Project.

A comprehensive marketing effort--using direct mail, telemarketing, and orientation seminars--to enroll elderly participants in a Medicare preventive health services demonstration project was undertaken in 1989. Out of the more than 11,000 eligible members in a large Medicare HMO plan in San Diego County, 1,800 (16.2%) agreed to participate. The authors describe the recruiting effort in detail and postulates reasons why the elderly resisted enrolling in the study. These results have important policy implications for the nation's Medicare program and are relevant to promoting other useful health care services in this population.

Aged↗
PubMed1992

Differences in the formation of poles of Enterococcus and Bacillus.

The pole of Enterococcus hirae (Streptococcus faecium) is more pointed than that of Bacillus subtilis; i.e. the pole of the former is prolate and the latter is oblate. Both species form their poles by constructing annular additions on the inside surface. In both cases, the thick septum starts to split from the outside before the septum is complete. Physiochemical considerations dictate that the peptidoglycan must be unstretched as laid down. However, it later becomes stressed and may stretch to increase its surface area or to change its shape. Our earlier analysis for B. subtilis demonstrated that, without the addition of new peptidoglycan, the nascent wall is stretched after it is externalized to 1.51 times the original area. The wall of partially formed poles that is already exteriorized continues to deform with further development. For E. hirae, Higgins & Shockman's measurements showed that the completed pole has a surface area 2.18 times larger than a completed septal disk and the wall changes shape very little after exteriorization. A model is presented here for the streptococcus in which the septal wall does not increase its surface area on exteriorization either by expansion or by murein insertion. Instead, the septal wall as it is split and exteriorized twists to become oblique, increasing the inner radius of the incomplete septum. In consequence of this rotation, extra layers of peptidoglycan are added to the inside face of the developing septum. This additional murein forms the more pointed pole shape for E. hirae. This "split-and-splay" model thus refines and extends the surface stress theory of E. hirae developed a decade ago by proposing a source of the extra wall needed for the formation of its prolate, more pointed, pole.

Animals↗
PubMed1992

Elasticity of the sacculus of Escherichia coli.

Preparations of purified peptidoglycan of Escherichia coli (i.e., sacculi) were studied by low-angle laser light scattering. Control experiments and theoretical calculations based on the Rayleigh-Gans theory showed that the mean sacculus surface area could be accurately inferred from measurements with our apparatus by using computer routines developed previously. Large changes in the mean saccular surface area resulted from alterations in the stress caused by varying the net charge on the sacculi. The net charge was affected by altering the suspending medium pH, causing carboxyl and amino groups in the peptidoglycan to gain or lose protons, or by acetylation or succinylation of the amino groups. A preponderance of either plus or minus charges caused an expansion of the mean sacculus surface area. The largest increase in area probably represents the elastic limit of the peptidoglycan and was 300% above the area of isoionic sacculi. This degree of expansion is consistent with possible conformations of the intact peptidoglycan structure without necessitating rupture of the wall fabric. Our findings concerning saccular elasticity provide support for the surface stress theory. It provides a mechanism so that bacteria can grow and divide while maintaining turgor pressure, without the necessity of having and using proteins to do the mechanical work.

Acetylation↗
PubMed1991

The first cellular bioenergetic process: primitive generation of a proton-motive force.

It is proposed that the energy-transducing system of the first cellular organism and its precursor was fueled by the oxidation of hydrogen sulfide and ferric sulfide to iron pyrites and two [H+] on the outside surface of a vesicle (the cell membrane), with the concomitant reduction of CO or CO2 on the interior. The resulting proton gradient across the cell membrane provides a proton-motive force, so that a variety of kinds of work can be done. It is envisioned as providing a selective advantage for cells capable of harvesting this potential. The proposed reactants for these reactions are consistent with the predicted composition of the Earth's early environment. Modern-day homologs of the ancestral components of the energy-transducing system are thought to be membrane-associated ferredoxins for the extracellular redox reaction, carbon monoxide dehydrogenase for the carbon fixation reaction, and ATPase for the harvesting of the proton gradient. With a source of consumable energy, the cell could drive chemical reactions and transport events in such a way as to be exploited by Darwinian evolution.

Adenosine Triphosphatases↗
PubMed1990

The relative rotation of the ends of Bacillus subtilis during growth.

Observation of long single filaments of Bacillus subtilis 168 in depression slide cultures demonstrated that one end rotated relative to the other during growth. This was observed with suspended filaments, filaments attached to glass surfaces and single stranded filaments folded back on themselves growing as a double stranded helix. This extends Mendelson's 1976 conclusion to cases with no alternative interpretation to the hypothesis that as each cell grows, the structure of the peptidoglycan changes to rotate one end relative to the other.

Bacillus subtilis↗