Binding of Dissolved Strontium by Micrococcus luteus.

Resting cells of Micrococcus luteus have been shown to remove strontium (Sr) from dilute aqueous solutions of SrCl(2) at pH 7. Loadings of 25 mg of Sr per g of cell dry weight were achieved by cells exposed to a solution containing 50 ppm (mg/liter) of Sr. Sr binding occurred in the absence of nutrients and did not require metabolic activity. Initial binding was quite rapid (<0.5 h), although a slow, spontaneous release of Sr was observed over time. Sr binding was inhibited in the presence of polyvalent cations but not monovalent cations. Ca and Sr were bound preferentially over all other cations tested. Sr-binding activity was localized on the cell envelope and was sensitive to various chemical and physical pretreatments. Bound Sr was displaced by divalent ions or by H. Other monovalent ions were less effective. Bound Sr was also removed by various chelating agents. It was concluded that Sr binding by M. luteus is a reversible equilibrium process. Both ion exchange mediated by acidic cell surface components and intracellular uptake may be involved in this activity.

Use of immobilized microbial membrane fragments to remove oxygen and favor the acetone-butanol fermentation.

Oxygen-reducing membrane fragments obtained from Escherichia coli were used with Clostridium acetobutylicum (C. acetobutylicum) to provide an oxygen-free microenvironment for the conversion of glucose to acetone, butanol, and ethanol (ABE). The batch fermentation of suspended C. acetobutylicum NRRL-B-643 and its ability to produce solvents in the presence of membranes as the oxygen-elimination agent are described and compared with the conventional sparging technique used to maintain anaerobiosis. The use of membrane fragments to remove oxygen for fermentation by C. acetobutylicum was successful and gave slightly improved results over the use of sparing with regard to lag, biomass, and solvent production (e.g., final butanol concentration of 3.25 and 2.7 g/L, respectively). Solvent production is also reported for a continuous columnar reactor with coimmobilized cells and membranes in kappa-carrageenan gel beads and air-saturated liquid feed.

The use of microbial membranes to achieve anaerobiosis.

The cytoplasmic membranes of many aerobic and facultative bacteria contain enzymes that catalyze the reduction of dissolved oxygen to water. Preparations of small particles derived from such membranes can be filter sterilized without loss of the oxygen-reducing enzymes. These particle preparations can be used to produce anaerobic conditions in a variety of biological environments. They have been shown to stimulate the growth of many anaerobic bacteria and can also be used to stabilize oxygen-sensitive chemical reagents. The particle preparations are stable for long periods of time. They are functional over a pH range and temperature range frequently encountered in biological systems. Various techniques for using the particles are presented. The advantages and limitations of this new approach to achieving oxygen-free conditions are discussed.

Mutations in Escherichia coli that effect sensitivity to oxygen.

Fifteen oxygen-sensitive (Oxys) mutants of Escherichia coli were isolated after exposure to UV light. The mutants did not form macroscopic colonies when plated aerobically. They did form macroscopic colonies anaerobically. Oxygen, introduced during log phase, inhibited the growth of liquid cultures. The degree of inhibition was used to separate the mutants into three classes. Class I mutants did not grow after exposure to oxygen. Class II mutants were able to grow, but at a reduced rate and to a reduced final titer, when compared with the wild-type parent. Class III mutants formed filaments in response to oxygen. Genetic experiments indicated that the mutations map to six different chromosomal regions. The results of enzymatic assays indicated that 7 of the 10 class I mutants have low levels of catalase, peroxidase, superoxide dismutase, and respiratory enzymes when compared with the wild-type parent. Mutations in five of the seven class I mutants which have the low enzyme activities mapped within the region 8 to 13.5 min. P1 transduction data indicated that mutations in three of these five mutants, Oxys-6, Oxys-14, and Oxys-17, mapped to 8.4 min. The correlation of low enzyme levels and mapping data suggests that a single gene may regulate several enzymes in response to oxygen. The remaining three class I mutants had wild-type levels of catalase, peroxidase, and superoxide dismutase, but decreased respiratory activity. The class II and III mutants had enzyme activities similar to those of the wild-type parent. Our results demonstrate that mutations in at least six genes can be expressed as oxygen sensitivity. Some of these genes may be involved in respiration or cell division or may regulate the expression of several enzymes.

A Technique for Predicting the Solvent-Producing Ability of Clostridium acetobutylicum.

Changes in colony morphology were associated with the degeneration of solvent-producing strains of Clostridium acetobutylicum. The most efficient solvent-producing strains gave rise exclusively to colonies with dense centers containing large numbers of spores. Many outgrowths of various morphologies developed from the perimeter of such colonies after several days of incubation. The most degenerate cultures did not produce solvents and gave rise to large diffuse colonies that did not contain spores. These diffuse colonies did not produce outgrowths. Intermediate colony types were also observed. These could be derived from liquid cultures that were relatively poor solvent producers or from the outgrowths of colonies of efficient solvent-producing strains. Some of these intermediate types produced spores but did so less frequently than the high-solvent-producing strains. The spores of the intermediate types could not be distinguished from those of the most efficient solvent producers on the basis of heat sensitivity. The relationship observed between colony morphology and solvent production provides a method for predicting the solvent-producing potential of C. acetobutylicum cultures.

Promotion of septation in irradiated Escherichia coli by a cytoplasmic membrane preparation.

Septation can be promoted in an X-irradiated lon mutant of Escherichia coli K-12 by the addition of an E. coli B/r cytoplasmic membrane preparation to the postirradiation plating medium. The promotion of septation was not associated with an inhibition of growth rate. Two distinct cytoplasmic membrane-associated properties were necessary to promote septation. One of these, the cytochrome-based electron transport system, produced anaerobic conditions by the reduction of oxygen dissolved in the medium. The second system, functioning independently from the first, altered substances found in the peptone and yeast extract components of the postirradiation plating medium. When both systems were operative, significant repair of the cell division mechanism occurred.

Cytoplasmic membrane fraction that promotes septation in an Escherichia coli lon mutant.

A particulate fraction derived from bacterial cells stimulates septation in irradiated Escherichia coli lon mutants when added to postirradiation plating media. It was established that the particles are derived from the cytoplasmic membrane and that they have been partially purified by sucrose density gradient centrifugation. These particles also contain the cytochrome-based respiratory activity of the cell. A variety of experiments established a correlation between the septation-promoting activity of the particles and their ability to remove oxygen from the postirradiation plating medium. It was suggested that the efficient removal of oxygen from the medium allowed the lon cells to repair radiation-induced damage to the septation mechanism.

Cell growth and division in Escherichia coli: a common genetic control involved in cell division and minicell formation.

Escherichia coli Div 124(ts) is a conditional-lethal cell division mutant formed from a cross between a mutant that produces polar anucleated minicells and a temperature-sensitive cell division mutant affected in a stage of cross-wall synthesis. Under permissive growth temperature (30 C), Div 124(ts) grows and produces normal progeny cells and anucleated minicells from its polar ends. When transferred to nonpermissive growth temperature (42 C), growth and macromolecular synthesis continue, but cell division and minicell formation are inhibited. Growth at 42 C results in formation of filamentous cells showing some constrictions along the length of the filaments. Return of the filaments from 42 to 30 C results in cell division and minicell formation in association with the constrictions and other areas along the length of the filaments. This gives rise to a "necklace-type" array of cells and minicells. Recovery of cell division is observed after a lag and is followed by a burst in cell division and finally by a return to the normal growth characteristic of 30 C cultures. Recovery of cell division takes place in the presence of chloramphenicol or nalidixic acid when these are added at the time of shift from 42 to 30 C, and indicates that a division potential for filament fragmentation is accumulated while the cells are at 42 C. This division potential is used for the production of both minicells and cells of normal length. The conditional-lethal temperature sensitive mutation controls a step(s) in cross-wall synthesis common to cell division and minicell formation.

Polyuridylic acid-directed phenylalanine incorporation in minicell extracts.

Cell-free extracts of miniature Escherichia coli cells deficient in deoxyribonucleic acid (DNA) and DNA-dependent ribonucleic acid polymerase have been shown to be capable of polyuridylic acid-directed [(14)C]phenylalanine incorporation.

Influence of the fertility episome on the survival of x-irradiated Escherichia coli.

The resistance of Escherichia coli to the lethal effects of X rays was increased by the presence of a fertility episome integrated into the chromosome.

Properties of a cell fraction that repairs damage to the cell division mechanism of Escherichia coli.

A factor in bacterial cell extracts which induces cell division in filaments of Escherichia coli produced by irradiation was found to be associated with a heat-labile particulate fraction which sediments at about 100S. Frozen or lyophilized samples of the cell extracts were stable for considerable periods of time. Fractions purified by centrifugation contained 2% of the total protein of the extract but no measurable ribonucleic acid or deoxyribonucleic acid. The extracts were inactivated by incubation with phospholipase A, lipases, and detergents, but not by incubation with selected nucleases and proteases.

Giant cells of Escherichia coli.

A mutant strain of Escherichia coli K-12 produced amorphous cells when grown in a variety of media. The lon(-) allele, known to increase the radiation sensitivity of the cytokinesis mechanism, was introduced into the mutant by means of conjugation. Cells of this recombinant strain grew, after exposure to radiation, into giant amorphous cells, approximately 500 to 1,000 times the volume of a normal E. coli cell. These giant cells are analogous to the filaments formed after the irradiation of lon(-) rod-shaped cells.

Repair of radiation-induced damage to the cell division mechanism of Escherichia coli.

Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), William D. Fisher, Alice A. Hardigree, and George E. Stapleton. Repair of radiation-induced damage to the cell division mechanism of Escherichia coli. J. Bacteriol. 91:737-742. 1966.-Microscopic observations of irradiated populations of filamentous Escherichia coli cells indicated that filaments can be induced to divide by a substance donated by neighboring cells. We have made this observation the basis for a quantitative technique in which filaments are incubated in the presence of nongrowing donor cells. The presence of "donor" organisms promotes division and subsequent colony formation in filaments. "Donor" bacteria do not affect nonfilamentous cells. An extract of "donor" cells retains the division-promoting activity. The extract has been partially fractionated, and consists of a heat-stable and a heat-labile component. The heat-stable component is inactive in promoting cell division, but enhances the activity of the heat-labile component. The division-promoting system is discussed as a radiation repair mechanism and as a normal component of the cell division system in E. coli.