Mechanism of Optical Effects in Suspensions of a Marine Pseudomonad

Abstract

When cells of a marine pseudomonad washed free of medium components with 0.05 M MgSO4 were suspended in solutions containing 200-mM concentrations of various salts, there was an immediate increase in optical density (OD), followed by a slow decrease. The decrease following the initial increase, but not the increase itself, could be prevented by omitting K+ from or by adding metabolic inhibitors to the suspending solution. With NaCl, the initial increase in OD rose to a maximum as the salt concentration was increased to 200 mm and then declined at 500 mM. There was a corresponding decrease in intracellular fluid volume to a minimum at 200-mM NaCl and then a rise. When the increased OD produced by NaCl was maintained, the internal Na+ and Clcould be shown to have reached essentially the same concentration in the cells as in the medium. Thus, the OD changes could not have been due to osmotic effects. No evidence was obtained of a salt-induced aggregation of nuclear material. The OD of suspensions of isolated cell envelopes increased in response to increases in NaCl concentration in the absence but not in the presence of 0.05 M MgSO4. The data was interpreted to indicate that the salt-induced increases in OD occurring in suspensions of the cells resulted from an interaction of salts with components of the cell envelope, causing contraction of the envelopes and shrinkage of the cells.Turbidity changes occur in suspensions of gram-negative bacteria when the solute concentration of the suspending medium is increased by the addition of electrolytes or nonelectrolytes. The changes can often be separated into two phases. The first is an increase in turbidity which is complete within seconds. The second, which may or may not occur depending on the species (6), is a slow decrease in turbidity which follows the initial increase. These effects, which have been observed by a number of workers (1, 2, 6, 14-16), have been ascribed generally to an initial rapid decrease in size of the cells caused by the sudden increase in osmotic pressure in the suspending medium followed by a slow restoration of the cells to normal size as the solutes come into equilibrium across the osmotic barrier. Gram-positive bacteria ordinarily do not show 1 From a thesis submitted by Tibor I. Matula in partial fulfillment of the requirements for the Ph.D. degree at McGill University, May 1967. A preliminary report of these findings was presented at such optical effects, although after incubation in phosphate buffer followed by washing in distilled water, the cells of a number of species of grampositive bacteria became susceptible to optical changes comparable to those of gram-negative organisms (6,9). Our interest in the mechanism of these optical effects was aroused when we observed that suspensions of a marine pseudomonad showed optical changes typical of those of other gram-negative bacteria upon the addition of NaCl to the suspending medium. Since previous studies had indicated that the cytoplasmic membrane of the marine pseudomonad presented no osmot...

Osmoregulation and compatible solutes in eubacteria

“…Little is known about the structure and composition of the periplasmic space, but it is generally assumed that the solute composition with regard to low-molecular-weight solutes is identical to that in the external medium, as these solutes can permeate through the outer membrane. First indications for some highly anionic non-penetrating compounds in the periplasm came from investigations with Alteromonas haloplanktis, which released such non-dialysable material when the cells were suspended at low solute concentrations [12]. It was proposed that these molecules, by their highly charged nature, would repel each other sufficiently tO stretch the envelope and cause the cell to swell.…”

“…It was proposed that these molecules, by their highly charged nature, would repel each other sufficiently tO stretch the envelope and cause the cell to swell. High concentrations of positively-charged ions would screen the negative charges, thereby causing the envelope to contract and the cell to shrink [12]. Thus, periplasmic osmotic pressure could originate from a Donnan equilibrium across the outer membrane, inside negative [10].…”

Transport and Retention of K + and Other Metabolites in a Marine Pseudomonad and Their Relation to the Mechanism of Optical Effects

“…Studies with a marine pseudomonad (12) and with Pseudomonas aeruginosa (13) showed that I Macdonald College Journal Series No. 608.…”

“…The second phase of the optical changes, the slow decrease in OD of the suspensions, could be shown in the case of the marine pseudomonad, but not in the case of P. aeruginosa, to be dependent on the presence of K+ in the medium. For both organisms, the second-phase change was prevented by metabolic inhibitors (12). The MATERIALS AND METHODS Culture.…”

“…The organism employed and referred to as marine pseudomonad B-16 (ATCC 19855) has been classified as a Pseudomonas species type IV. The medium used for its growth and the method of preparing cell suspensions have been described (12). Unless otherwise indicated, the harvested cells were washed free from medium components with 0.05 M MgSO4.…”