When cells of a marine pseudomonad were washed and suspended in 0.5 M sucrose, they retained their rod shape, but thin sections, when examined in an electron microscope, revealed that the outer layer of the cell wall had separated a considerable distance from the cytoplasmic membrane. Treatment of such cells with lysozyme alone produced no obvious change, but treatment with ethylenediaminetetraacetic acid (EDTA) alone caused the outer wall to disappear. A combination of EDTA and lysozyme resulted in the rapid formation of spheres essentially free from hexosamine and indistinguishable from protoplasts of gram-positive bacteria. When cells were washed with 0.5 M NaCl and then suspended in 0.5 m sucrose, they also retained their rod shape, but in this case the outer layer separated from the cells completely and could be recovered from the suspending medium. Such cells were converted to protoplasts by the action of lysozyme alone. Cells washed and finally suspended in 0.5 M NaCl, when treated with EDTA and lysozyme, slowly became spherical. Thin sections revealed typical spheroplasts of gram-negative bacteria in which the outer wall remained intact. Protoplasts took up a-aminoisobutyric acid by a Na+-dependent process. When certain species of gram-positive bacteria are treated with lysozyme, the entire outer wall of the cell is removed and the resulting osmotically sensitive structure is bounded only by its cytoplasmic membrane. Such a structure is referred to as a protoplast (3). Gram-negative bacteria are more resistant to the action of lysozyme and are converted to osmotically sensitive forms by this enzyme only if auxiliary agents or procedures are employed. The latter include heat treatment of the cells (L. E. Myerholtz and S. E. Hartsell, Bacteriol. Proc., p. 34, 1952), freezing and thawing (Ii), the addition of ethylenediaminetetraacetic acid (EDTA) in tris(hydroxymethyl)aminomethane (Tris) buffer (17), and plasmolysis (2). Such auxiliary treatments appear to increase the penetrability of the outer layers of the cell wall, permitting lysozyme to reach andattack the underlying mucopeptide 1 lssued as Macdonald College Journal Series No.

MATULA, AND ROBERT A. MAcLEOD. Nutrition and metabolism of marine bacteria. XV. Relation of Na+-activated transport to the Na+ requirement of a marine pseudomonad for growth. J. Bacteriol. 92:63-71. 1966.-A marine pseudomonad was found to require 50 to 100 mm Na+ for maximal rate of oxidation of D-galactose and for the transport of D-fucose-H3 into the cells. The same organism required 150 to 200 mm Na+ for the oxidation of L-alanine and for the transport of 4-aminoisobutyric acid-C'4 (AIB-C14) into the cells. Competition studies indicated that D-galactose and D-fucose on the one hand and L-alanine and AIB on the other shared common carriers for transporting the compounds into the cells. This parallelism in Na+ response for oxidation and transport extended to growth whenL-alanine was the sole carbon source in the medium. When D-galactose was the sole carbon source, an amount of Na+ equal to that with L-alanine was needed. KCN and dinitrophenol but not ouabain inhibited the uptake of AIB-C14 by the cells. K+ in addition to Na+ was required for transport, and both Mg++ and either Clor Brwere stimulatory. Photobacterium fischeri was also found to require Na+ specifically for the uptake of AIB-C14 by the cells. All marine bacteria investigated in detail have been found to have a highly specific requirement for Na+ for growth. In this respect, they differ from most terrestrial species examined [see MacLeod (8) for a review]. Previous efforts to MATERIALS AND METHODS Cultures. The organism referred to as B-16 has been classified as a Pseudomonas species type IV and is maintained as culture NCMB 19 at the Torry Research Station, Aberdeen, Scotland. Studies on the 63

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Five marine bacteria examined were found to differ considerably in lytic susceptibility. Some lysed completely below 0.15 M NaCl while suspensions of others contained some whole cells at 0.025 M NaCl. In general NaCl and LiCl were more effective than KCl or NH4Cl in protecting against lysis and the loss of ultraviolet-absorbing materials from the cells. KCl could spare the requirement for NaCl to prevent lysis. Mg++ and other divalent cations at 0.05 M or less completely prevented lysis of all but one of the organisms. Sulphate salts stabilized the cell suspensions better on incubation than did chlorides for four of the organisms. For the fifth the reverse was true. The lytic action of ethanol and glycerol could be prevented by NaCl or sucrose. The concentrations of the latter required to prevent lysis remained unchanged when ethanol or glycerol was included in the suspending medium. Cells washed essentially free of electrolytes with 0.5 M sucrose retained their morphological characteristics. The observations are considered in relation to the distinction between marine bacteria, halophiles, and terrestrial bacteria and to current theories of the mechanism of lysis of bacterial cells. The results are not consistent with the hypotheses either that primarily osmotic effects are involved or that electrolytes are required to maintain the cell wall.

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...

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