SUMMARY: An improved growth medium for lactobacilli is described. It supports good growth of lactobacilli generally and also is particularly useful for a number of fastidious strains which grow only poorly in other general media. In addition, tomato juice, a highly variable material, is not required. In a slightly modified form, it can also be used as a basal medium for fermentation tests.

A medium for the cultivation of lactobacilli

The absorption of inorganic salts by plant roots and other biological systems depends upon metabolic processes When absorption proceeds against a concentration gradient, an expenditure of metabolically derived energy is obviously required In some systems, such as barley roots, even absorption along a concentration gradient is negligible under conditions of arrested metabolism; and the concentration of the salt in the sap fails to reach the level of the outside solution (12, 13, 17) The absorption process is characterized by a considerable degree of selectivity The discrimination on the part of living cells between potassium and sodium is particularly striking Most living cells accumulate potassium in preference to sodium, even when the external environment is rich in sodium and poor in potassium The view has come to the fore in recent years that both the role of metabolism and the specificity in the absorption process can best be accounted for by assuming that ion binding compounds are generated by metabolic processes General reviews which make this point include those by Ussing (29), Robertson (25), and Steinbach (27) Osterhout (22) has succinctly stated some of the problems for whose solution such a concept would be useful Osterhout (22) and Jacobson and Overstreet (14) have outlined several properties which the ion binding compounds must possess to account for characteristic features of the absorption process These workers also stress the need for assuming that the complex is labile and breaks down again, releasing the ions Cowie et al (5), Roberts et al (23), Conway et al (4), Eddy et al (7), and Eddy and Hinshelwood (8, 9) have investigated the problem of ion binding in microorganisms, arriving at similar views Roberts and his co-workers (23, 24) and Eddy and co-workers (7, 8) have related the binding of alkali cations to carbohydrate metabolism, and Bolton and Roberts (1) have produced evidence that the hexose phosphates are responsible for the binding of potassium in Escherichia coli The hypothesis that the absorption of inorganic ions involves their combination with binding compounds forms the basis of the present study Evidence will be presented for the existence of several distinct reactive sites involved in the binding of alkali cations by barley roots It will be demonstrated that there are large differences in the affinity of these sites for various cations

http://www.plantphysiol.org/content/plantphysiol/27/3/457.full.pdf

A kinetic study of the absorption of alkali cations by barley roots.

It was shown by MacLeod and his co-workers that among bacteria only the true marine forms have a specific sodium requirement. These all require this ion in concentrations of the order of a few tenths of 1%. The non-marine bacteria studied by MacLeod were distinguished from marine forms by their lack of any requirement for sodium (MacLeod, Onofrey & Norris, 1954; MacLeod & Onofrey, 1956). In view of these facts, the following observation, which I made in the course of studying the amino acid nutrition of Rhodopseudomonas spheroides, seemed significant. This bacterium can grow very well in media of ordinary ionic strength and thus is not obviously marine. I observed that it did not grow in a medium in which an artificial mixture of amino acids had replaced an acid hydrolysate of casein. Since almost 40% of the hydrolysate used was sodium chloride, I added a little NaCl to the medium containing the artificial mixture of amino acids. I then found that the growth was as rapid as in the medium made with casein hydrolysate. It was easily shown that a very much simpler mixture of amino acids permitted rapid growth in the presence of as little as 100 μg. NaCl/ml. This requirement for a low concentration of sodium seemed to set this organism apart both from marine and from terrestrial bacteria and thus to merit a more detailed analysis, the first results of which are reported here.

A requirement for sodium in the growth of Rhodopseudomonas spheroides.

Lactobacillus plantarum is aerotolerant during log-phase growth on glucose, but is an obligate aerobe on polyols. Respiration was cyanide resistant and under certain conditions was associated with the accumulation of millimolar concentrations of H(2)O(2). On glucose, optimal growth was observed in the absence of O(2). Extracts of L. plantarum did not catalyze the reduction of paraquat by reduced nicotinamide adenine dinucleotide, but plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) was readily reduced. Such extracts produced O(2) (-) in the presence of NADH plus plumbagin. Plumbagin caused a 10-fold increase in the rate of respiration of intact cells in the presence of glucose and also imposed a loss of viability which was dependent upon both glucose and O(2). Although extracts of L. plantarum were devoid of true superoxide dismutase activity, this organism was comparable to superoxide dismutase-containing species in its resistance toward hyperbaric O(2) and toward the oxygen-dependent lethality of plumbagin. L. plantarum required Mn-rich media and actively accumulated Mn(II). Soluble extracts were found to contain approximately 9 mug of Mn per mg of protein and 75 to 90% of this Mn was dialyzable. Such extracts exhibited a dialyzable and ethylenediaminetetraacetic acid-inhibitable ability to scavenge O(2) (-). This O(2) (-)-scavenging activity was due to the dialyzable Mn(II) present in these extracts and could be mimicked by MnCl(2). Cells grown in Mn-rich media were enriched in dialyzable Mn and were more resistant toward oxygen toxicity and toward the oxygen-dependent plumbagin toxicity than were cells grown in Mn-deficient media. L. plantarum exhibited no nutritional requirement for iron and little or no iron was present in these cells, even when they were grown in iron-rich media. L. plantarum thus appears to use millimolar levels of Mn(II) to scavenge O(2) (-), much as most other organisms use micromolar levels of superoxide dismutases.

Manganese and Defenses against Oxygen Toxicity in Lactobacillus plantarum

A uniform medium for determination of amino acids with various microorganisms.

Some mineral requirements of the lactic acid bacteria.

The effect of related ions on the potassium requirement of lactic acid bacteria.

During a study of the specificity of the requirement of Lactobacillus arabinosus for Mn++, it was observed that ZnH was toxic for this organism and that its toxicity could be overcome by increasing the concentration of Mn++ in the medium. In addition to Mn++, several inorganic ions not previously implicated in the nutrition of these bacteria were found to counteract the Zn+ inhibition. The same ions also exerted a sparing action on the Mn+ requirement of this organism. These and related observations are presented below. An explanation of the observed antagonistic effects is offered based on the assumption that Zn++ inhibits growth by interfering with the formation of one or more metabolically essential metalloproteins from certain proteins (apoenzymes) of the cell and that those ions that counteract the inhibition do so by forming active complexes with these same proteins. This explanation represents an extension of the theory of competitive analogue-metabolite antagonism to ion antagonism in bacteria

The relation of ion antagonism to the inorganic nutrition of lactic acid bacteria

Observations on the function of sodium in the metabolism of a marine bacterium.

In 1882, Ringer’ showed that a solution of sodium chloride would not maintain the beat of a heart perfused with it unless additions of calcium and potassium chlorides were made. This initial observation of what is now called “ion antagonism” was extended to intact animals by Loeb12 to plants by Osterhout? and to bacteria by Flexner? E i ~ l e r , ~ and Lipmann.e Subsequently, results of a large number of investigators (cf. Falk7) have emphasized the importance of maintaining the proper ratio of ions for the proper functioning of biological systems, and a host of “antagonistic” or “synergistic” relationships between individual ions in particular biological systems has been observed. The trend of thought concerning ion antagonism can be summarized in the statement by Falk in 19227 that “the effect exerted by electrolytes appears to be primarily an effect upon external or internal membrane surfaces and upon surface interphases in colloidal systems.” Few attempts a t a more exact delineation of the mechanism of ion antagonism in living organisms have been made. Since knowledge of the inorganic requirements of the biological systems studied was either limited or nonexistent, no attempts to relate ion antagonism to nutritional requirements of the organism were made. Indeed, Loebs discounted the possibility that physiologically balanced ion solutions had nutritional significance, on the grounds that certain fish studied by him survived for long periods of time either in distilled water or in a solution containing correct proportions of NaC1, KCI, and CaCL, but not in “unbalanced” solutions of these ions. I t is the thesis of the present paper that many cases of ion antagonism can be explained on nutritional grounds, ie., that an ion which suppresses growth frequently does so by interfering with one or more of the essential metabolic roles played by an ion required for growth. Since the nutritionally essential trace elements function at least in part as necessary components of metabolically essential enzymes, a more exact picture of the mechanism of action of antagonistic ions might be to visualize a competition between the antagonists for an enzyme surface. An enzymatically active metalloprotein (MEE) results from the normal combination of the nutritionally essential ion (ME) and the apoenzyme (E) ; an enzymatically inactive metalloprotein (MTE) results when the “toxic” ion (MT) is thus combined. If combination of both metals with the protein is readily reversible, as diagrammed here, then the extent to which the enzyme can function (and growth of the organism proceed where the functional enzyme is required for growth) will depend onlyupon the ratio of ME to MT and not upon the absolute concentration of either, and a true competitive type of inhibition will result. Noncompetitive and intermediate types of inhibition could also result where reaction (2) was irreversible or only partially reversible.

Robert A. MacLeod

Papers

Some mineral requirements of the lactic acid bacteria.

The effect of related ions on the potassium requirement of lactic acid bacteria.

The relation of ion antagonism to the inorganic nutrition of lactic acid bacteria

Observations on the function of sodium in the metabolism of a marine bacterium.

Ion antagonism in bacteria as related to antimetabolites.