Prof. E. N. Mishustin

Environment and the Variability of Microorganisms

The session of the All-Union Academy of Agricultural Sciences named after V. I. Lenin (VASKhNIL), held in August 1948, marked a turning point in the development of biology. The session focused primarily on questions of variability and heredity in organisms, but the problem was resolved on a methodological basis of general significance and therefore applicable to other branches of science.

Soviet science is founded on Marxist philosophical materialism, which maintains, in the words of Comrade Stalin, “... that the world and its laws are fully knowable; that our knowledge of the laws of nature, tested by experience and practice, is reliable knowledge having the significance of objective truth; that there are no unknowable things in the world, but only things not yet known, which will be disclosed and known through the efforts of science and practice.”¹

The Marxist dialectical method opposes genuine science to the unscientific metaphysical view of the world as a chance accumulation of phenomena, and regards it instead as a unified whole in which objects and phenomena are organically connected and depend on one another.

The phenomena of variability and heredity have clearly revealed the fundamental difference between these views. The work of Timiryazev, Michurin, and Lysenko leads to recognition of the decisive influence of external conditions on organisms and of the inheritance of new properties acquired as a result of this influence. Lysenko notes that changes in heredity occur in a manner corresponding to the action of external factors. For the offspring of these organisms, such changes may be harmful, beneficial, or neutral, depending on the relationship of the new traits to all the conditions of life taken as a whole. The stamp of adaptation is imposed by the historical factor of selection—variability, heredity, and survival.

The representatives of formal genetics categorically reject the possibility that the nature of organisms can change under the influence of living conditions. In their view, the development of organisms is governed by “internal factors,” which essentially links such ideas to the idealist theory of the “self-development of the idea,” giving form to itself in inert matter.

¹ I. Stalin, Problems of Leninism, 11th ed., 1947, p. 543.

When autogeneticists speak of randomly occurring mutations as the basis of the evolutionary process, they overlook the fact that mutations are extremely rare and nonviable and therefore cannot become a source of speciation. The attempt to explain evolution on the basis of individual, not entirely favorable mutations that yield viable variants upon crossing appears largely illusory.

Matters are no better with the Morganists, who maintain that changes in heredity amount essentially to the recombination of genes. According to this conception, evolution cannot follow an ascending path and must be regarded as a process of impoverishment of the original gene pool as a result of the divergence of genes.

All these views lead to a rejection of the creative transformation of the nature of organisms, whereas optimism is characteristic of the Michurinist school of biology. Michurin said: “We cannot wait for favors from nature; our task is to take them from her.”

In his report marking the 31st anniversary of the Great October Socialist Revolution, Comrade Molotov, referring to Michurin’s words just quoted, observed: “This Michurinist precept, one might say, is imbued with the Bolshevik spirit and calls not only scientists but also millions of agricultural practitioners to living, creative work for the benefit and glory of our people.”

The discussion attracted such great attention among the broadest circles of working people in our country because of its general philosophical significance and because “...the guiding ideas of Comrade Stalin here, too, played a decisive role, opening up broad new prospects for scientific and practical work” (V. M. Molotov).

The vast body of material accumulated to date in microbiology clearly confirms the unquestionable correctness of the Michurinist school of biology. This applies to fundamental questions concerning the mechanism of hereditary phenomena, the formation of new hereditary properties in microbes under the influence of environmental conditions, the formation of microbial communities in natural settings, and so on.

It is quite clear that a short article can address only a few aspects of this broad and complex problem; we therefore confine ourselves chiefly to our own experimental material.

First, let us consider the role of various cellular structures in the transmission of hereditary properties. Certain microorganisms, such as bacteria, are exceptionally convenient subjects for investigating this problem. They possess an extraordinarily high capacity for reproduction. This makes it easy to trace the fate of a given trait in their progeny. It is also highly valuable that bacteria lack the sexual process, which complicates analysis of the mechanism by which traits are inherited. It is therefore unsurprising that many scientists have investigated general questions of heredity using bacteria. One may recall here the studies carried out in the Soviet Union by Imshenetsky, Krasilnikov, and other biologists.

If we set aside the extreme and unfounded assertions of a few scientists about the excessive lability of the nature of microbes, we must conclude, in general, that hereditary phenomena in bacteria do not differ fundamentally from those observed in higher organisms, although of course they must also have distinctive features of their own. In particular, we find the same conservatism of vitally necessary traits, among which the principal morphological and physiological characteristics of microbes must be included. The reasons for this conservatism were explained by Lysenko, who writes: “The situation is different

...with those organs, traits, and properties whose development plays an essential role in the life of the organism. Everything in the organism is directed toward ensuring that such organs or traits develop under conditions that do not depart from the norm. Their development therefore varies considerably less. Their heredity is usually more conservative and less amenable to change, because it is more strongly protected and safeguarded by the entire system of the organism as a whole.”

Lysenko indicated approaches to reshaping the nature of higher plants as rapidly as possible and in the desired direction. With due regard for the particular characteristics of microbes, these principles should also be extended to the world of lower organisms.

A legitimate question arises: is the transmission of hereditary properties in bacteria linked to nuclear material, as the followers of Weismann and Morgan maintain, or is it not? This explains the intense interest that the problem of the existence of a nucleus in bacteria has attracted over a long period, including in recent times.

A number of scientists, both abroad and in our country, have used highly refined staining techniques in an effort to demonstrate the presence of a distinct nucleus in all groups of bacteria. Some proponents of this view (Ravich-Birger and others) were inclined to attribute to the bacterial nucleus a structure analogous to that of higher organisms. Thus, attempts were made to link the phenomena of heredity in all living things to the characteristics of their nuclear apparatus and to explain them from the standpoint of formal genetics.

Meanwhile, a great deal of evidence, particularly striking in the work of Imshenetsky, has shown that at least the group of bacteria assigned to the order Eubacteriales has no nucleus. Nucleoproteins, which are constituents of the nucleus, are present in bacterial cells, but here they are dispersed and become separated from the rest of the cytoplasm only during degeneration or spore formation in bacilli. The structures that many microbiologists have taken for nuclei arise during cell staining and are artifacts.

Recently, in 1947, the author of this article, together with Biryuzova, had an opportunity to study the cell structure of a rather large filament-forming bacterium named Bac. longissimus. Careful examination of this microbe showed that its readily observable cells lack a nucleus.

More highly organized bacteria of the order Myxobacteriales, which have a complex developmental cycle, contain a distinct nucleus in their cells.

At the same time, in different groups of bacteria, both those with a nucleus and those without one, traits are inherited in essentially the same manner. This clearly demonstrates that the phenomena of heredity cannot be linked exclusively to the nuclear apparatus. It is also unsurprising that the school of formal geneticists has always sought to disregard the facts established by microbiologists, the substance of which contradicted that school’s assumptions.

Turning to the factors that determine the variability of microbes, we should point out that this question was raised as early as the 1870s, shortly after the appearance of Darwin’s classic works. The fundamental tenets of the schools of thought discussed above, which differed in principle, found expression in a wide variety of views that we cannot examine in detail here. Over time, the disagreements between them grew ever deeper.

Cohn allowed for the variability of microorganisms but placed it in the past. Under present conditions, he essentially denied the possibility that the fundamental properties of microorganisms could change. Later, Hansen revealed...

…clearly autogenetic concepts. He maintained that changes arise spontaneously in microbes and that the external environment, like a sieve, selects forms suited to the prevailing circumstances. More recently, various cyclogenic theories (Enderlein and others) have been advanced, reducing microbial variability to closed, predetermined cycles independent of environmental conditions.

The idealist essence of all these views has been exposed in the work of a number of Soviet microbiologists.

The examples given show how far some researchers had become detached from the realities, which at every turn testify that environmental conditions shape the organism.

As already noted, the greatest naturalists repeatedly emphasized this point. In addition to the statements quoted earlier, Engels, Darwin, V. Kovalevsky, Sechenov, and Pavlov expressed similar views. Engels wrote of the refinement of the human hand: “The hand is thus not only the organ of labor; it is also its product. Only through labor, through adaptation to ever new operations, through the inheritance of what was thus acquired, especially the development of the muscles ... did the human hand attain that high degree of perfection at which it could, as if by magic, call into being the paintings of Raphael, the statues of Thorvaldsen, the music of Paganini.”

It should be emphasized, however, that before Lysenko the experimental investigation of the immediate causes and sources of particular changes arising in the nature of the organism had not been undertaken on a broad scale. According to Lysenko, the heredity of an organism is determined by the relationship between its needs and the external conditions that help create specific properties of the organism that are passed on to its offspring. One method of studying heredity is therefore to identify the material conditions among those external environmental factors required by the nature of the developing organism. No trait can develop in an organism without the corresponding conditions in the external environment.

The evidence concerning changes in the properties of microorganisms under the influence of the environment is extensive and varied.

As early as Mechnikov’s time, he pointed to a number of facts indicating hereditary changes in bacterial traits under the influence of their living conditions. More than 60 years ago, Tsenkovsky cultivated the anthrax bacillus under different conditions and obtained strains that had permanently lost their virulence. Tsenkovsky’s vaccines came into widespread practical use.

The studies of Kedrovsky, Konev, and Gamaleya on the variability of a great many pathogenic microorganisms are well known.

In recent years, a large series of studies devoted to this question has appeared in the USSR. They are reviewed in an article by Vygodchikov and Grinbaum.¹

The influence of the environment on microbial virulence is also strikingly evident in nonpathogenic forms. This is vividly illustrated, for example, by the nodule bacteria, which play an enormous role in agriculture. Whole regions are often found in which, despite the presence of nodule bacteria, the root systems of leguminous plants are poorly infected. As a result, the bacteria do not perform their nitrogen-fixing function. Only the artificial inoculation of the soil with virulent races of nodule bacteria remedies the situation.

Each race of nodule bacteria can infect only a particular group of leguminous plants. Krasilnikov made the observa—

¹ Journal of Microbiology, Epidemiology and Immunobiology, no. 4, 13, 1949.

…showing that, when grown on certain media, cultures of root-nodule bacteria acquire the ability to infect plants on whose roots they had not previously formed nodules. Thus, in some cases, there are ways of conferring entirely new properties on microbes.

Related to these phenomena are observations concerning changes in toxigenic forms of bacteria. Thus, while some microbiologists recognize the existence of forms of Bact. vulgare that produce toxin and others that do not, Potkov (1947–1948) was able to demonstrate considerable variability in this characteristic. It can be either enhanced or diminished by appropriate methods of bacterial cultivation.

We shall not discuss here a number of recent studies demonstrating the possibility that saprophytic forms of microbes can become pathogenic. Without denying this possibility in principle, we must note, however, that in some cases investigators approach the problem too simplistically, forgetting the conservatism of heredity and the complexity of transforming the nature of living organisms.

Let us consider another group of phenomena: changes in the enzyme complex of microbes under the influence of the environment. As is well known, the set of enzymes in a microorganism’s cell is specific; it determines the nature of its metabolism and serves as a fairly characteristic diagnostic feature. At the same time, living conditions can substantially rearrange the biocatalysts present in the cell and induce the formation of new enzymes. We shall not discuss here the so-called adaptive enzymes in microbes, which manifest activity only when the corresponding substances are present in the medium. Otherwise, they are present in the cell in a zymogenic state. Thus, in this example, what is involved is essentially a change in the state of one enzyme or another. In a number of cases, however, it has been reliably established that new enzymes appear or old ones disappear when the living conditions of microorganisms change, and that these changes become hereditary.

Let us give a few examples. Kudryavtsev, for instance, cultivated yeasts that do not ferment maltose on a medium containing this carbohydrate and ultimately succeeded in obtaining an altered strain that breaks down this sugar.

Minkevich showed that the typical intestinal bacillus, after a prolonged stay in soil, acquires the ability to assimilate citrates; that is, its properties come to resemble those of Bact. aërogenes. In this case, there is some correlation between the adaptive process and the tendency to assimilate citric acid compounds, since citrates are, of course, not present in soil.

Very interesting observations in the field of interest to us have been made with azotobacter, a bacterium that assimilates molecular nitrogen. According to a number of studies, including those carried out in our laboratory, certain environmental conditions cause the complete and heritable inactivation of the enzyme complex that helps the microorganism assimilate gaseous nitrogen. This is achieved, in particular, by prolonged cultivation of azotobacter on media containing ammonium salts or nitrates. In such a case, the abundance of mineral nitrogen makes the presence in the cell of a catalytic system that fixes molecular nitrogen unnecessary, and the system gradually diminishes.

A similar effect is observed when azotobacter remains for a prolonged period in soils with a low pH. In this case, the loss of the enzyme complex is apparently explained by its…

sensitivity to acidity. As our experiments have shown, the new variant of Azotobacter transmits its properties by heredity.

As is well known, cultivation conditions have a marked effect on the ability of microorganisms to produce antibiotic substances. By establishing certain conditions, one can either enhance or entirely eliminate the ability of some microbial cultures to produce antibiotics. The practical significance of this is obvious.

Temperature adaptation in microorganisms provides particularly striking examples of directed variation. Microorganisms adapt to elevated temperatures only with great difficulty, and the cardinal temperature points of microbial cultures are a fairly stable and strongly inherited trait. The conservatism of this temperature trait and the difficulty of raising the maximum temperature are readily understandable. Life at high temperatures is possible only with substantial changes in the constituents of the cell’s colloidal systems, its permeability, and the chemical composition of its lipoids; with a reorganization of enzyme systems to replace heat-labile components with more heat-resistant ones; and so on (Imshenetsky, Mishustin, and others).

Thus, an organism acquires the ability to grow at elevated temperatures only through a fundamental reorganization of its physiological functions, essentially resulting in the formation of specific races. Nevertheless, by creating the necessary conditions, it is possible, for example, to obtain thermophiles from mesophilic microorganisms. There have been a considerable number of such successful attempts. We shall discuss only a few examples in greater detail here. Thus, Dallinger (1887) succeeded in acclimating protozoa to develop at fairly high temperatures. The experimentally obtained variants of these protozoa could grow at temperatures lethal to the original culture.

Darwin was greatly interested in Dallinger’s experiments. In a letter dated 2 July 1878, he wrote to Dallinger: “I did not know that you were studying the modification of lower organisms under the influence of changes in their conditions of life, and I have no doubt that your results will be extremely interesting and valuable. The fact you mention, that they live at different temperatures but can gradually become accustomed to considerably higher ones, is very remarkable. It explains the existence of algae in hot springs.”

In recent years, Loginova succeeded in obtaining a yeast culture that develops at an elevated temperature, a finding of undoubted interest to the fermentation industry.

We have shown that, in saprophytic soil bacteria living in different climatic zones, the optimum and maximum temperature points are functionally related to the mean annual air temperature. As noted above, this trait, which is linked to a number of biochemical characteristics of the cell, is hereditarily fixed and transmitted through a considerable number of generations. In such ecological races of bacteria, as we established in the case of Bac. mycoides, colony structure, rates of reproduction, the activity of different groups of enzymes, osmotic pressure, and other characteristics vary. All this supports the conclusion that the long-standing postulate of the vulgar cosmopolitanism of microorganisms in nature must be abandoned. Like higher organisms with a wide geographical range, microorganisms have ecological races whose characteristics have developed under the influence of particular environmental conditions.

The principles set out above are of fundamental importance for analyzing the processes by which components of the external environment purify themselves, and for finding

substrates for isolating pure cultures of microbes with specific properties, and so on.

To conclude the section on the influence of the external environment on the properties of microorganisms, we wish to say a few words about the artificial production of cultures of these organisms with prescribed properties. Such a need often arises in practice in medicine, agriculture, the fermentation industry, and other fields. The fundamentally different views on the nature of heredity discussed above lead to methodological approaches to this practical problem that are far from identical. Adherents of the Michurinist school address the problem by creating environmental conditions that induce the desired transformation of the organism. This is directed variation: the creation of forms of living organisms according to a predetermined plan. There is scarcely any need to demonstrate the logic of the approach we have briefly outlined.

Researchers who disregard or ignore Michurin’s teaching attempt to produce new traits in an organism by applying various powerful agents intended to induce mutational changes in hereditary properties. X-rays, light energy, poisons, and other agents are used in such work. Treatment of a microbial culture with appropriate stimuli does, of course, lead to the appearance of cells with novel potentialities; but because the variation employed is not directed, it is extremely difficult to find among the descendants cells with the properties of interest to the researcher. Such work, in Lysenko’s vivid expression, resembles treasure hunting.

The selection of microorganisms is now becoming a matter of great economic importance. Microbial cultures with specific properties are needed for the production of vaccines and antibiotics, for the fermentation industry, for agriculture, and so on. The use of correct approaches in this field will greatly facilitate the task.

The final subject we wish to address in this article, which is directly related to the problem of “environment and organism,” concerns the study of microbial communities in the external environment. Since our work has been directly concerned with soil, we shall confine ourselves to an analysis of this substrate.

An essentially anti-Darwinian view of the microbial population of soil has become established in microbiology. Microorganisms, including bacteria, are considered cosmopolitan, and consequently the microbial populations of soils even in different climatic zones are thought to differ primarily in quantitative rather than qualitative characteristics. Exceptions are made only for certain microorganisms, such as Azotobacter, which is highly demanding with respect to the composition of the substrate.

Yet the properties of the nutritive material available to microbes vary enormously among different soils, and, on general grounds, one would expect the composition of the environment to be reflected in the microbial population of the substrate. Observations to this effect can be found in the works of Academician Williams.

Our work in recent years has fully confirmed the view just mentioned. It has been found that each soil type possesses a highly distinctive microbial community, and that cultivation has a noticeable effect on its composition. The latter fact makes it possible to use microorganisms as indicators of soil type and condition.

Let us give a few examples. Thus, the Bac. subtilis–Bac. mesentericus group is found in large numbers only in the soils of the southern

zone (chestnut and sierozem soils). Another bacterium, Bac. mycoides, is found predominantly in soils of the podzolic zone. Bac. cereus is the dominant form in soils of the more northerly belt, and so on.

These observations are significant not only for agriculture. As is well known, the self-purification of soil from pathogenic bacteria depends to a considerable extent on the presence of antagonistic microbes. The latter are found in substantial numbers only among certain species. For example, they are particularly abundant in the Bac. subtilis–Bac. mesentericus group.

It is therefore evident that the potential capacity of soil for self-purification cannot be determined without taking into account the particular composition of its microflora.

In concluding our review, we must note that the Michurinist approach enriches the development of both new fields of microbiology and those that have already received considerable attention, fields capable of rendering valuable services to various sectors of the national economy.