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Part I, continued:

   Microorganisms manifest considerable variability. They are more variable and react to environmental factors more strongly and rapidly than higher plants. However, there is no basis for regarding them as highly polymorphic organisms. Bacteria, actinomycetes, fungi and other specimens of microbes undergo changes according to their species properties.

   An higher plants and animals, they have their evolution and philogeny which determine the development of cultures in ontogenesis and the formation of new forms and, in general, the nature of variability of the species.

   In this the microorganisms sharply differ from higher plants. Peculiarities of their structure, development, and many biochemical processes separate these creatures into independent groups or classes of organisms. Biological specificity of microorganisms is also undoubtedly reflected in the phenomenon of variability of their species. The regularities observed of variability processes in bacteria, actinomycetes and other microbes do not fit into the schemes and theories of variability of higher organisms.

   Not long ago, the followers of formal genetics refuted in general any regularity in hereditary variability of microorganisms only for the reason that the nature of alterations did not correspond to the "laws" of Mendel and to other established points of view. Microbes were regarded as defective organisms, as a result of which the whole factual material on variability was disregarded.

   Under the pressure of enormous material amassed in the literature, geneticists have recently also been forced to pay attention to microorganisms. Many species of bacteria, yeasts and fungi became the preferred subjects for attempts at the solution of a series of fundamental genetic problems. Many works, mono -graphs and reviews are devoted to the genetics of microbes.

   Reviewing this material, many foreign authors (Dubos, 1948, Lederberg, 1954, Braun, 1953, Catcheside, 1951, Hayes, 1953, and others) treat it from positions of pure genetic conceptions. According to their opinions, all alterations of a hereditary order in microbes are conditioned by genes. Genetic conceptions, applied to higher plants and animals are applied wholly into the microbial world. The authors take into account neither the specific structure and development of microbial cells, nor the manifestations of their life activity. Even the factual material which often speaks against these conceptions is used by them in attempts to prove their points of view.

   It should be noted that the changes observed in microbes were long considered as phenomena contradicting the universally recognized laws of formal genetics. However, microbiology was at that time deprived of theoretical generalization and could not offer its views against the existing genetical ones. Opinions of a conciliatory nature were put forward. The peculiarities of the variability phenomenon in microbes were regarded as a deviation from the general genetic law and other names were suggested for designation of separate manifestations of variability. Instead of mutation, the word saltation was used, etc.

   Together with the development of the theory of Michurin, microbiology in our country and in others generalizes this material in an entirely different way and draws conclusions according to the basic laws of Darwin on the variability of organisms.

   The large experience of microbiology shows that heredity is not a manifestation of some invariable particles of living substance, transferred from generation to generation during an infinite series of generations. Heredity cannot be regarded as a manifestation of idioplasm nor genes be regarded as carriers of hereditary features, not subjected to environmental influences,

   Heredity is a property of the whole organism, the whole protoplast or living substance, and not of separate particles located in chromosomes or in chromatin corpuscles of the nucleus, although the latter is of essential importance. The transmission of traits from one generation to the other, the ability to form new properties, develops in the organism during the whole historical process and depends upon the conditions under which the given microbe exists and develops. The property of inheritance of traits is formed during a long series of generations of one or another species. Formation of new species occurs under the influence of environmental factors and strictly according to the hereditary basis. New forms are produced due to the influence of various agents on the hereditary basis. Knowing the natural properties of the organism and its adaptive features, one may obtain, and in fact does obtain, useful variants by a choice of suitable conditions of growth and development.

   It is possible to create a new organism and to direct its development, varying the conditions of growth, following the history of its evolutionary development. This theory of Darwin was well confirmed by Michurin and his followers in numerous experiments in laboratories as well as in the fields.

   The enormous amount of microbiological material on adaptive variability at present induces many followers of the theory of corpuscular heredity to revise and alter their views on the variability and heredity of microorganisms. The investigations by foreign scientists (Ephrussi et al., 1949, 1956, Hinshelwood et al. , 1946. 1952; Monod et al., 1942, 1952, Slonimskii, 1956 and others) show the special importance of environmental conditions in the creation and formation of new species. In many instances they showed that the environment or substrate induced the formation of new features in the organism, and stabilized and transferred them into the constructive stage of living plasma.

   The spectfity of the development of microorganisms, and their unusually rapid proliferation enables us to follow a series of life activities in a large number of generations in a relatively short time and to establish regularities and particular features of species -forming processes. If, in order to establish the phenomenon of heredity in higher organisms many years are needed, in microorganisms it may be elucidated in one to two days. Thanks to the biological specificity of microbes, one succeeds in following a series of biochemical processes in them which are connected with the mechanism of variability and heredity. It is quite natural that many questions of genetics are solved on microbiological references.

   Variability in microorganisms is manifested in many ways.

   There is individual variability, age variability, species, adaptive, induced variability, etc.

   When observing cultures of microorganisms under the microscope, whether they are bacteria, yeasts or others, one may always notice a greater or lesser diversity of cells. Cells differ from each other in size, form and other external features and also in their internal structure. Some of them possess a homogeneus plasma without any apparent structure, others granular, vacuolized or without vacuoles. In some cells there is much reserve food--metachromatin, lipides or other substances; in still others--there is little or they lack it entirely (Krasil'nikov, 1943).

   Individual differences of cells of one and the same culture are manifested to various degrees depending on the age, species and growth conditions. Cells of old cultures differ most sharply from each other when their involution begins. In these cases, as was indicated above, the cells may assume diverse forms and sizes.

   Of great interest are very enlarged cells and cells of ultramicroscopic sizes or filterable forms.

   Formation of greatly enlarged, deformed cells as a reflection of polymorphism is noted in cultures of various bacteria and actinomycetes. The enlarged cells are of various sizes and patterns. They may be spherical, bottle-shaped, spindle-shaped, filamentous, amoeboid, etc. Often the long cells form side appendages resembling branches, and in swollen cells budlike formations often appear on the surface (Figure 30).

Figure 30, Involution cells of bacteria, greatly swollen and deformed:

A) Azotobacter unicapsa, strain A--5 days on must agar (pH = 6.5) 1:1,000; B) Azotobacter chroococcum--1-2 days culture on must agar; C) Achromobacter epsteinii (Peshkov, 1955). Growth of culture at 10° C, 1:2160.

   Such cells are tens and even hundreds of times larger than normal ones, often reaching gigantic dimensions. The protoplast of these greatly enlarged cells differs noticeably from the protoplast of normal ones. Their plasma is vacuolized with many inclusions of metachromatin, lipides, glycogen-like substances, etc. An accumulation of chromatin inclusions (see above) is characteristic of them.

   As a rule, these cells are less viable, they do not develop and multiply; usually they perish, disintegrate and undergo autolysis. Such cells are designated as involution forms. If the process of degeneration did not go far, the cells may under certain favorable conditions regenerate and produce normal progeny.

   With some bacteria, one may maintain a culture in a degenerative state for a long time, with characteristic, greatly enlarged and deformed cells. The latter multiply and give rise to organisms with degenerative features.

   We have preserved, In a degenerative form, cultures of Azotobacter, growing on must agar, or on a medium with meat-peptone broth, and a culture of root-nodule bacteria of pea, clover, vetch and other legumes, growing on meat-peptone agar. The indicated media are hardly suitable or not at all suitable for growing these bacteria. The development of cells is weak and as a rule they are of abnormal size and form. Though they do not lose the ability to proliferate, they do it in an unusual way--by budding and fragmentation. Consequently, any degeneration or deformation of cells does not necessarily lead to death.

   Strongly swollen cells are formed under the influence of physical, chemical and biological factors.

   Very large, deformed cells may be obtained under the influence of chemical substances. Gamaleya obtained them in a medium with lithium chloride. They are formed under the action of increased concentrations of ordinary salts, in the presence of certain dyes, by the effect of ultraviolet rays, supraoptimal temperature, antibiotics, phages, etc. As a rule, all unfavorable agents affecting the growth of microbes may provoke the indicated forms of degenerated cells Imshenetskii, 1940; Krasil'nikov, 1954 b; Iensen, 1954; Peshkov, 1955).

   The degree of morphological degeneration in various bacterial species and actinomycetes is different. In some it is strongly manifested, in others weakly, almost unnoticeable.

   Morphological polymorphism is very strongly manifested in Azotobacter. The diversity of cells in this species amounts to several tens of forms. Lönis and Smit (1916) counted 13 different types, we (1954b) and Bachinskii (1935) have found about 200 forms. The study of a large collection of Azotobacter showed that such polymorphism is not manifested in all strains.

   A great diversity of cellular forms of a degenerative nature is noted in some mycobacteria--Mycob. cyaneum and others. In actinomycetes this phenomenon is reflected mostly in Act. violaceus (Krasil'nikov, 1938a. 1954 c).

In sporeforming bacteria Bac. megatherium, deformed cells were studied by Kudryavtsev (1932). The diversity of the forms which he described was noted upon cultivation on conventional nutrient media, but more characteristic forms and better manifested forms occured on sugar media (must agar, etc).

   Peshkov (1955), studying the formation of reactive forms in the microbe Bact. epsteinii which he isolated, regards this bacterial species as an example of an unusual polymorphism.

   The formation of swollen forms is observed in various bacterial species--pathogenic and saprophytic (see Kalina, 1953, Muromtsev, 1953; Elin, 1954 and others). Numerous investigations show that bacteria react to various antibiotics--penicillin, streptomycin, aureomycin and many others--by a sharp alteration in their size and form. Deformations similar to those formed upon the action of other agents occur (Krasil'nikov, 1950, Troitskii and others, 1950).

   Aside from the formation of very large cells in cultures of bacteria and actinomycetes, the formation of tiny gonidial elements is observed frequently. Under the microscope in almost every culture, particularly in old ones, one may see among the usual cells very small bodies in the form of granules, which refract light weakly. These bodies often accumulate in a great quantity, forming a fine-grained sediment.

   The size of the fragmented cells is very small ranging from 0.5 u to a size hardly visible under good optical microscopes and even smaller --(beyond the limits of visibility).

   Formation of the diminutive forms may occur in various ways, It may be observed during the process of fragmentation of cells when division is not accompanied by growth or the latter is weak. With each successive division, cells of smaller size appear. In some bacteria (Azotobacter) such fragmented cells are found in a mucous capsule. As a result, it looks as if a large cell, filled with gonidial elements, resembling nanocytes of algae is produced, The size of such "nanocytes" vary within the limits of 0. 1 - 0. 5 u in diameter (See Figure 16).

   The fragmentation of cells is observed in sporeforming and nonsporeforming bacteria, in actinomycetes and mycobacteria, therefore the process is often accompanied by autolysis of the maternal cells.

   As was described above, tiny cells are also formed by budding.

   In separate cases inside the often deformed, and swollen cells, very small gonidia are formed. These are the so-called regenerative forms. Their formation proceeds from the protoplasm with the necessary participation of chromatin granules, as was stated above.

   Owing to their small size, all those forms pass through bacterial filters and may be regarded as filterable forms. Together with the latter they evidently constitute one group of reproductive elements. These forms are also close to each other in their properties. Both are slightly viable, and do not develop or develop weakly on the usual nutrient media.

   In recent years great attention has been attached to polymorphism which is connected with the formation of the highly altered, so-called L forms in some bacteria. The most detailed study was made on Streptobacillus moniliformis, Klieneberger-Nobel (1949). Oerskov (1942) and others; showed that in a given organism which is grown on artificial nutrient media, very different forms are manifested. Beside large, greatly swollen forms, there are extraordinarily small elements resembling the pleuropneumonia agent. The latter are so peculiar, that many investigators consider them a special branch of microbes having nothing in common with bacteria. Small forms of the pleuropneumonia type are described in many bacteria.

   Cells of one and the same culture differ from each other not only morphologically but also physiologically and biochemically, therefore the differences may be quantitative and qualitative. Although there are no methods for studying the physiology of separate cells, observations on hanging drops show that this difference exists. It is manifested in the reaction of cells to environmental stimuli. Depending on individual physiological properties, metachromatin accumulates rapidly and in large quantities in some cells, while in others it accumulates in a small quantity and slowly, in still others it is altogether lacking. Accumulation of fat. glycogen, glycogen-like substances and other inclusions, occurs in some cells and not in others. Some cells multiply rapidly, others slowly. Rapidly-growing cells produce two to five generations and more, while slow-growing cells produce one to two generations during the same period, or do not start division at all. If cells which are filled with reserve nutrient substances are transferred to a poor medium, the reserves proceed to disappear in various ways: in some cells it proceeds rapidly, in others, slowly.

   Bacterial cells react in a different way to the action of antibiotics--penicillin, streptomycin, aureomycin, or other preparations. Some die rapidly, others remain in a subvital condition; on a transfer to a fresh normal substrate they do not grow or grow only under special conditions of complementary nutrition; still others grow, but do not multiply or multiply slowly, and subsequently, swollen. degenerative forms appear. There are resistant cells, growing and proliferating more or less normally, without alteration of external form and size.

   Hughes (1953-1955) indicated that the two daughter cells of bacteria (Bac. coli Bac. proteus), formed after the division of the maternal cell, differ from each other in their sensitivity to antibiotics, lack of oxygen, and some other factors.

   Polymorphism of cells is also manifested in biological reactions. Cells of one and the same culture differ from each other in their resistance to various agents. Upon the action of increased temperature some cells die, while others are preserved but still others develop and multiply. This to also observed under the action of radiation, chemical reagents, etc.

   The sexual process is manifested in some cells (in yeasts), in others it is lost. Some cells form spores, and others do not.

   Consequently, cultures of microorganisms consist of cellular forms of great diversity; of organisms quantitatively nonhomogeneous.

   These differences--morphological, physiological and biochemical--are conditioned by the biological peculiarities of microbial cells. They are not fortuitous but biologically conform with natural laws in all microbial species. Qualitative diversity is of great biological importance. Owing to the diversity of cells, the over-all microbial culture of species possesses greater possibilities of adapting itself to various conditions. If this diversity is lacking. the whole culture would die upon the action of the first unfavorable condition.

   Diversity of a culture or the polymorphism of cells should be distinguished from the qualitative diversity of polymorphism of species. Polymorphism of cells in encompassed by the concept of the polymorphism of species but is not identical to it.

   The qualitative diversity of microbial cultures also determines to some degree the polymorphism of the species. The species in microorganisms is characterized not only by the polymorphism of cells but also by the polymorphism of the culture. One and the same species, even one and the same culture often produce various colonies on nutrient media. It is known that Azotobacter chroococcum on one and the same medium of Ashby (agar medium) grows either in the form of a mucoid spreading colony, or in a compact, pastelike form; it may be black, brown or colorless; in some cases it is wrinkled or rough, in others in the form of smooth, pastelike or mucoid colonies.

   In the sporeforming bacteria Bac. mesentericus, Bac. subtilis, Bac. mycoides, Bac. brevis, Bac. cereus and others, from five to ten types of colonies may be differentiated on agar media. One and the same culture of Bac. mesentericus upon inoculation into nutrient agar often forms a mixture. typical and atypical forms, rough and folded, finely-plicated; dry, spreading on the agar surface; flat, smooth, pellicle-like, glossy with a single radial fold (Figure 31).

Figure 31. Polymorphism of cultures of sporeforming bacteria Bac. mesentericus:

A) strain No 12, isolated from serozem of Central Asia; B) strain No 110, isolated from the turfy-podzol soil of Moscow Oblast'; variants of types of colonies obtained upon plating on nutrient medium (meat-peptone agar).

   In Bac. mycoides atypical, granular, anthracoidal, folded smooth and other colors have been described (Figure 32) (Lewis, 1932; Rautenshtein, 1946;Mishustin, 1947; Afrikyan, 1954a; and others). Considerable polymorphism of cultures is observed in nonsporeforming bacteria: the root-nodule bacteria specimens of Pseudomonas. Bacterium in lactic acid bacteria, Mycobacteria and others. A great diversity of colony structure is noted in actinomycetes particularly. In these organisms polymorphism is so great, that doubts arise among investigators as to the possibility of a species differentiation. Lieske (1921), for instance, refused to divide actinomycetes into species, being of the opinion that the diversity of forms observed is a result of polymorphism and not of species difference.

Figure 32. Polymorphism of a culture of Bac. mycoides. Types of colonies, obtained after plating on meat-poptone agar MPA. Strain isolated from the turfy-podsol soil of the Moscow Oblast,

   According to this point of view, the sporeforming bacteria--Bac. subtilis and Bac. mesentericus are extreme variants of one and the same species.

   According to our observations, Bac. subtilis, Bac. Mesentericus, Bac. cereus, Bac. brevis differ essentially from each other only by the form of colonies and character of growth on artificial nutrient media. Usual physiological indications, liquefaction of gelatin, curdling of milk, and decomposition of starch, are essentially the same for these organisms. More refined biochemical properties have not been revealed in them.

   The homogeneity of the mentioned bacteria is also confirmed by the method of experimental variability. By the ordinary cultivation and subsequent plating on various nutrient media of a typical culture of Bac. mesentericus, one may obtain cultures of the type Bac. subttlis, Bac. cereus or Bac. brevis, Bac. pumilis and some other forms in a relatively rapid manner.

   The so-called typical strains of Bac. cereus produce forms not differing from those of Bac. Subtilis, or Bac. brevis and Bac. licheniformis.

   From five species of sporeforming bacteria we obtained the following monotypical variants: a) wrinkled or plicate with a glossy or fatty-glossy surface, butterlike colonies; b) thin-filmy, fine-plicate, dry, mat; c) coarse-filmy with elevated edges, saucer-like with occasional radiate, separate small folds; d) dry colonies, fine-plicate. floury-white, often merging with the agar; e) grainy-plicate colonies, moist, moist-glistening fatty with uneven, slightly diffuse edges (Table 2).

   We observed the formation of monotypical variants in certain species of nonsporeforming bacteria of the genera Bacterium and Pseudomonas, in actinomycetes, mycobacteria and others. These manifestations of a monotypical character of variants in the process of variability of cultures is determined by homogeneity of the living substance and occurs in organisms which are closely related. By these variants one may judge the phylogenetic closeness of the initial cultures or whether the investigated organisms belong to, one and the same species. On this is based the method of experimental variability for the establishment of species in the classification of bacteria and other microbes (see further on).

   Diversity of forms and variants in cultures reflects the degree of polymorphism of species. The cellular as well as the cultural polymorphism has a defined organic connection with species variability. Among the multitude of unstable cellular elements or among unstable variants of a culture, separate organisms or colonies with hereditarily stabilized traits of the same property occur, In the yeast organisms, Saccharomyces cerevisiae we obtained (1934a) stable variants with properties which manifested themselves in cellular organisms as a reflection of individual variability or polymorphism. In a certain sense the species variability pre-determines individual variability. In species variability, the stable variants repeat or reproduce the traits which manifest themselves in polymorphism.

   Growth and development of the organisms and its reaction to environmental factors proceed within limits of a defined norm, separately characteristic of each species and conditioned by heredity. The alterations proceeding within the limits, of this norm do not touch the strain or species properties as long as the environment corresponds to the requirements of the organism. When the environment changes and it ceases to be suitable for the normal growth of the organism, the latter, either stops development and dies, sooner or later, or adapts itself to new conditions, altering its species properties. New variants with new hereditary features and requirements are obtained.

   In laboratory practice one may often observe the production of variants under the influence of the changing environment or the action of environmental factors. In the literature there is a great accumulation of material on the species variability of microorganisms. Variants stabilized in a hereditary way have been obtained in various specimens of bacteria, actinomycetes, fungi, algae, protozoa, plant and animal viruses, bacteriophages, actinophages, etc. They have various designations--mutations, long term modifications, saltations, adaptations, sudden and discontinuous variations, etc.

   All those forms of variability are of a hereditary nature, Due to this variability, the species properties of the microbe change, external morphological and cultural features as well as biochemical and biological features are thereby affected. The newly acquired properties and traits are transmitted to cells of the whole population.

   New hereditary variants are produced by the alteration of a separate cell in the culture. Among the individual differences in the population, such differences appear which cover species qualities and are stabilized in the progeny. Formation of hereditary variants occurs under the influence of environmental action and, as a rule, is of an adaptive nature. Variants are often obtained in old cultures without any special external action. In this case the altered medium constitutes the activating factor. The latter changes its composition and properties with the age of the culture in an essential way, the initial food elements disappear, now ones are synthesized, various products of metabolism are accumulated, its physical and chemical features change, etc. In short, the medium becomes entirely different, sometimes obviously unfavorable. In such a medium, the cells die in large numbers. There are many half-living or subvital cells, and also cells with a shattered heredity. In these cases, upon plating on a fresh nutrient substrate, colonies with new properties develop. Variants with diverse morphological and physiological properties are produced.

   With the first plating of the culture on an agar medium, as was indicated above, one may find variants with diverse cultural traits. Aside from the initial colonies, rough, wrinkled, mucoid, and hardly visible colonies which grow well appear. Under the microscope, an equivalent cell diversity in noted. The variants obtained, often differ from each other in biochemical properties. Some of them liquefy gelatin rapidly, others slowly, and still others do not liquefy it at all. The same is noted with respect to fermentation of milk, sugars and other fermentative processes.

   In Bac. coli one may observe the formation of stable variants, which lose the ability to produce gas from sugars. Some variants of root-nodule bacteria lose the ability to form nodules on the roots of leguminous plants, other variants lose the ability to ferment sugars. In Azotobacter variants are formed which are unable to fix nitrogen and to develop on a nitrogen-free medium. In pathogenic and phytopathogenic bacteria avirulent cultures are obtained. In actinomycetes one may succeed in obtaining new variants of great antibacterial activity, but the formation of entirely inactive variants is also observed.

   Hereditary alterations in microbes occur under the influence of various special factors--physical, chemical and biological. Resistant variants are obtained from the action of temperature, ultrasonic radiation energy, etc, Great attention in paid to X-rays, radium radiation and recently to nuclear radiations of uranium to well as to the effect of artificial isotopes. Ultraviolet rays are a very strong agent. Under the influence of radiation energy, many variants of practical importance which are of great industrial value have been obtained. For instance, very active variants of Penicillium chrysogenum--the producer of the antibiotic penicillin, cultures of the actinomycete Act. streptomycini, whose antibiotic properties exceed those of the initial strains by dozens of times. Under the influence of X rays numerous variants have been obtained in fungi and yeasts, with a complexity of features which differ from those of the initial strains. (Filippov, 1932; Rokhlina, 1930, 1954). When staphylococci are exposed to the action of these rays, the ability to produce the toxic factors dermonscrosin and hemolysin is lost. Under the influence of ß and gamma rays variants with a filamentous structure have been obtained from bacteria which never form them.

   Stable alterations. with respect to heredity have been obtained through the use of ultraviolet rays in various specimens of bacteria and fungi. Variants have been described with altered cultural, morphological and biochemical properties. Great attention to given to the so-called dependent or defective variants which have lost the ability to synthesize certain growth factors or vitamins. Thirty variants have been obtained from Bac. coli which require the supplementary growth substances--pyrimidine, purine, threonine, proline, phenylalanine, methionine, tryptophan, arginine, cystine and others (Table 3).

Note. Numbers designate the number of isolations of the variants, obtained by various investigators (from the book Microbial Genetics, Catcheside, 1951).

   A considerable number of dependent variants have been obtained in the fungus Neurospora (Kaplan, 1952).

   Similar results are obtained upon the action of chemical agents. Great attention to being paid to the action of colchtcin and mustard gas. These substances have a particular property for inducing variability in higher and lower organisms. However, investigations show that this property in characteristic of many other chemical compounds. There is no specificity in colchicin and in other chemical substances. Demerets and his collaborators distinguish three types of chemical substances with respect to the strength of their effect on the variability of microorganisms. Strongly active are: formalin, phenol, hydrogen-peroxide, a-dinitrophenol, manganese, iron; weakly active are: boric, acetic and formic acid, trinitrophenol, acriflavin, caffeine, necrosin, etc; of very weak activity or completely inactive are: ammonia, copper sulfate, trivalent iron, divalent cobalt, divalent strontium, etc, nonactive chemicals are: sodium hydroxide, potassium hydroxide, sublimate, lactic, sulfuric, phosphoric, nitric, and hydrochloric acids, silver nitrate and many other chemicals (cited from Kaplan, 1952).

   It should be noted that the production of hereditary stable and unstable variants also occurs in cultures without the special action of any agents, and essentially the same types of variants are thereby obtained as under the action of the mentioned agents. In our investigations (1933, 1934a) we compared the variants produced spontaneously and without special actions in the yeast Sporobolomyces and Saccharomyces with the variants simultaneously obtained by Nadson and Filippov (1932) by the action of radon and X-rays. In both cases monotypical groups of yeasts have been obtained (see also Nadson and Rokhlina, 1932; Rokhlina, 1954),

   Kurylovich and his collaborators subjected cultures of actinomycetes and fungi which produce antibiotics to the action of ultraviolet rays. Without exerting any influence, simultaneous analysis of the variability of cultures was carried out. In both cases variants were obtained, which were identical with respect to activity (Krasil'nikov, 1955c).

   The monotypical nature of the production of variants is often noted in microbiological laboratory practice and proves the great significance of the heredity of the organism.

   Among biological factors which induce the formation of variants, phages and antibiotics have recently been of special interest. The agents prove to have a great effect on the variability of microbes. Upon their action diverse variants are formed, whose characteristic property in the resistance to the given stimuli.

   Changes obtained in various ways in microorganisms are often of a correlative nature. When one feature is altered, other features or properties, as a rule, also become altered. This correlation may exist between the cultural, morphological and biochemical or physiological features, as well as between biochemical characteristics. For instance, in some lactic acid bacteria, the ability to ferment sorbital and mannitol in linked with a loss of the ability to synthese polysaccharide, upon which specific agglutination depends. Some strains of Staphylococcus aureus which are a to assimilate only normal proline, acquire the ability to assimilate both isomers, after their loss of pigment and transformation into colorless variants. A correlative connection of chemical and serological properties was noted in diverse variants of Vibrio cholerae, obtained under various conditions of cultivation on different media. Upon transition of smooth variants into rough forms, many bacteria lose biochemical properties, serological properties are altered, etc. Loss of the mucoid capsule in bacteria sharply alters their antigenic properties. This is also observed upon the loss of the flagellar apparatus. In microorganisms many other external properties correlate in various ways with internal biochemical processes. Alterations in the biochemical functions are accompanied by alteration of the external microscopic features and primarily of the state of the protoplasm (Meisell, 1950; Ierusalimskii, 1949, Stephenson, 1951; Dubos, 1948, Sakharov, 1952 and others).

   In the practice of investigating microbes one may not always note the correlative linkage between function and form. One often obtains variants which differ only in biochemical or physiological properties; by use of present methods one may not succeed in revealing any external alterations. In capsular bacteria, variants are obtained, having properties which do not depend upon the capsule or other cytomorphological features. Often variants are formed with different physiological functions but with the same external features. From one and the same culture of yeasts, actinomycetes and bacteria variants are obtained with red, pink. and yellow pigments as well as colorless variants, and those which ferment or do not ferment sugars, decomposing starch or do not, etc.

   The correlative connection is manifested in those cases when any function of the organism is related to certain particles of the protoplast, microsomes, chondriosomes or other corpuscles of living substance seen under the microscope. For instance, the capsular antigen is connected to the polysaccharide of the mucus envelope. The presence of the latter will determine the antigenic properties of the cell. It is known that some ferments are adsorbed on the surface of chondriosomes. Alteration of the latter indicates a change of a certain biochemical process. A series of properties is connected with chromatin structures. Alteration of these structures will be reflected in various ways in alteration of certain physiological functions and, in general, in biological manifestations of the organism.

   Correlation maybe manifested only between physiological and biochemical properties. For instance, in some lactic acid bacteria, the ability to ferment sorbitol and mannitol is linked with a loss of the ability to synthesize polysaccharides, which in turn affects specific agglutination. Some strains of Staphylococcus aureus, which have the ability to assimilate only natural proline, acquire the ability to assimilate both isomers. after a loss of the pigment and transformation into colorless variants.

   Correlative connection of chemical and serological properties manifests itself in diverse variants of Vibrio cholerae, obtained under various conditions of cultivation. There are also other manifestations of the correlative connections of physiological and biochemical processes to the variability of microorganisms. It should be assumed that variability always attacks a series of properties correlatively connected; but this connection is not always revealed by present methods of analysis. 

   Laboratory experience shows that after long cultivation of microbes in a medium which is unusual for them, and has unassimilated sources of nutrients, the microbes begin to assimilate the latter and the medium becomes an ordinary one and even indispensable. For instance, if yeasts which do not ferment maltose are cultivated on a medium with this sugar as the sole source of carbon, then after some time (it may be protracted after many passages) they acquire the ability to ferment this sugar, A new enzyme is formed in the yeasts--maltase; the cells are physiologically altered, they become new variants (Kosikov, 1950, Kudryavtsev, 1954).

   Such a physiological rebuilding may be induced in bacteria, actinomycetes, fungi and other microbes with respect to many sources of nutrients, carbon, nitrogen, organic and mineral sources.

   The first case of such adaptive variability in bacteria was described by Wortman: then a similar phenomenon was observed by Mechnikov. Kosyakov showed that bacteria may be "trained" to antiseptics--boric acid, sublimate, borax and others. He observed that the anthrax bacillus and other bacteria might be trained to high concentrations of the indicated poisons. This training was achieved by steps, starting with small doses. Neisser and Massini observed adaptation of the colon bacillus to lactose. By protracted cultivation on a medium with this sugar, bacteria start their fermentation and assimilation. The paratyphotd, bacillus Bac. paratyphi "B" adapts itself to raffinose, and the typhoid bacillus--Bac. Typhi--to lactose, saccharose, rhamnose, dulcitol and isodulcitol. Such an adaptation to sugars to also noted in the dysenteric and other bacteria of the colon group; (see Kalina, 1953; Kudlai, 1954; Elin, 1954; Muromtsev, 1953).

   A series of many sporeforming and nonsporeforming bacteria, an well as lactic acid bacteria were described as easily adaptable to rhamnose.

   Adaptive changes were also observed with respect to nitrogen sources of nutrition. Strains of Clostridium may be trained to decompose casein and gelatin by adopting the same method of cultivation. Typhoid and paratyphoid bacteria adapt themselves relatively easily to sources of mineral nutrition and to ammonia salts.

   The ability to liquefy gelatin may be induced in yeast of the Saccharomyces type. Some strains of Azotobacter, Az. chroococcum, do not grow on protein media (meat-peptone agar), but by a successive "training" they start assimilation. New forms or variants are obtained which develop well on media with organic nitrogen. If Azotobacter is cultivated for a long time on a medium containing nitrogen it loses the ability to fix molecular nitrogen.

   Many saprophytic bacteria from the genera Pseudomonas, Bacterium, Bacillus and others, which do not grow on media with mineral nitrogen, acquire the ability to assimilate it during the process of adaptation.

   In the literature numerous cases of quantitative and qualitative alterations of fermentative properties in bacteria are described. Under the influence of the nutritional substances of the medium, the cells of the microorganisms elaborate suitable enzymes. Production of such adaptive enzymes is noted in many bacteria, fungi, yeasts, actinomycetes and protozoa. For instance, in some variants of yeasts obtained experimentally which were deprived of the enzyme cytochromoxidase, the latter is synthesized under the influence of oxygen. Ephrussi and Slonimski showed that the mentioned enzyme is directly induced by oxygen molecules in the protoplasm. In this the novo process of formation, specific particles of the plasma which are not connected with nuclear elements take a direct part (Slonimski, 1956).

   Adaptive ferments such as galactosidase, maltase and others, were found in yeasts by various investigators. Synthesis of new enzymes under the influence of specific substrates to observed in specimens of various bacterial species--Bac. coli, Bac. typhi, Bac. typhi murium, Bac. lactis aerogenes in sporeforming bacteria, mycobacteria, actinomycetes and other forms of microorganisms.

   There are widely known microbes which produce the enzyme penicillinase in defense against penicillin. In penicillin-resistant variants experimentally obtained, this enzyme is produced in a strictly adaptive way. Some conditions were revealed under which the process of production of penicillinase is accelerated or decelerated. It was established that a dose of penicillin of 0.004 unit/ ml or 8 x 10 ^-9 M is sufficient to induce penicillinase formation. Cells treated with penicillin at 0°C and washed afterward, produce penicillinase, on subsequent incubation in a medium without penicillin, 30 times more rapidly than untreated cells.

   In separate cases of adaptation the authors attempted to elucidate the mechanism of production of enzymes. Of particular interest in this respect are the works of Monod and collaborators, Ephrussi, Hinshelwood and collaborators et al. Some stages of the consecutive synthesis and intermediary products of the formation of the enzyme ß-galactosidase in the colon bacillus, in Bac. lactis aerogenes and Saccharomyces cerevisiae were established. Separate factors affecting the process of enzyme synthesis were elucidated. Calcium, magnesium, iron and some other microelements were found to exert an essential influence on protease formation (liquefying gelatin) in Proteus, on phosphatase in propionic acid bacteria. It was proved that for synthesis of enzymatic systems special complementary substances coenzymes, vitamins, some amino acids and other compounds are needed. These substances enter into the composition of enzymes either as a functional part of the molecule or a binding component.

   A great influence is exerted on the formation of enzymes, by environmental conditions such as temperature, pH of the medium, and various chemical and physical agents. This effect may be of a direct or an indirect nature. Organisms react to external influences in various ways. depending upon the character and nature of the acting agent (Spiegelmann and Halvorson, 1956; Pollack, 1956; Knox, 1956; Stephenson, 1951 and others).

   Not all enzymes are formed with the same rapidity during the adaptation of the organism. Some of them are rapidly synthesized under the influence of the specific inducer, others slowly, and still others do not adapt themselves at all under conditions of laboratory experiments. On this basis, Karström subdivided ferments into adaptive and constitutive enzymes. The first, according to the author, are formed by the cells as a specific reaction to a corresponding substrate; the second are always in the cells as a constituent part of the living substance. They do not depend upon the substrate, their specific synthesis is not subjected to experimental investigation.

   This subdivision should be regarded as highly conventional. Investigations show that there is no sufficient basis for assuming essential differences between adaptive and constitutive enzymes. Probably all enzymes may be obtained by induction with specific substrates. If one does not succeed in obtaining some enzymes it is only due to the fact that conditions of their experimental synthesis have not yet been discovered.

   It was experimentally shown that one and the same enzyme may be adaptive and constitutive. In some microorganisms separately induced enzymes become constitutive and vice versa. Such transformations have been observed in variants of the colon bacillus: for arabinose by Cohen, for ß-galactosidase by Lederberg, for amylomaltase and ß-galactosidase by Cohen-Bazire and Jolly and others (Cohen and Monod, 1956).

   The enzymes which take part in the biosynthesis of basic metabolites (amino acids, proteins and other essential compounds) should be included in constitutive systems. Without them the organism cannot develop. One such enzyme in the colon bacillus is N-acetylornithase which hydrolyzes N-acetylornithine with the formation of ornithine. The latter constitutes an indispensable element for the growth of the mentioned bacterium.

   Some variants of the colon bacillus which are experimentally obtained, lack the indicated enzyme and do not develop on media without ornithine. In the presence of N-acetylornithine such variants synthesize N-acetylornithase. Consequently, the latter is a constitutive enzyme in the initial strain of Bac. coli and an adaptive enzyme in its variant. Similar phenomena were observed in experiments with other microbes. These data show the relative character of subdivision of enzymes into adaptive and constitutive.

   A comparative study of adaptive ß-galactosidase and constitutive ß-galactosidase in the colon bacillus or of adaptive and constitutive penicillinases in the sporeforming Bac. subtilis shows that there is no essential difference between them. The affinity for the substrate, the degree of activation by ions, the coefficient of thermal inactivation, the immunochemical specificity are entirely the same in both adaptive and constitutive enzymes.

   It was established that in many cases the cell reacted to the stimulation by the substrate, by the formation of the enzyme at once or almost at once. Production of some enzymes proceeds not during the process of protracted adaptation of the culture, not in successive generations of proliferating organisms but in the same cell which came into contact with the substrate. In such a cell a rebuilding of the protoplast or its parts occurs, under the influence of the specific substrate. As a result of this, the cells acquire new properties. In this way new variants appear.

   Only those substances which are able to evoke a suitable reaction in the metabolizing organism may induce the cell to form enzymes. Enzymes are formed in strict correspondence to the specificity of the substrate. Galactosidase is synthesized under the influence of galactose, maltozymase under the influence of maltose, arabinose induces formation of arabinase, etc.

   Substrates of the nutrient medium, and also substances synthesized in the cell may be inducers of enzymes. Compounds formed as a result of one enzymatic reaction, may serve as a substrate of other enzymatic processes or inducer for synthesis of new enzymes.

   The enzymes formed anew may also be preserved in the cultures of microbes after the disappearance of the inducing substance. The duration of hereditary transmission of the ability to form enzymes to subsequent generations varies depending on the organism, enzyme and environment. The production of adaptive nitrase, cytochromoxidase (under the influence of oxygen) and ß-galactosidase by bacteria ceases immediately after the removal of the inducers from the medium. In yeasts, which are adapted to galactose, the enzyme galactozymase is continuously produced for a long time in a series of numerous generations, which develop in the absence of galactose. Penicillinase is synthesized by cells of some adapted strains by Bac. subtilis when they are grown on media without penicillin.

   The longer the culture is subjected to the influence of a specific substrate, the stronger is the ability to produce a specific enzyme established. If the variant which was removed from the adaptive enzyme is grown anew on a medium with the same inducer, then the ability to form the enzyme again appears, but with greater rapidity, and is established in a more stable manner.

   Adaptive enzymes are very specific with respect to the substrate which induces their synthesis. Some of them are more specific than antibodies which are obtained upon the immunization of animals. With the aid of an adaptive enzyme one may sometimes succeed in subdividing bacterial strains which cannot be differentiated by antigenic indicators. Owing to the high specificity, adaptive enzymes are used as reagents in the analysis of many organic compounds for the differentiation and recognition of separate substances.

   Adaptive enzymes constitute the first manifestation of variability of the organism; with their aid, the nutritional substrate becomes an internal component of the living substance. These enzymes are like a gateway through which the milieu enters; and the nonliving becomes living. They determine the mechanism of the adaptive variability of the organism. It is possible that the study of the formation of adaptive enzymes in microorganisms will also solve some problems of species variability in general.

   With suitable conditioning one may alter the nature of the microbe in relation to those properties which do not seem to be connected with its enzymatic activity. For instance one may train a culture of bacteria or actinomycetes, to increased concentrations of salts or increased temperature and vice versa, thermophilic and halophilic bacteria may be transformed into mesophilic types.

   Referring to this type of adaptation, some investigators indicate that in this case also the great importance of enzymes is not excluded. The latter indirectly alter their properties or are synthesized anew under the influence of internal inducers which are formed in the cell under altered growth conditions. The presence of such inducers in cells was experimentally proved by Stainer (1956). A typical induced pyrocatecholase is formed by cells of Pseudomonas in the presence of tryptophan in the medium, but not in the presence of pyrocatechol. Tryptophan is a remote precursor of pyrocatechol. Formation of such internal inducers may occur in the cell under the influence of various physical, chemical and biological agents.

   Many organisms require complementary nutritional substances for their development and do not grow on special synthetic media without them. By the gradual "training" of the organisms to a medium with decreasing concentrations of these substances one may force the microorganisms to synthesize the latter and to grow on media without them. One succeeds in growing the typhoid bacillus on a medium without tryptophan, the dysentery bacillus without nicotinamide, i.e., without substances which are indispensable for the normal growth of these bacteria. Propionic acid bacteria--Bac. pentosaceum acquire the ability to synthesize thiamine after only several passages on media with substantial quantities of this growth factor. In this way their growth becomes independent of the presence of the given vitamin in the medium.

   Adaptation of microbes to phages is a widespread phenomenon often observed in laboratory practice. As was earlier noted, many bacteria and actinomycetes are subjected to the lytic action of phages. The latter penetrate into the cells of bacteria and actinomycetes and under suitable conditions dissolve and destroy them.

   Upon the interaction of microbial cells and phages, variants resistant to phages and not succumbing to their lytic effect are formed. Such variants are obtained in various specimens of bacteria (sporeforming and nonsporeforming) actinomycetes mycobacteria and micrococci. Phages, an was indicated above, are specific in their action on microbes of a given species. Those which lyse bacterial cells are designated as bacteriophages, those lysing actinomycetes--actinophages.

   Variants which are resistant to phages are endowed with a well-manifested specificity and are only resistant to those phages which induced their formation. Group specificity as well as species and strain specificity are noted. The phage of the colon bacillus induces formation of resistant variants only in cultures of the given bacterium. The phage of the typhoid bacillus evokes formation of resistant strains in Bac. typhi, in the diphtheria bacillus resistant variants are obtained under the influence of the phage of Mycob. diptheriae. In the tubercle bacillus--under the influence of the phage of Mycob. tuberculosis, etc.

   Such a specificity is not always manifested. There are phages known which lyse cultures of Bac. coli and Bac. dysentertae equally well. Phages of staphylococci are described, which attack the diphtheria bacillus. In actinomycetes phages are often found whose action to polyvalent, they lyse not only cultures of one and the same species but also strains of various species and even of different groups. Some phages of Act. streptomycini also actively lyse strains of Act. violaceus and Act. griseus.

   Experimentally obtained variants of actinomycetes or bacteria which are resistant to phages often acquire resistance to another phage species. However, the resistance to nonspecific phages is less strongly expressed.

   The phage-resistant variants are of differing stability, depending on the species of the microbe, the individual characteristics of the phage, and on the external conditions. In some cases, strains keep their resistance to the phage for a long time, and in others the resistancy is lost very soon.

   Variants which are resistant to phages differ from the initial cultures in several other properties; their antigenic characteristics, virulence and particular biochemical functions change. Often, with the appearance of resistant strains of actinomycetes, antagonists become less active or completely inactive against bacteria. Highly active strains of Act. streptomycini often lose the ability to synthesize streptomycin under the influence of phages.

   Adaptation of phages. During the interaction of phages, and cells of actinomycetes or bacteria, mutual adaptation takes place. An inactive phage, not lysing, and not attacking a culture of actinomycetes, acquires the ability to lyse its cells after prolonged common growth.

   Phages inducing the formation of adapted cultures of actinamycetes may undergo changes during the process of adaptation and adapt themselves to resistant variants. The latter are lysed under the influence of adapted phages; under certain conditions, they, in turn, form new strains which are resistant to adapted phages, etc. Thus, one may obtain a continuous series of adapted variants of microbial cultures on the one hand and phages on the other.

   The adaptive nature of phage variability may sharply change, depending on the culture of the host microbe on which the given phage is inoculated. Upon maintain_ Ing an actinophage on one culture of actinomycetes one obtains very active strains with a large range of action, and on cultivating it on another actinomycetes culture, adaptive variants of phages are produced with little activity and a narrow range of action.

   Adaptation of microbes to antibiotics. Adaptation of microorganisms to drugs and antibiotic substances is very strongly manifested. Adaptability of bacteria to penicillin, streptomycin, aureomycin, terramycin and many other antibiotics is widely known. Various species of sporeforming and nonsporeforming bacteria, cocci, mycobacteria, actinomycetes, fungi, yeasts, protozoa, and even insects, become adapted to antibiotics. Numerous cases of adaptation to antibiotics of various pathogenic microbes causing enteric diseases, anthrax, tuberculosis, diphtheria, pest, skin diseases, etc were described.

   Observations show that it is not the whole culture but individual cells which become adapted to antibiotics. The higher the concentration of the antibiotic in the medium, the lower the number of surviving and adapted cells. Cultures adapted to small doses of antibiotic are, as a rule, resistant, to small concentrations of antibiotics in the medium. Cultures adapted to high doses, develop on media with high concentrations of the antibiotic.

   Adaptation of microbes to antibiotics, as in other cases of adaptive variability, proceeds in a directed and specific manner. Variants are only resistant to that antibiotic which induced their formation. If bacteria are subjected to the action of streptomycin, variants resistant to streptomycin are obtained. Under the influence of penicillin, variants resistant to this antibiotic are formed; under the influence of aureomycin, strains resistant to aureomycin appear, etc. Such a specificity is constant and regular. It maybe used to some degree for the differentiation and identification of antibiotic substances and their producers.

   In many cases, the specificity of resistance in experimentally obtained variants to not an absolute one. In acquiring a resistance to one antibiotic, bacteria often become less sensitive to some other antibiotic. However, resistance to other preparations to considerably weaker than to that antibiotic which induced the formation of the variant.

   Microbes may simultaneously become adapted to two, three and more antibiotics, when exposed to suitable mixtures of the preparations. For instance, by the action of a mixture of streptomycin and pencillin on staphylococci, variants are obtained which are resistant to both antibiotics. Bacterial forms have been obtained which are simultaneously resistant to pencillin, streptomycin, and erythromycin, than to erythromycin, carbomycin, streptomycin, aureomycin, and to antibiotics in other combinations.

   The acquired resistance to antibiotics is stable and is transmitted hereditarily through a long series of microbial generations. Adapted variants are preserved during many subcultures on media not containing the antibiotic. The longer the action of the antibiotic on microbial cells. the stronger the acquired properties of resistance are established. The hereditary resistance to the antibiotic to rapidly lost by the action of some specially chosen antibiotic substances or chemical reagents on the culture. For instance, chloramphenicol abolishes the resistance of staphylococci to penicillin. An opposite phenomenon in also noted: an increase of resistance to antibiotics under the influence of particular substances.

   With the acquisition of resistance to antibiotics, microbial variants change some other properties. Often virulence and pathogenicity is lost, the ability to ferment various organic compounds, such as sugars, disappears, gram-positive bacteria become gra -negative, root-nodule bacteria lose their ability to form nodules on roots of leguminous plants, etc. Biochemical properties, an well as morphological properties are changed. Cultures with smooth colonies acquire a wrinkled, rough or granular structure, or become mucous.

   Formation of dependent variants. As a result of the prolonged adaptation of microbes to antibiotics, variants may be formed, which require the given antibiotic for their growth. Dependent cultures are obtained, which do not grow on the medium without the antibiotic, as when formation of vitamin-dependent variants takes place.

   A considerable number of dependent variants has been described in the literature. Such variants are often obtained in laboratory practice. They are very specific and require for their growth, only those antibiotics which induced their formation. Variants dependent upon antibiotics are more specific than resistant variants. This property of the given variants may be used for the differentiation of antibiotics as well as their producers.

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