HOME      AG LIBRARY       KRASILNIKOV TABLE OF CONTENTS

Part I, Section IV, continued

   Directed changes in microorganisms may also be obtained by the method of induction of properties. This is due to the fact that when two defined species are developing, some of their attributes and properties are transferred from one species to the other. These attributes are also transferred when the cultures are grown in a medium containing metabolic products or filtrates of the other culture. One of the organisms acquires the properties of the other.

   Such changes were first observed in the nonsporeforming bacillus, Bac. proteus by Zilber (1928), who obtained a para-agglutinating variant of Proteus under the influence of the typhoid bacillus. Gracheva (1946), obtained a strain of the colon bacillus, Bac. coli, with the properties of another species of the same group, Bac. breslau. Prokhorov (1950), transformed a colon bacillus into a culture with properties of the typhimurium bacillus by the same method. Timakov (1952) and his co-workers studied induced variability in different bacteria of the colon group for a number of years. He imparted the properties of the paratyphoid (the Breslau and Schotmüller types), typhoid and dysentery bacteria to a culture of Bac. coli, growing it in a medium with heat-killed cultures of these species. He suggested calling the culture that acquires the new properties, the "accepting" one, and that which transfers its properties, the "directing" one. Much data have been accumulated on the transfer of antigenic properties from one culture to the other (see Kalina, 1953; Dubos, 1943; Catcheside, 1951; Braun, 1953 and others). By the method of induction, variants were obtained in various bacterial species. In addition to the bacteria of the colon group, staphylococci, many nonsporeforming and sporeforming bacteria, mycobacteria and others may be induced. Legroux and Genevray (1933), transformed a nonpigmented, avirulent strain of Pa. pycoyanea into a pigmented virulent culture. We have observed the transformation of nonpigmented, nonfluorescent strains into pigmented fluorescent cultures of Pa. fluorencens, obtained from the rhizosphere of certain plants. Nonpigmented strains were grown, or merely kept in a medium with filtrates of fluorescent bacteria. After some time, upon plating, single colonies with well-expressed properties of the inducing culture were obtained.

   By use of this method Alexander and Leidy (1951) observed a change of smooth S-and rough R-forms of the pathogenic bacillus, Bac. influenzae.

   Similar transformations are also observed in sporeforming bacteria. Manninger and Nagradi (1948) kept a virulent encapsulated nonmotile culture of Bac. anthracis in a nitrate of the saprophytic, nonencapsulated motile soil bacillus, Bac. mesentericus, and obtained variants which differed from the original culture by properties characteristic of Bac. mesentericus. These new variants proved to be nonencapsulated, with flagella, motile and virulent. Tomcsik (1949) did not fully confirm these data, however, he observed at the same time that nonencapsulated variants of Bac. anthracis possessed an antigen identical with the antigen of the directing culture--Bac. mesentericus.

   The induction of properties by this method was observed with meningococci (Alexander and Redman, 1953) typhoid and paratyphoid bacteria, dysentery bacilli, diphtheria bacteria, micrococci and other specimens of bacteria.

   We have reported data of our investigations on the induction of new properties of virulence in root-nodule bacteria, i.e., the ability to form nodules on roots of leguminous plants foreign to these bacteria (Krasil'nikov, 1945 b).

   The root-nodule bacteria, as in well known, form nodules on roots of certain plants. For instance the nodule bacteria of soybean are capable of forming nodules only on roots of soybean; the bacteria of kidney beans, on roots of kidney beans, the lupine bacteria, on roots of lupine; etc. In accordance with this specificity these bacteria are subdivided into species: Rhizobium japonicum (sojas), Rh. phaseoli, Rh. lupini, etc.

   Not all the rhizobia have such a strict specificity. Some of them may form nodules on roots of plants belonging to different species or even to different, closely related genera. For example, Rh. leguminosarum forms nodules on roots of peas vetch, lentils and Lathyrus sativus; Rh. Meliloti--on roots of lucerne, Galeopsis ladanum and Frigonella. Consequently, among the rhozobia there are species strictly specialized toward a single plant species and bacteria with group specificity.

   By cultivating bacteria of this or that group on media with filtrates of the directing cultures, we have succeeded in changing their specificity and imparting to them properties of virulence of the directing culture. The root-nodule bacteria of peas, vetch and sweet clover acquire the ability to form nodules on roots of plants which are foreign to them: clover and lucerne; bacteria of acacia started to form nodules on roots of peas and lupine, and bacteria of broad beans on roots of kidney beans and Lucerne (Table 4).

Note: Plus means that the induction succeeded and the accepting cultures acquired the properties of the inducing bacteria; minus means that the properties were not imparted.

   Not all root-nodule bacteria can be changed in a given direction and not all directing cultures are able to transfer their specificity to other bacterial species.

   Out of nine species tested, four did not acquire the specificity of the inducing cultures. Many cultures belonging to the same species did not acquire new properties. For example, out of twelve cultures of root-nodule bacteria of beans isolated in different places, seven did not change their specificity. Moreover, not all the variants obtained experimentally from the same culture lend themselves to varation. Out of twenty variants of the root-nodule bacteria of clover, Rh. Trifolii, more than half did not acquire the virulence of the inducing culture which was the original strain of Rh. Trifolii. (Krasil'nikov, 1945 b, 1955 b).

   In subsequent studies we succeeded in imparting the properties of virulence to some nonroot-nodule bacteria of the genus Pseudomonas. Upon prolonged cultivation of these bacteria on media containing filtrates of root-nodule bacteria of clover and lucerne, strains were obtained which possessed the ability to form nodules on roots of clover or on roots of lucerne. It should be noted, that not many cultures change in this fashion. Out of 91 strains of different species and genera of bacteria, only two acquired the properties of the directing cultures, i.e., the ability to form nodules on roots of leguminous plants.

   The acquired virulence can be maintained indefinitely by means of passages from plant to plant. The newly acquired attributes of virulence often do not remove the former specificity. Root-nodule bacteria of peas which acquire the ability to form nodules on the roots of clover did not lose their ability to form nodules on the roots of peas. This was also observed in relation to other bacterial variants developing on vetch, acacia, sweet clover and broad beans.

   Change of properties of virulence in root-nodule bacteria have been observed by Rubenchik (1953) and Peterson (1955). Under the influence of the metabolic products of the root-nodule bacteria of clover, variants were obtained in other species of Rhozobium with the specificity of the directing culture.

   Balassa (1956) changed the specificity of virulence of root-nodule bacteria, acting upon them with DNA of the respective cultures of Rhizobium. He used Rh. meliloti as the directing culture. With the DNA obtained from this culture he acted on nodule bacteria of soybean (Rh. japonicum) and lupine (Rh. lupini). Then the cultures were tested for virulence toward lucerne. Nodule bacteria of soybean after being acted upon by the given metabolite of R. meliloti acquired the ability to form nodules on the roots of lucerne.

   As can be seen from the above-mentioned data, certain metabolic products exert a certain effect on the accepting species of bacteria, and lead to changes in their attributes and properties.

   Of special interest are the changes obtained by "transformation", described first by Griffith in 1928. This author found that pneumococci of type III possess substances which may be transferred under certain conditions to pneumococci of type II, imparting to it the properties of type III pneumococci. It was shown later that these transformations are quite specific and may occur not only in vivo but also in vitro on appropriate media under certain conditions.

   The substance causing these transformations was isolated in a chemically pure state and was thoroughly studied. It turned out to be present only in the pneumococcus type III (encapsulated, virulent type) and was acquired by the pneumococcus type II (nonpathogenic, nonencapsulated). The chemically pure substance possessed a high transforming capacity. It is sufficient to add 0.003µ g of this substance to 2 ml of the corresponding medium to transform an avirulent nonencapsulated culture of pneumococcus type II into a virulent encapsulated variant of pneumococcus type III. Chemical, enzymatic and serological analysis and data obtained by electrophoresis, ultraviolet spectroscopy, etc show that the active part of the substance contains neither protein nor free lipides or serologically active polysaccharides (Dubos, 1948; Braun, 1953; Catcheside, 1951 and others). This part consists mainly of DNA. The given acid causes the synthesis of the capsular substance. Studies have shown that the DNA of the pneumococcus S-variant is similar to the DNA obtained from other sources, for instance from cultures of the rough R-variant.

   However, their effects differ. The former possesses transforming ability, transforming the R-form of pneumococcus into the S-form but the latter is devoid of this ability. Consequently, the transforming action of DNA is obviously connected with the presence of extremely small quantities of other substances, the effect of which manifests itself in the presence of the given DNA.

   Not all the pneumococci are able to accept the transforming substance. Out of a great number of experimentally obtained nonencapsulated avirulent variants, only a very few show the ability to accept this substance. Consequently, the active part of the capsular substance of pneumococcus type III is effective only when the accepting culture possesses specific functionally active acceptors, determining the biochemical activity and specific nature of the pneumococcus cells.

   In 1952, Austrian and MacLeod observed that in addition to the variability of the polysaccharide, the pneumococci also show changes in their proteins. According to these authors, upon transformation of pneumococci a change takes place in a special somatic protein. Together with the above-mentioned substance this protein in various combinations may produce different variants in the process of changing of the culture.

   Hewitt (1956) transformed a nonencapsulated avirulent strain of the influenza bacillus into a capsulated strain. Hotchkiss (1951) obtained penicillin-resistant strains of pneumococci from a sensitive strain by growing the latter in a medium containing DNA isolated from cells of a strain adapted to penicillin.

   Streptomycin-sensitive pneumococci, resistant to penicillin may be changed into streptomycin-resistant by the use of a transforming substance which is liberated upon lysis of streptomycin-resistant strains of pneumococci by the action of penicillin. This substance which is capable of imparting streptomycin-resistance to the culture and of transferring other hereditary properties from one strain to another is not a DNA, but has a different chemical composition. It is not decomposed by the enzyme desoxyribonuclease. A transforming susbstance of the same type transforms streptomycin-sensitive strains of typhi murium into streptomycin-resistant ones (Zinder and Lederberg, 1952).

   The occurrence of transformation has been detected in certain nonsporeforming bacteria which possess slime capsules. By the action of corresponding substances they lose the capsules and with them some other properties are lost or new ones are acquired.

   According to Zinder and Lederberg, properties of cells can be transferred from one culture to another by special tiny corpuscules or particles of the protoplast. They lyse the directing cultures by a special phage. The products of lysis, in the form of the mentioned corpuscules, pass through fine glass ultrifilters and enter the calls of the accepting culture. The latter acquire the properties of the former. The particle carriers of the transferring substance are very small, not more then 0.1µ in diameter, and visible only in the electron microscope. The authors observed this method of changing the cultures in Bac. typhi murium. Two cultures differing in their nutritional requirements were used in the experiments. One of them required histidine and the other required tryptophan. They were placed in one vessel divided by a glass ultrafilter, each one of the two cultures being placed in a separate hall of the vessel. As a result, nonobligatory cultures were obtained. The authors called this method of changing cultures transduction.

   A similar picture of culture changes was observed by Brown and co-workers (Brown, Cherry, Moody and Gordon, 1955) in Bac. anthracis.

   They selected a special phage (Lambda phage) which in distinction to many other phages (in all 32 phages. were examined) possessed a wide range of action. It lysed bacteria of different species and genera: 9 strains of genus Bacillus anthrasicis, 3 strains of genus Bac. cereus, 4 strains of Bac. cereus var. mycoides and others.

   A motile culture of Bac. cereus was infected with this phage and after a twenty-four-hour incubation the culture was filtered through an ultra-bacterial filter; a nonmotile virulent strain of Bac. anthracis was infected by the filtrate or by the purified phage from this filtrate. After twenty-four hours motile bacilli appeared in the culture of the latter. These bacilli grew into a motile avirulent culture with certain properties of Bac. cereus.

   The authors assume that the phage elements transduce certain particles of living matter from Bac. cereus to Bac. anthracis, and introduce certain properties with them. According to the authors, changes, as were made in the above-mentioned bacillus, appear regularly, in all repeated experiments. They think that the mode of carrying over properties from one organism to the other, which they observed, differs from that described by Zinder and Lederberg. The latter claimed that the carriers were not the phage particles but special corpuscular elements.

   Spiegelman (1946) while studying induced changes in yeast, obtained a substance which evoked the ability to ferment galactose in species which were unable to ferment this sugar. He called this substance adaptin. Its nature remained obscure. By the method of transduction one may succeed in conferring on bacteria fermentative, antigenic, virulent and other properties possessed by the directing culture, and one may also obtain motile strains from nonmotile ones, to which the ability to form flagella is imparted by the phage particles (Baily, 1956).

   Changes which are called "recombinations" also belong to the phenomena of induced changes. The external part of these changes is based on the fact that, upon mixing two cells from different cultures, generations with the mixed or combined properties of the initial parental organisms are formed. The cells transfer their properties and attributes upon direct contact with each other. New variants appear which are called recombinants. The formation of such recombinants was described by Tatum and Lederberg (1947) in one strain of the colon bacillus K-12. From this strain defective variants were obtained which had lost the ability to synthesize certain amino acids and vitamins essential for growth. On these variants experiments of "vegetative crossing" were performed (Lederberg and Tatum, 1954).

   The colon bacillus, strain K-12, after irradiation by ultraviolet rays, forms strains which differ from the original culture by their inability to grow on simple synthetic media without additional substances. These defective variants are called auxotrophs or minus-variants. Some of them require a certain auxiliary substances these are monoauxotrophs. Others require two, three or more substances; these are polyauxotrophs.

   If one grows auxotroph strains separately on a full nutrient medium, and then inoculates 10-15 hour cultures on a deficient nutrient substrate, after thorough washing in saline, then one does not observe growth of the culture and the inoculation will be sterile. Only in rare cases do single colonies grow out; the new variants, the prototrophs, are able to develop on media without additional substances. The frequency of appearance of these variants, according to Lederberg and other investigators, is not higher than one cell per 1,000,000-10,000,000 of the initial auxotrophs.

   Observations show that auxotroph variants that were mixed before inoculation give a more intense growth on a noncomplete medium than that of the control inoculations.

   Tatum and Lederberg an well as some other investigators experimented mainly with two variants. One of the variants (58-161) required biotin for its growth (B-)and methionine (M-), and the other variant--W-1177 required threonine (T-) leucine (L-) and vitamin B ₁ (B ₁-) Both variants did not grow without the indicated substances on special mineral media. Mixing these variants, strains were obtained, which differed from the initial ones in that they developed on incomplete media in the absence of biotin and methionine and also variants which did not require threonine, leucine and vitamin B₁; they themselves synthesized these growth substances. This can be schematically represented in the following ways strain 58-161 (B-M-) and strain W-1177 (T-L-B _l-) give a variant B+M+T+L+B ₁+.

   The transfer of properties upon mixing cultures may be accomplished by various strains. Some attribute or property is transferred from cells of one strain to cells of another. This property or attribute will, for the sake of simplicity, be designated by the letter F. If the attribute is there, the cells or the whole culture is designated by its name with the addition of F ⁺. If the attribute is lacking the culture is designated by F ^-. Some attributes may be transferred from one F ⁺ strain to many other F ^- strains. According to Hayes (1953), a strain or, as he calls it, a mutant W-677 (-CA) (streptomycin- and azide-resistant) when present in a culture together with 58-161/F ⁺ acquires the properties of the latter and becomes strain W-677 CA/F ⁺. The number of such transformations in platings on agar media reaches 75% of the inoculated and tested colonies. A nonobligatory strain or a culture of 58-161/F ⁺ transmits the factor F ⁺ to the incomplete strains W-877/CA/F^-, 58-161/F^-, W-877/F^-, 58-161/CA/F ^-. Thereby new strains are obtained which are nonobligatory and retain the acquired properties quite well for a number of months on a nutrient egg Dorset agar medium at 4°C.

   Factor F, determining one or another attribute is not transmitted through the filtrate. A young broth culture of 58-161/F ⁺ was filtered through a colloid filter with pores of 0.74µ A. R. D. The incomplete strain W-677/F ^-, was kept in a filtrate after which colonies were plated on an agar medium. Out of 115 colonies sown, the acquisition of the properties of the directing culture F ⁺ was not observed in any one of them.

   As was noted earlier, when the cultures are mixed with an F ^- strain the planting is sterile. Upon mixing the F ⁺ and F ^- cultures the platings are of high productivity. For instance, upon plating the variants 58-161/F ⁺ and W-677/F ^-, 97 colonies of prototrophs were obtained out of a total number of 945 million auxotroph cells. Plating the strains 58-161/F⁺ and W-677/F⁺ only 4 prototroph colonies out of 477 million were observed and upon plating the strains 58-161/F ^- and W-677/F ⁺, 49 prototrophs appeared out of the total number of 450 million cells.

   As can be seen from the data given, the largest number of prototrophs is obtained when F ⁺ variants are plated together with F ^- variants.

   In the experiments of Hayes the incomplete variants of the colon bacillus were as follows: 58-101 required methionine and biotin (M-B-), fermented lactose, maltose, mannitol, galactose, xylose and arabinose, was sensitive to coli phage T₁ and resistant to T₃. The variants of W-677 required threonine, leucine and thiamine (T-L-B₁ - )for growth, did not ferment lactose, maltose, mannitol, galactose, xylose and arabinose, were resistant to phage T₁ and sensitive to phage T₃.

   In addition, these variants, according to Maccacaro (1955), differ from each other in certain other properties: their reaction to the acidity of the medium, their ability to absorb dyes, etc.

   In experiments of other investigators with the colon bacillus K-12 analogous results were obtained. Defective strains, that have lost the ability to synthesize a certain amino acid or vitamin, reacquire the ability upon contact with cells possessing these properties. Strain U-24 which in the experiments of Clark (1953) required biotin, phenylalanine and cyotine (B-Ph-C-), does not grow on a synthetic medium lacking these substances. Strain U-10 forms biotin, phenylalanine and cystine, but does not produce threonine, leucine, and thiamine (T- L- B1-) and does not develop on a synthetic medium without these compounds. After mixing two such organisms, strains are obtained, which grow well on the indicated media and synthesize the substances necessary for growth which are lacking in the initial cultures. (Catcheside, 1951; Hayes, 1953; Braun, 1953; Clark, 1953; Lwoff, 1955, etc).

   Sermonti and Spada-Sermonti (1955-56) described the formation of recombinants obtained upon crossing strains of blue actinomycetes--A. coelicolor, after irradiation by ultraviolet rays. One strain (5 me- hist-) required methionine and histidine and formed a blue pigment. Another requiring strain (14 pr.- glu- pigm.-) required proline and glutamic acid and did not form pigment. In mixed platings on complete agar media three types of colonies are formed. The initial ones: a) 5 me-hist-, b) 14 pr.- glu pigm, and c) recombinants nonrequiring cultures that do not require methionine. histidine, proline or glutamic acid.

   In this way Rizki (1954) observed the transmission of pigment formation ability in Bac. prodigiosium. Acting on a leuco-strain of this species of bacteria with a pigmented culture, the author observed that the former culture acquired the ability to form the pigment prodigiosin.

   Some investigators found similar transmissions of hereditary properties in pneumococci, in typhi-murium bacteria and. others (Ephrussi, Taylor 1951; Hotchkiss, 1954; Vavali and Lederberg 1953).

   Studying the process of formation of recombinants, Clark found that it was connected with the pleomorphism of the culture. As a rule, the external action that caused a degenerative development of an auxotroph culture with the formation of deformed gigantic cells and other cells, lowered the number of prototrophs formed. Haas and others noted a correlation between the formation of strongly enlarged, threadlike and other polymorphic cells and the increase of the number of prototroph variants among the auxotrophs. According to their observations, ultraviolet irradiation enhances the process of recombination in the colon bacillus with the simultaneous increase in the polymorphism of the culture. Certain chemical substances, hydrogen peroxide and others, stimulate the formation of prototrophs (Haas, Clark, Wyss, and Stone, 1952). Grigg (1952) stresses the importance of the dependence of the appearance of recombinants among auxotrophs upon the quantitative ra tios between the initial parental cells in the mixture.

   If there are more F ⁺ cells in the mixture of the strains crossed. all the F ^- cells acquire the properties of F ⁺. In cases where the numbers of cells of both types are equal there will be less recombinants. In case of a reversed ratio, i.e. , when there are less F ⁺ cells than F ^- cells, the number of F ^- transformed to F ⁺ will be the smallest (Table 5).

*Ratio of the number of F ^- cells transformed into F ⁺, after being mixed together (hours)

   As can be seen from this data at a ratio F ^-:F ⁺ of 1:25, out of 20 cells (colonies) tested, all were variants transformed from F ^- to F ⁺. At a 1:1 ratio of these strains, the number of transformed variants was 10 out of 20 and at a ratio of 4:1 it was 2 out of 20 cells studied. These numbers of variants are obtained half an hour after mixing; after one hour their number is smaller and after two hours it becomes even less. Ultraviolet rays increase the fertility of the F ⁺ cells but not that of F ^- cells and enhance the maturation of prophage into phage, an a result of which the number of gene carriers increases.

   In the formation of recombinant variants the following rules can be observed: variants B+M+ (not requiring biotin and methionine) are formed more often than variants B-M+, (requiring biotin). Upon planting a mixture their number reaches 14 %. B₁+L+variants (forming vitamin B1 and lecithin) are encountered more often than variants B₁-L+ or B₁+L-. Variants T-B+M+B₁+L+ (requiring threonine) are obtained more often than variants T+B+M+B₁+L+. All these data show that in the living matter of the organism there is a strong bond between various attributes.

   It should be noted that such variants cannot be obtained upon mixed cross platings of all cultures. Even in the same species of the colon bacillus only the K-12 strain gives variants capable of combined variability. Another such strain of the colon bacillus (strain B) does not give such variants. Numerous attempts to obtain variants from this strain, able to give recombinants, were unsuccessful.

   It was suggested that strain K-12 is a homothallic culture and strain B, a heterothallic one. The former possesses both the masculine and the feminine tendencies, whereas the other has only one of them. Therefore, the latter cannot dissociate according to this attribute and give combined variants upon mixing, as happens in K-12 strains. Strain B in monosexual or asexual, and is similar to certain imperfect fungi, for instance from the group of asporogenous yeasts: Torulopsis, Mycotorula, etc. According to the authors, upon contact between the minus variants of the homothallic culture a process of conjugation or exchange of substances takes place between them, similar to the sexual process in higher organisms. With these substances the carriers of hereditary properties, the genes, are also transmitted. (Lederberg and Tatum, 1954; Anderson et. al., 1957).

   On the mechanism of variation. As can be seen from the above-mentioned data, the phenomenon of variation in microorganisms is quite diverse both in its appearance and in the nature of its response to external influences. The mechanism of variation and the inheritance of properties remains unexplained. There are many points of view, and many suggestions were made all of which can be reduced to two basic theories: the theory of mutation" the theory of physiological adaptation. These theories were formulated long ago. and were already known during the period of the first discoveries in the field of variation in microbes in general. However, there have not been sufficient factual data to corroborate any of these theories.

   The studies performed in recent years in the field of variation are of great interest. Tatum and co-workers (1942) have shown that certain acquired fermentative properties in individual variants of the fungus Neurospora are regularly transmitted to the offsprings during sexual reproduction which is similar to that existing in higher plants. After these observations analogous data were obtained with yeast of the genus Saccharomyces, Saccharomycodes, etc. Gamete cells of the two cultures cross, unite and form one zygote with the properties of the parental organisms. In the subsequent multiplication of the zygote the properties of the original cultures segregate through the generations and combine in various groupings. As result, new variants are formed with combined properties. These variants are called combinants or recombinants.

   Variations of this type take place in the evolution of microorganisms. However, it is possible only in species which multiply sexually. In bacteria, actinomycetes and also in a great number of fungi (imperfect) and yeasts the sexual process in its full expression is nonexistent. Naturally, one cannot observe variations of the type produced by sexual mixing of properties, nor conduct a genetic analysis of any sort in these organisms.

   Data were given above, showing that in bacteria and other microbes hereditary changes which have a certain similarity to genetic changes of the combination and recombination type, characteristic of yeast may occur. The followers of the genic mutation theory assume that bacteria possess regular nuclei with a specific set of chromosomes and gene-like structures, which determine the rules of the process of variation. As a confirmation of this assumption the investigators use the observation of Tatum and him co-workers on the changes occurring In the K-12 variant of the colon bacillus upon crossing it with cells possessing different properties. The recombinations obtained are identified with the combinations in fungi and yeasts in sexual crosses.

   The phenomenon itself really resembles combinations formed during the process of sexual reproduction; however, the mechanism here is probably different. The bacteria mentioned do not have a sexual process of the form that is observed in fungi and yeasts. Direct conjugation and mixing of living matter of two cells is not possible here. It should be assumed that upon contact between calls in the above-described cases of transduction the transforming substance is transmitted from one cell to the other across intact membranes and if there are defects, in the latter, they are not detectable.

   The existence of transforming substances may now be regarded as proven. They are found in various microorganisms--bacteria, actinomycetes, yeasts and fungi. As previously noted, it was found in pneumococci, nonsporeforming bacteria, in sporeformers, etc. In certain cases the chemical structure of these substances was established or their chemical composition disclosed.

   In transduction the transforming substance may be carried over from cell to cell with the aid of phages. The latter build their body from the live matter of bacterial cells (mainly of DNA). When the cells lyse the phages are liberated and if they pass to cells of another culture they evidently carry over particles of living matter of the former cells. The mixing of protoplast particles of two different cultures takes place, and with it the hereditary properties are mixed, which later seggregate out with the formation of new variants--recombinants in the progeny.

   The ability of microbial cells to absorb complex organic compounds, metabolites of microorganisms, has been established by many investigators with different representatives of the lower organisms. It has been shown that compounds such as vitamins, auxins, amino acids, enzymes, antibiotics and other substances important to life are absorbed by microbial cells through an intact membrane.

   It has been proved that various metabolites of microbial origin can serve as inducers of the synthesis of specific biocatalysts: vitamins, ferments and other compounds. Penicillin. absorbed by cells of sensitive bacteria, induces biosynthesis of the enzyme penicillinase, sulfonamide preparations induce the formation of para-aminobenzoic acid (PABA), etc.

   Oparin and Yurkevich (1949) have shown that brewer's yeasts have the ability to absorb enzymes from the medium and use them. It was also established that absorbed enzymes stimulate the cells to form their own enzyme. Kosikov (1950) observed the formation of the induced enzyme, invertase, in yeast organisms. The cells that absorbed invertase from the nutrient medium soon began to synthesize their own invertase and used it for the splitting of saccharose. The variants thus obtained, maintained the ability to synthesize this enzyme for an indefinite length of time and transmitted this ability hereditarily through many generations.

   Antibiotic substances and other microbial toxins which are absorbed by the cells, lead to the formation of antitoxins, due to which new resistant variants are formed. Various metabolites formed by microorganisms may serve as antitoxins. For instance, PABA is synthesized in order to neutralize the poisonous effect of sulfonamide preparations; penicillinase and streptomycinase--as an antidote to the corresponding antibiotics, etc. The resistance of strains to sulfonamide is determined by the degree of formation of PABA. The higher the concentration of sulfonamide in the medium, the more PABA is necessary to counteract its effect. For the neutralization of 50 µ g of sulfonamide in the medium, the hemolytic streptococcus produces 0.007 microgram of PABA; when the dose of sulfonamide is thrice increased, the streptococcus also increases its production of PABA three times. Increasing the concentration of PABA in the medium tenfold, correspondingly requires more sulfonamide in order to exert an antibacterial action (Woods, 1940).

   It was observed that PABA is a specific metabolite which is antagonistic to sulfonamide preparations. It is part of the composition of the vitamin, folic acid and a vital substance for microbial cells.

   Many metabolites, as is well known, are of considerable importance in the life of microbial cells and some of them are quite indispensable: their function determines various processes of life in the cells. If for some reason any of these metabolites are blocked, the cells become different and their biochemical processes are not the same as those of the initial, normal cell. If the given change is stabilized and transmitted to subsequent generations, new variants are obtained during the process of multiplication.

   The blocking or change of metabolites may take place under the influence of antimetabolites or metabolites which are formed by other species of microorganisms. The interaction between metabolites and antimetabolites may manifest itself in different forms and degrees. After being absorbed by the cells from the medium, an antimetabolite may be fixed and combine with metabolites, disturbing their function. As a result of such blocking the formation of new variants of microorganisms is possible.

   Many metabolites are described in the literature, which have corresponding antimetabolites inactivating them. Vitamin B₁, or thiamine, has an analogous antimetabolite--pyrithiamine and neopyrithiamine. Pyrithiamine paralyzes the action of thiamine. Analogous antimetabolites have been found for biotin, choline, folic acid, ascorbic, and nicotinic acids and for the amino acids, tryptophan, methionine, glutamic acid and other compounds (cf. Woolley, 1954; Shive, 1952).

   If In all cases of induced variation--upon "induction" of properties, transformation, transduction, etc the acting substance or inducer is processed by the living protoplasm, and takes part in a series of various biochemical transformations of some metabolites, causing corresponding changes in them. The process of transmission of properties from one organism to another is evidently possible only in cases where there is a close phylogenetic relationship between them. In addition, the cells of the accepting culture must have a corresponding acceptor, capable of binding the inducing substance of the inducing culture. Loss of these acceptors deprive the cell of the ability to change in this manner.

   The phenomenon of adaptation to substrate and to external agents may be caused by other reasons and may consequently have another mechanism. Some investigators connect the resistance of bacteria to antibiotics or phages with the lose by the cells of the special substances, "receptors", which capture the molecules of the antibiotic and hold them within the cells or inactivate them, without disturbing the intactness of the cell.

   In a number of cases the mechanism of resistance formation to antibiotics is connected with a change in the permeability of the cell membranes or their protective barrier. Ehrlich has shown (1909), that normal trypanosomes take up acriflavin, while acriflavin-resistant strains which are adapted to it, do not absorb the drug. There are similar data concerning penicillin. Certain penicillin-resistant bacterial variants absorb less penicillin than the initial sensitive cultures. According to our data, chemically purified mycetin is absorbed by the diphtheroid bacteria of Mycobacterium sp. (initial culture) in the amount 32.5 units and the experimentally obtained drug-resistant variant absorbs only 16.0 units per same number of cells. According to different authors, streptomycin is absorbed by the membrane of certain bacteria and is bound within it by nucleic acids. As a result, the metabolism is disturbed and the activity of the cells differs from that of normal organisms.

   The adherents of the mutation theory maintain that any culture contains a small number of mutants which are undetectable by the usual methods of microbiological analysis. According to calculations of various workers, there is one mutation per 10⁷-10⁹ ordinary vegetative cells, developing on nutrient media under ordinary laboratory conditions. According to others. the number of mutations may reach 5 x 10² and also 10¹⁰ (cf. Braun, 1953; Catcheside, 1951).

   It should be noted that the authors determine the number of mutations only with respect to one characteristic. If one considers variations of different features. and they are quite numerous, then the number of mutations vastly increases. Variants which are resistant to various agents: antibiotics, phages, chemical reagents, and physical agents, like temperature, radiation energy, U.V., X-rays, etc, appear in a culture. Each type of these agents is in its term quite diverse. There exists a great number of antibiotics, phages and chemical reagents. Each acts on the cells in its particular way and against each of them ready microbial mutants ought to preexist. Furthermore, the culture should contain mutants adapted to different concentrations of each of the active factors, which are also quite numerous. The number of mutants formed for each agent is approximately equal. Therefore, if one accepts the point of view of the mutation theory, one should assume the pre-existence in each culture of mutants adapted to all possible agents and to various doses of the agents. In other words, an infinite number of mutants should exist; almost every cell of the culture is in some degree a mutant, which of course is hardly probable.

   It is assumed that the mutations are spontaneous, without any effect from external agents and independent of the environment. The environment only selects the already existing mutants from the culture. Upon action of one or another agent, be it antibiotic, phage, chemical or physical, the cells resistant to the agent, that is the mutants, survive, while all the sensitive forms are removed.

   The mutation theory cannot explain many phenomena of adaptive changes. It is known that microbial cells can adapt themselves to antibiotics (as well an to other agents), when they are in a resting state, in the lag phase of the culture. Under suitable conditions many adapted forms are simultaneously formed in the culture Their percentage may be increased considerably, to 1-10 per cent and more, whereas according to the mutation theory their number does not exceed 1 per 100-1,000 millions of normal cells.

   The phenomenon of stepwise adaptation of bacteria to ever-increasing concentrations of an antibiotic and of other substances remains unexplained. Upon inoculation of the active agents, into media with increasing concentrations the frequency of formation of resistant variants increases sharply and this process proceeds much faster.

   The appearance of forms which are simultaneously resistant to two, three or more substances is also not clear. It is even more difficult to understand the removal of acquired resistance of variants by the action of other antibiotics (or other substances). The mutation theory is also unable to explain many other aspects of variation.

   The lack of basic differences between mutants and adaptations was noted above. The so-called mutants which arise as a result of changes in the genes, may become adaptive forms. Hereditary fixation of properties is observed in quite obviously adaptive variants, whereby these properties are transmitted through thousands and millions of generations. On the other hand, mutants may be unstable and revert comparatively quickly to the initial microbial forms.

   The numerous observations and experiments of the recent years convince the investigators more and more that variation in microorganisms takes place by way of physiological adaptation to the environment. The living matter of the organisms specifically reacts to external agents, changing its properties to correspond to the quality of the acting substance. Under the influence of the substrate, inducer cells primarily react by changing their enzyme systems. Many investigators are of the opinion that when a substance induces the formation of an adaptive enzyme, it participates together with the specific component of the cell in organizing the synthesis of the given enzyme. Upon prolonged action of the inducer, the adaptive enzyme turns into a constitutive enzyme. The acquired ability may be carried through a long series of generations in the absence of the substance that caused its appearance.

   Due to vigorously acting agents (the so-called mutagens) a relaxing of the hereditary properties of the organism takes place. This also happens in old microbial cultures. Cells with relaxed hereditary properties are much more readily subject to the action of environment. They react more readily to specific substances and show the properties of variation that have been induced by different substrates. The specificity of the latter leaves its imprint on the formation of new variants, or mutants. Mutants are created by environment, as are adaptive variants. Both are phenomena of the same order.

   The problem of the variability of soil microbes under their natural conditions of habitation is little understood. Only a few works on this problem are available and those are of a speculative character.

   In laboratory practice, as was indicated above, one often encounters the phenomenon of variability, polymorphism and species variability in microorganisms. The sporeforming bacteria--Bac. mycoides, Bac. subtilis, Bac. mesentericus, and other typical cultures with the characteristic structure of colonies become atypical and form variants differing from the ordinary forms.

   Are similar variants indirectly formed in the soil or are they only formed under laboratory conditions?

   It is known that in some places typical variants of Bac. mycoides are not encountered but instead, atypical forms, with fine granular, evenly edged colonies, are found.

   We have found such forms in the chestnut soils of the Volga area. Typical variants of mycoid structure in their colonies were completely absent (Krasil'nikov et al., 1934b, 1936). In some soils typical forms together with atypical ones are found.

   We have performed a series of experiments in order to determine the genetic link of the typical cultures with the atypical variants,

   We did not succeed in obtaining typical cultures from atypical strains upon cultivation of the latter in artificial media under various growth conditions such an high or low temperatures, various pH, aerobic and anaerobic conditions. Nineteen strains, isolated from various places and various fields of the chestnut soils of the Volga area were studied. None of them produced typical mycoid variants. A certain resemblance to the mycoid outline of the colony was observed in the case of one strain. Six out of nineteen atypical strains studied were transformed into typical variants with mycold structure after a prolonged cultivation in spodsolic soil taken from fields of the Timiryazev Agricultural Academy. The remainder either did not change or gave variants of still another structure and type.

   The second series of expriments was performed with typical cultures of Bac. mycoides isolated from podsolic soils (4 strains) and chernozems (2 strains). The cultures were placed in soils in which these bacteria are not encountered, namely in chestnut soils. They were then incubated for 3 -6 months at room temperature. The soil humidity was maintained at the level of 60% soil capacity.

   Systematic platings from the soil had shown that the mycold forms soon began to disappear and with each successive plating their numbers decreased. After 1-3 months almost all the original strains disappeared. Instead, various atypical strains, characteristic of the given soil as well a strains which are not usually encountered in such soils, grew upon plating.

We have observed these transformations of Bac. mycoides in both sterile and nonsterile chestnut soils; in the latter the transformations proceed more rapidly.

   These data show that one form of bacterium maybe converted into another. The peculiarities of the soil influence the character of the variations.

   The specific effect of soils on the bacterial variability is also confirmed by experiments carried out with other cultures of soil bacteria.

   We have long been interested in a group of sporeforming bacteria which, according to the classification, are named Bac. subtilis, Bac. mesentericus, Bac. cerus, Bac. brevis, etc.

   Do they represent separate species or are they variants and forms of one and the same species? This question has been experimentally determined.

   Cultures of Bac. mesentericus were introduced into various soils--podsolic, forest, field and garden soils; chernozems, chestnut soils and red soils. The cells of Bac. mesentericus died out with relative rapidity in red soils and forest podsolic soils. In weakly cultivated fields of acid sod-podsolic soils and also in chestnut soils and chernozems, the culture did not die out completely. A small number of the cells survived. After some time the survivors began to multiply. However, upon plating on nutrient media they produced colonies which differed from the original ones.

   In the course of adaptation new variants were obtained in chernozems and in chestnut soils.

   In podsolic soils we obtained variants of Bac. cereus and Bac. subtilis and in chestnut soils variants of Bac. subtilis and Bac. licheniformis. In chernozems, all forms given in the table were obtained, i. e. , cultures, whose colonies resembled those of Bac. cereus, Bac. subtilis. Bac. licheniformis, Bac. brevis, and Bac. mesentericus.

   An can be seen from the aforesaid, one and the same culture of Bac. mesentericus manifests different morphological forms according to the properties of the soil. In some chernozem soils (Moldavian SSR) we have found only two types of this group of bacteria, namely, Bac. subtilis and Bac. cereus. We converted individual strains of one or another type into typical cultures of Bac. mesentericus.

   Considering only the external appearance of the culture, the difference between the mentioned species may be thought of as a reflection of polymorphism. Apparently, the classification of this bacterial group should not be based on external differences but on biochemical and biological properties. Of the latter, antagonism and antibiotic spectrum are the most indicative (in our opinion).

   Afrikyan (1954a), employing the principle of the specificity of antagonism had shown that the group of sporeforming bacteria classified as Bac. subtilis-mesentericus is a very diverse nonhomogenous group in its biological properties, and therefore, cannot be considered as a homogenous taxonomic entity.

   The principle of the specificity of antagonistic interactions makes it possible to reveal natural ecologic strains and types which actually exist, and to differentiate them.

   Some ecological pecularities of sporeforming bacteria which are in soils of the extreme north, in the Soils of the islands of the Arctic Ocean--Novaya Zemlya, Franz Joseph Land, Severnaya Zemlya and others, are devoid of spores. The cultures grow well on nutrient media under ordinary laboratory conditions and form large colonies, but spores are absent. When they are grown in many ordinary media, the cells do not form spores.

   The absence of spores in such cultures may mislead the investigators. Similar cultures can be classified as nonsporeforming bacteria. However, the structure of the colonies, the size of the cells, and the structure of the protoplast show that these organisms belong to the sporeforming group. The formation of spores in such bacteria can be induced. They form the usual endospores, but, as a rule, very late, after 2-3 weeks or later. Many of them form spores at elevated temperatures, 33-37° C. Some cultures begin to form spores after prolonged cultivation on protein-rich media, and others after they are kept in soils of the more southern regions such as the podsol of Moscow Oblast', chestnut soils of the Volga area and others. This peculiarity of these bacteria is apparently caused by long habitation In unusual soil-climatic conditions. Those conditions change the growth characteristics and the whole biology of the species. Bacteria lose the capacity to form spores. In some organisms, only the initial processes of spore formation are preserved. Cells of some strains form dense sectors, in the form of a prospore, at their ends, or in the middle part of the cells.

   Under the conditions of arctic climate where the soil is always frozen or thaws for a short period, spore formation does not constitute a protective reaction of species preservation and in gradually lost. The biological essence of such bacteria as a sporeforming species is nevertheless preserved.

   The capacity to form spores is lost, not only in bacteria of the arctic soils, but also in bacteria which inhabit more southern regions. The formation of asporogenic forms in general is a phenomenon quite frequently encountered in specimens of the genus Bacillus, and it takes place in laboratory cultures as well as in nature directly in the soil. One does not always succeed in revealing the asporogenic forms in the soil since they are differentiated only with difficulty from some nonsporeforming bacterial species.

   Much attention is being paid to Azotobacter, among the soil microflora. This microbe is widely distributed in soils.

   Azotobacter grows under various climatic conditions, from the extreme north to the tropics. It can be found in various soils, virgin and cultivated; it is found in podsols, serozems, chernozems, chestnut soils, brown soils and even in red soils. Notwithstanding the diverse conditions of the habitation of Azotobacter, only one species is described as widespread in the literature, namely, Azotobacter chroococcum. Some authors think it can be subdivided into 2 to 4 species. In our manual we have given 7 species, two of them being of an entirely different group.

   Laboratory experience shows that cultures of Azotobacter of whatever origin, are at first eight homogenous according to their culture characteristics. They all grow well on media devoid of nitrogen (Ashby agar) and form large slimy colonies. After some time the colonies become brown and eventually black. All cultures fix nitrogen, to a greater or lesser extent, and are diagnosed in the majority of cases as Azotobacter chroococcum. Detailed studies. however, show that the cultures of this species are far from being of one type.

   Upon studying a great number of Azotobacter strains collected from different soils of the Soviet Union it was found that they differ from each other, morphologically and biologically. The size of cells, their form and structure are far from similar, as described by many authors. We have described (Krasil'nikov, 1949 a) the characteristic differences of the, individual strains of Azotobacter, differences which served as the basis for dividing them into separate species such as Az. beijerinckii, v. jakutii, Az. galophilum and others. The cells of these and other organisms differ from each other in their size and form as well as in the internal structure of the protoplast. Cultures also differ in color, the nature of structure of the colonies and other factors.

Figure 33. Azotobacter chroococcum. Diversity of cultures and strains, according to formation of flagella, and cell forms:

a) strain 54, stock culture; b) strain 11 isolated from serozem (Central Asia); c) strain 13 from serozem of Tadzhik SSR; d) strain 14 from chestnut soil of the Volga area; e) strain 16 from chernozem of Khar'kov Oblast'; f) strain 19 from Crimean chernozem; g) strain 20 from red soils of Caucasus; h) strain 22 from podsol soil of Moscow Oblast', i) strain 25 from peat compost.

   Cultures of Azotobacter differ sharply from one another in their form, size and location of flagella. The flagella of some organisms are long and multiple and uniformly located along the periphery of the whole cell, the flagella of other organisms are short and few and located uniformly or not uniformly along the cell. There are forms with very short flagella which are in the form of bristles (Figure 33). These characteristics are very stable and can serve as means for their differentiation (Krasil'nikov, Khudyakova and Biryuzova, 1952).

   The main differentiating principle in this case as in other organisms is the specificity of antagonism phenomena. Khudyakova (1950) and then Babak (1956) showed that the cultures can be definitely divided into separate groups and subgroups according to their ability to suppress their competitors as well as according to their reaction to the action of antagonists. These groups and subgroups should be considered an independent taxonomic units--species and varieties.

   An was mentioned above the cultures of Azotobacter are characterized by the polymorphism of their cells. They may form very small hardly discernible cells or gontdia, or very large gigantic cellular elements. The latter are frequently considered lifeless involution forms, which are nevertheless capable of forming regenerative bodies in their protoplast.

   Under natural conditions of growth, Azotobacter is characterized by its great variability and polymorphism. This microbe changes under the influence of various soil factors, physical, chemical and biological.

   A typical culture of Az. chroococcum, which was isolated from the garden soil of Moscow Oblast' and kept for 2-6 months in a Caucasus red soil and in a chestnut soil of the Volga area, became polymorphous. Several stable variants different from the original strain were isolated. Among them. variants were found which had very small cells and had lost the capacity to form slime capsules and to accumulate a fatlike substance in their cells. Such variants persisted and even grew in chestnut soil while the original strains perished under those conditions. In red soils, variants with obvious degenerative signs were obtained. The cells of those variants were very small and polymorphous, their protoplasm was bright, hardly discernible under the microscope, almost optically empty and without granular inlets. The colonies were small, flat, pastelike and without slime. They ceased to grow and perished upon fifth or seventh planting.

   Small even filterable forms of Azotobacter were found in soils (Novogrudskii, 1935; Rybalkina, 1938b). Azotobacter forms which lost their ability to fix nitrogen are described in the literature (Rubenchik and Roisin, 1936, Stumbo and Gainey, 1938; Wyss, O., and Wyse, M., 1950).

   Considering the variability of microbes in the soil one should mention the roo -nodule bacteria.

   The root-nodule bacteria are more diverse and more widely distributed in the soil than Azotobacter. They are frequently encountered in soils where Azotobacter cannot grow. Each species of root-nodule bacteria to endowed with the capacity to form nodules on the roots of a given species of plants.

   Apart from this symbiotic specificity, many species of root -nodule bacteria can be subdivided into strains which are markedly different from each other in many other properties.

   We have studied numerous strains, of root-nodule bacteria of clover, lucerne, Onobrychis, kidney beans, vetch and peas, Strains of one and the same species differ from each other in cultural, physiological and biochemical properties and also in the extent of their activity and virulence. If their property of nodule formation were absent, they could have been classified as different species.

   Under the influence of soil and climatic factors other bacteria as well as fungi, actinomycetes, yeasts and other microorganisms also change. Some of them change their species' characteristics rapidly and sharply, others are more stable and change insignificantly.

   Ecological factors and not geographical locality, as some authors think, are the main reasons for such variability.

   The vegetative cover strongly affects the variability of soil microbes. Plants,through their root excretions, change the natural properties of some species of microorganisms.

   The different reaction of the various strains of Azotobacter upon the action of one and the same plant was frequently mentioned in the literature. Opinion was expressed as to the existence of the so-called local races of Azotobacter and on their specificity in regard to the root excretions of plants.

   Our observations have shown that this microbe changed its properties under the influence of the root excretion of wheat. Its cells become smaller and smaller until they are hardly discernible under a microscope. With the decrease in size their metabolism also changes, and the capacity to grow on ordinary nutrient media is lost. (Krasil'nikov. 1934b).

   Upon prolonged action of wheat-root excretion, the formation of new forms takes place which are markedly different from the original ones. We have obtained three such variants.

Figure 34. The variability of Azotobacter chroococcum under the influence of root excretion of wheat. The formation of stable variants in the rhizosphere in experiments under laboratory conditions:

a) original culture, b) variant A on Ashby medium, 24 -hour culture; c) variant B on Ashby medium, 72-hour culture; d) variant C, 24-hour culture.

   One of them, variant A, was of small cell size, the form of cells was bacillary, its protoplasm was homogeneous without fatlike inclusions. The presence of one or two small granules of chromophilic substance was noticed. They did not form sarcina-like packets, nor capsular resting forms (Figure 34). The cells in old cultures were shortened, often of oval or spherical form but without slime. Slime formation in this variant was either absent or weakly pronounced. The colonies were slightly convex of a pasty consistency, more rarely semislimy (Figure 35A). This variant resembles the culture of Az. vinelandii to a certain extent, but it does not form fluorescent pigment which is characteristic of the latter.

   The second variant (B) differed from the original cells by its somewhat smaller size and weak slime formation. The cells were of oval or spherical form, and contained a fatlike substance (Figure 34 C), The colonies on Ashby agar were slimy, diffuse and transparent (Figure 35 B).

Figure 35. Colonies of Azotobacter chroococcum variants obtained under the influence of wheat-root excretions:

A--variant A; B--variant B; C--variant C, 72-hour culture on Ashby agar.

   The third variant (C) differed from the former two by the polymorphism of its cells. Growing cells in a young culture (2-3 days) on Ashby agar, were of diverse form; spherical, oval, rodlike and sometimes threadlike of 15 µ and more in length (Figure 34d). Their diameter never exceeded 1. 5 µ being on the average 1-1.2 µ; cells with a diameter of 2-3 µ were rarely encountered.

   We found such cultures in the rhizosphere of wheat, in fields of the Experimental Station in Ershovo (Figure 36). One of them was sent to Dr. Bachinskaya, who studied it in detail. This culture was described as a new species of Azatobacter--Az. unicapsa (Bachinakaya and Kondratleva, 1941). This strain does not differ in morphological, cultural and physiological properties from variant A which we obtained.

Figure 36. Culture of Azotobacter unicapsa Batschin, isolated from the rhizosphere of wheat in Volga area: a) 24-hour culture; b) 48-72 hour culture on Ashby agar.

   The second culture corresponds to the experimental variant B and can be considered a new species or variety. The third strain resembles the experimental variant C in that it formed long threadlike cells reaching 50 µ in length and 1.5-2 µ in diameter. In its properties it differs sharply from the typical Azotobacter--Az. chroococcum and, according to the rules of taxonomy, can be considered as a separate variety if not as a species of the same genus.

   Bukatsch and Heitzer (1952) performed detailed experiments on numerous Azotobacter cultures isolated from the root zone and roots of 31 species of plants--legumes, cereals, grain, fruit and industrial cultures. Cultural, morphological, and physiological properties of the isolated strains were studied. The authors have shown that the studied strains differed from each other in appearance and biochemical properties to a greater or lesser degree. They differed in the rate of respiration, fermentation activity (in reference to catalyse), their response toward the pH of the medium, and high salt concentrations. They also markedly differed in their capacity to fix nitrogen and in their reaction to root excretion of different plants; finally, they exerted a different effect on the growth and yields of plants. The authors noticed that some strains promoted the production of high yields of peas and nettle (Lamium album L. ) but not of other plants. These active strains were isolated from the soil near the roots of the above-mentioned plants. This was the reason to think of specificity of the strains. Other strains did not show such a specificity with regard to plants.

   Petrenko (1949, 1953) reached the conclusion that Azotobacter forms variants which are strictly specialized with regard to some plant species. Each species of plants has its Azotobacter strain in the same manner that each species has its own strain of root-nodule bacteria. Hence, the effectiveness of employing a given microbe is manifested only in those cases when its appropriate specific strain is employed. Analogous data were obtained by Zinovlev (1950).

   Unfortunately, the authors did not confirm their assumptions by detailed laboratory experiments. The strain specificity was established only on the basis of the data of their effectiveness under field conditions without a corresponding microbiological analysis. The effectiveness of Azotobacter, as is well known, varies in different experiments. Even in experiments with one and the same culture of plants and in one field, the data on plant crop increments due to Azotobacter vary and are even contradictory.

   It should be remembered that Azotobacter, owing to its polymorphism under various conditions, in different parts of the same field, may manifest itself differently without changing its species identity.

   The sporeforming bacteria, Bac. mycoides, Bac. megatherium, Bac. mesentericus, and others, are subject to sharp variations when they grow in the rhizosphere of plants. Their cells lose the capacity to form spores, they become shorter, do not form threads--chains, and they decrease not only in length but also in diameter; as a result, cultures with very small, deformed elements, and pale, poorly stained plasma are obtained. The formation of asporogenic, deformed variants in the above-mentioned bacteria is described in the literature (Novogrudskii, Kononenko and Rybalkina, 1936; Clark F., 1940, 1949 and others).

   Arkhipov (1954) showed that the causative agent of anthrax, Bac. anthracis, markedly reacts to the action of certain plants. The cells of Bac. anthracis lose the capacity to form spores and become asporogenic and simultaneously avirulent. Degenerative forms, characterized by unusual structure and cell growth were obtained under the influence of plants.

   Data are available in the literature on the variability of root-nodule bacteria under the influence of plants. In our experiments, the root-nodule bacteria of clover and lucerne changed under the influence of the root excretion of peas, wheat, flax, corn, and cotton. The strongest influence was that of corn. After keeping the clover bacterium Rh. trifolii in the rhizosphere solution of the given plant for one and a half months the cells became sharply deformed. Upon plating on Petri dishes containing different media, several variants were obtained which differed from the original culture and from each other, morphologically, physiologically and culturally. Cells of some variants were swollen or spherical, spindlelike or threadlike in form, and the plasma was vacuolated with various inclusions (Figure 37). Such cells grew badly on the soy agar, they formed small colonies which were of pasty consistency and flat, without slime. They cannot be maintained for a long time in the laboratory; after several platings (5-8) they cease to grow (Krasil'nikov, 1949a, 1954b; Korenyako, 1942).

Figure 37. Rhizobium trifolii. Variation of culture under the influence of corn-root excretion:

a) original one-day culture on soy agar; b) variant A; c) variant B.

   Some strains acquired the ability to grow on meat-peptone medium, while the original culture cannot grow on this medium. The variants obtained also differed from each other in certain biochemical properties. They acidified milk (the original ones alkalized milk), fermented sugars, which were not attacked by the original strains, liquefied gelatin, etc (Table VI).

TABLE 6
Comparison of ability to ferment sugars displayed by variants of Rh.trifolii obtained under the influence of corn-root excretions

	External features	Glucose	Sacchrose	Maltose	Lactose	Mannitol
3	Pasty, smooth, small, rodlike cells	+	-	-	-	-
10	Slimy, normal cells	++	+++	-	+	-
12	Pasty, rough, very small and deformed cells	++++	+	-	++	+
13	Dwarf, small and deformed cells	-	-	-	-	-
	Original culture	-	-	-	-	-

Note: Plus-sugars fermented; minus-sugars unfermented

   The influence of plants on the variability of microorganisms was clearly shown in investigations in which root-nodule bacteria were grafted on roots of vegetatively coalesced soy cultures. If one kind of legume is grafted to another kind, for example a stock of kidney beans to soy or vice versa, and the coalesced plants are infected with root-nodule bacteria of the stock of the graft, then bacterial cells can be found in the formed nodules which are capable of growing and forming nodules in the roots of the graft. Employing this method (mentor method), we succeeded in changing the virulence properties of root-nodule bacteria of clover, peas, kidney beans and soy, and to confer on them the property of forming nodules foreign to them. The root-nodule bacteria isolated by us from the nodules of yellow acacia grafted to Haltmodendron halodendron were capable of forming nodules on the root of separately grown H. halodendron. The original cultures form nodules on roots of yellow acacia but do not form nodules on the roots of H. halodendron (Krasil'nikov 1954b).

   Similar results were obtained by Peterson (1953). She inoculated coalesced legumes with cultures of nodule bacteria of the stock and obtained variants with specific virulence characteristic of the graft bacteria.

    Wilson and Wagner (1936) have showed that root-nodule bacteria of clover can adapt themselves to the plant host. With the change of the plant the root-nodule bacteria change their properties accordingly.

   There are data in the literature on the adaptive variability of phytopathogenic bacteria and fungi. According to specialists, specific specialized forms of these microbes were formed as a result of adaptive variability (Naumov, 1940; Gorlenko, 1950).

   Gorlenko and co-workers showed that some strains of the sporeforming bacterium Bac. mesentericus and of the nonsporeforming Pseudomonas fluorescens could be converted into phytopathogenic forms by appropriate culture in artificial nutrient media and in vegetative substrates (Gorlenko, Voronkevich and Chumaevskaya, 1953).

   The given data provide a basis for the assumption that plants exert a definite effect on the variability of microorganisms. Unfortunately we are short of the means which are required for the detection of subtle changes which take place in many other species upon the action of root excretion or more strictly of the whole rhizosphere of this or another plant. We cannot say with certainty how this or another organism behaves when introduced under plants, whether it experiences any changes, or it remains in its original form.

   The microflora is the main factor of the variability of the microorganisms. Large numbers of antagonists supressing the growth of different species inhabit the soil. For example, many sporeforming bacteria, actinomycetes, fungi, protozoa and others have a harmful effect on Azotobacter. Under the action of these inhibitors, Azotobacter either dies or adapts itself by changing its properties. Forms of Azotobacter which are resistant to these forms are not infrequently encountered. They differ morphologically, culturally and physiologically from the original ones. Some of them are more vigorous nitrogen fixers, others, on the contrary, become less active or lose this capacity altogether. Xonokotina (1936) obtained Azotobacter variants under the influence of soil amoebae. These variants were no longer edible for the amoebae, the amoebae did not engulf them. These variants differed from the original strain in their pigment, character of growth on Ashby agar, cell structure, capacity to form slime capsules, etc.

   Afrikyan (1954) watched the formation of forms of Azotobacter resistant to microbial antagonists, bacteria, and actinomycetes.

   When obtained In this way, variants prove to be genetically stable in many cases. They can be maintained for years under laboratory conditions. We are dealing here not with polymorphism of bacteria but with species variability taking place in the natural conditions of their habitation.

   The root-nodule bacteria change their species properties under the influence of the soil microflora. Konokotina (1938) brings data of her observations on the variability of nodule bacteria of soy under the influence of pure culture of the sporeforming bacterium Bac. subtilis. The author obtained variants, among which three of them differed in their properties from the original form. Their activity was twice as high as that of the original culture.

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