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Part I, Section IV, continued

"Induced" Variation in Microorganisms

   Directed changes in microorganisms may also beobtained by the method of induction of properties. This is due to the fact that whentwo defined species are developing, some of their attributes and properties are transferredfrom one species to the other. These attributes are also transferred when the culturesare 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 nonsporeformingbacillus, Bac. proteus by Zilber (1928), who obtained a para-agglutinatingvariant of Proteus under the influence of the typhoid bacillus. Gracheva (1946),obtained a strain of the colon bacillus, Bac. coli, with the properties ofanother species of the same group, Bac. breslau. Prokhorov (1950), transformeda colon bacillus into a culture with properties of the typhimurium bacillus by thesame method. Timakov (1952) and his co-workers studied induced variability in differentbacteria of the colon group for a number of years. He imparted the properties ofthe paratyphoid (the Breslau and Schotmüller types), typhoid and dysentery bacteriato a culture of Bac. coli, growing it in a medium with heat-killed culturesof 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 fromone 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 bacterialspecies. In addition to the bacteria of the colon group, staphylococci, many nonsporeformingand sporeforming bacteria, mycobacteria and others may be induced. Legroux and Genevray(1933), transformed a nonpigmented, avirulent strain of Pa. pycoyanea intoa 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 culturewere 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 insporeforming bacteria. Manninger and Nagradi (1948) kept a virulent encapsulatednonmotile culture of Bac. anthracis in a nitrate of the saprophytic, nonencapsulatedmotile soil bacillus, Bac. mesentericus, and obtained variants which differedfrom 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 sametime that nonencapsulated variants of Bac. anthracis possessed an antigenidentical with the antigen of the directing culture--Bac. mesentericus.

   The induction of properties by this method wasobserved 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 onthe induction of new properties of virulence in root-nodule bacteria, i.e., the abilityto form nodules on roots of leguminous plants foreign to these bacteria (Krasil'nikov,1945 b).

   The root-nodule bacteria, as in well known, formnodules on roots of certain plants. For instance the nodule bacteria of soybean arecapable 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 accordancewith 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 oreven to different, closely related genera. For example, Rh. leguminosarumforms 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 asingle plant species and bacteria with group specificity.

   By cultivating bacteria of this or that groupon media with filtrates of the directing cultures, we have succeeded in changingtheir specificity and imparting to them properties of virulence of the directingculture. The root-nodule bacteria of peas, vetch and sweet clover acquire the abilityto 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 bacteriaof broad beans on roots of kidney beans and Lucerne (Table 4).

Table 4
Changes of specificity of virulence in root-nodule bacteria by induction
Directing
Cultures:
Accepting
Cultures: Peas
Accepting Culture: Vetch Accepting Culture: Lucerne Accepting Culture: Broad Beans Accepting Culture: Kidney beans Accepting Culture: Acacia Accepting Culture: Clover Accepting Culture: Sweet Clover
Rhizobium trifolii + + + - - - + +
Rhizobium meliloti + + + + - + + +
Rhizobium phaseoli - - - + + - - -
Rhizobium leguminosarum + + - - - + + -

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 ina given direction and not all directing cultures are able to transfer their specificityto other bacterial species.

   Out of nine species tested, four did not acquirethe specificity of the inducing cultures. Many cultures belonging to the same speciesdid not acquire new properties. For example, out of twelve cultures of root-nodulebacteria of beans isolated in different places, seven did not change their specificity.Moreover, not all the variants obtained experimentally from the same culture lendthemselves 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 inducingculture which was the original strain of Rh. Trifolii. (Krasil'nikov, 1945b, 1955 b).

   In subsequent studies we succeeded in impartingthe properties of virulence to some nonroot-nodule bacteria of the genus Pseudomonas.Upon prolonged cultivation of these bacteria on media containing filtrates of root-nodulebacteria of clover and lucerne, strains were obtained which possessed the abilityto form nodules on roots of clover or on roots of lucerne. It should be noted, thatnot many cultures change in this fashion. Out of 91 strains of different speciesand 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 indefinitelyby means of passages from plant to plant. The newly acquired attributes of virulenceoften do not remove the former specificity. Root-nodule bacteria of peas which acquirethe ability to form nodules on the roots of clover did not lose their ability toform nodules on the roots of peas. This was also observed in relation to other bacterialvariants developing on vetch, acacia, sweet clover and broad beans.

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

   Balassa (1956) changed the specificity of virulenceof root-nodule bacteria, acting upon them with DNA of the respective cultures ofRhizobium. He used Rh. meliloti as the directing culture. With theDNA obtained from this culture he acted on nodule bacteria of soybean (Rh. japonicum)and lupine (Rh. lupini). Then the cultures were tested for virulence towardlucerne. Nodule bacteria of soybean after being acted upon by the given metaboliteof 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 obtainedby "transformation", described first by Griffith in 1928. This author foundthat pneumococci of type III possess substances which may be transferred under certainconditions to pneumococci of type II, imparting to it the properties of type IIIpneumococci. It was shown later that these transformations are quite specific andmay occur not only in vivo but also in vitro on appropriate media under certain conditions.

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

   However, their effects differ. The former possessestransforming ability, transforming the R-form of pneumococcus into the S-form butthe latter is devoid of this ability. Consequently, the transforming action of DNAis 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 thetransforming substance. Out of a great number of experimentally obtained nonencapsulatedavirulent 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 onlywhen the accepting culture possesses specific functionally active acceptors, determiningthe biochemical activity and specific nature of the pneumococcus cells.

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

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

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

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

   According to Zinder and Lederberg, propertiesof cells can be transferred from one culture to another by special tiny corpusculesor 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 fineglass ultrifilters and enter the calls of the accepting culture. The latter acquirethe properties of the former. The particle carriers of the transferring substanceare very small, not more then 0.1µ in diameter, and visible only in the electronmicroscope. The authors observed this method of changing the cultures in Bac.typhi murium. Two cultures differing in their nutritional requirements were usedin 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 twocultures being placed in a separate hall of the vessel. As a result, nonobligatorycultures were obtained. The authors called this method of changing cultures transduction.

   A similar picture of culture changes was observedby 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) possesseda wide range of action. It lysed bacteria of different species and genera: 9 strainsof genus Bacillus anthrasicis, 3 strains of genus Bac. cereus, 4 strainsof Bac. cereus var. mycoides and others.

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

   The authors assume that the phage elements transducecertain particles of living matter from Bac. cereus to Bac. anthracis,and introduce certain properties with them. According to the authors, changes, aswere 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 latterclaimed that the carriers were not the phage particles but special corpuscular elements.

   Spiegelman (1946) while studying induced changesin yeast, obtained a substance which evoked the ability to ferment galactose in specieswhich were unable to ferment this sugar. He called this substance adaptin. Its natureremained obscure. By the method of transduction one may succeed in conferring onbacteria fermentative, antigenic, virulent and other properties possessed by thedirecting culture, and one may also obtain motile strains from nonmotile ones, towhich 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 changesis based on the fact that, upon mixing two cells from different cultures, generationswith the mixed or combined properties of the initial parental organisms are formed.The cells transfer their properties and attributes upon direct contact with eachother. New variants appear which are called recombinants. The formation of such recombinantswas 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 synthesizecertain amino acids and vitamins essential for growth. On these variants experimentsof "vegetative crossing" were performed (Lederberg and Tatum, 1954).

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

   If one grows auxotroph strains separately ona full nutrient medium, and then inoculates 10-15 hour cultures on a deficient nutrientsubstrate, after thorough washing in saline, then one does not observe growth ofthe culture and the inoculation will be sterile. Only in rare cases do single coloniesgrow out; the new variants, the prototrophs, are able to develop on media withoutadditional substances. The frequency of appearance of these variants, according toLederberg and other investigators, is not higher than one cell per 1,000,000-10,000,000of the initial auxotrophs.

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

   Tatum and Lederberg an well as some other investigatorsexperimented mainly with two variants. One of the variants (58-161) required biotinfor 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 notgrow without the indicated substances on special mineral media. Mixing these variants,strains were obtained, which differed from the initial ones in that they developedon incomplete media in the absence of biotin and methionine and also variants whichdid not require threonine, leucine and vitamin B1; they themselves synthesized these growth substances. This can be schematicallyrepresented 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 culturesmay be accomplished by various strains. Some attribute or property is transferredfrom cells of one strain to cells of another. This property or attribute will, forthe 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 cultureis designated by F -. Someattributes 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 propertiesof 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 areobtained which are nonobligatory and retain the acquired properties quite well fora number of months on a nutrient egg Dorset agar medium at 4°C.

   Factor F, determining one or another attributeis not transmitted through the filtrate. A young broth culture of 58-161/F + was filtered through a colloid filterwith 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 cultureF + was not observed in anyone of them.

   As was noted earlier, when the cultures are mixedwith an F - strain the plantingis sterile. Upon mixing the F +and F - cultures the platingsare 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 945million auxotroph cells. Plating the strains 58-161/F+ and W-677/F+ only4 prototroph colonies out of 477 million were observed and upon plating the strains58-161/F - and W-677/F +, 49 prototrophs appeared out of the totalnumber of 450 million cells.

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

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

   In experiments of other investigators with thecolon bacillus K-12 analogous results were obtained. Defective strains, that havelost the ability to synthesize a certain amino acid or vitamin, reacquire the abilityupon contact with cells possessing these properties. Strain U-24 which in the experimentsof Clark (1953) required biotin, phenylalanine and cyotine (B-Ph-C-), does not growon a synthetic medium lacking these substances. Strain U-10 forms biotin, phenylalanineand cystine, but does not produce threonine, leucine, and thiamine (T- L- B1-) anddoes not develop on a synthetic medium without these compounds. After mixing twosuch organisms, strains are obtained, which grow well on the indicated media andsynthesize 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) describedthe formation of recombinants obtained upon crossing strains of blue actinomycetes--A.coelicolor, after irradiation by ultraviolet rays. One strain (5 me- hist-) requiredmethionine and histidine and formed a blue pigment. Another requiring strain (14pr.- glu- pigm.-) required proline and glutamic acid and did not form pigment. Inmixed platings on complete agar media three types of colonies are formed. The initialones: a) 5 me-hist-, b) 14 pr.- glu pigm, and c) recombinants nonrequiring culturesthat do not require methionine. histidine, proline or glutamic acid.

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

   Some investigators found similar transmissionsof 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 culturewith the formation of deformed gigantic cells and other cells, lowered the numberof prototrophs formed. Haas and others noted a correlation between the formationof strongly enlarged, threadlike and other polymorphic cells and the increase ofthe number of prototroph variants among the auxotrophs. According to their observations,ultraviolet irradiation enhances the process of recombination in the colon bacilluswith the simultaneous increase in the polymorphism of the culture. Certain chemicalsubstances, hydrogen peroxide and others, stimulate the formation of prototrophs(Haas, Clark, Wyss, and Stone, 1952). Grigg (1952) stresses the importance of thedependence of the appearance of recombinants among auxotrophs upon the quantitativera 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 willbe 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 bethe smallest (Table 5).

Table 5
The effect of quantitative ratios between components of the mixture on formation of variation with transmission of properties of the F + cells to the F - cells

F -

F +

Ratio of F - : F +

0.5*

1*

2*

4 x 106 cells/ml

1 x 108 cells/ml

1:25

20/20

10/20

19/20

4 x 106 cells/ml

4 x 106 cells/ml

1:1

10/20

13/20

6/20

4 x 106 cells/ml

1 x 106 cells/ml

4:1

2/20

0/20

3/20

*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 transformedfrom F - to F +. At a 1:1 ratio of these strains, thenumber of transformed variants was 10 out of 20 and at a ratio of 4:1 it was 2 outof 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 ofwhich the number of gene carriers increases.

   In the formation of recombinant variants thefollowing 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 mixturetheir 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 thereis a strong bond between various attributes.

   It should be noted that such variants cannotbe obtained upon mixed cross platings of all cultures. Even in the same species ofthe 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 homothallicculture and strain B, a heterothallic one. The former possesses both the masculineand the feminine tendencies, whereas the other has only one of them. Therefore, thelatter cannot dissociate according to this attribute and give combined variants uponmixing, as happens in K-12 strains. Strain B in monosexual or asexual, and is similarto certain imperfect fungi, for instance from the group of asporogenous yeasts: Torulopsis,Mycotorula, etc. According to the authors, upon contact between the minus variantsof the homothallic culture a process of conjugation or exchange of substances takesplace between them, similar to the sexual process in higher organisms. With thesesubstances the carriers of hereditary properties, the genes, are also transmitted.(Lederberg and Tatum, 1954; Anderson et. al., 1957).

   On the mechanism of variation. As canbe seen from the above-mentioned data, the phenomenon of variation in microorganismsis quite diverse both in its appearance and in the nature of its response to externalinfluences. The mechanism of variation and the inheritance of properties remainsunexplained. There are many points of view, and many suggestions were made all ofwhich can be reduced to two basic theories: the theory of mutation" the theoryof physiological adaptation. These theories were formulated long ago. and were alreadyknown during the period of the first discoveries in the field of variation in microbesin general. However, there have not been sufficient factual data to corroborate anyof these theories.

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

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

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

   The phenomenon itself really resembles combinationsformed during the process of sexual reproduction; however, the mechanism here isprobably different. The bacteria mentioned do not have a sexual process of the formthat is observed in fungi and yeasts. Direct conjugation and mixing of living matterof two cells is not possible here. It should be assumed that upon contact betweencalls in the above-described cases of transduction the transforming substance istransmitted 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 maynow be regarded as proven. They are found in various microorganisms--bacteria, actinomycetes,yeasts and fungi. As previously noted, it was found in pneumococci, nonsporeformingbacteria, in sporeformers, etc. In certain cases the chemical structure of thesesubstances was established or their chemical composition disclosed.

   In transduction the transforming substance maybe carried over from cell to cell with the aid of phages. The latter build theirbody from the live matter of bacterial cells (mainly of DNA). When the cells lysethe phages are liberated and if they pass to cells of another culture they evidentlycarry over particles of living matter of the former cells. The mixing of protoplastparticles of two different cultures takes place, and with it the hereditary propertiesare mixed, which later seggregate out with the formation of new variants--recombinantsin the progeny.

   The ability of microbial cells to absorb complexorganic compounds, metabolites of microorganisms, has been established by many investigatorswith different representatives of the lower organisms. It has been shown that compoundssuch as vitamins, auxins, amino acids, enzymes, antibiotics and other substancesimportant to life are absorbed by microbial cells through an intact membrane.

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

   Oparin and Yurkevich (1949) have shown that brewer'syeasts have the ability to absorb enzymes from the medium and use them. It was alsoestablished 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 synthesizetheir own invertase and used it for the splitting of saccharose. The variants thusobtained, maintained the ability to synthesize this enzyme for an indefinite lengthof time and transmitted this ability hereditarily through many generations.

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

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

   Many metabolites, as is well known, are of considerableimportance 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 reasonany of these metabolites are blocked, the cells become different and their biochemicalprocesses are not the same as those of the initial, normal cell. If the given changeis stabilized and transmitted to subsequent generations, new variants are obtainedduring the process of multiplication.

   The blocking or change of metabolites may takeplace under the influence of antimetabolites or metabolites which are formed by otherspecies of microorganisms. The interaction between metabolites and antimetabolitesmay manifest itself in different forms and degrees. After being absorbed by the cellsfrom the medium, an antimetabolite may be fixed and combine with metabolites, disturbingtheir function. As a result of such blocking the formation of new variants of microorganismsis possible.

   Many metabolites are described in the literature,which have corresponding antimetabolites inactivating them. Vitamin B1, or thiamine, has an analogous antimetabolite--pyrithiamineand neopyrithiamine. Pyrithiamine paralyzes the action of thiamine. Analogous antimetaboliteshave been found for biotin, choline, folic acid, ascorbic, and nicotinic acids andfor 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 induceris processed by the living protoplasm, and takes part in a series of various biochemicaltransformations of some metabolites, causing corresponding changes in them. The processof transmission of properties from one organism to another is evidently possibleonly 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 ofbinding the inducing substance of the inducing culture. Loss of these acceptors deprivethe cell of the ability to change in this manner.

   The phenomenon of adaptation to substrate andto external agents may be caused by other reasons and may consequently have anothermechanism. Some investigators connect the resistance of bacteria to antibiotics orphages 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 inactivatethem, without disturbing the intactness of the cell.

   In a number of cases the mechanism of resistanceformation to antibiotics is connected with a change in the permeability of the cellmembranes or their protective barrier. Ehrlich has shown (1909), that normal trypanosomestake up acriflavin, while acriflavin-resistant strains which are adapted to it, donot absorb the drug. There are similar data concerning penicillin. Certain penicillin-resistantbacterial variants absorb less penicillin than the initial sensitive cultures. Accordingto our data, chemically purified mycetin is absorbed by the diphtheroid bacteriaof Mycobacterium sp. (initial culture) in the amount 32.5 units and the experimentallyobtained drug-resistant variant absorbs only 16.0 units per same number of cells.According to different authors, streptomycin is absorbed by the membrane of certainbacteria and is bound within it by nucleic acids. As a result, the metabolism isdisturbed and the activity of the cells differs from that of normal organisms.

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

   It should be noted that the authors determinethe number of mutations only with respect to one characteristic. If one considersvariations of different features. and they are quite numerous, then the number ofmutations 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 termquite 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 microbialmutants ought to preexist. Furthermore, the culture should contain mutants adaptedto 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, ifone accepts the point of view of the mutation theory, one should assume the pre-existencein each culture of mutants adapted to all possible agents and to various doses ofthe agents. In other words, an infinite number of mutants should exist; almost everycell 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 environmentonly selects the already existing mutants from the culture. Upon action of one oranother agent, be it antibiotic, phage, chemical or physical, the cells resistantto the agent, that is the mutants, survive, while all the sensitive forms are removed.

   The mutation theory cannot explain many phenomenaof 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 phaseof the culture. Under suitable conditions many adapted forms are simultaneously formedin the culture Their percentage may be increased considerably, to 1-10 per cent andmore, whereas according to the mutation theory their number does not exceed 1 per100-1,000 millions of normal cells.

   The phenomenon of stepwise adaptation of bacteriato ever-increasing concentrations of an antibiotic and of other substances remainsunexplained. Upon inoculation of the active agents, into media with increasing concentrationsthe frequency of formation of resistant variants increases sharply and this processproceeds much faster.

   The appearance of forms which are simultaneouslyresistant to two, three or more substances is also not clear. It is even more difficultto understand the removal of acquired resistance of variants by the action of otherantibiotics (or other substances). The mutation theory is also unable to explainmany other aspects of variation.

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

   The numerous observations and experiments ofthe recent years convince the investigators more and more that variation in microorganismstakes place by way of physiological adaptation to the environment. The living matterof the organisms specifically reacts to external agents, changing its propertiesto correspond to the quality of the acting substance. Under the influence of thesubstrate, inducer cells primarily react by changing their enzyme systems. Many investigatorsare 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 thesynthesis of the given enzyme. Upon prolonged action of the inducer, the adaptiveenzyme turns into a constitutive enzyme. The acquired ability may be carried througha long series of generations in the absence of the substance that caused its appearance.

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

Variability of Soil Microorganisms

   The problem of the variability of soil microbesunder their natural conditions of habitation is little understood. Only a few workson 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 variabilityin microorganisms. The sporeforming bacteria--Bac. mycoides, Bac. subtilis, Bac.mesentericus, and other typical cultures with the characteristic structure ofcolonies become atypical and form variants differing from the ordinary forms.

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

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

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

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

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

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

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

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

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

   The specific effect of soils on the bacterialvariability is also confirmed by experiments carried out with other cultures of soilbacteria.

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

   Do they represent separate species or are theyvariants and forms of one and the same species? This question has been experimentallydetermined.

   Cultures of Bac. mesentericus were introducedinto various soils--podsolic, forest, field and garden soils; chernozems, chestnutsoils and red soils. The cells of Bac. mesentericus died out with relativerapidity in red soils and forest podsolic soils. In weakly cultivated fields of acidsod-podsolic soils and also in chestnut soils and chernozems, the culture did notdie out completely. A small number of the cells survived. After some time the survivorsbegan to multiply. However, upon plating on nutrient media they produced colonieswhich differed from the original ones.

   In the course of adaptation new variants wereobtained 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. subtilisand 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 thesame culture of Bac. mesentericus manifests different morphological formsaccording 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. subtilisand Bac. cereus. We converted individual strains of one or another type intotypical cultures of Bac. mesentericus.

   Considering only the external appearance of theculture, the difference between the mentioned species may be thought of as a reflectionof polymorphism. Apparently, the classification of this bacterial group should notbe based on external differences but on biochemical and biological properties. Ofthe latter, antagonism and antibiotic spectrum are the most indicative (in our opinion).

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

   The principle of the specificity of antagonisticinteractions makes it possible to reveal natural ecologic strains and types whichactually exist, and to differentiate them.

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

   The absence of spores in such cultures may misleadthe investigators. Similar cultures can be classified as nonsporeforming bacteria.However, the structure of the colonies, the size of the cells, and the structureof the protoplast show that these organisms belong to the sporeforming group. Theformation 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 atelevated temperatures, 33-37° C. Some cultures begin to form spores after prolongedcultivation on protein-rich media, and others after they are kept in soils of themore southern regions such as the podsol of Moscow Oblast', chestnut soils of theVolga area and others. This peculiarity of these bacteria is apparently caused bylong habitation In unusual soil-climatic conditions. Those conditions change thegrowth characteristics and the whole biology of the species. Bacteria lose the capacityto form spores. In some organisms, only the initial processes of spore formationare 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 wherethe soil is always frozen or thaws for a short period, spore formation does not constitutea protective reaction of species preservation and in gradually lost. The biologicalessence of such bacteria as a sporeforming species is nevertheless preserved.

   The capacity to form spores is lost, not onlyin bacteria of the arctic soils, but also in bacteria which inhabit more southernregions. The formation of asporogenic forms in general is a phenomenon quite frequentlyencountered in specimens of the genus Bacillus, and it takes place in laboratorycultures as well as in nature directly in the soil. One does not always succeed inrevealing the asporogenic forms in the soil since they are differentiated only withdifficulty 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 climaticconditions, 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 thehabitation of Azotobacter, only one species is described as widespread inthe literature, namely, Azotobacter chroococcum. Some authors think it canbe subdivided into 2 to 4 species. In our manual we have given 7 species, two ofthem being of an entirely different group.

   Laboratory experience shows that cultures ofAzotobacter of whatever origin, are at first eight homogenous according totheir culture characteristics. They all grow well on media devoid of nitrogen (Ashbyagar) and form large slimy colonies. After some time the colonies become brown andeventually black. All cultures fix nitrogen, to a greater or lesser extent, and arediagnosed 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 strainscollected from different soils of the Soviet Union it was found that they differfrom each other, morphologically and biologically. The size of cells, their formand structure are far from similar, as described by many authors. We have described(Krasil'nikov, 1949 a) the characteristic differences of the, individual strainsof Azotobacter, differences which served as the basis for dividing them intoseparate species such as Az. beijerinckii, v. jakutii, Az. galophilum andothers. The cells of these and other organisms differ from each other in their sizeand form as well as in the internal structure of the protoplast. Cultures also differin 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 sharplyfrom one another in their form, size and location of flagella. The flagella of someorganisms are long and multiple and uniformly located along the periphery of thewhole cell, the flagella of other organisms are short and few and located uniformlyor not uniformly along the cell. There are forms with very short flagella which arein the form of bristles (Figure 33). These characteristics are very stable and canserve as means for their differentiation (Krasil'nikov, Khudyakova and Biryuzova,1952).

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

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

   Under natural conditions of growth, Azotobacteris characterized by its great variability and polymorphism. This microbe changesunder 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 monthsin 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 formslime capsules and to accumulate a fatlike substance in their cells. Such variantspersisted and even grew in chestnut soil while the original strains perished underthose conditions. In red soils, variants with obvious degenerative signs were obtained.The cells of those variants were very small and polymorphous, their protoplasm wasbright, hardly discernible under the microscope, almost optically empty and withoutgranular inlets. The colonies were small, flat, pastelike and without slime. Theyceased to grow and perished upon fifth or seventh planting.

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

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

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

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

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

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

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

Bacterial Variability under the Influence of Plants

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

   The different reaction of the various strainsof Azotobacter upon the action of one and the same plant was frequently mentionedin the literature. Opinion was expressed as to the existence of the so-called localraces of Azotobacter and on their specificity in regard to the root excretionsof plants.

   Our observations have shown that this microbechanged its properties under the influence of the root excretion of wheat. Its cellsbecome smaller and smaller until they are hardly discernible under a microscope.With the decrease in size their metabolism also changes, and the capacity to growon 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 originalones. 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). Thecells in old cultures were shortened, often of oval or spherical form but withoutslime. Slime formation in this variant was either absent or weakly pronounced. Thecolonies were slightly convex of a pasty consistency, more rarely semislimy (Figure35A). 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 originalcells by its somewhat smaller size and weak slime formation. The cells were of ovalor spherical form, and contained a fatlike substance (Figure 34 C), The colonieson 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 formertwo 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 threadlikeof 15 µ and more in length (Figure 34d). Their diameter never exceeded 1. 5µ being on the average 1-1.2 µ; cells with a diameter of 2-3 µ wererarely encountered.

   We found such cultures in the rhizosphere ofwheat, in fields of the Experimental Station in Ershovo (Figure 36). One of themwas sent to Dr. Bachinskaya, who studied it in detail. This culture was describedas a new species of Azatobacter--Az. unicapsa (Bachinakaya and Kondratleva,1941). This strain does not differ in morphological, cultural and physiological propertiesfrom 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 experimentalvariant B and can be considered a new species or variety. The third strain resemblesthe experimental variant C in that it formed long threadlike cells reaching 50 µin length and 1.5-2 µ in diameter. In its properties it differs sharply fromthe 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 detailedexperiments on numerous Azotobacter cultures isolated from the root zone androots of 31 species of plants--legumes, cereals, grain, fruit and industrial cultures.Cultural, morphological, and physiological properties of the isolated strains werestudied. The authors have shown that the studied strains differed from each otherin appearance and biochemical properties to a greater or lesser degree. They differedin the rate of respiration, fermentation activity (in reference to catalyse), theirresponse toward the pH of the medium, and high salt concentrations. They also markedlydiffered in their capacity to fix nitrogen and in their reaction to root excretionof different plants; finally, they exerted a different effect on the growth and yieldsof plants. The authors noticed that some strains promoted the production of highyields of peas and nettle (Lamium album L. ) but not of other plants. Theseactive strains were isolated from the soil near the roots of the above-mentionedplants. This was the reason to think of specificity of the strains. Other strainsdid not show such a specificity with regard to plants.

   Petrenko (1949, 1953) reached the conclusionthat Azotobacter forms variants which are strictly specialized with regardto some plant species. Each species of plants has its Azotobacter strain inthe 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 caseswhen its appropriate specific strain is employed. Analogous data were obtained byZinovlev (1950).

   Unfortunately, the authors did not confirm theirassumptions by detailed laboratory experiments. The strain specificity was establishedonly on the basis of the data of their effectiveness under field conditions withouta corresponding microbiological analysis. The effectiveness of Azotobacter,as is well known, varies in different experiments. Even in experiments with one andthe same culture of plants and in one field, the data on plant crop increments dueto Azotobacter vary and are even contradictory.

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

   The sporeforming bacteria, Bac. mycoides,Bac. megatherium, Bac. mesentericus, and others, are subject to sharp variationswhen they grow in the rhizosphere of plants. Their cells lose the capacity to formspores, they become shorter, do not form threads--chains, and they decrease not onlyin 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, deformedvariants 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 agentof anthrax, Bac. anthracis, markedly reacts to the action of certain plants.The cells of Bac. anthracis lose the capacity to form spores and become asporogenicand simultaneously avirulent. Degenerative forms, characterized by unusual structureand cell growth were obtained under the influence of plants.

   Data are available in the literature on the variabilityof root-nodule bacteria under the influence of plants. In our experiments, the root-nodulebacteria of clover and lucerne changed under the influence of the root excretionof peas, wheat, flax, corn, and cotton. The strongest influence was that of corn.After keeping the clover bacterium Rh. trifolii in the rhizosphere solutionof the given plant for one and a half months the cells became sharply deformed. Uponplating on Petri dishes containing different media, several variants were obtainedwhich differed from the original culture and from each other, morphologically, physiologicallyand culturally. Cells of some variants were swollen or spherical, spindlelike orthreadlike in form, and the plasma was vacuolated with various inclusions (Figure37). Such cells grew badly on the soy agar, they formed small colonies which wereof pasty consistency and flat, without slime. They cannot be maintained for a longtime 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 onmeat-peptone medium, while the original culture cannot grow on this medium. The variantsobtained also differed from each other in certain biochemical properties. They acidifiedmilk (the original ones alkalized milk), fermented sugars, which were not attackedby 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 ofmicroorganisms was clearly shown in investigations in which root-nodule bacteriawere grafted on roots of vegetatively coalesced soy cultures. If one kind of legumeis 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 thegraft, then bacterial cells can be found in the formed nodules which are capableof growing and forming nodules in the roots of the graft. Employing this method (mentormethod), we succeeded in changing the virulence properties of root-nodule bacteriaof clover, peas, kidney beans and soy, and to confer on them the property of formingnodules foreign to them. The root-nodule bacteria isolated by us from the nodulesof yellow acacia grafted to Haltmodendron halodendron were capable of formingnodules on the root of separately grown H. halodendron. The original culturesform 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 andobtained variants with specific virulence characteristic of the graft bacteria.

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

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

   Gorlenko and co-workers showed that some strainsof the sporeforming bacterium Bac. mesentericus and of the nonsporeformingPseudomonas fluorescens could be converted into phytopathogenic forms by appropriateculture in artificial nutrient media and in vegetative substrates (Gorlenko, Voronkevichand Chumaevskaya, 1953).

   The given data provide a basis for the assumptionthat plants exert a definite effect on the variability of microorganisms. Unfortunatelywe are short of the means which are required for the detection of subtle changeswhich take place in many other species upon the action of root excretion or morestrictly of the whole rhizosphere of this or another plant. We cannot say with certaintyhow this or another organism behaves when introduced under plants, whether it experiencesany changes, or it remains in its original form.

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

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

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

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



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