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Part I, continued:

Variability of Microorganisms

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

   An higher plants and animals, they have theirevolution and philogeny which determine the development of cultures in ontogenesisand the formation of new forms and, in general, the nature of variability of thespecies.

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

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

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

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

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

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

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

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

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

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

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

   Variability in microorganisms is manifested inmany ways.

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

Individual Variability or Polymorphism of Cells

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

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

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

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

 

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

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

   Such cells are tens and even hundreds of timeslarger than normal ones, often reaching gigantic dimensions. The protoplast of thesegreatly enlarged cells differs noticeably from the protoplast of normal ones. Theirplasma is vacuolized with many inclusions of metachromatin, lipides, glycogen-likesubstances, etc. An accumulation of chromatin inclusions (see above) is characteristicof them.

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

   With some bacteria, one may maintain a culturein a degenerative state for a long time, with characteristic, greatly enlarged anddeformed cells. The latter multiply and give rise to organisms with degenerativefeatures.

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

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

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

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

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

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

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

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

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

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

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

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

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

   As was described above, tiny cells are also formedby budding.

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

   Owing to their small size, all those forms passthrough bacterial filters and may be regarded as filterable forms. Together withthe latter they evidently constitute one group of reproductive elements. These formsare also close to each other in their properties. Both are slightly viable, and donot develop or develop weakly on the usual nutrient media.

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

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

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

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

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

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

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

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

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

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

   In the sporeforming bacteria Bac. mesentericus,Bac. subtilis, Bac. mycoides, Bac. brevis, Bac. cereus and others, from fiveto ten types of colonies may be differentiated on agar media. One and the same cultureof Bac. mesentericus upon inoculation into nutrient agar often forms a mixture.typical and atypical forms, rough and folded, finely-plicated; dry, spreading onthe agar surface; flat, smooth, pellicle-like, glossy with a single radial fold (Figure31).

 

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

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

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

 

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

   According to this point of view, the sporeformingbacteria--Bac. subtilis and Bac. mesentericus are extreme variantsof one and the same species.

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

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

   The so-called typical strains of Bac. cereusproduce forms not differing from those of Bac. Subtilis, or Bac. brevisand Bac. licheniformis.

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

Table 2
Formation of monotypical variants by various species of sproreforming bacteria
Species Initial strain Variants obtained
Bac. mesentericus wrinkled (a) (a), (c), (e)
Bac. lichenformis thin-filmy (b) (a), (b), (d), (e)
Bac.cereus thick, saucer-like (c) (a), (b), (c), (e)
Bac. subtilis dry, fine-plicate (d) (a), (c), (d), (e)
Bac. brevis smooth with hair-like edges (e) (a), (c), (e)

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

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

Species Variability of Microorganisms

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

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

   All those forms of variability are of a hereditarynature, Due to this variability, the species properties of the microbe change, externalmorphological and cultural features as well as biochemical and biological featuresare thereby affected. The newly acquired properties and traits are transmitted tocells of the whole population.

   New hereditary variants are produced by the alterationof a separate cell in the culture. Among the individual differences in the population,such differences appear which cover species qualities and are stabilized in the progeny.Formation of hereditary variants occurs under the influence of environmental actionand, as a rule, is of an adaptive nature. Variants are often obtained in old cultureswithout any special external action. In this case the altered medium constitutesthe activating factor. The latter changes its composition and properties with theage of the culture in an essential way, the initial food elements disappear, nowones are synthesized, various products of metabolism are accumulated, its physicaland chemical features change, etc. In short, the medium becomes entirely different,sometimes obviously unfavorable. In such a medium, the cells die in large numbers.There are many half-living or subvital cells, and also cells with a shattered heredity.In these cases, upon plating on a fresh nutrient substrate, colonies with new propertiesdevelop. Variants with diverse morphological and physiological properties are produced.

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

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

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

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

Table 3
Production of B. coli variants which require complementary nutrient substances and other forms
Complementary substance

Formed spontaneously

Formed with x-rays

Formed with ultraviolet rays

Formed with mustard gas

Pyrimidine

 

1

 

2

Purine

 

1

 

2

Threonine

1

1

 

2

Proline

2

2

1

2

Phenylalaine

 

2

 

2

Methionine

2

5

 

2

Tryptophan

2

 

 

2

Arginine

1

1

1

 

Cystine

 

4

1

 

Leucine

 

2

 

 

Lysine

 

1

 

 

Glutamic acid

 

 

 

 

Glutamine

 

1

 

 

Histidine

 

1

 

 

Isoleucine

 

2

 

 

Tyrosine

 

3

 

2

Homocystine

 

2

 

 

Thiamine

1

4

 

 

Nicotinamide

 

2

 

 

Biotin

 

2

 

 

PABA

 

1

 

 

Pantothenic acid

 

1

 

 

Pyroxidin

 

1

 

 

Sulfite-resistant

1

 

 

 

Sulfide-resistant

1

 

 

 

Resistant to sodium chloroacetate

2

 

 

 

Resistant to lithium chloride

2

 

 

 

Fermenting lactose

3

 

 

 

Resistant to mustard gas, X-rays and ultraviolet rays

2

 

 

 

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

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

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

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

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

   The monotypical nature of the production of variantsis often noted in microbiological laboratory practice and proves the great significanceof the heredity of the organism.

   Among biological factors which induce the formationof variants, phages and antibiotics have recently been of special interest. The agentsprove to have a great effect on the variability of microbes. Upon their action diversevariants are formed, whose characteristic property in the resistance to the givenstimuli.

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

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

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

   Correlation maybe manifested only between physiologicaland biochemical properties. For instance, in some lactic acid bacteria, the abilityto ferment sorbitol and mannitol is linked with a loss of the ability to synthesizepolysaccharides, which in turn affects specific agglutination. Some strains of Staphylococcusaureus, which have the ability to assimilate only natural proline, acquire theability to assimilate both isomers. after a loss of the pigment and transformationinto colorless variants.

   Correlative connection of chemical and serologicalproperties manifests itself in diverse variants of Vibrio cholerae, obtainedunder various conditions of cultivation. There are also other manifestations of thecorrelative connections of physiological and biochemical processes to the variabilityof microorganisms. It should be assumed that variability always attacks a seriesof properties correlatively connected; but this connection is not always revealedby present methods of analysis. 

Adaptive and Directed Variability

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

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

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

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

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

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

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

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

   Adaptive ferments such as galactosidase, maltaseand others, were found in yeasts by various investigators. Synthesis of new enzymesunder the influence of specific substrates to observed in specimens of various bacterialspecies--Bac. coli, Bac. typhi, Bac. typhi murium, Bac. lactis aerogenes insporeforming bacteria, mycobacteria, actinomycetes and other forms of microorganisms.

   There are widely known microbes which producethe enzyme penicillinase in defense against penicillin. In penicillin-resistant variantsexperimentally obtained, this enzyme is produced in a strictly adaptive way. Someconditions were revealed under which the process of production of penicillinase isaccelerated or decelerated. It was established that a dose of penicillin of 0.004unit/ ml or 8 x 10 -9 M issufficient to induce penicillinase formation. Cells treated with penicillin at 0°Cand washed afterward, produce penicillinase, on subsequent incubation in a mediumwithout penicillin, 30 times more rapidly than untreated cells.

   In separate cases of adaptation the authors attemptedto elucidate the mechanism of production of enzymes. Of particular interest in thisrespect are the works of Monod and collaborators, Ephrussi, Hinshelwood and collaboratorset al. Some stages of the consecutive synthesis and intermediary products of theformation of the enzyme ß-galactosidase in the colon bacillus, in Bac. lactisaerogenes and Saccharomyces cerevisiae were established. Separate factorsaffecting the process of enzyme synthesis were elucidated. Calcium, magnesium, ironand some other microelements were found to exert an essential influence on proteaseformation (liquefying gelatin) in Proteus, on phosphatase in propionic acidbacteria. It was proved that for synthesis of enzymatic systems special complementarysubstances coenzymes, vitamins, some amino acids and other compounds are needed.These substances enter into the composition of enzymes either as a functional partof the molecule or a binding component.

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

   Not all enzymes are formed with the same rapidityduring the adaptation of the organism. Some of them are rapidly synthesized underthe influence of the specific inducer, others slowly, and still others do not adaptthemselves at all under conditions of laboratory experiments. On this basis, Karströmsubdivided ferments into adaptive and constitutive enzymes. The first, accordingto the author, are formed by the cells as a specific reaction to a correspondingsubstrate; the second are always in the cells as a constituent part of the livingsubstance. They do not depend upon the substrate, their specific synthesis is notsubjected to experimental investigation.

   This subdivision should be regarded as highlyconventional. Investigations show that there is no sufficient basis for assumingessential differences between adaptive and constitutive enzymes. Probably all enzymesmay be obtained by induction with specific substrates. If one does not succeed inobtaining some enzymes it is only due to the fact that conditions of their experimentalsynthesis have not yet been discovered.

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

   The enzymes which take part in the biosynthesisof basic metabolites (amino acids, proteins and other essential compounds) shouldbe included in constitutive systems. Without them the organism cannot develop. Onesuch enzyme in the colon bacillus is N-acetylornithase which hydrolyzes N-acetylornithinewith the formation of ornithine. The latter constitutes an indispensable elementfor the growth of the mentioned bacterium.

   Some variants of the colon bacillus which areexperimentally obtained, lack the indicated enzyme and do not develop on media withoutornithine. In the presence of N-acetylornithine such variants synthesize N-acetylornithase.Consequently, the latter is a constitutive enzyme in the initial strain of Bac.coli and an adaptive enzyme in its variant. Similar phenomena were observed inexperiments with other microbes. These data show the relative character of subdivisionof enzymes into adaptive and constitutive.

   A comparative study of adaptive ß-galactosidaseand constitutive ß-galactosidase in the colon bacillus or of adaptive and constitutivepenicillinases in the sporeforming Bac. subtilis shows that there is no essentialdifference between them. The affinity for the substrate, the degree of activationby ions, the coefficient of thermal inactivation, the immunochemical specificityare entirely the same in both adaptive and constitutive enzymes.

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

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

   Substrates of the nutrient medium, and also substancessynthesized in the cell may be inducers of enzymes. Compounds formed as a resultof one enzymatic reaction, may serve as a substrate of other enzymatic processesor inducer for synthesis of new enzymes.

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

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

   Adaptive enzymes are very specific with respectto the substrate which induces their synthesis. Some of them are more specific thanantibodies which are obtained upon the immunization of animals. With the aid of anadaptive enzyme one may sometimes succeed in subdividing bacterial strains whichcannot be differentiated by antigenic indicators. Owing to the high specificity,adaptive enzymes are used as reagents in the analysis of many organic compounds forthe differentiation and recognition of separate substances.

   Adaptive enzymes constitute the first manifestationof variability of the organism; with their aid, the nutritional substrate becomesan internal component of the living substance. These enzymes are like a gateway throughwhich the milieu enters; and the nonliving becomes living. They determine the mechanismof the adaptive variability of the organism. It is possible that the study of theformation of adaptive enzymes in microorganisms will also solve some problems ofspecies variability in general.

   With suitable conditioning one may alter thenature of the microbe in relation to those properties which do not seem to be connectedwith its enzymatic activity. For instance one may train a culture of bacteria oractinomycetes, to increased concentrations of salts or increased temperature andvice versa, thermophilic and halophilic bacteria may be transformed into mesophilictypes.

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

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

   Adaptation of microbes to phages is awidespread phenomenon often observed in laboratory practice. As was earlier noted,many bacteria and actinomycetes are subjected to the lytic action of phages. Thelatter penetrate into the cells of bacteria and actinomycetes and under suitableconditions dissolve and destroy them.

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

   Variants which are resistant to phages are endowedwith a well-manifested specificity and are only resistant to those phages which inducedtheir formation. Group specificity as well as species and strain specificity arenoted. The phage of the colon bacillus induces formation of resistant variants onlyin cultures of the given bacterium. The phage of the typhoid bacillus evokes formationof resistant strains in Bac. typhi, in the diphtheria bacillus resistant variantsare obtained under the influence of the phage of Mycob. diptheriae. In thetubercle bacillus--under the influence of the phage of Mycob. tuberculosis,etc.

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

   Experimentally obtained variants of actinomycetesor bacteria which are resistant to phages often acquire resistance to another phagespecies. However, the resistance to nonspecific phages is less strongly expressed.

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

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

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

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

   The adaptive nature of phage variability maysharply change, depending on the culture of the host microbe on which the given phageis inoculated. Upon maintain_ Ing an actinophage on one culture of actinomycetesone obtains very active strains with a large range of action, and on cultivatingit on another actinomycetes culture, adaptive variants of phages are produced withlittle activity and a narrow range of action.

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

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

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

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

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

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

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

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

   A considerable number of dependent variants hasbeen described in the literature. Such variants are often obtained in laboratorypractice. They are very specific and require for their growth, only those antibioticswhich induced their formation. Variants dependent upon antibiotics are more specificthan resistant variants. This property of the given variants may be used for thedifferentiation of antibiotics as well as their producers.





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