MICROBIOLOGY: PROKARYOTE BIOLOGY, VOL. I — A. V. PINEVICH — 2006

CHAPTER 2. THE POSITION OF BACTERIA IN THE BIOLOGICAL MEGASYSTEM

FROM LEEUWENHOEK'S ANIMALCULES, THROUGH COHN'S SCHIZOMYCETES AND STANIER'S PROKARYOTES, TO THE PHYLOGENETIC DOMAIN BACTERIA

Class="center">A scientist must reckon with views that contradict their own scientific convictions; they must weigh all opinions and choose only those backed by the most compelling evidence.

L. S. Berg. Science: Its Content, Meaning, and Classification (Petrograd, 1920).

The Place of Bacteria within the biological megasystem was finally established only in the late 1970s, whereas the analogous task for plants and animals was solved as early as the first half of the 18th century by the Swedish botanist Carl Linnaeus (C. Linnaeus).

Linnaeus's system developed and deepened ideas already found in the writings of ancient philosophers and naturalists—Aristotle (Aristoteles), Theophrastus (Theophrastus), Titus Lucretius Carus (Titus Lucretius Carus), and Gaius Pliny the Elder. Its author also utilized all the valuable insights from the works of 16th- and 17th-century coryphaei of botany, anatomy, and physiology—Vesalius (A. Vesalius), Wotton (E. Wotton), Clusius (C. Clusius), Caesalpino (A. Caesalpino), Malpighi (M. Malpighi), and Swammerdam (J. Swammerdam). Today, the Linnaean system extends to all cellular organisms except prokaryotes.

The exceptionally long persistence of an "extra-systemic" classification of prokaryotes, particularly bacteria, has deep roots linked to their microscopic size and structural peculiarities. As bacteria were studied more closely, conceptions of their fundamental nature were repeatedly revised, which inevitably affected their megataxonomic status.

2.1. Early Views on the Biological Nature of Bacteria

The cornerstone of Carl Linnaeus's biological classification is the dichotomy between animals and plants. Therefore, it is unsurprising that researchers initially sought a place for bacteria within either the animal or plant kingdom.

Even in antiquity, the primary attribute of plants (Plantae) was considered to be passive growth—that is, an individual's life rooted in a fixed location (Lat. planto, to plant) accompanied by relatively rapid vegetative growth. Conversely, the defining characteristic of animals (Animalia) was active action driven by internal stimulus (Lat. animo, to rouse), representing a motile existence with relatively slow size increases. It is hardly surprising that the discoverer of bacteria, Leeuwenhoek, regarded them as "living animalcules" (Lat. vivi animalculi). Observing their rapid swimming, he had no doubt they possessed limbs (Lat. manusculi) so minute that they escaped microscopic detection. Leeuwenhoek also referred to bacteria as infusion animals, or "infusoria" (Lat. infusio, wetting), because they accumulated in soaked hay and other plant infusions.

Linnaeus was familiar with Leeuwenhoek's work, yet he found no morphological features among the "infusoria" sufficient to classify them as an independent group of animals. Consequently, he termed bacteria "doubtful species" (Lat. species dubia) and placed them in the "unknowable" genus Chaos, where protists and Flatworms also happened to end up.

Investigators in the latter half of the 18th century made no attempts to classify bacteria. It was not until the German protistologist Otto Müller (O. Müller), in his book Animalcula infusoria fluviatilia et marina (1786), subdivided "infusoria" into 18 genera based on criteria such as shape, motility, tendency to form cellular aggregates, and habitat. Among the genera he described were Monas and Vibrio—the first identified spherical and ellipsoidal bacteria, although protists with similar Morphology were inadvertently included.

In 1838, Christian Ehrenberg employed an improved Microscope model with achromatic lenses for the anatomical study of unicellular microorganisms. Due to the diminutive size of bacteria, he failed to discern their fine morphological details, yet he assumed they must mirror those of Ciliates. Based on external traits, he segregated bacteria into the genera Bacterium, Monas, Spirillum, Spirochaeta, and Vibrio. The names of several species he described have survived—for instance, Spirillum volutans and Spirochaeta plicatilis. Refining Ehrenberg's system, Félix Dujardin (F. Dujardin) was the first to demarcate bacterial species by shape and motility patterns. Specifically, he split the genus Vibrio into four species: V. bacillus, V. lineola, V. rugula, and V. serpens. In the early 1850s, Maximilian Perty continued research along these lines, proposing names for several new bacterial genera and species.

Until the final quarter of the 19th century, bacteria were considered lower animals. However, in 1878, Karl Nägeli (C. von Nägeli) discovered that Acetobacter xylinum and Sarcina ventriculi contain Cellulose, leading him to conclude that bacteria fundamentally differ from animals and instead align with plants (in which this polymer is universal and forms part of Cell walls).

Nägeli revised the prevailing views on the taxonomic position of bacteria, reassigning them to the plant kingdom as the class Schizomyceteae (from Gr. schizo, to split in two, and Lat. mycelium, fungus bed; literally, "Fungi reproducing by binary fission").

Soon, however, it became clear that Nägeli had chanced upon rare cases of bacterial cellulose synthesis (which, moreover, is deposited externally rather than integrated within the wall framework). Later investigations demonstrated that bacteria indeed possess a Cell wall, but it contains not cellulose, but rather a nitrogen-containing polymer known as murein.

Nevertheless, because of their possession of a cell wall, bacteria were placed within the group of lower plants (Thallophyta) and came to be viewed as a primitive group of "achorophyllous fission fungi" (Schizomycetes).

2.2. HISTORY OF BACTERIAL Systematics

Bacterial systematics is the youngest and most dynamic branch of biological classification. The streamlined cellular architecture of prokaryotes, often ignorantly termed "simplicity," stems from a limited repertoire of morphological traits. These traits are insufficient for a robust and detailed classification compared to that of higher eukaryotes, which relies heavily on morphology.

Unlike the systematics of Higher Plants and animals, bacterial systematics could not draw upon a paleontological record. Consequently, during The Development of taxonomic systems, the choice of criteria was dictated by the methodological arsenal available to researchers, which permitted the analysis of extant bacteria only.

A preeminent contribution to the development of bacterial classification in the 1870s was made by Ferdinand Cohn, a student of Christian Ehrenberg. He regarded bacteria as plants closely related to cyanobacteria (blue-green Algae), dividing them according to cell shape into four "tribes" encompassing six form-genera:

— Sphaerobacteria (spherical bacteria; genus Micrococcus);

— Microbacteria (rod-shaped bacteria; genus Bacterium);

— Desmobacteria (filamentous bacteria; genera Bacillus and Vibrio);

— Spirobacteria (spiral bacteria; genera Spirillum and Spirochaeta).

A more detailed Classification of Bacteria took into account specific morphological features as well as certain PHYSIOLOGICAL AND BIOCHEMICAL properties; for example, the genus Micrococcus was divided into three groups of species based on pigmentation (“chromogen”), enzymatic activity (“zymogen”), and infectiousness (“contagion”).

However, Cohn’s view that the botanical concept of a species applied to bacteria was not shared by all microbiologists. Many of them were convinced that bacteria were capable of spontaneous generation and exhibited pleomorphism (from the Greek plethos, meaning multitude, and morphe, meaning form)—that is, a propensity for unrestricted Structural and functional transformations, As a result of which an individual Organism appears in a multitude of life forms.

Bacterial cultures were essential for addressing the challenges of industrial and medical microbiology. Early sterilization Methods were largely inefficient, causing uninoculated media to become consistently overgrown. This, however, was attributed not to the accidental Introduction of microbes, but to their spontaneous generation, or “abiogenesis,” within the nutrient medium. It was assumed that bacteria arose spontaneously even during the chemical decomposition of PLANT AND ANIMAL Tissues.

The hypothesis that living creatures could originate spontaneously from “proto-elements” (Water, earth, and air) or decomposing organic remains dates back to antiquity. It is set forth, notably, in the writings of Aristotle and Lucretius. Its proponents believed that living organisms could arise spontaneously from inorganic matter (“abiogenesis”) or from the metabolic products of other organisms (“heterogenesis”). Because this process occurs randomly, parental involvement is not required.

One of the earliest opponents of the spontaneous generation hypothesis was Francesco Redi, who in the mid-17th century demonstrated that maggots do not appear in decaying meat de novo, but represent a larval stage in the development of insects. In the late 17th century, Leeuwenhoek rejected the spontaneous generation hypothesis, arguing that the germs of “animalcules” were distributed through the air. In the second half of the 18th century, J. T. Needham and L. Spallanzani used the boiling of nutrient media to test the possibility of spontaneous generation, yet they failed to reach a definitive Conclusion. Until the mid-19th century, the hypothesis of spontaneous generation was supported by such biological authorities as Gottfried Treviranus, Otto Müller, Jean-Baptiste de Lamarck, Friedrich Kützing, and Félix Dujardin. Specifically, Lamarck considered spontaneous generation to be the mechanism that served as the initial stage in the evolution of life from simpler to more complex forms.

After John Tyndall developed a method for the thermal sterilization of liquid media and proposed Physicochemical methods for sterilizing gases, it became conclusively clear that atmospheric air served as the primary vehicle for microbial dissemination. Louis Pasteur, who played The Role of the gravedigger of the spontaneous generation hypothesis, convincingly demonstrated that nutrient media remained sterile as long as they did not come into contact with non-sterile air. For such a strict determinist as Pasteur, the impossibility of spontaneous generation was of fundamental significance. Otherwise, any sterilization would be useless, and Fermentation industries and the combat against pathogenic microbes would depend entirely on chance. Ultimately, the hypothesis of spontaneous generation was discredited, although the fundamental impossibility of bacterial spontaneous generation cannot be proven by the results of any single specific experiment.

Alongside Structure/149.html">The problem of spontaneous generation, there was also the issue of the constancy of bacterial forms. Because the methods of elective media and bacterial cloning had not yet been developed, experimenters were dealing with cultures contaminated by extraneous bacteria, Yeasts, and Molds. In 1848, Carl Nägeli concluded that the biological species category did not apply to bacteria because they were pleomorphic and transformed into one another when external conditions changed. The cAMP of pleomorphists gathered prominent bacteriologists such as E. Hallier, R. Lankester, and Joseph Lister. A proponent of extreme pleomorphism, T. Billroth hypothesized in 1874 that all bacteria were manifestations of a single species, Coccobacillus septica. He and his supporters believed that this hypothetical microbe could adapt to different living conditions by altering its morphology, resulting in The Emergence of micro-, meso-, and megacocci, as well as micro-, meso-, and megabacteria (spiral-shaped bacteria were not considered in this context). Lister even entertained the fantastic possibility of the reversible transmutation of pathogenic bacteria into fungal spores. However, as early as the 1870s, Ray Lankester was compelled to acknowledge the reality of species in bacteria, even though he remained a proponent of pleomorphism. Conversely, the camp of monomorphists included Louis Pasteur, Ferdinand Cohn, and Robert Koch, as well as O. Brefeld and other prominent bacteriologists. It was only when Cohn published the results of his research on The life cycle of spore-forming bacilli, and Koch developed the method for isolating pure bacterial cultures from individual colonies on solid nutrient media, that the hypothesis of pleomorphism was finally discarded.

A tremendous impact on the development of bacteriology was made by the method of obtaining pure cultures through colony subculturing, invented by Robert Koch in 1881. In 1882, his associate W. Hesse suggested using Agar-agar—a water-soluble polysaccharide not metabolized by most bacteria, derived from the red alga Gelidium corneum—to solidify the medium. In 1887, R. Petri devised a brilliantly simple apparatus that allowed bacteria to be cultivated in the absence of airborne contamination, known as the Petri dish. Following the introduction of pure cultures into the daily practice of microbiological research, A large number of new bacterial species were described.

In 1884, the Danish physician H. C. Gram proposed a differential staining method later termed “Gram staining.” To this day, initial bacterial Diagnostics are based upon it.

During the 1890s and 1900s, several attempts were made to refine the systematics of bacteria. In the system proposed by K. B. Lehmann and R. Neumann in 1896, bacteria were divided into orders, families, and genera based on their appearance, the orientation of the plane of division, and the number and arrangement of flagella, among other features. Primary emphasis was placed on the Diagnosis of bacteria relevant to medicine and microbiological manufacturing. These authors also described the new genera Actinomyces, Corynebacterium, and Vibrio.

In 1897, W. Migula developed a system that encompassed all species known by the end of the 19th century. It was based on the principles of morphological classification proposed by Cohn back in the 1870s. However, this time, cultural characteristics—above all, nutritional requirements—were incorporated into the species diagnoses.

As cytophysiological and Biochemical Methods advanced, bacteriologists acquired tools previously employed solely in The Study of higher organisms. From that point on, bacterial classification began to rely not only on morphological traits but also on functional and chemotaxonomic characteristics.

However, in order to determine the genealogical relationships among bacterial groups and ultimately reconstruct the Phylogenetic Tree, it was necessary to rank traits according to their degree of archaism—that is, to determine which traits were acquired earlier in evolution and which later.

Initially, general physiology was assigned the leading role in solving this problem. In 1909, S. Orla-Jensen hypothesized that autotrophy represented a primitive physiological trait, and therefore autotrophs must be the most archaic among bacteria.

A contrasting viewpoint was held by E. G. Pringsheim, R. E. Buchanan, as well as Albert Kluyver and C. van Niel. In 1936, the latter two researchers developed a hypothetical scheme of bacterial evolution in which cocci were regarded as the primary morphotype from which rod-shaped bacteria, spiral bacteria, and others evolved. This position was succinctly formulated in 1933 by A. R. Prévôt: “Morphology dominates over physiology.”

From the second half of the 1950s, the “numerical” method—originally proposed back in 1763 by the French botanist M. Adanson—penetrated bacterial systematics. The underlying idea was that all characters are considered of equal weight, and the degree of relatedness is determined by the number of shared matches. When applying this method in the latter half of the 20th century, phenotypic dendrograms of relatedness were constructed by subjecting a series of parameters, determined for a large number of compared objects, to computer Processing. However, the postulate of character equivalence is inconsistent with the Genetic foundations of micro- and macroevolution and thus restricts the utility of numerical classification to low-rank taxa. In the early 1960s, total DNA characteristics—specifically the molar percentage of G+C pairs—were used for bacterial classification for the first time, and in the late 1960s, the DNA-DNA Hybridization method was applied for this purpose.

The first opportunity to establish a natural system of bacteria arose only in the mid-1970s. Prior to this, criteria for higher taxa were chosen subjectively, and compilers of bacterial systematics manuals divided bacteria into artificial groups convenient for identification. These groups were assigned trivial names rather than legitimate taxonomic designations.

Now, however, researchers began applying “molecular chronometry” methods—based on compiling catalogs or libraries of nucleotide sequences from short rRNA fragments—to reconstruct evolutionary relationships. Since the mid-1980s, the results of full rRNA sequencing have been utilized for this purpose.

Due to the avalanche-like growth of information regarding new bacterial species, describing them has become a task beyond the capacity not only of a “universal” author but even of multiple co-authors competent in several branches of bacteriology simultaneously.

Since the early 1920s, Bergey’s Manual of Determinative Bacteriology has served as the leading guide to bacterial identification and systematics, named after the American bacteriologist D. H. Bergey, who headed the editorial board for the first four editions. Although the Society of American Bacteriologists originally conceived the Manual as an internal departmental guide—and it continues to be published to this day with financial support from an independent foundation established in 1936—it quickly became evident that this was a scientific project of international scale.

As with botanical and zoological nomenclature, until the mid-20th century all bacterial classification systems utilized rules tracing back to Carl Linnaeus’s Species Plantarum (1753). At the First International Microbiological Congress, held in Paris in 1930, the Nomenclature and Taxonomy Committee of Bacteria was established, which in 1948 published the International Code of Nomenclature of Bacteria (ICNB; revised version in 1992). Its provisions, governed by the International Committee on Systematic Bacteriology (ICSB), mirror the rules of binary botanical nomenclature, albeit with certain modifications—specifically, a living culture rather than a herbarium specimen serves as the nomenclatural type, and a Latin diagnosis is not required to legitimize a new taxon.

Early descriptions of bacteria were fragmentary and often repetitive, with bacteria occasionally receiving multiple names simultaneously. Conversely, different bacteria were sometimes referred to under the same name. Consequently, it was decided to review all diagnoses, and following the 1980 publication of the Approved Lists of Bacterial Names, the birth date of bacterial nomenclature was shifted from May 1, 1753, to January 1, 1981.

Over the span of 50 years, Bergey’s Manual went through eight editions (1923, 1925, 1930, 1934, 1939, 1948, 1957, and 1974), which were consistently supplemented and revised.

In 1977, an abridged version of the 8th edition of Bergey’s Manual was published (with a Russian Translation appearing in 1980). It outlines the Principles of Bacterial classification, provides descriptions of all genera and higher-level taxa, offers identification keys and tables with species diagnoses, and reproduces all illustrative material.

Since the 9th edition, Bergey's Manual has been published as two independent guides pursuing different strategic goals and, accordingly, differing in structure and scope.

The 9th American edition of Bergey's Manual of Determinative Bacteriology (1994, with a Russian translation in 1997) was designed for practical identification using tables containing essential information on bacterial phenotypes. Independently of this, the four-volume Bergey's Manual of Systematic Bacteriology was published between 1984 and 1989. It provides fundamental information on the anatomy, physiology, and phenotypic classification of bacteria (at this stage, archaea were also classified as bacteria, though they were later separated into an independent group of prokaryotes).

Based on cell wall type, the kingdom Procaryotae was divided into four divisions:

— Gracilicutes (Gram-negative);

— Firmicutes (Gram-positive);

— Mollicutes (Mycoplasmas);

— Mendosicutes ("archaebacteria").

Independently, prokaryotes were divided into 33 quasi-taxonomic groups, or Sections. The diagnostic features of these Sections are based on phenotypic traits such as cell morphology, sheath formation, Gram reaction, type of METABOLISM/26.html">Energy Metabolism, oxygen requirements, and the presence of differentiated stages in the life cycle:

— spirochetes (1);

— aerobic/microaerophilic, motile, helical/vibrioid Gram-negative bacteria (2);

— non-motile (rarely motile) Gram-negative curved bacteria (3);

— Gram-negative aerobic rods and cocci (4);

— facultatively anaerobic Gram-negative rods (5);

— anaerobic Gram-negative straight, curved, and helical rods (6);

— sulfate- or sulfur-reducing bacteria (7);

— anaerobic Gram-negative cocci (8);

— rickettsias and chlamydias (9);

— mycoplasmas (10);

— endosymbiotic bacteria of protists, insects, and fungi (11);

— Gram-positive cocci (12);

— endospore-forming Gram-positive rods and cocci (13);

— regular, non-spore-forming Gram-positive rods (14);

— irregular, non-spore-forming Gram-positive rods (15);

— mycobacteria (16);

— nocardioform bacteria (17);

— anoxygenic phototrophic bacteria (18);

— oxygenic photosynthetic bacteria (19);

— aerobic chemolithotrophic bacteria and related organisms (20);

— budding and/or appendaged bacteria (21);

— sheathed bacteria (22);

— non-photosynthetic, non-fruiting gliding bacteria (23);

— fruiting gliding bacteria: myxobacteria (24);

— archaea (25);

— actinomycetes (26-33).

The fundamentally revised 2nd edition of Bergey's Manual of Systematic Bacteriology is planned as a five-volume work based on a novel concept of megataxa. Prokaryotes are subdivided into phylogenetic "domains" of bacteria and archaea. The domains, in turn, consist of "phyla". The primary criterion for distinguishing between domains and intradomain phyla is not phenotypic traits, but the divergence of rRNA Gene nucleotide sequences.

The advantage of this system is its objectivity, as it reflects evolutionary relationships.

However, phylogenetic classification does not coincide with practically convenient phenotypic classification. Many phyla represent a collection of contrasting forms, while similar forms are sometimes found within different phyla (Chapter 7).

Although modern bacterial classification increasingly relies on genomic analysis data, classification at lower taxonomic ranks, and primarily at the species level, is based on a compromise approach known as the "polyphasic" approach. It takes into account a diverse range of phenotypic characteristics as well as informational Features of the genotype, among which the Primary Structure of 168 rRNA holds the leading position.

2.3. Dichotomous Metasystem. Bacteria as Prokaryotes

At the turn of the 20th century, it finally became clear that there are Cells with a Nucleus and cells without a nucleus.

Ferdinand Cohn was the first to realize that the "nuclear" criterion is exceptionally vital for megasystematics. In the 1870s, he assigned all microorganisms reproducing by binary fission to the taxon "Schizophyta". This group included bacteria, or the class Schizomyceae (see Section 2.1), and blue-green algae, or the class Schizophyceae (from Greek schizo — to split in half, and phycos — sea weed; "algae reproducing by binary fission").

The leading characteristic of the taxon Schizophyta, apart from binary fission, was the absence of a nucleus. Thus, Cohn came very close to the idea of a global dichotomous classification, although its formulation in 1937 belongs to Édouard Chatton (see "Introduction"). However, Chatton's publication was known only to the few specialists who read the proceedings of the Sète marine biological station. Roger Stanier learned about it only in 1962 from André Lwoff, his colleague at the Pasteur Institute in Paris.

The term "nucleus" (Latin nucleus) was introduced in 1833 by the English botanist Robert Brown (R. Brown). Around the same time, the Austrian zoologist Johann Purkinje (J. Purkinje) proposed the term "protoplasm" (from Greek proteros — primary, and plasma — formed body; "primary formed body"), although it was actually introduced in 1844 by the German botanist Hugo von Mohl (H. von Mohl).

In the 1870s, it was demonstrated that the plant nucleus reproduces by division, and new nuclei pass into daughter cells. At the same time, at the suggestion of the German botanist Eduard Strasburger (E. Strasburger), the term "Cytoplasm" (from Greek kytos — vessel, and plasma — formed body; "cell protoplasm") began to be used as a synonym for protoplasm, or the substance surrounding The Nucleus.

In the early 1880s, the German histologist Walther Flemming (W. Flemming) provided the first description of karyokinesis in animals and proposed using the term "mitosis" (from Greek mitos — thread) to denote The process of indirect nuclear division. The terms denoting individual stages, or phases of mitosis (prophase, anaphase, etc.), were introduced by Eduard Strasburger.

In the late 1860s, the German biochemist Friedrich Miescher (F. Miescher) isolated protein markers of the nucleus — Histones, as well as "nuclein", a nuclear material possessing acidic properties — from pus. The fact that "nuclein" contains nitrogenous bases was established in the late 1870s by the German biochemist and physiologist Albrecht Kossel (A. Kossel, Nobel Prize in Physiology or Medicine, 1910). At the same time, Paul Ehrlich discovered an intra-nuclear substance with a high affinity for hematoxylin and aniline Dyes in situ. To denote it, Flemming proposed using the term "Chromatin" (from Greek chroma — color).

In 1888, the German cytologist Wilhelm Waldeyer (W. Waldeyer) named chromatin structures "Chromosomes" (from Greek chroma — color, and soma — body). Using genetic experiments in the 1920s, it was established that chromosomes are the bearers of genes. The American geneticist Thomas Morgan (T. H. Morgan, Nobel Prize in Physiology or Medicine, 1933) discovered the fundamental mechanism of genetic recombination in nucleated cells — meiotic Crossing Over.

Long searches for a nucleus in bacteria failed to yield the expected result. It remained to be assumed that the bacterial protoplast has a primitive structure and is not differentiated into a nucleus and cytoplasm.

Recall that the Cell Theory was established by the German zoologist Theodor Schwann in 1839. In his famous book Mikroskopische Untersuchungen über die Uebereinstimmung in der Struktur und dem Wachstum der Tiere und Pflanzen, the nucleus is regarded as an essential part of The Cell — a conditio sine qua non (Latin for an indispensable condition). Therefore, one should not be surprised that phyto- and zoomorphologists tried to experimentally prove that bacteria have a nucleus or at least its morphological equivalent. In the last quarter of the 19th century and the first half of the 20th century, work with bacteria involved special stains to reveal nuclear substance and light Microscopy preparation methods already refined on animals and plants. In doing so, researchers sought and "found" in bacteria not only a nucleus but also metaphase chromosomes, as well as individual phases of mitosis. Polyphosphate inclusions were often mistaken for the nucleus, and areas of more intense cytoplasmic staining were considered chromosomes. If a "nucleus" was not found, this was attributed to The small size of bacteria, damage during specimen preparation, and shortcomings in staining techniques.

However, general opinion gradually leaned toward the view that bacteria lack a nucleus, and its role is played by a nuclear substance distributed throughout the entire cell. Another possibility was also entertained — that the bacterial cell is differentiated into a thin peripheral layer of cytoplasm and a condensed "central body", an equivalent of the nucleus with chromatin aggregates that are distributed between daughter cells in the absence of mitotic figure formation.

Along with this, in the second half of the 19th century, more exotic hypotheses regarding the Internal Structure of bacteria were put forward. For example, the German cytologist Otto Bütschli believed that a bacterium was essentially a nucleus-cell devoid of cytoplasm.

As for microbiologists themselves, they intuitively perceived bacteria as an independent group of living organisms, separate from the kingdoms of protists, fungi, plants, and animals. Regardless of how the question of bacteria's place in the taxonomic system was resolved, most microbiologists preferred to view them as direct descendants of "cytodes"—anuclear protocells with a primitive level of Organization (from the Greek kitos, meaning vessel, and eidos, meaning likeness; "cell-like").

THE CONCEPT OF cytodes was originally proposed in the 1860s by the German naturalist Ernst Haeckel, who viewed them as an extinct transitional link between non-living and living matter.

However, at that stage of microbiological development, it was impossible to give a definitive answer as to whether bacteria possessed a nucleus. The main reason lay in the limited capabilities of the Light Microscope, whose resolving power was insufficient to reveal the ULTRASTRUCTURE OF THE bacterial cell.

A methodological breakthrough occurred in the 1930s–1950s with the advent of commercial electron microscopes, alongside specialized techniques for fixing, dehydrating, and embedding biological material in polymer resins. This made it possible to obtain ultrathin sections, the individual components of which scatter a focused beam of fast electrons to varying degrees. By the late 1960s, it was demonstrated that a true nucleus possesses a double-membrane envelope serving as a boundary between the DNA-containing structures and the cytoplasm. Furthermore, the nuclear cell was found not only to be membrane-bound but also to contain an internal system of elementary membranes.

It soon became clear that the physical boundary of the nucleus, formed by the double-membrane envelope, is absent in "true" bacteria and actinomycetes, as well as in blue-green algae (which at the time were classified under botany). In addition, it was established that prokaryotes do not undergo processes typical of nucleated cells—such as the reversible Condensation of chromosomes during the Cell Cycle with the segregation of sister chromatids in mitosis, or the segregation of homologous chromosomes in Meiosis. The low chromatin density of bacteria compared to that of nuclear organisms had already been noted via light microscopy as early as the late 19th and early 20th centuries.

In the 1930s, Édouard Chatton's idea of a global dichotomy between PROKARYOTES AND EUKARYOTES went largely unnoticed, partly because the role of membranes in cellular compartmentalization was not yet understood. When Cytology advanced to the ultrastructural level of research, it became evident that the nucleus possesses a membranous envelope that not only isolates chromatin but also regulates its behavior throughout ontogeny.

In the mid-1960s, Roger Stanier revisited the concept of a global dichotomy, but on a new level—incorporating the advancements of genetics, biochemistry, and Electron microscopy.

The criteria used by his predecessors (such as reproduction mode and nutritional type) failed to establish bacteria as an independent group of living organisms. The situation was objectively complicated by the vast Morphological diversity of bacteria. Some (filamentous actinomycetes) superficially resemble mycelial fungi; others (pigment-producing myxobacteria) look like myxomycetes; a third group (spherical or ellipsoidal cells growing on sugars) resembles yeasts; a fourth (cyanobacteria) resembles algae; and a fifth (large motile bacilli) resembles ciliates, among others. Consequently, at various stages in The history of microbiology, bacteria were alternately assigned to the kingdoms Protists, Fungi, Plantae, and Animalia. This apparent lack of cytological Specificity made it impossible to answer the question "quid est bacterium" (Latin for "WHAT IS A bacterium") or to clearly explain how bacteria differ from, say, ciliates or baker's Yeast.

Roger Stanier expanded upon Édouard Chatton's concept of two global morphotypes and was the first to elucidate the structural specifics of bacteria. At the same time, he defined the place of bacteria within the taxonomic hierarchy by proposing that the terms "prokaryotes" and "bacteria" be used as synonyms. Finally, he divided cellular organisms into two kingdoms: the kingdom of nucleated organisms (regnum Eucaryotae) and the kingdom of prokaryote-bacteria (regnum Procaryotae).

Thus, within the framework of the dichotomous megasystem, bacteria are classified as the taxon Procaryotae.

Stanier was forced to concede that most Characteristics of Bacteria are negative in nature (the absence of a nucleus, sexual reproduction, Golgi-mediated secretion, vacuoles, phagocytosis and pinocytosis, amoeboid movement and cytoplasmic streaming, cytoplasmic endosymbionts, and semiautonomous Organelles, etc.). Nevertheless, he did not view the bacterial cell as "primitive." On the contrary, he emphasized that prokaryotes and eukaryotes belong to fundamentally Different types of Cellular Organization, and that prokaryotes possess structures and Functions absent in eukaryotes (non-unit membranes, functional inclusions, Chemosynthesis, diazotrophy, etc.).

Because Stanier's arguments were highly persuasive, the "prokaryotic" concept extended far beyond bacteriology, and the terms "prokaryotes" and "eukaryotes," which he revived, were firmly integrated into the biological lexicon. However, taking these terms literally or superficially inevitably leads to erroneous Conclusions.

2.4. The Ambiguity of the Term "Prokaryotes"

The term "prokaryotes" is typically defined as "a type of cellular structure characterized by the absence of a defined nucleus." However, the Greek prefix pro- is polysemous, allowing the term "prokaryotes" to be interpreted in several ways:

— cells containing a "simpler structured nucleus";

— cells containing a structure that "replaces the nucleus";

— cells containing an "evolutionary precursor of the nucleus."

To what extent do these semantic variations align with cytological data and theories on the phylogeny of cellular organisms?

Can we truly say that prokaryotes contain a "simpler structured nucleus"? To answer this question, it is sufficient to compare the General Structure and chromatin compartmentalization of eukaryotes and prokaryotes.

The nucleus is a specialized compartment unique to organisms belonging to the phylogenetic domain Eukarya. It contains nucleoplasm and subnuclear structures (protein-rich chromatin, the nucleolar organizer, spliceosomes, the lamina, etc.), is bounded by a relatively rigid, pore-bearing envelope, and can be isolated in an intact state. The nucleus is a unique organelle that adheres to an "all-or-none" principle.

Prokaryotes lack all of these nuclear features. Their chromatin is protein-poor and consists of "naked" DNA that is compactly folded and situated directly within the cytoplasm. Therefore, the structure housing the genetic material in prokaryotes can be viewed as an alternative to a nucleus and, to a certain extent, its functional equivalent.

In 1892, the German phycologist Georg Hieronymus proposed calling the "central body" of bacteria and blue-green algae an "open nucleus" (German: offene Zellkern), in contrast to the "closed nucleus" of higher organisms (German: geschlossene Zellkern). From a logical standpoint, this terminology was flawed, as the very essence of a nucleus lies in its enclosure—namely, its relative isolation from the cytoplasm. Nevertheless, it was an intuitive step toward MODERN CONCEPTS OF bacterial cell compartmentalization.

Given that The structure of a nucleus is generally standardized, the prefix eu- (from the Greek eu, meaning true) in the term "eukaryotes" is arguably redundant. In contemporary literature, the corrected term "karyotes" (from the Greek karyon, meaning kernel or nucleus; "nucleated") is increasingly common.

Is it legitimate to attach the semantic connotation of "a structure replacing the nucleus" to the term "prokaryotes"? Yes, because the mode of genetic material compartmentalization in prokaryotes is fundamentally different from that in nucleated organisms. While the term "nucleoid" (from the Latin nucleus and Greek eidos) is widely used in bacteriological literature, one should not imply that it is equivalent to a true nucleus. Consequently, terms such as "nucleus," "nuclear equivalent," "nuclear body," "nuclear region," and "nucleoplasm" should strictly be avoided when referring to prokaryotes.

DNA-containing structures of bacteria are referred to as chromosomes. Another term used for bacteria is “genophore” (from the Greek genos meaning descent, and phoros meaning bearing; “gene carrier”), which was proposed in 1968 by the English geneticist W. Hayes. However, this term carries an informational rather than a cytological meaning.

Finally, can the term prokaryotes be endowed with the semantic connotation of “the evolutionary precursor of the nucleus”? In other words, is there an evolutionary continuity between prokaryotes and eukaryotes?

This question is answered in the negative. But although bacteria were not the evolutionary precursors of eukaryotes themselves, they are the evolutionary precursors of their cytoplasmic organelles—Mitochondria and Plastids.

Until the mid-1970s, the prevailing view was that the evolutionary tree of cellular organisms branches into two main trunks. One of these is the eukaryotic trunk, understood as nucleated cells with semiautonomous organelles (mitochondria and plastids). The other is the prokaryotic-bacterial trunk. It was assumed that prokaryotic bacteria formed a monophyletic group. The Unity of origin of nuclear organisms was not in doubt.

Archaic bacteria were considered the common prototype of cellular organisms. In the 1960s and 1970s, A number of hypothetical scenarios were proposed according to which eukaryotes originated through the gradual compartmentalization of a bacterial cell. Canadian botanist F. Taylor termed this “autogenesis” (from the Greek autos meaning self, and genos meaning origin). According to the autogenesis hypothesis, the nuclear envelope, Endoplasmic reticulum, Golgi apparatus, Lysosomes, and other eukaryotic compartments arose as a result of the differentiation of Regions of the cytoplasmic membrane that proliferated and invaginated into the cytoplasm. THE ORIGIN OF mitochondria and plastids was linked to the specialization of individual regions of the cytoplasmic membrane and their transformation into converters of chemical and light energy.

In the late 19th and early 20th centuries, long before the advent of the autogenesis hypothesis, the Russian botanist A. S. Famintzin, the Russian zoologist K. S. Mereschkowsky, and the German botanist A. Schimper proposed an alternative scenario for the evolutionary origin of plastids and mitochondria—the symbiogenesis hypothesis (from the Greek sym meaning together, bios meaning life, and genos meaning origin). Later, Francis Taylor called this scenario “xenogenesis” (from the Greek xenos meaning alien, and genos meaning origin).

According to the symbiogenesis scenario, plastids and Mitochondria are the descendants of endosymbiotic bacteria that lost their capacity for autonomous existence and specialized to supply the host cell with energy and Organic compounds.

For many years, symbiogenesis was regarded as a fantastic scenario. At best, it was considered to lack empirical evidence. It was superseded by the “more plausible” autogenesis scenario. However, at present, the symbiogenesis of plastids and mitochondria has been fully confirmed by the results of comparative 16S rRNA gene analysis.

In the early 1980s, the idea of symbiogenesis was revived by the American microbiologist L. Margulis. She overly detailed the symbiogenesis scenario and even named modern bacteria (mycoplasmas, spirochetes, cyanobacteria, prochlorophytes, and heliobacteria) which, in her opinion, are the precursors of the cytoplasmic compartment and flagella, as well as chlorophyll c-containing plastids, respectively. However, it was soon demonstrated that bacteria are indeed the evolutionary precursors of plastids and mitochondria, but not of other Eukaryotic Cell organelles.

On the one hand, data obtained using molecular biological methods confirmed the Famintzin–Mereschkowsky–Schimper hypothesis regarding the endosymbiotic origin of plastids and mitochondria. On the other hand, the hypothesis concerning the origin of eukaryotes from bacteria was proven untenable.

According to modern data, the global evolutionary tree branches not into two, but into three trunks. The two prokaryotic trunks, Bacteria and Archaea, formed independently of one another. As for eukaryotes, they did not arise as a separate branch of the bacterial trunk, but instead represent a third trunk.

2.5. Bacteria as the Phylogenetic Domain Bacteria

The collapse of the dichotomous system occurred in the mid-1960s. Data emerged that contradicted the postulate of prokaryote monophyly and failed to support the hypothesis of evolutionary continuity between bacteria and eukaryotes.

Whereas biochemical characteristics were previously used as routine taxonomic markers, PROTEIN AND NUCLEIC acid sequencing now became the primary weapons of chemosystematics. The obtained results made it possible to reconstruct phylogenetic relationships at all levels of the evolutionary tree—from the Base of the trunk to the tips of the crown.

In 1965, American biochemists L. Pauling (Nobel Prize in Chemistry, 1954) and E. Zuckerkandl arrived at a fundamentally new concept of “molecular paleontology.” They realized that phylogeny could be studied not only by comparing phenotypes, but also by comparing the Physical Structure of genomes. Such a task is difficult even today, and at that time, it was out of the question. Therefore, it was proposed to select the most conserved genes, or the Proteins encoded by them, as indicators of phylogeny.

Pauling and Zuckerkandl believed that the evolution of conserved proteins, occurring via the accumulation of random Mutations, is closely linked to the Evolution of the genotype as a whole. Therefore, the comparison of Amino acid sequences of such proteins across different organisms serves as a measure of the distance between species and higher-ranking taxa.

Assuming that the mutation rate is relatively constant, macromolecules whose primary structure carries hereditary information can be used as a chronometer. These are termed “semantides” (from the Greek semainein meaning to signify, and eidos meaning form). This chronometer objectively indicates the time elapsed since the divergence of objects A and B; that is, the evolutionary distance is estimated based on semantide Homology.

The basic requirements for a semantide are determined by The Nature of the task and the method of its solution. Depending on the range of objects, different types of semantides can be used. The top rank is held by archaic, universally distributed semantides. Semantides acquired later in the course of evolution have more limited application. Finally, the informational capacity of a semantide, which is roughly estimated by its molecular weight, must be sufficiently large (otherwise The Effect of random positional substitution will be too pronounced) and at the same time moderate (so as not to create technical problems during decoding).

Proteins and Polypeptides were initially analyzed as semantides, since molecular biology in the 1960s relied primarily on the achievements of Protein Chemistry. The choice of material for assessing molecular homology was not accidental. First and foremost, Cytochromes were sequenced—redox Enzymes that play a pivotal role in energy metabolism and are virtually universal.

With the development of nucleic acid molecular biology, polypeptides began to be used less frequently as semantides. Notable exceptions include the subunits of H⁺-translocating ATPases, ferredoxins, the recombination factor RecA, and molecular chaperones.

Comparative results obtained from protein sequencing found application in the specialized classification of bacteria, yet they had no significant impact on megasystematics.

In the mid-1970s, Carl Woese proposed a new method for analyzing molecular phylogeny based on sequencing Nucleic Acids rather than proteins. The indicator of divergence was not The amino acid sequences of proteins, but The nucleotide sequences of universally distributed genes.

Ribosomal RNAs (rRNAs), i.e., polynucleotides constituting Ribosomes, were chosen as semantides. The rRNAs of the small or large subunits were compared in pairs. In the case of prokaryotes, these are 16S rRNA and 23S rRNA, and in the case of eukaryotes, 18S rRNA and 28S rRNA.

Initially, Woese’s method consisted of sequencing oligonucleotides with 5–17 bases, which could be obtained from rRNA after Treatment with a fine-cleaving endonuclease. Next, nucleotide sequences were determined for fragments of equal length, or “isopliths” (from the Greek isos meaning equal, and Pleura meaning side). These “words” were used to compile “catalogs” for each object and compare them in pairs. The result of the comparison served as the “similarity quotient” (abbreviated as SAB), the maximum value of which was 1.0 (100%). Taking the similarity quotient into account, evolutionary trees—dendrograms—were constructed.

With the advancement of sequencing methods, this primitive approach was superseded by the recording of positional matches in two polynucleotide chains. To this end, an “alignment” Procedure is performed (referring to alignment in a military formation).

Following the invention of the Polymerase Chain Reaction (PCR), it became possible to amplify virtually any region of The Genome. This is achieved using specific primers, and the resulting amplicons are either analyzed immediately or subjected to additional cloning into a bacterial plasmid. In both cases, the process culminates in sequencing The nucleotide sequence using an automated Sequencer.

Thanks to methodological refinements, comparative analysis of full-sized structural rRNA genes ("rDNA") ranging from 500 to 2500 bp has been superseded by comparative Analysis of the entire rRNA Operon. Data processing has been streamlined using specialized software that aligns unfragmented nucleotide sequences to evaluate the identity between the analyzed amplicon and sequences deposited in the GenBank database.

To support the choice of rDNA as the primary phylogenetic marker, Woese and his followers put forward the following arguments:

— the emergence of ribosomes concurrently with the cell and their indispensable role in Protein Biosynthesis, which makes them universal;

— the presence of both highly conserved and relatively variable regions within the rRNA polynucleotide chain, enabling the assessment of both archaic and more recently established evolutionary relationships;

— "isochronicity," meaning a uniform rate of ribosomal gene divergence due to a constant mutation speed across different organisms. This prevents the misinterpretation of rapidly mutating "tachyletic" (from the Greek tachos — fast and telos — result) sequences as more archaic, or slowly mutating "bradyletic" (from the Greek bradis — slow and telos — result) sequences as less archaic;

— a rarely observed horizontal transfer of rDNA, which allows us to chronometricate divergent evolution only.

The First and Second arguments raise no fundamental objections. The other two contradict facts that indicate the heterochronous evolution of ribosomal genes, as well as the frequently observed phenomenon of "horizontal" gene transfer in prokaryotes. This recombination process can even affect genes encoding rRNA.

Nevertheless, the productivity of the approach to global phylogeny analysis proposed by Woese is beyond doubt, and the conclusions obtained serve as a cornerstone of modern megaclassification.

Even Carl Woese's earliest results showed that the dichotomy between prokaryotes and eukaryotes is an artificial construct that does not reflect actual genome evolution. It turned out that the global phylogenetic tree has at least a trichotomous structure (Fig. 2).

The main Branches of the global tree correspond to phylogenetic megataxa, which Woese called "domains" (from Greek domos — house, family). The term "domain" is completely unsatisfactory to those accustomed to the terms "kingdom" (Latin Regnum) and "super-

kingdom" (Latin Superregnum). Furthermore, it had been used much earlier in chemistry, crystallography, and the physics of electromagnetic phenomena. Nevertheless, it has firmly established itself in the biological vocabulary and was even officially introduced in 2001 into the 2nd edition of Bergey's Manual of Systematic Bacteriology.

The Bacteria domain formed earlier than the other two and therefore has a strong claim to being the most archaic of the three domains. Despite its name (derived from the Greek archaios, meaning ancient), the Archaea domain diverged from the Eucarya domain only after bacteria had branched off from the base of the phylogenetic tree trunk (Fig. 2).

Fig. 2. Schematic representation of the global phylogenetic tree. A — archaea; B — bacteria; C — eukaryotes.

Results from paleomicrobiological studies indicate that the age of the oldest fossil cyanobacteria approaches 3.5 billion years. This contradicts findings from molecular paleontology, which suggest that the three global phylogenetic domains diverged 1.5 billion years ago (this conclusion is based on calculations of the total number of nucleotide positional substitutions at a known average mutation rate—1% sequence divergence of the 16S rRNA gene is equivalent to an evolutionary distance of 50 million years). The true reason for such a large chronological discrepancy remains unclear. It is possible that early evolution was slower than previously thought, and therefore the fundamental postulate of molecular paleontology—the isochrony of gene lineages—should be approached with critical caution.

The Bacteria and Archaea domains consist of branches corresponding to phylogenetic taxa known as “phyla” (from the Greek phyle, meaning tribe). General characteristics of these phyla, along with their representative organisms, are discussed in Chapter 7.

Among prokaryotic phyla, one can find not only phenotypically characterized groups but also “phantom” phyla. The latter have been delineated solely through direct ANALYSIS OF GENE structures, with a complete absence of phenotypic data for the corresponding living organisms.

The method for identifying phantom phyla is based on screening both traditional and “exotic” niches using PCR assays. As noted previously, microquantities of “environmental” DNA are extracted from water, soil, and other samples. Fragments of this DNA are then amplified, and the resulting amplicons are sequenced. As a result of targeted searches conducted in recent years, the number of bacterial phantom phyla is constantly growing, and any predictions about how many ultimately exist remain premature.

Modern megaclassification of prokaryotic organisms—bacteria and archaea—is founded upon phylogenetics. In the 2nd edition of Bergey’s Manual of Systematic Bacteriology (2001), the Bacteria domain is divided into 23 phyla designated BI through BXXIII (abbreviated from Bacteria), each corresponding to a taxonomic group at the rank of phylum/division. Similarly, the Archaea domain comprises phyla AI and AII (abbreviated from Archaea).

It has been established that bacterial rRNA is only 10% homologous to the cytoplasmic ribosomal rRNA of nuclear organisms. At the same time, it exhibits high homology with the rRNA of mitochondria, Chloroplasts of green algae (Chlorophyceae) and higher plants, as well as the plastids of red algae (Rhodophyceae). This serves as one of the primary pieces of evidence supporting the endosymbiotic origin of these organelles. Mitochondria belong to the phylum BXII Proteobacteria (class Alphaproteobacteria), while the plastids of Chlorophyceae, higher plants, and Rhodophyceae belong to the phylum BX Cyanobacteria.

Thus, it has been proven that evolutionary continuity between prokaryotes and nucleated cells is a reality. However, this applies not to the entire nuclear cell, but strictly to its constituent parts—the endosymbiotic organelles.

According to 18S rRNA sequencing data, eukaryotes form a monophyletic domain, Eucarya. Conversely, the hypothesis of prokaryotic monophythood has not been supported: non-nuclear cells are at least diphyletic.

Consequently, following the discovery of bacteria, our understanding of the nature of these microorganisms evolved through the following stages:

— in the absence of knowledge regarding bacterial structure and physiology, they were placed outside the megasystem or lacked independent status within it, leading to their classification either as part of the unknowable (“Chaos” by Carl Linnaeus) or as a transitional realm between inanimate matter and living organisms (“cytodes” by Ernst Haeckel), as well as being grouped with animals, plants, or fungi on various grounds;

— as a result of studying bacterial ultrastructure in the 1960s, within the framework of the global dichotomy concept, they were defined as prokaryotes; for this reason, Archaea were also classified as bacteria;

— as a result of the reconstruction of the global tree in the 1970s, it was established that prokaryotic bacteria form an independent evolutionary Lineage — the domain Bacteria, while prokaryotic archaea form another evolutionary lineage — the domain Archaea.

To date, the genomes of over 60 bacterial species have been fully sequenced, and a number of projects are in the final stages. The analysis of bacterial genomes not only contributes to the study of The Diversity of the domain Bacteria, but also provides insight into the global patterns of the evolution of cellular structures, metabolic pathways, transport systems, and regulatory mechanisms.

According to current data, prokaryotes are diphyletic. This key conclusion reflects the role of molecular criteria, primarily genome infrastructure, in the CLASSIFICATION OF LIVING organisms at the highest taxonomic rank. However, it is possible that new data on prokaryotic biodiversity may lead us to reconsider our views on global phylogeny in the future.