MICROBIOLOGY PROKARYOTE BIOLOGY VOLUME I - A. V. PINEVICH - 2006

CHAPTER 1. HISTORY OF BACTERIOLOGICAL RESEARCH

Class="center">Every occurrence in Nature is preceded by other occurrences which are its causes, and succeeded by others which are its effects. The human mind is not satisfied with observing and studying any natural occurrence alone, but takes pleasure in connecting every natural fact with what has gone before it, and with what is to come after it.

(Every phenomenon in Nature is preceded by those that serve as its causes, and entails others that act as its consequences. The human mind is not content with merely observing and studying natural phenomena; it takes delight in linking every fact to what preceded it and what will follow it).

John Tyndall. The Forms of Water in clouds & rivers, ice & glaciers (London, 1885).

Cognition is inherently utilitarian, as it satisfies individual and collective needs for substrates, energy, and information. Both traditional and novel substrates serve as nutritional sources, as well as Materials for manufacturing clothing, tools, instruments, machinery, buildings, transport vehicles, and communication systems. In turn, traditional and alternative Energy Sources are utilized as fuel for homes and to power technical devices. Finally, informational needs, including aesthetic ones, account for human interest in elements of the surrounding reality.

Substrate, energetic, and informational aspects of comprehending macroorganisms are self-evident and have been traceable since prehistoric times. Conversely, the existence of Bacteria remained unknown until the late 17th century, and they were not intentionally utilized until the mid-19th century. Rare exceptions include sporadic superpopulations of pigmented bacteria. Notably, the dramatic phenomenon of marine water blooms caused by purple bacteria was described In the second half of the 1st century AD by the Roman encyclopedist Caius Plinius Secundus (Pliny the Elder). Prior to the Spanish conquest in the late 15th century, the Aztecs incorporated the biomass of the cyanobacterium Spirulina into their diet. A third example is provided by myxobacteria, which form brightly colored fruiting bodies (as late as the 19th century, they were misclassified as gasteromycetes or hyphomycetes). Bacterial biomass is not used as an energy source, although their Lipids represent a biological alternative to petroleum products. Until microbiology matured into a fundamental science at the beginning of the 20th century, the informational significance of bacteria was negligible. Finally, the aesthetic qualities of bacteria remain largely unappreciated, despite the fact that in perfection of form and brilliance of coloration, some of them rival aquarium fish.

In the latter half of the 19th century, it was definitively proven that bacteria can cause diseases in humans and animals. This marked the birth of medical microbiology—encompassing the Cytology, physiology, and systematics of pathogenic bacteria, along with its Structure/179.html">Practical Applications in Diagnostics, antisepsis, and therapy. Agronomic microbiology emerged in the second half of the 19th century, whereas biotechnology, based on the industrial exploitation of microbial enzymatic activity, arose only at the dawn of the 20th century.

Microbiology established itself as an independent science through the STUDY OF BACTERIAL biodiversity and ecological properties. Since the latter half of the 20th century, there has been a sharp increase in research conducted using Electron Cell/15.html">Microscopy, biochemistry, and molecular biology methodologies. Concurrently, the search for novel research subjects intensified—laboratory boundaries expanded, so to speak, as previously inaccessible corners of our planet and even other celestial bodies became the focus of scientific inquiry (exobiology is no longer science fiction, but reality).

While microbiology initially served medicine, the food industry, and agronomy, it has now evolved into a discipline with its own distinct concepts and Methods. Gone is the outdated view of it as a secondary science dealing not with "biologically familiar" animals and plants, but with invisible entities whose detrimental effects are more readily apparent than their utility.

Microorganisms, bacteria above all, are fundamental to the shaping of Earth's habitable biosphere. Higher forms of life emerged in the course of microbial evolution. Microbial communities possess the unique capacity to harness solar energy and inorganic substrates, rendering them accessible to plants and animals.

Our understanding of microbial properties has vastly expanded, the catalogue of studied representatives from the invisible world has multiplied exponentially, and humanity is increasingly harnessing them to serve its needs.

1.1. Discovery of Bacteria

Although bacteria were first observed through a microscope only in the late 17th century, the existence of microorganisms had already been suspected in antiquity. The founding father of medicine, the ancient Greek physician Hippocrates, brilliantly hypothesized in the 5th century BC that the wind carried invisible entities responsible for infectious diseases. Much later, in 36 BC, the Roman naturalist Marcus Terentius Varro suggested that the air contained microscopic animals (Lat. animalia quaedam minuta).

A millennium and a half later, in 1546, the Italian polymath Girolamo Fracastoro hypothesized in his treatise De contagione et contagiosis morbis... that Syphilis, plague, typhus, and smallpox were transmitted by contagion, and he also outlined rudimentary methods for preventing and treating these diseases. A century later, in 1658, the German naturalist Athanasius Kircher proposed that the plague was caused by a "living contagion" (Lat. contagium vivum) which, upon entering the body of healthy individuals, transformed into "living particles" (Lat. corpuscula animata). Since it was impossible to visualize the plague pathogen, the bacterium Yersinia pestis, using a primitive microscope, the particles described by Kircher were most likely formed Blood elements. Be that as it may, the idea

of microscopic agents of infectious diseases was favourably received by his contemporaries.

At the beginning of the 17th century, natural philosophy gave way to the empirical method substantiated by the English philosopher and statesman Francis Bacon, Baron Verulam. Science shifted its preference from deduction to sensory perception and inductive reasoning. The hunt for facts began, and bacteriology was no exception.

The progress of bacteriology is closely tied to The Development of instrumentation, with optical instruments—microscopes—occupying the foremost place among specialized equipment.

It is generally believed that the compound microscope was invented no later than 1590 in the Netherlands by the brothers Johannes and Zacharias Janssen. According to other sources, this occurred in the 1620s, with priority attributed to the Dutchman Cornelis Drebbel or the Italian Galileo Galilei. In the second half of the 17th century, the Dutch physicist Christiaan Huygens developed the optical theory of Glass lenses. However, the microscopes he designed provided a magnification of at most 100x. Furthermore, they suffered from low resolving power due to chromatic and spherical aberration, as light of different wavelengths was focused in different planes, and the image at the edge of the field of view was blurred. Such optical devices made it impossible not only to study the structure, physiological properties, and behavior of bacteria, but even simply to see them.

Bacteria were discovered in 1676 by the self-taught Dutch naturalist Antoni van Leeuwenhoek. The paradox is that the optical device he used was a step backward rather than forward in the development of microbiological instrumentation. It was a simple magnifying glass, whose uniqueness lay in having a tiny aperture and a very short focal length. As a result, in terms of linear magnification (approx. 300x) and resolving power (1.5 µm), it several times surpassed the microscopes of the time, which consisted of a tube (body), objective, and eyepiece. This allowed Leeuwenhoek not only to observe bacteria but also to describe their Morphology in broad outline; indeed, he was so precise and objective that we can readily infer which bacteria he was referring to. Leeuwenhoek can rightfully be considered the founding father of both prokaryotic cytology and physiology (among other things, he discovered that bacteria are immobilized upon heating or after Treatment with vinegar).

Unfortunately, Leeuwenhoek's magnifying glass never became an instrument for mass production and widespread use. It remains unclear how its core component—the lens—was manufactured: whether by grinding and polishing or by melting a small glass bead. In addition, the short-focus magnifier was extremely inconvenient for an observer with normal Vision (Leeuwenhoek himself was nearsighted) because it had to be held directly against the eye.

The design limitations of early microscopes were only overcome in the late 19th century, when the German optician Ernst Abbe developed The Theory of optical image formation. Building on this foundation, the German mechanic Carl Zeiss and his compatriot, the glass manufacturer Otto Schott, invented compound objectives, oil immersion systems, and condenser illumination. This new generation of microscopes boasted a maximum resolving power of 0.2 µm, and in the late 1870s Ferdinand Cohn and Robert Koch introduced them into routine microbiological research practice. After Koch proposed a technique for fixing smears on glass slides, replaced natural stains (such as indigo and hematoxylin) with synthetic aniline Dyes (such as gentian violet and methyl red), and applied photomicrography for research purposes, bacteria became easy to observe and the resulting data could be reliably documented.

Although bacteria were discovered as early as the late 17th century, General Concepts regarding their structure, physiology, and biodiversity began to take shape only by the mid-19th century, because earlier there was no proof that they could influence the life processes of other organisms or alter the physicochemical parameters of the environment.

The chronicles of bacteriology feature many names, including such luminaries as Louis Pasteur, Robert Koch, Ferdinand Cohn, Sergei Winogradsky, Martinus Beijerinck, Albert J. Kluyver, Roger Y. Stanier, Harland G. Wood, Ralph S. Wolfe, and many, many others. A profound mark on The history of microbiology was left by the French school (in Paris), the German school (in Berlin), the Dutch school (in Delft), and two American scientific schools (in Berkeley and San Diego).

Fig. 1. Tombstone of Antoni van Leeuwenhoek (1632–1723) in the Oude Kerk in Delft. Author's photograph.

Keeping pace with other biological sciences, bacteriology has constantly expanded The Scope of its subjects while gaining a deeper insight into the patterns of their Organization. The first evidence that bacteria possess unique physiological properties and exhibit biological activity was obtained through The Study of Fermentation and putrefaction.

1.2. Fermentation and Putrefaction

The PRACTICAL USE OF ethanol and lactic acid dates back to antiquity. However, until the early 19th century, The formation of these substances was not associated with the life activity of microorganisms such as Yeasts and bacteria.

The Chemical Nature of Alcoholic Fermentation, or "fermentation" (from Latin fermentum meaning bubbling), was proven in 1810 by the French physicist J. L. Gay-Lussac. Building on the prevailing view that sugar breaks down into "chalky" acid and wine alcohol, he came close to the modern understanding of fermentation as a redox reaction. In the late 1830s, C. Cagniard de Latour, T. Schwann, and F. T. Kützing independently established that alcoholic fermentation is a biological process caused by "sugar" Fungi, or yeasts of the genus Saccharomyces. However, this correct Conclusion met with sharp criticism from organic chemists and proponents of the "contact" theory of fermentation, namely J. J. Berzelius, F. Wöhler, J. von Liebig, and E. Mitscherlich. They viewed Yeast as a proteinaceous "ferment" that catalyzes The conversion of sugar into ethanol while remaining unchanged itself, much like an inorganic catalyst.

Louis Pasteur was a chemist by training, and his scientific career began with the discovery of stereoisomerism. Yet, he was not interested in the chemical side of fermentation, viewing it solely through a physiological lens. In the 1850s, he thoroughly investigated the microorganisms responsible for the accumulation of lactic and butyric acids, as well as ethanol. His first contribution to microbiology was proving the biological nature of Lactic acid fermentation. He established that the causative agents are microorganisms that thrive without air and differ morphologically from yeast (they are now classified as bacteria, specifically within the genus Lactobacillus).

In 1861, Pasteur investigated the microbial agents of Butyric acid fermentation (Clostridium butyricum). His findings led him to the paradoxical conclusion for that time that life is possible in the absence of oxygen. The discovery of anaerobiosis had a profound impact on the subsequent development of microbiology.

In 1858, M. Traube advanced the idea that sugar is fermented by a biological catalyst, or "enzyme" (from French en zymotique, meaning derived from fermentation), secreted by yeast. However, many years passed before E. Buchner (Nobel Prize in Chemistry, 1907) obtained a cell-free yeast extract in 1897 that retained The activity of "zymase," an enzyme complex that breaks down sucrose into ethanol and carbon dioxide. In 1905, A. Harden (Nobel Prize in Chemistry, 1929) established that alcoholic fermentation requires inorganic phosphate. Shortly thereafter, H. von Euler-Chelpin (Nobel Prize in Chemistry, 1929) determined the molecular nature of glucose breakdown products and identified the corresponding coenzyme, NAD. This marked the beginning of enzymology and its related applied science, biotechnology.

Another microbial process whose analysis became a milestone in understanding bacterial biodiversity is putrefaction. It is associated with the decomposition of protein-rich organic matter and is observed in animal diseases, necrotic lesions of Organs and Tissues, and the decay of corpses. The Mechanism of putrefaction is linked, in particular, to the formation of foul-smelling and toxic decarboxylation products of Lysine and Ornithine—putrescine (from Latin putor, meaning stench, and facio, meaning to make) and cadaverine (from Latin cadaver, meaning dead body). When methods for isolating bacteria and obtaining pure cultures on nutrient media were developed in the second half of the 19th century, Justus Liebig's idea that putrefaction is driven by the spontaneous breakdown of organic substances was discarded. Putrefaction was comprehensively studied in the 1870s by Louis Pasteur and Ferdinand Cohn. Specifically, they established that it is based on the decomposition of nitrogenous compounds under METABOLISM/18.html">The Influence of bacteria. Cohn drew a parallel between plant "diastase," which converts starch into sugar, and the "Enzymes" that bacteria use to break down Proteins into simpler nitrogenous compounds to support their growth. Louis Pasteur was the first to report on the ammonification of urea and to isolate the spherical bacteria responsible for this process (now classified as the species Sporosarcina ureae).

1.3. Infections

Beginning in the 1870s, putrefaction and toxigenic purulent-septic processes began to be viewed as the cause, rather than the consequence, of diseases in humans and animals infected with pathogenic bacteria.

Louis Pasteur, the founder of the French school of microbiology, proved The Link Between the life activity of specific groups of bacteria and various types of fermentations. In turn, Robert Koch, who established the German school of medical bacteriology, discovered that bacterial infections cause characteristic histopathological changes and that infected tissue harbors the pathogenic agent. Building upon the ideas of the anatomist J. Henle, who in 1840 outlined a strategy for searching for infectious disease agents, Koch formulated the minimum requirements—known as "Koch's postulates"—necessary to establish a causal relationship between a specific microbial agent and the pathological process it causes:

— using microscopic methods, to establish that the infectious agent is present in every case of the disease;

— to isolate the infectious agent from accompanying microbes and grow it in pure culture;

— to reproduce the disease in an experimental animal by inoculating it with a pure culture of the infectious agent; a control can be provided by reisolating the agent from the infected animal.

In 1876, Koch determined the etiological Nature of the anthrax agent (Bacillus anthracis). This was the first infectious bacterium found in the blood of a sick animal and, simultaneously, the first pathogenic bacterium obtained in culture. Thus, it was proven that a specific type of bacterium causes a disease with specific symptoms.

The period from the 1870s to the 1890s became the "golden age" of microbe hunting. In 1874, E. Hansen discovered the CAUSATIVE AGENT OF leprosy, Mycobacterium leprae, after which discoveries of pathogenic bacteria followed one after another.

In 1881, Louis Pasteur and G. Sternberg discovered the causative agent of bacterial Pneumonia, Streptococcus pneumoniae, and around the same time, A. Ogston described the pathogenic bacterium Staphylococcus aureus, which causes boils as well as conditions such as endocarditis and meningitis.

In 1882, Robert Koch isolated the bacterium responsible for Pulmonary Tuberculosis, Mycobacterium tuberculosis. During the epidemic that broke out in 1883 in Egypt and India, he discovered the cholera pathogen, Vibrio cholerae. That same year, E. Klebs and F. Loeffler reported the Discovery of the etiological agent of diphtheria, Corynebacterium diphtheriae.

In 1884, Koch's student G. Gaffky identified the causative agent of typhoid fever, Salmonella typhi. At the same time, F. J. Rosenbach described the spore-forming bacterium Clostridium tetani, which causes the formidable neurotropic disease tetanus. In 1885, T. Escherich discovered the colon bacillus, Escherichia coli.

In 1889, A. Neisser discovered the first agent of a sexually transmitted disease—the causative agent of Gonorrhea, Neisseria gonorrhoeae. In 1892, W. Welch and G. Nuttall identified the causative agent of gas gangrene, which turned out to be the spore-forming anaerobic bacterium Clostridium perfringens.

Plague is frequently mentioned in historical annals; notably, it was described in the 5th century BC by the ancient Greek writer Thucydides in his Historiae. Its causative agent, the plague bacterium Yersinia pestis, was independently discovered by A. Yersin and S. Kitasato during an epidemic in Hong Kong in 1894.

In 1895, E. van Ermengem discovered Clostridium botulinum, the causative agent of the fatal food poisoning known as botulism.

In 1898, K. Shiga isolated the agent of bacterial dysentery, Shigella dysenteriae. The syphilis pathogen, Treponema pallidum, was discovered in 1905 by F. Schaudinn and E. Hoffmann. In 1906, J. Bordet and O. Gengou identified the etiological agent of whooping cough, Bordetella pertussis.

Studying The properties of bacterial infection agents paved the way for the first victories over them. In 1867, the English physician J. Lister invented chemical antisepsis (from Latin anti-Sepsis, meaning counteracting putrefaction) and proposed using Phenolic Compounds to sterilize open wounds and surgical fields. In the 1880s and 1890s, Robert Koch and Ferdinand Cohn, along with P. Ehrlich (Nobel Prize in Physiology or Medicine, 1908), discovered the antibacterial action of other chemical substances, notably formalin. Thermal sterilization was invented in the second half of the 1870s by the prominent English naturalist J. Tyndall.

Concurrently with the Representatives of the German school who focused on discovering disease-causing agents, French bacteriologists sought biological means to combat infectious diseases. Louis Pasteur and his disciples introduced attenuation (Lat. *attenuo* — to weaken), a method for reducing the virulence of a pathogenic microbe through repeated subculturing on artificial media at a suboptimal Temperature (42°C) or following the infection of an animal that is not a primary host. The administration of an attenuated pathogen made it possible to induce active Immunity against a range of diseases, notably anthrax and plague.

In the early 1900s, observations of intracellular Digestion in invertebrates led É. Metchnikoff (Nobel Prize in Physiology or Medicine, 1908) to discover phagocytosis and formulate the cellular theory of immunity. Concurrently, E. von Behring (Nobel Prize in Physiology or Medicine, 1917) and Kitasato developed the humoral theory of immunity. Paul Ehrlich's hypothesis regarding the recognition of Antigens by specific Antibodies proved to be of paramount importance for immunology.

The first antibiotic, penicillin, was discovered in 1928 by A. Fleming (Nobel Prize in Physiology or Medicine, 1945). Its industrial production began in 1943. Over a relatively short period, between 1955 and 1962, up to 90% of Antibiotics produced by actinobacteria of the genus *Streptomyces* were described. Currently, the total number of antibiotics—including semi-synthetic, hybrid, and mutasynthetic ones—has reached tens of thousands. The successes of immunization and antibiotic therapy in the 1940s–1960s were so impressive that in 1969 U.S. Surgeon General W. H. Stewart announced to Congress the complete and final victory over infectious diseases. Unfortunately, however, this optimism proved premature. Although, according to WHO data, mortality from infectious diseases declined nearly 20-fold in the 1980s–1990s (from 797 cases per 100,000 population down to 36 cases per 100,000 population), at the end of the 20th century it still stood at 25%. The most frequent causes of fatal outcomes were pneumonia (3.5 million cases per year), acute intestinal diseases (2.2 million cases per year), and tuberculosis (1.5 million cases per year). At present, new etiological agents of acute and chronic infections have been identified, such as *Legionella pneumoniae* and *Helicobacter pylori*. The "counterattack" of pathogenic bacteria is driven by technological progress, anthropogenic environmental changes, demographic shifts, and the expansion of international contacts. Other factors negatively impacting the fight against pathogenic bacteria include adaptive Variability leading to The Emergence of resistant forms, as well as the ineffectiveness of preventive public health measures.

1.4. Bacterial Ecology

Bacteria occupy all sorts of niches—ranging from those favorable for mass proliferation to extreme environments where the degradation rate of ATP, NAD(P)H, proteins, and Nucleic Acids exceeds The rate of *de novo* Biosynthesis or repair. In terms of their ecological and geographical distribution, bacteria are cosmopolitans, a fact facilitated by their microscopic size, the diversity and lability of their metabolic systems, and their ability to withstand environmental stress.

The founder of ecological bacteriology was S. N. Winogradsky, who surpassed Louis Pasteur and Robert Koch in understanding the physiological specialization of microorganisms. He established the doctrine concerning The Role of individual bacterial groups in natural ecosystems and the participation of bacterial communities in biogeochemical cycles. Studying nitrifying bacteria, he formulated in the 1890s THE CONCEPT OF elective (Lat. *electio* — choice) conditions for development. According to this concept, specialized bacteria gain a physiological advantage over their ecocenosis partners and begin to outnumber them. The method of elective media forms the basis for screening bacterial biodiversity as well as for the laboratory analysis of bacterial communities and individual species.

In 1892, R. Thaxter isolated myxobacteria, which are among the major Components of the soil microbiota, and described their differentiated Cells and fruiting bodies. The distribution and ecological role of another typical soil inhabitant, the actinomycetes, were elucidated in the 1920s by S. A. Waksman (Nobel Prize in Physiology or Medicine, 1952) and Martinus Beijerinck.

During the 1900s–1940s, Beijerinck and his students refined Winogradsky’s method and were the first to obtain enrichment and pure cultures of bacteria belonging to various Ecological and Physiological groups. These included bacteria that hydrolyze urea; degrade Agar or Cellulose; oxidize hydrogen, sulfur, or methane; reduce sulfate or nitrate; synthesize cellulose; emit light (luminescent); anaerobic spore-forming; aerobic diazotrophic; lactic acid-producing; and acetic acid-producing bacteria.

At the beginning of the 20th century, independent disciplines branched out from general bacterial ecology: soil microbiology, aquatic microbiology, and geomicrobiology. The first microscopic observations of bacterial landscapes in soil samples collected from various regions around the globe were carried out in the early 1920s by S. N. Winogradsky at the Brie-Comte-Robert Biological Station near Paris. The results obtained enabled him to formulate the concept of autochthonous and allochthonous species as two main groups of bacteria in soil ecosystems. "Autochthonous" bacteria are those characteristic of a specific soil type (Gk. *autos* — self and *chthon* — earth; "living on one's own land"). They maintain a low, relatively stable population density and utilize poorly metabolizable soil substrates, primarily humus. This concept can also be applied to the normal microflora of animal integuments and the normal epiphytes of plants. Conversely, "allochthonous" bacteria are those that fortuitously colonize a specific soil type (Gk. *allos* — other and *chthon* — earth; "living on foreign land"). They are also referred to as "zymogenic" (Gk. *zyme* — leaven and *genea* — birth; "enzyme-producing"), as they proliferate when easily metabolizable substrates episodically enter the soil.

An important role in the study of soil bacterial biodiversity was played by the "immersed slide" method proposed by N. N. Kholodny in 1930 and the capillary method developed in 1961 by B. V. Perfiliev and D. R. Gabe. Outstanding representatives of the Russian school of soil microbiology—D. G. Zvyagintsev, N. A. Krasilnikov, E. N. Mishustin, and G. A. Nadson—along with their foreign colleagues, primarily Selman Waksman and A. G. Lochhead, made the principal contributions to the study of soil microorganism ecology.

The first investigations into the bacterial composition of continental water bodies were conducted in the late 19th century on carotenoid-pigmented purple bacteria, which had already been described in the late 1830s by the German protistologist C. Ehrenberg. Green anoxygenic bacteria, which are adapted to low light intensities and difficult to cultivate, were discovered by G. A. Nadson only in 1906.

The pioneer of marine microbiology is C. Zobell, who in the 1940s–1950s determined the species composition of bacterial populations inhabiting the water Column of the ocean. In the second half of the 1970s, H. W. Jannasch conducted studies on the microbiota of the World Ocean, which established that the Abundance of psychrophilic (Gk. *psychros* — cold and *phileo* — to love) bacteria increases with depth. In fact, the study of psychrophiles began as early as the late 19th century, after J. Foster demonstrated that bioluminescent bacteria can grow at 0°C. In the 1950s–1970s, the biology of psychrophiles was investigated by R. Y. Morita, J. A. Baross, and J. L. Ingraham. Later, interest in these subjects waned, and special attention shifted to extremely thermophilic bacteria and hyperthermophilic archaea.

Currently, bacteria inhabiting the coastal and continental areas of polar regions have once again become the subject of active research. Renowned hyperthermophile specialist J. W. Deming has in recent years focused on bacteria living in the water-ice phase transition zone. The results of analyses of bacterial communities in subglacial Lake Vostok in Antarctica have generated widespread Resonance.

During an investigation of marine ecosystems in 1979, A. A. Yayanos discovered barophilic (Gk.

baros — heavy and phileo — to love) bacteria adapted to high hydrostatic pressure. According to data obtained by Jannasch in the mid-1970s, water samples retrieved from ocean depths contain oligotrophic (Gk. *oligos* — few and *trophe* — nourishment) bacteria that thrive on low concentrations of nutrient substrates for their growth.

In 1988, S. W. Chisholm discovered the cyanobacterium *Prochlorococcus marinus* in the Atlantic Ocean, which turned out to be a champion among bacteria in terms of total population size and biological productivity. Cultivated strains of *P. marinus*, alongside the primary model Organism for cyanobacterial genetics *Synechocystis* sp. PCC 6803 (whose genome was completely sequenced in 1997), are becoming key models for studying the MOLECULAR MECHANISMS OF Photosynthesis.

An extensive section of geomicrobiology is dedicated to extremophilic bacteria, which include, in particular, thermophilic (Gk. *thermos* — warm and *phileo* — to love) forms. The first thermophilic bacillus was isolated by P. Miguel in 1888. Subsequently, various representatives of Gram-positive and Gram-negative bacteria possessing aerobic or anaerobic Types of Metabolism were described. Only prokaryotes are capable of growing at temperatures >60°C, and precisely such conditions are established in packed hay, compost heaps, and other organic materials, as well as in natural hydrothermal vents and certain hydraulic engineering structures.

The study of thermophilic bacteria in anthropogenic hydrothermal systems was carried out by J. G. Zeikus. In their natural habitats—thermal springs and geothermal fields of North America and Greenland—they were studied by T. D. Brock, R. W. Castenholz, and K. O. Stetter. Thermophiles from various regions and ecosystems of Eurasia are fruitfully investigated by G. A. Zavarzin, G. I. Karavaiko, and other domestic scientists.

In 1980, T. Kawasumi obtained in culture the aerobic hydrogen-oxidizing bacterium *Hydrogenobacter thermophilus*, which was soon followed in 1992 by G. Huber's Isolation of the aerobic thermophilic hydrogen bacterium *Aquifex pyrophilus*.

Halophilic (Gk. *alos* — salt and *phileo* — to love) bacteria, whose few species inhabit saline water bodies, attracted attention following the classical studies conducted by Claude Zobell in the 1940s and H. Larsen in the 1960s. Typical inhabitants of warm, saline water bodies include the extremely halophilic purple bacterium *Ectothiorhodospira halochloris*, described by H. G. Trüper in 1977, and extremely halophilic cyanobacteria of the genera *Synechocystis* and *Spirulina*. Between 1980 and 1998, new halophilic bacteria belonging to the genera *Antarctobacter*, *Haloanaerobium*, *Haloincola*, *Halomonas*, *Halothermothrix*, and others were discovered in saline water bodies of tropical, polar, and temperate latitudes. All of them are exquisitely adapted to high salinity, and their mechanisms of salt-stress resistance differ from those utilized by extremely halophilic archaea.

Most bacteria prefer neutral or slightly alkaline conditions, but mesophilic and thermophilic acidophiles (Lat. *acidus* — sour and Gk. *phileo* — to love) do exist. These are primarily specialized lithotrophs that utilize energy from The oxidation of substrates such as iron and sulfur. Many of them are of practical importance and were thoroughly studied by J. A. Brierley, G. I. Karavaiko, and R. S. Golovacheva in the late 1970s.

The logic of studying extremophilic bacteria poses A number of new challenges. In particular, at the initiative of G. A. Zavarzin, a comprehensive analysis has been undertaken of hydrothermal ecosystems, halophilic communities in marine lagoons, and alkaliphilic (Lat. *alcalinus* — alkaline and Gk. *phileo* — to love) bacteria in soda lakes, which serve as modern analogs of prehistoric biotopes.

Ever since Martinus Beijerinck first initiated the study of bacterial Symbiosis with leguminous plants in 1888, the list of ecological niches occupied by symbiotic bacteria (Gk. *symbiosis* — living together) has expanded to encompass the phyllosphere and rhizosphere of plants, the integuments and digestive tract of animals, and—in the case of endocytobiosis—the cytoplasmic and nuclear compartments of Eukaryotic cells.

In the late 20th century, ecological microbiology began to investigate "unconventional" niches and their inhabitants: endolithic (Gk. *endon* — within and *lithos* — stone) bacteria living inside rocks and cliffs; bacteria colonizing the Earth's crust, including inhabitants of oil-bearing strata; and bacteria from foulings, biofilms, and other extremophilic ecosystems.

The youngest branch of ecological microbiology is dedicated to studying the interrelated problems of hyperanabiosis and exobiology.

The analysis of rare situations where bacteria, existing in a state of complete physiological dormancy or anabiosis (from the Greek anabiosis — resurrection from the dead), remain viable over geological periods allows us not only to establish the chronological boundaries of an individual organism's existence, but also to study The structure of such "living fossils." The capacity for anabiosis underpins the dissemination of life through outer space, as posited by the hypothesis of panspermia (from the Greek pan-sperma — single seed). It is widely believed that this concept was proposed in the 1900s by Svante Arrhenius (S. A. Arrhenius, Nobel Prize in Chemistry, 1903), although it was actually formulated as early as 1821 by S.-G. de Montlivault, while H. E. Richter attempted to Supplement Charles Darwin's selectionist theory with similar considerations in 1865. Among prominent microbiologists, the panspermia hypothesis was supported in the 1870s by Ferdinand Cohn. Its adherents also included figures from other natural sciences, notably the renowned physicist Lord Kelvin (W. Thomson).

Indirect evidence supporting the existence of organic life on celestial bodies comes from data on the ability of bacterial endospores to enter anabiosis and the exceptional resistance of these differentiated cells to harsh environmental stressors.

Research in the field of exobiology received fresh momentum after water—a prerequisite for physiological processes—was discovered on Mars as well as on Europa, the Jovian moon. Another argument favoring the existence of extraterrestrial biological activity was provided by D.S. McKay's 2000 report on the discovery of magnetite particles inside the Martian meteorite ALH84001, which closely resemble bacterial magnetosomes, the cytoplasmic inclusions of biogenic iron minerals.

Bacterial ecology is moving away from the reductionism of Louis Pasteur and Robert Koch, which dominated the dawn of microbiology but now hinders its progressive development. Modern researchers successfully employ Sergei Winogradsky's principle of electivity alongside his Methods for the direct analysis of microbial communities. Laboratory modeling using pure cultures is gradually becoming a thing of the past. The future belongs to the in situ systems analysis of microbiota based on field observations and physiological tests, supported by molecular biological toolkits (such as the Polymerase Chain Reaction, probes, etc.). The Prospects for Microbial Ecology are vast, as are the challenges awaiting those who work in this field.

1.5. Bacterial Cytology

Following the advent of commercial laboratory microscopes in the latter half of the 19th century, analyzing bacterial morphology became a relatively straightforward task. Researchers characterized the primary shapes and cell aggregates of bacteria, appendage structures, and cytoplasmic inclusions, as well as structural transformations, particularly during the formation of differentiated cells. A monumental breakthrough was the discovery in 1876–1877 by Ferdinand Cohn, Robert Koch, and John Tyndall of dormant, resistant bacterial cells known as endospores. However, the internal architecture of bacteria remained elusive for a long time, especially regarding the bacterial "nucleus."

Initial insights into the functional cytology of bacteria began to accumulate in the 1950s and 1960s with the Introduction of electron microscopy to microbiology. Broad opportunities to study bacterial anatomy using the Electron microscope emerged after:

a) R. C. Williams integrated the shadowing of cells and subcellular structures with heavy metals into the bacteriological methodology;

b) G. A. Palade (Nobel Prize in Physiology or Medicine, 1974) began utilizing osmium tetroxide for fixation;

c) S. B. Newman developed a technique for preparing ultrathin sections from materials embedded in methacrylate resins;

d) E. Kellenberger proposed a fixation method using buffered glutaraldehyde alongside a contrasting technique employing uranyl acetate.

During the 1960s and 1970s, M. R. J. Salton and R. G. E. Murray obtained pioneering data on the STRUCTURE OF THE cell envelope and internal membranes; G. B. Chapman and C. Robinow described nucleoid morphology and Cell Division; P. C. Fitz-James investigated endospore differentiation; M. L. DePamphilis characterized the ULTRASTRUCTURE OF THE flagellum; and T. M. Schmidt studied sulfur globules and other functional inclusions.

In the latter half of the 1970s, when N. Nanninga introduced freeze-etching to electron microscopy, and particularly with the advent of ultra-fast cryofixation and organic solvent cryosubstitution, bacterial cytology largely overcame The problem of artifacts. The study of dynamic bacterial ultrastructure reached a new qualitative level in the early 1990s following the introduction of immunoprobes and methods for in situ autofluorescence and epifluorescence detection. This strongly stimulated research into the dynamic anisotropy of the bacterial cell, DNA Replication, daughter chromosome segregation, and the mechanism of binary fission.

1.6. Bacterial Genetics

Bacteria reproduce exclusively via vegetative means. Due to the absence of heterozygosity and meiotic Crossing Over, their genetic analysis was long restricted to recording Spontaneous and Induced Mutations, as well as the phenotypic manifestations of Homologous Recombination resulting from parasexual processes—namely, the vectorial transfer of genes from a donor to a recipient.

In 1943, S. Luria (Nobel Prize in Physiology or Medicine, 1969) and M. Delbrück (Nobel Prize in Physiology or Medicine, 1969) laid the foundations of bacterial genetics by demonstrating that bacteria adhere to Darwinian principles of natural Selection driven by the accumulation of random mutations. Intensive research into the Genetics of bacteria and Bacteriophages yielded insights into the material nature of hereditary information as early as the following decade.

In 1928, F. Griffith discovered the "transforming principle" that bacteria exchange with one another, leading to alterations in their genotype. In 1944, O. T. Avery, C. M. Macleod, and M. McCarty proved that the Genetic Transformation of non-capsulated, avirulent pneumococci is mediated by DNA extracted from cells of a virulent, encapsulated strain. Subsequently, A. Hershey (Nobel Prize in Physiology or Medicine, 1969) and M. Chase established that during T2 phage infection, only viral DNA enters the bacterial cell, thereby conclusively proving the role of DNA as the carrier of hereditary information.

In the early 1950s, extrachromosomal, autonomously replicating genetic elements were discovered in bacteria. At the suggestion of J. Lederberg (Nobel Prize in Physiology or Medicine, 1958), they were named Plasmids. Plasmids confer selectively advantageous traits upon bacteria, such as fertility, colicinogeny, multiple drug resistance, The ability to degrade xenobiotics, etc. The "horizontal" transfer of genes via plasmids drives the Genetic heterogeneity of bacterial populations.

In 1946, J. Lederberg and E. L. Tatum (Nobel Prize in Physiology or Medicine, 1958) discovered conjugation—a contact-mediated process ensuring plasmid-driven Gene transfer from a donor to a recipient. This represents the primary mode of "horizontal" gene transfer in bacteria. Throughout the 1940s and 1950s, the analysis of recombinant traits in exconjugants was successfully applied to map the E. coli genome.

In 1949, A. Lwoff (Nobel Prize in Physiology or Medicine, 1965) discovered Lysogeny in Bacillus megaterium, a phenomenon involving phage-induced cell lysis. Homologous recombination resulting from Transduction—The transfer of bacterial genes via a temperate phage chromosome—operates through mechanisms analogous to conjugative recombination. In 1952, J. Lederberg and N. G. Zinder observed this process in salmonellae.

In the early 1980s, R. R. Reed identified non-homologous recombination in bacteria. This process is mediated by Transposons, which integrate randomly into the recipient bacterial genome via migrating insertion sequences. In 1968, S. Linn and W. Arber (Nobel Prize in Physiology or Medicine, 1978) discovered restriction enzymes, which target and cleave foreign DNA invading The Cell. In 1970, H. Smith (Nobel Prize in Physiology or Medicine, 1978) isolated the first site-specific restriction endonuclease, Hind II, from Haemophilus influenzae. This breakthrough laid the groundwork for modern recombinant DNA technologies and Gene cloning methods, ultimately giving rise to the branch of cytogenetics that investigates the infrastructure, conservation, and variability of bacterial genomes.

One of the first to point out the ability of bacteria to mineralize a wide array of Organic compounds was the Dutch microbiologist L. E. den Dooren de Jong. In 1926, he proposed classifying members of the genus Pseudomonas According to the spectrum of substrates they utilize for growth.

Nevertheless, the discovery of adaptive enzyme synthesis did not contradict the concept of a conservative metabolic foundation. In 1926, Albert Kluyver formulated THE PRINCIPLE OF the biochemical unity of life: "From the butyric acid bacterium to the elephant, all organisms share the same biochemical foundation." In a modified version—where E. coli replaces Clostridium butyricum—this aphorism is often mistakenly attributed to Jacques Monod (J. Monod, Nobel Prize in Physiology or Medicine, 1965). Essentially, Kluyver advanced the idea that biochemistry rests on a common foundation comprising a universal Genetic Code, standard biopolymer monomers, global energy-transducing systems, and central pathways of Intermediary Metabolism.

Alongside this paradigm, Kluyver proposed the concept of metabolic pathways, suggesting that anabolic and catabolic processes consist of chains of mutually balanced oxidation-reduction reactions. Albert Kluyver can also be considered the "father" of comparative biochemistry, as he viewed microbes as model systems suited for reconstructing the metabolic networks of other biological entities, including higher animals and plants.

Decisive contributions to understanding the mechanisms of Amino Acid and nucleotide biosynthesis, as well as elucidating vitamin metabolism and the participation of their coenzyme forms in enzymatic reactions, were made by E. E. Snell in research conducted during the 1950s and 1960s using enteric bacteria, pseudomonads, and lower actinomycetes.

In the latter half of the 1950s, Roger Stanier conducted a classic study on carotenoids in purple bacteria. Based on his findings, the general pathway of polyisoprenoid biosynthesis was reconstructed, and the protective role of carotenoids under oxidative stress was elucidated.

Biotechnology is rooted in the fundamental knowledge of bacterial metabolism. Utilizing bacteria—frequently genetically engineered overproducing strains—makes it possible to manufacture foodstuffs, microbial biomass, Biopolymers, enzymes, primary metabolites (such as Amino acids and NUCLEOTIDES), secondary metabolites (including antibiotics), Vitamins, Surfactants, alcohols, ketones, and organic acids, among other products. Since the early 1980s, following the advent of Recombinant DNA technology and Genetic Engineering, it has become feasible to clone "foreign" genes into E. coli Cells under the control of strong or inducible promoters. This ensures the expression of valuable pharmacological products in E. coli cells, such as proteins from other bacteria (e.g., streptokinase, penicillinase) or from eukaryotic organisms (e.g., Insulin, interferon).

The metabolic potential of sulfur bacteria is harnessed in the industrial process of microbial metal leaching. Symbiotic associations involving pseudomonads, rhodococci, and other aerobically respiring bacteria purify water polluted by industrial, agricultural, municipal, and domestic waste. They also perform remediation (from Latin remediatio — healing), thereby eliminating the adverse environmental consequences of anthropogenic activity.

Aerobes. The high oxidation capacity of aerobic bacteria ensures maximum ENERGY EXTRACTION FROM nutrient substrates. This also accounts for non-utilitarian oxidation, or "cometabolism," of aliphatic and Aromatic Compounds via dehydrogenases and oxygenases. In the 1960s, Roger Stanier and R. P. Gunsalus carried out pioneering work using representatives of the genus Pseudomonas. Their results helped elucidate the degradation mechanisms of petroleum products, pesticides, and other xenobiotics (from Greek xenos — stranger and bios — life; "substances of non-natural origin that do not enter into the biological cycle") and explored the role played by "biodegradation" plasmids in this process.

Industrial wastewater treatment using bacterial aggregates, or activated sludge, was first established in 1913 in Manchester. This technology continues to be used today in an improved form. In parallel, new methods are being developed and implemented that rely on cascading treatment facilities, where water is freed from organic pollutants and biogenic elements capable of triggering eutrophication, commonly known as algal blooms in natural water bodies.

Anaerobes. The concept of anaerobiosis, or "life without oxygen," emerged as a major theoretical outcome of research conducted by Louis Pasteur between 1857 and 1877. He was the first to classify microorganisms based on their relationship with oxygen into aerobes (from French aérobies — "creatures living in the air") and anaerobes (from French anaérobies — "creatures living without air"). Furthermore, he drew attention to intermediate states of oxygen response, now referred to as facultative anaerobiosis, microaerobiosis, and microaerophilia. Using various methods to remove oxygen from the air and degassing nutrient media, he isolated the first culture of an anaerobic pathogenic bacterium (Clostridium septicum) in 1877.

In the mid-1930s, R. E. Hungate proposed the " roll-tube" method, which is still used today to obtain pure cultures of anaerobes. He isolated several new forms, including the cellulolytic bacteria Bacteroides succinogenes and Micromonospora propionici, which inhabit the rumen of ruminants. Hungate's work revealed that anaerobes are widespread in the digestive tracts of animals, sludge, waterlogged soils, and other environments, constituting over 90% of the total microbiota in all these niches.

A major contribution to the study of anaerobes was made by H. A. Barker, who obtained cultures capable of fermenting a diverse range of substrates. During the 1940s and 1950s, using radioisotope labeling, he established that propionic acid bacteria carry out heterotrophic fixation of CO₂ and that acetyl-CoA is not only a product of Pyruvate oxidation but can also serve as a metabolic precursor in The biosynthesis of acetoacetate and citrate.

The anaerobic digestion process—in which cellulolytic, amylolytic, and proteolytic bacteria, alongside acid-producing clostridia and heterotrophic acetogenic bacteria of the genera Syntrophomonas and Syntrophobacter, play key roles alongside methanogenic archaea—was studied in the 1970s in the laboratories of Zeikus and S. H. Zinder.

Bacterial photosynthesis. In the late 19th century, Ferdinand Cohn, E. Warming, E. Esmarch, M. Perty, W. Zopf, and S. N. Winogradsky first described purple bacteria and unicellular green bacteria. However, they did not consider them phototrophs, as photosynthesis at that time was inseparable from oxygen evolution. The light dependency of purple bacteria was first demonstrated between 1883 and 1888 by W. Engelmann, who discovered photokinesis and phototaxis using them as models—namely, the light-induced changes in cell movement speed and orientation. Proof that purple bacteria are capable of photosynthesis without oxygen evolution was obtained only between 1907 and 1919 through the works of H. Molisch and J. Buder. Around the turn of the 1960s and 1970s, N. Pfennig developed classical methods for the isolation and cultivation of anoxygenic phototrophic bacteria.

While investigating the anabolic metabolism of unicellular green bacteria, D. I. Arnon discovered a novel ferredoxin-dependent pathway of CO₂ assimilation in 1966, later designated as the "Evans–Buchanan–Arnon pathway." In 1974, Richard Castenholz first described filamentous green bacteria. The peculiarities of their anabolic metabolism eluded researchers for a long time, and it was not until 1992 that G. Fuchs demonstrated that they operate via a previously unknown "hydroxypropionate" cycle for autotrophic CO₂ assimilation.

In 1983, H. Gest discovered heliobacteria—anoxygenic bacteria containing bacteriochlorophyll $g$. In 1979, T. Shiba reported a new physiological group of quasi-phototrophic, or aerobic anoxygenic phototrophic bacteria, capable of growth and photosynthesis only in the presence of oxygen.

Until the early 1960s, cyanobacteria were regarded as "blue-green Algae" and, naturally, were not studied using bacteriological methods.

The fact that cyanobacteria were classified as botanical objects from the very beginning is easily explained: before Roger Stanier equated prokaryotes with bacteria, any oxygen-evolving phototrophic organism was placed in the kingdom Plantae. An additional factor was that cyanobacteria generally do not hydrolyze polymeric substrates, do not secrete specific exometabolites, and are not pathogenic—in other words, they lacked the very characteristics that early bacteriologists deemed typical of bacteria. Furthermore, cyanobacteria cannot be isolated into pure culture by plating onto selective media.

The realization that blue-green algae do not belong to plants followed directly from "Stanier's principle" that prokaryotes are synonymous with bacteria. Since the early 1980s, the term "cyanobacteria," proposed by him, has come into general use. After M. M. Allen obtained pure cultures of cyanobacteria in the 1960s, it became possible for the first time to study them using physiological, biochemical, and Genetic Methods. Today, this group of bacteria is arguably better characterized than any other.

In 1959, Howard Gest made a fundamentally important discovery: using the purple anoxygenic bacterium Rhodospirillum rubrum, he demonstrated that the photosynthetic apparatus is associated with chromatophores, or intracellular membranes. Throughout the 1950s to 1990s, purple bacteria and cyanobacteria became model organisms for studying photosynthesis.

Using spectroscopic methods in 1952, L. N. M. Duysens established that purple bacteria possess a "reaction center"—the structural unit of the photosynthetic apparatus where light-driven oxidation-reduction reactions take place. In 1968, D. W. Reed and R. K. Clayton isolated a reaction center preparation from Rhodopseudomonas sphaeroides using mild detergents, and G. Feher determined its composition in 1970. That same year, using EPR spectroscopy, Feher identified the quinone acting as the primary acceptor of the reaction center. In 1975, J. R. Norris discovered that

the primary donor in the reaction center of Rh. sphaeroides is a bacteriochlorophyll $a$ "dimer."

In the early 1980s, Hartmut Michel (Nobel Prize in Chemistry, 1989) grew crystals of the reaction center from the purple bacterium Rhodopseudomonas viridis. This achievement enabled the Construction of a three-dimensional model of its homolog—the reaction center of Photosystem II (PSII). Based on data obtained in 1996 for the cyanobacterium Synechococcus sp. by P. Fromme's group using X-ray crystallography at a 4.5 Å resolution, a model of the Photosystem I (PSI) reaction center was built. At the close of the 20th century, the Phylogenetic Tree of cyanobacteria and their descendant "simple" Plastids was reconstructed.

Chemosynthesis. In 1877, T. Schloesing and A. Müntz discovered that soil bacteria oxidize ammonia to nitrate. Subsequently, R. Warrington showed that nitrification proceeds in two stages, with nitrite as an intermediate product. However, it was not until 1886–1888 that S. N. Winogradsky established that bacteria can derive energy by oxidizing inorganic substrates (this mode of energy Nutrition was termed lithotrophy). As model subjects, he used the sulfur bacterium Beggiatoa alba and the iron bacterium Leptothrix ochracea. Continuing his studies on lithotrophy using nitrifying bacteria of the genera Nitrosomonas and Nitrobacter, he discovered chemosynthesis in 1890, which combines lithotrophy with carbon autotrophy.

In 1893, V. T. Shevyakov described the "giant" bacterium Achromatium oxaliferum, which oxidizes sulfide to elemental sulfur. In turn, R. Lauterborn described a "giant" filamentous sulfur bacterium belonging to the genus Thioploca in 1907. The credit for discovering sulfur-oxidizing bacteria (genus Thiobacillus), which oxidize sulfur to sulfate, belongs to Martinus Beijerinck, who reported on them in 1904.

In 1931, L. H. Stickland established that sulfate-reducing anaerobic bacteria can utilize molecular hydrogen for this process. It was later revealed that The energy released during hydrogen oxidation can also be harnessed by aerobic bacteria. In 1976, G. A. Zavarzin discovered aerobic carboxydotrophic bacteria, which use carbon monoxide simultaneously as an energy source and a building block. Later, in 1983, John Zeikus discovered anaerobic carboxydotrophs.

In the mid-1980s, Harland Wood traced The pathway of autotrophic acetate biosynthesis in the hydrogen-oxidizing bacterium Clostridium thermoaceticum, which was later named the "Wood pathway."

The discovery of two new types of lithotrophy came as a major surprise to microbiologists. In 1995, A. A. van de Graaf discovered the bacterial process of anaerobic ammonium Oxidation coupled with nitrite reduction (anammox), and simultaneously F. Widdel demonstrated that anaerobic bacteria can oxidize ferrous iron using nitrate.

Methylotrophy. In 1906, N. L. Söhngen first studied bacteria that use methane, methanol, or other single-carbon compounds as sources of energy and carbon. However, until the 1970s, little was known about their distribution in nature, their role in the carbon cycle, their physiological properties, or their biodiversity.

In the latter half of the 1970s, largely thanks to the research of J. R. Quayle, two formaldehyde assimilation mechanisms in methylotrophs were deciphered: the “ribulose monophosphate pathway” (or the “Quayle pathway”) and the “Serine pathway”.

Until recently, microbial methane oxidation by bacteria was considered to be an obligately aerobic process. However, in 2002, W. Michaelis reported that the microbiota of microbial mats from the floor of the Black Sea, which includes methanogenic archaea and sulfate-reducing bacteria, is capable of oxidizing methane in vitro in the absence of oxygen.

Diazotrophy. In 1885, H. Hellriegel and H. Wilfarth discovered that leguminous plants assimilate atmospheric nitrogen (this mode of nutrition is now known as diazotrophy). A few years later, Martinus Beijerinck revealed that the ability for diazotrophy is possessed not by the plants themselves, but by their endosymbionts—nodule bacteria of the genus Rhizobium.

The first free-living anaerobic diazotrophic bacterium, Clostridium pasteurianum, was isolated by S. N. Winogradsky in 1902. That same year, Beijerinck and A. van Delden described the first free-living aerobic bacterium, Azotobacter chroococcum.

In 1942, G. E. Fogg discovered diazotrophy in cyanobacteria, and in 1950, Howard Gest found it in purple bacteria.

1.8. Paleomicrobiology

Because bacteria possess a microscopic size and simple morphology, it was long believed that their fossils could not be distinguished from similar abiotic structures, or pseudofossils. Consequently, the topic of paleomicrobiology was not discussed, and practical tasks related to developing METHODS FOR STUDYING ancient microorganisms were not pursued. Doubts about the existence of reliable fossil remains of bacteria were dispelled in the mid-1970s as a result of research conducted by microbiologists in collaboration with geologists and geochemists.

Fossil stromatolites and microfossils. Fossil stromatolites and microfossils serve as the primary material for studying the Early stages of the evolution of organic life on Earth.

Fossil stromatolites (from the Greek stroma, meaning bed, and lithos, stone), or layered fossils, formed during past geological periods as a result of the in-vivo mineralization of microbial mats—stratified communities whose main component is cyanobacteria. Stromatolites also form in the current geological period (see Section 7.3.5); morphologically, they are similar to stromatolites that were widespread in marine and terrestrial Precambrian biotas. The earliest fossil remains of this type are up to 3 billion years old.

While stromatolites demonstrate the macrostructure of an ancient microbial consortium, extremely thin sections of microfossils (from the Latin fossicius, dug up), or individual fossilized microorganisms, viewed under a Light Microscope provide insight into the shape of fossil bacteria and even their ultrastructure.

In the early 1980s, J. W. Schopf, A. N. Knoll, and other paleomicrobiologists analyzed microfossils in thin sections of Precambrian sedimentary rocks dating back 1.5–3.5 billion years. As it turned out, these microfossils are nearly indistinguishable from modern cyanobacteria. Morphometric data characterizing cell shape and size, trichome structure, sheath architecture, microcolony organization, and other parameters have now been obtained for them. Comparing fossil unicellular and filamentous cyanobacteria (specifically, 1.4–10³ samples across 260 geological formations and 650 samples across 160 geological formations, respectively) with 600 samples of modern cyanobacteria revealed that microfossils can be identified down to the genus level. Therefore, they are assigned the names of modern cyanobacteria with The addition of the prefix palaeo- or eo-, as well as the suffixes -opsis or -ites (e.g., Palaeolyngbya and Aphanocapsaopsis). The majority of Precambrian microfossils (263 morphotypes) exhibiting external features of cyanobacteria correspond to the diagnoses of modern Chroococcales (25%) and Oscillatoriales (37%) strains.

Thus, cyanobacteria were widespread 2 billion years ago, which is supported by geological and geochemical data as well as the results of radiocarbon dating. Earlier microfossils from the Apex chert of Western Australia, dating back 3.5 billion years, also show similarities to oscillatorialean cyanobacteria. And since the age of the Earth is 4.5 billion years, this means that cyanobacteria are among the very first inhabitants of our planet.

Molecular fossils. Alongside stromatolites and microfossils, certain types of Biomolecules serve as paleomicrobiological records. They are, both figuratively and literally, “molecular fossils”.

Molecular fossils in the figurative sense are genomic nucleic acids—DNA in cellular organisms, and DNA or RNA in non-cellular life forms (Viruses). Thanks to continuous template copying combined with repair mechanisms, they preserve the most conservative regions of their structure over hundreds of millions and even billions of years. Therefore, by determining and comparing nucleotide sequences within DNA or RNA, we can construct the evolutionary tree (see Section 2.5).

Molecular fossils in the literal sense are biomolecules that persist after cell death with almost no changes across geological periods. This occurs because, unlike nucleic acids, proteins, and CARBOHYDRATES, they possess a rigid framework that is resistant to enzymatic or abiotic degradation.

A special place among such biomolecules is occupied by certain types of lipids, which are saturated Hydrocarbons with branched and/or cyclic skeletons. They are called recalcitrant biomolecules (from the Latin recalcitro, meaning kicking back; “resistant to destruction”).

Specific recalcitrant lipids can be identified for each of the three phylogenetic domains: phytanes for Archaea, Steroids for Eukarya, and hopanoids for Bacteria (see Section 8.3.3.1). Notably, sedimentary rocks dating back 2.5 billion years contain homologs of hopanoids found in modern cyanobacteria.

Reanimated ancient bacteria. Some prokaryotes remain viable over geological periods provided they are in a state of complete cryptobiosis. After reanimation, they can be studied using traditional cytophysiological methods.

In 1998, a report appeared on the ISOLATION OF A live culture of Staphylococcus succinus—a bacterium whose vegetative cells had been preserved for 25–35 million years in a piece of amber from the Dominican Republic (South America). However, even such longevity looks modest compared to the fantastic cryptobiosis lasting 250 million years. This is the exact age of vegetative cells of Bacillus sp. that were entombed in rock salt crystals discovered in 2000 in New Mexico, USA.

Unlike vegetative cells, endospores not only completely halt their metabolic activity upon entering a cryptobiotic state (dormancy), but also acquire exceptional resistance to environmental stresses (resistance). Thanks to these properties, they can germinate long after their formation. In the gut of a mummified bee found in a piece of amber, they remained intact for 25–40 million years.

1.9. “Virtual” Microbiology

At the end of the 20th century, not only the methods but also the conceptual approaches used to study the natural diversity of bacteria and their niche distribution underwent a transformation.

The classical strategy for studying biodiversity—“microbe hunting,” culturing, and comprehensive phenotypic analysis of accumulated material—is gradually fading into the Background. As an alternative, the polymorphism of specific genomic regions, primarily rRNA genes, is investigated. Simply put, researchers search for the ID cards rather than their owners.

To this end, DNA belonging to phenotypically uncharacterized entities is extracted from water, soil, or another substrate. To begin with, as little as 10^-8 g of DNA is sufficient. Then, 16S rRNA genes are amplified via the polymerase chain reaction using specific primers. The resulting amplicons are optionally cloned into bacterial plasmids, followed by automated sequencing. Finally, the reconstructed nucleotide sequence is compared against genetic Databases.

Using this method, it is possible to detect “phantom” bacteria that cannot be visualized by microscopy in natural samples and show no Signs of Life under laboratory conditions.

The advantage of this approach is that it eliminates the need to spend time and technical resources on collecting, purifying, and culturing bacterial strains, as well as on comprehensive analysis of their phenotypic properties.

However, by no means all bacteria observed under a microscope in natural samples can be induced to multiply in the laboratory. We are referring to the so-called "unculturable" bacteria (viable but nonculturable, VBNC). Such a latent state is triggered by stress when organisms enter an artificial environment.

There is another issue associated with cultivation, namely The Use of pure cultures, constant physicochemical conditions, and relatively rich nutrient media. For the opportunity to study the Properties of Individual objects quickly and without Interference, the modern researcher pays by analyzing monstrous phenotypes adapted to an artificial Homeostasis. Conditions in this "greenhouse" niche differ sharply from natural environments. In the absence of interspecific competition and under a fixed nutritional regime, Genetic Drift is enhanced, leading to the accumulation of genomic differences between museum strains and natural populations.

The future of experimental microbiology lies in mixed cultures and the simulation of natural environments featuring trophic webs, as well as established gradients of nutrient substrates and microbial metabolic products.