Microbial Ecology

The term “microbial ecology”is now used in a general way to describe the presence and contributions of microorganisms, through their activities, to the places where they are found. Students of microbiology should be aware that much of the information on microbial presence and contributions to soils, waters, and associations with plants, now described by this term, would have been considered as “environmental microbiology” in the past. Thomas D. Brock, the discoverer of Thermus aquaticus, which is known the world over as the source of Taq polymerase for the polymerase chain reaction (PCR), has given a definition of microbial ecology that may be useful: “Microbial ecology is the study of the behavior and activities of microorganisms in their natural environments.” The important operator in this sentence is their environment instead of the environment. To emphasize this point, Brock has noted that “microbes are small; their environments also are small.” In these small environments or “microenvironments,” other kinds of microorganisms (and macroorganisms) often also are present, a critical point that was emphasized by Sergei Winogradsky in 1947. Environmental microbiology, in comparison, relates primarily to all-over microbial processes that occur in a soil, water, or food, as examples. It is not concerned with the particular “microenvironment” where the microorganisms actually are functioning, but with the broader-scale effects of microbial presence and activities. One can study these microbially mediated processes and their possible global impacts at the scale of “environmental microbiology” without knowing about the specific microenvironment (and the organisms functioning there) where these processes actually take place. However, it is critical to be aware that microbes function in their localized environments and affect ecosystems at greater scales, including causing global-level effects. In the last decades the term “microbial ecology” largely has lost its original meaning, and recently the statement has been made that “microbial ecology has become a ‘catch-all’ term.” As you read various textbooks and scientific papers, possible differences between “microbial ecology” and “environmental microbiology” should be kept in mind.

Virology Early Development

Although the ancients did not understand the nature of their illnesses, they were acquainted with diseases, such as rabies, that are now known to be viral in origin. In fact, there is some evidence that the great epidemics of A.D. 165 to 180 and A.D. 251 to 266, which severely weakened the Roman Empire and aided its decline, may have been caused by measles and smallpox viruses. Smallpox had an equally profound impact on the New World. Hernán Cortés’s conquest of the Aztec Empire in Mexico was made possible by an epidemic that ravaged Mexico City. The virus was probably brought to Mexico in 1520 by the relief expedition sent to join Cortés. Before the smallpox epidemic subsided, it had killed the Aztec King Cuitlahuac (the nephew and son-in-law of the slain emperor, Montezuma II) and possibly 1/3 of the population. Since the Spaniards were not similarly afflicted, it appeared that God’s wrath was reserved for the Native Americans, and this disaster was viewed as divine support for the Spanish conquest The first progress in preventing viral diseases came years before the discovery of viruses. Early in the eighteenth century, Lady Wortley Montagu, wife of the English ambassador to Turkey, observed that Turkish women inoculated their children against smallpox. The children came down with a mild case and subsequently were immune. Lady Montagu tried to educate the English public about the procedure but without great success. Later in the century an English country doctor, Edward Jenner, stimulated by a girl’s claim that she could not catch smallpox because she had had cowpox, began inoculating humans with material from cowpox lesions. He published the results of 23 successful vaccinations in 1798. Although Jenner did not understand the nature of smallpox, he did manage to successfully protect his patients from the dread disease through exposure to the cowpox virus. Until well into the nineteenth century, harmful agents were often grouped together and sometimes called viruses [Latin virus, poison or venom]. Even Louis Pasteur used the term virus for any living infectious disease agent. The development in 1884 of the porcelain bacterial filter by Charles Chamberland, one of Pasteur’s collaborators and inventor of the autoclave, made possible the discovery of what are now called viruses. Tobacco mosaic disease was the first to be studied with Chamberland’s filter. In 1892 Dimitri Ivanowski published studies showing that leaf extracts from infected plants would induce tobacco mosaic disease even after filtration to remove bacteria. He attributed this to the presence of a toxin. Martinus W. Beijerinck, working independently of Ivanowski, published the results of extensive studies on tobacco mosaic disease in 1898 and 1900. Because the filtered sap of diseased plants was still infectious, he proposed that the disease was caused by an entity different from bacteria, a filterable virus. He observed that the virus would multiply only in living plant cells, but could survive for long periods in a dried state. At the same time Friedrich Loeffler and Paul Frosch in Germany found that the hoof-and-mouth disease of cattle was also caused by a filterable virus rather than by a toxin.

The Nucleoid

Probably the most striking difference between procaryotes and eucaryotes is the way in which their genetic material is packaged. Eucaryotic cells have two or more chromosomes contained within a membrane-delimited organelle, the nucleus. In contrast, procaryotes lack a membrane-delimited nucleus. The procaryotic chromosome is located in an irregularly shaped region called the nucleoid (other names are also used: the nuclear body, chromatin body, nuclear region). Usually procaryotes contain a single circle of double-stranded deoxyribonucleic acid (DNA), but some have a linear DNA chromosome. Recently it has been discovered that some bacteria such as Vibrio cholerae have more than one chromosome. Although nucleoid appearance varies with the method of fixation and staining, fibers often are seen in electron micrographs (figure 3.11 and figure 3.14) and are probably DNA. The nucleoid also is visible in the light microscope after staining with the Feulgen stain, which specifically reacts with DNA. A cell can have more than one nucleoid when cell division occurs after the genetic material has been duplicated (figure 3.14a). In actively growing bacteria, the nucleoid has projections that extend into the cytoplasmic matrix (figure 3.14b,c). Presumably these projections contain DNA that is being actively transcribed to produce mRNA. Careful electron microscopic studies often have shown the nucleoid in contact with either the mesosome or the plasma membrane. Membranes also are found attached to isolated nucleoids. Thus there is evidence that bacterial DNA is attached to cell membranes, and membranes may be involved in the separation of DNA into daughter cells during division. Nucleoids have been isolated intact and free from membranes. Chemical analysis reveals that they are composed of about 60% DNA, 30% RNA, and 10% protein by weight. In Escherichia coli, a rod-shaped cell about 2 to 6 m long, the closed DNA circle measures approximately 1,400 m. Obviously it must be very efficiently packaged to fit within the nucleoid. The DNA is looped and coiled extensively (see figure 11.8), probably with the aid of RNA and nucleoid proteins (these proteins differ from the histone proteins present in eucaryotic nuclei). There are a few exceptions to the above picture. Membranebound DNA-containing regions are present in two genera of planctomycetes. Pirellula has a single membrane that surrounds a region, the pirellulosome, which contains a fibrillar nucleoid and ribosome-like particles. The nuclear body of Gemmata obscuriglobus is bounded by two membranes (see figure 21.12). More work will be required to determine the functions of these membranes and how widespread this phenomenon is. The cell cycle and cell division (pp. 285–86). Procaryotic DNA and its function (chapters 11 and 12) Many bacteria possess plasmids in addition to their chromosome. These are double stranded DNA molecules, usually circular, that can exist and replicate independently of the chromosome or may be integrated with it; in either case they normally are inherited or passed on to the progeny. However, plasmids are not usually attached to the plasma membrane and sometimes are lost to one of the progeny cells during division. Plasmids are not required for host growth and reproduction, although they may carry genes that give their bacterial host a selective advantage. Plasmid genes can render bacteria drug-resistant, give them new metabolic abilities, make them pathogenic, or endow them with a number of other properties. Because plasmids often move between bacteria, properties such as drug resistance can spread throughout a population.

Living Magnets

Bacteria can respond to environmental factors other than chemicals. A fascinating example is that of the aquatic magnetotactic bacteria that orient themselves in the earth’s magnetic field. Most of these bacteria have intracellular chains of magnetite (Fe3O4) particles or magnetosomes, around 40 to 100 nm in diameter and bounded by a membrane (see Box figure). Some species from sulfidic habitats have magnetosomes containing greigite (Fe3S4) and pyrite (FeS2). Since each iron particle is a tiny magnet, the Northern Hemisphere bacteria use their magnetosome chain to determine northward and downward directions, and swim down to nutrient-rich sediments or locate the optimum depth in freshwater and marine habitats. Magnetotactic bacteria in the Southern Hemisphere generally orient southward and downward, with the same result. Magnetosomes also are present in the heads of birds, tuna, dolphins, green turtles, and other animals, presumably to aid navigation. Animals and bacteria share more in common behaviorally than previously imagined.

Bacteria & Fossil Fuels

F or many years there has been great interest in the origin of fossil fuels such as coal and petroleum. In the oceans, there is a constant “snow” of procaryotic membranes and other organic matter that settles on the bottom sediments. Fossil fuel formation begins when organic matter is buried before it can be oxidized to carbon dioxide by microorganisms. When organic matter is buried deeply and subjected to increasing temperature under anaerobic conditions, petroleum and coal are often formed. The quantities involved in these processes are enormous. It has been estimated that the earth contains about 1016 tons of carbon in its sediments. There is increasing evidence that much of the organic material in sediments is bacterial in origin. About 90% of this material is in the form of insoluble kerogen, an organic precursor of petroleum. Recently the hopanoid bacteriohopanetetrol (figure 3.6b) was isolated from kerogen, and evidence is accumulating that kerogen arises from bacterial activity. We may owe our supply of fossil fuels largely to bacteria that serve as the final degraders of the organic material in dead organisms.

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Monstrous Microbes

Biologists often have distinguished between procaryotes and eucaryotes based in part on cell size. Generally, procaryotic cellsare supposed to be smaller than eucaryotic cells. Procaryotes grow extremely rapidly compared to most eucaryotes and lack the complex vesicular transport systems of eucaryotic cells (see chapter 4). It has been assumed that they must be small because of the slowness of nutrient diffusion and the need for a large surface-to-volume ratio. Thus when Fishelson, Montgomery, and Myrberg discovered a large, cigarshaped microorganism in the intestinal tract of the Red Sea brown surgeonfish, Acanthurus nigrofuscus, they suggested in their 1985 publication that it was a protist. It seemed too large to be anything else. In 1993 Esther Angert, Kendall Clemens, and Norman Pace used rRNA sequence comparisons (see p. 432) to identify the microorganism, now called Epulopiscium fishelsoni, as a procaryote related to the gram-positive genus Clostridium. E. fishelsoni [Latin, epulum, a feast or banquet, and piscium, fish] can reach a size of 80 m by 600 m, and normally ranges from 200 to 500 m in length (see Box figure). It is about a million times larger in volume than Escherichia coli. Despite its huge size the organism does have procaryotic cell structure. It is motile and swims at about two body lengths a second (approximately 2.4 cm/min) using the bacterial-type flagella that cover its surface. The cytoplasm contains large nucleoids and many ribosomes, as would be required for such a large cell. Epulopiscium appears to overcome the size limits set by diffusion by having an outer layer consisting of a highly convoluted plasma membrane. This increases the cell’s surface area and aids in nutrient transport. It appears that Epulopiscium is transmitted between hosts through fecal contamination of the fish’s food. The bacterium can be eliminated by starving the surgeonfish for a few days. If juvenile fish that lack the bacterium are placed with infected hosts, they are reinoculated. Interestingly this does not work with uninfected adult surgeonfish. In 1997, Heidi Schulz discovered an even larger procaryote in the ocean sediment off the coast of Namibia. Thiomargarita namibiensis is a spherical bacterium, between 100 and 750 m in diameter, that often forms chains of cells. It is over 100 times larger in volume than E. fishelsoni. A vacuole occupies about 98 percent of the cell and contains fluid rich in nitrate; it is surrounded by a 0.5 to 2.0 m layer of cytoplasm filled with sulfur granules. The cytoplasmic layer is the same thickness as most bacteria and sufficiently thin for adequate diffusion rates. Nitrate is used as an electron acceptor for sulfur oxidation and energy production. The discovery of these procaryotes greatly weakens the distinction between procaryotes and eucaryotes based on cell size. They are certainly larger than a normal eucaryotic cell. In addition, some eucaryoticcells have been discovered that are smaller than previously thought possible. The best example is Nanochlorum eukaryotum. Nanochlorum is only about 1 to 2 mu.m in diameter, yet is truly eucaryotic and has a nucleus, a chloroplast, and a mitochondrion. Our understanding of the factors limiting procaryotic cell size must be reevaluated. It is no longer safe to assume that large cells are eucaryotic and small cells are procaryotic.

Microorganisms Disease Role

The importance of microorganisms in disease was not immediately obvious to people, and it took many years for scientists to establish the connection between microorganisms and illness. Recognition of the role of microorganisms depended greatly upon the development of new techniques for their study. Once it became clear that disease could be caused by microbial infections, microbiologists began to examine the way in which hosts defended themselves against microorganisms and to ask how disease might be prevented. The field of immunology was born.

Although Fracastoro and a few others had suggested that invisible organisms produced disease, most believed that disease was due to causes such as supernatural forces, poisonous vapors called miasmas, and imbalances between the four humors thought to be present in the body. The idea that an imbalance between the four humors (blood, phlegm, yellow bile [choler], and black bile [melancholy]) led to disease had been widely accepted since the time of the Greek physician Galen (129–199). Support for the germ theory of disease began to accumulate in the early nineteenth century. Agostino Bassi (1773–1856) first showed a microorganism could cause disease when he demonstrated in 1835 that a silkworm disease was due to a fungal infection. He also suggested that many diseases were due to microbial infections. In 1845 M. J. Berkeley proved that the great Potato Blight of Ireland was caused by a fungus. Following his successes with the study of fermentation, Pasteur was asked by the French government to investigate the pébrine disease of silkworms that was disrupting the silk industry. After several years of work, he showed that the disease was due to a protozoan parasite. The disease was controlled by raising caterpillars from eggs produced by healthy moths.

Indirect evidence that microorganisms were agents of human disease came from the work of the English surgeon Joseph Lister (1827–1912) on the prevention of wound infections. Lister impressed with Pasteur’s studies on the involvement of microorganisms in fermentation and putrefaction, developed a system of antiseptic surgery designed to prevent microorganisms from entering wounds. Instruments were heat sterilized, and phenol was used on surgical dressings and at times sprayed over the surgical area. The approach was remarkably successful and transformed surgery after Lister published his findings in 1867. It also provided strong indirect evidence for the role of microorganisms in disease because phenol, which killed bacteria, also prevented wound infections.

Discovery of Microorganisms

Even before microorganisms were seen, some investigators suspected their existence and responsibility for disease. Among others, the Roman philosopher Lucretius (about 98–55 B.C.)

and the physician Girolamo Fracastoro (1478–1553) suggested that disease was caused by invisible living creatures. The earliest microscopic observations appear to have been made between 1625 and 1630 on bees and weevils by the Italian Francesco Stelluti, using a microscope probably supplied by Galileo. However, the first person to observe and describe microorganisms accurately was the amateur microscopist Antony van Leeuwenhoek (1632–1723) of Delft, Holland (figure 1.1a).

Leeuwenhoek earned his living as a draper and haberdasher (a dealer in men’s clothing and accessories), but spent much of his spare time constructing simple microscopes composed of double convex glass lenses held between two silver plates His microscopes could magnify around 50 to 300 times, and he may have illuminated his liquid specimens by placing them between two pieces of glass and shining light on them at a 45° angle to the specimen plane. This would have provided a form of dark-field illumination (see chapter 2) and made bacteria clearly visible (figure 1.1c). Beginning in 1673 Leeuwenhoek sent detailed letters describing his discoveries to the Royal Society of London. It is clear from his descriptions that he saw both bacteria and protozoa.

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