Chapter 1: The Science of Microbiology

Microbiology is the study of microorganisms, a large and diverse group of microscopic organisms that exist as single cells or cell clusters; it also includes viruses, which are microscopic but not cellular. Microorganisms have a tremendous impact on all life and the physical and chemical makeup of our planet. They are responsible for cycling the chemical elements essential for life, including carbon, nitrogen, sulfur, hydrogen, and oxygen; more photosynthesis is carried out by microorganisms than by green plants. Furthermore, there are 100 million times as many bacteria in the oceans (13 × 10²⁸) as there are stars in the known universe. The rate of viral infections in the oceans is about 1 × 10²³ infections per second, and these infections remove 20–40% of all bacterial cells each day. It has been estimated that 5 × 10³⁰ microbial cells exist on earth; excluding cellulose, these cells constitute about 90% of the biomass of the entire biosphere. Humans also have an intimate relationship with microorganisms; more than 90% of the cells in our bodies are microbes. The bacteria present in the average human gut weigh about 1 kg, and a human adult will excrete his or her own weight in fecal bacteria each year. The number of genes contained within this gut flora outnumber that contained within our genome 150-fold, and even in our own genome, 8% of the DNA is derived from remnants of viral genomes.

Nowhere is biologic diversity demonstrated more dramatically than by microorganisms, creatures that are not directly visible to the unaided eye. In form and function, be it biochemical property or genetic mechanism, analysis of microorganisms takes us to the limits of biologic understanding. Thus, the need for originality—one test of the merit of a scientific hypothesis—can be fully met in microbiology. A useful hypothesis should provide a basis for generalization, and microbial diversity provides an arena in which this challenge is ever present.

Prediction, the practical outgrowth of science, is a product created by a blend of technique and theory. Biochemistry, molecular biology, and genetics provide the tools required for analysis of microorganisms. Microbiology, in turn, extends the horizons of these scientific disciplines. A biologist might describe such an exchange as mutualism, that is, one that benefits all of the contributing parties. Lichens are an example of microbial mutualism. Lichens consist of a fungus and phototropic partner, either an alga (a eukaryote) or a cyanobacterium (a prokaryote) (Figure 1-1). The phototropic component is the primary producer, and the fungus provides the phototroph with an anchor and protection from the elements. In biology, mutualism is called symbiosis, a continuing association of different organisms. If the exchange operates primarily to the benefit of one party, the association is described as parasitism, a relationship in which a host provides the primary benefit to the parasite. Isolation and characterization of a parasite—such as a pathogenic bacterium or virus—often require effective mimicry in the laboratory of the growth environment provided by host cells. This demand sometimes represents a major challenge to investigators.

The terms mutualism, symbiosis, and parasitism relate to the science of ecology, and the principles of environmental biology are implicit in microbiology. Microorganisms are the products of evolution, the biologic consequence of natural selection operating on a vast array of genetically diverse organisms. It is useful to keep the complexity of natural history in mind before generalizing about microorganisms, the most heterogeneous subset of all living creatures.

A major biologic division separates the eukaryotes, organisms containing a membrane-bound nucleus, from prokaryotes, organisms in which DNA is not physically separated from the cytoplasm. As described in this chapter and in Chapter 2, further major distinctions can be made between eukaryotes and prokaryotes. Eukaryotes, for example, are distinguished by their relatively large size and by the presence of specialized membrane-bound organelles such as mitochondria.

As described more fully later in this chapter, eukaryotic microorganisms—or, phylogenetically speaking, the Eukarya—are unified by their distinct cell structure and phylogenetic history. Among the groups of eukaryotic microorganisms are the algae, the protozoa, the fungi, and the slime molds.

The unique properties of viruses set them apart from living creatures. Viruses lack many of the attributes of cells, including the ability to replicate. Only when it infects a cell does a virus acquire the key attribute of a living system—reproduction. Viruses are known to infect all cells, including microbial cells. Recently, viruses called virophages have been discovered that infect other viruses. Host–virus interactions tend to be highly specific, and the biologic range of viruses mirrors the diversity of potential host cells. Further diversity of viruses is exhibited by their broad array of strategies for replication and survival.

Viral particles are generally small (eg, adenovirus is 90 nm) and consist of a nucleic acid molecule, either DNA or RNA, enclosed in a protein coat, or capsid (sometimes itself enclosed by an envelope of lipids, proteins, and carbohydrates). Proteins—frequently glycoproteins—in the capsid determine the specificity of interaction of a virus with its host cell. The capsid protects the nucleic acid and facilitates attachment and penetration of the host cell by the virus. Inside the cell, viral nucleic acid redirects the host’s enzymatic machinery to functions associated with replication of the virus. In some cases, genetic information from the virus can be incorporated as DNA into a host chromosome. In other instances, the viral genetic information can serve as a basis for cellular manufacture and release of copies of the virus. This process calls for replication of the viral nucleic acid and production of specific viral proteins. Maturation consists of assembling newly synthesized nucleic acid and protein subunits into mature viral particles, which are then liberated into the extracellular environment. Some very small viruses require the assistance of another virus in the host cell for their duplication. The delta agent, also known as hepatitis D virus, is too small to code for even a single capsid protein and needs help from hepatitis B virus for transmission. Viruses are known to infect a wide variety of plant and animal hosts as well as protists, fungi, and bacteria. However, most viruses are able to infect specific types of cells of only one host species.

Some viruses are large and complex. For example, Mimivirus, a DNA virus infecting Acanthamoeba, a free-living soil ameba, has a diameter of 400–500 nm and a genome that encodes 979 proteins, including the first four aminoacyl tRNA synthetases ever found outside of cellular organisms and enzymes for polysaccharide biosynthesis. An even larger marine virus has recently been discovered (Megavirus); its genome (1,259,197-bp) encodes 1120 putative proteins and is larger than that of some bacteria (see Table 7-1). Because of their large size, these viruses resemble bacteria when observed in stained preparations by light microscopy; however, they do not undergo cell division or contain ribosomes.

A number of transmissible plant diseases are caused by viroids—small, single-stranded, covalently closed circular RNA molecules existing as highly base-paired rodlike structures. They range in size from 246 to 375 nucleotides in length. The extracellular form of the viroid is naked RNA—there is no capsid of any kind. The RNA molecule contains no protein-encoding genes, and the viroid is therefore totally dependent on host functions for its replication. Viroid RNA is replicated by the DNA-dependent RNA polymerase of the plant host; preemption of this enzyme may contribute to viroid pathogenicity.

The RNAs of viroids have been shown to contain inverted repeated base sequences at their 3′ and 5′ ends, a characteristic of transposable elements (see Chapter 7) and retroviruses. Thus, it is likely that they have evolved from transposable elements or retroviruses by the deletion of internal sequences.

The general properties of animal viruses pathogenic for humans are described in Chapter 29. Bacterial viruses are described in Chapter 7.

A number of remarkable discoveries in the past three decades have led to the molecular and genetic characterization of the transmissible agent causing scrapie, a degenerative central nervous system disease of sheep. Studies have identified a scrapie-specific protein in preparations from scrapie-infected brains of sheep that is capable of reproducing the symptoms of scrapie in previously uninfected sheep (Figure 1-2). Attempts to identify additional components, such as nucleic acid, have been unsuccessful. To distinguish this agent from viruses and viroids, the term prion was introduced to emphasize its proteinaceous and infectious nature. The cellular form of the prion protein (PrP^c) is encoded by the host’s chromosomal DNA. PrP^c is a sialoglycoprotein with a molecular mass of 33,000–35,000 Da and a high content of α-helical secondary structure that is sensitive to proteases and soluble in detergent. PrP^c is expressed on the surface of neurons via a glycosylphosphatidyl inositol anchor in both infected and uninfected brains. A conformational change occurs in the prion protein, changing it from its normal or cellular form PrP^c to the disease-causing conformation, PrP^Sc (Figure 1-3). When PrP^Sc is present in an individual (owing to spontaneous conformational conversion or to infection), it is capable of recruiting PrP^c and converting it to the disease form. Thus, prions replicate using the PrP^c substrate that is present in the host.

There are additional prion diseases of importance (Table 1-1 and see Chapter 42). Kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease, and fatal familial insomnia affect humans. Bovine spongiform encephalopathy, which is thought to result from the ingestion of feeds and bone meal prepared from rendered sheep offal, has been responsible for the deaths of more than 184,000 cattle in Great Britain since its discovery in 1985. A new variant of CJD (vCJD) has been associated with human ingestion of prion-infected beef in the United Kingdom and France. A common feature of all of these diseases is the conversion of a host-encoded sialoglycoprotein to a protease-resistant form as a consequence of infection.

Human prion diseases are unique in that they manifest as sporadic, genetic, and infectious diseases. The study of prion biology is an important emerging area of biomedical investigation, and much remains to be learned.

The distinguishing features of the nonliving members of the microbial world are given in Table 1-2.

The primary distinguishing characteristics of the prokaryotes are their relatively small size, usually on the order of 1 μm in diameter, and the absence of a nuclear membrane. The DNA of almost all bacteria is a circle with a length of about 1 mm; this is the prokaryotic chromosome. Most prokaryotes have only a single chromosome. The chromosomal DNA must be folded more than 1000-fold just to fit within the prokaryotic cell membrane. Substantial evidence suggests that the folding may be orderly and may bring specified regions of the DNA into proximity. The specialized region of the cell containing DNA is termed the nucleoid and can be visualized by electron microscopy as well as by light microscopy after treatment of the cell to make the nucleoid visible. Thus, it would be a mistake to conclude that subcellular differentiation, clearly demarcated by membranes in eukaryotes, is lacking in prokaryotes. Indeed, some prokaryotes form membrane-bound subcellular structures with specialized function such as the chromatophores of photosynthetic bacteria (see Chapter 2).

Prokaryotic Diversity

The small size of the prokaryotic chromosome limits the amount of genetic information it can contain. Recent data based on genome sequencing indicate that the number of genes within a prokaryote may vary from 468 in Mycoplasma genitalium to 7825 in Streptomyces coelicolor, and many of these genes must be dedicated to essential functions such as energy generation, macromolecular synthesis, and cellular replication. Any one prokaryote carries relatively few genes that allow physiologic accommodation of the organism to its environment. The range of potential prokaryotic environments is unimaginably broad, and it follows that the prokaryotic group encompasses a heterogeneous range of specialists, each adapted to a rather narrowly circumscribed niche.

The range of prokaryotic niches is illustrated by consideration of strategies used for generation of metabolic energy. Light from the sun is the chief source of energy for life. Some prokaryotes such as the purple bacteria convert light energy to metabolic energy in the absence of oxygen production. Other prokaryotes, exemplified by the blue-green bacteria (Cyanobacteria), produce oxygen that can provide energy through respiration in the absence of light. Aerobic organisms depend on respiration with oxygen for their energy. Some anaerobic organisms can use electron acceptors other than oxygen in respiration. Many anaerobes carry out fermentations in which energy is derived by metabolic rearrangement of chemical growth substrates. The tremendous chemical range of potential growth substrates for aerobic or anaerobic growth is mirrored in the diversity of prokaryotes that have adapted to their utilization.

Prokaryotic Communities

A useful survival strategy for specialists is to enter into consortia, arrangements in which the physiologic characteristics of different organisms contribute to survival of the group as a whole. If the organisms within a physically interconnected community are directly derived from a single cell, the community is a clone that may contain up to 10⁸ cells. The biology of such a community differs substantially from that of a single cell. For example, the high cell number virtually ensures the presence within the clone of at least one cell carrying a variant of any gene on the chromosome. Thus, genetic variability—the wellspring of the evolutionary process called natural selection—is ensured within a clone. The high number of cells within clones also is likely to provide physiologic protection to at least some members of the group. Extracellular polysaccharides, for example, may afford protection against potentially lethal agents such as antibiotics or heavy metal ions. Large amounts of polysaccharides produced by the high number of cells within a clone may allow cells within the interior to survive exposure to a lethal agent at a concentration that might kill single cells.

Many bacteria exploit a cell–cell communication mechanism called quorum sensing to regulate the transcription of genes involved in diverse physiologic processes, including bioluminescence, plasmid conjugal transfer, and the production of virulence determinants. Quorum sensing depends on the production of one or more diffusible signal molecules (eg, acetylated homoserine lactone [AHL]) termed autoinducers or pheromones that enable a bacterium to monitor its own cell population density (Figure 1-4). The cooperative activities leading to biofilm formation are controlled by quorum sensing. It is an example of multicellular behavior in prokaryotes.

A distinguishing characteristic of prokaryotes is their capacity to exchange small packets of genetic information. This information may be carried on plasmids, small and specialized genetic elements that are capable of replication within at least one prokaryotic cell line. In some cases, plasmids may be transferred from one cell to another and thus may carry sets of specialized genetic information through a population. Some plasmids exhibit a broad host range that allows them to convey sets of genes to diverse organisms. Of particular concern are drug resistance plasmids that may render diverse bacteria resistant to antibiotic treatment.

The survival strategy of a single prokaryotic cell line may lead to a range of interactions with other organisms. These may include symbiotic relationships illustrated by complex nutritional exchanges among organisms within the human gut. These exchanges benefit both the microorganisms and their human host. Parasitic interactions can be quite deleterious to the host. Advanced symbiosis or parasitism can lead to loss of functions that may not allow growth of the symbiont or parasite independent of its host.

The mycoplasmas, for example, are parasitic prokaryotes that have lost the ability to form a cell wall. Adaptation of these organisms to their parasitic environment has resulted in incorporation of a substantial quantity of cholesterol into their cell membranes. Cholesterol, not found in other prokaryotes, is assimilated from the metabolic environment provided by the host. Loss of function is exemplified also by obligate intracellular parasites, the chlamydiae and rickettsiae. These bacteria are extremely small (0.2–0.5 μm in diameter) and depend on the host cell for many essential metabolites and coenzymes. This loss of function is reflected by the presence of a smaller genome with fewer genes (see Table 7-1).

The most widely distributed examples of bacterial symbionts appear to be chloroplasts and mitochondria, the energy-yielding organelles of eukaryotes. A substantial body of evidence points to the conclusion that ancestors of these organelles were endosymbionts, prokaryotes that established symbiosis within the cell membrane of the ancestral eukaryotic host. The presence of multiple copies of the organelles may have contributed to the relatively large size of eukaryotic cells and to their capacity for specialization, a trait ultimately reflected in the evolution of differentiated multicellular organisms.

Classification of the Prokaryotes

An understanding of any group of organisms requires their classification. An appropriate classification system allows a scientist to choose characteristics that allow swift and accurate categorization of a newly encountered organism. The categorization allows prediction of many additional traits shared by other members of the category. In a hospital setting, successful classification of a pathogenic organism may provide the most direct route to its elimination. Classification may also provide a broad understanding of relationships among different organisms, and such information may have great practical value. For example, elimination of a pathogenic organism will be relatively long-lasting if its habitat is occupied by a nonpathogenic variant.

The principles of prokaryotic classification are discussed in Chapter 3. At the outset, it should be recognized that any prokaryotic characteristic might serve as a potential criterion for classification. However, not all criteria are equally effective in grouping organisms. Possession of DNA, for example, is a useless criterion for distinguishing organisms because all cells contain DNA. The presence of a broad host range plasmid is not a useful criterion because such plasmids may be found in diverse hosts and need not be present all of the time. Useful criteria may be structural, physiologic, biochemical, or genetic. Spores—specialized cell structures that may allow survival in extreme environments—are useful structural criteria for classification because well-characterized subsets of bacteria form spores. Some bacterial groups can be effectively subdivided on the basis of their ability to ferment specified carbohydrates. Such criteria may be ineffective when applied to other bacterial groups that may lack any fermentative capability. A biochemical test, the Gram stain, is an effective criterion for classification because response to the stain reflects fundamental and complex differences in the bacterial cell surface that divide most bacteria into two major groups.

Genetic criteria are increasingly used in bacterial classification, and many of these advances are made possible by the development of DNA-based technologies. It is now possible to design DNA probe or DNA amplification assays (eg, polymerase chain reaction [PCR] assays) that swiftly identify organisms carrying specified genetic regions with common ancestry. Comparison of DNA sequences for some genes led to the elucidation of phylogenetic relationships among prokaryotes. Ancestral cell lines can be traced, and organisms can be grouped on the basis of their evolutionary affinities. These investigations have led to some striking conclusions. For example, comparison of cytochrome c sequences suggests that all eukaryotes, including humans, arose from one of three different groups of purple photosynthetic bacteria. This conclusion in part explains the evolutionary origin of eukaryotes, but it does not fully take into account the generally accepted view that the eukaryotic cell was derived from the evolutionary merger of different prokaryotic cell lines.

Bacteria and Archaebacteria: The Major Subdivisions Within the Prokaryotes

A major success in molecular phylogeny has been the demonstration that prokaryotes fall into two major groups. Most investigations have been directed to one group, the bacteria. The other group, the archaebacteria, has received relatively little attention until recently, partly because many of its representatives are difficult to study in the laboratory. Some archaebacteria, for example, are killed by contact with oxygen, and others grow at temperatures exceeding that of boiling water. Before molecular evidence became available, the major subgroupings of archaebacteria had seemed disparate. The methanogens carry out an anaerobic respiration that gives rise to methane, the halophiles demand extremely high salt concentrations for growth, and the thermoacidophiles require high temperature and acidity. It has now been established that these prokaryotes share biochemical traits such as cell wall or membrane components that set the group entirely apart from all other living organisms. An intriguing trait shared by archaebacteria and eukaryotes is the presence of introns within genes. The function of introns—segments of DNA that interrupts informational DNA within genes—is not established. What is known is that introns represent a fundamental characteristic shared by the DNA of archaebacteria and eukaryotes. This common trait has led to the suggestion that—just as mitochondria and chloroplasts appear to be evolutionary derivatives of the bacteria—the eukaryotic nucleus may have arisen from an archaebacterial ancestor.

The “true nucleus” of eukaryotes (from Gr karyon, “nucleus”) is only one of their distinguishing features. The membrane-bound organelles, the microtubules, and the microfilaments of eukaryotes form a complex intracellular structure unlike that found in prokaryotes. The agents of motility for eukaryotic cells are flagella or cilia—complex multistranded structures that do not resemble the flagella of prokaryotes. Gene expression in eukaryotes takes place through a series of events achieving physiologic integration of the nucleus with the endoplasmic reticulum, a structure that has no counterpart in prokaryotes. Eukaryotes are set apart by the organization of their cellular DNA in chromosomes separated by a distinctive mitotic apparatus during cell division.

In general, genetic transfer among eukaryotes depends on fusion of haploid gametes to form a diploid cell containing a full set of genes derived from each gamete. The life cycle of many eukaryotes is almost entirely in the diploid state, a form not encountered in prokaryotes. Fusion of gametes to form reproductive progeny is a highly specific event and establishes the basis for eukaryotic species. This term can be applied only metaphorically to the prokaryotes, which exchange fragments of DNA through recombination. Taxonomic groupings of eukaryotes frequently are based on shared morphologic properties, and it is noteworthy that many taxonomically useful determinants are those associated with reproduction. Almost all successful eukaryotic species are those in which closely related cells, members of the same species, can recombine to form viable offspring. Structures that contribute directly or indirectly to the reproductive event tend to be highly developed and—with minor modifications among closely related species—extensively conserved.

Microbial eukaryotes—protists—are members of the four following major groups: algae, protozoa, fungi, and slime molds. It should be noted that these groupings are not necessarily phylogenetic: Closely related organisms may have been categorized separately because underlying biochemical and genetic similarities may not have been recognized.

Algae

The term algae has long been used to denote all organisms that produce O₂ as a product of photosynthesis. One major subgroup of these organisms—the blue-green bacteria, or cyanobacteria—are prokaryotic and no longer are termed algae. This classification is reserved exclusively for photosynthetic eukaryotic organisms. All algae contain chlorophyll in the photosynthetic membrane of their subcellular chloroplast. Many algal species are unicellular microorganisms. Other algae may form extremely large multicellular structures. Kelps of brown algae sometimes are several hundred meters in length. A number of algae produce toxins that are poisonous to humans and other animals. Dinoflagellates, a unicellular alga, cause algal blooms, or red tides, in the ocean (Figure 1-5). Red tides caused by the dinoflagellate Gonyaulax species are serious because this organism produces neurotoxins such as saxitoxin and gonyautoxins, which accumulate in shellfish (eg, clams, mussels, scallops, oysters) that feed on this organism. Ingestion of these shellfish by humans results in symptoms of paralytic shellfish poisoning and can lead to death.

Protozoa

Protozoa are unicellular nonphotosynthetic protists. The most primitive protozoa appear to be flagellated forms that in many respects resemble representatives of the algae. It seems likely that the ancestors of these protozoa were algae that became heterotrophs—the nutritional requirements of such organisms are met by organic compounds. Adaptation to a heterotrophic mode of life was sometimes accompanied by loss of chloroplasts, and algae thus gave rise to the closely related protozoa. Similar events have been observed in the laboratory to be the result of either mutation or physiologic adaptation.

From flagellated protozoa appear to have evolved the ameboid and the ciliated types; intermediate forms are known that have flagella at one stage in the life cycle and pseudopodia (characteristic of the ameba) at another stage. A fourth major group of protozoa, the sporozoa, are strict parasites that are usually immobile; most of these reproduce sexually and asexually in alternate generations by means of spores. Protozoan parasites of humans are discussed in Chapter 46.

Fungi

The fungi are nonphotosynthetic protists growing as a mass of branching, interlacing filaments (“hyphae”) known as a mycelium. The largest known contiguous fungal mycelium covered an area of 2400 acres (9.7 km²) at a site in eastern Oregon. Although the hyphae exhibit cross walls, the cross walls are perforated and allow free passage of nuclei and cytoplasm. The entire organism is thus a coenocyte (a multinucleated mass of continuous cytoplasm) confined within a series of branching tubes. These tubes, made of polysaccharides such as chitin, are homologous with cell walls. The mycelial forms are called molds; a few types, yeasts, do not form a mycelium but are easily recognized as fungi by the nature of their sexual reproductive processes and by the presence of transitional forms.

The fungi probably represent an evolutionary offshoot of the protozoa; they are unrelated to the actinomycetes, mycelial bacteria that they superficially resemble. The major subdivisions (phyla) of fungi are Chytridiomycota, Zygomycota (the zygomycetes), Ascomycota (the ascomycetes), Basidiomycota (the basidiomycetes), and the “deuteromycetes” (or imperfect fungi).

The evolution of the ascomycetes from the phycomycetes is seen in a transitional group, whose members form a zygote but then transform this directly into an ascus. The basidiomycetes are believed to have evolved in turn from the ascomycetes. The classification of fungi and their medical significance are discussed further in Chapter 45.

Slime Molds

These organisms are characterized by the presence, as a stage in their life cycle, of an ameboid multinucleate mass of cytoplasm called a plasmodium. The plasmodium of a slime mold is analogous to the mycelium of a true fungus. Both are coenocytic. Whereas in the latter, cytoplasmic flow is confined to the branching network of chitinous tubes, in the former, the cytoplasm can flow in all directions. This flow causes the plasmodium to migrate in the direction of its food source, frequently bacteria. In response to a chemical signal, 3′, 5′-cyclic AMP (see Chapter 7), the plasmodium, which reaches macroscopic size, differentiates into a stalked body that can produce individual motile cells. These cells, flagellated or ameboid, initiate a new round in the life cycle of the slime mold (Figure 1-6). The cycle frequently is initiated by sexual fusion of single cells.

The life cycle of the slime molds illustrates a central theme of this chapter—the interdependency of living forms. The growth of slime molds depends on nutrients provided by bacterial or, in some cases, plant cells. Reproduction of the slime molds via plasmodia can depend on intercellular recognition and fusion of cells from the same species. Full understanding of a microorganism requires both knowledge of the other organisms with which it coevolved and an appreciation of the range of physiologic responses that may contribute to survival.

• Microorganisms are a large and diverse group of microorganisms existing as single cells or clusters; they also include viruses, which are microscopic but not cellular.

• A virus consists of a nucleic acid molecule, either DNA or RNA, enclosed in a protein coat, or capsid, sometimes enclosed by an envelope composed of lipids, proteins, and carbohydrates.

• A prion is an infectious protein, which is capable of causing chronic neurologic diseases.

• Prokaryotes consist of bacteria and archaebacteria.

• Prokaryotes are haploid.

• Microbial eukaryotes, or protists, are members of four major groups: algae, protozoa, fungi, and slime molds.

• Eukaryotes have a true nucleus and are diploid.