IDENTIFICATION OF BACTERIA
Classic MethodsThe criteria used for microscopic identification of procaryotes include cell shape and grouping, Gram-stain reaction, and motility. Bacterial cells almost invariably take one of three forms: rod (bacillus), sphere (coccus), or spiral (spirilla and spirochetes). Rods that are curved are called vibrios. Fixed bacterial cells stain either Gram-positive (purple) or Gram-negative (pink); motility is easily determined by observing living specimens. Bacilli may occur singly or form chains of cells; cocci may form chains (streptococci) or grape-like clusters (staphylococci); spiral shape cells are almost always motile; cocci are almost never motile. This nomenclature ignores the actinomycetes, a prominent group of branched bacteria which occur in the soil. But they are easily recognized by their colonies and their microscopic appearance.
Figure 10. Gram stain of Bacillus anthracis, the cause of anthrax. K. Todar.
Such easily-made microscopic observations, combined with knowing the natural environment of the organism, are important aids to identify the group, if not the exact genus, of a bacterium - providing, of course, that one has an effective key. Such a key is Bergey's Manual of Determinative Bacteriology 9th ed, the "field guide" to identification of the bacteria. Bergey's Manual describes affiliated groups of Bacteria and Archaea based on a few easily observed microscopic and physiologic characteristics. Further identification requires biochemical tests which will distinguish genera among families and species among genera. Strains within a single species are usually distinguished by genetic or immunological criteria.
A modification of the Bergey's criteria for bacterial identification, without a key, is used to organize the groups of procaryotes for discussion in a companion chapter Important Groups of Procaryotes
Figure 11. Size and fundamental shapes of procaryotes revealed by three genera of Bacteria (l to r): Staphylococcus (spheres), Lactobacillus (rods), and Aquaspirillum (spirals).
Figure 12. Chains of dividing streptococci. Electron micrograph of Streptococcus pyogenes by Maria Fazio and Vincent A. Fischetti, Ph.D. with permission. The Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University.
Molecular Techniques
The sciences of genomics and bioinformatics have led to a radical reclassification of procaryotes based on comparative analysis of organismal DNA. Genomics involves the study of all of the nucleotide sequences, including structural genes, regulatory sequences, and noncoding DNA segments, in the chromosomes of an organism. To date over 200 bacterial genomes have been sequenced. We have seen how highly conserved genetic sequences, such as those that encode for the small subunit ribosomal RNAs (16S rRNA) of an organism, can be analyzed to specifically relate two organisms. So can the identification of certain genes provide information about specific properties of an organism, and analysis of specific nucleotide sequences may be used to indicate identity and degrees of genetic relatedness among organisms.
The newest editions of Bergey's Manual are adapted to the new phylogenetic classification. This has resulted in the formation of several new taxa of bacteria and archaea at every hierarchical level. Occasionally, organisms thought to be more or less distantly related become unified; but more likely, organisms thought to be closely-related due to similar phenotypic properties are found to be genetically distinct and warrant separation into a new taxa.
Metagenomics. Sequencing of 16S rRNA genes obtained from environmental samples produces a broad profile of microbial diversity and reveals that the vast majority of microbes present have been missed by reliance on cultivation-based methods. This observation has given rise to the field of metagenomics. Metagenomics (also called environmental genomics) is the application of modern genomics techniques to the study of communities of microorganisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species. Metagenomics provides a means to identify and quantify microbes from environmental samples based on the presence of distinctive genes. This enables studies of organisms that are not easily cultured in the laboratory, as well as studies of organisms in their natural environment.
"Shotgun" metagenomics is capable of sequencing nearly complete microbial genomes directly from the environment. Because the collection of DNA from an environment is largely uncontrolled, the most abundant organisms in a sample are most highly represented in the resulting sequence data. To achieve the high coverage needed to fully resolve the genomes of underrepresented community members, large samples, often prohibitively so, may be needed. However, the random nature of shotgun sequencing ensures that many of these organisms will be represented by at least some small sequence segments. Due to the limitations of microbial isolation methods, the majority of these organisms would go unnoticed using traditional culturing techniques.
Shotgun sequencing and screens of clone libraries reveal genes present in environmental samples. This provides information both on which organisms are present and what metabolic processes are possible in the community. This can be helpful in understanding the ecology of a community, particularly if multiple samples are compared to one other.
BACTERIAL REPRODUCTION AND GENETICS
Most bacteria reproduce by a relatively simple asexual process called binary fission: each cell increases in size and divides into two cells. During this process there is an orderly increase in cellular structures and components, replication and segregation of the bacterial DNA, followed by formation of a septum or cross wall which divides the cell into two. The process is evidently coordinated by activities associated with the cell membrane. The DNA molecule is believed to be attached to a point on the membrane where it is replicated. The two DNA molecules remain attached at points side-by-side on the membrane while new membrane material is synthesized between the two points. This draws the DNA molecules in opposite directions while new cell wall and membrane are laid down as a septum between the two chromosomal compartments. When septum formation is complete the cell splits into two progeny cells. The time interval required for a bacterial cell to divide or for a population of cells to double is called the generation time. Generation times for bacterial species growing in nature may be as short as 15 minutes or as long as several days.
Figure 13. A pair of dividing streptococci. The chromosome has been replicated and is partially segregated as septum formation is beginning. Electron micrograph of Streptococcus pyogenes by Maria Fazio and Vincent A. Fischetti, Ph.D. with permission. The Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University.
Genetic Exchange in Bacteria
Although procaryotes do not undergo sexual reproduction, they are not without the ability to exchange genes and undergo genetic recombination. Bacteria are known to exchange genes in nature by three fundamental processes: conjugation, transduction and transformation. Conjugation requires cell-to-cell contact for DNA to be transferred from a donor to a recipient. During transduction, a virus transfers the genes between mating bacteria. In transformation, DNA is acquired directly from the environment, having been released from another cell. Genetic recombination can follow the transfer of DNA from one cell to another leading to the emergence of a new genotype (recombinant). It is common for DNA to be transferred as plasmids between mating bacteria. Since bacteria usually develop their genes for drug resistance on plasmids (called resistance transfer factors, or RTFs), they are able to spread drug resistance to other strains and species during genetic exchange processes. The genetic engineering of bacterial cells in the research or biotechnology laboratory is often based on the use of plasmids as vectors. The genetic systems of the Archaea are poorly characterized at this point, although the entire genome of Methanosarcina has been sequenced which opens up the possibilities for genetic analysis of the group.Evolution of Bacteria and Archaea
For most procaryotes, mutation is is a major source of variability that allows the species to adapt to new conditions. The mutation rate for most procaryotic genes is in the neighborhood of 10-8. This means that if a bacterial population doubles from 108 cells to 2 x 108 cells, there is likely to be a mutant present for any given gene. Since procaryotes grow to reach population densities far in excess of 109 cells, such a mutant could develop from a single generation during 15 minutes of growth. The evolution of procaryotes, driven by such Darwinian principles of evolution (mutation and selection) is called vertical evolution.However, as a result of the processes of genetic exchange described above, the bacteria and archaea can also undergo a process of horizontal evolution, also called horizontal gene transfer (HGT). In this case, genes are transferred laterally from one organism to another, including between members of different Kingdoms, which allows the recipient to experiment with a new genetic trait. Horizontal gene transfer is becoming realized to be a significant force in driving cellular evolution.
The combined effects of fast growth rates, high concentrations of cells, genetic processes of mutation and selection, and the ability to exchange genes, account for the extraordinary rates of adaptation and evolution that can be observed in the procaryotes.
ECOLOGY OF BACTERIA AND ARCHAEA
Bacteria and Archaea are present in all environments that support life. They may be free-living, or living in associations with "higher forms" of life (plants and animals), and they are found in environments that support no other form of life. Procaryotes have the usual nutritional requirements for growth of cells, but many of the ways that they utilize and transform their nutrients are unique. This bears directly on their habitat and their ecology.Nutritional Types of Organisms
In terms of carbon utilization a cell may be heterotrophic or autotrophic. Heterotrophs obtain their carbon and energy for growth from organic compounds in nature. Autotrophs use C02 as a sole source of carbon for growth and obtain their energy from light (e.g. photoautotrophs) or from the oxidation of inorganic compounds (e.g. lithoautotrophs).
Most heterotrophic bacteria are saprophytes, meaning that they obtain their nourishment from dead organic matter. In the soil, saprophytic bacteria and fungi are responsible for biodegradation of organic material. Ultimately, organic molecules, no matter how complex, can be degraded to CO2 (plus H2 and H2O). Probably no naturally-occurring organic substance cannot be degraded by the combined activities of the bacteria and fungi. Hence, most organic matter in nature is converted by heterotrophs to CO2, only to be converted back into organic material by autotrophs that die and nourish heterotrophs to complete the carbon cycle.
Lithotrophic procaryotes have a type of energy-producing metabolism which is unique. Lithotrophs (also called lithoautotrophs or chemoautotrophs) use inorganic compounds as sources of energy, i.e., they oxidize compounds such as H2 or H2S or NH3 to obtain electrons to feed in to an electron transport system and to produce ATP. Lithotrophs are found in soil and aquatic environments wherever their energy source is present. Most lithotrophs are autotrophs so they can grow in the absence of any organic material. Lithotrophic species are found among the Bacteria and the Archaea. Sulfur-oxidizing lithotrophs convert H2S to So and So to SO4. Nitrifying bacteria convert NH3 to NO2 and NO2 to NO3; methanogenic archaea strip electrons off of H2 as a source of energy and add them to CO2 to form CH4 (methane). Lithotrophs have an obvious impact on the sulfur, nitrogen and carbon cycles in the biosphere.
Photosynthetic bacteria convert light energy into chemical energy for growth. Most phototrophic bacteria are autotrophs so their role in the carbon cycle is analogous to that of plants. The planktonic cyanobacteria are the "grass of the sea" and their form of oxygenic photosynthesis generates a substantial amount of O2 in the biosphere. However, among the photosynthetic bacteria are types of photosynthetic metabolism not seen in eucaryotes, including photoheterotrophy (using light as an energy source while assimilating organic compounds as a source of carbon), anoxygenic photosynthesis, and unique mechanisms of CO2 fixation (autotrophy).
Photosynthesis has not been found to occur among the Archaea, but one archaeal species employs a light-driven non photosynthetic means of energy generation based on the use of a chromophore called bacteriorhodopsin.
Adaptations to Environmental Conditions
Most procaryotes, whether they have been cultured and studied in the laboratory, or observed growing in their natural habitats, seem to be highly adapted to their specific environment by means of their macromolecular structure and/or their physiologic (metabolic) capabilities. The nutritional quality of the environment determines whether a particular organism will be present, but so do various physical parameters such as the availability of light and O2, as well as the pH, temperature and salinity of the environment. As examples, the range of procaryotic responses to oxygen and temperature are discussed below.
Procaryotes vary widely in their response to O2 (molecular oxygen). Organisms that require O2 for growth are called obligate aerobes; those which are inhibited or killed by O2, and which grow only in its absence, are called obligate anaerobes; organisms which grow either in the presence or absence of O2 are called facultative anaerobes. Whether or not a particular organism can exist in the presence of O2 depends upon the distribution of certain enzymes such as superoxide dismutase and catalase that are required to detoxify lethal oxygen radicals that are always generated by living systems in the presence of O2
Procaryotes also vary widely in their response to temperature. Those that live at very cold temperatures (0 degrees or lower) are called psychrophiles; those which flourish at room temperature (25 degrees) or at the temperature of warm-blooded animals (37 degrees) are called mesophiles; those that live at high temperatures (greater than 45 degrees) are thermophiles. The only limit that seems to be placed on growth of certain procaryotes in nature relative to temperature is whether liquid water exists. Hence, growing procaryotic cells can be found in supercooled environments (ice does not form) as low as -20 degrees and superheated environments (steam does not form) as high as 120 degrees. Archaea have been detected around thermal vents on the ocean floor where the temperature is as high as 320 degrees!
Symbiosis
The biomass of procaryotic cells in the biosphere, their metabolic diversity, and their persistence in all habitats that support life, ensures that these microbe will play a crucial role in the cycles of elements and the functioning of the world ecosystem. However, the procaryotes affect the world ecology in another significant way through their inevitable interactions with insects, plants and animals. Some bacteria are required to associate with insects, animals or plants for the latter to survive. For example, the sex of offspring of certain insects is determined by endosymbiotic bacteria. Ruminant animals (cows, sheep, etc.), whose diet is mainly cellulose (plant material), must have cellulose-digesting bacteria in their intestine to convert the cellulose to a form of carbon that the animal can assimilate. Leguminous plants grow poorly in nitrogen-deprived soils unless they are colonized by nitrogen-fixing bacteria which can supply them with a biologically-useful form of nitrogen.
Bacterial Pathogenicity
Some bacteria are parasites of plants or animals, meaning that they grow at the expense of their eucaryotic host and may damage, harm, or even kill it in the process. Such bacteria that cause disease in plants or animals are pathogens. Human diseases caused by bacterial pathogens include tuberculosis, whooping cough, diphtheria, tetanus, gonorrhea, syphilis, pneumonia, cholera and typhoid fever, to name a few. The bacteria that cause these diseases have special structural or biochemical properties that determine their virulence or pathogenicity. These include: (1) ability to colonize and invade their host; (2) ability to resist or withstand the antibacterial defenses of the host; (3) ability to produce various toxic substances that damage the host. Plant diseases, likewise, may be caused by bacterial pathogens. More than 200 species of bacteria are associated with plant diseases, but a very small handful of genera are involved.
Figure 14. Borrelia burgdorferi. This spirochete is the bacterial parasite that causes Lyme disease. CDC.
APPLICATIONS OF BACTERIA IN INDUSTRY AND BIOTECHNOLOGY
Exploitation of Bacteria by HumansBacteria are used in industry in a number of ways that generally exploit their natural metabolic capabilities. They are used in manufacture of foods and production of antibiotics, probiotics, drugs, vaccines, starter cultures, insecticides, enzymes, fuels and solvents. In addition, with genetic engineering technology, bacteria can be programmed to make various substances used in food science, agriculture and medicine. The genetic systems of bacteria are the foundation of the biotechnology industry discussed below.
In the foods industry, lactic acid bacteria such as Lactobacillus, Lactococcus and Streptococcus are used in the manufacture of dairy products such as cheeses, including cottage cheese and cream cheese, cultured butter, sour cream, buttermilk, yogurt and kefir. Lactic acid bacteria and acetic acid bacteria are used in pickling processes such as olives, cucumber pickles and sauerkraut. Bacterial fermentations are used in processing of teas, coffee, cocoa, soy sauce, sausages and an amazing variety of foods in our everyday lives.
In the pharmaceutical industry, bacteria are used to produce antibiotics, vaccines, and medically-useful enzymes. Most antibiotics are made by bacteria that live in soil. Actinomycetes such as Streptomyces produce tetracyclines, erythromycin, streptomycin, rifamycin and ivermectin. Bacillus and Paenibacillus species produce bacitracin and polymyxin. Bacterial products are used in the manufacture of vaccines for immunization against infectious disease. Vaccines against diphtheria, whooping cough, tetanus, typhoid fever and cholera are made from components of the bacteria that cause the respective diseases. It is significant to note here that the use of antibiotics against infectious disease and the widespread practice of vaccination (immunization) against infectious disease are two twentieth-century developments that have drastically increased the quality of life and the average life expectancy of individuals in developed countries.
Biotechnology
The biotechnology industry uses bacterial cells for the production of biological substances that are useful to human existence, including fuels, foods, medicines, hormones, enzymes, proteins, and nucleic acids. The possibilities of biotechnology are endless considering the gene reservoirs and genetic capabilities within the bacteria. Pasteur said it best, "Never underestimate the power of the microbe."
Biotechnology has produced human hormones such as insulin, enzymes such as streptokinase, and human proteins such as interferon and tumor necrosis factor. These products are used for the treatment of a various medical conditions and diseases including diabetes, heart attack, tuberculosis, AIDS and SLE. Botulinum toxin and BT insecticide are bacterial products used in medicine and pest control, respectively
One biotechnological application of bacteria involves the genetic construction of super strains of organisms to perform particular metabolic tasks in the environment. For example, bacteria which have been engineered genetically to degrade petroleum products are used in cleanup of oil spills and other bioremediation efforts.
Another area of biotechnology involves improvement of the qualities of plants through genetic engineering. Genes can be introduced into plants by a bacterium Agrobacterium tumefaciens. Using A. tumefaciens, plants have been genetically engineered so that they are resistant to certain pests, herbicides, and diseases.
Finally, it should not be overlooked that industrial, pharmaceutical and food microbiology are applications of biotechnology. Archaea and bacteria are involved in production of biofuels. Bacteria are the main producers of clinically useful antibiotics; they are a source of vaccines against once dreaded diseases; they are probiotics that enhance our health; and they are primary participants in the fermentations of dairy products and many other foods.
Figure 15. Thermus aquaticus, the thermophilic bacterium that is the source of taq polymerase.
L wet mount; R electron micrograph. T.D. Brock. Life at High Temperatures. The polymerase chain reaction (PCR), a mainstay of the biotechnology industry because it allows duplication of genes starting with a single molecule of DNA, is based on the use of the DNA polymerase enzyme derived from Thermus aquaticus.
