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£coli is normally harmless, although occasional rogue strains occur. Even these few pathogenic E. coli strains mostly just cause diarrhea, by secreting a mild form of a toxin related to that found in cholera and dysentery bacteria. However, the notorious E. coli O157:H7 carries two extra toxins and causes bloody diarrhea that may be fatal, especially in children or the elderly. In outbreaks of E. coli, the bacteria typically contaminate ground meat used in making hamburgers. Several massive recalls of frozen meat harboring E. coli O157:H7 have occurred in the late 1990's. For example, in 1997 the Hudson Foods plant in Columbus, Nebraska was forced to shut down and 25 million pounds of ground beef were recalled.

4. Bacteria can be grown under strictly controlled conditions and many will grow in a chemically defined culture medium containing mineral salts and a simple organic nutrient such as glucose.

5. Bacteria grow fast and may divide in as little as 20 minutes, whereas higher organisms often take days or years for each generation (Fig. 2.11).

6. A bacterial culture contains around 109 cells per ml. Consequently genetic experiments that need to analyze large numbers of cells can be done conveniently.

7. Bacteria can be conveniently stored for short periods (a couple of weeks) by placing them in the refrigerator and for longer periods (20 years or more) in low temperature freezers at -70°C. Upon thawing, the bacteria resume growth. Thus it is not necessary to keep hundreds of cultures of bacterial mutants constantly growing just to keep them alive.

In practice, bacteria are usually cultured by growing them as a suspension in liquid inside tubes, flasks or bottles.They can also be grown as colonies (visible clusters of cells) on the surface of an agar layer in flat dishes, known as Petri dishes (Fig. 2.12).Agar is a carbohydrate polymer extracted from seaweed that sets, or solidifies, like gelatin.

It should be noted that the convenient properties noted above apply to commonly grown laboratory bacteria. In contrast, many bacterial species found in the wild are difficult or, by present techniques impossible, to culture in the laboratory. Many others have specialized growth requirements and most rarely grow to the density observed with the bacteria favored by laboratory researchers.

pathogenic Disease causing

Escherichia coli (E. coli) Is a Model Bacterium 31

FIGURE 2.12 Bacterial Colonies in a Petri Dish

A Petri dish showing colonies of Escherichia coli 0157:H7 growing on nutrient agar medium. This strain sometimes causes food-borne illness. It may cause bloody diarrhoea and occasionally kidney failure, particularly in the elderly or very young. This E. coli strain originates from the intestines of cattle and spreads to contaminate beef and milk. Provided by TEK Image, Science Photo Library.

FIGURE 2.12 Bacterial Colonies in a Petri Dish

A Petri dish showing colonies of Escherichia coli 0157:H7 growing on nutrient agar medium. This strain sometimes causes food-borne illness. It may cause bloody diarrhoea and occasionally kidney failure, particularly in the elderly or very young. This E. coli strain originates from the intestines of cattle and spreads to contaminate beef and milk. Provided by TEK Image, Science Photo Library.

The famous K-12 laboratory strain of E. coli was chosen as a research tool because of its fertility. In 1946, Joshua Lederberg was attempting to carry out genetic crosses with bacteria. Until then, no mechanisms for gene transfer had been demonstrated in bacteria, and genetic crosses were therefore thought to be restricted to higher organisms. Lederberg was lucky, as most bacterial strains, including most strains of E. coli, do not mate. But among those he tested was one strain (K-12) of E. coli that happened to give positive results. Mating in E. coli K-12 is actually due to a plasmid, an extra circular molecule of DNA within the bacterium that is separate from the chromosome. Because the plasmid carries the genes for fertility, it was named the F-plasmid.

According to Jaques Monod, who discovered the operon (see Ch. 9): "What applies to E. coli applies to E. lephant."

Escherichia coli (E. coli) Is a Model Bacterium

Although many different types of bacteria are used in laboratory investigations,the bacterium used most often in molecular biology research is Escherichia coli. E. coli is a rod-shaped bacterium of approximately 1 by 2.5 microns. Its natural habitat is the colon (hence "coli"), the lower part of the large intestine of mammals, including humans. The knowledge derived by examining E. coli has been used to untangle the genetic operation of other organisms. In addition, bacteria, together with their viruses and plasmids, have been used experimentally during the genetic analysis of higher organisms.

F-plasmid A particular plasmid which confers ability to mate on its bacterial host, Escherichia coli plasmid Circular molecule of double stranded helical DNA which replicates independently of the host cell's chromosomes. Rare linear plasmids have been discovered

Cytoplasmic membrane

FIGURE 2.13 GramNegative and GramPositive Bacteria

Gram-negative bacteria have an extra membrane surrounding the cell wall.

Cytoplasmic membrane

FIGURE 2.13 GramNegative and GramPositive Bacteria

Gram-negative bacteria have an extra membrane surrounding the cell wall.

Gram-negative

Gram-positive

E. coli is a gram-negative bacterium, which means that it possesses two membranes. Outside the cytoplasmic membrane possessed by all cells are the cell wall and a second, outer membrane (Fig. 2.13). (Although gram-negative bacteria do have two compartments, they are nonetheless genuine prokaryotes, as their chromosome is in the same compartment as the ribosomes and other metabolic machinery. They do not have a nucleus, the key characteristic of a eukaryote). The presence of an outer membrane provides an extra layer of protection to the bacteria. However, it can be inconvenient to the biotechnologist who wishes to manufacture genetically engineered proteins from genes cloned into E. coli. The outer membrane hinders protein secretion. Consequently there has been a recent upsurge of interest in gram-positive bacteria, such as Bacillus, which lack the outer membrane.

Familiar animals and plants are vastly outnumbered by microorganisms, in every natural habitat.

Where Are Bacteria Found in Nature?

Bacteria are found almost everywhere. Bacteria have been found 40 miles high in the atmosphere and seven miles deep beneath the ocean floor. Some bacteria live in the sea, others live in fresh water, and others are found growing happily in sewage. Some bacteria live in the soil, some are found living in the roots of plants, and some live inside animals. Most of the bacteria that live inside animals are harmless, and some are even of positive value in aiding digestion or synthesizing vitamins that are absorbed by their host animal.

The total number of bacteria on our planet is estimated at an unbelievable 5 x 1030. Over 90% are in the soil and subsurface layers below the oceans.The total amount of bacterial carbon is 5 x 1017 grams, nearly equal to the total amount of carbon found in plants. Probably over half of the living matter on Earth is microbial.

In addition to the "normal" habitats, some bacteria live in extreme environments where most other life forms cannot survive. Some bacteria can live in very concentrated salt solutions, such as the Dead Sea and the Great Salt Lake. Antarctic lakes that only thaw for a short period of each year contain bacteria. Other bacteria inhabit hot sulfur springs, where temperatures approach boiling point and the pH is close to 1. Bacteria even grow in some thermal deep sea vents where the temperature is above 100°C and the high pressure keeps the water liquid. Bacteria from these habitats may gram-negative bacterium Type of bacterium that has both an inner (cytoplasmic) membrane plus an outer membrane which is located outside the cell wall gram-positive bacterium Type of bacterium that has only an inner (cytoplasmic) membrane and lacks an outer membrane

Where Are Bacteria Found in Nature? 33

Patients are usually given antibiotics to treat bacterial infections. These are chemical substances capable of killing most bacteria by inhibiting specific biochemical processes, but which are relatively harmless to people. The most commonly used antibiotics, the penicillins and cephalosporins, are synthesized by a kind of fungus known as mold (see Fig. 2.14). However, many antibiotics are made by one kind of bacteria in order to kill other types of bacteria. The Strep-tomyces group of soil bacteria produces a wide range of antibiotics including streptomycin, kanamycin and neomycin. Some antibiotics, like chloramphenicol, were originally made by molds but nowadays can be chemically synthesized. Finally, some antibiotics, such as sulfonamides, are entirely artificial and are only synthesized by chemical corporations.

FIGURE 2.14 Bacterial Growth Is Suppressed by Bread Mold

The blue mold that often grows on bread makes penicillin. When penicillin is produced by molds grown on agar in a Petri dish, it will diffuse outwards and suppress the growth of bacteria in a circle around it.

provide products that are useful because of their resistance to extreme conditions. Thermus aquaticus, a bacterium from hot springs, has provided the heat stable DNA polymerase needed for the polymerase chain reaction (PCR), a widely used technique (see Ch. 23).

When different bacteria compete to live in the same habitat, they often resort to biological warfare. Some bacterial strains secrete toxic chemicals in order to kill off others that are competing for the same resources. Certain bacteria synthesize toxic proteins, known as bacteriocins. These proteins are designed to kill closely related bacterial strains, yet are harmless to the producer strain. Nisin, a bacteriocin produced by some strains of Lactococcus lactis acts as a food preservative and kills food-borne pathogens including Listeria monocytogenes and Staphylococcus aureus. Nisin and related bacteriocins are relatively short peptides of molecular weight 3.5 kDa. They are formed naturally by the strains of Lactococcus that are used to make silages and fermented foods such as wara, a Nigerian cheese product, and kimchi (Korean traditional fermented vegetables). Although scientists have found relatively few practical applications for bacteriocins, the plasmids which carry the genes for bacteriocins have provided the most widely used vectors for carrying genes in genetic engineering (described in Ch. 22).

Streptomycin and related antibiotics are also made by bacteria, especially those of the Streptomyces group, to kill competing bacteria in the soil environment. These antibiotics are not proteins (unlike the colicins) and have been widely used clinically.

antibiotics Chemical substances that inhibit specific biochemical processes and thereby stop bacterial growth selectively;that is, without killing the patient too.

bacteriocin A toxic protein made by bacteria to kill other, closely related, bacteria

DNA polymerase An enzyme that elongates strands of DNA, especially when chromosomes are being replicated penicillin An antibiotic made by a mold called Penicillium, which grows on bread producing a blue layer of fungus PCR See polymerase chain reaction vector (a) In molecular biology a vector is molecule of DNA which can replicate and is used to carry cloned genes or DNA fragments;(b) in general biology a vector is an organism (such as a mosquito) that carries and distributes a disease-causing microorganisms (such as yellow fever or malaria)

FIGURE 2.15 A Eukaryote Has Multiple Cell Compartments

False color transmission electron micrograph of a plasma cell from bone marrow. Multiple compartments surrounded by membranes, including a nucleus, are found in eukaryotic cells. Characteristic of plasma cells is the arrangement of heterochromatin (orange) in the nucleus, where it adheres to the inner nuclear membrane. Also typical is the network of rough endoplasmic reticulum (yellow dotted lines) in the cytoplasm. The oval or rounded crimson structures in the cytoplasm are mitochondria. Magnification x4,500. Provided by Dr. Gopal Murti, Science Photo Library.

If higher organisms disappeared from the Earth, the prokaryotes would survive and evolve. They do not need us although we need them.

Some Bacteria Cause Infectious Disease, but Most Are Beneficial

Bacteria are best known to the layman for causing infectious disease. Cholera, tuberculosis, bubonic plague ("Black Death"), anthrax, syphilis, gonorrhea, whooping cough, diphtheria and a variety of other diseases are caused by bacteria. These diseases were widespread before modern technology and hygiene largely eliminated them from advanced societies. This was mostly due to clean water, sewers, flush toilets and soap, rather than specifically "medical" advances such as the use of antibiotics or vaccinations.

Only a small proportion of bacteria causes disease. Many bacteria help maintain the ecosystem by degrading waste materials. For example, soil bacteria degrade the remains of dead plants and animals and take part in the breakdown of animal waste. Bacteria also degrade many man-made chemicals and pollutants. If "good" bacteria did not maintain the environment, higher life-forms could not survive.

Very occasionally bacteria which are even tinier than usual infect other, larger bacteria. This results in a bacterial disease of bacteria! The best known example of this is Bdellovibrio bacterivorus. This penetrates the outer membrane of a wide range of gram-negative bacteria, including E. coli, Pseudomonas, etc., and takes up residence in the space between the inner and outer membranes. Bdellovibrio lives on nutrients it steals from the host cell. After a few hours, the host cell bursts and releases half a dozen new Bdellovibrio cells.

Eukaryotic Cells Are Sub-Divided into Compartments

A eukaryotic cell has its genome inside a separate compartment, the nucleus. In fact, eukaryotic cells have multiple internal cell compartments surrounded by membranes (Fig 2.15). The nucleus itself is surrounded by a double membrane, the nuclear enve-

nuclear envelope Envelope consisting of two concentric membranes that surrounds the nucleus of eukaryotic cells

Eukaryotic Cells Are Sub-Divided into Compartments 35

Eukaryotic Cells Are Sub-Divided into Compartments 35

(crista)

FIGURE 2.16 Mitochondrion

(crista)

FIGURE 2.16 Mitochondrion

A mitochondrion is surrounded by two concentric membranes. The inner membrane is folded inward to form cristae. These are the site of the respiratory chain that generates energy for the cell.

Life is modular. Complex organisms are subdivided into organs. Large and complex cells are divided into organelles.

lope, which separates the nucleus from the cytoplasm, but allows some communication with the cytoplasm via nuclear pores (Fig 2.15).The genome of eukaryotes consists of 10,000-50,000 genes carried on several chromosomes. Eukaryotic chromosomes are linear, unlike the circular chromosomes of bacteria. Most eukaryotes are diploid, with two copies of each chromosome. Consequently, they possess at least two copies of each gene. In fact, eukaryotic cells often have multiple copies of certain genes as the result of gene duplication.

Eukaryotes possess a variety of other membranes and organelles. Organelles are subcellular structures that carry out specific tasks. Some are separated from the rest of the cell by membranes (so-called membrane-bound organelles) but others (e.g., the ribosome) are not. The endoplasmic reticulum is a membrane system that is continuous with the nuclear envelope and permeates the cytoplasm. The Golgi apparatus is a stack of flattened membrane sacs and associated vesicles that is involved in secretion of proteins, or other materials, to the outside of the cell. Lysosomes are membrane-bound structures specialized for digestion, containing degradative enzymes.

All except a very few eukaryotes contain mitochondria (singular, mitochondrion; Fig. 2.16). These are generally rod-shaped organelles, bounded by a double membrane. They resemble bacteria in their overall size and shape. As will be discussed in more detail (see Ch. 20), it is thought that mitochondria are indeed evolved from bacteria that took up residence in the primeval ancestor of eukaryotic cells. Like bacteria, mitochondria each contain a circular molecule of DNA. The mitochondrial genome is similar to a bacterial chromosome, though much smaller. The mitochondrial DNA has some genes needed for mitochondrial function.

Mitochondria are specialized for generating energy by respiration and are found in all eukaryotes. (A few eukaryotes are known that cannot respire; nonetheless these retain remnant mitochondrial organelles—see below.) In eukaryotes, the enzymes of respiration are located on the inner mitochondrial membrane, which has numerous infoldings to create more membrane area. This contrasts with bacteria, where the respiratory chain is located in the cytoplasmic membrane, as no mitochondria are present.

crista (plural cristae) Infolding of the photosynthetic membrane in chloroplast endoplasmic reticulum Internal system of membranes found in eukaryotic cells

Golgi apparatus A membrane bound organelle that takes part in export of materials from eukaryotic cells lysosome A membrane bound organelle of eukaryotic cells that contains degradative enzymes membrane-bound organelles Organelles that are separated from the rest of the cytoplasm by membranes mitochondrion Membrane-bound organelle found in eukaryotic cells that produces energy by respiration nuclear pore Pore in the nuclear membrane through which the nucleus communicates with the cytoplasm organelle Subcellular structure that carries out a specific task. Membrane-bound organelles are separated from the rest of the cytoplasm by membranes but other organelles such as the ribosome are not.

FIGURE 2.17 Chloroplast

The chloroplast is bound by a double membrane and contains infolded stacks of membrane specialized for photosynthesis. The chloroplast also contains ribosomes and DNA.

Chloroplasts are membrane-bound organelles specialized for photosynthesis (Fig. 2.17). They are found only in plants and some single-celled eukaryotes. They are oval to rod shaped and contain complex stacks of internal membranes that contain the green, light-absorbing pigment chlorophyll and other components needed for trapping light energy. Like mitochondria, chloroplasts contain a circular DNA molecule and are thought to have evolved from a photosynthetic bacterium.

The Diversity of Eukaryotes

Unlike prokaryotes that fall into two distinct genetic lineages (the eubacteria and archaebacteria), all eukaryotes are genetically related, in the sense of being ultimately derived from the same ancestor. Perhaps this is not surprising since all eukaryotes share many advanced features that the prokaryotes lack. When it is said that all eukary-otes are genetically related, it is in reference to the nuclear part of the eukaryotic genome, not the mitochondrial or chloroplast DNA molecules that have become part of the modern eukaryotic cell.

A wide variety of eukaryotes live as microscopic single cells. However, most eukaryotes are larger multicellular organisms that are visible to the naked eye. Traditionally, these higher organisms have been divided into the plant, fungus and animal kingdoms. This classification still holds, provided one remembers to include several new groups to account for the single-celled eukaryotes. Some single-celled eukaryotes may be viewed as plants, fungi or animals. Others are intermediate or possess a mixture of properties and need their own miniature kingdoms.

Eukaryotes Possess Two Basic Cell Lineages

The most primitive multicellular organisms are merely aggregates of more or less identical cells. However, most multicellular organisms consist of distinct tissues and organs containing a variety of specialized cells. Furthermore, most cells in higher organisms do not contribute to the next generation, but die when the multicellular individual of whom they are part dies. These are known as somatic cells (Fig. 2.20). Only the germ line cells take part in forming a new individual. This, of course, complicates genetic analysis. Although all cells in any multicellular organism start with an identical copy of the genome, they differentiate to give quite different structures that perform different functions. Understanding development is a major challenge facing molecular biology today. In animals there is a sharp division between somatic cells and germ line cells that persists throughout the life cycle. However, plants do not set aside special germ cells until close to the time that gametes are made.

germ line cells Reproductive cells producing eggs or sperm that take part in forming the next generation chlorophyll Green pigment that absorbs light during photosynthesis somatic cells Cells making up the body but which are not part of the germ cell line.

Chloroplast outer

Chloroplast outer

FIGURE 2.17 Chloroplast

The chloroplast is bound by a double membrane and contains infolded stacks of membrane specialized for photosynthesis. The chloroplast also contains ribosomes and DNA.

Eukaryotes Possess Two Basic Cell Lineages 37

The Symbiotic Theory of Organelle Origins

A well accepted theory of mitochondrial (and chloroplast) origin is that certain bacteria were ingested by ancestral eukaryotes and have lived in a symbiotic relationship with their descendents ever since. Figure 2.18 suggests how this could have occurred. The mitochondrion contains DNA and ribo-somes. The DNA of the mitochondria more closely resembles that of bacteria than of eukaryotes.

Certain primitive single-celled eukaryotes, such as Entamoeba and Giardia, lack the ability to respire and instead live by fermentation (Fig. 2.19). It was once believed that they lacked mitochondria and had branched off from the ancestral eukaryote before it had captured the bacterium that gave rise to the mitochondrion. More recently, it was suggested that the ancestors to these organisms did originally possess mitochondria, but lost them secondarily during the course of evolution. However, recent work has shown that even Entamoeba and Giardia retain small remnant organelles ("mitosomes") corresponding to mitochondria. Although the capability for respiration has indeed been completely lost, the remnant organelles function in assembling the iron sulfur clusters found in several essential proteins.

Respiring bacterium

Nucleus

Urkaryote

Urkaryote

Bacterium being engulfed

Bacterium (now mitochondrion) inside cell membrane

Bacterium (now mitochondrion) inside cell membrane

Mitochondrial membranes inner outer

Mitochondrial membranes inner outer

Mitochondrion divides to populate and respire inside eukaryote

Mitochondrion divides to populate and respire inside eukaryote

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