Allies and Enemies: How the World Depends on Bacteria (19 page)

Through Chemistry.” By 1964’s New York World’s Fair, DuPont had

rolled out a song-and-dance extravaganza on the power of chemistry.

The industry’s new drugs, pesticides, and plastics promised a better quality of life, but these products also required significant quality control at the manufacturing level. Wartime expertise in physics and chemistry turned toward making new analytical equipment to inspect a compound’s structure and measure its purity. Companies such as Hewlett-Packard, Varian Associates, and Perkin-Elmer filled the gap.

Alec Fleming’s legacy inspired a new interest in biology in the 1940s, but additional breakthroughs came slower than many people

might have expected. Antibiotic discovery involved laborious manual

tests. Microbiologists scooped up soil samples, recovered the soil’s fungi and bacteria, and then searched for extracts from the cultures to test against hundreds of bacteria. In addition to the tedium, microbiologists often saw variable results in laboratory tests. When a microbiologist inoculates ten tubes of broth with the same Staphylococcus, eight tubes might grow, one tube might not grow, and the tenth tube ends up contaminated. Chemists at drug companies sped up the process by

synthesizing new antibiotics based on the structures of known natural

antibiotics. By the 1950s, the chemical industry offered a faster way to find new drugs. To keep up with the chemists, microbiologists needed a dependable microbe that grew easily and quickly to large quantities.

E. coli

In the 1880s, outbreaks of infant diarrhea raged through European cities and killed hundreds of babies. Like other physicians, Austrian pediatrician Theodore von Escherich struggled to save his patients and simultaneously find the infection’s cause. He recovered various bacteria from stool samples without an idea as to their role, if any, in the illness. In 1885 von Escherich published a medical article describing 19

bacteria that dominated the infants’ digestive tracts. One in particular seemed to be consistently present and in high numbers. He named it (with a striking lack of creativity) Bacterium coli commune for “common colon bacterium.” In 1958, the microbe was renamed Escherichia coli in honor of its discoverer.

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E. coli’s physiology offers nothing remarkable. It does not pump out an excess of useful or unique enzymes or make antibiotics. It dominates the newborn’s intestinal tract but gradually other bacteria overtake it and carry out the important microbial reactions of digestion. For example, strict anaerobes produce copious amounts of

digestive enzymes that help break down proteins, fats, and carbohydrates. These bacteria also partially digest fibers and synthesize proteins and vitamins that are used in the host’s metabolism. E. coli does not contribute as much to digestive activities as the strict anaerobes, but because it is a facultative anaerobe that uses oxygen when present and lives without oxygen in anaerobic places, its main role is to deplete oxygen so that anaerobic bacteria can flourish.

Von Escherich probably noticed that E. coli grows fast to high numbers in laboratory cultures. The species flourishes on a wide variety of nutrients and does not need incubation. Leave a flask of E. coli on a lab bench overnight and a dense culture will greet the microbiologist the next morning. The strict anaerobes of the digestive tract take three days or longer to grow to densities that E. coli reaches in about 10 hours.

By the turn of the century, doctors had not solved the problem of

infant diarrhea—it remains a significant worldwide cause of infant mortality. They did, however, think that E. coli might be useful for treating intestinal ailments in adults. In Freiburg, Germany, physician Alfred Nissle planned to use E. coli for intestinal upsets such as diarrhea, abdominal cramping, and nausea. In this so-called “bacteria therapy” Nissle believed that dosing an ill person with live E. coli might drive the pathogenic microbes from the gut.

From 1915 to 1917, Nissle tested various mixtures of E. coli strains in Petri dishes against typhus-causing Salmonella. When a mixture appeared to be antagonistic toward the Salmonella, he tried it on other pathogens. Nissle finally concocted a “cocktail” of what he considered the strongest E. coli strains and with considerable courage he drank it.

When no harmful effects ensued, Nissle felt he was on the road to an

important medical discovery.

During the period Nissle had been conducting his E. coli experiments, Germany’s army suffered from severe dysentery as had others

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throughout Europe during the Great War. Dirty water, bad food, and

exhaustion conspired to weaken men in the foxholes as well as civil—

ians. In 1917 Nissle made his way to two field hospitals in search of a super E. coli that would work even better than the strains in his laboratory. In one tent, he found a non-commissioned officer who had suffered various injuries but never fell victim to diarrhea even when everyone around him had it. Nissle cultured some E. coli from the soldier and returned to Freiburg.

Alfred Nissle grew the special E. coli in flasks and then poured it into gelatin capsules. When the job of supplying an entire army overwhelmed him, he commissioned the production to a company in Danzig. The new antidiarrheal capsules were called Mutaflor. The wartime upheavals in Europe through 1945 forced Nissle to move the manufacturing more than once, but the production of Mutaflor never

ceased. Mutaflor remains commercially available today as a probiotic

treatment for digestive upset. The product contains “E. coli Nissle 1917” made from direct clones of the superbug Nissle isolated on the battlefield in 1917. The original strain that Nissle submitted to the

German Collection of Microorganisms remains in its depository in Braunschweig.

At Stanford University in 1922, microbiologists noted another

fast-growing E. coli strain with a curious trait: It did not cause illness in humans. The strain received a laboratory identification of “K-12.”

K-12 became a standard in teaching and research laboratories, soon

shared by Stanford with other universities. When eventual Nobel Prize awardees Joshua Lederberg and Edward Tatum began studies

on how genes carry information and the mechanisms organisms use

to exchange this information, they made the logical choice of K-12 as

an experimental workhorse. E. coli became forever linked with advances in genetics and biotechnology.

Since the first K-12 experiments, more than 3,000 different

mutants of this bacterium have been used in cell metabolism, physiology, and gene studies. One of the first bacterial genomes to be sequenced was that of K-12; the complete sequence of its 4,377

genes was published in 1997. In the last 50 years, 14 Nobel Prizes have been awarded based on work done with E. coli, mainly K-12.

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The power of cloning

By the 1970s, microbiologists were routinely taking apart E. coli to study its reproduction, enzymes, and virulence. The chemical industry had lost some luster with each new discovery of environmental pollution, and biology again looked like the science for the future: clean, quiet, and nonpolluting.

In biotech’s infancy, “cloning” became the buzzword for the

power of this new technology: The ability to take a single gene and

produce millions of identical copies. E. coli became a living staging area in which genes were cloned by the following general scheme: 1.
Extract DNA from an organism possessing a desirable trait (a

gene).

2.
Cleave the DNA into many smaller pieces with a specialized enzyme called restriction endonuclease (RE).

3.
Extract E. coli plasmids and open their circular structure with another RE.

4.
Insert the various DNA fragments into the many plasmids.

5.
Allow the bacteria to take the plasmid back into their cells.

6.
Grow all the bacteria and use screening procedures to identify the cells carrying the desirable gene.

7.
Grow large amounts of these gene-carrying cells, that is, cloning.

8.
Harvest the product that the gene controls.

In biotech’s infancy scientists painstakingly worked out each of the preceding steps (see Figure 5.1). Molecular biologists perfected the art of extracting DNA from cells without breaking the large molecule into pieces. They devised techniques for splicing new segments of one type of DNA into a second DNA molecule, and they developed methods for testing the activity in a new GMO. But the scientists also noticed that their favorite bacterium E. coli resisted taking plasmids into their cells, a key step in genetic engineering called transformation. Without an easy way to deliver DNA into E. coli, many genetic experiments might become impossible. In 1970, Morton Mandel and Akiko Higa solved this dilemma by showing that

calcium increased the permeability of cell membranes to DNA. By

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soaking E. coli in a chilled calcium chloride solution for 24 hours, biologists now make the bacterium 20 to 30 times more receptive to taking up plasmids. Bacteria that take in plasmids from the environment are called competent cells, and biotechnologists now use this simple soaking step to make E. coli competent for transformation.

Figure 5.1 Microbiologists in environmental study, medicine, industry, and academia use the same aseptic techniques. These disciplines use methods adapted from biotechnology for manipulating the genetic makeup of bacteria.

(Reproduced with permission of the American Society for Microbiology

MicrobeLibrary (http://www.microbelibrary.org))

In the early days of biotech research, bacterial cloning—it used to

be called gene splicing—served as the only way to make large

amounts of gene and gene products. Bacteria make millions of copies

of a target gene by replicating it each time a cell splits down the middle to make two new cells, a process called binary fission. In time, scientists developed methods for using bacteriophages to deliver genes directly into a bacterium’s DNA. Viruses’ modus operandi involves appropriating a cell’s DNA replication system, which is a perfect mechanism for delivering a foreign gene into bacterial DNA. PCR

would enter the picture next as a faster DNA-amplification method.

E. coli remains a major tool in biotechnology, but additional microbes such as the yeast Saccharomyces cerevisiae and the bacterium Bacillus subtilis also contribute a large share to recombinant DNA technology. Biotech companies use the basic cloning scheme

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described previously in yeasts and bacteria for making the drugs listed in Table 5.1.

Table 5.1 Major products made by biotechnology

Made by E. coli

Made by Other Microbes

Interferons as antiviral and antitumor

Antitrypsin emphysema treatment

drugs

Factor VIII for treatment of hemophilia

Colony-stimulating factor to counter—

Bone morphogenic proteins to induce

act effects of chemotherapy and treat

new bone formation

leukemia

Calcitonin for regulating blood calcium

Growth hormone

levels

Insulin for diabetes

Erythropoietin for anemia

Interleukins for treatment of tumors

Growth factor for wound recovery

and immune disorders

Hepatitis B vaccine

Relaxin as a childbirth aid

Macrophage colony-stimulating factor

Somatostatin treatment for

for cancer

acromegaly, a growth disorder of