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Great Expectations of Small Genomes.

Ongoing efforts to sequence the DNA of various microorganisms are fueled by the promise that the information gained will boost advances in such areas as medicine, energy production, environmental cleanup, and industrial processes.

In the heat of World War II, as the Nazis murdered millions and savaged countless others in labor camps, the small Polish village of Rozvadow (southwest of Warsaw) was largely spared from the brutality, thanks to the ingenuity of two physicians--Drs. Eugeniusz Lazowski and Stanislav Matulewicz. They injected many of their fellow villagers with the soil bacterium Proteus OX19, which is relatively benign but causes the body to produce antibodies that resemble those produced in response to the typhus bacterium, Rickettsia prowazekii. They then sent blood samples from the injected individuals to a laboratory for testing. As expected, the samples appeared to contain antibodies to typhus.

The Nazis were highly fearful of typhus, an extremely contagious disease that was often fatal. Based on just a minimal amount of checking, they quickly became convinced that the village was being ravaged by a typhus epidemic and stayed away from it for the most part. In this manner, the tiny Proteus bacterium became instrumental in tricking the Nazis and protecting the lives of many.

Microbes, microbes everywhere

Welcome to the amazing world of microbes! Scientists believe that the history of microorganisms on our planet stretches as far back as 3.8 billion years or so. Yet their invisibility to the unaided eye kept us ignorant of their existence until the seventeenth century, when the Dutchman Antonie van Leeuwenhoek discovered them through lenses that he produced with great skill.

According to some estimates, microorganisms currently account for about 60 percent of our planet's biomass, but it seems that no more than 1 percent of microbial species have been identified. Taken together, these tiny creatures live practically everywhere on our planet-- inhabiting the air, water, earth, plants, animals, and humans. Some thrive in extremely hot or cold environments; others live under conditions of high acidity or high salt content. Yet others survive extremes of radiation or pressure.

It is now known that microorganisms perform numerous vital functions that sustain all life-forms. For instance, they produce roughly half the oxygen in the atmosphere, and they lie at the base of the food web. They also recycle waste products from plants and animals. Many are involved in processes that affect geochemical changes and global climate.

Although a number of microbial species--such as the one that causes typhus--are pathogenic, most are not. Some live in our digestive tracts and help us metabolize food; others destroy toxins within our bodies. In addition, we routinely use certain species to brew alcohol, bake bread, and make dairy products.

Given the enormous variety of microbial species, they have been classified in all three major domains of life-forms: Bacteria, Archaea, and Eukarya [see "Classifying Living Things" on p. --]. In fact, the domains Bacteria and Archaea consist entirely of single-celled microorganisms. Eukarya incorporates certain single-celled organisms, such as yeasts and amoebas, as well as all multicellular species, including humans.

Among the smallest known single-celled creatures are parasitic bacteria of the genus Mycoplasma, measuring just 150--200 nanometers (a billionth of an inch). The largest bacterial species identified thus far is Thiomargarita namibiensis, which can grow up to nearly 1 millimeter (0.04 inch) in length.

In recent years, genetically engineered microbes have been employed to produce a wide range of commercially valuable products, from medications to industrial catalysts [see "The Gene Genie's Progeny," The World & I, January 2000, p. 172]. Now, with the aid of new techniques to rapidly sequence and analyze long stretches of DNA, researchers are probing microbial genomes in greater detail, seeking more information about microbial species and innovative ways to harness their power for human benefit.

Probing microbial genomes

Microbial genomes are relatively small--usually consisting of between one and four circular pieces of DNA, with a total length of one to five million base pairs (Mb). By comparison, the human genome is made up of about three billion base pairs of linear DNA distributed in 24 different types of chromosomes [see "Unraveling the Human Thread of Life," The World & I, September 2001, p. 136].

Thus, while scientists in many laboratories devoted about a decade's worth of coordinated efforts before obtaining working drafts of most of the human genome sequence, a small team of researchers can derive the full sequence of a microbial genome in a matter of weeks or months. Once the sequence of a genome has been determined, researchers use computational and experimental techniques to determine the locations and functions of the protein-coding genes [see "Which DNA Sequences Encode Proteins?" on p. --].

The largest bacterial genome sequenced so far is that of Pseudomonas aeruginosa. It is about 6.3 Mb in length and contains an estimated 5,570 genes. The smallest bacterial genome sequenced is that of Mycoplasma genitalium. It is about 580,000 base pairs (bp) long, with about 470 genes.

Comparison of the latter genome with the DNA of other microbes indicates that its smaller size does not mean that its genes are shorter or more densely packed. Rather, M. genitalium produces fewer proteins and operates on fewer biochemical pathways, making it dependent on nutrients from its host. Interestingly, one-third of the proteins predicted from the DNA sequence appear to be unrelated to proteins in other organisms.

The first archaeal genome to be completely sequenced was that of Methanococcus jannaschii, in 1996. This organism was discovered near a hydrothermal vent at a depth of more than 8,000 feet on the Pacific Ocean floor. The genome was found to consist of three separate DNA molecules: one large circular chromosome (1.66 Mb) and two extrachromosomal elements (about 58,400 bp and 16,550 bp).

Comparative studies showed that M. jannaschii DNA sequences associated with energy generation, various metabolic pathways, and cell division resemble genes present in bacterial cells. By contrast, sequences controlling DNA replication and RNA and protein synthesis resemble DNA sequences in eukaryotes. These findings support the theory that Archaea is a domain of life distinct from Bacteria and Eukarya.

Among eukaryotic organisms, the yeast species Saccharomyces cerevisiae is a fungal microbe that has been studied extensively. It has 16 types of chromosomes, with a cumulative DNA length exceeding 12 Mb. Its complete genome sequence, published in 1996, indicated the presence of over 6,000 potential genes extending across more than 70 percent of the genome. Earlier experiments had characterized the functions of roughly 30 percent of the genes of S. cerevisiae. By analyzing the genome sequence, specific proteins (or protein domains) have been associated with many additional DNA segments.

Analyses of microbial genomes have further revealed instances of gene duplication, gene loss, and possible horizontal (lateral) transfer of genes from one microbial species (or group) to another. For example, nearly one-fourth of the genes of the bacterium Thermotoga maritima closely resemble genes found in archaea, suggesting lateral gene transfer between bacteria and archaea. These dynamic changes within genomes indicate that one must use caution when attempting to derive evolutionary relationships from DNA sequences.

Applications

In this manner, genome sequences are giving scientists fresh insights into the genetics and biology of a variety of microorganisms. Based on these insights, the DNA sequences have become a gateway through which researchers can explore new applications in such areas as medicine, energy production, environmental cleanup (bioremediation), and industrial processes.

Medicine. The first bacterial genome to be sequenced in its entirety was that of the bacterium Haemophilus influenzae, which causes ear and upper respiratory infections. That project, relying on the whole-genome shotgun approach, was completed with unprecedented speed in 1995.

Since then, the complete genome sequences of many significant pathogens have been determined, including those of Borrelia burgdorferi, the causative agent of Lyme disease; Helicobacter pylori, associated with gastric diseases; Mycobacterium tuberculosis, responsible for tuberculosis; Vibrio cholerae, which causes cholera; and Treponema pallidum, which leads to syphilis. Much of the research to sequence the genomes of pathogenic microbes is funded by the National Institute of Allergy and Infectious Diseases, a branch of the National Institutes of Health.

The genome sequences will assist scientists in uncovering previously unknown proteins and biochemical pathways unique to each pathogen. Consequently, these studies will help clarify how the organism causes disease and will suggest ways of developing diagnostic tests, finding targets for drugs, and suggesting molecular candidates for vaccines. In addition, DNA sequence analyses can help identify mutations that confer drug resistance.

Comparison of the DNA sequences of variant strains of a particular microbial species can help locate genes associated with virulence. Take, for example, the bacterium Escherichia coli, which occurs as both benign and pathogenic strains. Benign strains of E. coli, along with other bacterial species, live in our intestines and produce many vitamins that we need. But the food-borne, pathogenic strain called E. coli O157:H7 causes bloody diarrhea and death from kidney failure.

The complete genome sequence of the pathogenic strain has been compared with that of a harmless strain, E. coli K12. Surprisingly, out of a total of over 5,000 genes in the harmful strain, more than 1,000 were absent from the benign one. It seems that many of the additional genes- -including toxin-producing genes--in E. coli O157:H7 were acquired from other bacterial species, with the transfer being initiated by bacteriophages (viruses that infect bacteria) and spreading through a population by direct exchange between bacteria.

Chlamydia trachomatis and Chlamydia pneumoniae are two bacterial species of the same genus but cause different types of diseases. C. trachomatis infects the genital tract of adults, leading to pelvic inflammation and infertility, and it can cause preventable blindness in infants. C. pneumoniae produces bronchitis and pneumonia. By looking for differences in the genome sequences of these two species, and in the genomes of variants of each species, researchers are identifying genes that are likely to be involved in the differing illnesses caused by the microbes.

Energy production. As the world's energy needs grow, many researchers are examining microbes as potential sources of fuels. A number of microorganisms, including the archaeon M. jannaschii, are "methanogens"--that is, they produce methane, a hydrocarbon that happens to be the main constituent of natural gas. M. jannaschii "fixes" (converts) carbon dioxide to methane.

Another archaeon that produces methane is Methanobacterium thermoautotrophicum. Originally isolated from a sewage-treatment plant, it is one of various methanogens present in animal manure and biodegradation facilities. Its genome has been entirely sequenced, and researchers are now seeking ways to genetically modify the microbe to grow more rapidly, generating larger amounts of methane. The conversion of wastes to fuels is becoming an increasingly attractive proposition.

The bacterium T. maritima and the archaeon Pyrococcus furiosus, discovered in hot marine sediments off Vulcano Island, Italy, both seem potentially useful for the production of fuels (such as ethanol or hydrogen) from biomass. T. maritima digests many carbohydrates, including the abundant plant polymers cellulose and xylan. P. furiosus obtains energy by fermentation of peptide, protein, and sugar mixtures. The genomes of both microbes have recently been sequenced in full.

Bioremediation. A number of microorganisms have been found capable of cleaning up various types of hazardous wastes. To explore ways of tapping the abilities of these microbes, the U.S. Department of Energy (DOE) has set up the Natural and Accelerated Bioremediation Research program.

As the genome sequences of several such microbes have been determined and additional genomes are being tackled, scientists are working to identify genes involved in environmental remediation. Selected genes may then be inserted into certain microbes, giving them the ability to process mixed wastes under varying conditions. Other microorganisms may be engineered to function as biosensors that call attention to the presence of specific chemicals in the soil, water, and air.

One promising soil bacterium is Deinococcus radiodurans, which has the extraordinary ability to survive damage to its DNA caused by radiation from radioactive materials, at doses that are thousands of times higher than amounts lethal to most other creatures. It can also resist the effects of ultraviolet radiation and various chemicals that produce genetic mutations.

The genome of D. radiodurans, fully sequenced in 1999, consists of three circular chromosomes and a plasmid (a small, self-replicating piece of DNA). Some researchers are now examining the genome sequences to find which genes speed up repair of damaged DNA. Others are attempting to insert genes from other microbes into this bacterium, to give it the added potential to break down organic solvents and remove heavy metals from solution by precipitating them.

The bacterium Shewanella oneidensis (or S. putrefaciens), which can grow in water and soil, can consume toxic organic wastes and precipitate certain heavy metals--including radioactive uranium--from solution. This ability could be used to trap and remove uranium from a contaminated stream. While sequencing of the S. oneidensis genome has not been completed as of this writing, researchers at Oak Ridge National Laboratory have begun placing hundreds of its DNA segments on microarrays, to find genes that might be useful for environmental remediation.

Industrial processes. A large number of microorganisms, dubbed "extremophiles," thrive under extremes of temperature, pressure, acidity, or salinity. They include the archaea M. jannaschii and P. furiosus and the bacteria T. maritima and Aquifex aeolicus, all of which grow at extremely high temperatures--between 80 and 100_C.

Researchers are investigating how the proteins (especially enzymes) of these organisms retain their three-dimensional shapes and functions at high temperatures, although enzymes from most species lose their shapes and activity when heated. The microbial genome sequences will be used to determine the structures of the proteins, which may reveal the reasons for their heat stability. Thereafter, the heat-stable enzymes could be used as environment-friendly catalysts in industrial processes.

Likewise, the genome sequences of microbes such as T. maritima and Archaeoglobus fulgidus are expected to help determine how their enzymes can function in high-pressure environments. And the DNA sequence of the archaeon Halobacterium halobium may help identify enzymes that function under high-salt conditions.

Additional organisms being investigated for industrial use include Magnetococcus MC-1, which produces the magnetic material magnetite, and Clostridium acetobutylicum, which produces the solvents acetone, butanol, and ethanol. Some microbes, including certain strains of Xylella fastidiosa and Pseudomonas syringae, are pathogens of agriculturally important plants. Their genomes are being sequenced as well.

Several microbial species--including Rhodopseudomonas palustris, Nostoc punctiforme, and Prochlorococcus marinus--directly contribute to the cycling of carbon in various forms, including converting carbon dioxide into organic matter. Given that carbon dioxide is a greenhouse gas that contributes to global warming, the DOE Joint Genome Institute has undertaken a program to determine the full genome sequences of these types of microbes as well. It is hoped that the research will lead to new ways of managing carbon in ecosystems.

From an overall perspective, the genome sequences of microbial species are clearly an important stepping-stone in advancing our understanding of how living things work. In addition, while the manipulation of life- forms at the genetic level always requires caution, it is exciting to think that the information gained may be put to use in so many beneficial ways.n

Additional Reading

Additional Reading:

Bernard Dixon, Power Unseen: How Microbes Rule the World, W.H. Freeman, New York, 1994.

"Engineering Genes," a special section in The World & I, January 2000, p. 164.

On the Internet

Additional Reading:

Microbial Genomics Gateway, DOE

www.microbialgenome.org/

Pathogen Genomics, NIAID

www.niaid.nih.gov/dmid/genomes/

The Institute for Genomic Research

www.tigr.org/

Dinshaw K. Dadachanji is an editor for the Natural Science section of The World & I.
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Author:Dadachanji, Dinshaw K.
Publication:World and I
Geographic Code:1USA
Date:Jan 1, 2002
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