Anthrax: "A Soil Bug Gone Bad"

Scientists Decipher, Analyze Genome of Bacillus anthracis

April 30, 2003

Rockville, MD - Scientists at The Institute for Genomic Research (TIGR) and collaborators have deciphered the genome of the notorious Ames strain of the bacterium that causes anthrax, Bacillus anthracis. It is the first anthrax strain to be completely sequenced and analyzed.

That genome analysis, published in the May 1, 2003, issue of Nature, found only small differences in gene content that distinguish the deadly anthrax bacillus from the common soil bacterium, Bacillus cereus, that thrives in gardens across the globe.

There may be no more than about 150 significant differences in the 5,000-plus genes in the chromosomes of those related bacterial species, says TIGR researcher Tim Read, the first author of the Nature letter. Other differences are found on small circular strands of DNA, called plasmids, which are key to the virulence of B. anthracis.

Those natural genomic differences - including the plasmid-encoded genes that make the anthrax bacterium toxic and virulent in attacking human and animal hosts - have made B. anthracis a potent pathogen. Over the last seven decades, the anthrax bacillus was developed and "weaponized" as a biowarfare/bioterror agent by several national programs, from Japan in the 1930s to Iraq in the 1980s.

TIGR's President, Claire M. Fraser, who supervised the anthrax genome project, says that the DNA sequence provides an invaluable tool for researchers who are seeking to develop new drugs or vaccines against anthrax, as well as for others who are investigating better ways to detect or trace the pathogen.

"Deciphering the anthrax genome is important to a wide range of biomedical and biodefense research," says Fraser. "The genome sequence will benefit research projects to find targets for new drugs and vaccines as well as to improve anthrax detection and diagnosis."

Since the B. anthracis sequencing project was started in 1999, TIGR had been posting preliminary anthrax genome data - already used by researchers in more than 20 published papers so far - on a public site that is freely available to other scientists.

The project was supported by the U.S. Office of Naval Research, the National Institute of Allergy and Infectious Diseases, the U.S. Department of Energy and the United Kingdom's Defense Sciences Technology Laboratory (DSTL). TIGR's scientific collaborators included researchers at the University of Oslo, in Norway; the University of Texas; the University of Michigan; the U.S. Army Medical Research Institute of Infectious Diseases and the University of Maryland Biotechnology Institute, both in Maryland; and the DSTL in Porton Down, U.K.

TIGR sequenced an isolate of the Ames strain, a particularly virulent strain of B. anthracis that was isolated from a Texas cow in 1981 and later was widely used for laboratory research. Other than the fact that its plasmids had been removed, the isolate that TIGR analyzed is closely similar to the Ames isolate that was used in the first anthrax bioterror attack in Florida in 2001, according to TIGR's comparative genome analysis, published in Science in May 2002. The Florida attack and parallel mail attacks in Washington, D.C., New York, and Connecticut led to five deaths and 17 injuries.

It has long been known that B. anthracis - a member of the Bacillus cereus family of soil bacteria - is distinguished from other B. cereus species mainly by its two plasmids, small circles of DNA that are outside of the cell's single chromosome. Those plasmids, called pX01 and pX02, carry genes related to virulence and toxicity that account for much of the lethal power of anthrax to kill animals, including humans.

The new study focused more on the B. anthracis chromosome, which comprises about 5 million DNA bases, in contrast to about 300,000 total bases in the plasmids. One finding is that virulence-related genes do not appear to be limited to the plasmids; the analysis found several genes on the chromosome that have a potential relationship to virulence. Even so, the gene content of the B. anthracis chromosome is closely similar to that of the chromosome of the garden-variety soil bacterium B. cereus, which is commonly found in soils and sometimes causes a form of food poisoning.

A genome comparison with another member of the same bacterial family, B. thuringiensis - a soil bug whose plasmids carry insect toxins - uncovered some clues indicating that a relatively recent ancestor of B. anthracis, which infects mammals, may have been an insect pathogen. In addition, researchers a gene in the anthrax bacillus that is similar to a gene in the plague bacterium, Yersinia pestis, which infects fleas as well as mammals.

Calling B. anthracis "a soil bug gone bad," Read says the anthrax bacillus has "a large metabolic tool kit" - similar to that of other soil bacteria - that gives the bacterium the versatility to import nutrients and respond to signals from its environment. "From a metabolic perspective, B. anthracis is quite nimble," he says.

In Nature, the researchers write: "The B. anthracis chromosome sequence portrays a soil-dwelling organism, possessing numerous potential virulence genes, which has possibly a preference for protein-rich environments. This is consistent with the evolution of B. anthracis from a B. cereus ancestor through acquisition of a key plasmid-encoded toxin, capsule and regulatory loci."

The virulence of B. anthracis in contrast to its bacterial cousins may also be related to how certain genes, present in both species, are "expressed" - that is, functioning. "Other major differences between B. anthracis and B. cereus may have been effected through altered gene expression rather than the loss or gain of genes," the researchers write. For example, the same genes in B. anthracis that have to do with penicillin resistance, motility, and various metabolic functions "may have slightly different functions or expression levels in B. cereus."

The genome analysis indicated that those changes in gene expression "may reflect recent adaptations" of the anthrax bacterium after it acquired what scientists call a "pathogenicity island" that includes the lethal toxin site on the pX01 plasmid. A regulatory gene in the same region, called atxA, controls the expression of that toxin gene, but it appears to be incompatible with another chromosomal regulator gene, called plcR, that is found in B. cereus.

TIGR scientist Scott N. Peterson, who led the comparative studies of genes in B. anthracis and closely-related bacteria in the B. cereus group, says his analysis indicate that lateral transfers of genes have occurred between the anthrax bacterium and its close relatives."This indicates that the virulence of B. anthracis may have come mainly from horizonal gene exchanges, more so than by mutations within the genome," he says.

In addition to deciphering the complete genome of the Ames strain, TIGR is now sequencing several other strains and isolates of the anthrax bacterium for comparative genomic studies that will help scientists explore the evolution and the biology of B. anthracis. "The genome analysis moves us closer to understanding every aspect of the anthrax bacterium," says Read.