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The Genome of Agrobacterium tumefaciens
A plant pathogen with a talent for transferring genes
  
By
Edward R. Winstead



Featured article.

The microbe Agrobacterium tumefaciens is harmful to plants and useful to scientists for the same reason: It transfers DNA into plant genomes. Found in soil worldwide, A. tumefaciens causes disease in plants by transferring its own DNA into plant cells. But in the laboratory, the ability to move all sorts of genes into plants has made the microbe the standard tool for investigating plant genetics and modifying crops.


Agrobacterium tumefaciens attached to a plant cell.

Now, two teams of researchers working independently have sequenced the A. tumefaciens genome. One group was led by Eugene W. Nester, of the University of Washington in Seattle, and the other by Steven Slater, of Cereon Genomics in Cambridge, Massachusetts. The projects were initiated and completed separately, but both groups published their findings in the same issue of Science.

"Our intent was to try to understand this important tool, and you can't do that fully without the genome sequence," says Slater. His group is interested in genes that can be modified or stimulated to give A. tumefaciens more versatility and efficiency as a biotechnology tool. Both groups are analyzing the sequence to identify genes that will reveal how the pathogen causes disease and interacts with the plant host.

Most A. tumefaciens infections in plants occur in wounds, like those that sometimes result from grafting together different plant stocks. Once in the plant cell, the bacterial DNA inserts randomly into the plant genome and leads to changes in the production of certain plant hormones. These, in turn, can lead to the growth of tumors, or galls, that weaken the plant and can be fatal.


Sunflower with crown gall disease.

Tumor caused by Agrobacterium on a tree (Fraxinus excelsior) in Austria.

Crown gall disease affects hundreds of species, particularly fruits, nuts and ornamental plants such as roses. The disease recently caused severe damage and economic losses among walnut tree growers in California. Once A. tumefaciens infects a plant, the bacterium travels throughout the root system, and can wipe out an entire crop. The only option for farmers is to destroy the plants.

The practice of replacing the tumor-inducing genes with other DNA began in the 1970s and led to the widespread use of the bacterium in research. The microbe is particularly well suited for testing the functions of individual plant genes because bacterial DNA disrupts the genome at the point of insertion.

"Agrobacterium can be used to insert a piece of DNA in the middle of a plant gene, thus inactivating the gene," says Derek W. Wood, who led part of the University of Washington project. "We can then analyze the mutation to see what it does to the plant."

This experiment has been done on a large scale. Many thousands of mutant potato and alfalfa plants are available to plant researchers from institutions like the University of Wisconsin in Madison. The microbe has also been used to create transgenic crops, including new strains of corn and soybeans.

The A. tumefaciens genome has a very unusual structure. Some 5,400 genes reside on four DNA elements—a circular chromosome, a linear chromosome, and two smaller circular structures called plasmids. Many bacteria have circular chromosomes and some have linear chromosomes, but Agrobacteria are the only species known to have both structures together.

The researchers expected to find two distinct chromosomes in part because of data published by Brad Goodner and his students at the University of Richmond in Virginia. In 1993, Goodner read a paper by French researchers reporting evidence that A. tumefaciens had a linear as well as a circular chromosome. He made testing the hypothesis a research project for his undergraduates.

Goodner and his students spent the next few years developing maps that supported the new theory, and they presented this evidence at a scientific conference in 1999. Slater was at the meeting, and he proposed a partnership with Goodner to complete the genome. Cereon had begun sequencing A. tumefaciens, but the company welcomed help during the later stages of the project.

"They had already initiated the shotgun sequencing, and our main job was to help in the assembly process," says Goodner. His laboratory knew the locations of more than 100 mutations in the genome, and they used these to place sequenced fragments where they belong on the chromosomes. The genome contains 5.67 million base pairs.

Agrobacterium tumefaciens is closely related to a bacterium that has a positive, symbiotic relationship with plants, Sinorhizobium meliloti. This bacterium lives on alfalfa and other legumes and helps its plant host acquire nitrogen. Comparisons of the two genomes suggest that very few genes determine whether a bacterium is by nature harmful or helpful.


Detail from schematic of A. tumefaciens genome. View larger

"It is a fine line between being a symbiont or a pathogen," says Ian Paulsen, of The Institute for Genomic Research in Rockville, Maryland, who worked with Nester's group on the annotation. "At the level of genes and proteins, the two look very similar."

Paulsen also says that A. tumefaciens has more ABC transporter genes than any other sequenced organism. ABC transporters are used to acquire nutrients, sugars and amino acids. Having so many of them may be an advantage in the competition among microbes for nutrients.

The bacterium is common enough that it can be brought into a hospital on someone's dirty hands or shoes. More than 50 cases have been reported in the literature of patients with severely compromised immune systems being infected by the microbe. Goodner has three strains isolated from patients and will be comparing them with the newly sequenced C58 strain.

A possible entry point for bacterial infection is through tiny wounds caused by, for example, a catheter that is inserted incorrectly. "The bacterium is pretty tenacious and can cause problems if a person's immune system is knocked down," Goodner says.

"We know very little about how A. tumefaciens causes disease in humans, but the mechanism of disease does not appear to be the same in animals and plants," says Goodner, now of Hiram College in Ohio. He points out that tumors in plants are caused by very specific changes in plant hormones, which would probably not affect humans.


Agrobacterium tumefaciens on plant cells.

Both projects were collaborations that benefited from significant contributions by undergraduate students. Nester's team also included Maynard Olsen, of the University of Washington Genome Center, who led the sequencing. Bioinformatics expertise was provided by Joao C. Setubal, of the University of Campinas in Brazil. His group analyzed the sequence and predicted functions for 3,475 genes.

Setubal joined the project unexpectedly while on sabbatical at the University of Washington. He led the Brazilian team that last year published the first genome sequence of a plant pathogen, Xylella fastidiosa.

See related GNN articles
»Modified tomato plants silence the genes of invaders
»In symbiosis with alfalfa: The complex genome sequence of Sinorhizobium meliloti
»Genome of bacteria Xylella fastidiosa, a threat to fruit and nut crops, is sequenced

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Wood, D.W. et al. The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294, 2317-2323 (December 14, 2001).
 
Goodner, B. et al. Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science 294, 2323-2328 (December 14, 2001).
 
Allardet-Servent, A. et al. Presence of one linear and one circular chromosome in the Agrobacterium tumefaciens C58 genome. J Bacteriol 175, 7869-7874 (December 1993).
 

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