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Sugar Transporters and Foreign DNA
The sequenced Streptococcus pneumoniae genome
Edward R. Winstead

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Researchers this week report the long-awaited genome sequence of Streptococcus pneumoniae, the bacterium that causes pneumonia, blood infections, and meningitis. As expected, the sequencing revealed a pathogen adept at acquiring DNA from other bacteria. But there were some surprises too, such as the bug's many genes devoted to transporting and metabolizing sugars.

S. pneumoniae

Streptococcus pneumoniae is the third completely sequenced bacterium that lives in the human throat and nasal passages. Of the three, S. pneumoniae can degrade the greatest number of sugars—at least 14 varieties. Thirty percent of the bacterium's genes are devoted to molecular transport, and most of these ferry sugars, according to the report in this week's Science.

"This bacterium is a real champion at using sugars, which gives it great versatility in feeding," says Hervé Tettelin, of The Institute for Genomic Research (TIGR), in Rockville, Maryland, where the sequencing was done. "Being able to degrade so many sugars could be an evolutionary advantage."

Comparisons of the three bacterial genomes suggest that S. pneumoniae occupies a niche related to its reliance on sugar, the researchers say. Rather than competing with its neighbors in the human upper respiratory tract, the bacterium may peacefully coexist with Haemophilus influenzae and Neisseria meningitidis.

"The three bacteria may be using different food sources," says Susan K. Hollingshead, of the University of Alabama at Birmingham and a collaborator on the project. "Streptococcus pneumoniae likes to exist on sugars, while the others prefer a more balanced diet, if you will."

Five percent of the sequenced S. pneumoniae genome consists of repetitive DNA and mobile elements, or DNA that hops around the genome, the researchers found. So much repetition is unprecedented. Other completely sequenced bacterial genomes have three percent or less repetitive DNA.

Detail of circular representation of the S. pneumoniae TIGR4 genome. View full

Streptococcus pneumoniae is known for acquiring new genes through contact with other bacteria. "This bacterium is highly efficient at taking up and integrating and maintaining foreign DNA in the genome," says Tettelin. "This provides for a lot of plasticity."

The acquisition of DNA is self-administered gene therapy, says Donald A. Morrison, of the University of Illinois at Chicago. An expert on genetic recombination in bacteria, Morrison has designed laboratory experiments based on the bug's tendency to pick up genes.

"You can construct a synthetic replacement for a gene and virtually overnight have a strain of the bacterium with the new gene," says Morrison, who consulted on the annotation of the S. pneumoniae genome. "These bacteria can recognize DNA around them, and they will use the foreign DNA to replace theirs if the two are similar."

"The complete genome sequence, plus the ability to do directed mutagenesis," he adds, "makes this a very nice model organism." The sequenced strain, known as TIGR4, has 2,236 genes arranged on a single chromosome.

The TIGR4 bacterium was isolated from a 30-year-old male patient in Norway in about 1987. Susan Hollingshead tracked down information about the isolate after realizing that it closely resembled a second strain used in mouse pathogenicity studies in her laboratory. She corresponded via email with Ingeborg Aaberg and Eric Falsen, the researchers who contributed the Norwegian and Swedish strains, respectively.

Ferritin-label shadows capsular material.

"We have a strain that came from Sweden a decade earlier than TIGR4 came from Norway," says Hollingshead. The strains are different but clearly related: A gene that is typically quite diverse is identical between the two strains except for one chemical base.

By the end of 2001—if not sooner—researchers will have three sequenced S. pneumoniae genomes to compare. Hollingshead, in collaboration with Tettelin at TIGR, has completed the draft sequencing of a type 6 strain renowned for its antibiotic resistance and global distribution. John I. Glass, of Eli Lilly & Co., in Indianapolis, Indiana, and colleagues will publish a third sequence in October.

Pharmaceutical companies have had private sequencing efforts underway for years. Earlier this summer, researchers at GlaxoSmithKline S.A., in Tres Cantos, Spain, made public a draft sequence of a S. pneumoniae 19F clinical isolate, reporting their findings in Microbial Drug Resistance.

The more sequenced genomes the better, says Hollingshead. In a "diversity genome sequencing project" at the University of Alabama at Birmingham, researchers are sequencing 20 chromosomal regions in 14 S. pneumoniae strains selected for their diverse traits. The goal is to identify genomic differences underlying invasiveness and virulence, for instance.

‘R6 is the flagship strain among researchers’

"Some strains have better ways of getting from the nasal passages to the blood stream, which is what we mean by invasiveness," says Hollingshead.

Individuals can carry multiple strains—some harmful, some not—at the same time. Contact among bacteria has probably contributed to the greater number of antibiotic-resistant strains seen in the last decade. Today, 25 to 80 percent of clinical isolates are resistant to penicillin, compared to 1 to 5 percent in 1990.

Streptococcus pneumoniae tends to affect the very young and very old, because the immune system is not fully developed early in life and can decline late in life. The pathogen is the most common bacterial cause of acute respiratory and ear infections, and is responsible for three million deaths among children worldwide each year.

The Eli Lilly group sequenced R6, an avirulent laboratory strain. R6 was derived from the bacterium used in the 1940s to show that DNA is the genetic material of life.

"R6 is probably the best characterized of all types of S. pneumoniae," says Glass. "It's the flagship strain among researchers." His partner on the sequencing project is Incyte Genomics, of Palo Alto, California.

The R6 strain lacks a capsule—the 'envelope' that surrounds the bacterium and offers protection against the host immune system. The genes responsible for the capsule are largely found in a cluster. Through DNA transfer, S. pneumoniae can acquire the capsule of a bug with a relatively different genome.

In the new study, the TIGR group identified genes outside the main cluster linked to the synthesis of the capsule. "Apparently, the gene cluster codes for a core capsular structure, and other genes contribute to the specific type of structure," says Tettelin, noting that there are more than 90 types of capsules.

Detail of overview of metabolism and transport in S. pneumoniae. View full

His team used bioinformatic tools and computer models to predict biological functions for more than half the genes. They identified, for instance, 69 genes that encode proteins on the surface of S. pneumoniae cells. Cell surface proteins are the interface between the bacterium and the host.

The computational analysis revealed a novel group of genes that appear to help move proteins to the cell surface. "These genes may constitute a previously uncharacterized mechanism involved in the transport of surface proteins," says Tettelin.

The bug's genome includes genes for novel enzymes that are secreted by the bacterium and essentially chew up carbohydrates in the host, the researchers say. The enzymes serve a dual role by damaging the host and providing the pathogen with nourishment.

Using a DNA microarray representing the complete genome sequence, the researchers compared the gene content of TIGR4 to that of R6 and a virulent strain, the D39 serotype 2 capsulated strain. They found nine gene clusters in TIGR4 that were absent in the other two. "The majority of loci that differ between the three strains are surface exposed and/or related to pathogenesis," the researchers write in Science.

"No one is better at microbial genome sequencing than TIGR," says Glass, who will work closely with the TIGR group on comparative analyses of R6 and TIGR4. "I think the Science paper is an elegant piece of work and one of their best genome papers. The annotation essentially shows how the phenotype of S. pneumoniae is an extension of the genome."

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Tettelin, H. et al. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293, 498-506 (July 20, 2001).
Dopazo, J. et al. Annotated draft genomic sequence from a Streptococcus pneumoniae type 19F clinical isolate. Microb Drug Resist 7, 99-125 (Summer 2001).

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