TIGR Introduces Vibrio cholerae Genome

The bacterium that causes cholera has joined the elite club of organisms whose genomes have been sequenced. Since 1817, seven pandemics of cholera have left millions dead from the dehydration caused by relentless diarrhea. But the disease has been noted for at least 1,000 years. "Cholera is one of the most ancient diseases we know about. Its home is the Ganges delta in India and Bangladesh; it has a long history there. It became a worldwide disease in 1817, when the first pandemic started

Aug 20, 2000
Ricki Lewis

The bacterium that causes cholera has joined the elite club of organisms whose genomes have been sequenced. Since 1817, seven pandemics of cholera have left millions dead from the dehydration caused by relentless diarrhea. But the disease has been noted for at least 1,000 years.

"Cholera is one of the most ancient diseases we know about. Its home is the Ganges delta in India and Bangladesh; it has a long history there. It became a worldwide disease in 1817, when the first pandemic started. The disease spread to the Middle East and Europe, to South and North America and Africa. We are now in the middle of the seventh pandemic. Cholera is everywhere, in 75 countries, on all continents," says Bradley Sack, a professor of international health at Johns Hopkins University.

Robert Koch identified the pathogen whose toxin causes cholera in 1883. But cholera left its stamp on human history at a time between the first pandemic and Koch's work, and today in London, the John Snow Pub stands in remembrance of that event. From 1849 to 1854, English physician John Snow deduced that the source of two cholera outbreaks in the Broad Street area was raw sewage in the drinking water. When the handle was removed from the water pump, the epidemic ceased, and Snow's waterborne disease hypothesis replaced the prevailing idea that cholera spread through casual contact or "bad air." The work is widely hailed as founding the field of epidemiology.

Despite its reputation, V. cholerae is not exclusively a bane of human existence. It persists quite unfettered in oceans and brackish waters, living on the chitinous outsides of plankton and other aquatic species. The genome organization and sequence may finally illuminate the fascinating ecology of this organism, for in its smaller chromosome--a genetic element that is more than a plasmid yet tiny as chromosomes go--may lie clues to how a resident of oceans and estuaries acquired, presumably through horizontal gene transfer, genes that enabled it to become a human pathogen.

Claire Fraser
The DNA sequences of the two circular chromosomes that are the genome of V. cholerae El Tor N16961 come courtesy of teamwork from The Institute for Genomic Research (TIGR) in Rockville, Md., veteran of more than 20 such efforts so far; Rita Colwell and colleagues at the Center of Marine Biotechnology at the University of Maryland; J. Craig Venter from Celera Genomics Group, Rockville, Md.; and John Mekalanos of Harvard Medical School.1 Following, in question-and-answer format, is how TIGR president Claire Fraser describes the work and what it may reveal.

Q: How did the presence of a second chromosome affect the progress of the sequencing project?

A: The project took a lot longer because of that. A 1996 paper had a physical map that suggested that V. cholerae had a single 2.8 megabase chromosome.2 We used that paper to write a grant for funds. Then toward the end of the random sequencing, we realized from continuous sequences that the genome size had been underestimated. We kept going and going and going, and it just didn't make any sense. We did a 15-fold coverage of a 2.8 megabase chromosome, but nothing we did could get it to assemble as one chromosome. Then another paper appeared from James Kaper at the University of Maryland School of Medicine, showing that V. cholerae has a two-chromosome genome.3

So the size we found was right. This says that the methodology is very robust. You can't generate artifacts through sequencing. But it was extremely frustrating to work on, for us and for scientists in the community saying that we were too slow--why weren't we making better progress? This wasn't the first time we started out with incorrect information on a genome's size or organization. We should know by now that when knowledge and information are confirmed, it's great, but we are not going to get our dander up when our data do not fit physical map data. We need to go back and look at the original cholera paper and figure out how those data were misinterpreted.

Vibrio cholerae GENOME AT A GLANCE

Total number of base pairs: 4,033,460

Number of protein-encoding genes: 3,885

Larger chromosome = 2,961,146 bases: housekeeping genes, pathogenicity factors

Smaller chromosome = 1,072,314 bases: many genes of unknown function, and plasmidlike "gene capture" and "host addiction" genes
Q: What is the significance of the second chromosome?

A: This isn't the first bacterium known to have a second chromosome by any means, but it is the first where the evidence is overwhelming that [the second chromosome] may have come from somewhere else. Several pieces of information led to our thinking of it as a genetic element acquired sometime in the history of cholera:

--Phylogenetic analysis of genes on the smaller chromosome, particularly the subset for DNA replication, segregate with the same kinds of genes that are on various plasmids in several species.

--Genes of various functions are distributed differently on the two chromosomes. Significantly more housekeeping genes, such as those involved in DNA replication, transcription and translation; cell wall synthesis; and pathogenicity are on the large chromosome. Only a small number of genes that we think are essential are on the small chromosome. Maybe they were transferred there from the large chromosome at some point in the history of the organism. There are also a larger number of genes of unknown function on the second chromosome.

--When we ask questions about the genes that genomes are most similar to, a diversity in origin shows up. V. cholerae is most similar to other gamma proteobacteria such as Hemophilus influenzae and Escherichia coli. But the second chromosome seems much more diverse in origin, which might be a function of its gene capture system. Through evolution, this genomic element may have acquired and internally stabilized various genes from other species.

All of this evidence helped us convince ourselves that Vibrio at some point was able to get along just fine without the second chromosome.

Q: Is the second chromosome a very big plasmid--or a very small chromosome?

A: We've seen multiple chromosomes before. Deinococcus radiodurans, the radiation-resistant organism, has plasmids. But they are clearly plasmids, not chromosomes. The distinction between a large plasmid and a small chromosome is a functional definition. According to our definition, chromosome implies essential genes are there. A chromosome must be replicated. Even if it has a limited number of essential genes, we call it a chromosome.

Cholera is the first example where such a large percentage of the total genome is represented on the genomic element. The second chromosome represents more than a quarter of the genome, but we certainly do not see a quarter of the essential genes in this organism in this element. It is quite striking.


Q: How will knowing the genome sequence affect the prevention and/or treatment of cholera?

A: We hope that the data will reveal additional genes that are involved in virulence and pathogenesis. Already we have identified new toxin and adhesin genes. The role that the genome sequence will play in medicine is in perhaps developing a new vaccine. Cholera is unusual with the genomes we've looked at in that it is self-limiting. There is debate even over whether antibiotics are necessary. As long as you can deal with the severe dehydration, it is a four-, five-, or six-day disease that clears itself. With the turnover of small intestinal cells, the organism is gone before you know it.

Still, there is hope that there can be a better cholera vaccine than the ones that are currently available. The vaccines out there are not terribly effective, not very long lived. In the case of the Rwandan refugees in 1994, for example, it was clear what was happening; it was almost inevitable there would be an outbreak--a large number of people, with poor sanitation. If a vaccine was inexpensive and on hand to deal with these types of crises, and we could immunize the people, vaccines could in the short term greatly limit the scope of an outbreak. More than 50,000 people had cholera in the Rwandan outbreak. Oral rehydration therapy is so incredibly effective, but we must get sufficient material to the people who need it over a fairly short time course.


Q: How will knowing the genome sequence help to reveal the ecological interactions of this microorganism?

A: During interepidemic periods, cholera exists in the open ocean associated with plankton. We found a number of metabolic pathways that make sense--for example, cholera uses chitin as the sole source of carbon and nitrogen. It is happy for long times in association with plankton.

Bigger ecological implications are suggested when considering global climate change. People have clearly mapped how changes in ocean temperature from El Niño have affected various blooms of plankton. There have been a number of cases examining climate conditions and effects on coastal waters, and blooms precede cholera outbreaks. It's possible that we will be able to use global climate surveillance to predict cholera outbreaks. Taking that kind of information, combined with a very efficient and inexpensive vaccine and the ability to mobilize forces to deal with potential outbreaks, could have tremendous impact. This tie-in with global climate is incredibly interesting. S

Ricki Lewis (rickilewis@nasw.org) is a contributing editor for The Scientist.


1. J.F. Heidelberg et al., "DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae," Nature, 406:477-83, Aug. 3, 2000.

2. R. Majumder et al., "Physical map of the genome of Vibrio cholerae 569B and localization of genetic markers," Journal of Bacteriology, 178:1105-12, 1996.

3. M. Trucksis et al., "The Vibrio cholerae genome contains two unique circular chromosomes," Proceedings of the National Academy of Sciences, 95:14464-9, 1998.