By David N. Leff

Russian scientists, a Chinese news agency reports, have developed a hydrogen-powered automobile engine that produces only water vapor as exhaust.

Nature did it first, at the dawn of life on earth.

A heat-tolerant, aerobic bacterium by the name of Acquifex aeolicus makes its living by burning hydrogen and oxygen to yield water.

As announced in today's issue of Nature, dated March 26, 1998, A. aeolicus is the 12th microorganism to have its genome completely sequenced — and the first in 1998. The report is titled: "The complete genome of the hyperthermophilic bacterium, Acquifex aeolicus."

The bacterial genome comprises 1,551,335 base pairs.

The Nature paper's senior author is molecular biologist Ronald Swanson, director for genomics for Diversa Corp., of San Diego, Calif.

"We're a company in enzymes and bioactive molecules," Swanson told BioWorld Today, "and we do a lot of discovery by activity screening. But as a complement to that, we decided to take on a project based just on sequence, so we could compare what we were discovering by activity, and also acquire a very broad range of hyperthermostable enzymes.

"We picked this particular organism," Swanson continued, "because it's one of the oldest and most heat-tolerant bacteria known. It can survive at temperatures as high as 95 degrees Centigrade [203 degrees Fahrenheit] — the highest of any known bacterial species — and in environments with only 7.5 parts per million in oxygen concentration. That's 28,000 times less oxygen than exists in the earth's atmosphere."

Commercialization Leapfrogs Genome Analysis

The company's chairman, president and CEO, Terrence Bruggeman, observed: "In attempting to determine the gene-encoded information responsible for the heat-loving qualities of A. aeolicus, Diversa scientists may also uncover the genetic origins of the first bacteria." He added: "In addition to the knowledge we are adding to evolution studies, we believe these specialty enzymes and bioactive compounds will have multiple applications in a variety of chemical industry processes and pharmaceutical development programs."

Microbiologist Robert Huber, a co-author of the paper, is at the University of Regensburg, in Germany, "where A. aeolicus was isolated," Swanson said. "He supplied us with information about the organism's physiology, which helped us interpret the sequence of its genome."

Regensburg scientists discovered A. aeolicus, Swanson recounted, "growing in a hot spring located in a couple of meters of ocean off a volcanic beach on the Italian island of Vulcano. The German team cultured it from there," he said, "so we were able to sequence it from a pure isolate culture.

"We'd already commercialized some enzymes from the organism before the sequencing was finished," Swanson recalled. "We're expressing a number of the more than 1,500 genes we discovered in there, and so far all the aminotransferases we've expressed are indeed thermostable, as we predicted. The sequencing was actually completed but not analyzed in February of 1997. After that, it took a long time to analyze and then get the results published.

"We're marketing some of them in small screening kits," Swanson continued. "Each kit might have seven to 20 different enzymes of the same type, but each with different properties in terms of their thermostability or pH optima, or substrate specificity."

One of the biggest industrial uses for these enzymes is in chiral resolution of pharmaceutical intermediates. That is, separating the optically left-handed (L) and right-handed (D) versions of a racemic (D/L) mixture in the precursor of a medicinal compound

"A. aeolicus is a rod-shaped bacterium;" Swanson said, "it doesn't look all that different from [Escherichia] coli. It attaches to rocks on the sides of the hot springs, and some related strains actually form filaments found in streams. You can see them near the geysers in Yellowstone National Park."

In August 1997, Diversa signed a five-year bioprospecting agreement with Yellowstone Park authorities. The firm pays an annual fee in return for access to certain areas within the park, looking for lead compounds in microorganisms collected and isolated from thermal springs.

In tackling the total sequencing of A. aeolicus genome, Swanson recounted, "we decided that the genome should be able to be done faster and cheaper. So we devised a strategy that we thought would be less labor-intensive and less expensive than those that had been used before to sequence genomes."

He explained: "Instead of sequencing 99.9% of the genome in the random phase of the shotgun process, we sequenced only 97%. That translates into about 10,000 fewer sequences. Then we took up a strategy of sequencing off large inserts to close all the gaps. Because if you sequence less than the random phase, you end up with more gaps in the sequence.

"So there's a trade-off," he went on. "It costs more to close the gaps than to do a single random sequence. But the information gained is obviously much higher. We did the entire genome with only 13,500 sequences."

A. aeolicus' Gene Housekeeping Is Something Else

Citing some of the insights afforded by the whole view of A. aeolicus' gene endowment, Swanson said: "On the scientific side, one thing that's very obvious is that the organization of genes here is very different from what's been seen in the genomes of Bacillus or E. coli. That's probably because this organism is an autotroph — that means it just grows on CO2 as the carbon source and doesn't grow on a lot of different sugars, doesn't take up a lot of amino acids, so it has very different requirements for what it does in terms of controlling gene expression.

"In that sense," Swanson continued, "it's much more like, say, Methanoccus, which is an archaebacterium, not a true bacterium, but is also an autotroph.

"One of the paradigms from classical molecular biology holds that bacteria organize their genes [and their operons] in certain ways. Archaebacteria do not.

"Another interesting thing about this organism," Swanson added, "is that it can build everything it needs from simple components, with a genome that's relatively small — only a third the size of E. coli's." *