Jurassic dinosaurs are a tough act to follow. How about TertiaryBacilli?

Movie-goers know by heart how scientists cloned dinosaur DNAfrom the gut of an insect entombed in amber 100 million years ago,and expressed living, breathing, roaring dinosaurs in a tropical park.That, they also know, is strictly science fiction.

What's science fact is that a California microbiologist RaLl Cano hasbrought back to life a microorganism, Bacillus sphaericus, thatinhabited the abdomen of a stingless honeybee, entrapped in amber25 to 40 million years ago.

This work marks the first proof that any organism approaching suchantiquity can be brought back to life, Cano said.

Today's issue of Science tells the story in an article by Cano titled"Revival and identification of bacterial spores in 25-to-40-million-year-old Dominican amber." That 15-million-year age bracket defines the Oligocene Epoch in the Tertiaryperiod of geologic time, just as Jurassic covers 250 to 195 millionyears ago. (See BioWorld Today, June 10, 1993, p. 1.)

Cano reawakened those ancient bacterial spores from their longhibernation not by genetic engineering, but simply by removing themfrom their hermetic amber time capsule to a livable, rehydratedenvironment. But it took a panoply of molecular genetic techniquesto do the job, as he relates in Science.

Cano told BioWorld Today that his paper reports only on one of themany species of amber-preserved microorganisms he has restoredfrom dormancy to activity. "We've already seen our ancient B.sphaericus produce antibiotics and industrial enzymes, as well ascarry out fermentations," he said.

He has tested these archeo-antibiotics in a pharmaceutical industryscreen of five pathogens, against which they showed activity. "Wehave seen that they produce antibacterial and antifungal antibiotics,"he added.

Cano, who chairs the microbiology program at California PolytechnicState University in San Luis Obispo, observed that "so much of thisdrug-discovery screening goes on in rain forests or elsewhere in theworld. We are actually sampling rain forests that are no longer here,screening in time as well as space _ adding another dimension."

Ambergene Inc., of San Carlos, Calif., founded in late 1992, "iscommercializing the potential of our organisms," he said. At the timeof the company's founding, as part of their investor due diligence,"the founders required independent verification of our methods. Sowe basically provided a biotechnology company of good standing inthe San Francisco Bay Area with our methodology and samples. Wesaid to them: `Here is how we do it. Can you do it?' And in fact thatcompany did reproduce it."

Ambergene, for which Cano serves as a consultant, "is funded byprivate investors. And they are looking for bucks," he said. "This isexpensive research. My university does not have a stake inAmbergene, which has purchased the technology from it in exchangefor royalties."

That acquisition included "rights to the entire collection of ourmicroorganisms, isolated from insects in various types of amber. Allthe organisms we had, 15 or 16, were freeze-dried and shipped overaccording to guidelines governing shipment of pathogens."

Pending Patent Goes For Broadest Protection

Now, "as a consultant, I've been asked to help in the design ofexperiments that will allow one to detect the presence of new anddifferent types of drugs."

When and as discovered, these products expect to be protected by "avery broad patent application," Cano noted, "which Ambergene filedprobably early last year. It covers basically any microorganism,bacterium, metabolite or fungus recovered from any fossil resin." Headded, "I don't know how much of that [patent] is going to issue, butit's going to be a fair amount. This is a pioneering invention, a brand-new source of life-saving pharmaceuticals or valuable industrialprocesses."

The Bacillus spores that Cano reincarnated started life in thedigestive tract of an extinct, stingless, Oligocene honeybee,Proplebeia dominicana. This hapless insect got stuck in the oozingsap of a tree, which solidified around it as amber.

Just as Escherichia coli flora populate the human digestive tract toaid food metabolism, so Bacillus species set up symbiotichousekeeping in the gut of honeybees, ancient and modern. Thisprokaryotic/eukaryotic partnership helps the bee produce andpreserve larval nutrients.

To extract their desiccated endospores from the amber-entombedbees, Cano and his graduate student, first author Monica Borucki,immersed the blob of hardened sap in liquid nitrogen, then dabbedthe super-cold surface with hot sterile saline solution. This crackedthe amber, and enabled them to tease out the insect's stomachcontents.

They date these fossilized bacteria by reference to microscopicfossils in the rock strata where the amber was buried. "Thecharacteristic enzymatic, biochemical and 16S [sedimentationmeasure] ribosomal DNA profiles," their Science paper states,"indicated that the ancient bacterium is most closely related to extant[i.e., modern] Bacillus sphaericus."

Besides assessing the utility of the ancient bacterial metabolites,Cano's program is now focusing on "assessing the organisms' geneticdiversity and uniqueness. We're going to be constructing genomiclibraries, doing sequencing and so on."

First R & D Ally Already On Line

He and Ambergene are now "looking for mutually beneficialpartnerships to help us go further in the pharmaceutical, industrialand fermentation areas." One ally they already have is MicrocidePharmaceuticals, of Mountain View, Calif. (See BioWorld Today,July 8, 1994, p. 3.)

That firm's chief operating officer and founder, Keith Bostian, toldBioWorld, "Microcide is focused on the discovery and developmentof new antibacterial drugs, specifically trying to find agents thatwould work against drug-resistant pathogens. We've formed arelationship with Ambergene, in which we are collaborating to usetheir ancient organisms to find new antibacterial compounds."

Bostian went on, "So we're applying our screening technology, whichis pretty substantial, to their diversity. Hopefully, out of that willcome new classes that will be developed either jointly or byMicrocide, as therapeutic agents.

"We're trying to help Ambergene right now demonstrate proof ofprinciple. Hopefully, it will position them to go forward and get thefunding for their company to grow and take advantage not only of ourcollaboration with them in the antibacterial area, but also their pursuitof other pharmaceutical indications, especially in the antifungalarea." n


-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.