By David N. Leff

Science Editor

Human beings' microbial friend and enemy, Escherichia coli, inhabits the intestines of mammals and the well plates of biotechnology laboratories.

Shaped like a medicinal capsule, this busy bacterium is about two microns long. That means it would take 10 million of them, lined up end to end, to span this 8.5-inch-wide page. Dividing every 20 minutes or so, E. coli wouldn't need more than one eight-hour working day to reach that 10-million mark. That is, assuming all its progeny survived every doubling cycle.

That's a dicey assumption.

Like all forms of life, bacteria have to compete for sustenance with their natural predators, mainly other microbes. And even though E. coli is one of the tiniest life forms on earth, it is a complex and sophisticated machine for getting food and fighting off foes.

Topping its shopping list of nutrients is the element iron.

"Iron," observed biochemist Phillip Klebba, "is fundamental to the physiology of bacterial cells. This metal is extremely important for all cells that breathe oxygen. There are two kinds of life forms on earth, the aerobes, which require oxygen, and the anaerobes, which do without it. By far the larger group of bacteria and other organisms are aerobic."

Klebba, who is on the University of Oklahoma faculty in Norman, Okla., continued: "In order to use oxygen, aerobes must go through the process of electron transport. That is, oxygen accepts electrons that are generated during metabolism in the cell. It's a way of generating energy."

E. coli spends much of that energy assuring itself of an iron-rich diet. What it's up against is a dilemma. The metal chelates that package its iron supply measure 750 daltons, much too big to pass through the two medium-size channels that stud the bacterial surface. They're both small enough to keep out most toxins, including antibiotics, that menace the microbe's existence.

That leaves the large-size channels, about 20 angstroms in diameter. (An angstrom is one-billionth of a meter.) They're wide enough to admit the iron ration -- but how to keep out those bacterial toxins?

Klebba reveals E. coli's answer to that life-and-death Hobson's choice in today's Science, dated May 23, 1997. The paper, of which he is senior author, bears the title: "Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria."

Microbe Practices Open-And-Shut Strategy

"This third type of pore," Klebba told BioWorld Today, "which we identified in 1992, is normally completely closed. It takes an expenditure of energy to open it, admit iron or other precious metals -- precious to E. coli, that is -- and then quickly shut again."

Below this closed surface, the channel traverses the microbe's bilayer membrane into the cell.

"All these iron-channel pores," Klebba pointed out, "are present on the bacteria's surface at levels that vary, depending on the environment the cells are growing in. If they are bathed in iron-rich surroundings, the microbes know enough to cut down on the number of energy-activated channels to a level of maybe 5,000.

"If not enough iron is present," Klebba continued, "the bacteria will actively secrete a small molecule to chelate the metal, and insert these iron channels, each consisting of a single trimeric porin protein, in their membrane at a level between 50,000 and 100,000 per cell."

In order to smuggle an antibody drug into the cell, that drug would have to contain iron, Klebba pointed out.

"In fact," he said, "a number of very interesting antibiotics have already been made along these lines. They have an iron-containing molecule on one end of the complex, and some kind of an effective antibiotic -- for example, a penicillin analogue -- tethered to the back of the molecule. It's been shown that they will enter through the iron channel and kill the cell."

But Klebba added: "At the present time, these Trojan-horse antibiotics are a little bit disappointing. They do enter the cell through the iron-specific pathway. But they don't seem to add anything new, because the mechanism of killing is still just the same. A cell that was resistant to penicillin would still be resistant to it.

"The main thing that needs to be done now," Klebba suggested, "is not to take a Trojan-horse approach, but to look for drugs that specifically interfere with the operation of the channels themselves. We're working on that, but it's really in the beginning."

He did confide: "Now that we have what's essentially an assay for the opening and closing of the channel, we are in contact with various pharmaceutical companies to obtain their entire spectrum of antibiotics. Then we would have to test each of these to see if it can specifically inhibit the functioning of the channel.

From Antibiotics To Antibodies

"When bacterial pathogens enter the human body," Klebba observed, "they find that all the iron it contains is sequestered, unavailable to them. Somehow, they must actively obtain iron from their human host."

He and his co-workers are working on this tug of war between microbe and man. "If we can shut down this iron-transporting channel, either through the use of chemical therapeutics or antagonists, then we can prevent bacteria from actively growing in the host. That would be of major interest.

"From the perspective of human disease," Klebba pointed out, "it's extremely relevant to consider generating antibodies that would recognize as antigens the iron channel's protein loops that become exposed when the channel opens up. The antibodies would either hold them in a permanently open position, or completely block them so the channels can no longer transport iron.

"This could have implications for disease prevention," he went on. "Suppose, for example, that we could generate these antibodies together with some kind of synthetic vaccine. Then, in populations subject to such deadly diseases as cholera, typhoid, dysentery, even gonorrhea, people could be immunized against these porin loops that open and close. So infectious bacteria would then be thwarted, because whenever the microorganisms tried to transport iron, these anti-loop antibodies in peoples' serum would block that process."

Molecular loops of protein that open and close on signal also suggest to Klebba a quite different potential application: "The use of these channels as biosensors." *