By David N. Leff

More and more harmful bacteria are developing resistance to more and more bactericidal antibiotics.

So what else is new?

Here's what: Harmful insects are on the brink of becoming resistant to an insecticidal bacterium. The bug-killer is Bt, a toxin synthesized by Bacillus thuringiensis.

Back in 1982, the U.S. Environmental Protection Agency approved the israelensis species of B. thuringiensis to fight mosquitoes. Its toxin, Bt, had proved it could kill 3,000 species of mosquito larvae, plus the black fly that causes African river blindness (onchocerciasis). At the time, the World Health Organization hailed Bt as "the most effective biological control agent against mosquito larvae."

This hailing recalled the DDT-chic euphoria of four decades earlier. But DDT (dichlorodiphenyltrichloroethane) is a chemically synthesized insecticide, whereas Bt toxin — extracted from a natural soil bacterium — is "organic."

Bt is widely sprayed on crops and in gardens, "but the major emphasis on insect control these days," observed entomological toxicologist Richard ffrench-Constant, "is taking these protein toxins and engineering them in plants.

"The whole driving force behind this work," he continued, "is to make crop plants transgenic for the Bt gene. It's the main thing out there right now, but B. thuringiensis has a limited number of cryotypes, and a limited range of activities against different insects."

As chemical companies genetically endow more and more cash crops with Bt genes, ffrench-Constant — a professor of insect toxicology at the University of Wisconsin-Madison — warned, "excessive exposure to the toxins can provide the evolutionary force necessary to cause the insects to become resistant to them."

To avoid being painted into this corner, he pointed out, "conventional pesticide usage has taught us that whether we like it or not, one of the best ways to manage resistance is just to make new insecticides with alternative modes of action."

His nominee as eventual alternative to Bt is "a rod-shaped bacterium called Photorhabdus luminescens, a member of the Enterobacteriaceae family, just like [Escherichia] coli. The place that P. luminescens would call home," ffrench-Constant added," is the gut of the nematode."

Home gardeners know nematodes — the lowly roundworm — as an organic insecticide. Few know why.

This barely visible creature makes its living by feeding on P. luminescens bacteria. "What happens in that bacterium's life cycle," ffrench-Constant recounted, "begins when a nematode invades an insect. There it releases its store of live bacteria, which replicate. The nematodes feed off these next-generation microorganisms in the insect.

"The bacterium has been adapted into this weird symbiosis," he continued, "where it gets released into the insect circulation. And it there secretes its insecticidal toxin, which we think is the factor that actually kills the insect, by destroying its midgut epithelium. The cadaver then begins to glow with bioluminescence."

Why Glow? Who Knows?

What survival function this light-show serves in the defunct insect is unknown. "My personal pet theory," ffrench-Constant theorized, "is that this luminescence is actually apomictic — a warning coloration. I think of it as a 'no vacancy' sign. It says to would-be scavengers that the motel is full, and warns them, 'don't devour the motel.' Because if a small mammal comes up and eats the cadaver, that's pretty much the end of the whole system."

To prove this concept, he recalled, "One guy painted mealworms with luminescent paint, and this caused mice to avoid eating them.

"It's a very tough call," ffrench-Constant mused, "as to which player in this drama gets what out of what. The bacterium gets a safe place to live; it's put in an environment where it can replicate rapidly and get transferred from host to host. But of course at the same time, a large portion of its population gets eaten by the nematodes."

P. luminescens toxins show insecticidal activity in Coleoptera (beetles) and Dictioptera (cockroaches), but the microbe is particularly partial to Lepidoptera (butterflies and moths). Among Lepidoptera, the large, crop-gobbling caterpillar of a moth called Manduca sexta is to research entomologists what white mice and rats are to medical microbiologists. That caterpillar is far better known as the tomato hornworm.

Ffrench-Constant is senior author of a paper in today's issue of Science, dated June 26, 1998. Its title: "Insecticidal toxins from the bacterium Photorhabdus luminescens."

In this research project, he and his co-authors set out to reveal the molecular mechanism of the bacterium's specificity for Lepidoptera and to isolate the microbe's protein toxins.

"We purified the high-molecular-weight protein fragments expressed by the genes for the bacterium's four toxin complexes — a, b, c and d," ffrench-Constant recounted. "Then, by raising antibodies against these complexes, we cloned the underlying toxin genes."

To prove their role in toxicity and specificity, the team knocked out each of the four genes in turn. They found that deleting toxin a or toxin d reduced the organism's oral activity against Mantuca larva. Knocking out the two together essentially abolished the toxicity altogether.

"So obviously," ffrench-Constant observed, "a and d are very important toxins to Lepidoptera. But it begs the question what these other genes do. We know that b is a direct homolog [similar gene sequence] to d. So, is it active, for example, against beetles or other groups of insects?"

That's what he and his team are working on now.

"I think the big attraction of P. luminescens," ffrench-Constant told BioWorld Today, "is that it represents one of the viable alternatives to B. thuringiensis. I think we're going to sorely need it next year, when 100 percent of the cotton crop will be planted out with a single transgenic Bt-expressing variety.

"With such total coverage," he commented, "you're just asking for resistance — and that's where P. luminescens comes in as an alternative.

Understudy To Bt

"The other major transgenic target of the agricultural companies," he went on, " is corn. The Novartis Agricultural Research Station [in Research Triangle Park, N.C.] has recently come out with a hybrid corn line that contains Bt as full control against the cornborer, and I think it's going to be agricultural product of the year 1998.

"Basically," ffrench-Constant observed, "cotton and corn are the two major places where people would like to see immediate return on their investment."

As for any concern that people may voice at the prospect of eating Bt on the cob, ffrench-Constant made the point: "The bottom line is that if you put Bt in these plants, people are going to end up ingesting a reasonable concentration of Bt. It's harmless to vertebrates."

He added: "If people are concerned about Bt pesticide residues on crops, what are they going to do when we tell them they're eating Photorhabdus luminescens?"

Ffrench-Constant is working with the research and development people at Dow AgroSciences Biotechnology, in Indianapolis, Ind., "looking forward to their engineering of P. luminescense toxin genes into crop plants."

He concluded: "I think that Photorhabdus luminescens is demonstrating that it is the first alternative to Bt on the transgene treadmill. I'm sure there are a lot more bacteria out there that kill insects, and purifying the genes that cause their virulence will really be a sort of gold mine for the biotech industry." *