91av

EVERYTHING is black. It’s cold, quiet and damp. If you could look around the
hole you’re sitting in, you’d see an intricate network of passageways twisting
off in all directions. Overhead, a natural ventilation shaft channels air down,
past the silvery sheets of water that cling to the soft walls. You have
companions in this dank pit with you, although not enough. But as soon as
reinforcements arrive, you’ll get the signal to attack . . .

This is war, but not quite as we know it. You’d probably never notice
skirmishes like these, although they go on every day. That’s because the shock
troops are bacteria, and their battlefield, the soil beneath your feet.

In recent years, microbiologists, the war correspondents for this microscopic
campaign, have made great progress in understanding how bacteria wage their
underground wars. Although a soil pore could be less than a millimetre across,
bacteria employ strategies that would fit right in on a conventional
battlefield. Most notably, disease-causing bacteria, like good generals, often
bide their time until they amass a force large enough to overwhelm their target
plant with ease. A special signalling system helps them decide exactly when to
strike. But the latest research has come up with some dirty tricks to help
plants ward off attack—such as spreading misinformation between bacteria
and cutting off their lines of communication.

The notion that bacteria can talk to each other emerged more than 30 years
ago, as scientists studied glow-in-the-dark bacteria that live in the
specialised “light organs” of certain squid and marine fish. The glowing
bacteria probably help mask their host’s shadow, making it harder for predators
to spot from below, but they don’t put on a show unless there are enough of them
around to create a decent light, the researchers learned. But how does a
bacterium know how many others are nearby?

It wasn’t until the 1980s that scientists finally identified the chemical
signal that the bacteria use to communicate: a small carbon and nitrogen-based
molecule of a type called acyl-homoserine lactone, or AHL. The bacteria
constantly pump out tiny amounts of this chemical signal. And because the
chemical molecules are small enough to move in and out of bacterial cells
easily, the microbes can monitor the strength of the signal. If the crowd of
bacteria is big enough, the signal will build up until it reaches a certain
level, at which point it prompts the microbes to change their behaviour. In the
case of the light-organ bacteria, a new set of genes switches on to make them
glow. Since the discovery of this group signalling system, or “quorum sensing”,
scientists have found a whole host of other bacteria that use AHL signals to
coordinate their actions (91av, 16 September 1995, p 30). But
the signal doesn’t always make bacteria glow—it often has more sinister
effects.

One common soil bacterium called Erwinia carotovora uses quorum
sensing to synchronise its attacks on plants. When the bacteria reach a critical
mass, they switch on a set of attack genes to produce enzymes that slice up the
plant’s cell walls and cause its tissues to soften and collapse into a
foul-smelling slime. “Erwinia is basically a brute force pathogen,”
says Rupert Fray of Nottingham University. “It’s like all the soldiers are
hiding in the trenches until there are enough of them to run over the top and
attack the plant. If there are enough of them, the plant’s defences can’t cope
with it.”

But suppose someone gave the order to attack too soon? What would happen
then? Late last year, Fray and his colleagues decided to find out. They
extracted a gene from a bacterium called Yersinia enterocolitica for an
enzyme that reacts with fatty acids to make two slightly different AHL
signalling molecules. They spliced this gene into the most bacterium-like part
of a tobacco plant, its chloroplasts. “They’re basically small photosynthetic
bacteria living within the plant cells,” says Fray. That means the chloroplasts
would probably have the necessary ingredients and machinery to put the new gene
to good use.

To find out if the plant could use its new-found AHL to communicate with
bacteria, Fray snipped off a leaf and pressed it briefly onto a plate of agar
gel. He then doused the plate with bacteria that glow in response to AHL. A
glowing leaf shape appeared in the gel, which told the team that AHL had seeped
out of the leaf and activated the bacteria (Nature Biotechnology, vol
17, p 1017). When Fray repeated the test with the plant’s roots, he found that
they also left a pattern of glowing bacteria. “This shows that we can get the
plants to make the bacterial signal molecules, and make them at high enough
levels,” he says. “And it’s not just the leaves—the roots do it too.”

And if you can talk to bacteria, says Fray, you can also tell them lies. By
sending out an AHL signal, a plant could trick a small but growing colony of
disease-causing bacteria into thinking they have ample reinforcements. The
hapless bacteria would then attack far too soon, while the plant’s natural
defences could still crush the invaders by ordering the suicide of plant cells
near the infection site and releasing a flood of antibiotics. Fray is now
testing the idea. He’s already engineered carrots and potatoes that can send
signals to bacteria. Next, he’ll see if they can send a timely signal to force a
premature bacterial attack.

If plants can talk to their attackers, perhaps they can also cry for help,
says Fray. With their new gene, Fray’s tobacco plants also produce a second AHL
molecule that another soil bacterium, Pseudomonas aureofaciens, uses
for a friendlier purpose—to time its attacks on a plant-damaging fungus.
Fray wondered if the tobacco plants could use their signal to persuade the
bacteria to come to their defence. He knocked out the AHL-producing machinery of
one Pseudomonas strain, so no matter how many of them huddled together,
they still wouldn’t produce the antifungal agent, phenazine. But when the
crippled bacteria grew right next to Fray’s tobacco plants, they pumped out
phenazine perfectly well.

This switch could prove useful. “What this means,” says Fray, “is that you
could have a bacterial strain that would only kill fungi if it was close to your
plant.” For example, farmers could sprinkle AHL-deficient Pseudomonas
uniformly across their fields. But the bacterium would only produce the
antifungal agent where the plants were, so farmers could reduce the likelihood
that the fungi would become resistant to it. So far, though, Fray’s idea remains
on the drawing-board. Indeed, he cautions that you’d have to be very careful
using either of these techniques in fields, because other bacteria may also
communicate via the same AHL signals. Until researchers work out exactly which
bacteria use which signals, any talking plant could be issuing unknown orders to
other bacteria at the same time that it’s confounding its main enemy.

Silent treatment

Spreading misinformation is just one way of influencing the course of a war,
though. A less subtle way is simply to cut all lines of communications. And in
March, Lian-Hui Zhang and his group at the National University of Singapore
found a way to do just that with an enzyme from a bacterium called
Bacillus 240B1 that chops up AHLs. After isolating the gene for this
enzyme, Zhang set about using it to stifle bacterial conversations.

His target bacterium was Erwinia carotovora, the same strain Fray’s
plant fooled into launching premature attacks. Zhang spliced the AHL-chopping
gene into Erwinia and poured a suspension of the modified bacteria onto
cuts in cabbages, cauliflowers and tobacco plants. A week later, the plants
showed little or no sign of disease—even though normal Erwinia
would have reduced the plants to foul-smelling mush by then (Proceedings of
the National Academy of Sciences, vol 97, p 3526).

This signal-breaking enzyme could stop bacteria in their tracks, Zhang
believes. If the bacteria can’t receive their AHL signal, they’ll have no way of
knowing when there are enough of them to infect a plant successfully. Taking a
leaf out of Fray’s book, Zhang suggests the gene could even be added to plants
to silence the bacteria in the soil around them.

Indeed, some bacteria not only destroy AHLs, they dine out on them. “If
bacteria are giving out these signalling molecules all the time, why aren’t
we up to our kneecaps in them?” wondered Jared Leadbetter at the University of
Iowa. Leadbetter suspected that since AHLs are rich in carbon and
nitrogen—vital nutrients for bacteria—it was entirely possible that
bacteria were munching up each other’s signals. “My approach was to look for a
bacterium that would eat homo-serine lactones,” he says.

Leadbetter began his search by mixing assorted bacteria into solutions with
only tiny amounts of AHLs as a food source. Then he sat back and watched what
grew. Most of the bacteria died, but one species, Variovorax paradoxus,
grew quite happily. When Leadbetter mixed Variovorax with a
quorum-sensing bacterium that destroys corn, he found that it kept AHL at a very
low level—far below the threshold needed to switch the pathogen into
attack mode. Variovorax was literally eating up the signals between the
other bacteria—and so preventing them from turning nasty.

While Leadbetter wants to find out if Variovorax really can prevent
infections in plants, that’s not his first priority. Instead, he’s interested in
the complex crosstalk that goes on when several species of bacteria depend on
the same signalling molecule for communication. Among other effects, Leadbetter
suspects that different genes will be switched on when the signalling reaches
different levels. “That would give rise to some interesting nuances and give us
a better idea of exactly what is going on,” he says.

Rallying allies, spreading false information and blocking
communications—these tactics of modern warfare could all play a part in
helping our domestic plants keep the upper hand in their underground wars. But
they might not work forever. Eventually, bacteria could get wise to these dirty
tricks and get around them by changing their language, or making their words
just a little less appetising. Still, it offers agricultural scientists at least
a temporary advantage in their long, tough war against crop diseases.