'Zombie bacteria' key to drug therapy

Even seemingly simple single-celled organisms must have the means to meticulously duplicate their DNA, carefully separate the newly copied genetic material, and delicately divide in two to ensure their offspring survive.

In eukaryotic cells such as those in plants and animals, an elaborate molecular circuitry coordinates duplication and separation of genetic material with division. The cellular control system has sensors that detect anomalies and shut things down if something is wrong.

In terms of bacteria, Heidi A. Arjes, a doctoral student in the lab of Petra Levin, associate professor of biology in Arts & Sciences at Washington University in St. Louis, has presented the first experimental data that show there are at least two fail-safe points in the bacterial cell cycle that tie DNA replication to cell division.

“When I talk to people who study bacterial cells, they’re astonished. It’s a completely novel idea

Heidi A. Arjes

A cell that stumbles at either division or DNA replication can repair itself and re-enter the cell cycle. But if it does not do so quickly, the fail-safes are activated, forcing the cell to exit the cell cycle forever. It then enters a zombie-like state and is unable to reproduce even under the most favorable of conditions.

When nutrients are scarce, bacteria grow slowly, working through the cell cycle step-by-step. In nutrient rich conditions, however, bacteria take advantage of the situation to multiply as rapidly as possible, overlapping two of the steps: cell division and DNA replication – something that never happens in eukaryotic cells.

Because of their ability to overlap these two essential processes, bacteria have been thought to lack the fail-safe mechanisms that ensure stepwise progression through reproduction in eukaryotic cells.

Understanding the mechanisms of bacterial cell-cycle control may have major payoffs for medicine. Drugs that shut down the cell cycle could be used to fight bacterial infections. Not only would they prevent bacteria from multiplying, they would prevent them from passing on resistance genes or recovering once the drug had been metabolised.

The bacterial cell cycle begins when a newborn daughter cell elongates and begins to make another copy of its DNA. As DNA replication proceeds, a division ring, called the Z-ring, starts to assemble at mid-cell. The Z-ring recruits other molecules to form a contractile ring and, once there are at least two complete copies of the DNA, pinches the bacterial cell in two to form daughter cells.

When bacteria find themselves in nutrient-rich conditions, they shift into high gear, doubling in size and dividing as often as once every 20 minutes. To make everything come out right, bacteria employ ’multifork replication’: initiating new rounds of DNA copying before the first round finishes.

Getting a head start on DNA replication ensures at least one set of genetic material will be ready before they divide. Only the most mature DNA round must be complete before the cell divides.

The other replication forks will finish in subsequent generations. Although it takes a bacterium 40 minutes to completely copy its DNA, multifork replication enables it to divide once every 20 minutes.

In a recent article, Arjes and co-workers described experiments that show cell division and DNA replication are not independent. New rounds of DNA replication depend on the successful completion of cell division and assembly of the division machinery at midcell depends on the initiation of DNA replication.

One set of experiments showed that after division is blocked, DNA replication gradually diminishes and, after about five generations, the bacterium reached the point of no return. In other experiments DNA replication was blocked directly.

In that case, it took about three generations for the bacterium to reach the point of no return. The timing suggests DNA replication might be the event that shunts bacteria into the state of suspended animation.

“It might actually be a form of altruism,” Arjes said.

“In nature, bacteria often exist not in isolation, but in communities. An aged or unhealthy cell that removed itself from the population would benefit the community as a whole because it would no longer compete for nutrients or produce defective daughter cells.”

Although the research firmly establishes the existence of two fail-safe points in the bacterial cell cycle, the mechanisms that ensure proper cell-cycle progression are still a mystery.

“That’s the next thing we have to do,” Levin said.

“Figure out how the division machinery is telling the DNA replication machinery something is wrong,” and how the “information that DNA replication isn’t working is communicated to the division machinery.”

These signaling pathways will be great targets for new antibiotic therapies. Drugs that drove bacteria past the point of no return would prevent them from proliferating, stalling an infection. Blocking DNA replication would prevent bacteria from sharing mutations that confer antibiotic resistance.

“Most importantly,” said Arjes, “if a bacterium has encountered a terminal cell-cycle arrest, it cannot recover even after the drug has been metabolised.”

“People are already working on drugs that hit the division machinery,” said Arjes.

“They’ve done a lot of screens; in fact in our experiments we used a new drug called PC190723 that blocks division in Staphylococcus aureus. It was synthesised for us by Jared Shaw, a chemist at the University of California at Davis, and is being tested against MRSA (methicillin-resistant Staphylococcus aureus) isolates.

“Combination therapy with PC190723 and other drugs such as methicillin, an extended-spectrum penicillin antibiotic, appear to be effective against MRSA even though methicillin alone is no longer effective. The division blocker somehow sensitizes the bacteria to drugs to which they have become resistant.”

The eukaryotic cell cycle has been studied for more than a century. Three scientists won the Nobel Prize for Physiology and Medicine in 2001 for figuring out the regulators that prevent the cell from dividing promiscuously and endlessly. It is even possible to play a cell cycle control game at the Nobel site, Nobelprize.org.

“When I talk to people who study eukaryotic cells about our work with the bacterial cell cycle, they say, ’What? This is new? People don’t know this?’” said Arjes.

“But when I talk to people who study bacterial cells, they’re astonished. It’s a completely novel idea.”