BY CHARLES CLAWSON

To better understand the life cycle of a bacterial cell, Stanford researchers took a unique approach. They applied systems-engineering principles to a study of the bacterium Caulobacter crescentus, revealing a surprisingly sophisticated organism made up of multiple circuits and subroutines that run on a tight temporal sequencing.

The process enabled the researchers to monitor 3,000 genes at once, laying a foundation for the understanding of a complete genome.

"For years we studied individual events during the cell cycle, such as the construction process for Caulobacter's motor-driven flagellum and the DNA replication process," said Lucy Shapiro, PhD, professor of developmental biology and the Virginia and D. K. Ludwig Professor for Cancer Research. "We always had to examine one gene or one protein at a time. This is a breakthrough in being able to look at the activity of all the genes simultaneously through a cell cycle."

Shapiro and two Stanford colleagues reported their work in a study in the Dec. 15 issue of Science. The study was funded by the National Institutes of Health, the Ludwig Institute for Cancer Research and the U.S. Department of Defense's Advanced Research Projects Agency.

The ultimate aim of the research is twofold: to fully understand the regulatory machinery of this benign bacterial cell and its parallels in pathogenic bacterial cells; and to understand the molecular mechanism of asymmetrical cell division. Unlike most bacterial cells, Caulobacter doesn't replicate by dividing into two identical daughter cells. Instead it divides asymmetrically into one cell propelled by a flagellum and a second that is stationary.

"How a cell can divide and give the same genome to both daughter cells but have the genomic instructions interpreted differently is a central mystery of life," said Shapiro. "Our goal is to understand molecular details of the genetic network governing this asymmetric process."

The study demonstrates the existence of genetically coded circuits with subroutines in a cell cycle that is short and includes a relatively small gene pool. "By looking at 3,000 of the 3,700 genes in the genome at different times during the cell cycle, we found that genes are transcribed from the DNA at the time they're needed to do specific jobs, such as copying DNA, pulling the chromosome apart or starting cell division," Shapiro said. "They're turned on, they work and they're turned off. Nobody expected to see this kind of very tight, temporal transcription control."

The temporal functioning of Caulobacter genes forms a parallel to actions in yeast cells, suggesting the system has been conserved in higher cells and making its understanding all the more important.

Harley McAdams, PhD, senior research scientist in developmental biology, likened the cell workings to a factory. "Machinery turns on to make the chromosome and then machinery turns on to make a motor that enables the cell to swim around. It just goes ka-chunk, ka-chunk, ka-chunk -- turning things on and off in a very orderly way," he said. "It's amazing."

The study also found that a "master regulator" protein controls a quarter of the genes regulating the cell cycle. That finding could be crucial if researchers find a similar regulatory system in the cells of pathogenic bacteria.

"The regulator works like a contractor who's organizing all the different builders," said Michael Laub, graduate student and lead author of the study. "Knowing who the contractor is makes it easier to reach in and knock out the critical nodes. The surprising thing is there seems to be a small number of these master regulators."

"We have a credible potential within the next five years for getting a complete molecular-level understanding of how this regulation happens," said McAdams. He added that scientists are several decades away from being able to fully understand the regulatory process of human cells because they are vastly more complex, containing 50,000-100,000 genes compared to Caulobacter's 3,700 genes.

A systemic analysis of the bacterium was made possible by the microarray, a technology developed in recent years at Stanford by Patrick Brown, PhD, associate professor of biochemistry. A microarray is a thumbnail-sized device containing a grid of tiny spots that correspond with each gene in a particular genome. Because RNA is produced as cells turn on genes, it can be harvested to provide a snapshot of which genes have been turned on or off at a given instant.

For the study Laub disabled a critical regulating protein in Caulobacter and followed changes in gene behavior through the cell's cycle. He took samples at 15-minute intervals, applying the harvested RNA to a microarray and analyzing it under a laser-illuminated microscope. Brighter spots indicated higher levels of RNA, meaning the gene was more fully expressed at that particular instant.

"It's like taking a snapshot of the RNA levels in the cells at particular time points," said Laub. "You can see how the RNA fluctuates in each gene as a function of time."

McAdams said the approach yielded an overwhelming amount of data. "You get more data in three months than people traditionally would have gotten in a lifetime," he said.

Another essential technology was the availability of Caulobacter's DNA blueprint from The Institute for Genomic Research.