Issue Date: February 16, 2009
Bacteria Boast Unexpected Order
FOR DECADES, researchers believed that bacterial cells lack the tightly controlled internal architecture seen in eukaryotic cells, the type that animals, plants, fungi, and protists such as algae are made of.
Viewed through an electron microscope, eukaryotic cells are like the order-obsessed Felix Ungars of the cellular world in that their organization appears downright fastidious. Inside one of these cells, the nucleus neatly encapsulates DNA—whose own molecular order is legendary—within a membrane. Other organelles such as mitochondria, chloroplasts, and Golgi bodies are also tidily wrapped in membranes. The cytoskeleton provides additional subcellular structure and organization. Consisting of minuscule filaments and hollow tubes, the cytoskeleton supports the cell's shape and serves as a set of roadways for shuttling proteins and organelles around the cell. Furthermore, proteins associated with a given function localize within specific regions of the cell.
The bacterial or prokaryotic cell, on the other hand, appears to have been assembled by Oscar Madison, Felix' disheveled partner of the "Odd Couple," with its innards scattered about with no apparent order. It possesses almost no organelles, and it has no nucleus to contain its chromosome.
"The traditional view of a bacterium was that it was an amorphous vessel with proteins floating around freely in it," says Harvard University biology professor Richard M. Losick.
Because bacterial cells are typically less than a micrometer in diameter—compared with eukaryotic cells, which are tens of micrometers in diameter—researchers figured that "diffusion of molecules would happen in milliseconds," says Stanford University developmental biology professor Lucy Shapiro. "So the conventional wisdom was that there was no need for a bacterial cell to localize anything because everything could be anywhere."
But technological advances in the past 15 years or so have shown that view to be "completely incorrect," says Christine Jacobs-Wagner, an associate professor of molecular, cellular, and developmental biology at Yale University. And although these organisms have been slow to reveal their subtle complexity, and many of the details are still murky, every passing week yields more proof that "the level of spatial organization in bacterial cells is quite exquisite," she says.
The organization of eukaryotic cells "has been appreciated for a long time because they have conspicuous organelles like mitochondria and a nucleus," Losick notes. "It took longer to realize that bacteria are highly organized because they are so tiny and there weren't obvious structures."
Shapiro says the first hints that bacterial cells were more complicated than they originally seemed came from three seminal papers published independently in the early 1990s by her group and that of Joe Lutkenhaus, a professor in the microbiology, molecular genetics, and immunology department at the University of Kansas Medical Center, Kansas City.
The Lutkenhaus lab reported that FtsZ, a protein required for cell division, aggregates to form a ring structure at the site where a cell is going to divide (Nature 1991, 354, 161). Lutkenhaus detected this activity using the intestinal bacterium Escherichia coli.
Shapiro's papers concerned chemoreceptors, which bacteria use to sense chemicals, including nutrients and toxins, in their environment. The researchers reported that chemoreceptor membrane proteins and associated chemotaxis proteins cluster predominantly at the ends or "poles" of crescent-shaped Caulobacter crescentus cells (Genes Dev. 1992, 6, 825) and rod-shaped E. coli cells (Science 1993, 259, 1717).
Researchers have since uncovered more and more evidence of subcellular organization in bacteria as imaging techniques have grown more sophisticated. One key advance was the mid-1990s development of green fluorescent protein as a tool for molecular biologists, Losick says. The fluorescent marker made it possible to tag individual proteins and then visualize their location inside bacterial cells.
"Before that, people used other methods like immunofluorescence microscopy that were very damaging to the cell," Losick says. "The beauty of the green fluorescent protein is you can look in living cells, and if a protein is dynamic, you can see it move."
THE OTHER CRUCIAL ADVANCE was the improvement in the spatial resolution of the microscopes and cameras used to capture such images, Jacobs-Wagner says. In particular, deconvolution microscopy and photoactivated localization microscopy have significantly enhanced the ability of researchers to visualize single molecules and follow their path inside living cells, Shapiro notes.
These techniques have revealed an astonishing degree of bacterial organization and architecture. Consider bacterial shapes, which range from spheres and rods to spirals, commas, and stars, depending on the species. These shapes are "clearly genetically programmed," notes Jeff Errington, a microbiology professor at the Newcastle University, in the U.K. "The question is: How do you determine shape? We're starting to understand how that works."
One of the breakthroughs, he says, was finding that bacteria have cytoskeletal proteins analogous to those found in eukaryotic cells. The eukaryotic cytoskeleton is based on microtubules, actin microfilaments, and "intermediate filaments."
Bacteria boast numerous counterparts of these proteins, including the tubulin homolog FtsZ and the actin homolog MreB, Jacobs-Wagner says. Her lab discovered the first homolog for proteins comprising intermediate filaments. The group named it crescentin for its role in creating the curved shape of the aquatic C. crescentus bacteria from which it was obtained (Cell 2003, 115, 705). The molecule is found only along the inner curve of these cells.
MreB is also "a pivotal player in cell shape," says Errington, whose team identified MreB's role (Cell 2001, 104, 913). "It forms helical elements that run around the membrane just under the surface of the cell. It's responsible for elongating rod-shaped cells and for giving them a cylindrical shape."
And as Lutkenhaus reported a decade earlier, FtsZ is necessary for cell division, Errington says. The protein polymerizes into a "Z ring" structure at the midpoint of the cell, where cell division then begins. Before cell division, the circular chromosome is replicated beginning at a specific point called the origin. Replication then proceeds in both directions along the circle away from the origin. Once the entire chromosome has been duplicated, the two complete circles disconnect and move away from each other toward opposite ends of the cell. Correct placement of the Z ring in the middle of the dividing cell ensures that the two daughter cells are generally equal in size and that each receives a complete chromosome.
HOW DOES A CELL find its middle? "There's a protein—Min—that sweeps from pole to pole like a ping-pong ball," Losick says. "If you average where it is over time, it's at the lowest concentration in the middle and the highest concentration at the poles." Min inhibits Z-ring formation, so this distribution means that Z-ring formation is thwarted at the poles, but allowed in the cell's middle.
DivIVA, which is found in the soil bacterium Bacillus subtilis, also helps divert the Z ring toward the middle of the cell. The protein "can self-assemble into a kind of lattice that in some way that we don't fully understand is targeted to highly curved areas of the membrane at the ends of the cells," Errington says.
Many other proteins also make their way to the bacterial cell's poles, or at least to one of them. The Caulobacter protease ClpXP, for instance, responds to a phospho-signaling cascade by scuttling to the pole right before DNA replication begins, Shapiro says. Once there, ClpXP degrades the replication inhibitor CtrA (Cell 2006, 124, 535; Proc. Natl. Acad. Sci. USA 2006, 103, 10935). "You can't start replicating until you clear CtrA out of the cell, and this can only happen when both the protease and the CtrA substrate are positioned at the cell pole—which is utterly remarkable," she says.
Shapiro and Jacobs-Wagner recently identified another protein, PopZ, which assembles and oligomerizes into a sort of Velcro at one cell pole in the bacterium C. crescentus. There PopZ grasps ParB, a protein bound near the origin point of the chromosome. During cell division, a second pool of PopZ collects at the cell's other pole, where it fastens on to ParB on the newly replicated chromosome. By anchoring the origin of one chromosome at each pole, PopZ keeps the chromosomes correctly oriented while the cell divides (Cell 2008, 134, 945, and 956).
Other proteins help ensure that cell division doesn't slice through a chromosome. In B. subtilis, for instance, as Errington's team has shown, the protein Noc binds to the chromosome and inhibits cell division in its vicinity (Cell 2004, 117, 915).
ONE OF THE MOST intriguing outcomes of subcellular localization is the bacterial production of daughter cells that differ from one another. When a Caulobacter bacterium divides, for example, it produces a stationary "stalked" cell and a "swarmer" cell that moves around with the help of a rotary tail. The stalked cell goes on to divide into a stalked and a swarmer cell. The swarmer cell can't reproduce until it sheds its tail and transforms into a stalked cell.
The different cell types result from asymmetrical transcription and subcellular positioning of regulatory and structural proteins in the parental and daughter cells, Shapiro notes. For instance, the "MS ring," which is a component of the rotary tail, is only distributed to the daughter cell that is destined to become the swarmer cell.
Asymmetric partitioning also occurs when bacteria form spores, tough cells that can withstand stresses such as radiation or lack of food. During sporulation in B. subtilis, the cell partitions into a small compartment that forms the spore and a larger compartment that initially engulfs the spore and then disintegrates when the spore matures. This transformation requires hundreds of proteins to assemble at specific places and times in the cell, Losick says, and most of the biochemical cues that direct the process haven't yet been identified.
What happens if subcellular organization is disrupted by some sort of error? If the error can't be fixed, it could impair the cell's function—or it could prove fatal. "You need spatial organization for many different bacterial properties," Jacobs-Wagner explains. "This can include motility, chemotaxis, even virulence. But it also affects processes that are absolutely fundamental and essential for life, such as cell division and DNA segregation. So if you disrupt this organization, then the cells are not going to make it."
Researchers are beginning to probe this vulnerability as a novel mechanism for destroying pathogenic bacteria. Shapiro, for example, is working to identify additional proteins that orient the chromosome within the cell. "Those proteins are targets for new antibiotics," she says.
Errington and his colleagues have already designed a lead for a new antibiotic. Their experimental compound prevents FtsZ from forming a Z ring, thereby preventing cell division in bacteria, including the notoriously dangerous methicillin-resistant Staphylococcus aureus strain known as MRSA (Science 2008, 321, 1673). The compound, PC190723, cured mice infected with a lethal dose of S. aureus. The antibacterial drug firm Prolysis, based in Oxford, England, is optimizing the structure of this compound and plans to begin clinical trials in about a year's time.
Although "people often view bacteria as the bad guys," Jacobs-Wagner points out that many of these microorganisms are beneficial in applications such as bioremediation or pharmaceutical production. So leveraging the emerging new knowledge about bacterial cell organization to find new ways of killing microbes is just part of the excitement. "We really want to know as much as we can about bacteria so we can exploit them," Jacobs-Wagner says.
- Chemical & Engineering News
- ISSN 0009-2347
- Copyright © American Chemical Society