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 Despite lacking organelles, microbial cells muster an tiny and there weren’t obvious structures.” extraordinary degree of INTERNAL ORGANIZATION Shapiro says the first hints that bacteSOPHIE L. ROVNER, C&EN WASHINGTON rial 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 no apparent order. It possesses almost no FOR DECADES, researchers believed that Lutkenhaus, a professor in the microbiolorganelles, and it has no nucleus to contain bacterial cells lack the tightly controlled inogy, molecular genetics, and immunology its chromosome. ternal architecture seen in eukaryotic cells, department at the University of Kansas “The traditional view of a bacterium the type that animals, plants, fungi, and Medical Center, Kansas City. was that it was an amorphous vessel with protists such as algae are made of. The Lutkenhaus lab reported that FtsZ, a proteins floating around freely in it,” says Viewed through an electron microscope, protein required for cell division, aggregates Harvard University biology professor Richeukaryotic cells are like the order-obsessed to form a ring structure at the site where a ard M. Losick. Felix Ungars of the cellular world in that their cell is going to divide (Nature 1991, 354, 161). Because bacterial cells are typically less organization appears downright fastidious. Lutkenhaus detected this activity using the than a micrometer in diameter—compared Inside one of these cells, the nucleus neatly intestinal bacterium Escherichia coli. with eukaryotic cells, which are tens of encapsulates DNA—whose own molecular Shapiro’s papers concerned chemoremicrometers in diameter—researchers order is legendary—within a membrane. ceptors, which bacteria use to sense chemifigured that “diffusion of molecules would Other organelles such as mitochondria, cals, including nutrients and toxins, in happen in milliseconds,” says Stanford chloroplasts, and Golgi bodies are also tidily their environment. The researchers reportUniversity developmental biology profeswrapped in membranes. The cytoskeleton ed that chemoreceptor membrane proteins sor Lucy Shapiro. “So the conventional provides additional subcellular structure and associated chemotaxis proteins cluster wisdom was that there was no need for a and organization. Consisting of minuscule predominantly at the ends or “poles” of bacterial cell to localize anything because filaments and hollow tubes, the cytoskeleton crescent-shaped Caulobacter cressupports the cell’s shape and serves centus cells (Genes Dev. 1992, 6, 825) as a set of roadways for shuttling SUPPORT STRUCTURE A helix of MreB (green) and rod-shaped E. coli cells (Science proteins and organelles around the helps shape this 1.5-µm-long B. subtilis cell. 1993, 259, 1717). cell. Furthermore, proteins associResearchers have since uncovated with a given function localize ered more and more evidence of within specific regions of the cell. subcellular organization in bacteria The bacterial or prokaryotic as imaging techniques have grown cell, on the other hand, appears more sophisticated. One key adto have been assembled by Oscar vance was the mid-1990s developMadison, Felix’ disheveled partment of green fluorescent protein ner of the “Odd Couple,” with as a tool for molecular biologists, its innards scattered about with KNOW YOUR ENEMY
Insights into the inner workings of bacteria could help in the battle against species such as Salmonella typhi, which causes typhoid fever. These cells are almost 2 µm long.
BACTERIA BOAST UNEXPECTED ORDER
JE FF ERR INGTON / U O F N EWCASTLE
VOLK E R B RI N K MAN N / M AX PLAN C K I N STI TU TE FO R IN FECTIO N BIO LO GY
SCI E N C E & TEC HN O LO GY
W W W.C EN - ON L INE .ORG
42
F E B RUA RY 16, 20 09
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
Bruker Optics
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
Jacobs-Wagner
F R E D E R I C K B ECK H A M
COURTESY O F CHRI STI N E JACO BS -WAG N E R/ YALE
karyotic 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
Losick says. The fluorescent marker made it Fluorescence possible to tag microscopy reveals individual proPopZ (labeled in teins and then red) clustered at the visualize their poles of C. crescentus location inside cells. The protein bacterial cells. keeps chromosomes “Before that, correctly oriented people used during cell division. 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.”
POLE TO POLE
Affordable FT-IR without compromises 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 eu-
About the foot-print of a lab notebook, ALPHA is a small FT-IR spectrometer that will play a big part in your daily routine. Plug & play set-up, easy-to-use software, combined with QuickSnapTM accessory modules assure powerful and reliable FT-IR analysis you expect from Bruker. See us at PITTCON 2009 - Booth # 4254 www.brukeroptics.com
[email protected] 1-888-4BRUKER
think forward
W W W.C E N -O N LIN E .O RG
43
F E B RUA RY 16, 20 0 9
F T-IR
COURTESY O F J E FF E RRIN GTO N / U O F N EWCAST LE
SCI E N C E & TEC HN O LO GY
“It took longer to realize that bacteria are highly organized because they are so tiny and there weren’t obvious structures.” 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. Be-
fore 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
TOUGHING IT OUT
In the face of inhospitable conditions, bacteria such as B. subtilis (red) undergo a complex transformation to form a tough-coated spore (green), which can better survive stresses such as radiation or a lack of food. The Bacillus cell is 2 µm long.
W W W.C EN - ON L INE .ORG
44
F E B RUA RY 16, 20 09
COU RTESY O F RIC HARD LOSIC K/H A RVA R D
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
vide 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
of them. The Cau- CHROMOSOME SHIELD Errington has lobacter protease found a protein that ClpXP, for inprevents cell division stance, responds in the vicinity of the to a phosphochromosome. 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 di-
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
Get there faster
RegisSEP® GMP SFC Separations Services s Fast Project Execution
s Chiral Expertise
s GMP Compliant
s Bulk Chiral Phase
s 30+ Years of
Separation Expertise
Manufacturer
s Green Technology
s Custom Synthesis
8 0 0 . 323 . 814 4 w w w. R e g i s Te c h . c o m
SERVING
W W W.C E N -O N LIN E .O RG
THE
45
SCIENTIFIC
F E B RUA RY 16, 20 0 9
COMMUNITY
SINCE
1956
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.”
BACTERIA BUSTER
FtsZ (green) normally coalesces into a ring in the middle of cells (blue) at the site where the cells will divide (right). When cells are treated with the experimental antibiotic PC190723 (shown), FtsZ can’t coalesce, and the cells fail to divide (bottom). O F
NH2 F S
O N
LEAN
GREEN
Density & Refractometry
MACHINE
Also introducing, our NEW Green Electrodes: s RoHS and WEEE compliant electrodes (pH, cond., ISE, DO) s ENERGY STAR® compliant electrochemistry meters
Titration
Balances
Cl
ANALYTICAL
'PGTI[GHƂEKGPVCPFGPXKTQPOGPVCNN[ HTKGPFN[RTQFWEVUHTQO/'66.'461.'&1
Thermal Analysis
N
Visit www.mt.com/chem, or call 1-800-METTLER
W W W.C EN - ON L INE .ORG
46
F E B RUA RY 16, 20 09
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. ■
DAV I D ADAMS & JE FF E RRI N GTO N /U O F N EWCASTLE
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
