Michael Hoppert & Frank Mayer. American Scientist. Volume 87, Issue 6. Nov/Dec 1999.
What’s in a name? A lot, sometimes. A name might betray an underlying assumption-some might even call it a prejudice-as it does in cell biology.
Take, for example, the names assigned to the two major classes of cells. The cells found in plants and animals are called eukaryotes, literally, “true nucleus.” In contrast, bacterial cells are prokaryotes, a designation intended to convey their relatively primitive status as proto-cells and possible forerunners to the eukaryotes.
The basis for this nomenclature lies in the organizational complexity of the eukaryotes on the one hand and the relative simplicity of the prokaryotes on the other. Such differences became evident early in the 20th century, when improvements in microscopy allowed scientists to get a detailed view inside cells. They noted that both cell types contain a large cellular compartment, known as the cytoplasm, which is surrounded by a membrane. But they also noted additional membranous compartments within the eukaryotic cytoplasm that were absent from the prokaryotic cytoplasm. With more sophisticated microscopic techniques, scientists found an additional distinction between the two cell classes. Eukaryotic cells gain structural support from an internal network of fibrous proteins called a cytoskeleton, whereas prokaryotes gain their primary structural support from a rigid wall that surrounds the cell. But for purposes of defining the two classes, scientists still focus mainly on the issue of compartments.
One compartment in particular serves as the defining distinction between eukaryotes and prokaryotes. This is the nucleus, the membrane-delineated compartment that houses eukaryotic DNA. There are other organizational distinctions: Prokaryotes do not contain any of the other specialized membrane-bounded compartments found in the typical eukaryote. They do not, for example, contain mitochondria, lysosomes or peroxisomes.
Throughout this century cell biologists have refined their understanding of the eukaryotic compartments. Each compartment is a sort of subcellular organ-in fact the compartments are called organelles-housing all of the elements necessary to perform a specific metabolic function. For example, mitochondria generate chemical energy with which to power all the cell’s other activities. Lysosomes and peroxisomes contain enzymes that degrade macromolecules. Each organelle operates efficiently because it bundles together all of the biomolecules required to perform a particular task.
In contrast, prokaryotes lack membranous organelles and a typical eukaryotic cytoskeleton. Therefore biomolecules are commonly supposed to be scattered randomly throughout the prokaryotic cytoplasm. From this notion comes another: Prokaryotic metabolism is extremely helter-skelter and inefficient. In truth, this is not the case.
Prokaryotic cells perform their biological functions with stunning efficiency (Anyone who has ever had a badly infected cut can appreciate just how rapidly bacteria multiply.) So it seems illogical to think of prokaryotes as a bag of randomly distributed chemicals. It makes more sense to suppose that, even without membrane-bounded compartments and a eukaryotic-like cytoskeleton, the molecules required for a particular metabolic activity are grouped together into areas that we like to call functional compartments. The idea of a functional compartment is somewhat analogous to functional areas in a loft apartment. The apartment lacks walls, yet it is possible to identify a “kitchen area” that contains all of the appliances and utensils required to efficiently prepare and eat food. Of course, in the case of the loft, someone has deliberately placed the appliances and utensils in the functional kitchen area. Since the loft does not offer an aqueous environment, these objects are not expected to drift off.
The same cannot be said of the prokaryotic cell. Although cell biologists can discern a sophisticated cellular architecture, cellular components are not deliberately grouped together. Furthermore, the internal environment is aqueous, so compartments could conceivably drift off. Given these factors, how do functional compartments arise and how are they maintained? Studies in our laboratory and others suggest that functional compartments arise spontaneously as a result of the intrinsic properties of the biomolecules themselves and the way they interact with water in the cytoplasm We have also found that the specific structure of water itself can influence the level of enzyme activity in particular microenvironments.
Until the middle of the 20th century, scientists did not clearly distinguish between prokaryotes and eukaryotes. Based on their observations of these cells under the light microscope, they classified bacteria as very primitive eukaryotic cells. Under the increased magnification of the electron microscope, they first glimpsed the profound organizational differences that now distinguish the two cell types. It was then that the terms “prokaryote” and “eukaryote” came to be widely used by scientists.
More modem electron microscopes have increasingly refined the picture of the prokaryotic cell. They have also made cell biologists aware that some prokaryotes-specifically, a class of bacteria known as Gram-negative bacteriaare actually encircled by two membranes. Just a few millionths of a meter inside the outer membrane there is the cytoplasmic membrane. Between these two membranes are the murein sacculus, a major cell-wall component, and an aqueous compartment called the periplasmic space. The periplasmic space is involved in various biochemical pathways, including, for example, the degradation or export of proteins and the extrusion of noxious compounds from the cell.
This refined picture of the prokaryotic cell also extends to structures involved in cell locomotion. Some prokaryotes swim through their environment with the aid of a whiplike appendage called a flagellum. Powered by molecular motors, flagella rotate and in this way propel bacteria through their environments. All parts of the flagellum, including its molecular motor, are composed of protein. The motor is anchored in the cytoplasmic membrane and exposed to the cytoplasm. A part of the flagellum that connects the motor with the flagellar filament protrudes across the periplasmic space, the murein sacculus and the outer membrane. The flagellar filament is located in the cell’s external environment. Hence the flagellum, an organelle per se, spans a number of compartments: the cytoplasm, the cytoplasmic membrane (a functional compartment), the periplasmic space, the outer membrane (again, a functional compartment) and the external environment.
With the exception of the cytoplasm proper and the periplasmic space, no membranous compartments can regularly be found in prokaryotes. But that is not to say that identifiable compartments are entirely lacking, either. Inside the cell, several more distinct compartments can be described. Some of these were evident even in the earliest days of prokaryotic research. Using light microscopes, cell biologists could detect small, highly refractive granules in the cytoplasm of certain prokaryotes. These granules are now known as inclusion bodies. They appear to be storage granules, enclosing large aggregates of water-insoluble materials, such as fats and starches. Materials inside the inclusion bodies are degraded into smaller fragments that are released into the cytoplasm when they are required to fuel a metabolic activity.
Scientists now understand something about the biochemistry of inclusion bodies. Among the fats often found inside are long chains of fatty acids called polyhydroxyalkanoates (PHAs). Like most fats, PHAs repel water, so, naturally, they repel the aqueous cytoplasm.
However, the enzymes that synthesize PHAs are soluble in water, which leads to an interesting situation. While they are being synthesized, PHAs are linked to the enzymes and form a complex, part of which is water-soluble and part of which is not. Eventually, the complex organizes itself into a sphere in which the water-soluble enzymes form the shell, shielding the water-insoluble fatty molecules within. Water is expelled from the interior of this sphere, creating a water-free fatty internal compartment separated from the aqueous cytoplasm by a boundary of water-soluble enzymes. Research conducted in the laboratory of Alexander Steinbichel, now at the University of Munster in Germany, and his colleagues demonstrated that as the PHA inclusion bodies mature, other water-soluble molecules-including small proteins called phasins-are added to the growing circumference of the boundary layer, while more PHAs are added to the interior compartment.
A big part of the cell’s interior space is given over to the bacterial chromosome. This single, very long and circular piece of DNA-on which lie all of the bacterium s essential genes-is found in an area that serves as a nucleus-equivalent. This area is termed the nucleoid. (Additional genes are situated on smaller circles or linear strands of DNA, called plasmids, found in the cytoplasm.) Although the nucleoid is not delimited by a membrane, it nevertheless exhibits an organizational structure of the highest complexity.
In most bacteria, the chromosome attaches to the cytoplasmic membrane at one point and branches out from there, so that the nucleoid gives the overall impression of being shaped like coral. This shape is maintained by proteins that bind to the DNA, causing certain regions to be highly coiled, whereas others are more loosely wound. Other proteins are also associated with the nucleoid, and these are required to replicate the DNA, regulate gene expression and help apportion DNA into daughter cells during cell division.
The differentially coiled regions give rise to distinct domains within the chromosome. The typical bacterial chromosome has about 100 such domains. It is possible that these domains correspond to groups of genes that all need to be either expressed or repressed at the same time, and so they have important functional significance.
Under the microscope, the cytoplasm of bacteria appears uniform, save for the occasional inclusion body. No structure is visible that corrals biomolecules into a particular spot. And yet few metabolic enzymes in the prokaryote are found to be alone. Instead, enzymes that act together in a sequence of metabolic events tend to join together into larger functional units. Victor Norris at the University of Rouen in France coined the term “enzoskeleton” to describe such enzyme assemblages. There exists evidence to suggest that enzymes may well be arrayed within these complexes in the same sequence in which they perform their functions. This would lead to a high metabolic efficiency, as the product of one enzymatic reaction would immediately serve as the substrate for the next, with very little loss of product between steps.
One well-documented example is the multiprotein complex called the cellulosome found on the surface of the bacterium Clostridium thermocellum. This complex allows the bacterium to adhere to and degrade cellulose, the main polymer found in the cell walls and woody stalks of plants. Under conditions where oxygen is scarce, as it is in lake sediments, bacteria such as Clostridium can obtain energy by degrading cellulose and other biological polymers from decayed plants. The cellulosome includes over 14 degradative enzymes called cellulases bound together onto a proteinaceous scaffold (known variously as scaffoldin or CipA), which is anchored to the bacterial cell surface.
The nucleoid, the flagellum and the cellulosome provide examples of associations of many different protein species, some of which are structural and some of which are catalytic. Several other protein aggregates that are built up of large numbers of similar units can be found within the cytoplasm. One of these is the proteasome. This barrel-shaped structure is composed of digestive enzymes, which degrade proteins into their constituent parts. Another barrel-shaped structure called a chaperonin contains repeats of an enzyme whose function is to fold freshly synthesized proteins into their mature conformation.
Responses to Water
The notion that enzymes could be grouped into defined functional compartments has inspired us to explore other types of organization. In particular, we are interested in learning how the microenvironments of the aqueous cytoplasm affect enzyme organization. We have been particularly interested in the interaction between enzymes and the water in the cytoplasm, since we believe this interaction to be of the utmost importance in defining another type of functional compartment.
We have come to our proposition by considering how closely enzymatic studies conducted in vitro approximate the actual interactions that may be important in vivo. Some people will correctly point out that several complex metabolic systems can function, disaggregated, in vitro. But we do not believe that this is an accurate simulation of what actually takes place in living cells. In vitro, the enzymes making up the system are the only ones in the test tube, so they are free from confounding interactions with other components. But this is not realistic. In the living cell, enzymatic systems have to work in a cytoplasm replete with proteins and other compounds. We strongly believe that in this situation, defined enzymatic activities are only possible when the enzymes are maintained at defined distances from other macromolecules. And that is only possible in a structured cytoplasm. Our studies suggest that water itself is an important part of this structure. In 1984 James Clegg at the Scripps Institution of Oceanography in La Jolla, California wrote, “in virtually all interactions, the participating macromolecules first ‘see’ each other through their water structures.”
In other words, the behavior of enzymes-the speed and efficiency with which they carry out their functions and the nature of their interactions-is influenced by the aqueous environment in which they function. This at first may seem a trivial statement, because all agents that are dissolved or suspended in water can modify enzyme activities. Salts, amino acids, proteins and organic solvents can alter the stability and activity of enzymes. What is less evident is that dissolved components may influence enzymes indirectly by altering the actual structure of the water surrounding the enzymes.
To study further the interactions between water and enzyme activities, we have developed a versatile model system for structured cytoplasm. Water can exist in various states. It can be a solid or a liquid, for example. But even liquid water can take on various forms. This becomes particularly evident inside the artificial system of a reversed micelle. A conventional micelle is formed when water surrounds a sphere of amphiphilic molecules. These are molecules in which one end is water soluble, while the other is water insoluble. To form a micelle, water-soluble heads of amphiphilic molecules maintain contact with the water, while the water-insoluble tails are tucked inside. A reversed micelle is just the opposite. The water is held inside a sphere of amphiphilic molecules. A model of cellular water structure developed by Philippa Wiggins at the University of Auckland in New Zealand helped us to understand the role of water in reversed micelles.
Depending on the size of the reversed micelle and the location of the water molecules within it, the water exhibits at least two structures. Water that is close to the periphery of the micelle, in direct contact with the barrier molecules (called surfactants), differs from water nearer the center of the reversed micelle. And both of these structures differ from free, chemically pure water.
The water molecules closest to the surfactants in the boundary layer are less free to move about and are more densely packed than are the molecules in free water. Densely packed water molecules are less likely to form hydrogen bonds with neighboring water molecules than are the molecules in free water.
In contrast, water farther away from the surfactant boundary is less densely packed than is free water. The low-density water balances out the effects of the high-density water and brings the entire system into thermodynamic equilibrium. Low-density water forms a greater number of hydrogen bonds than does pure water. The highly bonded low-density water results in a regular network of water molecules, resembling somewhat the molecular lattice of ice. In addition to maintaining a regular structure, low-density water is less electrically charged, less reactive and more viscous than regular water.
In living cells, all cellular compartments and macromolecular assemblies affect water structure and create a framework of low- and high-density water. This in turn introduces some heterogeneity into the cytoplasm. Thus water structures are important parts of cellular compartmentalization. The water structure in an area of the cytoplasm determines the physical properties of a compartment and in this way influences the activity of enzymes in that area.
It is possible to measure the vibrational frequency of proteins dissolved in low-density water, using the reversed-micelle system. In this system proteins surrounded by low-density water do indeed experience a decrease in vibrational frequency compared with proteins dissolved in free water. The vibrational frequency influences the activity of the enzymatic reaction. For example, lowering the vibrational frequency of an enzyme may shift upwards the temperature at which the enzyme achieves its optimal rate of reaction.
The reversed micelle system has allowed us to get at some additional facets of the relation between enzyme activity and water structure. Our experimental setup is quite simple. The reversed micelle will form spontaneously when we supply the proper, experimentally determined, proportions of water, surfactant and solvent. We can alter the size of the reversed micelle by varying the water concentration, and we can add enzymes and other water-soluble compounds to the internal aqueous compartment, until we achieve the conditions we are trying to study.
In our studies, we have varied the size of the reversed micelles and have discovered that all of the enzymes we tested achieve their optimal efficiency when the reversed micelle reaches a particular size. Interestingly enough, that size roughly corresponds to the dimensions of the water-filled spaces, such as the periplasm, found in the average prokaryote. Furthermore, physicochemical properties of the surface provided by the surfactant structures resemble the surface properties of macromolecules in the living cell. So it seems reasonable to us that water within these structures assumes an array similar to that seen in reversed micelles. Given that we feel comfortable that reversed micelles mimic the actual conditions inside the prokaryotic cell, we conclude that most enzymes have evolved to operate best within a particular water structure.
At the moment, we are considering ways to expand our studies of water structure into the living cell. Were such experiments possible, we would not be surprised to discover that enzymes are positioned inside the prokaryotes so that their active sites sit within an area whose water structure promotes optimal activity. The level of enzymatic activities we measure inside the reverse micelles may well reflect the real level of activity inside the living cell. These activity levels cannot be observed under conditions that promote the inappropriate water structures.
We therefore believe that when considering prokaryote organization, it is important to think about possible microenvironments within the cytoplasm, one form of which may be brought about by differential structures of water. We propose that a boundary of structured water approximately 2 to 10 nanometers thick (one nanometer is one-billionth of a meter) can be found abutting a cell membrane and other macromolecular cell structures.
Within this boundary, we would expect high-density water to lie closest to the cell membrane, followed by a layer of low-density water. Further inside the cell, but only at some distance from other macromolecular surfaces, we would expect to find a region of free water. Furthermore, we would expect enzymes to be preferentially positioned in layers of structured water. In our experiments, enzymes seem to function best in low-density water, and this is where we would predict finding enzymes in living cells. Therefore, we predict that the layers of structured water are likewise a functional compartment, such as inclusion bodies, the nucleoid and other specialized macromolecular structures.