Figure 2-1 The generalized bacterium
This cartoon displays many of the common structure found in prokaryotic microorganisms, though not every one will have every one of these structures.
Microorganisms typically face the world as single cells rather than as the multi-cellular assemblies of higher organisms. Each single cell [1] must therefore contain all the structures necessary for managing its internal state and dealing with the outside environment. Not surprisingly, this evolutionary process results in the use of rather similar structures and processes to solve similar need in different microorganisms. However, prokaryotes have been on this Earth for a long period of time and this has allowed them to differentiate into a dizzying number of different species [2]. Eukaryotic [3] microbes are not quite so diverse, but they still display a remarkable range of properties. The diversity of the microbial organisms also means that this survey of structures is not exhaustive. No one cell contains all the structures that we describe here, but we will explore the more common structures that have been observed by scientists in the past 150 years as show in Figure 2-1 [4]. A distinction in this discussion must be made between the two types of prokaryotes: the Archaea [5] and their cousins, the Bacteria [6]. We will initially focus on the Bacteria, since that is what we know the most about. Many of the structures we will examine are found in both the Bacteria and the Archaea, but there are some significant differences and these will be covered at the end of the chapter. Finally, we will talk about the features that are distinctive among the microbial eukaryotes.
This cartoon displays many of the common structure found in prokaryotic microorganisms, though not every one will have every one of these structures.
So how did scientists find out so much about such very small organisms? As you might guess, many techniques come into play when tackling a subject as complex as bacterial structure. Electron microscopes [7] have been important, of course, but so have genetics, molecular biology and biochemistry. Microscopes help scientists to visualize where these structures are located and how they are arranged spatially in the microbe. Bacterial genetics and molecular biology identify and analyze the genes necessary for the synthesis and regulation of these structures. Biochemistry permits the detailed examination of each part separately, with implications for its role in the living bacterium. The powerful combination of these disciplines has provided a deep understanding of how a bacterium is put together, but there is still much to learn.
This chapter on microbial structure is separated into two sections [8]. In the first section, we describe the chemical nature of the types of molecules and polymers that are important in carrying out the business of biology. In the second section, we examine the functional units in the cell, describing how the various chemical structures in the cell interact to carry out important cellular functions. In this discussion we assume that the student has had an introductory chemistry course (at least in high school) and is somewhat familiar with chemical notation.
First and foremost, it is important to point out that there are some universal structures that all living cells contain. They are the basic building blocks of life: DNA [9], RNA, protein [10] and cellular membranes. Most, but not all, bacteria also possess cell walls. Beyond these essentials, the frequency of the rest of the structures we mention here ranges in the bacterial world from quite common to very rare.
Sugars serve three basic purposes in the cell: as carbon and energy sources, as reservoirs of carbon and energy, and as parts of cellular structures. Large amounts of energy can be extracted from sugars by processes referred to as catabolism [11], as discussed in Chapter 9. This may explain why many microorganisms show a preference for sugars if given a choice of energy sources. The term carbohydrate is often used to refer to them because their chemical formula can be broken down into [C(H2O)]x where x is any number greater than three.
Single sugar molecules are typically 3 to 7 carbons long and are termed Figure 2-2 [12] shows that each carbon on the sugar molecule is decorated with a hydroxyl group (OH) except for one carbon that forms a carbonyl group. If the carbonyl group is at the end of the molecule, it forms an aldehyde [13]; if the carbonyl group is in the middle of the molecule, it forms a ketone [14] as indicated in Figure 2-2 [15]. All sugars can exist as linear molecules in solution and those greater than 5 carbons long can also circularize with the carbonyl group attacking a hydroxyl on one of the other carbons. The circular sugar contains 5 to 7 members in the ring with one of the members being oxygen. Glucose, fructose and ribose are some of the more common sugars found in the cell.
The structures of some important monosaccharides are shown. These often serve as building blocks for major structures in the cell, such as the cell wall and the genome. The top row shows the linear forms and the bottom row shows their circular forms.
Sugars readily polymerize: the combination of two sugars is called a disaccharide, while the combination of three is called a tri saccharide, and polymers of greater than three sugars are referred to as polysaccharides. Sugars are connected by α or β linkages. If the hydrogen is pointing up, it is a α linkage, if the hydrogen is pointing down, it is a β linkage as shown in Figure 2-3 [16]. This distinction may seem trivial, but it greatly influences the properties of the molecule. (The name of the linkage further depends upon the orientation of the hydrogen on the lowest numbered carbon that forms the bond. The lowest numbered carbon is decided by the rules of organic chemistry, but this is really not important for our discussion. Just realize there is a set pattern of numbering for each organic molecule and this helps determines the α or β linkage.) For example, starch is made up of glucose linked by α-1,4 bonds. Starch is also water-soluble and can serve as a food source for many organisms. In contrast, cellulose, containing glucose linked by β-1,4 bonds, is insoluble in water and is not as readily degraded by most microbes. Polysaccharides might contain only one type of sugar monomer or a variety of different ones, sometimes in repeating units.
Sugars can be linked together to form more complex polymers. Lactose is a common sugar in milk, while maltose is found in many grains. Starch found in potatoes and other vegetables is a long polymer of glucose units. A common form of starch contains 20% amylose and 80% amylopectin.
Polymers of sugars can serve as storage products for the cell. The breakdown of sugar yields a huge amount of energy, which means that they are terrific molecules for effectively storing energy for later utilization. Starch and glycogen are two examples of sugar polymers that are used in this way. Polysaccharides also serve as structural components of many different molecules in the cell including nucleic acids and the cell wall.
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are involved in information storage and processing. DNA [17] serves as the cell [18]'s hereditary information, while RNA is involved in converting that information into functional products, such as proteins. RNA and DNA are long polymers of only 4 nucleotides [19]: adenine, guanine, cytosine and thymine [20] (or uracil for RNA). Figure 2-4 [21] lists the structure of the five nucleotides found in nucleic acids.
The nucleotides for adenosine (A), guanine (G), cytosine (C ) and thymine (T) as found in DNA are shown. The first three are also found in RNA, but when incorporated into that polymer, the associated sugar has two hydroxyls, as shown in the model of uracil (U). Uracil is the RNA equivalent of the DNA nucleotide thymine.
The nucleotide [22] structure can be broken down into two parts: the sugar-phosphate backbone and the base [23]. All nucleotides share the sugar-phosphate backbone, while the base distinguishes each type of nucleotide. Linking these monomer units together using a 5’-oxygen on the phosphate and the 3’-hydroxyl group on the sugar forms the nucleotide polymer as shown in Figure 2-5 [24]. Many thousands of DNA nucleotides are strung together to form genes and chromosomes.
In this picture the bases of the two anti-parallel (running in opposite direction) strands of a DNA double helix are shown. Note the sugar-phosphate backbone from which the bases extend and pair with matching bases from the other strand. The dashed lines show hydrogen bonds between the base pairs.
The bases of the four nucleotides are different, but there is also a pattern. Adenine (A) and guanine (G) are purines [25], and therefore have a distinctive two-ring structure; they differ in the chemical groups attached to the rings. Likewise, cytosine (C), thymine (T) and uracil (U) are all pyrimidines [26] and share a single-ringed structure, but also differ in their attached groups. Not surprisingly, as these extra chemical groups distinguish the different purines and pyrimidines structurally, they are also responsible for their important functional differences as well.
The bases in a nucleic acid polymer are capable of forming hydrogen bonds with neighboring bases on a second strand of nucleic acid, a process termed base pairing [27]. However, there are rules to this association. Adenine is capable of forming two hydrogen bonds with thymine (or uracil) and cytosine can base pair with guanine, forming three hydrogen bonds. Some of these bonds require the extra chemical groups mentioned above. If two single strands of nucleic acid have sequences that can base pair along the polymer (such sequences are sometimes said to complement [28]), they will generate a long double-stranded polymer that has a staircase topology as shown in Figure 2-6 [29]. This structure is termed a double helix, since two strands form the molecule and it spirals around an axis in a regular pattern. Such a reaction between two complementary [30] DNA strands is spontaneous: if you mix two complementary single strands of nucleic acid together in a test tube at a reasonable temperature, pH [31] and salt concentration, they will find each other and anneal to form a double-stranded polymer.
Note how the two nucleic acid strands spiral around each other in a regular repeating pattern. There are 10 bases per turn of the double helix. Bases pair with one another in the center of the helix cylinder and form what some have likened to a spiral staircase. The above figure can be rotated by clicking on the arrows.
DNA almost always exists in cells as a double-stranded structure of complementing strands. It happens that this double-stranded form is rather stable [32], and resists tight twists and turns. It is often assumed that the stability is due to the hydrogen bonding between the bases, but this is not the case (the bases would hydrogen bond just as well to water). Instead, it is largely due to the interaction between adjacent base pairs along the helix, which is termed "base stacking [33]" Figure 2-7 [34] for an example. Finally, the larger organization of the DNA strands with respect to each other, termed the tertiary structure [35], is also fairly similar in all DNA molecules. One implication is that proteins that want to distinguish between different DNA molecules must do so by reading different primary structure [36] sequences by interacting with the outer surface of the base pairs.
The repressor binding to its recognition sequence. Any DNA-binding protein that recognize specific DNA sequences must make specific contacts with that recognition sequence by reading the pattern of bases in the DNA. These proteins do this by making very specific contacts between themselves and those atoms on DNA that are different in the different bases. In this figure, the DNA helix is shown by the ball-and-stick figure running along the bottom and the protein is depicted by cylinders (which refer to alpha helices) and other "squiggles" (which refer to any other structure in the protein). The point of the figure are that the DNA is not opened up and that the recognition regions of the proteins are very often alpha helices that lay parallel with or perpendicular to the DNA helix. Not shown are the side chains of the amino acids that actual make the specific contacts with the DNA. In this and subsequent molecular figures, of three different displays will be shown: ball and stick, which shows all atoms larger than hydrogen; ribbon models, in which a ribbon that runs along the carbon backbone of the protein is displayed; and space-filling models, where the actual atomic surface of the molecule is shown. Sometimes, as in this figure, the ribbon form is modified slightly be showing cylinders for alpha helices instead of an actual helix as in Fig. 2-11. Most of the structures shown in this text are the product of X-ray crystallography, which essentially displays the position of every atom (larger than hydrogen) in a molecule. Increasingly all biological questions are being thought about in terms of these structures at the atomic scale.
In composition and therefore primary structure, RNA is similar to DNA, except that uracil (U) takes the place of thymine in the molecule and the ribose unit on each sugar contains a additional hydroxyl group. However, most RNA in cells exists as single-stranded molecules and not a complex of two different strands as with DNA. Now if complementary base sequences are present in an RNA molecule, it can fold back upon itself and base pair, so that many RNA molecules have at least some double-stranded regions. However, this bending and folding means that RNA molecules typically have much more complicated tertiary structures than does DNA. Both the single-stranded loops and the double-stranded stems are critical for the function of most RNA molecules. Many are involved in creating physical structures, such as ribosomes, that are involved in processing information. The other general class of RNAs are messenger RNAs, which represent a version of the DNA primary structure that is suitable for translation into protein.
Proteins and peptides (small proteins) are essential to the cell and serve two major functions. Many proteins are enzymes that catalyze almost all biological reactions in a living organism. Other proteins perform a structural role for the cell - either in the cell wall, the cell membrane or in the cytoplasm [37]. In this section, we will look at the basic structural elements shared by all proteins.
Proteins are polymers of amino acids.. Amino acids, with rare exception, contain an α carbon that is connected to an amino (NH3) group, a carboxyl group (COOH), and a variable side group (R). Figure 2-8 [38] shows this generic amino acid structure. The side group gives each amino acid its distinctive properties and helps to dictate the folding of the protein.
Each amino acid contains a carboxyl group, an amino group and a variable side group (R). These all connect to a central carbon, termed the α-carbon, identified by the arrow.
As with nucleic acids, primary structure refers to the ordered sequence of the different amino acids in a protein. The carboxyl group and the amino group of amino acids are reactive. As shown in Figure 2-9 [39], cells synthesize proteins by attaching the carboxyl group of one amino acid to the amine group the next, with polymerization taking place at the ribosome. This is termed a peptide bond. Since each amino acid has a carboxyl group and an amino group, hundreds of amino acids can be linked together.
The peptide bond between an alanine residue and a valine residue is identified by the arrow. Peptide bonds can form between any two of the 20 amino acids and link the carboxyl group of one amino acid and the amine group of the next.
There are 20 common amino acids found in proteins and these amino acids can be roughly classified into 3 groups: polar, non-polar and charged. Polar and charged amino acids are hydrophilic and are often found on the surface of a protein, interacting with the surrounding water. In contrast, non-polar (or hydrophobic) amino acids avoid water. While this categorization is adequate for most purposes, you should recognize that it is a bit simplistic. For example, arginine does have a charged hydrophilic group at one end, but the -CH2- backbone that makes up most of the amino acid is actually quite hydrophobic. Figure 2-10 [40] shows the chemical structure of all 20 common amino acids.
In the figure, the amino acids are organized into their chemical type and characteristics. Acidic and basic amino acids carry a charge, amino acids with a sulfur or hydroxyl comonent are polar, and aliphatic and aromatic amino acids are non-polar.
Peptides and proteins are formed when a ribosome and the rest of the translation machinery link amino acids together in polymers that range from 10 to 10,000 residues in length. During and after protein synthesis, the residues of the primary sequence dictate how the protein folds. The simplest aspect of protein folding is termed its secondary structure, which refers to the geometry of the local polypeptide chain with respect to their immediate neighbors. How a protein folds is dictated by the primary sequence of amino acids, but predicting the overall structure from the primary sequence remains one of the most important unsolved problems in biology. Nevertheless, it is clear that the major determinants of this final structure are hydrophobic interactions. During protein folding, hydrophobic amino acids must be hidden from the water interface by being buried in the interior of the protein. This burying defines the protein core which then influences the immediate structure around it and greatly affects the protein's overall structure.
Peptide bonds between adjacent amino acids can rotate and twist to allow a large number of interactions, but two local organization schemes, the α helix and the β sheet, are found in many proteins. Their prevalence is certainly because they happen to form particularly energetically favorable structures. Formation of these structures is driven by favorable hydrogen bonding and hydrophobic interactions between nearby amino acids in the protein. The α helix resembles a ribbon of adjacent amino acids wrapped around a tube to form a staircase-like structure. Figure 2-11 [41] shows different representations of an α helix. This structure is very stable, yet cn be flexible in specific cases and is sometimes seen in parts of a protein that may need to bend or move.
This figure shows two common depictions of an α-helix, an extremely common and important motif in proteins. The ball-and-stick depiction on the left shows the various side chains, but the helical nature of the structure is not so obvious. The ribbon representation on the right only traces the backbone of the peptide bonds and overemphasizes the symmetry a bit, but shows the helical nature of the structure.
In the β sheet, two protein chains (perhaps different segments of the same protein) align themselves in a planar structure such that hydrogen bonds can form between facing amino acids in each sheet. Figure 2-12 [42] shows different representations of this structure. The β sheet is different from the α helix in that it can involve amino acids from different sections of the protein, which come together to form this structure. Also, the structure tends to be rigid and less flexible than the α helix.
Two views of a β-sheet. (a) A diagram of β sheet showing hydrogen bonding between protein strands (b) A ribbon representation of a β-sheet.
The relationships mentioned above do not fully define the structure of proteins. For example, we need a way to describe larger organizations of α helices and β sheets, as well as other parts of proteins that do not fit these two patterns. This larger organization is termed tertiary structure.
As noted before, the most important stabilizing force in proteins is burying hydrophobic residues from the surrounding water, but there are other chemical features that are important in creating and stabilizing tertiary structure. These include hydrogen bonds, ionic interactions, and sulfhydryl bonds. Ionic interactions are attractions between groups of opposite charge. There are amino acids with negatively charged side groups (aspartate, glutamate) and amino acids with positively charged side groups (typically lysine and arginine). If opposite charges are brought close enough together, there will be an attractive force that can contribute to protein structure. It also follows that like charges repel each other and this can also dictate protein structure. Sulfhydryl linkages are covalent bonds between cysteine groups. Cysteine is a unique amino acid in that it has a sulfur group at the end of its variable side group that is available for binding to other groups. Often in proteins, nearby sulfhydryl groups on cysteines form a covalent bond and these are often crucial for the stabilization of the mature protein or for it to perform its function. Figure 2-13 [43] shows some representations of sulfhydryl bond in proteins.
Several views of sulfhydryl linkages are shown. In the top left panel, the covalent sulfhydryl linkage between two cysteine residues is shown. In the top right panel, this sort of Cys-Cys bond is shown in the context of a section of a protein. The bottom panel shows a ribbon diagram of a similar protein loop, but where the interacting sulfhydryl groups on the side chains are shown in yellow.
Figure 2-14 [44] shows the tertiary structure of ribulose bisphosphate carboxylase (RubisCo), one of the most abundant enzymes on this planet. This enzyme takes energy, obtained most often from the sun, and uses it to convert carbon dioxide into carbohydrate. Its abundance reflects the fact that virtually all photosynthetic organisms, from bacteria to plants, use this enzyme to produce much of the cell's carbon. Note the α helices throughout the protein and the β sheet near the bottom. These structures help to hold the protein in its proper conformation so that it can carry out its enzymatic activity.
The figure shows the structure of ribulose bisphosphate carboxylase, typically abbreviated Rubisco, from the bacterium Rhodospirillum rubrum. As described in the text, this complex structure has alpha helices (shown as cylinders) and beta sheets (shown as the flat ribbons) throughout.
Now we have talked about certain secondary structures that recur in many proteins, but it also happens that there are conserved tertiary structures as well. In other words, there are regions of proteins, which are sometimes termed motifs or domains, that are structurally identical in two or more different proteins. Figure 2-15 [45] shows some examples of protein motifs. By "structurally identical," we mean that they have similarly sized α helices and β sheets that are organized in a similar overall way. Interestingly, these domains typically do not have identical primary structures. Instead, it appears that a variety of different sequences can fold to give the same overall tertiary form. It is usually clear from the sequence that structurally similar domains from different proteins are evolutionarily related. That is, at one time there was a single protein with the domain and through evolution, an extra copy of the gene was created. Subsequent random mutations in each gene altered the primary sequences of the proteins, but the only changes that were tolerated were those that maintained the structure, and therefore the function, of each copy of the domain. This implies that these motifs often have a specific function. For example, there is a very common motif that proteins use for binding ATP. A second class of examples would be the set of motifs that are used to bind DNA. Through genetic changes, these motifs have been grafted onto other protein regions, so that the same ATP-binding (or the DNA-binding) domain performs a similar function for proteins with completely different overall activities. Of course the unique functions are defined by those portions of the protein that are not in common.
Three examples of protein motifs are shown. The top left panel shows two zinc-finger motifs (the red helices and thin gray lines), each bound to a Zinc atom shown in pale blue. This is a common motif found in DNA-binding proteins and the interaction is shown here (the ball-and-stick figure running from left to right in this panel is a section of DNA). Note that the Zinc does not interact with the DNA, but instead stabilizes the protein motif to allow it to form the precise shape to bind to DNA. The top right panel shows an ATP-binding domain, with the ATP displayed as the space-filling model and the protein shown in a ribbon format. The bottom panel shows another common DNA-binding motif, termed the helix-turn-helix, as found in the lac repressor. Here the DNA is shown in space-filling form and the two helices after which the structure is named are shown in red and white (the latter is shown end-on and is interacting with the DNA).
The existence of these conserved motifs was revealed by the analysis of the crystal structures of many different proteins. One saw similar structural regions performing similar functions in proteins that otherwise were quite dissimilar. Not surprisingly, since there are some residues that need to be conserved to form these structures, we can now predict some of these motifs based on the sequence of the proteins as well. The explosion of DNA sequence information and tools that can find these motifs has allowed the prediction of protein functions solely from sequences to be done with ever greater accuracy.
From this description you can see that proteins can often be looked at as a collection of "protein pieces," each of which serves a function and has an evolutionary history. We refer to these as functional domains. The enzyme pyruvate kinase, which generates ATP by removing it from phosphoenol pyruvate, has three domains, termed A, B and C. The A domain is responsible for binding substrate (phosphoenolpyruvate and ADP) while the C domain serves a regulatory role, binding fructose-6-bisphosphate and stimulating activity.
Up to this point, we have talked as if proteins are static rigid structures, but this is certainly wrong. Instead, most proteins are flexible, dynamic structures that respond to the conditions around them. Movement is typically essential to their function, especially for enzymes that need to grab a substrate, react with it and release product. In some cases, the flexibility is over very small distances; moving the residues of an active site just a few tenths of an -10m')">angstrom [46] (10-10 meters) would have a major effect on binding and reacting with a substrate that is itself only a few angstroms in size. However, some protein movements are huge, where one domain changes its position relative to that of the rest of the protein. This obviously changes the tertiary structure of that protein. Thus, when we talk about tertiary structures, typically determined by crystallography, we are really referring to only one of the possible structures that the protein can exist in while performing its biological function. The structure we see through crystallography is simply the form of the protein that happened to form crystals and whose structure was then solved.
This flexibility has many important implications for a protein. At too high a temperature the protein might become too dynamic. This might overwhelm the stabilizing energy that maintains the protein, so that it unfolds or denatures. At too low a temperature, a protein might be so stable that it locks into a single structure and lacks the flexibility necessary to carry out its function. Interestingly, thermophilic microbes have been found to have more rigid proteins that can tolerate the turbulent environment of higher temperatures.
Many proteins are actually complexes of several polypeptides. The arrangement of more than a single polypeptide into one protein is termed the quaternary structure of that protein. Protein complexes might contain two or more copies of the same protein or they may consist of any number of different polypeptides in various ratios. Such complexes are certainly not random, but reflect precise interactions among the protein subunits based on the same sorts of interactions (e.g. hydrophobic, hydrogen bonds, etc) described above.
Figure 2-16 [47] shows the catabolite activator protein, an example of a protein containing identical subunits. In this case is has two subunits, so it is termed a dimer. Each subunit has a cyclic adenosine monophosphate molecule bound that activates the protein. In the active state this protein binds DNA and activates various genes in the cell.
Two depictions of the catabolite activator protein (CAP) of E. coli are shown. The left panel shows a ribbon depiction, with the two identical subunits shown in red and blue. CAP senses the presence of cyclic AMP (cAMP) in the cell and a molecule of cAMP bound to each monomer as indicated. The dimer is actually symmetrical, but that is not obvious here because the model is rotated slightly. The right panel shows a ball-and-stick representation of the same dimer and gives a better sense of the overall shape. Note that the bound cAMP molecules are buried within the protein (so how do they ever come and go?).
Dinitrogenase is an example of a protein containing non-identical subunits. As shown in Figure 2-17 [48], the active protein has two copies of one protein (termed α) and two copies of a second protein (termed β), and therefore is also referred to as a α2β2 tetramer. This protein is responsible for the reduction of N2 gas to ammonia and is critical to global nitrogen cycling. The importance of the quaternary structure of dinitrogenase is only partially clear. The α subunits contain the metal cluster where N2 is actually reduced (or "fixed") and the β subunit helps form another metal center that helps transfer electrons to that active site. We can therefore understand why there might need to be an αβ dimer. However, it is not clear why there are two such αβ dimers hooked together in the tetramer found in nature. There is, for example, no apparent communication between the two active sites. Quite possibly, the fact that dinitrogenase is a tetramer is simply a relic of evolution; perhaps a precursor to dinitrogenase really did have a need to be a tetramer and there is simply no disadvantage to the protein retaining that organization.
Nitrogenase is made up of four protein chains, with two copies each of different proteins, termed the α and β subunits. Though not apparent in this view, the two α subunits are identical to each other, as are the two β subunits. The quaternary structure is referred to as an α2β2 tetramer.
Glycoproteins are proteins with attached sugars that are typically important for the proper function of the protein. In most cases the sugars are added to the protein after it has been translated. Proteins that encounter the outside environment of the cell are sometimes glycosylated to stabilize the protein to attack from degradative enzymes and destructive physical forces. Glycolipids [49] are combinations of lipids and polysaccharides. In these cases the third glycerol hydroxyl attaches to a polysaccharide instead of a small polar molecule. These polysaccharides can be quite long, extending several microns into the outside environment. Lipopolysaccharides [50] (one type of glycolipid and usually abbreviated LPS) impart several unique properties to the surface of gram-negative bacteria and we will have more to say about them when examining the cell wall.
Lipids are molecules with two personalities. One part of the molecule wants to associate with water and the other does not. Molecules with these properties are termed amphipathic [51]. Figure 2-18 [52] shows that the backbone of the lipid consists of a three-carbon glycerol molecule. Hydrophhobic, long-chain fatty acids attach to two hydroxyl groups on the glycerol. To the third hydroxyl group, a polar, and therefore hydrophilic, group is attached. Many bacteria contain phospholipids in which this third group contains a phosphate connected to a carbon molecule. The amphipathic nature of lipids is important in their function in the cell.
The chemical structure (left) and a space-filling model (right) of phosphatidylethanolamine.
There are also a number of important small molecules that shuttle protons, electrons or small carbon moieties around the cell. These small entities typically do their job in association with proteins to which they can be either loosely or tightly bound. All of life on this planet seems to have settled on a surprisingly small set of molecules to perform these tasks. Almost certainly this is because the use of these molecules evolved early and has been maintained through evolution.
Most amino acids are not particularly good at either donating or accepting electrons and when they do, it is under a limited range of conditions. As you will read in the chapter on metabolism, the ability to move electrons among proteins is critical to all life, so two general types of prosthetic group associated with proteins have evolved for this purpose. Figure 2-19 [53] shows the first type, which are organic multi-ring structures and the other type are iron-sulfur clusters. In both cases, these carriers have characteristic affinities for accepting and donating electrons and protons, but these affinities are also affected by the proteins in which they are found. Thus, a wide range of electron carriers with different properties has evolved. By organizing these carriers in precise patterns in the cell, the cell is able to use the transfer of electrons to do work.
The chemical structures of quinone (left) and nicotinamide adenine dinucleotide (NAD) (right). In each case, both structures the oxidized and reduced forms are depicted.
There are also small molecules in the cell that serve as carriers of important carbon compounds. Essentially, these carriers have the right chemical properties that make it relatively easy for enzymes to add or remove a particular carbon unit. Tetrahydrofolate and cobalamin (vitamin B12) are often involved in adding or removing one-carbon units during the synthesis of various structures in the cell. Coenzyme A is necessary for the transfer of small 2 to 4 carbon units (acetyl, propyl) from one enzyme to another. It finds utility in both the synthesis and breakdown of organic molecules. The beauty of using a small set of carriers is that it allows the easy movement of carbon from one pathway to another.
Many types of minerals are important for the proper functioning of enzymes. For example, magnesium ions are essential for ATP-binding by many enzymes. Zinc is important in the proper folding of some enzymes and iron, in the form of iron-sulfur centers and hemes, is critical in many electron transport proteins. Minerals also help bind structures in the cell together. For example, magnesium and calcium are necessary for the stabilization of membranes. Potassium ions in the cell shield the large amount of negative charge on the DNA allowing it to pack more tightly together. More will be said in later chapters about their specific roles, but some of the more important ions include K+, PO4-3, Mg+2, Zn+2, Ca+2, Mn+2, Fe+2 and Fe+3.
Now that you have had an introduction to the chemicals that make up the typical cell, we will now look at how this chemistry combines to form major functional units. These can be thought of as the organizations that carry out the major business of the cell: growth, replication, feeding and movement. We will first start with membranes because so many things interact with them. Next internal structures in the cytoplasm will be described and finally structures outside of the membrane.
First, however, we should describe an important evolutionary hypothesis that will make sense of much of the following details. As you will see, there are a number of curious similarities and differences in the details of cellular structure among bacteria, archaea and eukaryotes. In general much of the machinery in a eukaryotic nucleus [54] and in the cytoplasm looks rather a lot like what is present in the archaea. However, the organelles [55] of eukaryotes, such as the mitochondria and chloroplasts, have properties that are much more similar to those of bacteria. How is this possible? One clue comes from observing organisms in nature. It is very common to find cooperative relationships between different species and this is also true in the microbial world. In some instances these relationships involved close physical contact between their participants, sometimes with one participant engulfing the other. In 1968 Dr. Lynn Margulis extended this observation and proposed that some of the organelles found in eukaryotes, specifically mitochondria and chloroplasts, were originally endosymbionts of their host. Originally these two microbes probably were able to live independently, but over time, the endosymbiont lost functionality that its host was already providing and then became dependent. Over the years ample evidence has accumulated to support this exceptional insight.
In summary, it seems clear that the eukaryotic cell was born by the merger of an archaeal cell with a gram-negative proteobacteria. Photosynthetic eukaryotes arose from a second endosymbiosis, where the eukaryotic cell engulfed a cyanobacterium. Clearly, eukaryotes, including us, were the result of the cooperation of several bacterial species in the long distant past.
The cytoplasmic membrane immediately surrounds the inside of the cell and is perhaps the most conserved structure in living cells. Membranes are thin structures, measuring about 8 µm thick and every living thing on this planet has some type of membrane They are the major barrier separating the inside of the cell from the outside and allow cells to selectively interact with their environment. Membranes are highly organized and asymmetric. This asymmetry comes from the fact that the membrane that faces the environment performs very different functions than does the side that faces the cytoplasm. Membranes are also dynamic, constantly adapting to changing environmental conditions.
Membranes are composed of lipids and proteins. The majority of lipids are phospholipids as described earlier, but about 50% of all know bacterial species also contain hopanoids as shown in Figure 2-75 [56]. These molecules have a similar structure to sterols found in eukaryotic membranes and serve to help stabilize the membrane. Proteins are more numerous in bacterial membranes than in eukaryotic membranes. This is because bacteria in general only contain a single membrane in contact with the cytoplasm and this has to carry out all the functions of the cell. In eukaryotes these functions are divided amongst the cytoplasmic membrane and the other organelles.
The chemical structure and space-filling model of a hopanoid, which is found in many different bacterial membranes.
Much of the general behavior of membranes is dictated by the behavior of lipids in water. Because phospholipids are amphipathic, they tend to congregate when placed in an aqueous environment. This is done in a very specific fashion such that the hydrophilic portions face the water and the hydrophobic portions are buried inside. Under the cell's direction lipids are organized into a bilayer, where there are two sheets of lipids oriented so that the hydrophobic faces of each sheet face each other as shown in Figure 2-20 [57]. Lipid bilayers can be almost any size and can form vesicles spontaneously, if lipids are placed in an aqueous environment. In the cell, however, the synthesis of membranes is performed by specific enzymes and is tightly controlled.
A space-filling representation of a lipid bilayer as developed by H. Heller et al. (J. Phys. Chem. 1993. 97:8343-60).
The cytoplasmic membrane is held together by a number of forces. Hydrophobic interactions between the alkyl chains of neighboring lipids are a major component of membrane stability. Hydrogen bonds between lipids and between membrane proteins and lipids also hold a membrane together. Further stability comes from negative charges on proteins that form ionic interactions with divalent cations such as Mg+2 and Ca+2 and the hydrophilic head of lipids.
The hydrophobic region of the membrane provides a critical function: it prevents polar compounds, such as ions and most biological molecules, from passing through it. This allows the cell to create and maintain gradients of ions and small molecules across the membrane by mechanisms described below.
So how do polar molecules ever cross this membrane, since this represents an important interaction between the cell and its environment? This transfer across the membrane comes about through the specific functioning of proteins that are imbedded in the membrane. Some proteins span the membrane while others are exposed on the outside or the inside. These proteins may move within the plane of the membrane or they may be anchored to structures in or near the membrane. Many of the membrane-spanning proteins are involved in transport of the polar molecules that must pass through the membranes. A subset of these proteins are also involved in energy generation as discussed below.
The membrane is fluid and has the consistency of a light grade oil. It has been termed a fluid mosaic: "fluid" because the lipids are free to move about on each side of the membrane and "mosaic" because there is a definite pattern to it. Lipids do not generally switch from outside to inside or vice versa, because of the problem of trying to move the hydrophilic group through the hydrophobic core of the bilayer.
The concentration of solutes, sugars, and most ions is generally much higher within the cell than outside. A fundamental principle of nature is that different concentrations of a given solute tend to equilibrate across a boundary due to diffusion [58]. However, the cell boundary is the membrane and its hydrophobic core prevents this diffusion for polar molecules. Compounds such as amino acids, organic acids and inorganic salts must therefore be specifically transported across the membrane by proteins and once inside these molecules cannot escape. The cell can therefore control the nature and amount of these compounds that enter or leave the cell.
One of the basic ingredients of life is water, and this must be present inside the cell for it to function. Water can diffuse relatively slowly across the membrane, but it was a matter of debate whether this was fast enough for cellular processes. Studies of adaptation to osmotic stress suggested that passive diffusion of water was too slow to explain the rapid changes observed in some bacteria. A search began for the elusive protein that was carrying out this process. The first water channel was discovered by Peter Agre in 1988. These proteins were first decsribed in eukaryotic cells, but a wide variety of living systems are now know to contain them and the term aquaporins was coined to describe them. Over 100 aquaporins have been discovered in bacterial systems and while these do not share a high degree of sequence homology with eukaryotic aquaporins, they share significant structural similarity. Aquaporins can be divided into two large classes, those that only allow water to flow through them, and those that will also transport glycerol and a few other small uncharged molecules. Aquaporins have not been found in every microorganism, in fact some species appear to lack them. In these cases the microbe may rely solely on diffusion across the membrane, or its aquaporin has a novel sequence.
Aquaporins (Figure 2-76) are intrinsic membrane proteins of about 23 kDA that contain 6 α-helices arranged around a central core channel. Each aquaporin will associate with three others to form a homotetramer in the membrane. The central channel is narrow and contains a conserved stucture that can selectively allow only the passage of the desired molecule. In this way, water can rapidly flow into and out of the cell, speeding up its transport through the membrane.
A ribbon diagram of the 23 kDA Aquaporin from E. coli. Each of the 6 α helices that line the water channel are shown. The arrows indicate the path of water through the protein. The two sequences of 3 amino acids (Asparagine-Proline-Alanine) are shown in pink. This area is thought to be important in discriminating, only allowing water molecules to pass through the pore. Thus, serving as a gate.
While aquaporins are essential to cell function they also create a serious problem. The inside of the cell is full of many types of solutes: proteins, nucleic acids, other small molecules and ions. In comparison the outside environment, in most cases, is very dilute. Because of this there is a higher concentration of water outside the cell than inside the cell. Nature hates imbalances such as this and in an effort to correct the problem; water tends to flow into the cell, by a process called osmosis [59]. Osmosis causes a high pressure against the cell membrane. This pressure would rapidly cause lysis of most cells and one of the major purposes of the peptidoglycan of the cell wall (discussed below) is to prevent the cell membrane from bursting.
For molecules that are soluble in both the lipid membrane and the surrounding aqueous environment, the law of simple diffusion directs transport. The membrane is not a barrier for such molecules. These types of molecules are uncommon since solubility in both a hydrophobic and a hydrophilic environment is unusual. There is no transport protein for such compounds, so there is no specificity of control or energy cost. The cell cannot create a concentration gradient of these molecules. One important example is water. Water can pass freely into and out of cells.
There are three basic types of transport systems
Many of the proteins in the membrane function to help carry out selective transport, particularly of polar compounds. These proteins typically span the entire membrane, making contact with the outside environment and the cytoplasm. They often require the expenditure of energy to help compounds move across the membrane, though cells can also use concentration gradients of these compounds to generate energy, as described below
This process involves a protein that binds the molecule to be transported and physically moves that compound through the membrane. Binding of the molecule to the protein causes a conformational change in the protein so that the molecule now faces the opposite side from where it was. Facilitated diffusion, as shown in Figure 2-21 [60], is therefore specific because a protein must bind the molecule. However, these small molecules are readily moved in and out of the cell, so a gradient cannot be formed nor is energy required. One example of a protein involved in facilitated diffusion is the glycerol facilitator protein. In E. coli this enzyme binds glycerol and a few other polyalcohols and allows their diffusion into the cell. Once inside, the glycerol is immediately phosphorylated, preventing its diffusion back outside the cell.
An animation of the migration of solutes in and out of the cell as facilitated by a protein. Notice that this mechanism does not lead to a solute concentration inside the cell that is higher than outside. Rather, it leads to an equilibrium of that solute across the gradient.
In this process, a protein specifically binds the target molecule and transports it inside the cell while simultaneously modifying it chemically. Most group translocations require energy and tend to be unidirectional, unlike facilitated diffusion. The substrates of catabolic pathways, such as sugars, are sometimes transported by group translocation. This is an efficient way to both bring substrate into the cell and begin the breakdown process. Figure 2-22 [61] shows an animation of group translocation.
An animation of group translocation. The glucose molecule that is being transported into the cell is modified by the addition of a phosphate from phosphoenolpyruvate to form glucose-6-phosphate.
In active transport, energy is expended to transport the small molecules, but they are not chemically altered. The process is efficient enough to cause the internal concentration in the cell to reach many times its external concentration. Active transport proteins are molecular pumps that expend energy to pump their substrates against a concentration gradient. This energy comes in two forms: ATP and ion gradients (both ATP and ion gradients are made by central metabolism and we will cover their formation in the chapters on metabolism). In ATP-based active transport, ATP hydrolysis is coupled to the movement of the small molecule across the membrane. One large group of proteins involved in this type of transport is the ATP binding cassette (ABC) transporters. ABC transporters have been found in all living species with 80 identified in the E. coli genome and 48 in the human genome. The mechanism of ABC transporters is exemplified by the maltose binding protein of E. coli.
Three separate types of transport are shown. In the first, an antiporter moves two different small molecules across the cell membrane, but in the opposite directions. In the second, a symporter moves two or more different molecules to move into the cell simultaneously. Typically, the desired molecule is being concentrated against a gradient and that transport is driven by the transport of the other molecule, which is moving with a gradient. Finally, a uniporter binds and transports the target molecule only. Energy is required for these processes and the cell can accumulate molecules inside the cell using this mechanism.
Ion gradient active transport uses the energy of one chemical gradient, that of the specific ion, to drive the creation of a different gradient, the uptake of the small molecule. The ion gradient that supports the work is at a higher concentration on one side of the membrane than the other. The transport protein both binds its small molecule to transport and provides a gateway for this ion to fall down its concentration gradient. When the ion moves through its gateway, it causes a conformational change in the protein and this change is used to transport the target small molecule into the cell.
Active transport proteins may be highly specific for only one molecule or may be able to carry a class of chemically related molecules. The animation in Figure 2-23 [62] shows several different types of transport molecules. An example of a more general transport protein is the branch chain amino acid transporter of Pseudomonas aeruginosa, which transports leucine, valine, and isoleucine. Figure 2-63 [63] summarizes the various properties of transport mechanisms.
| Property | Passive Diffusion | Facilitated Diffusion | Active Transport | Group Translocation |
| Carrier Mediated | - | + | + | + |
| Concentration Against Gradient | - | - | + | Not Applicable |
| Specificity | - | + | + | + |
| Energy Expended | - | - | + | + |
| Solute Modified During Transport | - | - | - | + |
A comparison of the methods for transporting molecules through the membrane and into the cell.
Many cells use respiratory processes to obtain their energy. During respiration, organic or inorganic compounds that contain energy are oxidized, releasing electrons to do work. In many microorganisms these electrons find their way to the membrane where they are passed down a series of electron carriers as shown in Figure 2-24 [64]. During this operation, protons are transported outside the cell. This creates a gradient of protons across the cell membrane, energizing it, in a fashion similar to charging a battery. The energy of this gradient can then be used to do work directly, a process known as the proton motive force, or can be channeled into a special protein known as ATP synthase. ATP synthase can convert: ADP to ATP, and the ATP can itself do work.
Membranes are critical in many cells for the generation of usable energy. The cartoon shows the various membrane proteins involved in converting high-energy electrons from photoreceptors into useful energy. They do so by forming a proton gradient across the cell membrane, termed a proton motive force, which is in turn used by other proteins to synthesize ATP. This will be discussed in greater detail in the chapter on metabolism.
The prokaryotic cells performing photosynthesis have membrane systems specific to that process. Light excites electrons found in pigmented proteins in the membrane and the electrons are again passed down through a series of electron carriers. As above, a proton motive force is generated and used to synthesize ATP. The specifics of these systems are discussed in the chapter on metabolism.
Membranes also contain specialized enzymes that carry out certain biosynthetic functions. For example, the last few steps of lipid synthesis take place inside the membrane. Another example is cell wall synthesis and assembly. Much of the synthesis of cell wall monomers occurs there and the stitching together of the cell wall polymer takes places while it is anchored to the membrane. In addition, any cellular protein that carries out its function outside the cell membrane (such as outer membrane and extracellular proteins) must pass through that membrane. During their synthesis the ribosome is guided to the cytoplasmic face of the membrane and the growing peptide chain is synthesized directly into the lipid bilayer. Integral membrane proteins then fold up and stay in the membrane while extracellular proteins move through the membrane and take on their final shape on the other side.
Infoldings of the membrane are found in some photosynthetic bacteria. These bacteria use pigments in their membranes to capture light energy. Under low light, they need to increase the surface area to catch more light. They cannot make the membrane thicker, but they can increase the surface area by creating regions where the membrane folds into the cytoplasm. These invaginations are still attached to the cytoplasmic membrane and a picture of such structures, termed the intracytoplasmic membrane in the case of Rhodobacter sphaeroides, is shown in Figure 2-25 [65].
This electron micrograph shows the complicated infolding of the cytoplasmic membrane of R. sphaeroides when it is performing photosynthesis. This infolding creates a larger membrane surface area into which light-harvesting complexes can be inserted. Under low-light conditions many light-harvesting complexes are needed to capture the small number of photons striking the microbe. (Source: Samuel Kaplan, University of Texas - Houston Medical School)
The cytoplasm or protoplasm [66] is the portion of the cell that lies within the cytoplasmic membrane. The cytoplasmic matrix is defined as substances within this membrane, excluding the genetic material. In most prokaryotes, it appears to be relatively featureless by electron microscope, but that simply means that there are no large structures in the matrix. This is in contrast to eukaryotic cells, which have mitochondria [67], and typically other visible organelles [68] that exist for different specific functions. Despite this visual simplicity, the prokaryotic cytoplasm is the site of almost all of the important metabolic functions in the cell.
The cytoplasm has a gel-like consistency, with rather different properties than the simple solutions that we typically make up in the laboratory. This is because there is surprisingly little free water in the cell. Rather than picturing the cytoplasm as a pool of water with the occasional large molecule floating around, it is better to think of it as a bag of proteins and other macromolecules, each coated with a layer of water, and with a modest number of free water molecules bouncing around in between. In fact, there is so little free water in the cell that one-third of all water molecules are making hydrophilic contacts with the macromolecules in the cell. Given this difference between our lab solutions and the actual nature of the cytoplasm, it is a bit surprising that the biochemical analyses we perform in the lab mimic the behavior observed in the cell.
The cytoplasm is the site of the majority of metabolism of the cell and the details of these processes are covered in the chapters on metabolism and photosynthesis. However, there are a few general issues concerning enzymes in the cytoplasm that should be mentioned here.
Though the cytoplasm appears featureless, there is actually a significant amount of local organization. A good illustration of this can be found by examining the enzymes of DNA replication. Though too small to be seen, the proteins that perform replication are in complex assemblies of many proteins. This is much more efficient that having each protein float around, and simply finding the right time and place to perform its function by random chance. For example, DNA gyrase, which unwinds and opens the DNA for copying, has to function in coordination with DNA polymerase, which inserts each new nucleotide in the growing strand. Without this coordination, the DNA would not open up for replication and the process would simply not occur. In some other processes it would certainly be possible for the enzymes to float around in the cytoplasm without any interaction, but often it is much more efficient to organize in some fashion. The glycolytic enzymes (enzymes that oxidize sugars for energy) are an example of this type of multi-enzyme complex. One enzyme directly hands over its product to the next enzyme for which it is the substrate - a sort of molecular assembly line. This is much more efficient, because substrate does not accumulate where it should not and the local concentration of substrate for each enzyme is very high.
The nucleoid [69] is a bit difficult to define because unlike eukaryotes with their nuclei, prokaryotic chromosomes are not confined to a specific organelle. The nucleoid is sometimes referred to as the region of the bacterial cell that contains the chromosome, but that suggests there is a specific region in any cell where the chromosomes is always found, and this is probably not true. A better definition might be that the nucleoid is the region of the cell that is currently occupied by the DNA-protein complex that makes up the chromosome. So what do we know about prokaryotic chromosomes? What does a typical chromosome look like?
It is a little awkward to talk about "typical" anything in prokaryotes, since there is always such a large range for any given property. In the case of prokaryotic chromosomes, the smallest known is about 160 kB, while the largest is about 10,000 kB. E. coli is certainly well-studied and might well be considered typical, with strain K12 having a chromosome of 4,639,221 bp containing 4405 genes. The chromosome for this bacterium is circular and this is a common arrangement, but there are a number of species with linear chromosomes. If stretched out, this material would be about 1400 µm long or about 1/16th of an inch. The E. coli cell is about 1-5 µm in length so it is clear that an amazing degree of packing is necessary to fit the chromosome inside its tiny host. The nucleoid occupies about half of the bacterial cytoplasm and has a density of 20-50 mg/ml, similar to what is observed for the nucleus of a non-dividing eukaryotic cell. The following description of the nucleoid refers specifically to that of E. coli, but is probably generally similar to that of other prokaryotes. Figure 2-26 [70] shows the nucleoid of E. coli.
The nucleoid as seen in thin sections of growing E. coli. Panels A and B show the same section; in panel B the ribosome-free spaces were enhanced by coloring by hand. Panel C shows a similar cell stained with antibodies specific for DNA. (Source: E. Kellenberger).
The nucleoid is composed of DNA in association with a number of DNA-binding proteins that help it maintain its structure. The protein HU non-specifically binds to DNA and bends it, with the DNA apparently wrapping around the HU protein. Another protein found on DNA is IHF, whose sequence is reminiscent of that of HU and is therefore evolutionarily related. It also facilitates bending of DNA, but it does so by binding to specific DNA sites. A third protein, H-NS, binds to DNA non-specifically and is apparently involved in compacting DNA structure. It is found throughout the nucleoid and is likely the major DNA-binding protein that organizes the chromosome. The nucleoid also contains a large amount of RNA polymerase and RNA, as well as small amounts of many different proteins that regulate the expression of specific genes. These seem not to perform any structural role, but reflect the importance of RNA transcription in the nucleoid in growing cells.
The DNA double helix typically has a bit of twisting tension in the opposite direction of the helix ladder (the twists typically "unwind" the left-handed DNA helix). Negative supercoiling, as it is called, is produced by the action of enzymes termed topoisomerases. Figure 2-27 [71] shows supercoiling of the DNA. This negative supercoiling makes it slightly easier to separate the two strands of the double helix, as must be done to start transcription and replication.
Supercoiling is very difficult to show in a static picture and it is hard to create a cartoon that adequately describes it. Instead consider this: Take a few feet of tubing or a garden hose and hold one end in your left hand. Then with your right hand, rotate the other end along its long axis. Since the tubing/hose is resistant to this twisting, this energy will cause a section of the tubing to form a new structure, a bit like the middle picture above. This "supertwisting" is supercoiling. Now the analogy between tubing and a DNA double-helix is not bad - both objects are able to bend but cannot really rotate much along their long axis, so if one could do the same thing with a piece of DNA, the same supertwisting would result. Of course no one is "holding one end of the DNA" in the cell, but the fact that the DNA is a closed circle has the same effect. In this picture, double-stranded DNA is shown, but for clarity the helical nature of the DNA is not depicted (though it certainly IS helical). Twist refers to the number of helical turns in the DNA and writhe to the number of times the double helix crosses over on itself (these are the supercoils). The bottom two figures shows how the supercoils could be concentrated in a single region or dispersed.
As shown in Figure 2-28 [72], the chromosome is further folded into 50 or so loops of about 100,000 base pairs. These domains supercoil independently and indeed, even small sections within the same loop can transiently have different degrees of supercoiling. Because supercoiling changes fairly rapidly, it has been extremely difficult to study in living cells. Nevertheless, it is clear that a variety of environmental effects and even the act of transcription itself can affect the supercoiling of a region. In turn, the specific supercoiling of a region can affect the ability of the cell to express genes in that region. Because supercoiling is affected by so many things and in turn can affect expression of much of the genome, it will be important for microbiologists to develop a better understanding of this phenomenon in the future.
A model of the overall structure of the bacterial chromosome. (A) The unfolded, circular chromosome of E. coli depicted as a single line for simplicity, though of course it is a double-stranded helix. (B) The DNA folded into chromosomal domains by protein-DNA associations. The proteins are depicted as the black circles, interacting with both the DNA and with each other. Six domains are shown, but the actual number for E. coli is about 50. (C) Supercoiling and other interactions cause the chromosome to compact greatly.
So we have told you that the DNA in the cell has a very compact structure because of DNA-binding proteins, but we have also said that that structure is very dynamic because gene transcription is going on all of the time. This paradox creates a very difficult situation for the cell that only becomes worse when it is time to replicate the chromosome. Remember that prokaryotes continue to perform gene expression throughout replication, in contrast to eukaryotes. As you will learn below, transcription and translation are coupled in bacteria - the beginning of the messenger RNA (mRNA), termed the 5' end, is being actively translated while last portion, termed the 3' end, is still being synthesized. This process is depicted in Figure 2-29 [73]. Yet the internal regions of the nucleoid appear to be devoid of ribosomes and non DNA-binding proteins, suggesting that all transcription and translation must occur on the surface of the nucleoid. Therefore the cell must shift this large tangle of DNA around by some unknown mechanism as gene expression of certain buried sequences is needed. It is a marvel that the whole process of transcription, translation and replication works at all, especially within the very small confines of the cell.
In prokaryotes the process of transcription and translation are tightly coupled. This increases the rate at which proteins can be expressed and is one reason that some bacteria can multiply so quickly.
Translation is the process of converting the instructions coded in the DNA into the proteins that actually carry out the work. The macromolecules that perform this task consist of mRNA, transfer RNA [74] (tRNA) and the ribosome, which is made of ribosomal RNA [75] (rRNA) and ribosomal proteins. In brief, this process consists of making an mRNA copy of a region in the DNA that gives directions for the synthesis of a protein or proteins. The mRNA is then bound by a ribosome that translates the mRNA into a amino acid sequence. The amino acids necessary for the protein are carried to the ribosome by tRNA that actually read the information in the mRNA and add the appropriate amino acid to the nascent protein chain. This is described in much greater detail in the chapter on the central dogma.
We will now examine the structure of the molecules involved in translation, starting with mRNA, where the primary structure is simple - merely unmodified A, G, C and U bases. In almost all prokaryotic mRNAs there is not a great deal of secondary and tertiary structure, since they are typically being translated by ribosomes and the translating ribosome certainly removes any structure as it moves along the mRNA. What structure there is tends to be in the regions that are not translated, notably the 5' and 3' ends of the mRNA. One of the roles of structure, especially at the 3' end, is to stabilize the mRNA. Now it happens that most prokaryotic mRNAs are not very stable in the cell because they are rapidly degraded by RNases. However, different types of RNA structures can impede the progress of nucleases, particularly the type that degrades from the 3' end of the mRNA (termed exonucleases, because they attack from the exterior ends). As a complication, however, there are some RNA structures that actually serve as a specific target for other types of RNases (termed endonucleases, because they cut within an RNA) and thus lead to destabilization of the mRNA. This is biologically important because a more stable mRNA is translated by more ribosomes and therefore leads to more protein product.
As a final structural feature, most if not all prokaryotic mRNAs have short stretches of adenosine residues added to the 3' end after transcription. The presence of these also tends to lead to mRNA degradation. This is a bit surprising because the presence of long adenosine stretches on the 3' ends of eukaryotic mRNAs actually tends to stabilize those mRNAs. This appears to be a case where evolution has taken a single feature, addition of adenosines to mRNAs, and changed its functional importance through evolution.
In eukaryotes, the fact that mRNA is transcribed in the nucleus and must be exported to the cytoplasm for translation changes some details. One of these is that eukaryotic mRNAs are rather more stable than in prokaryotes.
In contrast to the case with mRNAs, the other RNAs involved in translation, tRNA and rRNA, have very distinct structures. Each rRNA folds into a known secondary structure and has a complex tertiary structure containing many short helical regions and long range base pair interactions. These structures are also maintained by interactions between the RNAs and protein.
The composition of ribosomes is 62 % RNA and 38 % protein by weight. Two complexes of RNA and protein make up the ribosome, the 30S subunit and the 50S subunit. (The S stands for Svedberg [76] units, a measure of how fast something sediments in solution. For our purposes all you need to know is that larger molecules sediment faster and have larger Svedberg units. The unit is named after Theodor Svedberg who won the Nobel Prize in Chemistry in 1926 for his work on suspensions of large molecules and other compounds in solutions.) The 30S subunit is composed of 21 proteins and a single-stranded rRNA molecule of about 1,500 nucleotides, termed the 16S rRNA. The 50S subunit contains 31 proteins and two RNA species, a 5S rRNA of 150 nucleotides and a 23S rRNA of about 2,900 nucleotides. Several of the nucleotides on the 16S and 23S rRNAs have been modified by methylation and these modifications are probably critical to the function of the rRNAs since they always happen in regions conserved through evolution.
The ribosome-associated proteins are positively charged, with a high proportion of lysine and arginine residues. This facilitates complex formation between the acidic RNA and these basic proteins. The crystal structure of the entire 70S ribosome has been solved as shown in Figure 2-30 [77], which is quite remarkable because solving such structures becomes more difficult as the size of the complex increases and the ribosome is huge. We are still trying to fully understand what that structure tells us about ribosome function. We also know that the ribosome is not static, but dynamic, changing shape during a translation cycle. It is also clear that this complex mix of protein and rRNAs can self-assemble when mixed in the right order, though the process of assembly in the cell is certainly more carefully controlled.
A molecular model of the 70S ribosome of Thermus thermophilis showing the position of proteins (ball-and stick) and RNA (Gray and blue-green ribbons) within the structure. Note the prominence of RNA in the ribosome, which constitutes over 60% of the molecular weight. (Source: adaption from S. Petry et al. 2005. Cell 123:1255-1266)
Transfer RNA (tRNA) is the ferry that transports the amino acids to the ribosome. There are one or more different tRNA molecules for each of the 20 amino acids. Each consists of 70 to 80 nucleotides of single-stranded RNA that is extensively base-paired to form four short helical domains. These structures are commonly represented as a two-dimensional cloverleaf, but look more like an "L" in the native three-dimensional structure as shown in Figure 2-31 [78]. Now the tertiary structures of tRNAs are all rather similar, so the critical features that make each appropriate to a specific amino acid are largely found in the primary structure itself. Many bases in tRNA molecules are chemically modified by enzymes to help the molecule carry out its function.
The two-dimensional structure of tRNA looks like a cloverleaf, but in the actual three-dimensional form, it has a surprising L-shaped structure as shown. The bottom of the structure as shown here contains the anticodon that interacts with the mRNA. The top right of the structure is where the appropriate amino acid residue is attached by synthetases.
The aminoacyl-tRNA synthetases are the enzymes that add the amino acid to the tRNAs. Figure 2-32 [79] depicts a complex between an aminoacyl tRNA synthetase and tRNA. There is a single synthetase for each amino acid and it binds each of its appropriate tRNA molecules and charges it with its appropriate amino acid. The synthetases avoid both the wrong tRNAs as well as the wrong amino acids. However, the process is somewhat trickier than it first appears. First, one might expect that the synthetase might recognize the proper tRNAs by examining the anticodon [80] loop, the part recognized by the ribosome to match the tRNA to the mRNA. After all, the anticodon certainly does define the amino acid in translation. This is not the case, however, perhaps because the anticodon is very far away from the end of the tRNA that is charged with the amino acid. In any event, it is clear that most of the basis for proper synthetase-tRNA recognition lies elsewhere in the tRNA. This then raises another problem: when one looks at the different tRNAs that all carry a given amino acid and are therefore all recognized by a single synthetase, no obvious pattern emerges. In other words, the important features that allow the alanine synthetase to recognize only alanine tRNAs are not completely clear, though great strides in understanding this process have been made.
A molecular model of aminoacyl tRNA synthetase binding its tRNA. The tRNA is shown as ball-and-stick, while the synthetase is depicted in the ribbon form. In this picture, the anticodon is at the top of the figure and the site of amino acid attachment is at the bottom right. Note that the synthetase does not "sense" the anticodon directly.
One final thought before we move on. Think about the chicken-and-egg conundrum that translation brings up. This whole process has the express purpose of synthesizing proteins, yet it involves proteins at every step. How could these proteins have evolved to serve this function when they are needed for their own synthesis? In other words, how could you synthesize any protein until a complete set of translation proteins had evolved? Part of the answer is that primordial translation was probably much simpler, though less accurate and efficient. Perhaps only very few and somewhat general protein functions were actually required. Alternatively, perhaps early translation used no proteins at all: Some scientists believe that early life employed RNA molecules that were capable of both carrying out necessary enzymatic functions and storing hereditary information. They posit that it was only later that proteins came along and started to assist in their own synthesis. However, this hypothesis still does not explain how one simultaneously evolved functional proteins and a process for creating them.
We said before that there were few structures in the prokaryotic cytoplasm that are visible by microscopy. Some of those that are seen are discussed below. In general, they serve specific purposes in the cell and are often found only in certain cell types or under certain growth conditions.
Inclusions are dense aggregates of specific chemical compounds in the cell. Typically, the aggregated chemical serves as a reservoir of either energy-rich compounds or building blocks for the cell. Forming polymers costs energy and it may seem wiser for the cell to keep the excess monomers around for when they are needed. The benefit of polymerization is that it decreases the osmotic pressure on the cell, a serious problem as described later. Inclusions often accumulate under laboratory conditions when a cell is grown in the presence of excess nutrients. However, the role of some inclusions is unclear. Growth on rich medium causes their creation, but subsequent starvation in the test tube does not always result in the use of these reserves. This suggests that these inclusions, at least, are not storage bodies.
One of the more common storage inclusions involves poly-β-hydroxyalkanoate (PHA). It is a long polymer of repeating hydrophobic units that can have various carbon chains attached to it. The most common form of this class of polymers is poly-β-hydroxybutyrate, which has a methyl group as the side chain to the molecule as shown in Figure 2-33 [81]. The function of PHA in bacteria is as a carbon and energy storage product. Just as we store fat, some bacteria store PHA. Some PHA polymers have plastic-like qualities and there is some interest in exploiting them as a form of biodegradable plastic.
The figure shows an electron micrograph of inclusion bodies of poly-β-hydroxyalkanoate inside a cell of a Rhodobacter sphaeroides. The specific chemical here is poly-β-hydroxybutyrate (PHB). In the generalized poly-β-hydroxyalkanoate structure shown at the left, the R group is a methyl in PHB. (Source, Sam Kaplan, University of Texas - Houston Medical School)
Glycogen is another common carbon and energy storage product. Humans also synthesize and utilize glycogen, which is a polymer of repeating glucose units.
Given the opportunity, many organisms accumulate granules containing long chains of phosphate, since this is often a limiting nutrient in the environment. These polyphosphate polymers, also called volutin, form visible granules in some microbes. These granules are readily stained by many basic dyes such as toluidine blue and turn reddish violet in color. These inclusions are often referred to as metachromatic granules because they become visible by "metachromasy" (a color change). Polyphosphate is found in all known cells (eukaryotes, bacteria and archaea) and appears to serve many important roles.
Figure 2-34 [82] depicts another visible structure, termed a sulfur globule, which is found in a variety of bacteria capable of oxidizing reduced sulfur compounds such as hydrogen sulfide and thiosulfate. Oxidation of these compounds is linked either to energy metabolism or photosynthesis. Oxidation of sulfide initially yields elemental sulfur, which accumulates in globules inside or outside the cell. If the sulfide is exhausted the sulfur may be further oxidized to sulfate.
Sulfur globules found in Thiomargarita namibiensis. This large microbe (100 to 750 µm in size) is found in Walvis Bay off the coast of Namibia. (Source: copyright Max Planck Institute for Marine Microbiology, Bremen, Germany)
Figure 2-35 [83] shows an example of gas vesicles [84], also known as gas vacuoles, that are found in cyanobacteria. Cyanobacteria are photosynthetic and live in lakes and oceans. In these environments, the cyanobacteria use gas vesicles to control their position in the water column to obtain the optimum amount of light and nutrients.
The hexagonal forms inside the cytoplasm of this cyanobacterium are the gas vesicles. These actively dividing cells are Microscystis sp.(Source: A. E. Walsby, 1994. Microbiol. Rev. 58:94-144)
Gas vesicles are often aggregates of hollow cylindrical structures composed of rigid proteins. They are impermeable to water, but permeable to gas. The amount of gas in the vesicle is under the control of the microorganism. Release of gas from the cell causes the bacteria to fall in the water column, while filling the vesicle with gas causes the cells to rise.
magnetosomes [85] are intracellular crystals of iron magnetite (Fe3O4) that impart a permanent magnetic dipole to prokaryotic cells that have them. They allow these microbes to orient themselves in a magnetic field. This process does not appear to involve any special machinery beside the magnetosome, Each microbe can be thought of as having a tiny magnet that is responding to the magnetic field in the environment. These magnetosomes allow the microbes to follow the magnetic field of the earth. Some species of magnetotatic bacteria have the following behavior. In the northern hemisphere magnetotatic bacteria swim north along the magnetic field, while in the southern hemisphere they swim south. Because of the inclination of the earth's magnetic field, this causes the microbes to swim downward. Many microbes containing magnetosomes are aquatic organisms that do not grow well in the presence of atmospheric concentrations of oxygen and they move away from the oxygen higher up in the water column by detecting the magnetic field and swimming downward.
A special membrane surrounds magnetosomes that confines the magnetite to a defined area. The membrane likely plays a role in precipitating the iron as Fe3O4 in the developing magnetosome. Magnetosomes can be square, rectangular or even spike-shaped. Magnetosomes are primarily found in aquatic bacteria and in some unicellular algae (eukaryotes).
The periplasm [86] is found in gram-negative bacteria and is the space in between the cytoplasmic and outer membranes. (Many feel a periplasm is also present in gram-positive bacteria in between the cytoplasmic membrane and the peptidoglycan.) The periplasm is filled with water and proteins and is therefore somewhat reminiscent of the cytoplasm. However, pools of small molecules in the periplasm are not like those in the cytoplasm because the membrane prevents the free exchange between these two compartments. Also, the proteins found in the periplasm are distinct from those in the cytoplasm and are specifically guided to this site during translation through specific signal sequences typically near their N-termini. Figure 2-64 [87] lists some examples of these proteins.
The peptidoglycan shell that provides the strength to prokaryotic membranes is also found in the periplasmic space of gram-negative bacteria, while in gram-positive bacteria it provides the outside border to the periplasm.
| Enzyme Type | Examples | Function |
| Hydrolytic enzymes | phophatases | Degrading phosphate-containing compounds. |
| proteases | Degrading proteins and peptides. | |
| endonucleases | Degrading nucleic acids. | |
| Binding proteins | sugars, amino acids, norganic ions, vitamins | Binding substrates and docking with transport protein in membrane. |
| Chemoreceptors | Chemotaxis, ermination | Sensing the environment and changing cell behavior in response. |
| Detoxifying enzymes | β-lactamase | Degrading penicillin and related compounds before they get into the cell. |
Periplasmic enzymes have several main functions, detecting nutrients in the environment, degradation of polymers, and protection from harmful compounds.
This section will restrict itself to the bacterial cell wall, but at the end of the chapter we will compare this to archaeal cell walls. The cell wall is essential to the survival of most microorganisms. Many microbes live in environments in relatively dilute environments and the cell wall's most important function is to prevent the cell from bursting due to the osmotic stress placed upon it as discussed previously in section 2-13. The cell wall also determines the shape of the cell. Any cell that has lost its cell wall, either artificially or naturally, becomes roughly spherical and lyses due to osmotic pressure, unless placed in certain concentrated solutions. Finally, the cell wall helps to support any structure that penetrates from the cell out into the environment.
A Gram stain of the gram-positive bacterium Bacillus cereus
The structure and synthesis of prokaryotic cell walls is unique and many compounds found in the bacterial cell wall are found nowhere else in nature. It is true that plants also make cell walls, but they are chemically and structurally different. There are two basic types of bacterial cell wall structures that have been studied in detail: gram-positive and gram-negative. These two classes of bacterial cells look very different following staining with the Gram stain and this has been a standard basis for starting to identify different bacterial species. Figures 2-36 and 2-37 show Gram stains of gram-positive and gram-negative bacteria, respectively.
A Gram stain of the gram-negative bacterium Serratia marcescens.
When the Gram stain was developed by Hans Christian Gram in 1884 the molecular basis of the stain was unknown. In fact very little was understood about bacteria in general. He just determined empirically that when bacterial smears were run through a four-step staining procedure using two different dyes, some cells retained the first dye and stained purple, while other only retained the second dye and stained pink. Years later it was discovered that the basis for this differential reaction relates to the cell wall as shown in Figure 2-38 [88].
The different Gram reactions occur because of structural differences between the bacterial cell walls. Gram-positive cells (Group B streptococci) appear smooth in a scanning electron micrograph (A) and are composed of a single layer of peptidoglycan (B). Gram-negative cells (E. coli) have an undulating surface and have three layers (C and D). (Sources: S. H. Pincus, et al. 1992. J. Bacteriol 174:3739-3749 [panels A and B]; M. E. Bayer and C. C. Remsen. 1970. J. Bacteriol. 101:304-313 [panel C]; T. J. Beveridge. 1999. J. Bacteriol. 181:4725-4733 [panel D])
As shown in Figure 2-38 [89], the gram-negative cell has an additional layer and the outside of the cell appears convoluted when compared to the gram-positive cell. The gram-positive wall is much thicker than is the gram-negative wall and its external appearance is smoother. gram-positive and gram-negative cells do share one thing in common that is unique to bacteria - peptidoglycan. We will talk about the structure of this and then move on to examine the various structures found in each cell wall type.
Peptidoglycan is a thick rigid layer composed of an overlapping lattice of two sugars, N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM), that are cross-linked by amino acid bridges as shown in Figure 2-39 [90]. The exact molecular makeup of these cross-bridges is species-specific. NAM is only found in the cell walls of bacteria and nowhere else. Attached to NAM is a side chain generally composed of four amino acids. In the best-studied bacterial cell walls (E. coli) the cross-bridge is most commonly composed of L-alanine, D-alanine, D-glutamic acid and diaminopimelic acid (DPA).
The generalized peptidoglycan monomer showing the two sugars that make up the backbone. The R group consists of four amino acids, with the best-studied cell walls containing L-alanine, D-alanine, D-glutamic acid and diaminopimelic acid.
Note that peptidoglycan contains D-amino acids, which are different than the L-amino acids found in proteins. D-amino acids have the identical composition as L-amino acids, but are their mirror images. The use of D-amino acids is unusual in biology and bacteria have enzymes called racemases to convert between D and L forms specifically for this use.
The NAM, NAG and amino acid side chain form a single peptidoglycan unit that can link with other units via covalent bonds to form a repeating polymer. The polymer is further strengthened by covalent bonds between cross-bridges and the degree of cross-linking determines the degree of rigidity. In the E. coli, the penultimate D-alanine of one unit is linked to DPA of the next cross-bridge. In some gram-positive microbes there is a peptide composed of various amino acids that serves as a link between the cross-bridges. For example, in Staphylococcus aureus strains, five glycines make up the linker between peptidoglycan monomers. The sequence of these linkers varies considerably between species. The completed peptidoglycan layer forms a strong mesh that can be thought of as a chain link fence. The complete cell wall contains one or more layers of peptidoglycan one atop the other, providing much of the strength of the cell wall.
While both gram-negative and gram-positive bacteria have peptidoglycan, its physical arrangement in the cell wall is different. In gram-positive cells the peptidoglycan is a heavily cross-linked woven structure that encircles the cell in many layers. It is very thick with peptidoglycan accounting for 50% of weight of cell and 90% of the weight of the cell wall. Electron micrographs show the peptidoglycan to be 20-80 µm thick. In gram-negative bacteria the peptidoglycan is much thinner with only 15-20% of the cell wall being peptidoglycan and it is only intermittently cross-linked. In both cases peptidoglycan is not a barrier to solutes, as the openings in the mesh are large enough for most molecules including proteins to pass through. Figure 2-40 [91] shows an artist's rendering of what the structure might look like.
The peptidoglycan polymers then crosslink with other peptidoglycan chains to form a complex mesh that wraps the cell in a structure a kin to chicken wire.
There are numerous antibacterial agents that target the bacterial cell wall because mammals do not synthesize walls and therefore are not susceptible to the toxic effects of these agents. Penicillin inhibits the linking of the amino acid side chains of peptidoglycan units, which therefore weakens the stability of the wall eventually, causing the cells to rupture. Humans and other animals even synthesize an enzyme that specifically attacks bacterial cell walls. The enzyme lysozyme is found in many body fluids and hydrolyzes the NAM-NAG bond in the cell wall. It serves as a critical part of the mammalian defense against bacterial invasion. Figure 2-41 [92] shows a depiction of the gram-positive cell wall.
The cell wall is made mostly of peptidoglycan, interspersed with teichoic acid which knits the different layers together. The amount of crosslinking is higher and the wall is thicker than in gram-negative cell walls.
Another structure in the gram-positive cell wall is teichoic acid. It is a phosphodiester polymer of glycerol or ribitol joined by phosphate groups. Amino acids such as D-alanine are attached. Teichoic acid is covalently linked to muramic acid and stitches various layers of the peptidoglycan mesh together. Teichoic acid stabilizes the cell wall and makes it stronger. The chemical formula of teichoic acid is shown in Figure 2-42 [93]
Teichoic acid is a long, thin molecule that weaves through the peptidoglycan.
Gram-negative cell walls have a more complicated structure than do those of gram-positive organisms. Outside the cytoplasmic membrane is the periplasm, which contains the thin layer of peptidoglycan. The peptidoglycan in gram-negative cells contains less cross-linking than in gram-positive cells with no peptide linker. Covalently bound to the peptidoglycan is Braun's lipoprotein, which has a hydrophobic anchor in the outer membrane that helps to strongly bind the peptidoglycan to the outer membrane. Figure 2-43 [94] shows the arrangement of the gram-negative cell wall.
The cell wall in gram-negative bacteria contains much less peptidoglycan and is surrounded by an outer membrane. There is much less crosslinking between the peptidoglycan. LPS is also present in the outer membrane and penetrates into the surrounding environment.
The outer membrane of gram-negative bacteria is another lipid bilayer similar to the cytoplasmic membrane, and contains lipids, proteins, and also lipopolysaccharides (LPS). The membrane has distinctive sides, with the side that faces the outside containing all the LPS. LPS is composed of two parts: Lipid A and the polysaccharide chain that reaches out into the environment. Lipid A is a derivative of two NAG units with up to 7 hydrophobic fatty acids connected to it that anchor the LPS in the membrane as shown in Figure 2-44 [95]. Attached to Lipid A is a conserved core polysaccharide that contains KDO, heptose, glucose and glucosamine sugars. The rest of the polysaccharide consists of repeating sugar units and this is called the O-antigen. The O-antigen varies among bacterial species and even among various isolates of the same species. Many bacterial pathogens vary the make-up of the O-antigen in an effort to avoid recognition by the host's immune system.
LPS is composed of three sections: the lipid A region, a conserved core polysaccharide, and a highly variable O-polysaccharide. (A) The chemical structure of LPS. (B) A molecular model of the membrane from Pseudomonas aeruginosa. Source (T. P. Straatsma, Pacific Northwest National Laboratory)
LPS confers a negative charge and also repels hydrophobic compounds including certain drugs and disinfectants that would otherwise kill the cell. Some gram-negative species live in the gut of mammals and LPS repels fat-solubilizing molecules such as bile that the gal bladder secretes. This repulsion enables these bacteria to survive in this environment. The O-antigen and other molecules on the outer membrane are also used by certain viruses that infect bacteria, as a means to identify the correct hosts for infection.
LPS is medically important because when LPS is released from bacterial cells it is toxic to mammals and is therefore called endotoxin [96]. It creates a wide spectrum of physiological reactions including the induction of a fever (endotoxins are said to be pyrogenic [97]), changes in white blood cell counts, leakage from blood vessels, tumor necrosis and lowered blood pressure leading to vascular collapse and eventually shock. At high enough concentrations the LPS endotoxin is lethal. Finally, the outer membrane keeps the enzymes in the periplasm from floating away from the cell.
There are fewer total proteins and fewer unique types of proteins in the outer membrane than in the cytoplasmic membrane. Porins are particularly important components because of their role in the permeability of the outer membrane to small molecules. Porins are proteins that form pores in the outer membrane large enough to allow passage of most small hydrophilic molecules. Figure 2-45 [98] shows the structure of a porin at the molecular scale. All known porins have a similar structure, with the protein containing a central channel that allows the passage of molecules. This allows migration of these molecules into the periplasmic space for possible transport across the cytoplasmic membrane. Some porins in the outer membrane are general, doing simple discrimination on size and charge, but having little substrate specificity. Examples include OmpF that is selective for positively charged molecules and PhoE that is permeable to negatively charged molecules. Other porins are more specific. The best studied is LamB, which recognizes the sugar polymer maltooligosaccharide and transports it through the outer membrane. Very large or hydrophobic molecules cannot penetrate the outer membrane, so the outer membrane serves as a permeability barrier to at least some molecules.
The molecular structure of a porin. The view in (A) is from the outside of the cell looking at the membrane surface. The view in (B) is the perspective from the side (i.e. from the membrane). The porin has three protein subunits and the actual pore is the central triangular area in the top panel formed by the three subunits.
There are also other types of outer membrane proteins that are involved in various functions. OmpA in E. coli seems to connect the outer membrane to the peptidoglycan. Some pathogens contain outer membrane proteins that help them neutralize host defenses. Finally all gram-negative bacteria contain high molecular weight proteins involved in the uptake of large substrates such as iron-complexes and vitamin B12.
The differences between the cell walls of gram-positive and gram-negative bacteria greatly influence the success of the microbes in their environments. The thick cell wall of gram-positive cells allows them to do better in dry conditions because it reduces water loss. The outer membrane and its LPS helps gram-negative cells excel in the intestines and other host environments. Figure 2-65 [99] summarizes the difference between gram-negative and gram-positive cell walls.
| Property | gram-positive | gram-negative |
| Thickness of wall | 20-80 nm | 10 nm |
| Number of layers in wall | 1 | 2 |
| Peptidoglycan content | >50% | 10-20% |
| Teichoic acid in wall | + | - |
| Lipid and lipoprotein content | 0-3% | 58% |
| Protein content | 0% | 9% |
| Lipopolysaccharide | 0 | 13% |
| Sensitive to penicillin | Yes | Less sensitive |
| Digested by lysozyme | Yes | Weakly |
A summary of the differences between gram-positive and gram-negative cell walls.
For most bacterial cells, the cell wall is critical to cell survival, yet there are some bacteria that do not have cell walls. Mycoplasma species are widespread examples and some can be intracellular pathogens that grow inside their hosts. Cell walls are unnecessary here because the cells only live in the controlled osmotic environment of other cells. It is likely they had the ability to form a cell wall at some point in the past, but as their lifestyle became one of existence inside other cells, they lost the ability to form walls. Consistent with this very limited lifestyle within other cells, these microbes also have very small genomes. They have no need for the genes for all sorts of biosynthetic enzymes, as they can steal the final components of these pathways from the host. Similarly, they have no need for genes encoding many different pathways for various carbon, nitrogen and energy sources, since their intracellular environment is completely predictable. Because of the absence of cell walls, Mycoplasma have a spherical shape and are quickly killed if placed in an environment with very high or very low salt concentrations. However, Mycoplasma do have unusually tough membranes that are more resistant to rupture than other bacteria since this cellular membrane has to contend with the host cell factors. The presence of sterols in the membrane contributes to their durability by helping to increase the forces that hold the membrane together.
Other bacterial species occasionally mutate or respond to extreme nutritional conditions by forming cells lacking walls, termed L-forms. This phenomenon is observed in both gram-positive and gram-negative species. L-forms have varied shapes and are sensitive to osmotic shock.
Surface structures are typically attached to a membrane, and extend into the environment. Important structures include flagella [100], pili [101], fimbriae [102], and glycocalyx [103]. These protrusions and surfaces interact with the environment around the microorganism and therefore are pivotal in how the microbe sees the world and how we see the microbe.
Surface structures are typically attached to a membrane and extend into the environment. Important structures include flagella [104], pili [105], fimbriae [106], and glycocalyx [107]. These protrusions and surfaces interact with the environment around the microorganism and therefore are pivotal in how the microbe sees the world and how we see the microbe.
In many bacteria, flagella are responsible for motility in liquid. There is also a loose correlation between cell shape and the presence of flagella. Almost all spirilla, half of all rod-shaped bacteria, and only a few of the cocci are motile by flagella. In fact, most cocci are non-motile. One rationale for this correlation might be that spherical cells such as the cocci simply do not have the best geometry for directional movement by flagella, unlike more linear bacteria.
Flagella can be thought of as little semi-rigid propellers that are free at one end and attached to a cell at the other. Flagella are thin (20 µm) and long with some having a length 2-3 times (about 10 µm) the length of the cell. Due to their small diameter, flagella cannot be seen in the light microscope unless a special stain is applied. Bacteria can have one or more flagella arranged in clumps or spread over the cell surface. Figure 2-46 demonstrates some of the more common arrangements.
A cartoon of several common flagellar arrangements.
Flagella are mostly composed of the protein flagellin, which is bound in long chains and wraps around itself in a left-handed helix as shown in Figure 2-47 [108]. The number of protein monomers that it takes to make a single turn of the helix is determined by the protein subunits themselves.
Flagella are attached by a hook and rings that anchor it to the cell wall of the microorganism. In gram-positive bacteria (A) the rings are located in the cytoplasmic membrane and the flagella passes through the peptidoglycan to the outside environment. In gram-negative bacteria (B) there are additional protein rings in the outer membrane.
The flagellum is attached to the cell through complex protein structures termed the hook and the basal body. One ring in the basal body rotates relative to the other causing the flagellum to rotate. The energy to drive the basal body is obtained from the proton motive force. In some fashion the translocation of protons from outside to inside the membrane causes the rotation of the flagellum. In a sense, the protons move through the wheel-like structure of the basal body (similar to a water wheel, except using protons) and this causes the rotation of the assembly including the flagellum. When E. coli is swimming through a solution the flagella turn counter-clockwise and push the microbe through solution. This behavior is termed smooth swimming. It is possible for E. coli to also reverse the direction of flagellar rotation and when the flagella turn clockwise, they pull against the bacterial cell. Since E. coli is flagellated peritrichously (that is, at many positions), it is pulled in all directions and tumbles.
How fast do bacterial cells move? They average 50 µm/sec, which is about 0.00015 kilometers/hr. This may seems slow but remember their tiny size. Figure 2-66 [109] shows a better comparison and indicates that relatively speaking, bacteria are faster than humans. Also remember that this motility happens in water, which is much more viscous than air.
| Organism | Kilometers per hour | Body lengths per second |
| Cheetah | 111 | 25 |
| Human | 37.5 | 5.4 |
| Bacteria | 0.00015 | 10 |
Bacteria seem slow, until you consider relative size. Then they are quite fast.
If a flagellum breaks off it is resynthesized until it reaches the appropriate length. This growth actually occurs from the tip. The flagellar filament is hollow and flagellin monomers are passed up through this space until they reach the growing tip and are added to the structure.
Typically microbes in aqueous environments continually move around looking for nutrients. Even microorganisms in the soil have uses and opportunities for movement. Sometimes this movement is random, but in other cases it is directed toward or away from something. As a rough guide, bacteria want to move towards food or energy sources and away from toxic compounds. In other words, bacteria are capable of showing simple behavior that depends upon various stimuli.
There are several classifications of tactic responses and the category is based upon the stimulus that the movement is responding to.
The purpose of chemotaxis should be fairly evident: attraction to nutrients or avoidance of damaging compounds. A cell distinguishes chemicals that fall into either of these classes with receptor molecules near their surfaces that can tell if specific chemicals are in the environment. Many of these receptors are also involved in transport of their target molecules. Now it actually is trickier than it sounds. A cell does not merely want to sense the presence of nutrient, but wants to move toward the highest concentration of it. It does this by an amazing process that is sort of a primitive memory, where it essentially asks if its receptors are binding more or less of the compound than they were a moment ago ("a moment ago" being about 200 milliseconds.). If they are binding more, the cell keeps swimming, but if they are binding less, it tumbles and then swims in a random direction. This tumbling is the direct result of E. coli's ability to reverse the rotation of its flagella.
Phototaxis is somewhat different, but again the cells move toward optimal levels of specific wavelengths of light. Rhodobacter sphaeroides (a photosynthetic microbe) performs phototaxis by a mechanism analogous to chemotaxis in E. coli, but there are important differences. R. sphaeroides contains only one polar flagella, which rotates during a run toward light. If, during a run, light conditions worsen, the flagella stop rotating (instead of reversing its rotation). The microbe instead depends upon Brownian movement in the environment to turn it in a new direction. R. sphaeroides is also capable of modulating the speed of rotation of the flagella in response to the environment, swimming faster when conditions are improving and slower if conditions are deteriorating.
Aerotaxis refers to the ability of some bacteria to be respond to the presence of oxygen. It is mechanistically generally similar to what has just been described and depends on levels of dissolved oxygen in the environment, but whether a bacterium swims toward it or away depends on the type of metabolism that it has.
Flagella are not the only means bacteria have developed for moving about the environment. A large collection of phylogenetically diverse bacteria have developed gliding motility [110], which is an energy-requiring process by which bacteria move smoothly over a solid surface. Gliding microbes tend to be predominately gram-negative, but there are examples of gram-positive bacteria. This type of motility is not as well understood as flagellar propulsion, and there appear to actually be several different mechanisms employed to accomplish it.
In one mechanism the microbes use pili (type iv pili [111] specifically) that are extended away from the cell and stick to the surrounding surface. The microbe then pulls itself toward the tethered end by retracting the pilus back inside the cell. By repeating this process the cell drags itself along a surface. This type of motility is observed in a number of microorganisms including Pseudomonas aeruginosa, Neisseria gonorrhoeae, and Myxococcus xanthus. Movement in this manner requires energy in the form of ATP.
There are also gliding bacteria that use other mechanisms. Some filamentous cyanobacteria seem to extrude an extracellular polysaccharide through small pores on the surface of the organism. Figure 2-48 [112] depicts this type of gliding motility. It is thought that the polysaccharide exiting from the cell propels the microbe along the surface by some means.
Colonies of vegetative cells of a Paenibacillus isolate moving across an agar surface using gliding motility. (Source: Jon Roll, University of Wisconsin-Madison)
A third groups of microbes (Cytophaga and Flavobacterium) seem to use yet another mechanism that is dependent upon proton motive force. Observation of these microbes in the presence of very small latex beads show the beads moving along the surface of the microbe in a directional manner. The bead moves from one pole of the rod-shaped bacteria to the other, sometimes reversing direction. One model to explain this behavior is the concerted movement of cytoplasmic membrane proteins that are coupled to outer membrane proteins. These membrane proteins might form a kind of conveyor belt. The outer membrane proteins in this mechanism are in contact with the environmental surface and their movement propels the microbe forward.
Interestingly, Myxococcus xanthus uses several of these mechanisms in its motility. M. xanthus is a social predator. It glides around in large groups of cells secreting toxins and degradative enzymes that kill other microbes. The leftovers of these dead microbes then provide nutrients for the marauding Myxococcus. Investigations by Dale Kaiser and others have revealed that this microbe actually has two types of gliding motility, social and adventurous. Social motility occurs in groups while adventurous motility involves single cells. It turns out that social motility is dependent upon pili as described above, while adventurous motility involves the extrusion of slime from pores in the microbe's cell wall. Dr. Kaiser has a collection of interesting images and movies [113] that describe this interesting microbe
Pili and fimbriae are structurally similar to flagella and are composed of one or a few proteins arranged in a helical fashion. Figure 2-49 [114] shows pili isolated from Neisseria gonorrhea. Each protein subunit assembles on the growing structure at the tip, as is the case with flagella. There are a number of genes necessary for the successful construction of pili and their products might perform functions such as moving the structural proteins across the membrane, methylating the structural proteins or retracting the pilus. The same is generally true for fimbriae.
The pili shown in this micrograph are those of Neisseria gonorrhea with Tobacco Mosaic virus (the thicker structures) added as a size reference. The length of the Tobacco Mosaic virus is 0.05 µm. (Source: Katrina Forest, University of Wisconsin-Madison)
Fimbriae are found on many bacteria and are shorter and straighter than flagella and are more numerous. Not all bacteria synthesize them. Fimbriae do not function in motility, but are thought to be important in attachment to surfaces. Some microbes attach to hosts by fimbriae, and successful colonization of many surfaces is totally dependent upon the ability to make fimbriae. Swarming microbes such as Myxococcus use them to sense the presence of similar microbes, which helps keep their "hunting packs" together.
Pili are longer than fimbriae and there are only a few per cell. They are known to be receptors for certain bacterial viruses, but certainly the bacterium makes them for another purpose. There are two basic functions for pili: gene transfer and attachment to surfaces. In genetic transfer in a broad variety of bacteria, a donor bacterium attaches to a recipient by the sex pilus. Then the donor cell depolymerizes the pilus at the end that is attached to itself, which draws the cells together and eventually a small pore is created between the two cells. DNA is then transferred through that pore from the donor to the recipient and the cells separate. For a long time, it was thought that the donor bacterium's genome passed through the sex pilus into the recipient, but this is certainly not the case. Transfer of genes this way is not restricted to related species, which implies that a pilus from one organism can attach to a variety of others. Conjugation, as this transfer process is known, is one explanation for the rapid spread of drug resistance in many different species of bacteria and is covered in depth in the Chapter on Genetics and Genomics.
Pili have also been show to be important for the attachment of many types of microorganisms to surfaces. For example, Neisseria gonorrhoeae, the causative agent of gonorrhea, has a special pilus that helps it adhere to the urogenital tract of its host. The microbe is much more virulent when able to synthesize pili.
The general term for any network of polysaccharide or protein containing material extending outside of the cell is the glycocalyx [115]. Many bacteria produce such a coating on the outside of their cell, and they come in two types: capsules and slime layers. The difference between the two is somewhat arbitrary. A capsule is closely associated with cells and does not wash off easily, while a slime layer is more diffuse and is easily washed away. Figure 2-50 [116] shows the capsule surrounding Klebsiella planticola.
The capsule (made of polysaccharide) in the figure is colorless and about the diameter of the cell. The background is darker.
There are many different types of proteins, polysaccharides, polyalcohols and amino sugars in glycocalyx and the exact makeup is species-specific. The structure can be thick or thin, rigid or flexible. Observing cells stained with India ink in the microscope shows dark cells with an outline around them, as the stain does not penetrate the glycocalyx.
There are several functions attributed to glycocalyx, one of which is to help cells attach to their target's environment. Streptococcus mutans produces a slime layer in the presence of sucrose. This forms a surface that allows other bacteria to aggregate on tooth surfaces and results in dental plaque. Vibrio cholerae, the cause of cholera, also produces a glycocalyx that helps it attach to the intestinal villi [117] of the host. Glycocalyx can play other roles in pathogenesis as well. Bacteria that enter the body are always in danger of being attacked by phagocytes (host cells that protect you from invaders). Often the capsule makes it more difficult for phagocytes [118] to attach to and engulf pathogens. As one example, Streptococcus pneumoniae, when encapsulated by a glycocalyx, is able to kill 90% of infected animals, while non-encapsulated forms cannot kill. In addition capsules and slime layers are largely hydrophilic, so they can bind extra water in the environment and contribute to the protection of the cell from desiccation. Capsules and slime layers can also provide protection from the loss of nutrients by holding them within the layer. These extra layers coating the surface of the cell may also potentially mask viral receptors making it more difficult for viruses to attach. Many of these functions manifest themselves in the form of biofilms, which allow the formation of communities of microorganisms all held together by glycocalyx. Biofilms are covered in more detail in the chapter on Environmental Microbiology.
For obvious reasons we have focused on growing cells, but there are non-growing states of microbes that are important to both microbes and humans. In these states, termed spores and cysts, the cells remain dormant for long periods of time. Part of the relevance of these states is that the very properties that allow the cell to survive extended time periods also happen to make the cells resistant to our typical efforts to kill them. As a consequence, the attempt to sterilize a sample can be thwarted by the presence of bacterial spores or cysts. In this section we examine some of the properties of these structures.
Spores and cysts are resting structures. That is, these states have very low to nonexistent rates of metabolism. They are common in organisms that live in soil and may need to survive some rough conditions such as lack of nutrients, high heat, radiation, or drying.
Sporulation is a unique developmental cycle. After the decision to sporulate is made, creation of a different type of cell needs to take place, which requires turning on a large collection of genes in a tightly coordinated fashion. In addition, all of this expression has to be finished before the microbe runs out of energy. There are several types of spores. Some are highly resistant structures that are formed under conditions of cell stress and are created inside a supportive cell and are termed endospores [119]. Others are part of the normal reproductive cycle, being created by differentiation of a vegetative cell and we will refer to these as spores. In this section we talk generally about the structure of spores and in the chapter on Regulation we will examine the regulation of sporulation.
Endospores are refractile - light cannot penetrate them - so that they are very easy to see in the phase microscope and this makes it easy to detect them. Most endospores [120] have no measurable metabolism [121] and are really a form of suspended animation. Endospores can survive for a very long time, and then return to a growing state, a process termed germination. Endospores that were dormant for thousands of years in the great tombs of the Egyptian Pharaohs were able to germinate and grow when placed in appropriate medium [122]. Several scientists have been able to recover viable endospores from bees trapped in amber that is 25-40 million years old. The microbe isolated was found to be most closely related to Bacillus sphaericus. There are even claims of endospores that are over 250 million years old being able to germinate when placed in appropriate medium, but these claims still need to be verified. Endospores are found everywhere, are easily dispersed throughout the environment and can be difficult to remove. The anthrax scare of 2001 in the United States is ample evidence of the insidiousness of endospores and their impressive resistance.
Endospores are resistant to heat (>100 °C), radiation, many chemicals (i.e. acids, bases, alcohol, chloroform), and desiccation. The mechanisms that account for this resistance include the impermeability of the endospore coat, the dehydration of the cytoplasm [123] and the production of special proteins that protect the spores DNA [124]. Figure 2-51 [125] shows the major structures of an endospore.
An electron micrograph of an endospore of Bacillus subtilis showing the core, cortex and coat. (Source: M. Serrano, et al. 1999. J. Bacteriol. 181:3632-3643)
The formation of an endospore is clearly a great advantage for these bacteria [126] and enables them to endure extreme stress. At a later time, even much later, when conditions are favorable, they can reemerge and flourish. Endospores enable a species [127] to spread easily from one suitable environment to another an many endospore-forming bacteria are ubiquitous in the environment. Endospores are a particular problem in the food industry where great care must be taken to insure either the destruction of endospores or suitable preservation methods so that endospore-forming bacteria (and other microbes) cannot grow.
Endospores can be divided into several important parts (Fig 2-51). The center of the endospores contains the core and it consists of the cytoplasm, DNA, ribosomes, enzymes and everything that is needed to function once returned to the vegetative state. The core is dehydrated, which is essential for heat resistance, long-term dormancy and full chemical resistance. Calcium dipicolinate is a major component of the core and has been shown to play a role in resistance to wet heat and UV [128] light. The cortex surrounds the core and is composed of two layers, a thin more densely staining layer that is similar in structure to the vegetative cell [129] wall and a thicker less dense layer containing modified peptidoglycan. Two major modifications are present. First, there is less cross-linking with only 3% of the muramic acid present in the peptidoglycan of the cortex participating, in comparison to 40% of muramic acid in the vegetative cell wall. Second, much of the muramic acid is modified to a muramic-β-lactam structure. Both of these modifications of the cortex appear to be important in germination. Muramic-β-lactam serves as a specific target for lytic enzymes that are activated during germination and the lower cross-linking enables easier outgrowth. Outside of the cortex is the spore coat [130] containing several protein [131] layers that are impermeable to most chemicals. The coat is composed of more than two dozen different types of proteins and there is some evidence that these proteins are connected by cross-links. This covalent connection between coat proteins probably contributes to the spores' resistance.
Endospores are not the only type of spore made by microorganisms. Azotobacter species and several others are know to form cysts, which are dormant cells with thickened cells walls. Cysts are resistant to desiccation and some chemicals, but cannot withstand high temperatures as endospores can. The actinomycetes are a large group of spore-forming, gram-positive bacteria that grow by forming long tubules called filaments. Under nutrient poor conditions these filaments differentiate into round resting structures termed spores. In contrast to endospores, these structures are part of the reproductive process. The developmental process to create an actinomycete spore is less complex than that of the endospore. It involves the simple formation of cross walls that divide the filament into sections, each containing a chromosome. These then differentiate into mature spores. During this process a tougher cell wall is laid down and there is conversion of the cytoplasm to a dormant state so that the spore becomes more resistant to heat and chemicals, though not as hardy as an endospore. Actinomycete spores are capable of surviving for long periods of time (for years) and can germinate into vegetative cells when appropriate growth conditions are present. Many different genera are capable of forming this type of spore and the ability to form these structures does not seem to correlate with any group of microorganisms.
The micrograph shows cysts and cells of Azotobacter vinelandii at 1000 X magnification. The cysts are the phase-bright objects, while the cells are darker.
Some photosynthetic filamentous cyanobacteria are capable of forming specialized structures called heterocysts [132]. These are rounded structures distributed at regular intervals along the string of vegetative cells as shown in Figure 2-53 [133] or at one end. Heterocysts evolved to solve the problem of performing plant-like photosynthesis (which produces oxygen) and at the same time fixing N2 to ammonia (a process that involves enzymes that are inactivated by O2). The single focus of the heterocysts is to fix N2, while the rest of the cells perform photosynthesis (and divide), thus keeping the two processes separate. Heterocysts develop a surface that is impermeable to gasses, and begin synthesizing large amounts of nitrogenase, the protein that fixes N2. Importantly, these heterocysts maintain some permeability to the cells on either side. Neighboring cells take N2 from the atmosphere and pass it along to the heterocyst, which reduces it to NH3 and returns fixed nitrogen to its neighbors. The neighboring cells also take up and utilize O2, but they prevent it from reaching the heterocyst. Heterocysts are essentially specialized organs for the "multi-cellular organism" represented by a chain of cyanobacterial cells and they are only formed when nitrogen is limiting. The regulation of this developmental cycle is intriguing and serves as a simple example of multicellular development in a unicellular organism.
Heterocysts are common in several different groups of cyanobacteria and are the site of nitrogen fixation. Note the slightly enlarged size and distinct shape of the heterocyst when compared to the vegetative cells on either side. (Source: Michael Clayton, University of Wisconsin-Madison)
This ends our survey of the cellular structure of bacteria. In the remaining sections we take a look at the major structural differences that distinguish the archaea and eukaryotes from the bacteria.
Before 1977 prokaryotes were phylogenetically organized into one group termed the Monera. The discovery of the extreme thermophile Thermus aquaticus in the hot springs of Yellowstone National Park by Tom Brock and the subsequent analysis by Carl Woese began a series of experiments that would change how scientists think about the organization of life on this planet. This revolution is the subject of later chapters, but here we will discuss the structural differences between this new group, the Archaea, and the Bacteria. "Bacteria" and "Archaea" refers to the formal phylogenetic classification of organisms, but we will typically refer to them more casually as groups of organisms and then use "bacteria [134]" and "archaea [135]". The third domain consists of eukaryotic organisms, both microbes and multi-cellular and is referred to as Eukarya [136]. As explained early in this chapter, it has become evident that eukaryotes arose when certain bacteria became engulfed in archaeal cells, eventually becoming organelles. Not surprisingly then, Archaea is a group of microbes that share some things in common with Bacteria, others with Eukarya and have still other properties that are all their own. In the following section we highlight some of the major structural features of the Archaea.
| Property | Bacteria | Archaea | Eukarya |
| RNA polymerase | 4 proteins Rifampicin sensitive | 8-10 proteins Rifampicin resistant | 12 proteins (RNA pol II) Rifampicin resistant |
| Transcription start site | Variable often contains a -35 and -10 region | TATA box | TATA box |
| Starting amino acid | formylmethionine | methionine | methionine |
| Lipids | ester-linked | ether-linked | ester-linked |
| Cell wall composition |
G+ peptidoglycan G- peptidoglycan and outer membrane |
pseudopeptidoglycan or S-layer of proteins, glycoproteins, or polysaccharides |
none or cellulose |
Archaea are a unique form of life, as different phylogenetically from bacteria as they are from eukaryotes.
DNA-dependent RNA polymerases in bacteria and archaea are different from each other. The primary RNA polymerase of all organisms is responsible for creating messenger RNA that is then translated into proteins at the ribosome. Bacterial RNA polymerase is relatively simple, containing 4 different proteins. In contrast, RNA polymerase from methanogens and halophiles (both Archaea) contains 8 proteins. In hyperthermophiles (Archaea) RNA polymerase is even more complex, containing 10 proteins. None of the archaeal RNA polymerases are affected by the antibiotic rifampicin, a known inhibitor of the bacterial RNA polymerase. The eukaryotic RNA polymerase responsible for most mRNA transcription (termed RNA polymerase II) contains 12 proteins and is similar to the RNA polymerase in the archaea.
RNA polymerases from all organisms recognize a variety of start sequences or promoters. A promoter for mRNA transcription in bacteria is recognized by the (sigma [137]) protein and has two recognition zones about 10 and 35 bases before the transcription start site. The exact sequence recognized by RNA polymerase depends upon the σ factor that has bound to the enzyme, and cells have different numbers of σ factors that bind to different types of promoter sequences. This allows regulation of mRNA expression by changing the levels of σ factors inside the cell. In archaea and eukaryotes the transcription recognition sequence is a TATA sequence (termed the TATA box [138]) and transcription is regulated by various protein transcription factors that bind to regions near the TATA box and then recruit RNA polymerase.
The best-studied bacteria have promoters at two positions upstream of the transcription start site that are recognized by the σ factors associated with RNA polymerase. The transcriptional control elements in the archaea are much more like those found in eukaryotes, where RNA polymerase is recruited to a promoter by interaction with protein regulatory factors, and transcription begins downstream of a TATA box.
The structural make-up of the ribosomes of bacteria and archaea are similar in many respects. The ribosomes of archaea and bacteria are of the same size (70S) and are smaller than those of eukaryotes (80S). However, most of the ribosomal proteins, translation factors and tRNAs of archaea more closely resemble their counterparts in eukaryotes. In fact, mixing experiments with ribosomes from E. coli (Bacteria), Sulfolobus (Archaea) and yeast (Eukarya) have shown functional substitution between Archaea and Eukarya. Specifically, when the small subunit from a yeast ribosome was mixed with the large subunit from Sulfolobus, the hybrid ribosome was capable of translation in vitro. When the small subunit from yeast was mixed with the large subunit of E. coli, translation did not occur. Also, in all organisms translation begins at a start codon (typically an AUG in the mRNA). In bacteria, this codon causes the insertion of formylmethionine [139], while in archaea and eukaryotes, it results in insertion of an unmodified methionine.
Archaeal membranes have features unlike those found in either eukaryotes or bacteria. The lipids in archaea have a different chemical make-up in the following way. Remember that lipids in bacteria are amphipathic molecules (i.e. having both hydrophobic and hydrophilic portions) containing a backbone of glycerol connected to a hydrophilic head group and two hydrophobic long-chain fatty acids. In eukaryotes and bacteria the fatty acids are attached to the glycerol backbone by ester bonds, while in archaea ether linkages are used. Also, the stereochemistry of lipids from archaea is primarily of the S form, while that of bacteria and eukaryotes is of the R form. In some archaea the hydrophobic chains attached to the glycerol backbone are twice normal length and pass completely through the membrane, attaching to a second backbone on the opposite side. This adds extra stability to the membrane and these dual lipids are often found in archaea living in extreme environments.
Archaea have an ether linkage between their fatty acids and the glycerol backbone, which is in contrast to the ester linkage seen in bacteria and eukaryotes. (A) An ether-linked lipid show as the chemical structure and a space-filling model. (B) A tetraether lipid, showing a general chemical structure. An example of a tetraether lipid from the thermophilic archaeal species Thermoplasma acidophilium is shown as a space-filling model.
As we have discussed, almost all bacteria have cell walls. These walls contain peptidoglycan, with N-acetyl muramic acid being the signature molecule for the presence of peptidoglycan. Archaea are considerably more diverse in the composition of their cell walls. They lack peptidoglycan, but some contain pseudomurein that has a similar structure as shown in Figure 2-54 [140]. N-acetylalosaminuronic acid replaces N-acetylmuramic acid in the backbone of the molecule and each glycan unit is linked together using 1,3 glycosidic bonds, instead of the 1,4 glycosidic bonds seen in peptidoglycan. Many archaea do not contain a peptidoglycan molecule in any form, instead covering the outside of the membrane with proteins, glycoproteins or polysaccharides. Scientists refer to these cell walls as S-layers. In any case the function of the cell wall remains the same, containing the cytoplasm and giving the microbe its shape.
Archaea have several different types of cell wall. Some contain a structure reminiscent of peptidoglycan called pseudomurein. The chemical formula is pictured on the left. Other microbes will have a surface layer (S-layer) composed of repeating units of one or a few proteins, glycoproteins or sugar. These crystal lattices serve to protect the cell. The micrograph on the right shows the surface of Methanospirillum hungatei cells. Note the regular repetition of the pattern on the outside surface. (Source: M. Firtel, et al. 1993. J. Bacteriol. 175:7550-7560)
These various differences between archaea, bacteria and eukaryotes indicate major differences between these life forms, and phylogenetic analysis bears out this conclusion. It is clear that the archaea are more divergent from bacteria than we are from an amoeba. This in turn indicates that the common progenitor of these groups existed very early in evolution. Analysis and classification of the archaea has fundamentally changed how biologists think about organismal diversity.
After the recent journey through the bacterial cell, you may have started to wonder about your own cells or other eukaryotic cells. How many properties do we share with bacteria? How are we different? It turns out, as you might expect, we share some basic things in common, but other structures are very different.
It turns out that much of what you now know about bacterial cells also applies to those of eukaryotes. It is safe to say we are more similar than we are different. The basic building blocks of the cell, such as nucleic acids, amino acids and sugars are identical. Macromolecular organizations such as chromosomes and membranes have many similarities. Many proteins in eukaryotes, especially those that carry out essential cell functions, have homologs in bacteria that share a high degree of sequence and structural similarity. An example that illustrates this point is the respiratory enzyme cytochrome oxidase. As shown in Figures 2-55 and 2-61, a comparison of cytochrome oxidase from the bovine and Rhodobacter sphaeroides reveals a near identical arrangement of the catalytic proteins and high sequence similarity. However, the cytochrome oxidase in the bovine has a number of other polypeptides that serve a structural role.
Molecular models of cytochrome oxidase from Rhodobacter sphaeroides (A) and bovine (B) are compared. Each protein is a complex of several distinct proteins, but the four polypeptides shown in color have a high degree of similarity in both their sequence (see Figure 2-61) and structure. The structural similarity should be obvious in this view. Such structurally similarity cannot have arisen by chance, but must reflect the evolution of each from a single ancestor. The gray polypeptides in the bovine cytochrome oxidase are not found in the bacterial protein.
A sequence comparison of cytochrome oxidase showing the high degree of identical amino acids between these very different species: cows and two different bacteria. The colored boxed indicate where the amino acids are identical or similar among the three sequences and the different colors refer to different classes of amino acids.
The DNA in all organisms is chemically similar but the organization of the helix into higher order structures varies. Eukaryotes contain a larger number of histones (chromosome-binding proteins) and in many cases during cell division, compaction of the chromosome takes place, so that they are visible using light microscopy.
The basic mechanism of converting genetic information into proteins is also rather similar. Most all of the central components of this process show sequence similarities across biology, and substantial functional similarities as well. The fact that eukaryotes have nuclei means that mRNA must be transported outside of that structure before translation can begin, but the process is otherwise rather similar.
The most conserved of all structures is probably the membrane. Membranes enclose all living systems and every membrane contains amphipathic lipids. The exact chemical structure of the lipids is often different depending upon the species and its environment, but the overall arrangement of the membrane is the same. Membranes perform remarkably similar functions in all species: keeping the cytoplasm in and the environment out. Cholesterol is common in the membranes of eukaryotes, but is uncommon in bacterial and archaeal membranes.
Eukaryotes are typically more complex than prokaryotes and appear more organized when examined in the microscopic. This organization into different intracellular compartments likely reflects the demands of a more complex cellular system. It may also be that the formation of these separate organelles allowed the subsequent evolution of more elaborate cells. Figure 2-70 [141] diagrams the various structures found in the typical eukaryotic cells. Note that chloroplasts are only found in photosynthetic organisms. The organelles are the nucleus [142], the mitochondria [143], the endoplasmic reticulum [144] and the golgi apparatus [145]. Internally eukaryotic cells have a cytoskeleton that determines cell structure and in plants this is supplemented by cell walls. We discuss these structures and their functions in eukaryotic cells in the following sections.
A diagram of the common structures found in eukaryotic cells.
The cytoskeleton is a network of filaments and fibers found in the cytoplasm of many eukaryotic cells. It serves four known roles in cells.
The cytoskeleton components can be divided into three classes based upon the size, distribution and function of the filaments. Microfilaments are the smallest at 4 to 6 µm in diameter and are made of actin. These lie beneath the surface of the cell membrane and are anchored to it, forming a web inside the cell. They dictate the cell's shape and can also be involved in motility by contraction or expansion of the filament. Filaments may also tether organelles to the membrane and help move them around the cell. This movement can be important for modulation of organelle function. Intermediate filaments are 10 µm in diameter and are made of keratin, which is the same protein found in hair and fingernails. These filaments take different forms and are found in many types of cells, but their exact function is unknown. They may play a structural role similar to that of some microfilaments. Figure 2-56 [146] shows some examples of cytoskeletal elements
Eukaryotic cells have several different types of scaffolding proteins to help them keep their shape. Microfilaments, microtubules, and centrioles are all important structural elements.
In addition to the above, there are more complex fibers and structures. Microtubules are hollow cylindrical structures that are 20-25 µm in diameter containing tubulin as the major structural protein. Tubulin polymerizes into a helical cylindrical structure and thirteen of these protofilaments then combine to make a microtubule. Microtubules can form the basis of a number of different structures. Many of these structures form to perform a necessary function and are then disassembled afterwards. Often microtubules form centrioles that contain nine sets of microtubules arranged in a circular matrix. These are 400 µm long and 150 µm wide and are usually found in pairs at right angles to each other. Centrioles are important in proper chromosome segregation and cell division as discussed in the section on the nucleus below. Microtubules are also part of the basal bodies of flagella and cilia.
Cilia and flagella are examples of more permanent structures that contain microtubules. Both cilia and flagella have a similar structure of nine pairs of microtubules arranged in a circular fashion around a tenth pair that runs down the center. Both are attached to the membrane and project into the environment. In a process that requires energy, the microtubules in the outer ring are moved with respect to each other, causing the cilia or flagella to bend and snap back in a whip-like fashion. This bending causes the movement of liquid near the structures such that spent liquid with few nutrients and waste products is moved away from the cell and is replaced by fresh liquid containing nutrients and oxygen. The beating of cilia and flagella can also push the cell through its environment. Cilia are 2 to 10 µm long and 0.5 µm wide and are shorter and typically more numerous than flagella with hundreds of them on some types of cells. An example of a ciliated organism is the unicellular protist Paramecium, which can be found in fresh water ponds. The microbe is a predator of bacteria and motility is vital in this life-style, both for chasing down prey and moving away from danger. Cilia cover the surface of Paramecium and move the organism through the environment by beating in a coordinated fashion. Figure 2-57 [147] shows one example of a protozoan.
Flagella are 50-100 µm in length and there are typically only one or two per cell. Eukaryotic flagella are larger than those found on bacteria or archaea and have a more complex structure. Flagella are found in many unicellular creatures with one example being the dinoflagellates and their primary role is cell motility. These aquatic creatures contain two flagella; one encircling the body of the organism while the other is attached in a perpendicular fashion to the first. Dinoflagellates are often photosynthetic and important as primary producers in the oceans.
The endoplasmic reticulum (ER) is a finely divided system of interconnected membranes, consisting of tubules and vesicles that loop through the cell and are contiguous with the nuclear membrane. A drawing of the ER is shown in Figure 2-58 [148]. It functions in the synthesis of membranes and membrane proteins and is also involved in protein secretion. Not surprisingly, the ER is especially prominent in cells doing a large amount of protein secretion. The ER works very closely with the Golgi apparatus (see below) to carry out these functions. There is no structure in bacterial cells that is analogous to the ER, but many of the same functions are carried out on the inside surface of the cellular membrane in bacteria. ER comes in two types: rough ER and smooth ER.
Eukaryotic cells contain a network of passages that connect various elements of the cell and are also important in secretion.
Rough ER gets its appearance from the presence of ribosomes on its surface as seen in the electron microscope, and its function is the production, processing and export of proteins. During translation an appropriate signal guides the ribosome to the ER membrane and causes the protein to be synthesized directly across the membrane into the lumen of the ER. There proteins may be processed or modified by the addition of carbohydrates to form glycoproteins. After processing proteins move slowly through the ER and are packaged into vesicles of ER membrane called transition vesicles. These release from the ends of the ER and move by elements of the cytoskeleton either to the Golgi apparatus or to the plasma membrane. Once contact is made between the transition vesicle and the Golgi or the plasma membrane, the two fuse and release the contents of the vesicle into the target compartment.
Smooth ER does not contain ribosomes and the lumen and membrane of smooth ER contain a variety of enzymes that perform many functions including modification of toxins and synthesis of steroids.
The Golgi apparatus is an organelle containing a double membrane and it is mainly devoted to the processing of proteins synthesized in the ER. A drawing of the Golgi apparatus is shown in Figure 2-59 [149]. It is found in many eukaryotic cells, but it lacks a well-formed structure in many fungi and ciliate protozoa. It consists of regions of stacked contiguous membranes containing no ribosomes. Each membrane sac is 15 to 20 µm thick and separated from the next stack by about 30 µm. A complex network of tubes and vesicles extend from the edges of these sacs into the surrounding cytoplasm. The stack of membranes has a definite polarity with those near the ER (the cis face) having a different shape and enzyme content than those at the opposite end (the trans or maturing face). Studies of the Golgi apparatus appear to show material flowing into the cis face from vesicles, through the apparatus and then exiting at the trans face.
The Golgi apparatus is involved in the glycosylation and proteolytic processing of proteins that are to be secreted into various cellular organelles or to the outside environment. Proteins often pass through the Golgi apparatus as part of their maturation.
The role of the Golgi apparatus is to package material for export, but its exact function varies depending upon the organism. For example, Giardia and Entamoeba utilize Golgi apparatus to form cell walls during cyst formation. It often participates in the synthesis of cell membranes and the final processing of proteins before export. In many cases the Golgi apparatus contains glycosylation enzymes [150] that add sugars to proteins as they move through its lumen. The type of glycosylation that takes place is dependent upon signals contained within the protein sequence. The Golgi also processes enzymes using proteases, which clip at specific amino acids sequences to form mature proteins and hormones. These mature proteins then move to their final destinations, which may be in the membrane, in lysosomes or secreted into the environment.
One of the most important functions of the Golgi apparatus is the synthesis of lysosomes [151], an organelle that is found in a variety of eukaryotic cells. Lysosomes are spherical structures enclosed in a single membrane that can vary in size from 50 µm to several µm. They are involved in intracellular digestion and contain enzymes (called hydrolases) that digest many types of macromolecules. Hydrolases function best under acidic conditions (pH 3.5 to 5.0) and the lysosome maintains this pH by membrane proteins that pump protons into its interior. Enzymes bound for lysosomes are synthesized on ribosomes that deposit them in the rough ER. They then move though the smooth ER and Golgi apparatus before being package into lysosomes. In some cases lysosomes can also bud off from the smooth ER.
Lysosomes serve a variety of functions depending upon the cell type. In unicellular eukaryotes they are often digestive structures that take bacteria or other substances from the outside environment and degrade them into usable nutrients. In mammals, lysosomes serve to eliminate unwanted particles, either cell structures that are no longer needed or foreign macromolecules (from viruses or bacteria) that have invaded the cell. In any case the hydrolytic enzymes and low pH typically inactivate and then degrade any particle that enters the lysosome
Lysosomes are an important part of endocytosis, a process that eukaryotic cells use to take up particles from the environment. Endocytosis is illustrated in Figure 2-60 [152]. The process creates membrane-bound cavities filled with fluid and solid materials. Larger membrane-enclosed cavities are called vacuoles while smaller ones are called vesicles. Endocytosis comes in two forms, phagocytosis and pinocytosis. Phagocytosis involves the engulfment of large particles, even microorganisms, into membrane-bound compartments. It is a process used most often in the immune system and is described in detail in the chapter on infection and immunity. Pinocytosis involves the recognition of specific particles in the environment as described below. The process is used by unicellular eukaryotic microbes to ingest food and by multicellular organisms to take in certain macromolecules traveling from other parts of the organism.
Eukaryotic cells absorb materials from the outside by using endocytosis. This is a standard pathway through which most material enters the cell.
Pinocytosis begins when protein receptors on the cell surface bind to the target molecule to be ingested. The bound particle migrates to an area of the membrane that is rich in a protein called clathrin. This protein forms a matrix and causes an indentation in the membrane called a clathrin-coated pit. The entrance of a receptor with a bound particle begins a process of invagination at the pit that results in the internalization of the pit and the engulfment of the incoming particle inside a membrane vesicle. This vesicle is called an endosome and once inside the cell, proteins in the endosome membrane begin to pump protons inside, dropping the internal pH of the endosome. The clathrin on the membrane of the endosome then migrates back to the plasma membrane to repeat the cycle. The endosome fuses with a lysosome to form an endolysosome, which causes the ingested particle to be degraded. The valuable breakdown products are transported out of the endolysosome into the cell. The spent endolysosome is called a residual body and sometimes fuses with the plasma membrane to release remaining compounds into the environment.
The ER, Golgi apparatus, lysosomes and endosomes seem to operate as a coordinated whole, functioning in the import and export of materials. It is probably correct to think of these structures as a functional unit and the term vacuome [153] has been coined to describe them. Ribosomes in the ER manufacture proteins and these are modified in the ER and Golgi. The mature proteins eventually find their way to the plasma membrane for export or become part of lysosomes. Lysosomes serve in the endocytotic pathway to take up materials and process them for cell use. They also digest spent cell constituents. All of these processes occur within membrane structures and this carefully controls the import and export of materials from the cell. Considering the extent of these structures in the cell it is remarkable that the membranes of these structures, especially lysosomes, never rupture. If they did it would be catastrophic to the cell and would rapidly lead to cell death.
While the bacterial cell does seem to sequester its chromosome to a portion of the cytoplasm, there is no demarcation that divides the nucleoid from the rest of the cell. In eukaryotes the nuclear membrane separates the cell's DNA from the cytoplasm. The nucleus is the largest and most clearly visible organelle of eukaryotic cells. It contains almost all the cell's DNA and is the site of chromosome replication and transcription. It has two layers of membrane encircling it called the nuclear envelope, with the outer layer being contiguous with the ER. Scattered throughout this nuclear envelope are circular openings known as nuclear pores. These pores are highly discriminatory, allowing easy movement in and out of the nucleus of only appropriate macromolecules such as proteins with specific sequences.
In eukaryotes, the chromosome is not a single circular piece of DNA as in most prokaryotes. Rather, it is split into a number of linear chromosomes with each cell containing at least two copies of each chromosome. The exceptions are those cells that specialize in reproduction and only contain one copy of the cell's chromosomes. Each piece of DNA is complexed with special basic structural proteins called histones that seem to be important in keeping the DNA organized. The DNA is also bound by other proteins involved in its maintenance and the entire set of DNA and associated proteins is called chromatin. For much of the cell cycle chromatin consists of long DNA strands formed into beads by association of histones along it length. Figure 2-71 [154] shows a nucleus in the midst of mitosis, with the chromosomes visible.
Eukaryotic DNA replication takes place during the cell phase called mitosis. At this time, protein synthesis is halted and the chromosomes condense. The sister chromatids meet at the middle of the cell and then migrate to two separate poles. This movement is coordinated by centrosomes, kinetochores and microtubules.
In actively growing cells the DNA is replicated from numerous sites, rather than the single bi-directional origin in prokaryotes. This is necessary due to the much larger amount of DNA found in most eukaryotic cells. During division in prokaryotes, the cell appears to simply split in two with each daughter cell receiving a chromosome. In contrast, eukaryotic cells go through a morphologically distinct phase, mitosis, to achieve separation of the chromosomes. One of the more important events of mitosis is the binding of additional histones and the contraction of the chromatin into compact structures that were called chromosomes due to their staining properties. (The original meaning of the term chromosomes is a colored body, but is now synonymous with a cell's DNA). The two daughter chromosomes formed during replication are physically separated into the separate daughter cells by the filaments called microtubules [155]. These attach at one end to the chromosome at a region termed the kinetochores and at the other end they attach to one of two regions of the cell called a centrosome [156]. By depolymerization of the microtubules at each centrosome, each daughter chromosome is pulled away from its partner and toward a region that eventually reforms as a new nucleus.
There are also a number of important differences in transcription between eukaryotes and prokaryotes. In eukaryotes, mRNA transcription takes place in the nucleus and the finished mRNA moves through the nuclear pores and into the cytoplasm for translation by the ribosomes. The genes of eukaryotic cells also contain regions of largely unimportant DNA, termed introns [157], that do not code for protein. After a gene is transcribed into mRNA these introns are removed before translation. One set of nuclear proteins removes these sequences and splices the actual coding sequences (exons) together to make the finished mRNA. The finished mRNA then travels from the nucleus to ribosomes in the cytoplasm. The mRNA of eukaryotic cells is also decorated with modifications at each end that affect the stability of the mRNA. At the front end is usually a 5’-cap made of 7-methylguanine attached to the mRNA by a triphosphate linkage. At the 3' end of the mRNA is a long stretch of A bases (a poly-A tail) that have a role in mRNA stability as mentioned earlier in this chapter. Finally, while it is quite common in bacteria to have a number of genes on each mRNA transcript, the vast majority of mRNAs in eukaryotes code for only a single protein product. Figure 2-72 [158] shows the steps in gene expression in eukaryotes.
Processing genetic information into protein is more complex in eukaryotes than in prokaryotes. In part this is due to the existence of introns in most genes of "higher eukaryotes" (though introns are rare in yeast). These have to be removed, a poly-A tail added to the 3' end and a cap added to the 5' end of the mRNA before it reaches the cytoplasm for translation.
Eukaryotic cells also contain one or more dark-staining structures within the nucleus called nucleoli. Although they are not enclosed within a separate membrane the nucleoli are complexes with separate granular and fibrillar regions. They are present in non-dividing cells, but frequently disappear during mitosis and again reappear after cell division is over. The nucleolus is the initial site of ribosome synthesis. This structure contains the DNA that codes for the ribosomal RNA genes. The ribosomal RNAs are synthesized and then processed to form the final rRNA molecules. These are then combined with several ribosomal proteins synthesized in the cytoplasm to form an initial ribosomal complex. The entire complex then migrates out of the nucleus into the cytoplasm where it combines with other proteins to form a ribosome.
Mitochondria are involved in energy generation through respiration. Mitochondria have no fixed shape, but often look like short rods in transmission electron micrographs when viewed along their long axis (Figure 2-73). Each mitochondrion contains two membranes. The outer membrane is smooth and serves as a selective barrier. The inner membrane is highly convoluted and folded and contains high numbers of membrane complexes. Nutrients are oxidized inside the mitochondria by catabolic enzymes and the high-energy electrons extracted are donated to a respiratory chain in the inner membrane. These enzymes then create a proton gradient and this gradient is then used to synthesize ATP. ATP leaves the mitochondria and it serves as a source of energy for the rest of the cell's machinery.
Mitochondria are rod-shaped structures that resemble the shape of common bacteria. They contain two membranes, similar to what is found in gram-negative bacteria, and 70S ribosomes. Energy generation occurs at the inner membrane.
Plastids are specialized organelles involved in metabolism that are unique to plants and come in several forms. Amyloplasts are starch storage containers found in some plants. Chloroplasts are oval-shaped structures inside of plant and algal cells that contain an outer and inner membrane as shown in Figure 2-74 [159]. The outer membrane serves a similar function to the outer membrane of mitochondria, while the inner membrane consists of a network of stacks of membranous disks, called thykaloids, which are attached together by narrow tubes of membrane. The thykaloid membranes are the centers of photosynthesis in eukaryotes. They contain enzyme complexes that capture light and produce ATP and high-energy electrons that are used to form sugar from carbon dioxide.
Chloroplasts are the site of the light reactions of photosynthesis. Light striking the chloroplast is converted into a proton motive force and this is used to generate energy. Chloroplasts contain two membranes, again similar to gram-negative bacteria, and the chloroplast itself is a relative of cyanobacteria.
Mitochondria and chloroplasts each have a single circular chromosome and large numbers of ribosomes that are of bacterial (70S) and not eukaryotic (80S) form. The presence of two lipid bilayers, a circular chromosome and 70S ribosomes is consistent with the evolutionary hypothesis that we have already explained [160] near the beginning of this chapter.
Eukaryotic cells are generally much larger than prokaryotic ones and this difference in volume has several implications. First bigger cells can afford to have more things stored in the cytoplasm. This means it is not as costly to a eukaryotic cell to have structures taking up space. In a prokaryote, space is at a premium and anything not being used is pretty rapidly degraded. This may be one reason that organelles are possible. Second, larger cells have a lower surface-to-volume ratio than do smaller cells and therefore prokaryotes effectively have more contact with their environment. This greater exposure can mean a more rapid response to changing environmental conditions. Finally bigger cells have more of a challenge moving molecules within themselves. Prokaryotes can often depend on simple diffusion to move molecules around the cell, but this process might be too slow and inefficient in much larger cells. Eukaryotes overcome this by having specific transport mechanisms (i.e. microtubules) inside the cell.
Size constrains eukaryotes in two important ways: how fast they can grow and what environments they can tolerate. The compartmentalization of the genome inside the nucleus limits the rate at which eukaryotic cells can divide. The complete cell division cycle in a multicellular eukaryotic organism depends upon the cell type, but even in rapidly dividing skin cells it takes at least 8 hours. In unicellular yeast cultures, the shortest cell cycle is about 1.7 hours under ideal conditions. Due to their smaller genomes, lack of a nucleus and the ability to couple transcription and translation, bacteria can grow much faster. Clostridium perfringens has been shown to go through a complete division cycle in at little as 6.6 minutes at 43 °C in beef cubes!
There are environments that have one or more physical characteristics that prevent the growth of most organisms. These extreme environments might be too hot, cold, acidic or alkaline for typical organisms to grow. However, a small subset of prokaryotes has evolved to take advantage of these environments and thrive, and prokaryotes always define the extremes of where life can exist. In part this is probably due to the fact that simpler cells have fewer "body parts" that must be changed in order for growth under very different conditions. In eukaryotes, they would need to modify not only their cytoplasmic contents to tolerate the extreme environment, but also the makeup of their organelles.
We have moved from within prokaryotic and eukaryotic cells to the outside, and this should provide a framework for thinking about the make-up of microbes. Some ideas that you should take away from this chapter are:
In subsequent chapters we will learn what all these various parts are doing.