Cell Structure and Organisation PDF

3 Cell Structure and Organisation The basic unit of all living things is the cell. The cell theory is one of the fundamental concepts of biology; it states that: ! all organisms are made up of cells, and that ! all cells derive from other, pre-existing cells. As we shall see...

3 Cell Structure and Organisation The basic unit of all living things is the cell. The cell theory is one of the fundamental concepts of biology; it states that: ! all organisms are made up of cells, and that ! all cells derive from other, pre-existing cells. As we shall see in this chapter, there may exist within a cell many smaller, subcellular structures, each with its own characteristics and function, but these are not capable of independent life. An organism may comprise just a single cell (unicellular), a collection of cells that are not morphologically or functionally differentiated (colonial), or several dis- tinct cell types with specialised functions (multicellular). Among microorganisms, all bacteria and protozoans are unicellular; fungi may be unicellular or multicellular, while algae may exist in all The names given to the three forms. There is, however, one way that organisms two cell types derive can be differentiated from each other that is even more from Greek words: fundamental than whether they are uni- or multicellular. Procaryotic = ‘before It is a difference that is greater than that between a lion and a mushroom or between an earthworm and an oak nucleus’ tree, and it exists at the level of the individual cell. All or- Eucaryotic = ‘true ganisms are made up of one or other (definitely not both!) nucleus’ of two very distinct cell types, which we call procaryotic and eucaryotic cells, both of which exist in the microbial world. These differ from each other in many ways, including size, structural complexity and organisation of genetic material (Table 3.1). The most fundamental difference between procaryotic and eucaryotic cells is reflected in their names; eucaryotic cells possess a true nucleus, and several other distinct subcel- lular organelles that are bounded by a membrane. Procaryotes have no such organelles. Most of these differences only became apparent after the development of electron mi- croscopy techniques. As can be seen from Table 3.2, the procaryotes comprise the simpler and more prim- itive types of microorganisms; they are generally single celled, and arose much earlier in evolutionary history than the eucaryotes. Indeed, as discussed later in this chapter, it 52 CELL STRUCTURE AND ORGANISATION Table 3.1 Similarities and differences between procaryotic and eucaryotic cell structure Similarities Cell contents bounded by a plasma membrane Genetic information encoded on DNA Ribosomes act as site of protein synthesis Differences Procaryotic Eucaryotic Size Typically 1–5 µm Typically 10–100 µm Genetic material Free in cytoplasm Contained within a membrane-bound nucleus Single circular chromosome or Multiple chromosomes, generally in pairs nucleoid Histones absent. DNA complexed with histone proteins Internal features Membrane-bound organelles absent Several membrane-bound organelles present, including mitochondria, Golgi body, endoplasmic reticulum and (in plants & algae) chloroplasts Ribosomes smaller (70S), free in Ribosomes larger (80S), free in cytoplasm cytoplasm or attached to membranes Respiratory enzymes bound to plasma Respiratory enzymes located in membrane mitochondria Cell wall Usually based on peptidoglycan (not When present, based on cellulose or chitin Archaea) External features Cilia absent Cilia may be present Flagella, if present, composed of Flagella, if present, have complex (9 + 2) flagellin. Provide rotating motility structure. Provide ‘whiplash’ motility Pili may be present Pili absent Outside layer (slime layer, capsule, Pellicle or test present in some types glycocalyx) present in some types is widely accepted that eucaryotic cells actually arose from their more primitive coun- terparts. Note that the viruses do not appear in Table 3.2, because they do not have a cellular structure at all, and are not therefore considered to be living organisms. (See Chapter 10 for further discussion of the viruses). The use of DNA sequencing methods to determine phylogenetic relationships between organisms has re- Phylogenetic: pertaining vealed that within the procaryotes there is another fun- to the evolutionary rela- damental division. One group of bacteria were shown to tionship between organ- differ greatly from all the others; we now call these the isms. Archaea, to differentiate them from the true Bacteria.  CELL STRUCTURE AND ORGANISATION 53 Table 3.2 Principal groups of procaryotic and eucaryotic organisms Procaryotes Eucaryotes Bacteria Fungi Blue-green ‘algae’∗ Algae Protozoa Plants Animals ∗ An old-fashioned term: this group are in fact a specialized form of bacteria, and are known more correctly as the Cyanobacteria, or simply the blue-greens. They are discussed in more detail in Chapter 7. Animals and plants fall outside the scope of this book. These two groups, together with the eucaryotes, are thought to have evolved from a common ancestor, and Despite their differences, represent the three domains of life (Figure 3.1). The Archaea and Bacteria Archaea comprise a wide range of mostly anaerobic bac- are both procaryotes. teria, including many of those that inhabit extreme en- vironments such as hot springs. In this book we shall Taxonomy is the science largely confine our discussions to the Bacteria, however of classifying living (and in Chapter 7 there is a discussion of the principal features of the Archaea and their main taxonomic groupings. once-living) organisms. Figure 3.1 The three domains of life. All life forms can be assigned to one of three domains on the basis of their ribosomal RNA sequences. The Archaea are quite distinct from the true bacteria and are thought to have diverged from a common ancestral line at a very early stage, before the evolution of eucaryotic organisms. The scheme above is the one most widely accepted by microbiologists, but alternative models have been proposed 54 CELL STRUCTURE AND ORGANISATION Figure 3.2 Bacterial shapes. Most bacteria are (a) rod shaped, (b) spherical or (c) curved. These basic shapes may join to form (d) pairs, (e and f) chains, (g) sheets, (h) packets or (i) irregular aggregates The procaryotic cell Bacteria are much smaller than eucaryotic cells; most fall into a size range of about 1–5µm, although some may be larger than this. Some of the smallest bacteria, such as the mycoplasma measure less than 1µm, and are too small to be resolved clearly by an ordinary light microscope. Because of their extremely small size, it was only with the advent of the electron microscope that we were able to In recent years, square, learn about the detailed structure of bacterial cells. Using triangular and star- the light microscope however, it is possible to recognise shaped bacteria have differences in the shape and arrangement of bacteria. Al- all been discovered! though a good deal of variation is possible, most have one of three basic shapes (Figure 3.2): ! rod shaped (bacillus) ! spherical (coccus) ! curved: these range from comma-shaped (vibrio) to corkscrew-shaped (spirochaete)  THE PROCARYOTIC CELL 55 All these shapes confer certain advantages to their owners; rods, with a large surface area are better able to take up nutrients from the environment, while the cocci are less prone to drying out. The spiral forms are usually motile; their shape aids their movement through an aqueous medium. As well as these characteristic cell shapes, bacteria may also be found grouped to- gether in particular formations. When they divide, they may remain attached to one another, and the shape the groups of cells assume reflects the way the cell divides. Cocci, for example, are frequently found as chains of cells, a reflection of repeated division in one plane (Figure 3.2(f)). Other cocci may form regular sheets or packets of cells, as a result of division in two or three planes. Yet others, such as the staphylococci, divide in several planes, producing the irregular and characteristic ‘bunch of grapes’ appear- ance. Rod-shaped bacteria only divide in a single plane and may therefore be found in chains, while spiral forms also divide in one plane, but tend not to stick together. Blue–greens form filaments; these are regarded as truly multicellular rather than as a loose association of individuals. Procaryotic cell structure When compared with the profusion of elaborate organelles encountered inside a typical eucaryotic cell, the interior of a typical bacterium looks rather empty. The only internal structural features are: ! a bacterial chromosome or nucleoid, comprising a closed loop of double stranded, supercoiled DNA. In addition, there may be additional DNA in the form of a plasmid ! thousands of granular ribosomes ! a variety of granular inclusions associated with nutrient storage. All of these are contained in a thick aqueous soup of carbohydrates, proteins, lipids and inorganic salts known as the cytoplasm, which is surrounded by a plasma membrane. This in turn is wrapped in a cell wall, whose rigidity gives the bacterial cell its charac- teristic shape. Depending on the type of bacterium, there may be a further surrounding layer such as a capsule or slime layer and/or structures external to the cell associated with motility (flagella) or attachment (pili/fimbriae). Figure 3.3 shows these features in a generalised bacterial cell. In the following pages we shall examine these features in a little more detail, noting how each has a crucial role to play in the survival or reproduction of the cell. Not all bacteria con- form to the model of a Genetic material single circular chromo- Although it occupies a well defined area within the cell, some; some have been the genetic material of procaryotes is not present as a shown to possess two true nucleus, as it lacks a surrounding nuclear membrane with genes shared be- (c.f. the eucaryotic nucleus, Figure 3.12). The nucleoid or tween them, while ex- bacterial chromosome comprises a closed circle of dou- amples of linear chromo- ble stranded DNA, many times the length of the cell and somes are also known. highly folded and compacted. (The common laboratory 56 CELL STRUCTURE AND ORGANISATION Plasma membrane Outer membrane Mesosome Inclusion body Cell wall Plasmid Nucleoid Pili Flagellum Endospore Ribosome Figure 3.3 Structure of a generalised procaryotic cell. Note the lack of complex internal organelles (c.f. Figure 3.12). Gram-positive and Gram-negative bacteria differ in the details of their cell wall structure (see Figures 3.7 & 3.8) bacterium Escherichia coli is around 3–4 µm in length, but contains a DNA molecule some 1400 µm in length!) Plasmids are small loops The DNA may be associated with certain bacterial pro- of DNA independent of teins, but these are not the same as the histones found the chromosome. They in eucaryotic chromosomes. Some bacteria contain ad- are capable of directing ditional DNA in the form of small, self-replicating ex- their own replication. trachromosomal elements called plasmids. These do not carry any genes essential for growth and reproduction, and thus the cell may survive without them. They can be very important however, as they may include genes encoding toxins or resistance to antibiotics, and can be passed from cell to cell (see Chapter 12). Ribosomes Apart from the nucleoid, the principal internal structures of procaryotic cells are the ribosomes. These are the site of protein synthesis, and there may be many thousands of these in an active cell, lending a speckled appearance to the cytoplasm. Ribosomes are composed of a complex of protein and RNA, and are the site of protein synthesis in the cell. Although they carry out a similar function, the ribosomes of procaryotic cells are smaller and lighter than their eucaryotic counterparts. Ribosomes are mea- sured in Svedberg units (S), a function of their size and shape, and determined by their rate of sedimentation in a centrifuge; procaryotic ribosomes are 70S, while those of eucaryotes are 80S. Some types of antibiotic exploit this difference by  THE PROCARYOTIC CELL 57 Table 3.3 Comparison of procaryotic and eucaryotic ribosomes Procaryotic Eucaryotic Overall size 70S 80S Large subunit size 50S 60S Large subunit RNA 23S & 5S 28S, 5.8S & 5S Small subunit size 30S 40S Small subunit RNA 16S 18S targeting the procaryotic form and selectively disrupting bacterial protein synthesis (see Chapter 14). All ribosomes comprise two unequal subunits (in pro- caryotes, these are 50S and 30S, in eucaryotes 60S and A polyribosome (poly- 40S: Table 3.3)). Each subunit contains its own RNA and some) is a chain of ri- a number of proteins (Figure 3.4). Many ribosomes may bosomes attached to simultaneously be attached to a single mRNA molecule, the same molecule of forming a threadlike polysome. The role of ribosomes in mRNA. bacterial protein synthesis is discussed in Chapter 11. Inclusion bodies Within the cytoplasm of certain bacteria may be found granular structures known as inclusion bodies. These act as food reserves, and may contain organic compounds such as starch, glycogen or lipid. In addition, sulphur and polyphosphate can be stored as inclusion bodies, the latter being known as volutin or metachromatic granules. Two special types of inclusion body are worthy of mention. Magnetosomes, which contain a form of iron oxide, help some types of bacteria to orientate themselves downwards into favourable conditions, whilst gas vacuoles maintain bouyancy of the cell in blue greens and some halobacteria. 16S rRNA 30S Subunit 70 S 21 proteins Ribosome 5S rRNA 50S 23S rRNA subunit 34 proteins Figure 3.4 The bacterial ribosome. Each subunit comprises rRNA and proteins. The nu- cleotide sequence of small subunit (16S) rRNA is widely used in determining the phylogenetic (evolutionary) relationship between bacteria (see Chapter 7) 58 CELL STRUCTURE AND ORGANISATION Endospores Endospores of pathogens Certain bacteria such as Bacillus and Clostridium pro- such as Clostridium bo- duce endospores. They are dormant forms of the cell tulinum can resist boiling that are highly resistant to extremes of temperature, pH for several hours. It is this and other environmental factors, and germinate into new resistance that makes bacterial cells when conditions become more favourable. The spore’s resistance is due to the thick coat that sur- it necessary to auto- rounds it. clave at 121◦ C in order to ensure complete sterility. The plasma membrane The cytoplasm and its contents are surrounded by a plasma membrane, which can be thought of as a bilayer of phospholipid arranged like a sandwich, together with associated proteins (Figure 3.5). The function of the plasma membrane is to keep the contents in, while at the same time allowing the selective passage of certain substances in and out of the cell (it is a semipermeable membrane). Phospholipids comprise a compact, hydrophilic (= water-loving) head and a long hydrophobic tail region (Figure 2.27); this results in a highly ordered structure when the membrane is surrounded by water. The tails ‘hide’ from the water to form the inside of the membrane, while the heads project outwards. Also included in the membrane are a variety of proteins; these may pass right through the bilayer or be associated with the inner (cytoplasmic) or outer surface only. These proteins may play structural or functional roles in the life of the cell. Many enzymes associated with the metabolism of nutrients and the production of energy are associated with the plasma membrane in procaryotes. As we will see later in this chapter, this is fundamentally different from Figure 3.5 The plasma membrane. Phospholipid molecules form a bilayer, with the hy- drophobic hydrocarbon chains pointing in towards each other, leaving the hydrophilic phos- phate groups to face outwards. Proteins embedded in the membrane are known as integral proteins, and may pass part of the way or all of the way through the phospholipid bilayer. The amino acid composition of such proteins reflects their location; the part actually embed- ded among the lipid component of the membrane comprises non-polar (hydrophobic) amino acids, while polar ones are found in the aqueous environment at either side. Singleton, P: Bacteria in Biology, Biotechnology and Medicine, 5th edn, John Wiley & Sons, 1999. Re- produced by permission of the publishers  THE PROCARYOTIC CELL 59 eucaryotic cells, where these reactions are carried out on specialised internal organelles. Proteins involved in the active transport of nutrients (see Chapter 4) are also to be found associated with the plasma membrane. The model of membrane structure as depicted in Figure 3.5 must not be thought of as static; in the widely accepted fluid mosaic model, the lipid is seen as a fluid state, in which proteins float around, rather like icebergs in an ocean. The majority of bacterial membranes do not contain sterols (c.f. eucaryotes: see below), however many do contain molecules called hopanoids that are derive from the same precursors. Like sterols, they are thought to assist in maintaining membrane stability. A comparison of the lipid components of plasma membranes reveals a distinct difference between members of the Archaea and the Bacteria. The bacterial cell wall Bacteria have a thick, rigid cell wall, which maintains the integrity of the cell, and determines its characteristic shape. Since the cytoplasm of bacteria contains high concentrations of dissolved substances, they generally live in a hypotonic environment (i.e. one that is more di- A protoplast is a cell that lute than their own cytoplasm). There is therefore a nat- has had its cell wall re- ural tendency for water to flow into the cell, and without moved. the cell wall the cell would fill and burst (you can demon- strate this by using enzymes to strip off the cell wall, leaving the naked protoplast). The major component of the cell wall, which is re- sponsible for its rigidity, is a substance unique to bacte- Proteases are enzymes ria, called peptidoglycan (murein). This is a high molec- that digest proteins. ular weight polymer whose basic subunit is made up of three parts: N-acetylglucosamine, N-acetylmuramic acid and a short peptide chain (Fig- ure 3.6). The latter comprises the amino acids l-alanine, d-alanine, d-glutamic acid and either l-lysine or diaminopimelic acid (DAP). DAP is a rare amino acid, only found in the cell walls of procaryotes. Note that some of the amino acids of peptidoglycan are found in the d-configuration. This is contrary to the situation in proteins, as you may recall from Chapter 2, and confers protection against proteases specifically directed against l-amino acids. Precursor molecules for peptidoglycan are synthesised inside the cell, and transported across the plasma membrane by a carrier called bactoprenol phosphate before being in- corporated into the cell wall structure. Enzymes called transpeptidases then covalently bond the tetrapeptide chains to one another, giving rise to a complex network (Figure 3.7); it is this cross-linking that gives the wall its mechanical strength. A number of antimicrobial agents exert their effect by inhibiting cell wall synthesis; β-lactam an- tibiotics such as penicillin inhibit the transpeptidases, thereby weakening the cell wall, whilst bacitracin prevents transport of peptidoglycan precursors out of the cell. The action of antibiotics will be discussed further in Chapter 14. Although all bacteria (with a few exceptions) have a cell wall containing peptidoglycan, there are two distinct struc- tural types. These are known as Gram-positive and Gram-negative. The names derive from the Danish scientist Christian Gram, who, in the 1880s developed a rapid staining technique that could differentiate bacteria as belonging to one of two basic types (see Box 1.2). Although the usefulness of the Gram stain was recognised for many years, it 60 CELL STRUCTURE AND ORGANISATION N-acetylmuramic N-acetylglucosamine acid residue residue 6 CH2OH CH2OH H 5 O H O H H H O 4 1 O O H OH H H 3 2 H H H NH C O H NH C O O H3C CH C O CH3 CH3 NH CH3 C H L–Alanine C O NH H C CH2 CH2 C O D –Glutamic acid COOH NH H C (CH2)3 CH COOH meso –Diaminopimelic acid C O NH2 NH H C CH3 D –Alanine C O OH NAM NAG Simplified depiction Figure 3.6 Peptidoglycan structure. Peptidoglycan is a polymer made up of alternating molecules of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). A short pep- tide chain is linked to the NAM residues (see text for details). This is important in the cross-linking of the straight chain polymers to form a rigid network (Figure 3.6). The com- position of E. coli peptidoglycan is shown; the peptide chain may contain different amino acids in other bacteria. Partly from Hardy, SP: Human Microbiology, Taylor and Francis, 2002. Reproduced by permission of Thomson Publishing Services  THE PROCARYOTIC CELL 61 Figure 3.7 Cross-linking of peptidoglycan chains in E. coli. (a) The d-alanine on the short peptide chain attached to the N-acetylmuramic acid cross-links to a diaminopimelic acid residue on another chain. In other bacteria, the precise nature of the cross-linking may differ. From Hardy, SP: Human Microbiology, Taylor and Francis, 2002. Reproduced by permis- sion of Thomson Publishing Services. (b) Further cross-linking produces a rigid network of peptidoglycan. The antibiotic penicillin acts by inhibiting the transpeptidase enzymes responsible for the cross-linking reaction (see Chapter 15) was only with the age of electron microscopy that the underlying molecular basis of the test could be explained, in terms of cell wall structure. Gram-positive cell walls are relatively simple in structure, comprising several layers of peptidoglycan connected to each other by cross-linkages to form a strong, rigid scaffolding. In addition, they contain acidic polysaccharides called teichoic acids; these contain phosphate groups that impart an overall negative charge to the cell surface. A diagram of the gram-positive cell wall is shown in Figure 3.8. Gram-negative cells have a much thinner layer of peptidoglycan, making the wall less sturdy, however the structure is made more complex by the presence of a layer of lipoprotein, polysaccharide and phospholipid known as the outer membrane 62 CELL STRUCTURE AND ORGANISATION Figure 3.8 The Gram-positive cell wall. Peptidoglycan is many layers thick in the Gram- positive cell wall and may account for 30–70% of its dry weight. Teichoic acids are neg- atively charged polysaccharides; they are polymers of ribitol phosphate and cross-link to peptidoglycan. Lipoteichoic acids are teichoic acids found in association with glycolipids. From Henderson, B, Wilson, M, McNab, R & Lax, AJ: Cellular Microbiology: Bacteria- Host Interactions in Health and Disease, John Wiley & Sons Inc., 1999. Reproduced by permission of the publishers (Figure 3.9). This misleading name derives from the fact that it superficially resembles the bilayer of the plasma membrane; however, instead of two layers of phospholipid, it has only one, the outer layer being made up of lipopolysaccharide. This has three parts: lipid A, core polysaccharide and an O-specific side chain. The lipid A component may act as an endotoxin, which, if released into the bloodstream, can lead to serious con- ditions such as fever and toxic shock. The O-specific antigens are carbohydrate chains whose composition often varies between strains of the same species. Serological meth- ods can distinguish between these, a valuable tool in the investigation, for example, of the origin of an outbreak of an infectious disease. Proteins incorporated into the outer membrane and penetrating its entire thickness form channels that allow the passage of water and small molecules to enter the cell. Unlike the plasma membrane, the outer membrane plays no part in cellular respiration. Box 3.1 Mesosomes − the structures that never were? When looked at under the electron microscope, Gram-positive bacteria often con- tained localised in-foldings of the plasma membrane. These were given the name mesosomes, and were thought by some to act as attachment points for DNA dur- ing cell division, or to play a role in the formation of cross-walls. Others thought they were nothing more than artefacts produced by the rather elaborate sample preparation procedures necessary for electron microscopy. Nowadays, most micro- biologists support the latter view.  THE PROCARYOTIC CELL 63 Figure 3.9 The Gram-negative cell wall. Note the thinner layer of peptidoglycan compared to the gram-positive cell wall (Figure 3.8). It accounts for