BT-Sem 1 topic -1.2.pdf

1.2. ULTRASTRUCTURE OF PROKARYOTIC CELL Prokaryotes can be divided into two different groups: Bacteria and Archaea. Bacteria vary in size and shape. For example, Escherichia coli, a rod shaped bacteria, measures an average size of 1.1 to 1.5 µm wide and 2.0 to 6.0 µm long. A prokar...

1.2. ULTRASTRUCTURE OF PROKARYOTIC CELL Prokaryotes can be divided into two different groups: Bacteria and Archaea. Bacteria vary in size and shape. For example, Escherichia coli, a rod shaped bacteria, measures an average size of 1.1 to 1.5 µm wide and 2.0 to 6.0 µm long. A prokaryotic cell can be described for having the following structures: Cell envelope, cell wall, periplasmic space, plasma membrane, bacterial cytoskeleton, nucleoid, plasmid, vacuoles and inclusion bodies, ribosomes. CELL ENVELOPE Many bacteria have a protective layer outside the cell wall. This layer is given different names depending on its makeup and how it is organized. The cell envelope may be called capsule, slime layer, glycocalyx, or S-layer. Capsules: Capsules are layers that are well organized and not easily washed off. They are most often composed of polysaccharides. Some bacteria show proteinaceous capsule, which is made of poly D-glutamic acid (Eg: Bacillus anthracis). They are usually not required for growth and reproduction. Capsules are clearly visible in the light microscope when negative stains or specific capsule stains are employed. Capsule confers several advantages when bacteria grow in their normal habitats. For example, the pathogenic bacteria Streptococcus pneumoniae resist phagocytosis by host phagocytes, but when the bacteria lack capsule, it is easily phagocytosed. Further, the capsulated variant quickly kills mice. A Capsule can also protect a bacterium against desiccation. Further, capsules can also exclude viruses and most hydrophobic toxic materials such as detergents. Slime layer Slime layer is a diffuse, unorganized material that can be removed easily. It is usually composed of polysaccharides. Gliding bacteria often produce slime layer, which in some cases has been shown to facilitate motility. Glycocalyx: Glycocalyx is a layer consisting of a network of polysaccharides extending from the surface of some bacterial cells. The glycocalyx aids in attachment to solid surfaces, including tissue surfaces in plant and animal hosts. S-Layers Many bacteria have a regularly structured layer called an S-layer on their surface. It is composed of protein or glycoprotein. It can be seen in Gram-negative and Gram-positive bacteria. The S-layer helps maintain the shape and envelope rigidity of some cells, promote cell adhesion to surfaces, protect some bacterial pathogens against host defences, etc. CELL WALL The cell wall is the layer that lies just outside the plasma membrane. It is one of the most important structures for several reasons: (1) it helps maintain cell shape and protect the cell from osmotic lysis; (2) it can protect the cell from toxic substances; (3) For pathogenic microbes, it can contribute to pathogenicity. Cell walls are so important that most bacteria have them. Antibiotics have been identified that target most pathogenic bacteria’ cell wall synthesis. Overview of Bacterial Cell Wall Structure After Christian Gram developed a procedure to stain a bacterium cell, which is now popularly called the ‘Gram stain’ in 1884, it soon became evident that most bacteria could be divided into two major groups based on their response to the Gram-staining procedure: (1) Gram- positive bacteria, which stain purple, and (2) Gram negative bacteria, which stain pink or red. The cell walls of typical Gram-positive bacteria (Bacillus subtilis) consist of a single, 20- to 80-nm-thick homogeneous layer of peptidoglycan (murein) lying outside the plasma membrane. In contrast, the cell walls of typical Gram-negative bacteria (E. Coli) have two distinct layers: an inner layer which is 2nm to 7nm-thick peptidoglycan layer, and an outer membrane of 7- to 8-nm-thickness. Peptidoglycan Structure The presence of peptidoglycan is a common feature in nearly all bacterial cell walls. Peptidoglycan forms an enormous mesh like structure often referred to as the peptidoglycan sacculus. Peptidoglycan is composed of a helical arrangement of two alternating sugars; N- acetylglucosamine (NAG) and N- acetylmuramic acid (NAM). Short peptides are linked to the carboxyl group of NAM and extend out of the helical structure. The amino acids of the short peptide consisting of four alternating D- and L-amino acids. D-amino-acids are generally not found in cellular proteins. It is a protective mechanism adopted by bacteria to escape the proteolytic action by the enzymes secreted by their hosts or other pathogenic bacteria. (Generally proetolytic enzymes recognize only L amino acids and hence cleave only such proteins which have L amino aicds). The three common D- amino acids found in bacterial cell wall peptides are : D-glutamic acid, D- alanine, and meso-diaminopimelic acid. The alternating NAG and NAM residues form a helical backbone from which the peptides extend outward and make cross-links with other peptidoglycan backbones finally resulting in a mesh like structure. Inter-peptide bridges also appear connecting the peptidoglycan strands. A Typical Gram-Positive Cell Walls has thick cell wall composed of peptidoglycan and large amounts of other polymers such as teichoic acids. Teichoic acids are polymers of glycerol or ribitol joined by phosphate groups. The teichoic acids are covalently connected to peptidoglycan or to plasma membrane lipids. Teichoic acids are negatively charged and hence give the cell wall a negative charge. Teichoic acids are present only in Gram positive Bacteria. A typical Gram-negative cell wall is much more complex than a typical Gram-positive wall. Gram negative cell wall contains two layers of cell walls. The inner layer is composed of peptidoglycan, and is very thin (2 to 7 nm, depending on the bacterium). The outer layer is composed of lipopolysaccharides (LPSs). These large, complex molecules contain both lipid and carbohydrate, and consist of three parts: (1) lipid A, (2) the core polysaccharide, and (3) the O-side chain. LPS has many important functions. (1) It contributes to the negative charge on the bacterial surface. (2) It helps stabilize outer membrane structure (3) It helps create a permeability barrier. Restrict the entry of bile salts, antibiotics, and other toxic substances that might kill or injure the bacterium. (4) LPS also plays a role in protecting pathogenic bacteria from host defences. A lipoprotein called Braun's lipoprotein, is also most abundant protein in the outer membrane. This small lipoprotein is covalently joined to the underlying peptidoglycan and is embedded in the outer membrane by its hydrophobic end. PERIPLASMIC SPACE The space between the cell wall and plasma membrane is called the periplasmic space. The periplasmic space in Gram negative cell wall is usually 30 to 70 nm wide. It may constitute about 20 to 40% of the total cell volume., and is much larger than that observed in typical Gram-positive cells. The substance that occupies the periplasmic space is the periplasm. PROTOPLAST: The plasma membrane and everything within is called protoplast. PLASMA MEMBRANE The most widely accepted model for membrane structure is the fluid mosaic model of Singer and Nicholson, which proposes that plasma membranes are lipid bilayers within which proteins float. Plasma membrane is a selectively permeable barrier: it allows particular ions and molecules to pass, either into or out of the cell, while preventing the movement of others. Bacterial plasma membranes are similar to eukaryotic plasma membranes in that many of their lipids are phospholipids, but they usually differ from eukaryotic membranes in lacking sterols (steroid-containing lipids) such as cholesterol. However, many bacterial membranes contain sterol-like molecules called hopanoids. Hopanoids are synthesized from the same precursors as steroids, and like the sterols in eukaryotic membranes, they probably stabilize the membrane. Distinct transport systems exist in plasma membrane, that perform tasks such as nutrient uptake, waste excretion, and protein secretion. The prokaryotic plasma membrane also is the location of a variety of crucial metabolic processes: respiration, photosynthesis, and the synthesis of lipids and cell wall constituents. Finally, the membrane contains special receptor molecules that help prokaryotes detect and respond to chemicals in their surroundings. CYTOPLASMIC MATRIX Internal to plasma membrane, is the cytoplasmic matrix, the substance in which the nucleoid, ribosomes, and inclusion bodies are suspended. It lacks organelles bound by lipid bilayers (often called unit membranes), and is largely water (about 70% of bacterial mass is water). BACTERIAL CYTOSKELETON The bacterial cytoskeletal proteins are structurally similar to their eukaryotic counterparts and carry out similar functions: they participate in cell division, localize proteins to certain sites in the cell, and determine cell shape. Eukaryotes possess three cytoskeletal elements: actin filaments, microtubules, and intermediate filaments. Actin filaments are made from the protein actin; Microtubules are made from the protein, tubulin; and Intermediate filaments are composed of a mixture of one or more members of different classes of proteins. Homologues of all three types of eukaryotic proteins have been identified in bacteria. INTRA-CYTOPLASMIC MEMBRANES Although Bacteria do not contain complex membranous organelles like mitochondria or chloroplasts, internal membranous structures are observed in some bacteria, which support such activities. The internal membranous structures observed in bacteria may be aggregates of spherical vesicles, flattened vesicles, or tubular membranes. They are often connected to the plasma membrane and are thought to arise from it by invagination. INCLUSIONS They are formed by the aggregation of substances that may be either organic or inorganic. The first bacterial inclusions were discovered in the late 1800s. Inclusions can take the form of granules, crystals, or globules; some are amorphous. Some inclusions may lie free in the cytoplasm or are enclosed by a shell that is single-layered and may consist of proteins or of both proteins and phospholipids. Some inclusions are surrounded by invaginations of the plasma membrane. Many inclusions are used for storage (e.g., of carbon compounds, inorganic substances, and energy) or to reduce osmotic pressure by tying up molecules in particulate form. The quantity of inclusions used for storage varies with the nutritional status of the cell. Some inclusions are so distinctive that they are increasingly being referred to as micro compartments. A brief description of several important inclusions follows. Storage Inclusions Cells have a wide variety of storage inclusions. The most common storage inclusions are glycogen inclusions, polyhydroxyalkonate granules, sulfur globules, and polyphosphate granules. Some storage inclusions are observed only in certain organisms, such as the cyanophycin granules in cyanobacteria. Carbon is often stored as polyhydroxyalkonate (PHA) granules. Several types of PHA granules have been identified, but the most common contain poly-(3-hydroxybutyrate (PHB). PHB granules are now known to be surrounded by a single-layered shell composed of proteins and a small amount of phospholipids. Much of the interest in PHB and other PHA granules is due to their industrial use in making biodegradeable plastics. Polyphosphate granules and sulfur globules are inorganic inclusions observed in many organisms. Polyphosphate granules store the phosphate needed for synthesis of important cell constituents such as nucleic acids. In some cells, they act as an energy reserve, and polyphosphate also can serve as an energy source in some reactions, when the bond linking the final phosphate in the polyphosphate chain is hydrolyzed. Sulfur globules are formed by bacteria that use reduced sulphur containing compounds as a source of electrons during their energy-conserving metabolic processes. For example, some photosynthetic bacteria can use hydrogen sulfide (rather than water) as an electron donor and accumulate the resulting sulfur either externally or internally. Micro-compartments Some bacterial inclusions are unique and serve functions other than simply storing substances. Micro-compartments are relatively large polyhedrons formed by one or more different proteins. Enclosed within the protein shell are one or more enzymes. Micro-compartments include the ethanolamine utilization (Eut) micro-compartment, the propanediol utilization (Pdu) micro-compartment, and carboxysomes. Carboxysomes are present in many cyanobacteria and other CO2-fixing bacteria. Their polyhedral coat is about 100 nm in diameter. It contains the enzyme carbonic anhydrase, which converts carbonic acid into CO2. Carboxysome shell prevents CO2 from escaping so it can accumulate within. Also enclosed within the carboysome is the enzyme ribulose-1, 5- bisphosphate carboxylase/oxygenase (RubisCO). RubisCO is the critical enzyme for CO2 fixation, the process of converting CO2 into sugar. Thus the carboxysome serves as a site for CO2 fixation. ParA, a cytoskeletal protein, helps ensure appropriate segregation of carboxysomes during bacterial cell division. Other Inclusions Two of the most remarkable inclusions are gas vacuoles and magnetosomes. Both are involved in bacterial movement. The gas vacuole provides buoyancy to some aquatic bacteria, many of which are photosynthetic. Gas vacuoles are aggregates of enormous numbers of small, hollow, cylindrical structures called gas vesicles. Gas vesicle walls are composed entirely of a single small protein. These protein subunits assemble to form a rigid cylinder that is impermeable to water but freely permeable to atmospheric gases. Cells with gas vacuoles can regulate their buoyancy to float at the depth necessary for proper light intensity, oxygen concentration, and nutrient levels. They descend by simply collapsing vesicles and float upward when new ones are constructed. Magnetosomes: Aquatic magnetotactic bacteria use magnetosomes to orient themselves in Earth's magnetic field. Magnetosomes are intra-cellular chains of magnetite (Fe3O4) particles. They are around 35 to 125 nm in diameter and enclosed within invaginations of the plasma membrane. The invaginations have been shown to contain distinctive proteins that are not found in the rest of the plasma membrane. Each iron particle is a tiny magnet: the Northern Hemisphere bacteria use their magnetosome chain to determine northward and downward directions, and swim down to nutrient rich sediments or locate the optimum depth in freshwater and marine habitats. Magnetotactic bacteria in the Southern Hemisphere generally orient southward and downward, with the same result. For the cell to move properly within a magnetic field, magnetosomes must be arranged in a chain. A cytoskeletal protein called MamK is thought to be responsible for establishing a framework upon which the chain can form. BACTERIAL RIBOSOMES Ribosomes are the site of protein synthesis, and large numbers of them are found in all cells. The cytoplasm of bacterial cells is often packed with ribosomes. They may also be loosely attached to the plasma membrane. The cytoplasmic ribosomes synthesize proteins destined to remain within the cell, whereas plasma membrane-associated ribosomes make proteins that will reside in the cell envelope or are transported to the outside. Bacterial ribosomes are called 70S ribosomes and are constructed of a 50S and a 30S subunit. The S in 70S and similar values stands for Svedberg unit. This is the unit of the sedimentation coefficient, a measure of sedimentation velocity in a centrifuge; the faster a particle travels when centrifuged, the greater its Svedberg value or sedimentation coefficient. The sedimentation coefficient is a function of a particle's molecular weight, volume, and shape. Heavier and more compact particles normally have larger Svedberg numbers and sediment faster. Bacterial ribosomes are composed primarily of ribosomal RNA (rRNA) molecules. The small subunit contains 16S rRNA; large subunit contains 23S and 5S rRNA molecules. Approximately 55 proteins make up the rest of the mass of the ribosome: 21 in the small subunit, and 34 in the large subunit. NUCLEOID The nucleoid is an irregularly shaped region that contains the cell's chromosome and numerous proteins. The chromosomes of most bacteria are a single circle of double stranded deoxyribonucleic acid (DNA). Chromosomes are longer than that of the lengths of cells. E. coli's circular chromosome measures approximately 1,400 µm. It is about 230-700 times longer than the cell. For this reason the chromosome is organized and packaged in a manner that decreases its overall size. This structure, also called the folded genome, or Nucleiod, is the functional state of a bacterial chromosome. Within the folded genome, the large DNA molecule in an E. coli chromosome is organized into 50 to 100 domains or loops, each of which is independently negatively super-coiled. RNA and protein are both components of the folded genome, which are thought to maintain the compactness. Several nucleoid-associated proteins (NAPs) cause the chromosome to bend and fold, thereby helping to pack the DNA into a smaller space. One NAP found in many bacteria is the protein called HU. HU is a bacterial histone-like protein that resembles the eukaryotic Histone H2B. HU acts similarly to a histone by inducing negative supercoiling into circular DNA with the assistance of topoisomerase (HU: H for histone, and U for the E.coli strain, U93). During cell division, bacterial chromosomes are further compacted by proteins called Condensins. This extra level of packing is important for proper segregation of daughter chromosomes during cell division. PLASMIDS In addition to the genetic material present in the nucleoid, many bacteria contain extra-chromosomal DNA molecules called plasmids. Plasmids are small, double-stranded DNA molecules that can exist independently of the chromosome. Both circular and linear plasmids have been documented, but most known plasmids are circular. Plasmids have relatively few genes, generally less than 30. Their genetic information is not essential to the bacterium, and cells that lack them usually function normally. However, many plasmids carry genes that confer a selective advantage to the bacterium in certain environments. Plasmids use the cell's DNA-synthesizing machinery to replicate, but their replication is not linked to any particular stage of the cell cycle. Thus regulation of plasmid and chromosomal replication are independent. However, some plasmids are able to integrate into the chromosome. Such plasmids are called episomes and when integrated are replicated as part of the chromosome. Plasmids are inherited stably during cell division, but they are not always equally apportioned into daughter cells and sometimes are lost. The loss of a plasmid is called curing. It can occur spontaneously or be induced by treatments that inhibit plasmid replication but not host cell reproduction. Some commonly used curing treatments are acridine mutagens, ultraviolet and ionizing radiation, thymine starvation, antibiotics, and growth above optimal temperatures. Plasmids may be classified in terms of their mode of existence, spread, and function, as given below. These various types of plasmids can also differ in terms of the number of copies found within the cell. Single copy plasmids produce only one copy per host cell. Multicopy plasmids may be present at concentrations of 40 or more per cell. EXTERNAL STRUCTURES Many bacteria have structures that extend beyond the cell envelope and are involved in either attachment to surfaces or motility. In addition, these external structures can function in protection and horizontal gene transfer. Bacterial Pili and Fimbriae Many bacteria have fine, hairlike appendages that are thinner and typically shorter than flagella. These are usually called fimbriae (s., fimbria) or pili (s., pilus). They are slender tubes composed of helically arranged protein subunits and are about 3 to 10nm in diameter and up to several micrometers long. Several different types of fimbriae have been identified in Gram- negative bacteria. Most function to attach cells to solid surfaces such as rocks in streams and host tissues. One type, called type IV pili, are involved in motility and the uptake of DNA during the process of bacterial transformation. Gram- positive bacteria have at least two types of pili; both are involved in attaching the bacteria to surfaces. Many bacteria have up to 10 sex pili (s., sex pilus) per cell. These hair like structures differ from other pili in the following ways. Sex pili often are larger than other pili (around 9 to 10 nm in diameter). They are genetically determined by conjugative plasmids and are required for conjugation. Some bacterial viruses attach specifically to sex pili at the start of their multiplication cycle. Bacterial Flagella Many motile bacteria move by use of flagella (s., flagellum), threadlike locomotor appendages extending outward from the plasma membrane and cell wall. Although the main function of flagella is motility, they can have other roles. They can be involved in attachment to surfaces, and in some bacteria, they are virulence factors. Bacterial flagella are slender, rigid structures about 20 nm thick and up to 20 µm long. Bacterial species often differ in their patterns of flagella distribution, and these patterns are useful in identifying bacteria. Monotrichous bacteria (trichous means hair) have one flagellum; if it is located at an end, it is said to be a polar flagellum. Amphitrichous bacteria (amphi means on both sides) have a single flagellum at each pole. Lophotrichous bacteria (lopho means tuft) have a cluster of flagella at one or both ends. Peritrichous bacteria (peri means around) have flagella that are spread evenly over the whole surface. The hook and basal body are quite different from the filament. Slightly wider than the filament, the hook is made of different protein subunits. The basal body is the most complex part of a flagellum. The basal bodies of E. coli and most other typical Gram-negative bacteria have four rings: L, P, MS, and C, which are connected to a central rod. The L, P, and MS rings are embedded in the cell envelope, and the C ring is on the cytoplasmic side of the MS ring. Typical Gram-positive bacteria have only two rings: an inner ring connected to the plasma membrane and an outer one probably attached to the peptidoglycan. Flagellar Movement The filament of a bacterial flagellum is in the shape of a rigid helix, and the cell moves when this helix rotates like a propeller on a boat. The flagellar motor can rotate very rapidly. When bacteria are in an aquatic environment, flagellar rotation results in two types of movement: a smooth swimming movement often called a run, which actually moves the cell from one spot to another, and a tumble, which serves to reorient the cell. Swarming This motility occurs on moist surfaces and is a type of group behaviour in which cells move in unison across the surface. Most bacteria that swarm have peritrichous flagella. Many also produce and secrete molecules that help them move across the substrate. When bacteria that swarm are cultured in the laboratory on appropriate solid media, they produce characteristic colony morphologies. Spirochete Motility The flagella of Spirochete bacteria do not extend outside the cell wall but rather remain in the periplasmic space and are covered by the outer membrane. They are called periplasmic flagella and are thought to rotate like the external flagella of other bacteria, causing the corkscrew- shaped outer membrane to rotate and move the cell through the surrounding liquid, even very viscous liquids. Flagellar rotation may also flex or bend the cell and account for the creeping or crawling movement on solid surfaces. Twitching and gliding motility are similar in that both occur on moist surfaces and can involve type IV pili and the secretion of slime. Twitching motility is a jerky movement, whereas gliding motility is smooth. chemotaxis Motile cells can respond to gradients of attractants and repellents, a phenomenon known as chemotaxis. A peritrichously flagellated bacterium accomplishes movement toward an attractant by increasing the length of time it spends moving toward the attractant and shortening the time it spends tumbling. Conversely, a bacterium increases its run time when it moves away from a repellent.