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MICROBIAL METABOLISM AND METABOLIC
DIVERSITY - charry Role of Metabolism in Biosynthesis and Growth
- Requires >the polymerization of biochemical building blocks into proteins, nucleic acids, polysaccharides, and lipids.
- Building blocks > must come preformed in the growth medium > must be synthesized by the growing cells.
- Growth > demands a source of metabolic energy for the synthesis of anhydride bonds > maintenance of transmembrane gradients of ions and metabolites.
- Metabolism has two components: catabolism and anabolism.
- Catabolism > processes that harvest energy released from the breakdown of compounds and using that energy to synthesize ATP.
- Anabolism (biosynthesis) > includes processes that utilize the energy stored in ATP to synthesize and assemble the subunits, or building blocks, of macromolecules that make up the cell.
- Catabolism encompasses processes that harvest energy released during disassembly of compounds using it to synthesize adenosine triphosphate (ATP)
- Anabolism , or biosynthesis, includes processes that utilize ATP and precursor metabolites to and assemble subunits of macromolecules that make up the cell. - The primary principle underlying metabolic regulation is that enzymes tend to be called into play only when their catalytic activity is required. - The activity of an enzyme may be changed by varying either the amount of enzyme or the amount of substrate. - In some cases, the activity of enzymes may be altered by the binding of specific effectors, metabolites that modulate enzyme activity. - Determined in one of two ways:
In nucleic acids and proteins it is template-directed: DNA serves as the template for its own synthesis synthesis of the various types of RNA messenger RNA serves as the template for the synthesis of proteins. In carbohydrates and lipids the arrangement of building blocks is determined entirely by enzyme specificities. macromolecules have been synthesized they form the supramolecular structures of the cell, ribosomes, membranes, cell wall, flagella, pili. FOCAL METABOLITES AND THEIR CONVERSION Glucose 6-Phosphate and Carbohydrate Interconversions
- The biosynthetic origins of building blocks and coenzymes can be traced to relatively few precursors, called focal metabolites. Biosynthetic end products formed from glucose 6- phosphate. Carbohydrate phosphate esters of varying chain length serve as intermediates in the biosynthetic pathways.
Biosynthetic end products formed from phosphoenolpyruvate. Biosynthetic end products formed from oxaloacetate. The end products aspartate, threonine, and pyrimidines serve as intermediates in the synthesis of additional compounds. Biosynthetic end products formed from α-ketoglutarate. ● Figure 2 illustrates how G6PD is converted to a range of biosynthetic end products via phosphate esters of carbohydrates with different chain lengths. ● Carbohydrates > empirical formula (CH 2 O) n ,
primary objective is to change n = the length of the carbon chain. ● Oxidative reactions > used to remove a single carbon from G6PD producing the pentose derivative ribulose 5-phosphate. ● **Isomerase and epimerase reactions ** interconvert the biochemical forms of the pentoses: ● > ribulose 5-phosphate, ribose 5-phosphate, and xylulose 5-phosphate. Transketolases transfer a two-carbon fragment from a donor to an acceptor molecule. ● Reactions allows: pentoses to form or to be formed from carbohydrates of varying chain lengths. ● Biochemical mechanisms for changing the length of carbohydrate molecules. ● The six-carbon hexose chain of fructose 6- phosphate can be converted to two three-carbon triose derivatives by: the consecutive action of a kinase and an aldolase on fructose 6-phosphate. ● Aldolases > can be used to lengthen carbohydrate molecules: Triose phosphates → fructose 6-phosphate Triose phosphate and tetrose 4-phosphate → heptose 7-phosphate. ● The final form of carbohydrate chain length interconversion is the transaldolase reaction , which interconverts heptose 7-phosphate and triose 3-phosphate with tetrose 4-phosphate and hexose 6- phosphate. The hexose monophosphate shunt. Oxidative reactions reduce NAD+^ and produce CO 2 , resulting in the shortening of the six hexose phosphates to six pentose phosphates. Carbohydrate rearrangements convert the pentose phosphates to hexose phosphates so that the oxidative cycle may continue. ● In sum, G6PD can be regarded as a focal metabolite because: it serves as a direct precursor for metabolic building blocks A source of carbohydrates of varying length that are used for biosynthetic purposes.
● The amino acids of the pentapeptide are sequentially added, each addition catalyzed by a different enzyme and each involving the split of ATP to ADP + ● The UDP- N - acetylmuramic acid-pentapeptide
attached to bactoprenol (a lipid of the cell membrane) receives a molecule of N - acetylglucosamine from UDP. form a pentaglycine derivative in a series of reactions using glycyl-tRNA as the donor the completed disaccharide is polymerized to an oligomeric intermediate before being transferred to the growing end of a glycopeptide polymer in the cell wall. ● Final cross-linking is accomplished by: a transpeptidation reaction ● Transpeptidation is catalyzed by one of a set of enzymes called penicillin-binding proteins (PBPs). ● PBPs bind penicillin and other β-lactam antibiotics covalently because of a structural similarity between these antibiotics and the pentapeptide precursor. ● Some PBPs have transpeptidase or carboxypeptidase activities their relative rates perhaps controlling the
degree of cross-linking in peptidoglycan Peptidoglycan synthesis
The pentapeptide contains l-lysine in Staphylococcus aureus peptidoglycan and diaminopimelic acid (DAP) in E coli. Inhibition by bacitracin, cycloserine, and vancomycin is also shown. Synthesis of Extracellular Capsular Polymers ● The capsular polymers are enzymatically synthesized from activated subunits. ● No membrane-bound lipid carriers have been implicated in this process. ● The presence of a capsule is often environmentally determined: Dextrans and levans can only be synthesized using the disaccharide sucrose (fructose–glucose) as the source of the appropriate subunit ● Their synthesis depends on the presence of sucrose in the growth environment. Synthesis of Reserve Food Granules ● When nutrients are present in excess of the requirements for growth: bacteria convert certain of them to intracellular reserve food granules. starch, glycogen, poly-β-hydroxybutyrate, and volutin which consists mainly of inorganic polyphosphate. ● The type of granule formed is species specific. ● The granules are degraded when exogenous nutrients are depleted. PATTERNS OF MICROBIAL ENERGY-YIELDING METABOLISM Pathways of Fermentation ● A. Strategies for Substrate Phosphorylation cells are entirely dependent on substrate phosphorylation for their energy (when respiration is absent): Generation of ATP must be coupled to chemical rearrangement of organic compounds. ● Three general stages: (1) Conversion of the fermentable compound to the phosphate donor for substrate phosphorylation. (2) Phosphorylation of ADP by the energy-rich phosphate donor. (3) Metabolic steps that bring the products of the fermentation into chemical balance with the starting materials. B. Fermentation of Glucose The diversity of fermentative pathways is illustrated by consideration of some of the mechanisms used by microorganisms to achieve substrate phosphorylation at the expense of glucose.
- In principle, the phosphorylation of ADP to ATP can be coupled to either of two chemically balanced transformations:
- There are two mechanisms by which this can be achieved:
(1) Extracellular glucose may be:
transported across the cytoplasmic membrane into the cell phosphorylated by ATP to yield G6PD and ADP. (2) Extracellular glucose is phosphorylated as it is being transported across the cytoplasmic membrane producing: intracellular G6PD and pyruvate. C. The Embden-Meyerhof Pathway ● A commonly encountered mechanism for the fermentation of glucose uses a kinase and an aldolase =to transform the hexose (C 6 ) phosphate → two molecules of triose (C 3 ) phosphate. ● Produces a net yield of two ATP pyrophosphate bonds. ● Direct reduction of pyruvate by NADH produces: lactate as the end product of fermentation results in acidification of the medium ● pyruvate may be decarboxylated to acetaldehyde which is then used to oxidize NADH resulting in production of the neutral product ethanol. The pathways yield only a single molecule of triose phosphate from glucose The energy yield is correspondingly low The Entner-Doudoroff and heterolactate pathways yield only a single net substrate phosphorylation of ADP per molecule of glucose fermented. D. The Entner-Doudoroff and Heterolactate Fermentations ● Alternative pathways for glucose fermentation include some specialized enzyme reactions. ● The Entner-Doudoroff pathway: diverges from other pathways of carbohydrate metabolism by a dehydration of 6-phosphogluconate followed by an aldolase reaction. Produces: Pyruvate and triose phosphate ● The heterolactate fermentation and some other fermentative pathways: depend upon a phosphoketolase reaction that phosphorolytically cleaves a ketosephosphate to produce: acetyl phosphate and triose phosphate. Table 1. Microbial Fermentation based on the Embden- Meyerhof Pathway E. Additional Variations in Carbohydrate Fermentations ● Pathways for carbohydrate fermentation can accommodate more substrates reductive formation of succinate. clinically significant bacteria form pyruvate from glucose via the Embden-Meyerhof pathway they may be distinguished on the basis of reduction products formed from pyruvate reflecting the enzymatic constitution of different species. F. Fermentation of Other Substrates ● Carbohydrates are by no means the only fermentable substrates. ● Metabolism of amino acids, purines, and pyrimidines: may allow substrate phosphorylations to occur.
D. Cooperativity ● The binding of substrate by one catalytic site increases the affinity of the other sites for additional substrate molecules. ● The net effect of this interaction is to produce an exponential increase in catalytic activity in response to an arithmetic increase in substrate concentration. E. Covalent Modification of Enzymes ● The regulatory properties of some enzymes are altered by covalent modification of the protein
For example, the response of glutamine synthetase to metabolic effectors is altered by adenylylation, the covalent attachment of ADP to a specific tyrosyl side chain within each enzyme subunit. ● The enzymes controlling adenylylation also are controlled by covalent modification. ● The activity of other enzymes is altered by their phosphorylation. F. Enzyme Inactivation ● The activity of some enzymes is removed by their hydrolysis. ● This process can be regulated and sometimes is signaled by covalent modification of the enzyme targeted for removal.
MICROBIAL GROWTH, NUTRITION AND CONTROL
TOPIC 1 : Requirements For Growth And Sources Of Metabolic Energy - Jenn Microbiologists use the term growth to indicate an increase in a population of microbes rather than an increase in size. Microbial growth depends on the metabolism of nutrients, and results in the formation of a discrete colony, what is colony? colony is an aggregation of cells arising from a single parent cell. A nutrient is any chemical required for growth of microbial populations. The most important of these are compounds containing carbon, oxygen, nitrogen, and/or hydrogen. WHY IS NUTRITION IMPORTANT? The hundreds of chemical compounds present inside a living cell are formed from nutrients. Macronutrients: elements required in fairly large amounts Micronutrients: metals and organic compounds needed in very small amounts GROWTH REQUIREMENTS CHEMICAL AND ENERGY REQUIREMENTS ✓ Source of carbon ✓ Use of chemical or light as a source ofenergy ✓ Source of electrons or hydrogen atoms PHOTOAUTOTROPHS ➢ use carbon dioxide as a carbon source and light energy from CHEMOAUTOTROPHS ➢ use carbon dioxide as a carbon source but catabolize organic molecules forenergy. PHOTOHETEROTROPHS ➢ are photosynthetic organisms that acquire energy from light and acquire nutrients via catabolism of organic compounds. CHEMOHETEROTROPHS use organic compounds for both energy and carbon. NOTE: first word tells us the energy source Photo source of energy= light Chemo source of energy= organic chemical compounds (proteins, lipids, and carbohydrates) SECOND word tells us the CARBON source Autotrophs inorganic chemical like carbon dioxide= carbon/nutrient source (makes their own food) Heterotrophs organic compounds= carbon/nutrient source (other- eat other- consuming organic molecules PHYSICALREQUIREMENTS/ ENVIRONMENTAL FACTORS AFFECTING GROWTH ✓ Temperature ✓ pH ✓ Osmolarity ✓ Pressure ENVI FACTOR 1: TEMPERATURE Temperature is a major environmental factor controlling microbial growth. CARDINAL TEMPERATURES ✓ minimum: Temperature below which growth ceases , or lowest temperature at which microbes will grow. ✓ Optimum: Temperature at which its growth rate is the fastest. ✓ Maximum: Temperature above which growth ceases , or highest temperature at which microbes will grow. Since both proteins and lipids are temperature- sensitive, different temperatures have different effects on the survival and growth rates of microbes. Though microbes survive within the limits imposed by a minimum growth temperature and a maximum growth temperature, an organism's metabolic activities produce the highest growth rate at the optimum growth temperature. CLASSIFICATION OF MICROORGANISMS BYTEMPERATURE REQUIREMENTS Psychrophiles ( 0 - 20C) ✓ Cold temperature optima ✓ Most extreme representatives inhabit permanently cold environments Mesophiles ( 20 – 45C) ✓ Midrange temperature optima ✓ Found in warm-blooded animals and in terrestrial and aquatic environments in temperate and tropical latitudes Thermophiles ( 50 - 80C) ✓ Growth temperature optima between 45ºC and 80ºC Hyperthermophiles ✓ Optima greater than 80°C ✓ These organisms inhabit hot environments including boiling hot springs, as well as undersea hydrothermal vents that can have temperatures in excess of 100ºC
MICROBIAL GROWTH
✓ Microbes grow via binary fission , resultingin exponential increases in numbers ✓ Generation time is the time it takes for asingle cell to grow and divide Microbes grow via binary fission , resulting in exponential increases in numbers. The time required for a bacterial cell to grow and divide is its generation time. Viewed another way, generation time is the time required for a population of cells to double in number. Most bacteria have a generation time of 1 - 3 hours. BINARY FISSION binary fission is the process that bacteria use to carry out cell division. Most unicellular microorganisms reproduce by binary fission, a process in which a cell grows to twice its normal size and then divides in half to produce two equally sized daughter cells. Binary fission is similar in concept to the mitosis that happens in multicellular organisms (such as plants and animals), but its purpose isdifferent. When cells divide by mitosis in the body of a multicellular organism, they cause the organism to grow larger or replace old, worn- out cells with new ones. In the case of a bacterium, however, cell division isn’t just a means of making more cells for the body. Instead, it’s actually how bacteria reproduce, or add more bacteria to the population. Binary fission has features in common with mitosis, but also differs from mitosis in some important ways. STEPS OF BINARY FISSION Like a human cell, a dividing bacterium needs to copy its DNA. Unlike human cells, which have multiple linear (rod-like) chromosomes enclosed in a membrane-bound nucleus, bacterial cells usually have a single, circular chromosome and always lack a nucleus. However, the bacterial chromosome is found in a specialized region of the cell called the nucleoid. Copying of DNA by replication enzymes begins at a spot on the chromosome called the origin of replication. The origin is the first part of the DNA to be copied. As replication continues, the two origins move towards opposite ends of the cell, pulling the rest of the chromosome along with them. The cell also gets longer, adding to the separation of the newly forming chromosomes. Replication continues until the entire chromosome is copied and the replication enzymes meet at the far side. Once the new chromosomes have moved to opposite cell ends and cleared the center of the cell, divisionof the cytoplasm can take place. In this process, the membrane pinches inward and a septum , or new dividing wall, forms down the middle of the cell. (Bacteria have a cell wall, so they must regenerate this wall when they undergo cell division.) Finally, the septum itself splits down the middle, and the two cells are released to continue their lives as individual bacteria. With binary fission, any given cell divides to form two cells; then each of these new cells divides in two, to make four, and then four becomes eight, and so on. his type of growth, called logarithmic growth or exponential growth, produces dramatically greater yields than simple addition, known as arithmetic growth. Microbiologists use scientific notation to deal with the huge numbers involved in expressingmicrobial population size. GROWTH CURVE ✓ A graph that plots the number of bacteria growing in a population over time is called a growth curve. When microbial growth is plotted on a semilogarithmic scale (which uses a
logarithmic scale for the y-axis), the plot of the population's growth results in a straight line. When bacteria are grown in a broth, the typical microbial growth curve has four distinct phases: ✓ During lag phase, cells are recovering from a period of no growth and are making macromolecules in preparation for growth ✓ During log phase cultures are growing maximally ✓ Stationary phase occurs when nutrients are depleted and wastes accumulate (Growth rate = death rate) ✓ During death phase death rate is greater than growth rate CALCULATING GENERATION TIME SOURCE: https://www.youtube.com/watch?v= 8 AhDxAQ aDOA&ab_channel=MicrobiologyMantra Measuring Microbial Growth DIRECT METHODS ✓ viable plate counts ✓ Membrane Filtration ✓ Microscopic Counts ✓ Electronic Counters ✓ Most-Probable Number INDIRECT METHODS ✓ measurements of metabolic activity ✓ measurements of a population's dry weight ✓ measurement of the turbidity of a broth (spectrophotometer)
SMALL SPACE BETWEEN GENES - Perhaps due to the space constraints of packing so many essential genes onto a single chromosome, prokaryotes can be highly efficient in terms of genomic organization. Very little space is left between prokaryotic genes. Summary – Prokaryotic vs Eukaryotic Genome Prokaryotes are two types such as bacteria and archaea. On the other hand, eukaryotes include plants, animals, fungi, algae and protozoa. Prokaryotes lack membrane-bound organelles such as the nucleus, mitochondria, etc. In contrast, eukaryotes have extensive internal compartments that separate by membranes. According to these differences in the cellular organization, prokaryotic and eukaryotic genomes also differ from each other. Thus, the main difference between prokaryotic and eukaryotic genome is that the prokaryotic genome floats in the cytoplasm while the eukaryotic genome protects inside the nucleus. Furthermore, the prokaryotic genome is more compact and has no repetitive DNA, introns, and spacer DNA compared to the eukaryotic genome. In contrast, the eukaryotic genome has many genes, more repetitive DNA and introns. Hence, this is the difference between prokaryotic and eukaryotic genome. Overview
- Each species has a uniquely fundamental set of genetic information, its genome.
- The genome is composed of one or more DNA molecules, each organized as a chromosome.
- The prokaryotic genomes are mostly single circular chromosomes.
- Eukaryotic genomes consist of one or two sets of linear chromosomes confined to the nucleus.
- A gene is a segment of DNA that is transcribed into a functional RNA molecule.
- Introns interrupt many eukaryote genes.
- Viral genomes consist of either DNA or RNA. VIRAL GENOME Viral genomes consist of DNA or RNA only, never both. DNA and RNA molecules can be double stranded or single stranded, linear or circular (Fig. 1.6), segmented (composed of multiple pieces of nucleic acid) or Non segmented. The meaning of the term genomic segment could be confusing as its use is driven by both nomenclature principles and historic reasons. Strictly speaking, a genome segment is an individual and unique piece of nucleic acid among multiple pieces comprising one whole viral genome. For example, the influenza A virus has a segmented genome comprised of eight ssRNA segments (Fig. 1.5). Herpesviruses, which have Non segmented genomes composed of one linear dsDNA molecule, have the so- called UL (unique long) and US (unique short) segments, corresponding to regions/portions of their genomes flanked by repeats. Where is the genome in a virus? The viral genome is packed inside a symmetric protein capsid, composed of either a single or multiple proteins, each of them is encoding a single viral gene. Introns are found in the genes of most organisms and many viruses, and they can be in both protein-coding genes and genes that function as RNA (noncoding genes). In many viruses the genome ends contain repeated sequences, chemical modifications, or secondary structures, which often have regulatory functions. THE DIFFERENCE between VIRUS AND PHAGE : Virus - microbes that infect cells. Human viruses affect human cells. Plant virus affects plant cells. Phage - are virus but this type of virus solely kills and selectively kill bacteria. They infect and replicate within bacterium and archaea. The term was derived from "bacteria" and the Greek (phagein), meaning "to devour". T-phages Early research mostly focused on phages that infect Escherichia coli. A series of 7 phages that infect Escherichia coli are known as T-phages ( T for type ). The T2, T4, and T6 phages are called T-even phages. The T1, T3, and T7 phages are known as dependent virulent, while the T5 phage is called "autonomously virulent". Out of all the T-phages, T4 bacteriophage is well studied.
PARTS OF A PHAGE:
I. Head The head is made up of capsid head and nucleic acid. a. Capsid head The head of the T4 phage is 120 nm long and 86 nm wide having hexagonal and prism shapes. The capsid head stores and protects the genome. b. Nucleic acid T4 phage contains double-stranded DNA that contains approximately 170000 bases. The modified bases present in T4 DNA protect the genome from breakdown by bacterial restriction endonucleases. II. Tail The structure of the tail is more complicated compared to the head. It contains a tail tube, sheath, collar, whisker, spike, base plate, and tail fiber. a. Tail tube It is an inner non-contractile tube made up of protein. It provides the passage for the movement of DNA from the head to the bacterial cell. b. Sheath The tail tube is enclosed by a contractile envelope known as a sheath which is made up of tail sheath proteins. The contraction of the sheath pushes the tail tube through the outer membrane of bacteria, forming a canal for the delivery of the viral genome. c. Collar and whisker The collar and whisker are made up of fibers present near the head-to-tail interference of the page. They control the withdrawals of the long tail fibers and help in the attachment of tail fibers to phage particles during assembly. d. Baseplate It is made up of multiple protein molecules. It controls sheath contraction, host recognition, DNA ejection, and attachment. e. Spike A spike pierces the cell membrane of bacteria and makes an opening for the entrance of the tail tube. f. Tail fiber It is like appendages. It is a thin rod-like structure used for the attachment of bacteriophage on the surface of bacteria.
INITIAL STEPS IN THE LIFE CYCLE OF A
BACTERIOPHAGE:
The first step involved in the replication of bacteriophages is the injection of its genetic material into the bacterial cell. Attachment or absorption Bacteriophage attaches to the surface of the bacterial cell wall at a specific receptor site by their tail fibers. This attachment is the courtesy of week chemical bonding between receptor site and bacteriophage. Penetration The tail of the bacteriophage discharges lysozyme which is an antibacterial enzyme to digest the small part of the cell wall. After digestion, the tail sheath contracts, and a rigid tube is propelled out of the sheath to puncture a hole in the bacterial cell membrane. Genetic material is then passed through the tail tube and injected into the bacterial cell. The protein coat and tail of the bacteriophage do not enter the cell and remain outside. LYTIC PHASE a. DNA replication and protein synthesis After the penetration, viral genetic material hijacks the biosynthetic machinery of the bacterial cell and starts multiplying by synthesizing its components such as DNA, protein, and tail structures. b. Assembly of phages The new genetic material is packed into the head and then the tail is attached to complete the construction of the bacteriophage. It takes 25 minutes for a bacteriophage to make about 200 new bacteriophages after entry into the cell. c. Lysis As the number of daughter bacteriophages increases in the bacterial cell, it starts to weaken, and newly formed bacteriophages are released causing lysis of the bacterial cell. Such a phage which causes lysis of bacterial cell is known as lytic phage. LYSOGENIC In the lysogenic cycle of the temperate cycle, no bursting of the bacterial cell takes place, and it keeps on living normally.
Viral Structure and Replication
VIRAL STRUCTURE AND REPLICATION –
Claudine I. Introduction to Viral Structure and Replicatio n Viruses are inert outside the host cell. Small viruses, e.g., polio and tobacco mosaic virus, can even be crystallized. Viruses are unable to generate energy. As obligate intracellular parasites, during replication, they fully depend on the complicated biochemical machinery of eukaryotic or prokaryotic cells. The main purpose of a virus is to deliver its genome into the host cell to allow its expression (transcription and translation) by the host cell. A fully assembled infectious virus is called a virion. The simplest virions consist of two basic components: nucleic acid (single- or double-stranded RNA or DNA) and a protein coat, the capsid, which functions as a shell to protect the viral genome from nucleases and which during infection attaches the virion to specific receptors exposed on the prospective host cell. Capsid proteins are coded for by the virus genome. Because of its limited size the genome codes for only a few structural proteins (besides non- structural regulatory proteins involved in virus replication). Capsids are formed as single or double protein shells and consist of only one or a few structural protein species. Therefore, multiple protein copies must self assemble to form the continuous three-dimensional capsid structure. Self assembly of virus capsids follows two basic patterns: helical symmetry, in which the protein subunits and the nucleic acid are arranged in a helix, and icosahedral symmetry, in which the protein subunits assemble into a symmetric shell that covers the nucleic acid- containing core. Some virus families have an additional covering, called the envelope, which is usually derived in part from modified host cell membranes. Viral envelopes consist of a lipid bilayer that closely surrounds a shell of virus-encoded membrane- associated proteins. The exterior of the bilayer is studded with virus-coded, glycosylated (trans-) membrane proteins. Therefore, enveloped viruses often exhibit a fringe of glycoprotein spikes or knobs, also called peplomers. In viruses that acquire their envelope by budding through the plasma or another intracellular cell membrane, the lipid composition of the viral envelope closely reflects that of the particular host membrane. The outer capsid and the envelope proteins of viruses are glycosylated and important in determining the host range and antigenic composition of the virion. In addition to virus-specified envelope proteins, budding viruses carry also certain host cell proteins as integral constituents of the viral envelope. Virus envelopes can be considered an additional protective coat. Larger viruses often have a complex architecture consisting of both helical and isometric symmetries confined to different structural components. Small viruses, e.g., hepatitis B virus or the members of the picornavirus or parvovirus family, are orders of magnitude more resistant than are the larger complex viruses, e.g. members of the herpes or retrovirus families. Figure 1.1 The structures of a naked icosahedral capsid virus (top left) and enveloped viruses (bottom) with an icosahedral (left) nucleocapsid or a helical (right) ribonucleocapsid. Helical nucleo-capsids are always enveloped for human viruses.
Viral Structure and Replication
Table 1.1 Classitication of major viral families based on genome structure and virion morphology. A, ONA viruses, L, linear genome: C, circular genome. B, RNA viruses. S, segmented genome. (Rosenthal KS and Tan MJ. Rapid Review Microbiology and Immunology. 3rd Edition. (2011). Mosby, Inc., an affiliate of Elsevier Inc). II. Viral Morphology A. Helical Symmetry In the replication of viruses with helical symmetry, identical protein subunits (protomeres) self-assemble into a helical array surrounding the nucleic acid, which follows a similar spiral path. Such nucleocapsids form rigid, highly elongated rods or flexible filaments; in either case, details of the capsid structure are often discernible by electron microscopy. In addition to classification as flexible or rigid and as naked or enveloped, helical nucleocapsids are characterized by length, width, pitch of the helix, and number of protomeres per helical turn. The most extensively studied helical virus is tobacco mosaic virus. Many important structural features of this plant virus have been detected by x-ray diffraction studies. Figure 2.1. The helical structure of the rigid tobacco mosaic virus rod. About 5 percent of the length of the virion is depicted.cylindrical core 4 nm in diameter. B. Icosahedral Symmetry An icosahedron is a polyhedron having 20 equilateral triangular faces and 12 vertices. Lines through opposite vertices define axes of fivefold rotational symmetry: all structural features of the polyhedron repeat five times within each 360° of rotation about any of the fivefold axes. Lines through the centers of opposite triangular faces form axes of threefold rotational symmetry; twofold rotational symmetry axes are formed by lines through midpoints of opposite edges. An icosaheron (polyhedral or spherical) with fivefold, threefold, and twofold axes of rotational symmetry is defined as having 532 symmetry (read as 5,3,2). Figure 2.2. Icosahedral models seen, left to right, on fivefold, threefold, and twofold axes of rotational symmetry. These axes are perpendicular to the plane of the page and pass through the centers of each figure. Both polyhedral (upper) and spherical (lower) forms are represented by different virus families Viruses were first found to have 532 symmetry by x- ray diffraction studies and subsequently by electron microscopy with negative-staining techniques. In most icosahedral viruses, the protomers, i.e. the structural polypeptide chains, are arranged in oligomeric clusters called capsomeres, which are readily delineated by negative staining electron microscopy and form the closed capsid shell. The arrangement of capsomeres into an icosahedral shell (compare Fig. 2 - 3 with the upper right model in Fig. 2 - 2 ) permits the classification of such viruses by capsomere number and pattern. This requires the identification of the nearest pair of vertex capsomeres (called penton: those through which the fivefold symmetry axes pass) and the distribution of capsomeres between them. Figure 2.3. Adenovirus after negative stain electron microscopy. The capsid reveals the typical isometric shell made up from 20 equilateral triangular faces. The 252 capsomeres, 12 pentons and the 240 hollow hexon capsomeres are arranged in a T = 25 symmetry pattern vetite (x 400,000).
Viral Structure and Replication
Figure 3.1. A virulent phage shows only the lytic cycle pictured here. In the lytic cycle, the phage replicates and lyses the host cell. The third stage of infection is biosynthesis of new viral components. After entering the host cell, the virus synthesizes virus-encoded endonucleases to degrade the bacterial chromosome. It then hijacks the host cell to replicate, transcribe, and translate the necessary viral components (capsomeres, sheath, base plates, tail fibers, and viral enzymes) for the assembly of new viruses. Polymerase genes are usually expressed early in the cycle, while capsid and tail proteins are expressed later. During the maturation phase, new virions are created. To liberate free phages, the bacterial cell wall is disrupted by phage proteins such as holin or lysozyme. The final stage is release. Mature viruses burst out of the host cell in a process called lysis and the progeny viruses are liberated into the environment to infect new cells. The Lysogenic Cycle In a lysogenic cycle, the phage genome also enters the cell through attachment and penetration. A prime example of a phage with this type of life cycle is the lambda phage. During the lysogenic cycle, instead of killing the host, the phage genome integrates into the bacterial chromosome and becomes part of the host. The integrated phage genome is called a prophage. A bacterial host with a prophage is called a lysogen. The process in which a bacterium is infected by a temperate phage is called lysogeny. It is typical of temperate phages to be latent or inactive within the cell. As the bacterium replicates its chromosome, it also replicates the phage’s DNA and passes it on to new daughter cells during reproduction. The presence of the phage may alter the phenotype of the bacterium, since it can bring in extra genes (e.g., toxin genes that can increase bacterial virulence). This change in the host phenotype is called lysogenic conversion or phage conversion. Some bacteria, such as Vibrio cholerae and Clostridium botulinum, are less virulent in the absence of the prophage. The phages infecting these bacteria carry the toxin genes in their genome and enhance the virulence of the host when the toxin genes are expressed. In the case of V. cholera, phage encoded toxin can cause severe diarrhea; in C. botulinum, the toxin can cause paralysis. During lysogeny, the prophage will persist in the host chromosome until induction, which results in the excision of the viral genome from the host chromosome. After induction has occurred the temperate phage can proceed through a lytic cycle and then undergo lysogeny in a newly infected cell Figure 3.2 A temperate bacteriophage has both lytic and lysogenic cycles. In the lysogenic cycle, phage DNA is incorporated into the host genome, forming a prophage, which is passed on to subsequent generations of cells. Environmental stressors such as starvation or exposure to toxic chemicals may cause the prophage to be excised and enter the lytic cycle. Transduction Transduction occurs when a bacteriophage transfers bacterial DNA from one bacterium to another during sequential infections. There are two types of transduction: generalized and specialized transduction. During the lytic cycle of viral replication, the virus hijacks the host cell, degrades the host chromosome, and makes more viral genomes. As it assembles and packages DNA into the phage head, packaging occasionally makes a mistake. Instead of packaging viral DNA, it takes a random piece of host DNA and inserts it into the capsid. Once released, this virion will then inject the former host’s DNA into a newly infected host. The asexual transfer of genetic
Viral Structure and Replication
information can allow for DNA recombination to occur, thus providing the new host with new genes (e.g., an antibiotic-resistance gene, or a sugar- metabolizing gene). Generalized transduction occurs when a random piece of bacterial chromosomal DNA is transferred by the phage during the lytic cycle. Specialized transduction occurs at the end of the lysogenic cycle, when the prophage is excised and the bacteriophage enters the lytic cycle. Since the phage is integrated into the host genome, the prophage can replicate as part of the host. However, some conditions (e.g., ultraviolet light exposure or chemical exposure) stimulate the prophage to undergo induction, causing the phage to excise from the genome, enter the lytic cycle, and produce new phages to leave host cells. During the process of excision from the host chromosome, a phage may occasionally remove some bacterial DNA near the site of viral integration. The phage and host DNA from one end or both ends of the integration site are packaged within the capsid and are transferred to the new, infected host. Since the DNA transferred by the phage is not randomly packaged but is instead a specific piece of DNA near the site of integration, this mechanism of gene transfer is referred to as specialized transduction (see Figure 3.3). The DNA can then recombine with host chromosome, giving the latter new characteristics. Transduction seems to play an important role in the evolutionary process of bacteria, giving them a mechanism for asexual exchange of genetic information. Figure 3.3 This flowchart illustrates the mechanism of specialized transduction. An integrated phage excises, bringing with it a piece of the DNA adjacent to its insertion point. On reinfection of a new bacterium, the phage DNA integrates along with the genetic material acquired from the previous host. Life Cycle of Viruses with Animal Hosts Lytic animal viruses follow similar infection stages to bacteriophages: attachment, penetration, biosynthesis, maturation, and release (see Figure 3.4). However, the mechanisms of penetration, nucleic-acid biosynthesis, and release differ between bacterial and animal viruses. After binding to host receptors, animal viruses enter through endocytosis (engulfment by the host cell) or through membrane fusion (viral envelope with the host cell membrane). Many viruses are host specific, meaning they only infect a certain type of host; and most viruses only infect certain types of cells within tissues. This specificity is called a tissue tropism. Examples of this are demonstrated by the poliovirus, which exhibits tropism for the tissues of the brain and spinal cord, or the influenza virus, which has a primary tropism for the respiratory tract. Figure 3.4 In influenza virus infection, viral glycoproteins attach the virus to a host epithelial cell. As a result, the virus is engulfed. Viral RNA and viral proteins are made and assembled into new virions that are released by budding.
