MICROBIOLOGY

General organization and Classification of bacteria

Prokaryotic Cell organization

 Prokaryotic cells almost always are bounded by a chemicallycomplex cell wall. Inside this wall, and
separated from it by a periplasmic space, lies the plasma membrane. This membrane can beinvaginated to
form simple internal membranous structures. Sincethe prokaryotic cell does not contain internal
membrane-bound organelles,its interior appears morphologically simple.

Prokaryotic cell membranes

 Membranes are an absolute requirement for all living organisms.Cells must interact in a selective fashion
with their environment,whether it is the internal environment of a multicellularorganism or a less protected
and more variable externalenvironment.
 Cells must not only be able to acquire nutrientsand eliminate wastes, but they also have to maintain their
interiorin a constant, highly organized state in the face of externalchanges. The plasma membrane
encompasses the cytoplasm of both prokaryotic and eukaryotic cells.
 This membrane is thechief point of contact with the cell’s environment and thus is responsiblefor much of
its relationship with the outside world.

The Plasma Membrane

 Membranes contain both proteins and lipids, although the exactproportions of protein and lipid vary
widely. Bacterial plasmamembranes usually have a higher proportion of protein than do eukaryotic

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 membranes, presumably because they fulfill so manydifferent functions that are carried out by other
organelle membranes in eukaryotes.
 Most membrane-associated lipids are structurallyasymmetric with polar and nonpolar endsandare called
amphipathic. The polar ends interact with water and arehydrophilic; the nonpolarhydrophobic ends are
insoluble inwater and tend to associate with one another.
 This property oflipids enables them to form a bilayer in membranes. The outersurfaces are hydrophilic,
whereas hydrophobic ends are buried inthe interior away from the surrounding water.
 Many of these amphipathic lipids are phospholipids. Bacterial membranes usually differ from eukaryotic
membranes in lackingsterols such as cholesterol.

Phosphatidylethanolamine, an amphipathic
phospholipid often found in bacterial membranes.

 The most widely accepted current model for membranestructure is the fluid mosaic model of S. Jonathan
Singer and Garth Nicholson. They distinguish between two typesof membrane proteins. Peripheral
proteins are loosely connectedto the membrane and can be easily removed.
 They are solublein aqueous solutions and make up about 20 to 30% of totalmembrane protein. About 70 to
80% of membrane proteins areintegral proteins. These are not easily extracted from membranesand are
insoluble in aqueous solutions when freed oflipids.
 Integral proteins, like membrane lipids, are amphipathic; theirhydrophobic regions are buried in the lipid
while the hydrophilicportions project from the membrane surface. Some ofthese proteins even extend all
the way through the lipid layer.
 Integral proteins can diffuse laterally around the surface to new locations, but do not flip-flop or rotate
through the lipid layer. Often carbohydrates are attached to the outer surface of plasma membrane proteins
and seem to have important functions.
 The plasma membrane retains the cytoplasm, particularly in cells without cell walls, and separates it from
the surroundings. The plasma membrane also serves as 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. Thus the
membrane prevents the loss of essential components through leakage while allowing the movement of
other molecules.
 Transport systems can be used for 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,

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 photosynthesis, the synthesis of lipids and cell wall constituents, and probably chromosome segregation.
The membrane contains special receptor molecules that help prokaryotes detect and respond to chemicals
in their surroundings.

Plasma Membrane Structure.This diagram of the fluid mosaic model of bacterial membranestructure shows the integral
proteins (blue) floating in a lipid bilayer. Peripheral proteins (purple) are associated loosely with the inner membrane surface.
Small spheres represent the hydrophilic ends of membrane phospholipids and wiggly tails, the hydrophobic fatty acid chains.
Other membrane lipids such as hopanoids (pink) may be present.

Internal Membrane Systems

 Although procaryotic cytoplasm does not contain complex membranousorganelles like mitochondria or
chloroplasts, membranousstructures of several kinds can be observed. A commonlyobserved structure is
the mesosome. Mesosomes are invaginationsof the plasma membrane in the shape of vesicles, tubules,
orlamellae.
 They are seen in both grampositiveand gram-negative bacteria, although they are generallymore prominent
in the former.
 Mesosomes often are found next to septa or cross-walls in dividingbacteria and sometimes seem attached
to the bacterialchromosome. Thus they may be involved in cell wall formation during division or play a
role in chromosome replication and distributionto daughter cells.

The Cytoplasmic Matrix

 Procaryotic cytoplasm, unlike that of eukaryotes, lacks unitmembrane-bound organelles. The cytoplasmic
matrix is the substancelying between the plasma membrane and the nucleoid.
 The matrix is largely water (about 70% of bacterial massis water). It is featureless in electron micrographs
but often ispacked with ribosomes and highly organized. Specificproteins are positioned at particular sites
such as the cell poleand the place where the bacterial cell will divide.
 Thus althoughbacteria may lack a true cytoskeleton, they do have a cytoskeletonlikesystem of proteins in
their cytoplasmic matrix.

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  The plasmamembrane and everything within is called the protoplast; thus thecytoplasmic matrix is a
major part of the protoplast.

Inclusion Bodies

 A variety of inclusion bodies, granules of organic or inorganicmaterial that often are clearly visible in a
light microscope, ispresent in the cytoplasmic matrix. These bodies usually are usedfor storage (e.g.,
carbon compounds, inorganic substances, andenergy), and also reduce osmotic pressure by tying up
moleculesin particulate form.
 Some inclusion bodies are notbounded by a membrane and lie free in the cytoplasm—for
example,polyphosphate granules, cyanophycin granules, andsome glycogen granules.
 Other inclusion bodies are enclosed bya membrane about 2.0 to 4.0 nm thick, which is single-layeredand
not a typical bilayer membrane. Examples of membrane-enclosed inclusion bodies are poly β
hydroxybutyrate granules,some glycogen and sulfur granules, carboxysomes, and gas vacuoles.
 Inclusion body membranes vary in composition. Someare protein in nature, whereas others contain lipid.
Because inclusionbodies are used for storage, their quantity will vary withthe nutritional status of the cell.
 Organic inclusion bodies usually contain either glycogen or poly β hydroxybutyrate.
 Glycogen is a polymer of glucose unitscomposed of long chains formed by α(1→4) glycosidic bondsand
branching chains connected to them by α(1→6) glycosidicbonds.
 Poly β hydroxybutyrate (PHB) containshydroxybutyrate molecules joined by ester bonds betweenthe
carboxyl and hydroxyl groups of adjacent molecules.
 Glycogen and PHB inclusion bodies arecarbon storage reservoirs providing material for energy
andbiosynthesis. Many bacteria also store carbon as lipid droplets.
 Carboxysomesare present in manycyanobacteria, nitrifying bacteria, and thiobacilli.
 A most remarkable organic inclusion body, the gas vacuole,is present in many cyanobacteria purple
andgreen photosynthetic bacteria,These bacteria float at or near thesurface, because gas vacuoles give them
buoyancy.
 Gas vacuoles are aggregates of enormous numbers of small,hollow, cylindrical structures called gas
vesicles. Gas vesicle walls do not contain lipid and are composed entirely of a single small protein. These
protein subunits assemble to forma rigid enclosed cylinder that is hollow and impermeable to waterbut
freely permeable to atmospheric gases.
 Bacteria with gas vacuolescan 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
upwardwhen new ones are constructed.
 Two major types of inorganic inclusion bodies are seen.Many bacteria store phosphate as polyphosphate
granules orvolutin granules.
 Polyphosphate is a linear polymerof orthophosphates joined by ester bonds. Thus volutin granulesfunction
as storage reservoirs for phosphate, an importantcomponent of cell constituents such as nucleic acids.

Ribosomes
 As mentioned earlier, the cytoplasmic matrix often is packed withribosomes; they also may be loosely
attached to the plasmamembrane. Ribosomes look like small, featureless particles atlow magnification in
electron micrographs but areactually very complex objects made of both protein and ribonucleicacid
(RNA).
 They are the site of protein synthesis; matrix ribosomessynthesize proteins destined to remain within the
cell,whereas the plasma membrane ribosomes make proteins fortransport to the outside.
4 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr
  The newly formed polypeptide folds intoits final shape either as it is synthesized by the ribosome or
shortlyafter completion of protein synthesis. The shape of each proteinis determined by its amino acid
sequence. Special proteins calledmolecular chaperones, or chaperones, aid the polypeptide in foldingto its
proper shape.
 The procaryotic ribosomes are smaller than eukaryotic ribosomes. They commonly are called 70S
ribosomes, have dimensionsof about 14 to 15 nm by 20 nm, a molecular weight ofapproximately 2.7
million, and are constructed of a 50S and a30S subunit. The S in 70S and similar values stands for
Svedbergunit.
 Ribosomesin the cytoplasmic matrix of eucaryotic cells are 80S ribosomesand about 22 nm in diameter.
Despite their overalldifference in size, both types of ribosomes are similarly composedof a large and a
small subunit.

The Nucleoid

 Probably the most striking difference between prokaryotes andeukaryotes is the way in which their genetic
material is packaged.
 Eucaryotic cells have two or more chromosomes containedwithin a membrane-delimited organelle, the
nucleus. In contrast,prokaryotes lack a membrane-delimited nucleus. The prokaryotic chromosome is
located in an irregularly shaped region called thenucleoid.
 Usually prokaryotes contain a single circleof double-stranded deoxyribonucleic acid (DNA), but some
have a linear DNA chromosome.
 The nucleoid is visible in the light microscope after stainingwith the Feulgen stain, which specifically
reacts with DNA. Acell can have more than one nucleoid when cell division occursafter the genetic
material has been duplicated.
 Inactively growing bacteria, the nucleoid has projections that extendinto the cytoplasmic matrix.
Presumablythese projections contain DNA that is being actively transcribedto produce mRNA. The
nucleoid is seen in contact with either the mesosome or the plasmamembrane.
 Membranes also are found attached to isolated nucleoids.Thus there is evidence that bacterial DNA is
attached tocell membranes, and membranes may be involved in the separationof DNA into daughter cells
during division.
 Many bacteria possess plasmids in addition to their chromosome.These are double-stranded DNA
molecules, usually circular,that can exist and replicate independently of the chromosomeor may be
integrated with it; in either case they normally are inheritedor passed on to the progeny. However, plasmids
are notusually attached to the plasma membrane and sometimes are lost to one of the progeny cells during
division.
 Plasmids are not requiredfor host growth and reproduction, although they may carrygenes that give their
bacterial host a selective advantage. Plasmidgenes can render bacteria drug-resistant, give them new
metabolicabilities, make them pathogenic, or endow them with anumber of other properties.

The Prokaryotic Cell Wall

 The cell wall is the layer, usually fairly rigid, that lies just outsidethe plasma membrane. It is one of the
most important parts of aprocaryotic cell for several reasons. The cell walls ofmany pathogens have
components that contribute to their pathogenicity.
 The wall can protect a cell from toxic substances and isthe site of action of several antibiotics.

5 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

GRAM POSITIVE AND GRAM NEGATIVE

 After Christian Gram developed the Gram stain in 1884, itsoon became evident that bacteria could be
divided into two majorgroups based on their response to the Gram-stain procedure.
 Gram-positive bacteria stained purple, whereasgram-negative bacteria were colored pink or red by the
technique
 The gram-positive cell wall consists of a single 20 to 80 nm thickhomogeneous peptidoglycan or
mureinlayer lying outside theplasma membrane.
 In contrast, the gram-negativecell wall is quite complex. It has a 2 to 7 nm peptidoglycan layersurrounded
by a 7 to 8 nm thick outer membrane. Because of thethicker peptidoglycan layer, the walls of gram-
positive cells arestronger than those of gram-negative bacteria.

 Microbiologistsoften call all the structures from the plasma membrane outwardthe envelope or cell
envelope. This includes the wall and structures like capsules when present.
 A space is seen between the plasma membraneand the outer membrane in electron micrographs of gram
negativebacteria, and sometimes a similar but smaller gap may be observed between the plasma membrane
and wall in gram positivebacteria. This space is called the periplasmic space.
 Evidence indicates that the periplasmic space may be filledwith a loose network of peptidoglycan. Possibly
it is more a gelthan a fluid-filled space.
 The substance that occupies theperiplasmic space is the periplasm.
 Gram-positive cells mayhave periplasm even if they lack a discrete, obvious periplasmicspace. Size
estimates of the periplasmic space in gram-negativebacteria range from 1 nm to as great as 71 nm.
 The periplasmic space of gram-negative bacteriacontains many proteins that participate in nutrient
acquisition.
 The periplasmic space alsocontains enzymes involved in peptidoglycan synthesis and themodification of
toxic compounds that could harm the cell.
 Gram positivebacteria may not have a visible periplasmic space and donot appear to have as many
periplasmic proteins; rather, they secreteseveral enzymes that ordinarily would be periplasmic ingram-

6 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

 negative bacteria. Such secreted enzymes are often calledexoenzymes. Some enzymes remain in the
periplasm and are attachedto the plasma membrane.

Peptidoglycan Structure

 Peptidoglycan or murein is an enormous polymer composed of manyidentical subunits. The polymer

contains two sugar derivatives,N-acetylglucosamine and N-acetylmuramic acid (the lactyl ether ofN-
acetylglucosamine), and several different amino acids, three ofwhich—D-glutamic acid, D-alanine, and
meso-diaminopimelicacid—are not found in proteins.
 The presence of D-amino acids protectsagainst attack by most peptidases. The backbone of this polymer is
composedof alternating N-acetylglucosamine and N-acetylmuramic acidresidues. A peptide chain of four
alternating D- and L-amino acids isconnected to the carboxyl group of N-acetylmuramic acid.
 Chains of linked peptidoglycan subunits are joined by crosslinksbetween the peptides. Most gram-negative
cell wall peptidoglycanlacks the peptide interbridge.
 This cross-linking results in anenormous peptidoglycan sac that is actually one dense,
interconnectednetwork. These sacs have been isolated fromgram-positive bacteria and are strong enough to
retain their shapeand integrity, yet they are elastic and somewhatstretchable, unlike cellulose. They also
must be porous, as moleculescan penetrate them.

Peptidoglycan Cross-Links. (a) E. coli peptidoglycanwith direct

cross-linking, typical of many gram-negative bacteria.
(b) Staphylococcus aureuspeptidoglycan. S. aureusis a gram-positive
bacterium. NAM is N-acetylmuramic acid. NAG is N-
acetylglucosamine.
Gly is glycine.

7 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

Gram-Positive Cell Walls
 Normally the thick, homogeneous cell wall of gram-positive bacteriais composed primarily of
peptidoglycan, which often containsa peptide interbridge. Howevergram-positive cell walls usually also
contain large amounts of teichoicacids, polymers of glycerol or ribitol joined by phosphate groups.
 Amino acids such as D-alanine orsugars like glucose are attached to the glycerol and ribitol groups.
 The teichoic acids are connected to either the peptidoglycan itselfby a covalent bond with the six hydroxyl
of N-acetylmuramicacid or to plasma membrane lipids; in the latter case they arecalled lipoteichoic acids.
 Teichoic acids appear to extend to thesurface of the peptidoglycan, and, because they are
negativelycharged, help give the gram-positive cell wall its negative charge.
 The functions of these molecules are unclear, but they maybe important in maintaining the structure of the
wall. Teichoicacids are not present in gram-negative bacteria.

1. Gram positive envelope 2. Teichoic Acid Structure. The segment of a teichoic acid made of phosphate, glycerol, and a
side chain, R. R may represent D-alanine, glucose, or other molecules.

Gram-Negative Cell Walls

 Gram-negative cellwalls are much more complex than gram-positive walls. The thinpeptidoglycan layer
next to the plasma membrane may constitute notmore than 5 to 10% of the wall weight. In E. coli it is
about 2 nm thickand contains only one or two layers or sheets of peptidoglycan.
 The outer membrane lies outside the thin peptidoglycanlayer. The most abundant membrane proteinis
Braun’s lipoprotein, a small lipoprotein covalently joinedto the underlying peptidoglycan and embedded in
the outer membraneby its hydrophobic end.
 The outer membrane and peptidoglycanare so firmly linked by this lipoprotein that they can beisolated as
one unit.
 Another structure that may strengthen the gram-negativewall and hold the outer membrane in place is the
adhesion site. The outer membrane and plasma membrane appear to be in directcontact at many locations
in the gram-negative wall.
 Adhesion sites may be regions of directcontact or possibly true membrane fusions. Possibly the most
unusual constituents of the outer membraneare its lipopolysaccharides (LPSs). These large,

8 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

 complexmolecules contain both lipid and carbohydrate, and consistof three parts: (1) lipid A, (2) the core
polysaccharide, and(3) the O side chain.
 The lipid Aregion contains two glucosaminesugar derivatives, each with three fatty acids and phosphate
orpyrophosphate attached. It is buried in the outer membrane andthe remainder of the LPS molecule
projects from the surface.
 The core polysaccharide is joined to lipid A. The O side chain or O antigen is a polysaccharide chain
extendingoutward from the core. It has several peculiar sugarsand varies in composition between bacterial
strains.
 Although Oside chains are readily recognized by host antibodies, gramnegativebacteria may thwart host
defenses by rapidly changingthe nature of their O side chains to avoid detection.
 Antibody interactionwith the LPS before reaching the outer membraneproper may also protect the cell wall
from direct attack.
 The LPS is important for several reasons other than the avoidanceof host defenses. Since the core
polysaccharide usually containscharged sugars and phosphate, LPS contributesto the negative charge on
the bacterial surface.
 Lipid A is a majorconstituent of the outer membrane, and the LPS helps stabilizemembrane structure. Lipid
A often is toxic; as a resultthe LPS can act as an endotoxin and cause someof the symptoms that arise in
gram-negative bacterial infections.
 A most important outer membrane function is to serve as a protectivebarrier. It prevents or slows the entry
of bile salts, antibiotics,and other toxic substances that might kill or injure the bacterium.
 Even so, the outer membrane is more permeable than the plasmamembrane and permits the passage of
small molecules like glucoseand other monosaccharides.
 This is due to the presence of specialporin proteins. Three porin molecules clustertogether and span the
outer membrane to form a narrow channelthrough which molecules smaller than about 600 to 700 daltons
canpass.

1. The Gram-Negative Envelope. 2. Lipopolysaccharide Structure. Abe, abequose; Gal, galactose; Glc, glucose;
9 Departments
GlcN, glucosamine;of Biochemistry,
Hep, S.P. College
heptulose; KDO, / Women’s College
2-keto-3-deoxyoctonate; Man, M.A. RoadNAG,
mannose; Sgr N-acetylglucosamine;
P, phosphate; Rha, L-rhamnose. Lipid A is buried in the outer membrane.
The Mechanism of Gram Staining
 The difference betweengram-positive and gram-negative bacteria is due to the physicalnature of their cell
walls. If the cell wall is removed from grampositivebacteria, they become gram negative.
 The peptidoglycanitself is not stained; instead it seems to act as a permeability barrierpreventing loss of
crystal violet.
 During the procedure thebacteria are first stained with crystal violet and next treated withiodine to promote
dye retention.
 When gram-positive bacteriathen are decolorized with ethanol, the alcohol is thought to shrinkthe pores of
the thick peptidoglycan. Thus the dye-iodine complexis retained during the short decolorization step and
the bacteriaremain purple.
 In contrast, gram-negative peptidoglycan isvery thin, not as highly cross-linked, and has larger pores.
Alcoholtreatment also may extract enough lipids from the gramnegativewall to increase its porosity further.
For these reasons, alcoholmore readily removes the purple crystal violet-iodinecomplex from gram-
negative bacteria.
The Cell Wall and Osmotic Protection
 The cell wall usually is required to protect bacteria against destructionby osmotic pressure. Solutes are
much more concentratedin bacterial cytoplasm than in most microbial habitats, which are hypotonic.
 During osmosis, water moves across selectivelypermeable membranes such as the plasma membrane
fromdilute solutions (higher water concentration) to more concentratedsolutions (lower water
concentration). Thus water normallyenters bacterial cells and the osmotic pressure may reach 20
atmospheresor 300 pounds/square inch.
 The plasma membranecannot withstand such pressures and the cell will swell and bephysically disrupted
and destroyed, a process called lysis, withoutthe wall that resists cell swelling and protects it.
 Solutes aremore concentrated in hypertonic habitats than in the cell. Thuswater flows outward, and the
cytoplasm shrivels up and pullsaway from the cell wall.
 This phenomenon is known as plasmolysisand is useful in food preservation because many
microorganismscannot grow in dried foods and jellies as they cannotavoid plasmolysis.
Components External to the Cell Wall
 Bacteria have a variety of structures outside the cell wall that canfunction in protection, attachment to
objects, and cell movement.
Capsules, Slime Layers, and S-Layers
 Some bacteria have a layer of material lying outside the cell wall.When the layer is well organized and
not easily washed off, it iscalled a capsule.
 A slime layer is a zone of diffuse, unorganizedmaterial that is removed easily.
 A glycocalyx is anetwork of polysaccharides extending from the surface of bacteria and other cells.
 Capsules and slime layers usually are composedof polysaccharides, but they may be constructed of
othermaterials. Capsules are not required for bacterial growth and reproduction in laboratory cultures;
they do confer several advantageswhen bacteria grow in their normal habitats.
 They helpbacteria resist phagocytosis by host phagocytic cells. Capsules contain a greatdeal of water
and can protect bacteria against desiccation. They exclude bacterial viruses and most hydrophobic toxic
materialssuch as detergents.

10 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

  The glycocalyx also aids bacterial attachmentto surfaces of solid objects in aquatic environments or to
tissuesurfaces in plant and animal hosts. Gliding bacteriaoften produce slime, which presumably aids in
their motility.
 Many gram-positive and gram-negative bacteria have a regularly structured layer called an S-layer on
their surface. The Slayerhas a pattern something like floor tiles and is composed ofprotein or
glycoprotein.
 In gram-negative bacteriathe S-layer adheres directly to the outer membrane; it is associatedwith the
peptidoglycan surface in gram-positive bacteria. Itmay protect the cell against ion and pH fluctuations,
osmoticstress, enzymes
 The S-layer also helps maintain the shape and enveloperigidity of at least some bacterial cells. It can
promote celladhesion to surfaces. Finally, the layer seems to protect somepathogens against
complement attack and phagocytosis, thuscontributing to their virulence.
Pili and Fimbriae
 Many gram-negative bacteria have short, fine, hairlike appendagesthat are thinner than flagella and not
involved in motility. These areusually called fimbriae (s., fimbria). They seem to beslender tubes
composed of helically arranged protein subunits andare about 3 to 10 nm in diameter and up to several
micrometerslong.
 Sex pili(s., pilus) are similar appendages, about 1 to 10 percell, that differ from fimbriae in the
following ways. Pili often arelarger than fimbriae (around 9 to 10 nm in diameter).
 They are geneticallydetermined by sex factors or conjugative plasmids andare required for bacterial
mating. Some bacterialviruses attach specifically to receptors on sex pili at the startof their reproductive
cycle.
Flagella and Motility
 Most motile bacteria move by use of flagella (s., flagellum),threadlike locomotor appendages
extending outward from theplasma membrane and cell wall. They are slender, rigid structures,about 20
nm across and up to 15 or 20 µm long.

11 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

Flagellar Ultrastructure
 Transmission electron microscope studies have shown that the bacterial flagellum is composed of three
parts. (1) The longest and most obvious portion is the filament, which extends from the cell surface to
the tip. (2) A basal body is embedded in the cell;and (3) a short, curved segment, the hook, links the
filament to itsbasal body and acts as a flexible coupling.
 The filament is a hollow,rigid cylinder constructed of a single protein called flagellin,which ranges in
molecular weight from 30,000 to 60,000. The filamentends with a capping protein. Some bacteria have
sheathssurrounding their flagella.
 The hook and basal body are quite different from the filament. Slightly wider than the filament, the
hook ismade of different protein subunits. The basal body is the mostcomplex part of a flagellum.
 In E.coli and most gram-negative bacteria, the body has four ringsconnected to a central rod. The outer
L and P rings associatewith the lipopolysaccharide and peptidoglycan layers, respectively.
 The inner M ring contacts the plasma membrane. Grampositive bacteria have only two basal body
rings, an inner ring connected to the plasma membrane and an outer one probablyattached to the
peptidoglycan.

12 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

Chemotaxis
 Bacteria do not always swim aimlessly but are attracted by suchnutrients as sugars and amino acids, and
are repelled by manyharmful substances and bacterial waste products. Bacteria alsocan respond to other
environmental cues such as temperature, light, and gravity.
 Movement toward chemical attractantsand away from repellents is known as chemotaxis. Such behavioris
of obvious advantage to bacteria.

The Bacterial Endospore

 A number of gram-positive bacteria can form a special resistant,dormant structure called an endospore.
Endospores developwithin vegetative bacterial cells of several genera: Bacillus andClostridium (rods),
Sporosarcina(cocci), and others. Thesestructures are extraordinarily resistant to environmental stressessuch
as heat, ultraviolet radiation, gamma radiation, chemical disinfectants,and desiccation.
 Because of their resistance andthe fact that several species of endospore-forming bacteria aredangerous
pathogens, endospores are of great practical importancein food, industrial, and medical microbiology. This
is becauseit is essential to be able to sterilize solutions and solid objects.
 The spore often is surrounded by a thin, delicatecovering called the exosporium.
 A spore coat lies beneath the exosporium,is composed of several protein layers, and may be fairlythick. It
is impermeable and responsible for the spore’s resistanceto chemicals.
 The cortex, which may occupy as much as half thespore volume, rests beneath the spore coat. It is made of
a peptidoglycanthat is less cross-linked than that in vegetative cells.
 Thespore cell wall (or core wall) is inside the cortex and surrounds theprotoplast or core. The core has the
normal cell structures such asribosomes and a nucleoid, but is metabolically inactive.

CLASSIFICATION

 Historically, prokaryotes were classified on their basis of phenotypic characteristics. Prokaryotic taxonomy
therefore involved measuring a large number of characteristics, including morphology and biochemical
characteristics (e.g. ability to grow on different substrates, cell wall structure, and antibiotic sensitivities).

13 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

 A major revolution occurred with the realization that evolutionary relationships could be deduced on the
basis of differences in gene sequence.
 The most important gene for Prokaryotic phylogeny is the 16s rRNA gene, which is present in all cells. The
gene is 1500bp in length and possesses signature sequences. Based on the RNA signature sequences, Carl
Woeseproposed a radical reorganization of the five kingdoms into three domains.
 In his classification system, Woese placed all four eukaryotic kingdoms into a single domain calledeukarya
also called Eukaryotes. He split the rest into Eubacteria and Archeae.

14 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

Culture media and types
 For any media to be propagated for a purpose it is necessary to provide the appropriate biochemical and
biophysical environment. A culture media is a solid or liquid medium that contain all the nutrients a
microorganism requires for growth.
 It is used to grow, transport and store microorganisms. Depending upon the special needs of particular
bacteria a large variety and types of culture media have been developed with different purpose and uses.
 Culture media are employed in the isolation and maintenance of pure cultures of bacteria and are also used
for identification of bacteria according to their biochemical and physiological properties.
 Although all microorganisms need sources of energy,carbon, nitrogen, phosphorus, sulfur, and various
minerals, theprecise composition of a satisfactory medium will depend onthe species one is trying to
cultivate because nutritional requirementsvary.
 Culture media can be classified on the basis of
1. physical nature (liquid, semi-solid and solid),
2. chemical composition (synthetic and complex) and
3. Functional type (supportive, enriched, selective and differential).

Minimal and supplementary media

 Minimal media are those that contain the minimum nutrients possible for growth of wild type organisms.
Minimal media typically contains: a carbon source, which may be sugar such as glucose, various inorganic
salts and water.
 Supplementary media are a type of minimal media that also contains a single selected agent, usually an
amino acid or sugar, for the culturing of specific auxotroph.

Synthetic and complex media

 A synthetic medium is a chemically defined medium in which the exact chemical composition is known.
Many chemoorganotrophicheterotrophs also can be grown in defined media withglucose as a carbon source
and an ammonium salt as a nitrogensource.
 A complex medium is undefined medium in which the exact chemical composition is unknown. Such
media are very useful, as a single complex medium may be sufficiently rich and complete tomeet the
nutritional requirements of many different microorganisms.
 Complex media contain undefined components like peptones,meat extract, and yeast extract. Peptones are
protein hydrolysatesprepared by partial proteolytic digestion of meat, casein, soyameal, gelatin, and other
protein sources.
 They serve as sources of carbon, energy, and nitrogen. Beef extract and yeast extract are aqueous extracts
of lean beef and brewer’s yeast, respectively. Beef extract contains amino acids, peptides, nucleotides,
organic acids, vitamins, and minerals.
15 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr
  Yeast extract is an excellent source of B vitamins as well as nitrogen and carbon compounds.
Threecommonly used complex media are (1) nutrient broth, (2) trypticsoy broth, and (3) MacConkey agar
Agar
 Agar is a sulfated polymercomposed mainly of D-galactose, 3,6-anhydro-L-galactose,and D-glucuronic
acid. It usually is extracted from red algae.
 Agar is well suited as a solidifying agent becauseafter it has been melted in boiling water, it can be cooled
toabout 40 to 42°C before hardening and will not melt again until thetemperature rises to about 80 to 90°C.
Agar is also an excellenthardening agent because most microorganisms cannot degrade it.
 Other solidifying agents are sometimes employed. For example,silica gel is used to grow autotrophic
bacteria on solid mediain the absence of organic substances and to determine carbonsources for
heterotrophic bacteria by supplementing the mediumwith various organic compounds.
Enriched media

 An enriched medium contains some component that permits the growth of specific types or species of
bacteria, usually because they alone can utilize the component from the environment.
 However an enrichment medium may have selective features. Blood agar is an example of enriched media
because it encourages the growth of many fastidious microbes.

Selective media

 A selective medium is one which has a component(s) added to it which will inhibit or prevent the growth
of certain types or species of bacteria and/or promote the growth of desired species. For example bile salts
and crystal violet favor the growth of gram negative bacteria by inhibiting the growth of gram positive
bacteria.
 Eosin methylene blue agar, MacConkey agar and mannitolsuagr agar are commonly used as selective
media. MacConkey agar also contains bile salts. A medium containingonly cellulose as a carbon and
energy source is quite effectivein the isolation of cellulose-digesting bacteria.
Differential media

 A culture medium is described as a differential medium if it allows the investigator to distinguish between
different types of bacteria based on some observable trait in their pattern of growth on the medium.
 Blood agar is an example of differential media and is used to distinguish between hemolytic and non-
hemolytic bacteria. Eosin methylene blue agar, MacConkey agar and mannitolsuagr agar are commonly
used as selective media.
 MacConkey agar is bothdifferential and selective. Since it contains lactose and neutral reddye, lactose-
fermenting colonies appear pink to red in color andare easily distinguished from colonies of nonfermenters.
Assay media

 Media of prescribed composition are used for the assay of vitamins, amino acids and antibiotics. Media of
special composition are also available for testing disinfectants.

Pure culture

 A pure culture is the one that contains only a single kind of microbial population grown from a single cell.
 A pure culture is usually derived from a mixed culture (containing many species) by methods that separate
the individual cells so that when they multiply, each will form a distinct colony, which may then be used to
establish new cultures with assurance that only one type of organism will be present.

16 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

  Pure cultures may be more easily isolated if the growth medium of the original mixed culture favors the
growth of one organism to the exclusion of others.
 There are several waysto prepare pure cultures; a few of them areSpread Plate and Streak Plate, Pour Plate
method. Etc

Sterilization and methods

 Heat and other physical agents are normally used to control microbialgrowth and sterilize objects, as can be
seen from the continualoperation of the autoclave in every microbiology laboratory. Thefour most
frequently employed physical agents are heat, low temperatures, filtration, and radiation.
HEAT
 Fire and boiling water have been used for sterilization and disinfection since the time of the Greeks, and
heating is still one of the most popular ways to destroy microorganisms. Either moist ordry heat may be
applied.
 Moist heat readily kills viruses, bacteria, and fungi. Exposure to boiling water for 10 minutes is sufficient
to destroy vegetative cells and eukaryotic spores. Unfortunately thetemperature of boiling water (100°C or
212°F) is not high enoughto destroy bacterial endospores that may survive hours of boiling.
 Therefore boiling can be used for disinfection of drinking waterand objects not harmed by water, but
boiling does not sterilize.Because heat is so useful in controlling microorganisms, it isessential to have a
precise measure of the heat-killing efficiency.
 Initially effectiveness was expressed in terms of thermal deathpoint (TDP), the lowest temperature at which
a microbial suspensionis killed in 10 minutes. Because TDP implies that a certaintemperature is
immediately lethal despite the conditions,thermal death time (TDT) is now more commonly used.
 This isthe shortest time needed to kill all organisms in a microbial suspensionat a specific temperature and
under defined conditions.
 However, such destruction is logarithmic, and it is theoreticallynot possible to “completely destroy”
microorganisms in a sample,even with extended heating. Therefore an even more precise figure,the
decimal reduction time (D) or D value has gained wideacceptance.
 The decimal reduction time is the time required tokill 90% of the microorganisms or spores in a sample at a
specifiedtemperature.
 Moist heat sterilization must be carried out at temperaturesabove 100°C in order to destroy bacterial
endospores, and this requiresthe use of saturated steam under pressure.
 Steam sterilization is carried out with an autoclave, a device somewhat like a fancy pressure cooker. The
development of the autoclave by Chamberland in 1884 tremendously stimulated the growth of
microbiology.
 Water is boiled to produce steam, which isreleased through the jacket and into the autoclave’s chamber.
Theair initially present in the chamber is forced out until the chamberis filled with saturated steam and the
outlets are closed. Hot, saturatedsteam continues to enter until the chamber reaches the desiredtemperature
and pressure, usually 121°C and 15 pounds of pressure.
 At this temperature saturated steam destroys all vegetativecells and endospores in a small volume of liquid
within 10 to 12minutes. Treatment is continued for about 15 minutes to provide amargin of safety.

17 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

  Moist heat is thought to kill so effectively by degrading nucleicacids and by denaturing enzymes and other
essential proteins.It also may disrupt cell membranes.
 Autoclaving must be carried out properly or the processedmaterials will not be sterile. If all air has not been
flushed out ofthe chamber, it will not reach 121°C even though it may reach a pressure of 15 pounds.
 The chamber should not be packed too tightly because the steam needs to circulate freely and
contacteverything in the autoclave. Bacterial endospores will be killedonly if they are kept at 121°C for 10
to 12 minutes.
 When a largevolume of liquid must be sterilized, an extended sterilization timewill be needed because it
will take longer for the center of the liquidto reach 121°C; 5 liters of liquid may require about 70 minutes.

Pasteurization
 Many substances, such as milk, are treated with controlledheating at temperatures well below boiling, a
process known as pasteurizationin honor of its developer Louis Pasteur.
 Pasteur examinedspoiled wine under the microscope and detected microorganismsthat looked like the
bacteria responsible for lactic acid and aceticacid fermentations.
 He then discovered that a brief heating at 55 to60°C would destroy these microorganisms and preserve
wine forlong periods.
 In 1886 the German chemists V. H. and F. Soxhletadapted the technique for preserving milk and reducing
milktransmissiblediseases.
 Pasteurization does not sterilize a beverage, but itdoes kill any pathogens present and drastically slows
spoilage by reducingthe level of nonpathogenic spoilage microorganisms.
 Milk can be pasteurized in two ways. In the older method themilk is held at 63°C for 30 minutes. Large
quantities of milk arenow usually subjected to flash pasteurization or high-temperatureshort-term (HTST)
pasteurization, which consists of quickheating to about 72°C for 15 seconds, then rapid cooling.
 Thedairy industry also sometimes uses ultrahigh-temperature(UHT) sterilization. Milk and milk
products are heated at 140 to150°C for 1 to 3 seconds.
 UHT-processed milk does not requirerefrigeration and can be stored at room temperature for about
2months without flavor changes. The small coffee creamer portionsprovided by restaurants often are
prepared using UHT sterilization.

Dry heat sterilization

 Many objects are best sterilized in the absence of water bydry heat sterilization. The items to be sterilized
are placed in anoven at 160 to 170°C for 2 to 3 hours. Microbial death apparentlyresults from the oxidation
of cell constituents and denaturation ofproteins.
 Dry heat does not corrode glasswareand metal instruments as moist heat does, and it can be used tosterilize
powders, oils, and similar items. Most laboratories sterilizeglass petri dishes and pipettes with dry heat.
Despite theseadvantages, dry heat sterilization is slow and not suitable for heatsensitivematerials like many
plastic and rubber items.
Incineration
 Destruction of microorganisms by burning is practiced in the lab when the transfer needle is introduced
into the flame of the Bunsen burner. When the transfer needle is sterilized care has to be exercised to
prevent splattering since the droplets which fly off are likely to carry viable organisms.
 Incineration is used for the destruction of carcasses, infected laboratory animals, and other infected
materials to be disposed of.

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LOW TEMPERATURES
 Although our emphasis is on the destruction of microorganisms,often the most convenient control
technique is to inhibit theirgrowth and reproduction by the use of either freezing or refrigeration.
 This approach is particularly important in food microbiology. Freezing items at -20°C or lower stops
microbial growth because of the low temperature and the absence of liquid water.
 Some microorganisms will be killed by ice crystaldisruption of cell membranes, but freezing does not
destroy contaminatingmicrobes.
 In fact, freezing is a very good method forlong-term storage of microbial samples when carried out
properly,and many laboratories have a low-temperature freezer forculture storage at -30 or -70°C.
 Because frozen food can containmany microorganisms, it should be prepared and consumedpromptly after
thawing in order to avoid spoilage and pathogengrowth.
 Refrigeration greatly slows microbial growth and reproduction,but does not halt it completely. Fortunately
most pathogensare mesophilic and do not grow well at temperatures around 4°C.Refrigerated items may be
ruined by growth of psychrophilic andpsychrotrophic microorganisms, particularly if water is present.
 Thus refrigeration is a good technique only for shorter-term storageof food and other items.

Filtration
 Filtration is an excellent way to reduce the microbial population insolutions of heat-sensitive material, and
sometimes it can be usedto sterilize solutions. Rather than directly destroying
contaminatingmicroorganisms, the filter simply removes them.
 There are twotypes of filters. Depth filters consist of fibrous or granular materialsthat have been bonded
into a thick layer filled with twistingchannels of small diameter. The solution containing microorganismsis
sucked through this layer under vacuum, and microbialcells are removed by physical screening or
entrapment and also byadsorption to the surface of the filter material.
 Depth filters aremade of diatomaceous earth (Berkefield filters), unglazed porcelain(Chamberlain filters),
asbestos, or other similar materials.
 Membrane filters have replaced depth filters for many purposes.These circular filters are porous
membranes, a little over0.1 mm thick, made of cellulose acetate, cellulose nitrate,
polycarbonate,polyvinylidene fluoride, or other synthetic materials.
 Although a wide variety of pore sizes are available, membraneswith pores about 0.2 µm in diameter are
used to remove most vegetativecells, but not viruses, from solutions ranging in volumefrom 1 ml to many
liters.
 The membranes are held in special holdersand often preceded by depth filters made of glassfibers to
remove larger particles that might clog the membrane filter.
 These filters are used to sterilize pharmaceuticals, ophthalmic solutions,culture media, oils, antibiotics, and
other heat-sensitive solutions.
 Air also can be sterilized by filtration. Two common examplesare surgical masks and cotton plugs on
culture vessels that let air in but keep microorganisms out.
 Laminar flow biological safety cabinetsemploying high-efficiency particulate air (HEPA) filters,which
remove 99.97% of 0.3 µm particles, are one of the most importantair filtration systems.
 This protects a worker from microorganismsbeing handled within the cabinet and prevents contaminationof
the room. A person uses these cabinets whenworking with dangerous agents such as Mycobacterium
tuberculosis tumor viruses, and recombinant DNA.
 They are also employed in researchlabs and industries, such as the pharmaceutical industry, whena sterile
working surface is needed for conducting assays, preparingmedia, examining tissue cultures, and the like.

19 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

Radiation

 Ultraviolet (UV) radiation around 260 nm is quite lethal but does not penetrate glass, dirt films, water,
andother substances very effectively. Because of this disadvantage,UVradiation is used as a sterilizing
agent only in a few specific situations.
 UV lamps are sometimes placed on the ceilings of rooms orin biological safety cabinets to sterilize the air
and any exposed surfaces.
 Because UV radiation burns the skin and damages eyes, people working in such areas must be certain the
UV lamps are off when the areas are in use. Commercial UV units are available for water treatment.
Pathogens and other microorganisms are destroyed when a thin layer of water is passed under the lamps.
 Ionizing radiation is an excellent sterilizing agent and penetratesdeep into objects. It will destroy bacterial
endospores andvegetative cells, both procaryotic and eucaryotic; however, ionizingradiation is not always
as effective against viruses.
 Gamma radiationfrom a cobalt 60 source is used in the cold sterilization ofantibiotics, hormones, sutures,
and plastic disposable supplies such as syringes. Gamma radiation has also been used to sterilize
and“pasteurize” meat and other food.
 Both the Food and Drug Administration and the World Health Organization have approved food irradiation
and declared it safe. The U.S. government currently approves the use of radiation to treat poultry, beef,
pork, veal, lamb, fruits, vegetables, and spices.

Growth of bacterial cells

 Growth may be defined as an increase in cellular constituents. Itleads to a rise in cell number when
microorganisms reproduce byprocesses like budding or binary fission. In the latter, individual cellsenlarge
and divide to yield two progeny of approximately equal size.
 Growth also results when cells simply become longer or larger. If themicroorganism is coenocytic—that is,
a multinucleate organism inwhich nuclear divisions are not accompanied by cell divisionsgrowth results in
an increase in cell size but not cell number.
 It is usuallynot convenient to investigate the growth and reproduction of individualmicroorganisms because
of their small size. Therefore,when studying growth, microbiologists normally follow changes inthe total
population number.
The Growth Curve
 Population growth is studied by analyzing the growth curve of amicrobial culture. When microorganisms
are cultivated in liquidmedium, they usually are grown in a batch culture or closed system—that is, they
are incubated in a closed culture vessel with asingle batch of medium.
 Because no fresh medium is providedduring incubation, nutrient concentrations decline and
concentrationsof wastes increase. The growth of microorganisms reproducingby binary fission can be
plotted as the logarithm of thenumber of viable cells versus the incubation time. The resultingcurve has
four distinct phases:

20 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

Lag Phase
 When microorganisms are introduced into fresh culture medium,usually no immediate increase in cell
number occurs, and thereforethis period is called the lag phase.
 The cells which may be old and depleted of ATP, essential cofactors,and ribosomes; these must be
synthesized before growth canbegin. The medium may be different from the one the microorganismwas
growing in previously. Here new enzymes would beneeded to use different nutrients.
 Possibly the microorganismshave been injured and require time to recover. Whatever thecauses, eventually
the cells retool, replicate their DNA, begin toincrease in mass, and finally divide.
 The lag phase varies considerably in length with the conditionof the microorganisms and the nature of the
medium. Thisphase may be quite long if the inoculum is from an old culture orone that has been
refrigerated. Inoculation of a culture into achemically different medium also results in a longer lag phase.
 On the other hand, when a young, vigorously growing exponentialphase culture is transferred to fresh
medium of the same composition, the lag phase will be short or absent.
Exponential Phase
 During the exponential or log phase, microorganisms aregrowing and dividing at the maximal rate
possible given theirgenetic potential, the nature of the medium, and the conditionsunder which they are
growing.
 Their rate of growth is constantduring the exponential phase; that is, the microorganisms aredividing and
doubling in number at regular intervals. The population is most uniform in terms of chemicaland
physiological properties during this phase; therefore exponentialphase cultures are usually used in
biochemical and physiologicalstudies.
 Exponential growth is balanced growth. That is, all cellularconstituents are manufactured at constant rates
relative to eachother. If nutrient levels or other environmental conditions change,unbalanced growth
results.
 Unbalanced growth also results when a bacterial populationis shifted down from a rich medium to a poor
one. The organismsmay previously have been able to obtain many cell componentsdirectly from the
medium.
 The rate of growth also increaseswith nutrient concentration. At sufficiently high nutrient levelsthe
transport systems are saturated, and the growth rate doesnot rise further with increasing nutrient
concentration

21 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

Stationary Phase

 In the stationary phase the total number of viablemicroorganisms remains constant. This may result from a
balancebetween cell division and cell death, or the population maysimply cease to divide though remaining
metabolically active.
 Microbial populations enter the stationary phase for severalreasons. One obvious factor is nutrient
limitation; if an essentialnutrient is severely depleted, population growth will slow. Aerobicorganisms often
are limited by O2 availability.
 The cells beneath the surface will not be able to grow unless theculture is shaken or aerated in another way.
 Population growthalso may cease due to the accumulation of toxic waste products.This factor seems to
limit the growth of many anaerobic cultures(cultures growing in the absence of O2).
 Accumulation of inhibitory metabolites or end products can be one of the factors or exhaustion of space in
this case called lack of biological space.
Death Phase
 Detrimental environmental changes like nutrient deprivation andthe buildup of toxic wastes lead to the
decline in the number ofviable cells characteristic of the death phase.
 The death of a microbialpopulation, like its growth during the exponential phase,is usually logarithmic
(that is, a constant proportion of cells dieevery hour). This pattern in viable cell count holds even when
thetotal cell number remains constant because the cells simply fail tolyse after dying.
 Often the only way of deciding whether a bacterialcell is viable is by incubating it in fresh medium; if it
does notgrow and reproduce, it is assumed to be dead. That is, death is definedto be the irreversible loss of
the ability to reproduce.
 Although most of a microbial population usually dies in alogarithmic fashion, the death rate may decrease
after the populationhas been drastically reduced. This is due to the extendedsurvival of particularly resistant
cells. For this and other reasons,the death phase curve may be complex.

STRUCTURE OF A TYPICAL VIRUS

 Virus morphology has been intensely studied over the pastdecades because of the importance of viruses
and the realizationthat virus structure was simple enough to be understood. Progresshas come from the use
of several different techniques: electron microscopy,X-ray diffraction, biochemical analysis, and
immunology.

22 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

Virion Size
 Virions range in size from about 10 to 300 or 400 nm in diameter. The smallest viruses are a little larger
than ribosomes,whereas the poxviruses, like vaccinia, are about the samesize as the smallest bacteria and
can be seen in the light microscope.
 Most viruses, however, are too small to be visible in thelight microscope and must be viewed with the
scanning and transmissionelectron microscopes
General Structural Properties
 All virions, even if they possess other constituents, are constructedaround a nucleocapsidcore (indeed,
some viruses consistonly of a nucleocapsid). The nucleocapsid is composed of anucleic acid, either DNA
or RNA, held within a protein coatcalled the capsid, which protects viral genetic material and aidsin its
transfer between host cells.
 There are four general morphological types of capsids andvirion structure.
1. Some capsids are icosahedral in shape. An icosahedron is aregular polyhedron with 20 equilateral
triangular faces and12 vertices. These capsids appear sphericalwhen viewed at low power in the electron
microscope.
2. Other capsids are helical and shaped like hollow proteincylinders, which may be either rigid or flexible.
3. Many viruses have an envelope, an outer membranouslayer surrounding the nucleocapsid. Enveloped
viruses havea roughly spherical but somewhat variable shape eventhough their nucleocapsid can be either
icosahedral orhelical.
4. Complex viruses have capsid symmetry that is neither purelyicosahedral nor helical. They may possess
tails and other structures (e.g., many bacteriophages)or have complex, multilayered walls surrounding the
nucleicacid (e.g., poxviruses such as vaccinia).
 Both helical and icosahedral capsids are large macromolecularstructures constructed from many copies of
one or a fewtypes of protein subunits or protomers.
 For example, the tobacco mosaic virus (TMV) capsid contains asingle type of small subunit possessing 158
amino acids. Onlyabout 474 nucleotides out of 6,000 in the virus RNA are requiredto code for coat protein
amino acids.
 The capsid is constructed withoutany outside aid, the process is called self-assembly. Some more complex
viruses possess genes for special factorsthat are not incorporated into the virion but are required for
itsassembly.
Helical Capsids
 Helical capsids are shaped much like hollow tubes with proteinwalls. The tobacco mosaic virus provides a
well-studied exampleof helical capsid structure. A single type of protomerassociates together in a helical or
spiral arrangement to produce along, rigid tube, 15 to 18 nm in diameter by 300 nm long.
 The RNAgenetic material is wound in a spiral and positioned toward the insideof the capsid where it lies
within a groove formed by the proteinsubunits. Not all helical capsids are as rigid as the TMV capsid.
 Influenza virus RNAs are enclosed in thin, flexible helicalcapsids folded within an envelope.

23 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

  The diameterof the capsid is a function of the size, shape, and interactionsof the protomers. The nucleic
acid determines helical capsidlength because the capsid does not seem to extend much beyondthe end of
the DNA or RNA.
Icosahedral Capsids
 The icosahedron is one of nature’s favorite shapes (the helix isprobably most popular). Viruses employ the
icosahedral shapebecause it is the most efficient way to enclose a space.
 Certain requirementsmust be met to construct an icosahedron. Hexagonspack together in planes and cannot
enclose a space, and thereforepentagons must also be used.
 When icosahedral viruses are negatively stained and viewedin the transmission electron microscope, a
complex icosahedralcapsid structure is revealed.
 The capsids are constructedfrom ring- or knob-shaped units called capsomers, eachusually made of five or
six protomers.
 Pentamers (pentons) havefive subunits; hexamers (hexons) possess six. Pentamers are atthe vertices of
the icosahedron, whereas hexamers form its edgesand triangular faces.
 The icosahedrons is constructed of 42 capsomers; larger icosahedral are madeif more hexamers.
 In many plant and bacterial RNA viruses, both the pentamersand hexamers of a capsid are constructed with
only onetype of subunit, whereas adenovirus pentamers are composed ofdifferent proteins than are
adenovirus hexamers.
 Protomers join to form capsomers through noncovalentbonding. The bonds between proteins within
pentamers andhexamers are stronger than those between separate capsomers.Empty capsids can even
dissociate into separate capsomers.
Nucleic Acids
 Viruses are exceptionally flexible with respect to the nature of theirgenetic material. They employ all four
possible nucleic acid types:single-stranded DNA, double-stranded DNA, single-stranded RNA,and double-
stranded RNA.
 All four types are found in animal viruses.Plant viruses most often have single-stranded RNA genomes.
Althoughphages may have single-stranded DNA or single-strandedRNA, bacterial viruses usually contain
double-stranded DNA. Most RNA viruses employ single-stranded RNA (ssRNA) as their genetic material.
 The RNA base sequence may be identicalwith that of viral mRNA, in which case the RNA strand is
calledthe plus strand or positive strand (viral mRNA is defined asplus or positive). However, the viral
RNA genome may instead becomplementary to viral mRNA, and then it is called a minus ornegative
strand.
 Polio, tobacco mosaic, brome mosaic, and Roussarcoma viruses are all positive strand RNA viruses;
rabies,mumps, measles, and influenza viruses are examples of negativestrand RNA viruses. Many of these
RNA genomes are segmentedgenomes—that is, they are divided into separate parts.
Viral Envelopes and Enzymes
 Many animal viruses, some plant viruses, and at least one bacterialvirus are bounded by an outer
membranous layer called an envelope. Animal virus envelopes usually arise fromhost cell nuclear or
plasma membranes; their lipids and carbohydratesare normal host constituents.
 In contrast, envelope proteinsare coded for by virus genes and may even project from the envelopesurface
as spikes or peplomers.
 Thesespikes may be involved in virus attachment to the host cell surface.Since they differ among viruses,
they also can be used toidentify some viruses.
 Influenza virus is a well-studied exampleof an enveloped virus. Spikes project about 10 nm from the
surfaceat 7 to 8 nm intervals.

24 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr

 Some spikes possess the enzyme neuraminidase,which may aid the virus in penetrating mucous layersof
the respiratory epithelium to reach host cells. Other spikeshave hemagglutinin proteins, so named because
they can bind thevirions to red blood cell membranes and cause hemagglutination
 Hemagglutinins participate in virion attachmentto host cells. Proteins, like the spike proteins that are
exposed on the outer envelope surface, are generally glycoproteins that is, the proteins have carbohydrate
attached to them. A nonglycosylatedprotein, the M or matrix protein is found on the innersurface of the
envelope and helps stabilize it.

Diagram of the influenza virion.

TMV

Vaccinia Virus Morphology.

The structure of the T4 bacteriophage.

25 Departments of Biochemistry, S.P. College / Women’s College M.A. Road Sgr