MICROBIOLOGY NOTES
Bacteriology
Fundamental Unit of Life:
A cell is isolated by a cell membrane and perhaps a cell wall. Within this unit is a
variety of chemical and sub-cellular structures. The key to the functioning of any cell
is the genetic information and macromolecules it contains. Cells are dynamic and
have open systems. It is estimated that they first evolved about 3.8 billion years ago.
All cells are descendants from this universal ancestor.
All/most cells have the following:
- Compartmentalization and metabolism.
- Reproduction (growth)
- Differentiation
- Communication
- Movement
- Evolution
COMPARTMENTALIZATION AND METABOLISM:
Cells take up nutrients from the environment, transform
them and then release wastes back into the environment.
Thus, the cell is an open system.
REPRODUCTION (GROWTH):
Chemicals from the environment are turned into
new cells under the genetic direction of pre-
existing cells.
DIFFERENTIATION
Some cells can form new cell structures such as a
spore, usually as part of a cellular life cycle.
COMMUNICATION
Cells communicate or interact by means of chemicals that are released or taken
up.
MOVEMENT
Some cells are capable of self-propulsion.
EVOLUTION
Cells contain genes and evolve to display new biological properties.
Phylogenetic trees show the evolutionary relationship between cells.
PROKARYOTES VS EUKARYOTES
Prokaryotes comprise Archae and Bacteria.
Eukaryotes comprise Fungi, Algae, Protozoa, Plants and Animals.
Characterist Prokaryotic Eukaryotic
ics
Size Typically, 1-5 µm Typically, 10-100 µm
Genetic Free in cytoplasm (no true Contained within membrane bound
Material nucleus) nucleus
Singular circular Multiple chromosomes generally in
chromosome or nucleoid pairs
Histones absent DNA complexed with histone
proteins
Internal Membrane bound organelles Several membrane bound
features absent organelles present
Ribosomes smaller (70S) free Ribosomes larger (80S) free in the
in the cytoplasm cytoplasm or attached to
Respiratory enzymes bound to membrane
plasma membrane Respiratory enzymes located in
mitochondria
Cell wall Usually based on When present based on cellulose
peptidoglycan or chitin
External Cilia absent Cilia may be present
features Flagella if present, composed Flagella, if present, have complex
of flagellin; provide rotating (9+2) structure; provide whiplash
motility motility
Pilli absent
Pilli may be present Pellicle present in some types.
Outside layer (slime layer,
capsule, glycocalyx) present in
some
Bacteriology
Bacteriology is concerned with prokaryotic diversity. Morphological features are
limited so the metabolic properties, pathogenicity, nutritional requirements,
staining reactions and antigenic properties are studied. Regulated by Bergey’s
Manual of Systematic Bacteriology which is based on phenotypic characteristic
combined with genotypic characteristics (16S ribosomal RNA genes are
conserved).
CELL MORPHOLOGY (SHAPE)
Coccus (round)
Bacillus (rod)
Spirillum
Spirochete
Budding and appendaged
Filamentous
Bacteria are unicellular but after cell division some cells may not separate
forming chains, sheets, cube-like clusters etc. (irregular
aggregates). Unusual shapes include spirochetes
(tightly coiled), appendage (extensions of tubes or
stalks) and filamentous (thin cells or chains of cells).
CAPSULE:
The capsule is an exopolymer secrete by the cell that
surrounds and adheres to the cell wall. Not all cells
have a capsule and it is generally associated with
virulence as it protects cells from phagocytosis. Other
functions of the capsule are protection against
antimicrobial compounds and desiccation, attachment of the bacteria to
surfaces and contributes to biofilms.
A biofilm is a slime that can be significant in waste water treatment, industrial
water-cooling systems where it may result in corrosion, insert catheters and
inside your mouth. Slime is also produced during spoilage of food products.
You can stain for the capsule using crystal violet and decolorize with copper
sulphate.
PLASMA MEMBRANE:
Majority of bacterial membranes do not contain sterols but may contain
hopanoids (derived from the same precursors), these molecules assist in
maintaining membrane stability. Archaea and bacteria have different lipid
components.
CELL WALL:
Cells wall gives shape and rigidity to cells and protects them against osmotic
shock. Bacteria live in hypotonic environments, which is more dilute than
cytoplasm thus, allowing water to flow into the cell. Without a cell wall
(protoplast) the cell would burst.
The main component of
the cell wall is
peptidoglycan.
Alternating and repeating
units NAG-NAM-NAG-NAM
are held together by beta
1,4 glycoside bonds.
Precursor molecules of peptidoglycan are synthesized inside the cell and
transported across the plasma membrane by a carrier protein (bactoprenol
phosphate) before being incorporated into the cell wall. The enzyme
transpeptidases covalently bonds tetrapeptide chains to one another, giving rise
to a cross-linked complex network.
Some antimicrobial agents inhibit cell wall synthesis. Penicillin and
cephalosporins block the formation of peptide cross linkages.
Absent in walls of Archaea which may have pseudomurein-N-acetylglucosamine
and N-acetyltalosaminuronic acid (replaces NAM)
TYPES OF CELL WALLS:
Two structural types of
cell walls: Gram positive
and Gram negative. Gram
positive cell walls are a
single layer and much
thicker; gram negative
cell walls are multilayered
and complexed. Can distinguish between them by using gram staining. Gram
staining is a differential staining technique developed by Christian Gram in
1880s.
Graim staining:
Step 1: flood the heat-fixed smear with crystal violet for 1 min. All cells are
purple.
Step 2: Add iodine solution for 3 min. All cells remain purple.
Step 3: decolorize with alcohol briefly (about 20 sec). Gram-positive cells are
purple; gram-negative cells are colourless.
Step 4: counterstain with safranin for 1-2 min. Gram-positive cells are purple;
gram-negative cells are pink to red.
GRAM POSITIVE CELL WALLS:
Teichoic acids may bind and regulate movement of cations (Mg 2+ and Ca2+) into
and out of the cell; prevent extensive wall breakdown and possible lysis during
cell growth; provide much of the cell wall’s antigenicity.
Lipoteichoic acids are teichoic acids found in association with glycolipids.
GRAM NEGATIVE CELL WALLS:
Outer membrane:
Different to the inner cell plasma membrane in that it contains lipoproteins,
phospholipids and lipopolysaccharides. It surround a thin, sometimes single
layer, of peptidoglycan. Outer membrane protects cells from phagocytosis,
lysozyme and other chemicals.
The outer membrane has one LPS layer with 3 parts.
1. Lipid A: may act as an endotoxin which if released into the bloodstream
causes fever and toxic shock. Not a typical glycerol but is connected to the
amine group of through a core polysaccharide – ketodeoxyoctonate.
2. Core polysaccharides: attached to lipid A.
3. O-polysaccharide specific side chain: attached to the core polysaccharides.
They are antigens which are carbohydrate chains which vary between strains
of the same bacteria.
Proteins called porins penetrate the entire thickness of the outer membrane to
allow for the passage of water and small molecules into the cell. The outer
membrane protects that cell from antibiotics and lysozyme (enzyme) which
reacts with peptidoglycans.
In gram-negative bacteria peptidoglycan comprises 10-20% of the cell wall.
Gram negative bacteria have a periplasmic space between the plasma
membrane and the cell wall. It is the site of the metabolic activity and it
contains a number of enzymes (distinct from cytoplasm) and transports
proteins. Periplasmic enzymes have several functions, such as detecting
nutrients in the environment, degradation of polymers and protection from
harmful compounds.
Features Gram-Positive Gram-Negative
Gram reaction Retains crystal violet dye Can be decolorized to
and stains blue or purple accept counterstain
(safranin) and stain
red/pink
Peptidoglycan layer Thick (multilayered) Thing (single-layered)
Teichoic acids Present in many Absent
Periplasmic Space Absent Present
Outer membrane Absent Present
Lipopolysaccharide (LPS) Virtually none High
Content
Lipid and Lipoprotein Low (acid-test bacteria High (because of presence
Content have lipids linked to of outer membrane)
peptidoglycan)
Flagellar structure 2 rings in the basal body 4 rings in basal body
Toxins produced Exotoxins Endotoxins and exotoxins
Resistance to Physical High Low
Digestion
Cell wall disruption by High Low (required
Lysozyme pretreatment to
destabilize outer
membrane)
Susceptibility to Penicillin High Low
and Sulfonamide
Susceptibility to Low High
Streptomycin,
Chloramphenicol and
Tetracycline
Inhibition by Basic Dyes High Low
Susceptibility to Anionic High Low
Detergents
Resistance to Sodium High Low
Azide
Resistance to drying High low
THIRD TYPE OF CELL WALL:
Acid fast: the acid-fast stain retains carbol fuchsin and appears red. Cannot be
stained by gram staining. An example of this bacteria is Mycobacterium tubercolosis.
Mycolic acids make up 60% of the acid- fast cell wall. It is resistant to chemical agents
and phagocytes. The organisms grow slowly.
Some bacteria do not have a cell wall at all. Examples include mycoplasmas
(Mycoplasma pneumoniae); chlamydias (most common bacterial STI) and
rickettsias (tick bite fever).
OTHER CELL SURFACE STRUCTURES:
Fimbriae are filamentous proteins that extend from the
cell surface, they enable bacteria to stick to surfaces.
They also form thin sheets of cells on liquid surface
and biofilms on surfaces.
Pili (pilus) are longer structures and generally cells only
have one or few pili. Pathogenic bacteria have proteins
called adhesions on their pili (fimbriae
for attachment) which adhere to
specific receptors on host tissue. They
are receptors for viruses and facilitate
genetic exchange between bacteria
during conjugation.
FLAGELLA
Some bacteria are motile due to
structures called flagella. Motile
bacteria have one or more
flagella, which can be used as a
diagnostic feature. The position of
the flagella can be used to
distinguish certain bacteria.
Monotrichous – a single flagellum
from one pole
Amphitrichous – a single
flagellum at each pole
Lophotrichous- two or more flagella located at one or both poles
Peritrichous – flagella surround the entire bacterial cell.
Flagella are composed of a protein subunit called flagellin. The basal body is the
portion of the flagellum that is embedded into the membrane, it is surrounded
by two pairs of rings.
Gram-negative bacteria have an outer ring which is associated with the outer
membrane (L ring), the peptidoglycan layer (P ring) and an inner pair of rings
(MS and C rings) which are located within or just above the plasma membrane.
Gram positive bacteria (lack the outer membrane) only have the inner pair of
rings (MA and C rings) present.
Flagella are
rigid
structures
which do not
flex but they
rotate, this
movement is
driven by the
basal body.
Surrounding
the inner
rings and
anchored to the plasma membrane are Mot proteins which drive the motor as
well as Fli proteins which function as a motor switch reversing the rotation of
the flagella.
The motor consists of a central rod that passes through the rings. The rings are
thought to act together to generate rotational movement. The driving force
comes from the proton motive force, which is the dissipation of the proton
gradient releasing energy which causes the rotation. The wider base of the
flagella is called the hook, it consists
of a single type of protein and
functions to connect the filament to
the motor portion of the flagellum.
The direction of rotation determine
the type of movement, cells with
single flagellum move forward during
counterclockwise rotation (pushing)
and move backwards (pulling) with
clockwise rotation. Flagella of
peritrichous organisms behave as a
single bundle during
counterclockwise rotation and thus
move forward. During clockwise
rotation the flagella act
independently, and the organism tumbles.
DNA
DNA consists of single chromosomes which vary in sizes between different
species. It is circular, tightly supercoiled and associated with bacterial proteins
and it is found in the discreet area called the nucleoid. Many bacteria contain
small molecules of extra-chromosomal DNA called plasmids. These plasmids
carry non-essential genes and confer some advantage such as antibiotic
resistance, they are self-replicating.
RIBOSOMES
Cells contain many thousand 70S ribosomes. They are composed of protein and
RNA and are the site of protein synthesis. They comprise of two unequal
subunits of 50S and 30S. the nucleotide sequence of the small subunit (16S)
rRNA is widely used in determining phylogenetic relationships between bacteria
INCLUSION BODIES
There are several granular structures that are used for storage. Organic
inclusions are lipid droplets, glycogen, polyhydroxybuturate, and starch which
serves as C and as an energy source; polyphosphate granules (volutin or
metachromatic granules); Sulphur granules found in purple photosynthetic
bacteria; gas vacuole (cyanobacteria and other photosynthetic bacteria that live
in aqueous environments) provides buoyancy; and magnetosomes which
contain a form of iron oxide that orientate bacteria downwards.
CELL DIVISION
Cell division is growth which leads
to an increase in the number of
cells. When bacteria are inoculated
into a media containing all the
essential nutrients, the bacteria
will accumulate the nutrients and
use them to synthesize new cell
wall materials and then divide into
two. This is cell division by binary
fission. The generation time is the
time required for one bacterial cell
to become 2 cells.
Cells grow until they reach a
critical size. During this time a
copy of each chromosome is made
(DNA replication), this occurs during the C phase (chromosomal replication). Cell
division is initiated by the formation of a contractile ring or divisome (anchors
the chromosomes to the plasma membrane). It triggers an inwards growth of
the plasma membrane and cell wall to form a partition or septum which will
eventually separate the two daughter cells. The segregation of the chromosome
occurs during the G (gap) phase. The cell finally divides into 2 cells during the D
phase and one copy of the genetic material is present in each daughter cell.
ENDOSPORES
Some gram-positive bacteria produce endospores (Bacillus & Clostridium).
Spores are survival structures
that enable the bacteria to
withstand adverse
environmental conditions.
Spores can survive UV
radiation, desiccation, most
disinfectants, antibiotics and
heat. They are sometime
responsible for food spoilage
because they are heat
resistant (e.g. C. botulinium).
One endospore is produced
per vegetative cell; once
produced the vegetative cell disintegrates thus releasing the endospore.
Step 1: DNA
replication and
cytoplasmic
membrane septum
forms around one
end of the cell.
Step 2: second layer
of cytoplasmic
membrane forms
around one of the
DNA molecules
(forespore).
Step 3: synthesis of
peptidoglycan in
space between two
membranes
providing protective coat (cortex).
Step 4: calcium dipicolinates incorporated into endospore.
Step 5: spore coat formed around cortex, contains keratin-like protein.
Sometime an outer membrane of lipid and protein is formed (exosporium).
Step 6: vegetative cell degrades, and endospore is releases
(takes about 15 hours)
GLIDING MOTILITY
Many bacteria move without flagella, this is known as gliding; it is a slower,
smoother movement than ‘swimming’. Gliding requires contact with solid
surfaces. Cyanobacteria are the most well-known gliding bacteria.
More than one method results in gliding: the cells secrete a polysaccharide
slime on the outer surface, the movement is driven by type IV pili or the
movement of the cell surface proteins move the cell forward using a ratchetting
mechanism.
The slime connects the cell to the surface against which the bacteria moves. As
the slime adheres to the surface, the bacteria are pulled along.
CHEMOTAXIS
Chemotaxis is the movement of an organism towards or away from a chemical.
It is the response bacteria have to chemicals in the environment and it can be
positive or negative. Positive chemotaxis is when the bacteria move towards an
attractant or substrate and negative chemotaxis is when bacteria move away
from a repellent. For bacteria to undergo chemotaxis, sensory systems are
required in the cells.
Prokaryotic movement can be divided into runs, where the organism moves in
one direction, or twiddles where it randomly tumbles. There is no gradient of
either attractant or repellent and the organisms moves in random ways with
many twiddles. Gradient means the runs become longer with less frequent
twiddles as the concentration increases.
CHEMORECEPTORS
The link between flagellar movement and concentration of attractant or
repellent are the proteins located in the periplasm. These are chemoreceptors
that can be specific for the compounds that they combine with. However,
galactose receptors also recognise glucose and fructose and mannose receptors
also recognise glucose. Methyl-accepting chemotaxis proteins (MCPs) are
involved in translating signals from chemoreceptors to the flagella motor. Their
transducers become methylated or demethylated depending on detection of the
attractant.
E.coli
MCP proteins – 4 types (I,II,III,IV)
- Each respond to a different attractant or repellent.
Following the sensory recognition of an attractant, MCPs become increasingly
methylated causing an excitatory signal to the flagellar motor (rotation). If counter
clockwise movement occurs, the cell continuous to move and will continue into a
‘run’. If chemoreceptors no longer sense an increasing gradient it triggers a
process called adaptation. This results in the complete methylation of the MCPs,
which decreases the sensitivity of the compound and caused a clockwise rotation;
the cell starts to twiddle.
Demethylation returns the MCPs to a state in which it can excite the flagella motor
once more.
In the presence of a repellent, increasing concentration results in increasing
demethylation; causing it to go the other way.
Phototaxis
Phototaxis is the movement towards light rather than chemical concentrations.
There are 2 main types amongst phototrophic bacteria, namely Scotophobotaxis &
true phototaxis.
Scotophobotaxis is only observed under the microscope. When bacteria leave the
field of view, they receive a signal to reverse the flagella and re-enter the light.
True phototaxis is like chemotaxis but it is stimulated by light.
Other Taxa
Aerotaxis movement towards or away from oxygen
Osmotaxis movement away or towards conditions of high ionic strength
Hydrotaxis undertaken by gliding cyanobacteria which move towards increasing
gradients of hydration.
