Download Lecture 10: Introduction to Bacteria (Structure, Growth

Lecture 10: Introduction to Bacteria (Structure, Growth & Physiology)
Study Objectives
•Explain bacterial classification schemes and nomenclature.
•Describe the structural components of bacteria.
•Explain cellular and colony morphologies, Gram staining, motility and spore formation.
•Give examples of encapsulated microorganisms and discuss their importance to human health.
•Describe various structures of the bacterial cell (including the cellular membrane, cell wall, internal and
external structures) and how they affect growth, identification and virulence of these organisms.
•Describe the metabolic requirements for energy and biosynthesis in bacteria.
•Explain the diversity of bacterial metabolism and how metabolic properties of bacteria have impacted
classification/identification.
•Describe bacterial growth (growth curves, dynamics and sporulation).
•Describe the various effects of oxygen, temperature, pH on bacterial growth.
•Define aerobes, obligate anaerobes and facultative anaerobes.
Feature
Prokaryotes
Eukaryotes
Internal
Structure
Simple
Complex
Nucleus
No membrane or
nucleoli
True nucleus:
membrane bound and
contains nucleoli
Organelles
(membrane
bound)
No
Yes
Cell wall
Usually present.
Chemically
complex
If present, chemically
simple.
Filamentous
cytoskeleton
No
Yes
Cell division
Binary fission
Mitosis and meiosis
mRNA
processing
Little
Extensive
Ribosomes
70S
80S (70S in
mitochondria and
chloroplasts)
DNA
(chromosomes)
Usually single.
Circular. Naked.
Multiple. Linear.
Histones.
Division rate
20-30 minutes
10-24 hours
Bacteria (Prokaryotes)Features
•Anucleated; nucleic acid intermingles with cytoplasm – not membrane bound
•Lack organelles (membrane-bound compartments)
Classification & Nomenclature
•Medically important bacteria only account for a fraction of total
bacteria
•Binomial = Genus and Species
•Names should be italicized or underlined
•Genus capitalized and species name not capitalized
•Can be abbreviate Genus name but not species name
•Correct: Bacillus anthracis, B. anthracis
•Incorrect: Bacillus Anthracis, Bacillus Anthracis, Bacillus a.
Bacterial Cell Structures




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Pilli- for attachment and
locomotion
Nucleoid- contains DNA
or RNA
Bacterial cells are
constituted by a
cytoplasmic mass where
the nucleic acid,
ribosomes, storage
granules, and other
cellular components are
found and a protective
cell envelope of variable
complexity.
The bacterial cell envelope consists of a cell wall and an underlying cytoplasmic membrane.
The rigid cell wall which provides protection and imparts shape to most bacterial cells is entirely
absent in a few unusual bacteria (ex mycoplasmas).

o Peptidoglycan (murein) is the principle structural component of the cell wall. This compound
is found in both Gram-positive and Gram-negative organisms although it is more abundant in
Gram-positive bacteria.
Plasma Membrane is selectively permeable phospholipid bi-layer,
Bacterial Cell Walls (Gram +: purple; note techoic and lipotechoic acid; Gram -: pink; note more
polysaccharides
Peptidoglycan Structure & Synthesis



Peptidoglycan polymers consist of repeating
disaccharides formed by Nacetylglucosamine(NAG) and N-acetylmuramic
acid(NAM).
Makes a lattice structure
NAM and NAG associate in the cytoplasm and then end up in the outer membrane
o It does this by attaching to a Bactoprenol which flips the NAM+NAG into the outer membrane
o These are things that drugs target
Gram-Positive Bacteria
Teichoic (TA) & Lipoteichoic Acids (LTA)
•TA linked directly to the peptidoglycan layer
•LTA possess a transmembrane domain so anchored into the bacterial cell
membrane
•Expression of both essential for bacterial viability
•Provide a channel of negative charges so as to attract positively charged
substances to the cell membrane (through the thick peptidoglycan)
•Both function in adherence to surfaces
Gram-Negative Bacteria
Porins
•Channels in outer membrane which permit transport of small
hydrophilic materials into the periplasm
•Antibiotics (Ab) can place selection pressure on the bacteria
resulting in mutation of porins to exclude drug passage
Gram-Negative Bacteria
Lipopolysaccharide (LPS)
•~750,000 sepsis cases/y (30%
mortality)
•~40% of septic patients
progress to shock
•TNF/IL-1 induce tissue
factor release by
endothelial cells  clots
form
•Clots lodge in blood vessels
lowering profusion of
organs  organ failure
•Clot formation depletes
blood of clotting factors
(coagulopathy)  DIC
***TLR-4 recognizes!
LPS is an endotoxin!
Variations from Gram + or 1.Acid Fast Organisms
2.Mycoplasmas and Chlamydia
Acid-Fast Cell Wall
Mycolic Acid (on outer most layer)
•Acid-fast microbes contain this waxy material
•Prevents Gram staining
•Expressed by Mycobacterium and Nocardia
•Use an Acid-fast staining protocol to detect these cell walls
•Takes up initial stain (carbol fuchsin) while other bacteria decolorize and
take on counterstain (methylene blue)
Bacteria Without Cell Walls
Mycoplasma
•Smallest known bacteria (agent of pneumonia)
•Does not take up the Gram stain
•Not easily visible under the light microscope
Bacterial Replication (Binary Fission) (Make split-pea babies! Only takes 20-30min)!!!
The bacterial cell lacks a nuclear
membrane; instead, DNA is
concentrated in the cytoplasm as a
nucleoid (one or multiple copies of a
double-stranded, circular closed,
supercoiled DNA).
In many bacteria, a small portion of the
DNA persists as extrachromosomal
elements referred to as plasmids,
which are also circular but are much
smaller than bacterial chromosomes.
Plasmids encode variable numbers of
genes and often determine virulent
behavior.
Bacterial Ribosomes
Note below: Top is for Prokaryotes; bottom is eukaryotic ribosomes.
Description is for Eukaryotes, but applies to prokaryotes!
The tetracyclines (tetracycline, doxycycline, demeclocycline, minocycline,
etc.) block bacterial translation by binding reversibly to the 16S rRNA in the
30S subunit and distorting it in such a way that the anticodons of the charged
tRNAs cannot align properly with the codons of the mRNA.
-antibiotics bind to 16s
Flagella: motility; made of
flagellin; rotary action for
movement
Flagella Arrangements
 Peritrichous flagella-distributed over the surface
of the bacterium
 Monotrichous flagellum-some bacteria only have a single flagellum
 Polar flagella or Lophotrichous-bundled at one or both ends of the bacterium
Pili: attachment onto mucosal surfaces
F-pili: sex pili for bacterial
conjugation
Pili are protein fibers cover the entire surface of Gram-negative bacteria.
Motility
•Some bacteria are motile.
•Bacteria may use flagella or other means for motility.
•Motility may also be by pili (fimbria) or by gliding.
Capsules (protection; made of polysaccharides)
(Glycocalyx-Slime Layer)
•A polysaccharide material secreted by cells and associated with its surface.
•Slime layers are less organized and more loosely attached to surface.
•Highly immunogenic (K antigen).
Negative staining used to observe.
•Considered a virulence factor:
–Helps bacteria avoid phagocytosis.
–Helps bacteria attach to surfaces.
–Capsules aid in adhesion to surfaces.
Examples capsule of pathogens:
***Streptococcus pneumoniae
***Pseudomonas aeruginosa
***Bacillus anthracis
–Klebsiella pneumoniae
–Haemophilus influenzae
–Neisseria meningitides
–Cryptococcus neoformans
Biofilm (community of bacteria in GI tract compete with pathogenic bacteria for surface area)
 Biofilms formed by aggregation of bacteria secreting protein or carbohydrate coats (capsules) which
combine to surround a colony of bacteria.
 Some bacteria can form aggregates by pili interactions leading to biofilm formation. These biofilms
allow bacteria to persist in environments despite body responses such as immunity and chemicals
that would remove them.
 Biofilms are helpful for some environments such as enriching sand or soils but harmful for animal
tissues (dental plaque). For animals, if biofilms remain, excess wastes in the form of acids build up
and cause tissue loss (cavities).
Endospore
•Dormant form of certain bacteria that remain viable for decades
•Endospores found in soil and tissues/fluids of dead animals but
not in tissues/fluids of living animals
•Resists heat, desiccation, extremes of pH and radiation (makes
hard to kill, like Anthrax)
Examples capsule of pathogens:
–Bacillus spp. (e.g. B. anthracis, B. subtilis).
–Clostridium spp. (e.g. C. tetani, C. difficile ).
Endospore Formation: endospore is formed from original cell
Bacterial Physiology
Chemical/Nutritional Requirements
•Carbon, Nitrogen, Sulfur, Phosphorus
 Carbon is backbone of all living matter
 Nitrogen, sulfur, phosphorus are required for synthesis of proteins, DNA, RNA and ATP
•Oxygen
 May be needed for energy production through respiration
 Toxic to some bacteria
 If required (or if tolerated), bacteria have mechanisms to remove oxygen byproducts
 Use of oxygen may lead to formation of superoxides, hydrogen peroxide and hydroxyl radicals –
enzymes will be needed to protect from these toxins (Respiratory bursts)
Oxygen Requirements for Bacterial Growth
Bacteria can be divided into
five groups on the basis of
their oxygen requirements.
1)Obligate aerobes: The
growth of aerobic bacteria in
a nutrient-rich medium is
restricted by limited
availability of oxygen.
2)Obligate anaerobes:
Conversely, the growth of
strict anaerobes may be
inhibited by an oxygen
tension as low as 10-5
atmospheres (atm).
3)Facultative anaerobes are
able to use molecular oxygen,
organic and inorganic
compounds as terminal
electron acceptors.
4)Microaerophilic bacteria grow best under decreased oxygen tension (generally 2-5%-air is 20%) as
obtained in a candle jar.
5)Aerotolerant bacteria can survive (but not grow) for a short period of time in the presence of
atmospheric oxygen.
The tolerance to oxygen is related to the ability of the bacterium to detoxify superoxide and hydrogen
peroxide, produced as by-products of aerobic respiration. Two key enzymes are involved in this
detoxification:
1)Superoxide dismutase: which converts superoxide (the most toxic metabolite) into hydrogen peroxide,
is present in aerobic and aerotolerant bacteria.
2)Catalase: which converts hydrogen peroxide into water and oxygen is also present in all aerobic
bacteria but is lacking in aerotolerant organisms. Strict anaerobes lack both enzymes.
•Energy metabolism
–3 pathways: 1.Fermentative metabolism 2.Respiratory metabolism 3.Autotrophic metabolism
1.Fermentative
•Uses organic compounds as both electron donors and acceptors
•Includes the
–Glycolytic (Embden-Meyerhof pathway)-is the major glucose utilization pathway used by most aerobic
and anaerobic bacteria

Pathway divided into 2 phases
o Phase I glycolysis
o Phase II glycolysis
– Enter-Doudoroff pathway (alternative pathway; doesn’t make enough ATP – would only make 1 ATP)-is
the major hexose-degrading pathway in organisms that lack phosphofructokinase (ex. Members of
Pseudomonas genus).
–Pentose-Phosphate pathway
Autotrophic Metabolism
•Anaerobes can use a variety of inorganic sources for energy and reducing power
•Can use the ETC but they just need to find an alternative terminal electron acceptor; instead of O2
•Examples of alternates:
–Nitrate/Nitrite
–Sulfate/Sulfite
–Carbon Dioxide
-Iron
Iron Acquisition (Lactoferrin takes up free iron)
•Iron is an essential element for bacterial growth and pathogenesis
•Require free iron
•Have evolved mechanisms to acquire iron
–Siderophores
–Iron reductase-reduces ferrrin iron (insoluble) to ferrous iron (soluble)
–Hemolysins-lyse red blood cells thereby releasing hemoglobin
–Exotoxins-may lyse eukaryotic cells
Bacterial Growth
Physical Requirements
•Temperature
−Required for normal enzymatic function
−Most medically important are mesophiles
−Food spoilage usually occurs by psychrotrophs
•pH
−Range from 6.5 to 7.5
−Preserved food with high pH prevents bacterial growth
−Acidophiles may survive in high acidity
−Bacterial products result in change in pH
oBreakdown of carbohydrates produce acids
−Buffers are added to growth media
Generation Time
•Interval of time between successive
replication events
•Highly variable for different species:
−Staphylococcus aureus generation time = 30
min
−Treponema pallidum generation time = 33 h
•Uncontrolled growth prevented only by
depletion of food, buildup of waste or some
other limiting physical requirement
Phases
•Lag Phase
−First few hours of growth
−No cell division occurs
−Bacteria adapting to the environment
•Log Phase
−Active stage of exponential growth
•Stationary Phase
−Reproduction = Cell death
•Decline Phase
−Death > Reproduction
Bacterial Identification
•Staining Properties: Gram Pos: Purple; Gram Neg: Pink/Red
•Morphological: SHAPE: Spirilla, bacilli, cocci; ARRANGEMENT: diplo, strept, tetrad, staph, sarcina
•Metabolic Activity.
•Serological.
•Molecular
Examples of morphological:
•Macroscopic:
–Colony size and shape.
–Colony color and texture.
–Colony elevation and margin.
•Microscopic:
–Cell shape.
–Arrangement.
–Differential staining
–Motility.
–Spore formation.
Classification by Metabolic Activity
1.Oxygen requirements:
–Aerobic
–Anaerobic
–Facultative
2.Carbohydrate utilization
–Identified by the variety of carbohydrates they use as energy sources
3.Enzyme Production
Carbohydrate Utilization
•Identification of microbes based upon pathways involved in ATP synthesis (specific enzymes used or
metabolic products)
Examples of tests for Enzyme Production
Identification by Serologic Reactivity
•Determined by using specific antisera that are directed against
antigenic determinants of the bacterial cell
•Carried out by agglutination techniques – test blood types
Serotyping is usually carried out by agglutination techniques that entail
mixing a drop of antiserum with a drop of bacterial suspension. A
positive reaction results in clumping of the bacterial suspension.
Genetic relatedness established by:
–The ability to exchange genetic information (i.e. via transformation or conjugation) which is
•only possible between related organisms
–Nucleotide base composition
•G-C:A-T ratio
–Nucleic acid homology among bacteria
•Determined by hybridization studies
–Nucleic acid sequences
–Homology of 16s rRNA sequences