Microbiology
31.03.2015
Helmut Pospiech
The cell
wall of
Eubacteria
Brock Biology of Microorganisms, 13th ed.
Other cell surface structures of
prokaryotes – fimbriae and pili
Fimbriae:
• Many per cell
• Used mainly for attachment
Pili
• Only one or few per cell
• More extended than
fimbriae
• Also used for attachment and
intercellular communication
Brock Biology of Microorganisms, 13th ed.
Other cell surface
structures of prokaryotes –
capsules and slime layers
• Gel-like matrix around the cell
• Polymers
– often polysaccharides
– Sometimes amino acid polymers
• Evasion of immune system
• Biofilm formation (attachment)
• Increasing resistence against
desiccation
Brock Biology of Microorganisms, 13th ed.
Flagella
• Used for motility
• Bacteria differ in terms
of number, size, position
and function of flagella
Brock Biology of Microorganisms, 13th ed.
The structure and
function of a
eubacterial flagellum
Brock Biology of Microorganisms, 13th ed.
Flagella Biosynthesis
Brock Biology of Microorganisms, 13th ed.
Chemotaxis – moving relative to some
concentration gradient of a chemical compound
How chemotaxis is achieved?
• Cell must have a ”memory” system
Different ways
to change
direction while
swimming
Brock Biology of Microorganisms, 13th ed.
Gliding Motility
Several mechanisms exist:
• Slime excretion
 cell pulled along by adherence
• Type IV pili
• Ratcheting movement
Brock Biology of Microorganisms, 13th ed.
Secretion of proteins
• The sec system is the most common and important protein
translocase in bacteria
• Structurally and functionally related to the secretion of
proteins in the eukaryotic ER
• Depends on a signal sequence
Also used for
integral membrane
proteins
Mori & Ito (2001) Trends Microbiol. 9:494-500.
Several protein translocases are
operational in bacteria
Dependent on final destiny of the protein
Desvaux et al. (2009) Trends Microbiol 17:139-45
Secretion of proteins across the outer
membrane – ”many way lead to Rome”
• Type 1 secretion system
(T1SS)
• e.g. E. coli haemolysin
(HlyA)
– TolC forms a pore in the outer
membrane
– HlyD can connect to TolC to
form a continuous channel
through the periplasm
– HlyB is an ABC transporter,
that exports its target
proteins under expense of
ATP hydrolysis
– Signal sequence in the Cterminal region of the target
proteins
Gentschev I et al. (2002) Trends Microbiol. 10:39-45.
Type 2 Secretion system (T2SS)
– the major way out from the periplasm
•
•
•
•
•
•
The type II secretion system (T2SS) is a doublemembrane-spanning protein secretion system
composed of 12–15 different general secretory pathway
(Gsp) proteins
It is found in a large number of pathogenic and nonpathogenic Gram-negative bacteria.
The T2SSs of different species secrete a wide variety of
folded exoproteins of different functions, shapes, sizes
and quaternary structures.
The T2SS secretion signal is still unknown, but it has
been suggested that β-complementation is a feature of
this signal.
The T2SS contains several subassemblies: the outermembrane secretin, the periplasmic pseudopilus and a
cytoplasmic ATPase all interact with components of the
inner-membrane platform.
The current hypothesis regarding the mode of T2SS
action is that an exoprotein is captured in the
periplasmic vestibule of the outer-membrane secretin,
possibly assisted by the inner-membrane protein GspC,
and that this induces ATP hydrolysis by the ATPase,
leading to conformational changes in the ATPase and
the inner-membrane platform. This, in turn, results in
elongation of the pseudopilus, which then functions as a
piston, opening the periplasmic gate in the outermembrane secretin to form a channel and then
expelling the exoprotein.
Korotkov et al. (2012) Nat Rev Microbiol 10:336-51
Type 3 secretion system (T3SS)
- An organic nanosyringe
• Used by many
pathogenic bacteria
to deliver proteins
into host cells
– e.g. Salmonella,
Yersinia pestis or
Shigella
• Secretion coupled
with translation
• Signal in the 5’ end
of mRNA, not in the
protein
Marlovits & Stebbins (2010) Current Opinion in Microbiology 13, 47 - 52
Desvaux et al. (2009) Trends Microbiol 17:139-45
Desvaux et al. (2009) Trends Microbiol 17:139-45
Endospores
• Endospores are formed by certain
bacteria as dormant (retsing) stage
that is resistant to unfavourable
environmental conditions like
–
–
–
–
–
Heat
Drying
Acid
Chemical disinfectants or radiation
Nutrient exhaustion
• They can remain viable for
extremely long periods of time
• Most endospore formers belong to
the genera Bacillus and Clostridium
– Common
– Can be found in every soil sample
Brock Biology of Microorganisms, 13th ed.
Structure of the endospore
• Very low water content
• Extremely little metabolic activity
• The core contains large amounts
of small acid-soluble spore
proteins (SASPs)
– Binds and protects DNA
– Serves as carbon source during
germination)
• The core also contains large
amounts of dipicolinic acid
complexed with Ca2+
Brock Biology of Microorganisms, 13th ed.
Endospore formation
• Takes about 8 hours
in B. subtilis
• 8 stages can be
separated by
microscope
• Is highly regulated
– More than 200
genes involved
– Transcription
cascade
Brock Biology of Microorganisms, 13th ed.
Endospores – the ultimate survival package
Brock Biology of Microorganisms, 13th ed.
Spore Germination
Very rapid – takes only few minutes
1. Activation
•
Heat shock or prolonged rest phase
2. Germination
•
•
•
Loss of dipicolinic acid
SASP cleavage
Water uptake
3. Outgrowth
How long can an endospore survive?
Thermoactinomyces sp. from lake sediments and archaeological
sites
Several 1000 years – Gest & Mandelstam 1987
Bacillus sp. from insect guts
embedded in amber?
25-40 million years
– Cano & Borucki 1995
Cano lab home page
Halotolerant endospore former from
primary salt crystals??
250 million years
– Vreeland et al. 2000
from Nature 407, 844
Nutrition and Growth
of Bacteria
Composition of the bacterial cell
Brock Biology of Microorganisms, 13th ed.
Nutrition
Macronutrient:
• Required in large amounts
–
–
–
–
–
Carbon
Nitrogen
Phosphorus (HPO42-)
Sulfur (HS-, HSO4-)
K, Mg, Ca, Na
•
•
Micronutrients:
Required in small amounts
– Trace elements:
• Iron - Fe2+ is soluble, whereas
Fe3+ requires siderophores
(chelate that makes Fe3+ soluble)
• Copper, zinc, manganese and
other metals
– Growth factors
• vitamins, amino acids etc.
Brock Biology of Microorganisms, 13th ed.
Nutritional Categories of Organisms
Organisms are described according to
1. their requirements for the energy source
2. their requirements for the (principle) carbon
source
3. their “hydrogen” donator
4. their relationship towards oxygen
the requirements for the energy
source
Phototrophs
 use light as an energy
source (e.g. plants)
Chemotrophs
 are dependent on a
chemical energy
source (e.g. animals)
Brock Biology of Microorganisms, 13th ed.
the requirements for the (principle)
carbon source
•
Many microorganism can use a single organic
compound as carbon and energy source.
They synthesize all cellular constituents from
this compound. These are called
prototroph (e.g. Escherichia coli)
•
Other organisms require additional
compounds which cannot be synthesized by
the cell itself. These compounds are called
growth factors
and the organisms requiring growth factors
are called
auxotrophs (e.g. humans)
•
Typically growth factors are
–
–
–
amino acids (required for protein synthesis)
purines and pyrimidines (required for nucleic
acid synthesis)
vitamins (required as prosthetic groups or
cofactors of enzymes)
Autotrophs
• use CO2 as a principle
carbon source (e.g.
plants)
Heterotrophs
• are dependent on an
organic carbon source
(e.g. animals)
The requirement for the principle carbon source and additional growth factor is very
different for different microorganisms, but
 all naturally produced organic compounds can be used as a source of
carbon and energy by some microorganism.
the requirements for the
“Hydrogen”/reduction equivalent donor
Lithotrophs
• use anorganic “hydrogen”
donors (e.g. H2, NH3, H2S,
S, CO, Fe2+, H2O)
(e.g. plants)
Organotrophs
• are dependent on organic
“hydrogen” donators (e.g.
glucose or other sugars,
amino acids etc.)
(e.g. animals, fungi)
Brock Biology of Microorganisms, 13th ed.
Catabolic diversity of microbial life
Brock Biology of Microorganisms, 13th ed.
Relation of Microorganisms
to Oxygen
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•
•
•
•
O2 and its metabolism is toxic for every cell
Some important microbial enzymes are very oxygen-sensitive
(e.g. nitrogenase and hydrogenase).
The hydroxyl radical is the most reactive and toxic oxygen species in the
cell.
The toxic effect of reactive oxygen is due to modification or destruction
of cellular macromolecules like proteins, lipids or nucleic acids.
Microorganisms can be divided into 2 major categories according to
their requirement for oxygen:
Brock Biology of Microorganisms, 13th ed.
Formation and detoxification of
reactive oxygen
H2O
Brock Biology of Microorganisms, 13th ed.
Environmental Factors Affecting
Microbial Life
The cardinal temperatures:
Brock Biology of Microorganisms, 13th ed.
Classification of microbes based on
their temperature preferences
Brock Biology of Microorganisms, 13th ed.
Classification of microbes based on
their temperature preferences
Brock Biology of Microorganisms, 13th ed.
Life at extreme temperatures requires
adaptation
• Protein stability and flexbility
– E.g. proteins with more α-helices and polar side
chains in psychrophiles
– More S-S bounds in thermophiles
• Lipids – membrane have to maintained in a
liquid state
– E.g. more unsaturated fatty acids in psychrophiles
– Bifunctional lipids spanning the complete bilayer
Microbes and pH
• Intracellular pH is always
close to neutral
• As for temperature, growth
at extreme pH requires
extensive adaptation
Effect of Salts and Osmotic
Compounds on Microbial Growth
•
•
•
•
salts have effect on
* ionic strength
* osmotic strength
the intramolecular salt concentration (ionic
strength) influences strongly the stability of
enzymes and other macromolecules
divalent cations (e.g. Mg 2+, Ca2+) have a
stronger effect on the ionic strength than
monovalent
⇒ the cell has to keep the intracellular ionic
strength constant and at the same time adjust
to the osmotic strength of the medium
How is this done?
•
the use of compatible solutes:
compounds that adjust the osmolarityinside the
cell to that of the medium having little effect on
cellular functions (e.g. enzyme activity)
(e.g. K+, sugars, glycerol, betains etc.)
•
adjustment of intracellular di- and polyvalent
cations to kept the ionic strength constant (by
decrease of intracellular concentrations of e.g.
Mg2+, Ca2+, putrescine2+ or other polyamines
with increasing salt concentration of the
medium)
Classification of microbes based on
their salt preferences
• Halophiles – high salt concentration
• Osmophiles – high osmotic pressure (e.g. honey, jam)
• Xerophiles – low water content (e.g. dried fruit)